Biopharmaceutics & Pharmacokinetics (Thakur)

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BIOPHARMACEUTICS &
PHARMACOKINETICS
B.Pharm, Semester-VI
According to the syllabus based on ‘Pharmacy Council of India’
Dr. N. Deattu
M. Pharm, Ph.D
Assistant Professor
College of Pharmacy, Madras Medical College, Chennai
Prof. (Dr.) N.B. Santha Sheela

M. Pharm, Ph.D
Professor & Head (Department of Pharmaceutics)
Mohamed Sathak A. J. College of Pharmacy, Chennai
Mr. Sunil Kumar

Assistant Professor
M. Pharm, (Ph.D)
Vaish Institute of Pharmaceutical Education & Research, Rohtak,
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Biopharmaceutics & Pharmacokinetics

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Preface
It gives us immense pleasure to place before the B.Pharm Sixth Semester
pharmacy students the book on “Biopharmaceutics & Pharmacokinetics”.
This book has been written strictly in accordance with the current syllabus
prescribed by Pharmacy Council of India, for B.Pharm students. Keeping in view
the requirements of students and teachers, this book has been written to cover all
the topics in an easy -to-comprehend manner within desired limits of the
prescribed syllabus, and it provides the students fundamentals of
pharmacokinetics of drugs, different pharmacokinetic parameters to be
determined from compartment models, multi -compartment models, and non -
linear pharmacokinetics which are required by them during their pharmaceutical
career.
All efforts have been made to keep the text error -free and to present the subject
in a student friendly and easy to understand. However, any suggestions and
constructive comments would be highly appreciated and incorporated in the
future edition.
Please e-mail us at, thakurpublication@gmail.com
Website, www.tppl.org.in
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Acknowledgement
It is with great privilege that I acknowledge my sincere gratitude to Dr. R.
Jayanthi, Dean, Madras Medical College, Chennai and Prof. K. Chinnaswamy
for their constant motivation and support. I take this opportunity to express my
heartfelt thanks to Prof. Dr. M. Nagaraj , Dr. S. Manivannan , Prof. Dr. A.
Jerad Suresh , Prof. Dr. N. Narayanan and Dr. Narmada Sambasivam , for
their suggestions and encouragement. I sincerely thank Mr. J. Jayseelan who
has always been an inspiration for me. I convey my gratitude wholeheartedly to
Thakur Publication Pvt . Ltd. for the efforts taken in the publication of this
book.
-Dr. N. Deattu
I wish to thank Thakur Publication for giving me this opportunity for writing
this book. I thank the Management of Mohamed Sathak A.J.College of
Pharmacy, Sholinganallur, Chennai , for providing me suitable working
environment to carry out my work in a smooth and efficient manner.
I am extremel y thankful to Dr. R. Sundhararajan , M.Pharm, Ph.D., Principal,

Mohamed Sathak , A. J. College of Pharmacy, Sholinganallur, Chennai, for
guiding me to reach this milestone and also providing valuable suggestions in
writing this book. I am extremely grateful to bestow my honor and respect to all
my Teachers who have guided me throughout my career. I also thank Mrs. G.
Rajalakshmi, M.Pharm, Ph.D., Associate Professor, Department of
Pharmaceutics, for sharing her suitable suggestions. Above all I thank God for
providing me this opportunity and helping me to overcome all hindrances and
obstacles to reach my goal in a smooth and efficient manner.
- Prof. (Dr.) N.B. Santha Sheela
I am very thankful to my teachers, parents and family members for encouraging

in all the efforts. I make extending their support, guiding at each and every step.
I thank almighty God, for showing his infinite mercies on me every day for
achieving all the desire goal of my life.
I would like to thank a lot to Thakur Publication Pvt. Ltd. and their entire team
for their timely assistance and coordination
-Mr. Sunil Kumar
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Syllabus
Module 01 10Hours
Introduction to Biopharmaceutics
Absorption
 Mechanisms of drug absorption through GIT, factors influencing drug
absorption though GIT, absorption of drug from Non per oral extra -
vascular routes.
Distribution
 Tissue permeability of drugs, binding of drugs, apparent, volume of drug
distribution, plasma and tissue protein binding of drugs, factors affecting
protein-drug binding. Kinetics of protein binding, Clinical significance of
protein binding of drugs.
Module 02 10Hours
Elimination
 Drug metabolism and basic understanding metabolic pathways renal
excretion of drugs, factors affecting renal excretion of drugs, renal
clearance, Non-renal routes of drug excretion of drugs.
Bioavailability and Bioequivalence

 Definition and Object ives of bioavailability, absolute and relative
bioavailability, measurement of bioavailability, in -vitro drug dissolution
models, in-vitro-in-vivo correlations, bioequivalence studies, methods to
enhance the dissolution rates and bioavailability of poorly soluble drugs.
Module 03 10Hours
Pharmacokinetics
 Definition and introduction to Pharmacokinetics, Compartment models,
Non compartment models, physiological models, One compartment open
model. Intravenous Injection (Bolus). Intravenous infusion. Extra
vascular administrations.
 Pharmacokinetics parameters – KE ,t1/2,Vd,AUC,Ka, Clt and CLR -
definitions methods of eliminations, understanding of their significance
and application.
Module 04 08 Hours
Multicompartment Models
 Two compartment open model. IV bolus.
 Kinetics of multiple dosing, steady state drug levels, calculation of
loading and mainetnance doses and their significance in clinical settins.
Module 05 07 Hours
Nonlinear Pharmacokinetics
 Introduction.
 Factors causing Non-linearity.
 Michaelis-menton method of estimating parameters, Explanation with
example of drugs.
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Contents
Chapter 1: Introduction to Chapter 2: Introduction to
Biopharmaceutics and Absorption Distribution
1.1. Biopharmaceutics 9 2.1. Distribution 45
1.1.1. Introduction 9 2.1.1. Introduction 45
1.1.2. Applications of 9 2.1.2. Tissue Permeability of Drugs 46
Biopharmaceutics 2.1.3. Physiological Barriers 48
1.2. Absorption 10 2.1.4. Factors Affecting Drug 51
1.2.1. Introduction 10 Distribution
1.2.2. Mechanisms of Drug 10 2.1.5. Volume of Distribution 52
Absorption Through GIT 2.1.6. Apparent Volume of 52
1.2.2.1. Passive Diffusion 11 Distribution
1.2.2.2. Carrier-Mediated Transport 12 2.1.7. Binding of Drugs 53
1.2.2.3. Facilitated Diffusion 12 2.1.7.1. Plasma Protein Binding of 55
1.2.2.4. Active Transport 13 Drugs
1.2.2.5. Pore Transport 14 2.1.7.2. Binding of Drugs to Blood 56
1.2.2.6. Ionic or Electrochemical 14 Cells
Diffusion 2.1.7.3. Tissue Binding of Drugs 57
1.2.2.7. Ion-Pair Transport 15 2.1.8. Factors Affecting Protein - 59
1.2.2.8. Endocytosis 15 Drug Binding
1.3. Factors Influencing Drug 16 2.1.8.1. Drug-Related Factors 59
Absorption through GIT 2.1.8.2. Protein/Tissue Binding - 59
1.3.1. Introduction 16 Related Factors
1.3.2. Physicochemical Factors 16 2.1.8.3. Drug Interactions 60
1.3.3. Pharmaceutical Factors 26 2.1.8.4. Patient-Related Factors 62
1.4. Absorption of Drugs from 31 2.1.9. Kinetics of Protein Binding 62
Non per Oral Extra - 2.1.10. Clinical Significance of 64
Vascular Routes Protein Binding of Drugs
1.4.1. Introduction 31 2.2. Summary 65
1.4.2. Sublingual/Buccal Route 31 2.3. Exercise 67
1.4.3. Intravenous (IV) Route 32
1.4.4. Intramuscular (IM) Route 33 Chapter 3: Introduction to
1.4.5. Subcutaneous (SC) Route 34 Elimination
1.4.6. Inhalational Route 34 3.1. Drug Metabolism 67
1.4.7. Transdermal Route 35 3.1.1. Introduction 67
1.4.8. Topical Route 35 3.1.2. Organs Involved in Drug 68
1.4.9. Rectal Route 37 Metabolism
1.4.10. Vaginal Route 37 3.1.3. Enzymes Involved in Drug 68
1.4.11. Intraocular Administration 38 Metabolism
1.4.12. Absorption of Drugs from 39 3.1.4. Factors Affecting Drug 68
Common Non per Oral Metabolism
Routes of Drug 3.2. Basic Understanding of 69
Administration Metabolic Pathways
1.5. Summary 40 3.2.1. Introduction 69
1.6. Exercise 43 3.2.2. Phase I Metabolic Pathways 70
3.2.2.1. Hydrolysis Reaction 70
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3.2.2.2. Reduction Reaction 73 4.2.2. Use of Salt Forms 108

3.2.2.3. Oxidation Reaction 75 4.2.3. Co-Crystallisation 109
3.2.3. Phase II Metabolic Pathways 82 4.2.4. Cosolvency 109
3.2.3.1. Glucuronidation 82 4.2.5. Hydrotrophy 110
3.2.3.2. Sulphation 84 4.2.6. Solubilising Agents 110
3.2.3.3. Acetylation 84 4.2.7. Nanotechnology Approaches 110
3.2.3.4. Methylation 85 4.2.8. Particle Size Reduction 110
3.2.3.5. Glutathione Conjugation 85 4.2.9. Modification of the Crystal 111
3.2.3.6. Amino Acid Conjugation 87 Habit
3.3. Drug Excretion 87 4.2.10. Complexation 112
3.3.1. Introduction 87 4.2.11. Solubilisation by Surfactants 112
3.3.2. Clearance and Renal 87 4.2.12. Drug Dispersion in Carriers 113
Clearance 4.3. In Vitro-In Vivo 115
3.3.3. Renal Excretion of Drugs 89 Correlations (IVIVC)
3.3.3.1. Glomerular Filtration 90 4.3.1. Introduction and Definition 115
3.3.3.2. Active Tubular Secretion 90 4.3.2. Purpose of IVIVC 115
3.3.3.3. Tubular Re-Absorption 91 4.3.3. Levels of IVIVC 116
3.3.4. Factors Affecting Renal 91 4.3.3.1. Level A Correlation 116
Excretion of Drugs 4.3.3.2. Level B Correlation 116
3.3.5. Non-Renal Routes of Drug 93 4.3.3.3. Level C Correlation 116
Excretion 4.3.3.4. Multiple Level C Correlations 117
3.4. Summary 96 4.3.3.5. Level D Correlation 117
3.5. Exercise 98 4.3.4. In vitro Drug Dissolution 117
Models
Chapter 4: Bioavailability and 4.3.5. Applications of an IVIVC 120
Bioequivalence 4.3.6. Limitations in the IVIVC 122
4.1. Bioavailability 100 Arising from In Vivo Data
4.1.1. Introduction and Definition 100 4.4. Bioequivalence Studies 122
4.1.2. Objectives of Bioavailability 100 4.4.1. Introduction and Definition 122
Studies 4.4.2. Objectives for Bioequivalence 123
4.1.3. Types of Bioavailability 100 Studies
4.1.3.1. Absolute Bioavailability 101 4.4.3. Types of Bioequivalence 123
4.1.3.2. Relative Bioavailability 102 Studies
4.1.4. Measurement of 103 4.4.4. Bioequivalence Study 124
Bioavailability Parameters
4.1.4.1. Plasma Level-Time Studies 104 4.4.5. Evaluation of Bioequivalence 125
Data
4.1.4.2. Urinary Excretion Studies 105
4.4.6. Significance of 127
4.1.4.3. Acute Pharmacologic 107
Bioequivalence Studies
Response
4.1.4.4. Therapeutic Response 107 4.5. Summary 128
4.1.5. Significance of 107 4.6. Exercise 131
Bioavailability Studies
4.2. Methods to Enhance the 108 Chapter 5: Pharmacokinetics
Dissolution Rates and 5.1. Pharmacokinetics 133
Bioavailability of Poorly 5.1.1. Definition and Introduction 133
Soluble Drugs 5.1.2. Objectives 133
4.2.1. Introduction 108 5.1.3. Applications 134
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5.2. Pharmacokinetic Models 134 Intravenous Infusion

5.2.1. Introduction 134 5.3.5.3. Determination of 163
5.2.2. Compartment Models 135 Pharmacokinetic Parameters
5.2.2.1. Types 135 from Urine Data after
5.2.2.2. Advantages 136 Extravascular Administration
5.2.2.3. Disadvantages 137 5.4. Summary 164
5.2.3. Non-Compartmental Models 137 5.5. Exercise 166
5.2.3.1. Statistical Moment Theory 138
5.2.3.2. Advantages 139 Chapter 6: Multicompartment
5.2.3.3. Disadvantages 139 Models
5.2.4. Physiological Models 139 6.1. Multi-Compartment Models 168
5.2.4.1. Types 141 6.1.1. Introduction 168
5.2.4.2. Advantages 141 6.1.2. Two Compartment Open 169
5.2.4.3. Disadvantages 141 Model
5.3. One Compartment Open 142 6.1.3. Two Compartment Open 170
Model – I.V. Bolus
Model
5.3.1. Introduction 142 6.1.4. Two Compartment Open 174
Model – I.V. Infusion
5.3.2. Intravenous Injection (Bolus) 143
6.1.5. Two Compartment Open 175
5.3.2.1. Elimination Rate Constant 143
Model - Extravascular
(KE)
Administration
5.3.2.2. Elimination Half-Life (t1/2) 145
6.1.6. Kinetics of Multiple Dosing 176
5.3.2.3. Apparent Volume of 145
6.1.7. Steady State Drug Levels 177
Distribution (Vd)
6.1.8. Calculation of Loading and 178
5.3.2.4. Clearance (ClR) 146
Maintenance Doses and their
5.3.2.5. Total Body Clearance (Cl T) 146 Significance in Clinical
5.3.2.6. Area Under Curve (AUC) 147 Settings
5.3.3. Intravenous Infusion 148 6.2. Summary 180
5.3.4. Extravascular Administration 150 6.3. Exercise 181
5.3.4.1. Determination of 152
Pharmacokinetic Parameters -
Chapter 7: Non-Linear
Cmax and tmax
Pharmacokinetics
5.3.4.2. Elimination Rate Constant 153
7.1. Non-Linear 182
5.3.4.3. Absorption Rate Constant 153
Pharmacokinetics
(Ka)
7.1.1. Introduction 182
5.3.5. Methods of Elimination 158
7.1.2. Factors Causing Non - 182
5.3.5.1. Determination of 160 Linearity
Pharmacokinetic Parameters
7.1.3. Michaelis-Menten Method of 184
from Urine Data after
Estimating Parameters
Intravenous Bolus
Administration 7.2. Summary 190
5.3.5.2. Determination of 162 7.3. Exercise 190
Pharmacokinetic Parameters
from Urine Data after
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Introduction to Biopharmaceutics and Absorption (Chapter 1) 9
CHAPTER Introduction to Biopharmaceutics

1 and Absorption
1.1. BIOPHARMACEUTICS
1.1.1. Introduction
Drugs are used in the diagnosis, cure, mitigation, treatment, or prevention of
diseases. They are given in various dosage forms, such as solids (tablets,
capsules, etc.), semisolids (ointments, creams, etc.), liquids, suspensions,
emulsions, etc., for systemic or local therapeutic activity. Drug products are drug
delivery systems that deliver and release drug at the action site so that they
produce the desired therapeutic effect. These products are also designed to ful fil
the patient’s requirements including palatability, convenience, and safety.
Release of drug substance from the drug product either for local drug action or
for plasma drug absorption for systemic therapeutic action defines the drug
product performance . Advancements in pharmaceutical technology and
manufacturing have led to the development of safer, more effective, and more
patient convenient quality drug products.
Biopharmaceutics is defined as, ―study of the interrelationship of

physicochemical properties of the drug, dosage form in which the drug is
given and the administration route on the rate and extent of systemic drug
absorption.‖
Biopharmaceutics is also defined as ―study of the factors influencing the rate

and amount of drug that reaches the systemic circulation and the use of this
information to optimise the therapeutic efficacy of drug products.‖
1.1.2. Applications of Biopharmaceutics

The field of biopharmaceutics has the following applications:
1) When a newly developed dosage form by a company is given to human beings,
sometimes the drug is released slowly and sometimes the entire drug is released
at a time; both these situations are not useful. Thus, the principles of
biopharmaceutics are utilised to obtain the required action from the formulation.
2) When a company wishes to change the ingredients of tablet dosage forms, the
altered ingredients will be approved by the FDA only if the bioavailability is
equal to the initial formulation. Thus, the principles of biopharmaceutics are
utilised to study the bioavailability of the new ingredients.
3) When a company wishes to change a drug’s administration route from oral to
transdermal, bioavailability of the drug is compared from both the routes and
will be released only if the bioavailability is similar. Thus, the principles of
biopharmaceutics are utilised to study the bioavailability of the drug.
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10 Biopharmaceutics & Pharmacokinetics
1.2. ABSORPTION
1.2.1. Introduction
The desired therapeutic objective can be achieved if the drug product delivers the
active drug at an optimal rate and extent. If a proper biopharmaceutical design is
maintained, the rate and extent of drug absorption (or bioavailability) or the
systemic delivery of drug into the body can be altered from rapid and complete
absorption to slow and sustained absorption; however, this depends on the
desired therapeutic action. The events that occur after a solid dosage form (a
tablet or a capsule) is administered until its absorption in the systemic circulation
are shown in the figure 1.1.
GI lumen GI barrier Blood
Solid dosage Dissolution Non-ionic Non-ionic

forms minor (3) drug drug
Disintegration
Drug in solution
(1)
at the absorption
Granules or Dissolution site Absorption (4)
aggregates major (3)
Deaggregation
(2)
Dissolution Ionic Ionic
Fine particles major (3) drug drug
Figure 1.1: Sequence of Events in the Absorption of Drugs after Orally Administered
Solid Dosage Forms
These events comprise of the following four steps:
1) Disintegration of the drug product,
2) De-aggregation and release of the drug,
3) Dissolution of the drug in aqueous fluids at the absorption site, and
4) Movement of the dissolved drug throug h the gastrointestinal membrane into
the systemic circulation and away from the absorption site.
A drug molecule gets absorbed from the GIT and enters the systemic circulation
only if it effectively penetrates all the intestinal regions. Once the drug ent ers the
solution, its absorption is governed by the following three important factors:
1) The physicochemical properties of the drug molecule,
2) The properties and components of the GI fluids, and
3) The nature of the absorbing membrane.
1.2.2. Mechanisms of Drug Absorption Through GIT

The drug after entering the GI fluids exists in the form of a solution and can get
absorbed. The drug’s physicochemical properties (its inherent absorbability) and
the environmental properties around i t (such as pH, presence of interfering
materials, and local properties of the absorbing membrane) decide whether or not
the drug is in absorbable form. If there are no interfering materials (that affect
* *
drug absorption), the drug molecule diffuses from the GI fluids to enter the
absorbing membrane surface. A drug molecule gets absorbed from the GIT and
enters the systemic circulation only if it effectively penetrates all the intestinal
regions.
The pathways of drug transport show that drug absorption from the GI lumen
into the systemic circulation involves the passage of drug molecules across
several cellular membranes and fluid regions in the mucosa, i.e., the
gastrointestinal-blood barrier. The GIT epithelium lining is the major cellular
barrier to the a bsorption of drugs from the GIT. The drug molecules in GI fluids
should cross the unstirred aqueous layer, mucus layer, and glycocalyx to reach
the apical cell membrane.
The following mechanisms are involved in the transport of drug molecules

across the cell membrane:
1) Passive diffusion,
2) Carrier-mediated transport:
i) Facilitated diffusion, and
ii) Active transport,
3) Pore transport,
4) Ionic or electrochemical diffusion,
5) Ion-pair transport, and
6) Endocytosis.
1.2.2.1. Passive Diffusion

Passive diffusion (or non-ionic diffusion) is the major process for absorption of
more than 90% of drugs. It is defined as the difference in the drug concentration
on either side of the membrane. Concentration or electrochemical gradient is
the driving force for this process.
Extracellular space
Lipid Bilayer
(Cell Membrane)
Intracellular space
Time
Figure 1.2: Passive Diffusion of a Drug
The kinetic energy of drug molecules is responsible for the movement of drug.
Since no energy source is required, the process is called passive diffusion, during
which the drug in the aqueous solution at the absorption site, partitions and
dissolves in the lipid material of the membrane and leaves it by dissolving in an
aqueous medium at the inside of the membrane.
* *
Passive diffusion can be expressed by Fick’s first law of diffusion, according to

which the drug molecules diffuse from a region of higher concentration to lower
concentration until equilibrium is achieved, and the rate of diffusion is directly
proportional to the concentration gradient across the membrane. Fick’s first law
of diffusion is mathematically expressed as:
dQ DAKm/w
 (CGIT  C) …..(1)
dt h
Where,
dQ/dt = Rate of drug diffusion (amount/time) or the rate of appearance of
drug in blood.
D = Diffusion coefficient of the drug through the membrane (area/time).
A = Surface area of the absorbing membrane for drug diffusion (area).
Km/w = Partition coefficient of the drug between the lipid membrane and the
aqueous GI fluids (no units).
(CGIT – C) = Concentration gradient (amount/volume), i.e., difference in the
concentration of drug in the GI fluids and plasma.
h = Membrane thickness (length).
1.2.2.2. Carrier-Mediated Transport

Some polar drugs pass through the membrane more rapidly than can be predicted
from their concentration gradient and p artition coefficient values. This is due to
the presence of a specialised transport mechanism, without which
monosaccharides, amino acids, and vitamins (essential water -soluble nutrients)
will undergo poor absorption.
The mechanism involves a membrane Outside of cell
component, called the carrier that Higher concentration
reversibly or non -covalently binds to
the solute molecules to be transported. Glucose
This carrier -solute complex crosses
the membrane to reach the other side,
where it dissociates and releases the
Carrier protein
solute molecule; after which, th e
carrier returns to its original site and
accepts a fresh solute molecule, Membrane
thereby completing the cycle.
Carrier-mediated transport system is

of two types: Inside of cell
Lower concentration
1) Facilitated diffusion, and
2) Active transport. Figure 1.3: Carrier-Mediated Transport
1.2.2.3. Facilitated Diffusion

Facilitated diffusion is a carrier -mediated transport system that works at a much
faster rate than the passive diffusion. Concentration gradient is the driving force
for this process that operates down the hill and thus is a passive process.
* *
Intestinal Membrane Blood

lumen
B12 IF
Carrie
Carrier
r
Dissociation
of complex
B12-IF-carrier Free
complex vitamin B12
Figure 1.4: Facilitated Diffusion of Vitamin B12

Since the process involves down-hill transport, there is no expenditure of energy,
and the system is not inhibited by metabolic poisons. Some applications of
facilitated diffusion system in drug absorption are:
1) Its major application involves entry of glucose into RBCs.
2) Another application involves intestinal absorption of vitamins B1 and B2.
3) The most significant application involves gastrointestinal absorption of
vitamin B 12 (figure 1. 4), which forms a complex with glycopro tein [an
Intrinsic Factor (IF) produced by gastric parietal cells]; and then this complex
is transported across the intestinal membrane through a carrier system.
1.2.2.4. Active Transport

Active transport i nvolves movement of a substance from a region of low
concentration to high concentration, i.e., against its concentration gradient. In all
the cells, this is concerned with accumulating high concentrations of ions,
glucose, and amino acids that the cell requires (figure 1.5).
Active transport utilises energy, unlike passive transport that does not use any
type of energy. If it uses chemical energy from ATP, it is termed as primary
active transport. If it uses an electrochemical gradient, it is termed as secondary
active transport.
The significance of active transport pro cess in the absorption of nutrients and
drugs is more than the facilitated diffusion, and both the processes differ in the
following aspects:
1) In active transport, drug is transported from a region of lower concentration
to a region of higher concentration, i.e., against the concentration gradient or
uphill transport.
2) Active transport is an uphill process, thus energy is required in the work done
by the carrier.
3) Active transport process requires energy, thus it can be inhibited by
metabolic poisons (like flu orides, cyanides, di -nitrophenol, lack of oxygen,
etc.) that interfere with energy production.
* *
GI lumen Membrane Blood
Carrier
Drug
Drug-carrier Dissociation Free

complex of complex drug
ATP
Figure 1.5: Active Absorption of a Drug

Sodium, potassium, calcium, iron, glucose, certain amino acids, and vitamins like
niacin, pyridoxine and ascorbic acid are the endogenous subs tances that are actively
transported. Drugs that are structurally similar to such agents (mainly the agents
used in cancer chemotherapy) are actively absorbed.For example, absorption of 5-
fluorouracil and 5-bromouracil through pyrimidine transport system;absorption of
methyldopa and levodopa through L-amino acid transport system; and absorption of
enalapril (ACE inhibitor) through small peptide carrier system.
An example of competitive inhibition of drug absorption through active transport

is the impaire d absorption of levodopa when taken with protein -rich meals.
Active transport is also important in renal and biliary excretion of many drugs
and their metabolites and secretion of certain acids out of the CNS.
1.2.2.5. Pore Transport

Pore transport (or connective transport , bulk flow or filtration) involves the
absorption of low molecular weight, low molecular size, and water -soluble drugs
(e.g., urea, water, and sugar) through narrow, aqueous filled channels o r pores in
the membrane structure. This mechanism facilitates the transport of molecules
into the cell through protein channels present in the cell membrane. Chain-like or
linear compounds of 400 Daltons molecular weight can be observed by filtration.
Hydrostatic pressure or the osmotic differences across the membrane is the
driving force for this process. Due to this, bulk flow of water along with small
solid molecules occurs through such aqueous channels. Water flux promoting
such a transport is called as solvent drag. Drug permeation through water -filled
channels is significant in renal excretion, removal of drug from the cerebrospinal
fluid, and entry of drugs into the liver.
1.2.2.6. Ionic or Electrochemical Diffusion

Ionic or electrochemical diffusion involves the diffusion of ionic molecules
across the membrane as a function of potential difference or electrical gradient;
while non-ionic diffusion involves the diffusion of uncharged mol ecules. No
matter ionic drug molecules diffuse through the membrane at a slower rate than
the lipid-soluble, uncharged molecules, still they undergo significant absorption.
The outer surface of membrane is positively charged, while its intracellular
* *
surface carries negative charge. Cationic drugs, due to the repulsion between
similarly charged molecules, show electrostatic repulsion on the outer surface of
the membrane. Thus, the molecules with a high kinetic energy can only cross the
membrane barrier. On t he other hand, cationic drugs inside the membrane
undergo significant interaction with the negatively charged intracellular
membrane, and create the electrical gradient, causing electrical diffusion.
Electrochemical diffusion is the function of electrical field as well as the
concentration difference across the membrane, and this process lasts till
equilibrium is achieved.
1.2.2.7. Ion-Pair Transport

Drugs with a Zwitter ion undergo ionisation over the enti re pH range of the GIT,
e.g., ampicillin, amoxicillin, and tetracycline. Smaller ionic drugs travel through
the water -filled pores; but, the Zwitter ionic drugs are large enough to pass
through the water -filled pores and are also highly lipid insoluble to partition
through lipoidal membrane. Thus, these drugs utilise their charge to pair with the
endogenous ions of the GIT and cross the membrane. The resulting paired
molecule partitions into the lipoidal membrane (figure 1. 6). These drugs are
ionic, but sho w passive absorption and maximum partition coefficient when the
net charge on the molecule is minimum.
+ – – +
Drug Endogenous Ion-pair
Drug
ions
Membrane Blood
Figure 1.6: Ion Pair Transport of Cationic Drug
1.2.2.8. Endocytosis
Endocytosis is a minor transport mechanism in which the extracellular materials
are engulfed within a segment of the cell membrane to form a saccule or a
vesicle, which is then pinched -off intracellularly ( figure 1.7). Thus, this process
is also called as corpuscular or vesicular transport.
Outside Membrane Inside
cell cell
Macromolecule
Process of Vesicle
engulfing
Figure 1.7: Endocytic Uptake of Macromolecules

The process of endocytosis helps in the cellular uptake of macromolecular
nutrients (like fats and starch), oil -soluble vitamins (like A, D, E, and K), and
drugs (like insulin). This process is also significant because since the drug is
absorbed into the lymphatic circulation, it bypasse s first-pass hepatic metabolism.
* *
Types of Endocytosis
The process of endocytosis is of the following two types:
1) Phagocytosis (Cell Eating): This process involves adsorptive uptake of
solid particulates.
2) Pinocytosis (Cell Drinking ): This process i nvolves uptake of fluid solute;
for example , orally administered Sabin polio vaccine and large protein
molecules are absorbed by pinocytosis.
At times, transcytosis occurs in which an endocytic vesicle is transferred from
one extracellular compartment to another.
1.3. FACTORS INFLUENCING DRUG

ABSORPTION THROUGH GIT
1.3.1. Introduction
The desired therapeutic objective is achieved when the drug product delivers the
active drug at an optimal rate and extent, a nd at the target site. If a proper
biopharmaceutical design is adopted, the rate and extent of drug absorption (i.e.,
bioavailability) or the systemic delivery of drug to the body can be varied from
rapid and complete absorption to slow and sustained absorption depending on the
desired therapeutic activity. The following factors influence drug absorption:
1) Physicochemical factors, and 2) Pharmaceutical factors.
1.3.2. Physicochemical Factors

Drug absorption through GIT is influenced by the following physicochemical
factors: Physicochemical Factors
Drug Solubility
Dissolution Rate
Wetting
Chemical Forms
Drug pKa and Lipophilicity and GI pH
Drug pKa and Gastrointestinal pH
Lipophilicity and Drug Absorption
Particle Size and Effective Surface Area
of the Drug
Polymorphism and Amorphism
Hydrates/Solvates
Salt Form of the Drug
1) Drug Solubility: Almost all the factors that influence dissolution rate, also
affect the drug solubility either directly or indirectly. The dissolution rate is
considered to be directly related to drug solubility. An empirical relation
used for predicting the dissolution rate of a drug from its solubility is:
dc
R= = 2.24 Cs Where R = Dissolution rate of the drug.
dt
Bioavailability problems can be avoided if the dr ug exhibits a minimum
aqueous solubility of 1%.
* *
An orally administered drug undergoes degradation due to the acidic or

alkaline nature of gastrointestinal content, and the presence of enzymes and
bacteria. For example, antibiotics are relatively stable be tween pH 6 -8 but
get rapidly destroyed at gastric pH (1.5 -3.5). Longer is the gastric emptying
time, more the drug is at risk to get degraded by acidic pH of the stomach.
This problem of degradation can be avoided by enteric coating, but it delays
drug rel ease and absorption. As an alter native, the drug is administered via
other routes such as sublingual, transdermal, or rectal.
Due to degradation of drug, the following consequences may occur:

i) Little drug is absorbed, so reduced bioavailability.
ii) Toxicity increases upon degradation, e.g., salicylic acid is more irritant
than aspirin.
iii) Degradation process is essential for pro -drugs, because they release the
therapeutically active molecule after degradation in the GIT.
The compromise between solubility and s tability of drug in gastrointestinal

content is a key factor to determine the bioavailability of the orally
administered drug. For example, Progabide is a weak base with pK a value
of 3.41; it is highly soluble below pH 3 and poorly soluble above pH 4; it
undergoes hydrolysis at acidic pH to release GABA and benzophenone; it
has maximum solubility and undergoes rapid absorption from small intestine
at pH 6.3. In such cases, the dosage form design and bioavailability is
decided by considering the following drug characteristics:
i) Drug micronisation increases the drug solubility in small intestine (a
better absorption site), but also increases the drug degradation rate in
stomach due to increased surface area.
ii) Enteric coating protects the drug from acidic enviro nment, but the
insoluble drug reaches the small intestine.
iii) The rapid gastric emptying of micronised drug ensures faster drug
dissolution and minimum drug degradation.
2) Dissolution Rate: For the absorption of drug, it should be in solution state at
the absorption site. Therefore, the factors influencing dissolution rate of the
drug in gastrointestinal fluid also influence the drug bioavailability.
[C]
x Drug [Cs]
Blood
GI Fluid Membrane
Figure 1.8: Dissolution Process of Drug in GIT
* *
Dissolution rate is the amount of substance that goes into the solution per
unit time under standard conditions of temperature, pressure, and solvent
composition. Dissolution is a dynamic process and involves mass transfer.
The following theories explain the process of drug dissolution:

i) Noyes-Whitney Theory or Stagnant Film Theory: Noyes-Whitney
equation describes the process of dissolution, and states that the
dissolution rate of drug (d C/dt) is directly proportional to the diffusion
coefficient of the drug in solution state in gastrointestinal fluid (D),
effective surface area of the drug particle ava ilable for drug dissolution
(A), difference in the saturation solubility of the drug in the diffusion
layer (C s), and the concentration of drug in solution state in the bulk of
gastrointestinal fluid (C); while the dissolution rate of drug is inversely
proportional to the thickness of diffusion layer (x) (equation 2):
dC DA (Cs  C)
 ….. (2)
dt x
This equation is based on diffusion controlled dissolution process. The
driving force for this first -order process is (C s  C), but the in vivo
conditions are different. Gastrointestinal motility affects the diffusion
layer thickness by disturbing it. Rapid and continuous absorption of drug
in blood circulation increases the concentration gradient. Equation (2)
may be expressed as:
dc D  A  CS
 ..... (3)
dt x
This theory assumes that the surface area of the dissolving solute remains
constant during dissolution; however, this is practically not possible.
Hixson and Crowell modified the Noyes -Whitney equation to explain
the effects of change in the surface area:
W01/3  W1/3 = Kt ….. (4)
Where,
W0 = Initial powder weight.
W = Powder weight at time t.
K = Constant which is function of particle density, viscosity,
diffusion coefficient, Cs and x.
Equation (4) is also termed as Hixson and Crowell’s Cubic Root Law.
ii) Danckwert’s Model: This model of dissolution does not consider the
formation of diffusion layer and the solid surface is constantly exposed
to fresh solvent causing mass transfer. It is assumed that the dissolution
medium and the solid surface are under turbulence. T he agitated
dissolution medium comprises of a mass of eddies or pockets, which are
continuously exposed to the solid surface, absorb the solute by diffusion,
and carry it to the bulk of the solution. The contact of fresh pockets with
the solid surface tran sfers the mass to the bulk, and does not allow the
formation of diffusion layer.
* *
Since this dissolution process involves the contact of fresh pockets of

solvent with new solid surface, it is termed as surface renewal process.
VdC dm
 A D (C S  C )
dt dt
….. (5)
Where,
V = Volume of dissolution medium.
m = Mass of solid dissolved.
 = Rate of surface renewal or interfacial tension.
: Fresh pocket
: Drug dissolved in
pocket
Figure 1.9: Surface Renewal Process of Dissolution
iii) Limited So lvation Theory: This theory states that an intermediate
concentration less than saturation exist at the interface due to salvation
mechanism, which is the function of solubility than that of the diffusion.
In the dissolution of crystals, the different faces of a crystal have
different interfacial barrier. Limited solvation theory is demonstrated by
the following equation:
G = Ki  (Cs – C) ….. (6)
Were,
G = Dissolution rate per unit area.
Ki = Effective interfacial transport constant.
3) Wetting: Good wettability results in particle size reduction. Aggregation of
powder and air adsorption on powder surface is minimised by adding a
wetting agent. Surfactants and hydrophilic polymers have been added in
dosage forms for enhancing drug dissolution and bioavailability.
2.5
Plasma drug concentration
Agglomerate with PEG

2.0
Agglomerate without PEG
1.5
1.0
0.5
0 2 4 6 8 10 12
Time
Figure 1.10: Effect of PEG on Bioavailability of Phenytoin
* *
When phenytoin is agglomerated by spherical crystallisation, the formed

agglomerates containing PEG show higher Area Under the blood
concentration-time Curve (AUC) and C max. A drug with poor wettability in
water or in conventional dissolution media may have good wetting by
gastrointestinal fluid. The native surfactants in GIT, such as bile salts, may
also help in the wetting of drug.
4) Chemical Forms: The desired effect can be achieved by selecting an
appropriate chemical form of drug. Sometimes, chemical modifications are
made in the structure of drugs to have better therapeutic response or effect
than the parent drugs. These modified drugs exhibit the same therapeutic
value as they breakdown in the body into active forms of the parent drugs.
The ideal conditions for a chemical form to act as a drug are:
i) It should have sufficient water solubility for dissolution.
ii) It should have optimum o/w partition coefficient for diffusion through
lipid layers.
iii) Chemical modifications are made in the part of drug molecule that
obstructs absorption.
Better therapeutic response can be achieved by implementing the following
chemical modifications in the chemical forms:
i) Specific Chemical Modifications: These involve chemical modifications
of drugs, e.g., sulphonamides to succinyl sulphonamide or phthalyl
sulphonamide. These chemical modifications in sulphonamides can
ionise in the gut. Therefore, their lipid/water partition coefficient
decreases, thus reducing the absorption of these chemically modified
drugs. The antibacterial activity begins when the amide links are broken
down by hydrolysis. S
HOOC—CH2—CH2—CONH— —SO2NH—
CH2—
CONH— N
Succinyl sulphonamide
COOH
S
—CONH— —SO2NH—
CH2—
CONH— N
Phthalyl sulphonamide
In contrast, decreased ionisation results in better absorption; for
example, hexa -methonium chloride (a quaternary amine) and
mecamylamine hydrochloride (a tertiary amine) are steadily and
completely absorbed as ionic species.
NHCH3
+ + – CH3
[(CH3)3 N(CH2)6 N(CH3)3 ] 2Cl . HCl
CH3
CH3
Hexamethonium chloride Mecamylamine hydrochloride
ii) Chemical Modifications to Increase Lipid Solubility: In some drugs,
chemical modifications are done to enhance their lipid solubility. For
example, increase in lipid solubility of barbiturates is directly
* *
proportional to their absorption from colon; doxycycline is better

absorbed than its parent drug tetracycline; erythromycin is better
absorbed than its estolate form.
Drugs Partition Coefficient CHCl 3/H2O % of Absorption
Barbital 0.69 12.2
Apobarbital 4.88 17.2
Phenobarbital 4.78 20.1
Butabarbital 10.48 23.1
Butenal 11.67 23.9
Pentobarbital 28.02 29.9
Secobarbital 50.68 39.9
Hexabarbital 100.0 44.1
iii) Salt Formation for Increasing Absorption: Drugs are mostly weak
acids or bases. The salts of such drugs have different solubility than their
parent drugs. The solubilit y of such drugs can be easily enhanced by
converting them into their salt forms. Some examples are:
Drugs Salts of the Drug
Chloramphenicol Chloramphenicol succinate
Menadiol Menadiol phosphate
Oxazepam Oxazepam semisuccinate
Testosterone Testosterone phosphate
Tetracycline Tetralysine
Theophylline Diprophylline
a) The aluminium salt of aspirin undergoes very slow dissolution in the
GIT due to the deposition of insoluble aluminium on the surface of
solid particles. Due to this reason, the drug is only partially absorbed.
b) Dissolution rate of phenobarbital tablets is better than that of the
tablets of sodium salt of phenobarbital because the former undergoes
rapid disintegration while the latter swell and dissolve slowly from
the surface.
c) Heptabarbital in tablet form attains the peak plasma level (C max) in
1.5-5 hours (t max). However, its sodium salt attains C max at 0.4 -2
hours (t max). Bioavailability of heptabarbital is up to 17% more than
that of its sodium salt due to the precipitation of sodium salt of the
drug in crystalline form.
5) Drug pKa and Lipophilicity and GI pH (pH Partition Theory): Brodie
gave the pH partition theory to explain the absorption process of drug from
GIT and its distribution across the biological membranes. This theory states
that the absorption of drug compounds with molecular weight more than 100
Daltons involves transportation across the biomembrane by passive diffusion,
and this process depends on:
i) The dissociation constant (pKa) of the drug,
ii) The lipid solubility (Ko/w) of unionised drug, and
iii) The pH at absorption site.
* *
Drugs are mostly either weak acids or weak bases; thus, their ionisation
degree is influenced by the pH of biological fluid. If the pH on either side of
the membrane is different, the compartment whose pH favour s greater
ionisation of drug will have greater amount of drug. Also, the unionised or
un-dissociated fraction of the drug, if is sufficiently lipid -soluble, will
permeate the membrane by passive diffusion until equilibrium is attained
between the concentra tions of unionised drug on either side of the
membrane.
The pH partition theory lies on the following assumptions:
i) GIT acts as a lipoidal barrier to the transport of drug.
ii) The absorption rate of a drug is directly proportional to the fraction of
unionised drug.
iii) Higher the lipophilicity (K o/w) of the unionised drug, better is its
absorption.
Limitations of pH-Partition Theory

i) The pH -partition theory is based on the assumption that equilibrium is
achieved by the distribution between the ionised and unionise d forms of
a drug. However, drug is continuously swept away due to blood
circulation, thus maintaining sink condition. This theory therefore failed
in calculating the absorption from equilibrium distribution.
ii) This theory describes absorption of weak acids and weak bases more
suitably.
iii) Ionisation of drug in the lumen occur s similarly as in blood, but ion
trapping may also occur.
iv) Absorption from GIT depends on pH of all sites, drug solubility in lipid,
gastric residence, absorption surface area, drug degradation, etc.
v) Weak acidic drugs are better absorbed from small intestine, because:
a) Poorly soluble weakly acidic drug has a high dissolution rate in an
alkaline environment. This results in availability of higher surface
area for absorption of the dissolved drug.
b) The weakly acidic pH (pH 5.3) at surface of the intestinal mucosa is
responsible for effective absorption of weakly acidic drug that exists
in unionised state. Thus, this hypothesis raises questions regarding
the absorption of weakly basic drug that ex ists in unionised state.
These weak bases can interact with organic cations and are secreted
from blood into the intestinal lumen.
A poorly water-soluble, weakly basic drug,which dissociates and dissolves
in stomach to a greater extent, also show s better absorption, when it
reaches to small intestine. The delayed gastric emptying rate would permit
a longer time for dissolution of weak ly basic drug in acidic fluid and in
turn would increase its absorption when it reaches the small intestine. For
example, absorption of nitrofurantoin is increased inthe presence of food.
vi) Solubility of unionised form of drugs is the rate determining step in the
absorption of a drug, but presence of unionised drug is not the sole
* *
concept, e.g., derivatives of barbituric acid h ave almost same pK a, but

have different lipid solubilities, which can be ranked as barbital <
apobarbital < pentobarbital.
vii) Few drugs show good solubility in all parts of GI T, despite of good
ionisation, e.g., theophylline and tetracycline.
6) Drug pKa and Gas trointestinal pH: The dissociation constant (pKa) of
drug and the pH of body fluid at the absorption site influence the amount of
unionised drug. The unionised form of drug is also most suitable for
absorption in GIT. The relative amounts of ionised and un ionised drug in a
solution at a particular pH can be obtained from the pKa value of drug and
pH of body fluid at the absorption site, by using the Henderson and
Hasselbalch equation:
For Weak Acids
Ionizeddrug concentration
pH = pKa + log …..(7)
Unionizeddrug concentration
10pH  pKa
% of drug ionised = 100 …..(8)
1 10pH  pKa
10pH pKa
% of drug unionised = 100 – 100 ….. (9)
110pH pKa
For Weak Bases

Unionizeddrug concentration
pH = pKa + log ….. (10)
Ionizeddrug concentration
10 pKa  pH
% of drug ionised = 100 ….. (11)
1 10pka  pH
pKa  pH
10
% of drug unionised = 100 – pKa  pH
100 ….. (12)
1  10
If the concentrations of ionised and unionised drug are equal, the second term
of equations (8) and (11) becomes zero (  log 1 = 0), and therefore pKa =
pH. Thus, pKa is the characteristic of the drug. If a membrane barrier
separates the aqueous solution of different pH (such as the GIT and plasma),
the theoretica l ratio of drug concentration (Ra or R b) on either side of the
membrane can be obtained by the equations developed by Shore et al.
For Weak Acids
 pKa
C GIT 1  10 pHGrt
Git
Ra =  ….. (13)
C Plasma 1  10pHPlasma  pKa
For Weak Bases
C GIT 1  10 pKa  pHGit
Grt
Rb =  pKa  pHPlasma
….. (14)
C Plasma 1  10
* *
If the pH ranges from 1 to 8 in the GIT, from 1 to 3 in the stomach, and from
5 to 8 in the intestine (duodenum to colon), certain generalisations about
ionisation and absorption of drugs can be made from the pH -partition
hypothesis.
For Weak Acids

i) Weakly acidic drugs with pKa > 8, e.g., phenytoin, ethosuximide , and
several barbiturates, are unionised at all pH values and hence their
absorption rate is rapid and independent of gastrointestinal pH.
ii) Weakly acidic drugs with pKa ranging from 2.5 to 7.5, e.g., aspirin,
ibuprofen, ph enylbutazone, and penicillin analogues, undergo pH -
dependent absorption. At acidic conditions of stomach (pH > pKa), they
exist in unionised form and undergo better absorption.
iii) Strongly acidic drugs with pKa < 2.5, e.g., cromolyn sodium, are ionised
in the entire pH range of GIT and hence are poorly absorbed.
For Basic Drugs

i) Weakly basic drugs with pKa < 5.0, e.g., caffeine, theophylline,
diazepam, oxazepam , and nitrazepam, are ionised at all pH values and
hence undergo rapid and pH-independent absorption.
ii) Weakly basic drugs with pKa ranging from 5 to 11 , e.g., morphine
analogues, chloroquine, imipramine, and amitriptyline, undergo pH -
dependent absorption. At alkaline conditions of the intestine, they exist
in unionised form and undergo better absorption.
iii) Strongly basic drugs with pKa > 11, e.g., mecamylamine and
guanethidine, remain ionised in the entire pH range of GIT and hence are
poorly absorbed.
7) Lipophilicity and Drug Absorption: If a drug exists in unionised form, it
will still undergo poor absorption , provided its lipid solubility is low, i.e., it
has a low value of K o/w. Thus, for a drug to undergo optimum absorption, it
should be sufficiently soluble in aqueous medium at the absorption site and
also highly lipid-soluble to bring about partitioning of the drug in the lipoidal
biomembrane and into systemic circulation. Thus, for optimum
bioavailability a perfect hydrophilic -lipophilic balance should exist in the
drug structure.
The lipid solubility of a drug can be determined from its oil/water partit ion
coefficient value (Ko/w).
8) Particle Size and Effective Surface Area of the Drug: A solid drug’s
particle size and surface area are inversely proportional. Smaller the drug
particle, greater is its surface area. Two types of surface area are:
i) Absolute Surface Area: Total area of solid surface of any particle, and
ii) Effective Surface Area: Area of solid surface exposed to the dissolution
medium.
With micronisation , the dose of certain drugs can be decreased because of
increased absorption efficiency, for example, griseofulvin dose was reduced
to half and spironolactone dose was reduced 20 times by micronisation.
* *
However in hydrophobic drugs (like aspirin, phenacetin , and phenobarbital),

micronisation reduces the effective surface area and hence the dissoluti on
rate due to the following three reasons:
i) The hydrophobic drugs adsorb air on th eir surface, and thus inhibit their
wettability and allow them to float on the dissolution medium.
ii) The particles, due to their high surface free energy, re -aggregate into
larger particles, which either float on the surface or settle at the bottom of
the dissolution medium.
iii) Extreme particle size reduction imparts surface charges that prevent
wetting; electrically-induced agglomeration prevents intimate contact of
the drug with the dissolution medium.
9) Polymorphism and Amorphism: A solid can exist either in a crystalline or
amorphous form depending on its internal structure. The phenomenon in
which a substance exists in more than one crystalline form is termed as
polymorphism and the different forms are termed polymorphs. Polymorphs
are of two types:
i) Enantiotropic polymorphs can be reversibly changed into another form
by altering the temperature or pressure, e.g., sulphur, and
ii) Monotropic polymorphs are unstable at all tem peratures and pressures,
e.g., glyceryl stearates.
Polymorphs differ from each other on the basis of their physical properties,
such as solubility, melting point, density, hardness, and compression
characteristics. They can be prepared by crystallising the drug f rom different
solvents under varied conditions. Existence of polymorphs can be determined
by techniques of optical crys tallography, X -ray diffraction, differential
scanning calorimetry, etc.
10) Hydrates/Solvates (Pseudopolymorphism): The crystalline form of a drug
can be a polymorph or a molecu lar adduct or both. Stoichiometric type of
adducts in which the solvent molecules are incorporated in the crystal lattice
of the solid are termed solvates, and the trapped solvent is termed solvent of
crystallisation. Solvates can exist in different crystalline forms, which are
known as pseudopolymorphs, and the phenomenon is termed
pseudopolymorphism. If the solvent in association with the drug is water,
the solvates are termed hydrates, which are the most common solva te forms
of drugs.
Aqueous solubility of the anhydrous form of a drug , i.e., anhydrates, is more
than that of the hydrates. This is because the latter are in interaction with
water and have less energy for crystal break -up for further interaction with
water in compari son to the former that are in thermodynamically higher
energy state.
Theophylline and ampicillin in anhydrous form have higher aqueous
solubility, dissolve at a faster rate, and show better bioavailability in
comparison to when they are in mo nohydrate and trihydrate forms,
respectively. In contrast, the aqueous solubility of organic (non -aqueous)
* *
solvates is more than the non -solvates, for example, n-pentanol solvate of
fluorocortisone and succinyl sulfathiazole and the chloroform solvate of
griseofulvin have greater water solubility than their non -solvated forms like
polymorphs. Solvates differ from each other in terms of their physical
properties.
11) Salt Form of the Drug: Drugs are mostly weak acids or bases. The salts of
such drugs have diffe rent solubility than their parent drugs. The solubility of
such drugs can be easily enhanced by converting them into their salt forms.
With weakly acidic drugs, a strong base salt is prepared ( e.g., sodium and
potassium salts of barbiturates and sulphonamides). With weakly basic drugs,
a strong acid salt is prepared ( e.g., hydrochloride or sulphate salts of several
alkaloidal drugs).
1.3.3. Pharmaceutical Factors

Various processing variables can affect dissolution by altering effective surface
area of drug particles. Drug dissolution is the single most important factor in the
absorption of drugs.
The dosage form factors that influence dissolution and hence absorption of a drug
are discussed below:
1) Dosage Form Considerations: The dosage form and its properties greatly
affect the absorption rate and bioavailability, which also depend on the rate
of drug release from dosage form , i.e., the rate of availability of the drug to
the biological fluids. The decreasing order of drug availability is as follows:
Solutions > Emulsions > Suspensions > Capsules > Compressed Tablets
> Coated Tablets > Enteric-coated tablets.
i) Solutions: Aqueous solutions of drugs are absorbed by a faster rate
through GIT, but s ometimes drug solution in the gastric fluids may
precipitate, where the extremely fine precipitate permits rapid re -
dissolution.
ii) Emulsions: An emulsion provides large surface area of oil to GIT that
elevates rate of partitioning. Few drugs are absorbed fas t in an emulsion
dosage form as compared to aqueous suspension (viscosity of the
emulsion should not be limiting factor). If oils are digestible , absorption
rate may increase further.
iii) Suspensions: An aqueous suspension is one of most efficient dosage
form. Mostly, dissolution rate limits the absorption of drug from a
suspension; however, large surface area is immediately presented to the
fluids at the absorption sites.
iv) Capsules: Release of drug from hard gelatin capsule mainly depends on
the formulation. The fine particles present in capsule are not compressed
and may be fused, resulting in a reduced effective surface area. A large
effective surface area will be available for di ssolution if particles in a
capsule are intimately wet by the biological fluid s. Absorption from
capsules is affected by particle size, selection of diluents and fillers.
* *
v) Compressed Tablets: These are the most common dosage form, but
simultaneously, the most difficult problem is with respect to availability
of a drug for absorption. The main problems arise during the transfer of a
solid drug from a compressed tablet to a solution in the GIT fluid as the
effective surface area of drug is reduced to a greater extent during the
tablet manufacturing process.
In an intact tablet, the sur face area is very limited, i.e., the dissolution
rate is negligible, except for very water -soluble drugs. The absorption of
drug is affected by primary disintegrat ion that ultimately influences the
dissolution process. Tablet fragmentation increases the su rface area in
contact with fluids at the absorption site, resulting in an increased rate of
dissolution. In granular form , the effective surface area of drug is more
than in the intact tablet, however, it is never less than the effective
surface area of the primary drug particles. Compression force also affects
the bioavailability of drug from compressed tablets.
vi) Coated Tablets: Commonly used coating technique for tablets are sugar
coating, film coating, and press coating. Coating forms a physical barrier
between the GI fluid and the tablet (drug). For proper drug dissolution , it
is important that the film of coating dissolves before tablet disintegration.
The disintegration is rate limiting process of drug absorption in coated
tablets, because in vitro disintegration time affects bioavailability.
Film-coated tablets are compressed tablets coated with water -soluble
material as a thin layer or film providing rapid solubility of the film.
Coatings of the tablet have almost negligible effect on the drug
availability as compared to the availability of drug from uncoated core
tablet. Independent of GIT pH, the film coating should dissolve quickly
in the GI fluids.
vii) Enteric-Coated Tablets: An enteric -coat is a special type of film
coating designed to protect the tablet from gastric fluid and to promote or
allow the dissolution of drug in the intes tine. The most important factor
for enteric-coated tablet is the high liability of the coated tablet passing
through the GIT. This problem mainly arises when enteric -coated
products descend to release the drug at some finite time after
administration. Lack of availability of drug can be decreased by the use
of formulations that are based on pH difference between the stomach and
intestine (a realistic assessment of intestina l pH should be provided).
High intra- and inter-subject variability is observed in the enteric-coated
preparation due to the variability in gastric emptying.
2) Manufacturing Processes: Processes of tablet manufacturing include:
i) Granulation Method: Conventional method for preparing granules is
wet granulation process. Limitations of granulation are:
a) The liquid used may cause chemical reactions like hydrolysis.
b) Formation of crystal bridge due to the presence of liquid.
c) Harmful effects on thermo degradable drugs due to the drying
process.
* *
In granulation, many steps are involved that influence drug dissolution:

a) Method and duration of blending, and
b) Method, time and temperature of drying.
The method of direct compression is used to yield tablets that dissolve at
a faster rate.
ii) Compression Force: This force is required in the tableting process and
affects density, hardness, porosity, disintegration time , and dissolution
rate. The dissolution rate is affected in the ways shown in figure 4.14
when compression force is applied on drug-excipient mixture to form
tablets.
Dissolution
A C D
B
Rate
Compression Force
Figure 1.11: Effect of Compression Force on Dissolution Rate
On increasing the compression force, particles bind more tightly to each
other (Curve A). High pressures may also break the particles and form
smaller particles (Curve C). Sometime, a sum of an y of these two may
result. So, it can be concluded that the effect of the compression force on
the dissolution rate of a tablet would appear to be unpredictable.
Therefore, a proper study should be performed on each formulation to
ensure better dissolution as well as bioavailability.
3) Pharmaceutical Ingredients/Excipients (Formulation Factors): Many
excipients (non-drug components) are added in a formulation. Excipients are
used to enhance acceptability and physicochemical stability during shelf-life,
uniform composition and dosage, optimum bioavailability and function -
ability drug product. Important excipients are:
i) Vehicles: These are mainly:
a) Aqueous vehicles (e.g., water and syrup),
b) Non-aqueous water miscible vehicles ( e.g., propylene glycol,
glycerol, and sorbitol), and
c) Non-aqueous water immiscible vehicles (e.g., vegetable oils).
Miscibility of vehicles with biological fluids is an important factor for
bioavailability. Aqueous and water mis cible vehicles are miscible with
the body fluids imparting quick absorption of drug.
ii) Diluents (Fillers): Hydrophilic diluents ( e.g., starch, lactose, and
microcrystalline cellulose) are used to enhance the dissolution of poorly
water-soluble, hydrophobic drugs ( e.g., steroids). Hydrophobic diluents
(e.g., dicalcium phosphate) are used in the formulation of tablets.
iii) Binders and Granulating Agents: Poor wettable drugs ( e.g.,
phenacetin) can improve dissolution by imparting hydrophilic properties
to the granule surface through hydrophilic binders. Proportion of stro ng
*
binders during tablet formulation is very critical. If binders are used in *
greater amount, it may increase hardness and decrease disintegration or

dissolution rates of tablets. For example, PEG-6000 forms a poorly
soluble complex with phenobarbital, and non-aqueous binders like ethyl
cellulose also decrease drug dissolution.
iv) Disintegrants: These are mainly hydrophilic in nature. Amount of
disintegrant directly affect the bioavailability of drug, e.g., lower amount
of disintegrant significantly decrease s bioavailability. Microcrystalline
cellulose is a good disintegrant, but when compression force is increased,
it may decrease the drug dissolution.
v) Lubricants/Antifriction Agents: These agents are mainly hydrophobic
and form a coating over the drug particle. So, they should be used in such
a way that they do not hinder or reduce drug dissolution rate and
bioavailability. Increasing hydrophilicity of formulation also increases its
dissolution rate in an aqueous medium, but, increase in hydrophobicity
may decrease the rate of dissolution.
vi) Suspending Agents/Viscosity Imparters: These agents stabilise solid
drug particles by decreasing the settling rate by increasing viscosity of
the medium. Commonly used hydrophilic polymers are vegetable gums
(e.g., acacia and tragacanth), semi-synthetic gums ( e.g., CMC and MC),
and synthetic gums. Increased viscosity functions as mechanical barrier
to drug diffusion process from the dosage form into GI fluids as well as
from GI fluids to mucosal lining (forms a viscid layer on the GI mucosa).
These drugs also reduce the GI transit of drugs.
vii) Surfactants: In any formulation, surfactants are used as wetting agents,
solubilisers, emulsifiers, etc. Surfactants involve the given mechanisms
to increase drug absorption:
a) Drug dissolution and wetting are enhanced by increasing effective
surface area, e.g., Tween-80 with phenacetin.
b) Drug and membrane contactis modified for increasing drugabsorption.
c) Membrane permeability of drug is enhanced.
viii) Buffers: Potassium cations containing buffer systems can inhibit drug
absorption, e.g., as in sulfanilamide and vitamin B2. It is because of
uptake of fluids by the intestinal epithelial cells due to which the
effective drug concentration reduced in the tissues decreases the rate of
absorption.
Example of order of an inhibitory effect of various buffer catio ns on the
drug transfer rate is K+ > NH 4+ > Li + >Na+ >TRIS+. Therefore, a buffer
system for a salt of a drug should have the same cation as the drug salt
and there should not be any additional cations.
ix) Complexing Agents: The physicochemical and biopharmaceutical
properties of drug can be altered by complexing agents. Few examples
where drug bioavailability is enhanced by complexation are:
a) Formation of a soluble complex ( e.g., ergotamine tartrate-caffeine
complex and hydroquinone-digoxin complex) increases dissolution.
* *
b) Membrane permeability is enhanced by increasing lipophilicity (e.g.,

caffeine-PABA complex).
c) Enhanced membrane permeability (e.g., GI absorption of heparin ,
which normally is not a bsorbed from the GIT is enhanced) by using
EDTA that chelates magnesium and calcium ions of the membrane.
x) Colourants: An inhibitory effect is produced on dissolution rate of
crystalline drugs by water-soluble dyes, even when used in little amount.
This is because dyes are adsorbed on the crystal faces, and inhibit drug
dissolution, for example, brilliant blue inhibits dissolution of sulfathiazole.
Dyes also inhibit micellar solubilisation effect of bile acids and retard the
absorption of hydrophobic drugs(e.g., steroids). Cationic dyes have greater
adsorption on particles, hence are more reactive than the anionic one s.
xi) Crystal Growth Inhibitors: These are used to maintain the initial
physical properties of drug in suspension. Few crystal growth inhibitors
(e.g., PVP and PEG) hinder the conversion of high energy metastable
polymorph into less soluble and stable polymorph.
xii) Disintegration Rate: Disintegration time for solid dosage form ( e.g.,
tablets and capsules) is significant. In vitro disintegration test r elates to
bioavailability because if the drug is not disintegrating, it will not
dissolve, and there will not be any possibility of absorption.
If any drug does not confirm for the disintegra tion time, its
bioavailability is directly affected , a s the sub sequent process of
dissolution will be much slower and absorption may be insufficient. A
solid dosage form disintegrate into fine particles or granules, which
further deaggregate into fine particles; and dissolution of these fine
particles is faster than that of granules.
Disintegration time of coated tablets is more than that of the compressed
tablets. Disintegration can be enhanced by incorporating suitable
amounts of disintegrants during formulation. Disintegration time directly
depends on the concentration of disintegrant, fillers, and binders.
4) Dissolution Time of Drug in Dosage Form: Prediction of bioavailability
can be done more preciously by dissolution test as compared to disintegration
test. Bioavailability can be pre -determined by dissolution if these two
conditions are met:
i) In GIT, the dissolved drug should remain free and intact. If the dissolved
drug forms a complex with any GIT component or if drug decomposes in
the GIT, dissolution test will not give a clear estimation of bioavailability.
ii) If absorption is not the rate limiting step, the drug solution formed in the
GIT will be absorbed quickly, and the amount absorbed can be correlated
with in vitro dissolution rate. However, if the absorption is limited or
slow, bioavailability may not be proportional to the dissolution rate.
Drug d issolution is directly dependent on the particle size of drug.
Dissolution rates of hydrophobic drugs can be increased by adding
surfactants as they remove the air pockets around the particles that facilitate
the contact of the dissolution medium with the drug. In gastric fluids, the
* *
primary cause of surface activity is the reflux of intestinal contents into the
stomach. Dissolution of drug is also affected by fillers and diluents used in
formulation.
5) Product Age and Storage Conditions: Any changes in physicochemical
properties of a drug in dosage form affect the drug absorption. Changes in
physicochemical properties of drug can result due to ageing and alterations in
storage conditions that adversely affect the b ioavailability of drug. In
solutions, drug is precipitated due to alteration in solubility, conversion of
metastable into poorly soluble, stable polym orph during the shelf -life of the
product. In suspensions, changes in particle size distribution decrease the rate
of drug dissolution and absorption.
Ageing and storage conditions greatly affect the disintegration and
dissolution rates of solid dosage forms, especially in tablets. It occurs due to
the hardening of excipients ( e.g., polyvinyl pyrrolidone, aca cia etc.) on
storage, whereas decrease in these parameters is due to the softening of tablet
binder (e.g., carboxymethyl cellulose) during storage. Alterations that occur
during the shelf -life of a dosage form are mainly affected by variations in
humidity and temperature.
1.4. ABSORPTION OF DRUGS FROM NON

PER ORAL EXTRA-VASCULAR ROUTES
1.4.1. Introduction
Drugs are most often administered extravascularly and are mainly intended to
produce systemic action; for this reason, absorption is a prerequisite for
pharmacological effects. Delays or drug loss during absorption contribute to
variability in drug response, and sometimes may result in drug therapy failure.
The absorption site is separated from blo od via gastrointestinal membrane.
Therefore, a drug to get absorbed have to pass through the membrane, and this is
possible only when the drug is in solution form and dissolution becomes very
critical for the absorption of a drug.
Non per Os or Oral indicates drug administration routes other than the oral route,
which bypasses the GIT and enter the systemic circulation. A major advantage
of drug administration through non-invasive transmucosal and transdermal routes
(like nasal, buccal, rectal, etc.) is that a greater systemic availability is attained.
1.4.2. Sublingual/Buccal Route

Drug (small size d tablet) is kept beneath the tongue (without water) to
disintegrate and absorb in mouth, e.g., nitroglycerine tablets. The drug enters the
systemic circulation through diffusion into the capillary network. In buccal route,
the drug is kept within the mouth around the cheeks or buccal cavity, where it
disintegrates and absorbs. The following factors should be considered in the oral
mucosal delivery of drugs:
1) Lipophilicity of Drug: For passive permeation, slightly higher lipid
solubility than that required for gastrointestinal absorption is necessary.
* *
2) Salivary Secretion: The drug should be highly lipid-soluble, and should also
be soluble in aqueous buccal fluids. Biphasic solubility of drug is required
for absorption, because absorption will be delayed if the mouth is dry.
3) Saliva pH: Buccal pH of 6 assists the absorption of unionised drugs.
4) Binding to Oral Mucosa: Drugs that bind to oral mucosa have a poor
systemic availability.
5) Storage Compartment: A storage compartment exists in the buccal mucosa
for the slow absorption of drugs, like buprenorphine.
6) Thickness of Oral Epithelium : Sublingual absorption is faster than buccal
absorption since the epithelium region of the former is thinner and immersed
in a larger volume of saliva.
Merits of Drug Administration by Sublingual/Buccal Route

1) Rapid absorption of drugs due to highly vascu larised site, therefore fast onset
of action.
2) Stomach enzymes and acids are not involved so the drug remains stable.
3) Drugs do not undergo first-pass metabolism.
4) Portal circulation is by-passed.
5) In case of any side effects, drugs can be withdrawn.
6) Drugs can be administered easily.
7) Less chances of infection.
8) No involvement of harsh gastrointestinal environment.
Demerits of Drug Administration by Sublingual/Buccal Route

1) It is sometimes inconvenient to keep drugs in mouth.
2) Small doses of drugs are required to keep in mouth.
3) Drugs having high molecular weight cannot be absorbed (e.g., insulin).
4) Unpleasant, distasteful, irritant drugs cannot be administered.
1.4.3. Intravenous (IV) Route

Injections are preferred for orally unabsorbed drugs like atrac urium
(neuromuscular blocker). First pass metabolism by the liver can be avoided by IV
route because drugs do not enter into GI T. Intravenous route show s rapid effect
and maintains level of drug in circulation.
Bacterial or other microbial infections may occur due to the use of syringe and
contamination at the site of injection. Injected drugs cannot be removed from
circulation through emesis or using activated charcoal like in oral delivery.
Merits of Drug Administration by Intravenous (IV) Route
1) 100% bioavailability is achieved.
2) Desired blood concentrations are achieved.
3) Large quantities.
4) Helpful in emergency situations.
5) No first-pass metabolism occurs.
6) Prevent gastric manipulation.
* *
Demerits of Drug Administration by Intravenous (IV) Route

1) Inconvenient, painful and cause irritation, cellulitis and thrombophlebitis.
2) Repeated injections are not suitable.
3) Safety level is very low.
4) Technical and trained person are required to inject drugs.
5) Infection may occur.
6) Costly.
1.4.4. Intramuscular (IM) Route

In intramuscular (IM) route, drug is delivered in the form of aqueous solutions or
depot prepara tions, i.e., drug suspension in non -aqueous vehicle (polyethylene
glycol). By this route, aqueous preparation gets rapidly absorbed. Depots are
used when slow release of drug is needed. Diffusion of vehicle from muscle and
subsequent precipitation of drug at the site of injection occur providing a
sustained release of drug. For example, sustained-release haloperidol decanoate
slowly diffuses from the muscle and gives prolonged neuroleptic effect.
The following factors determine the absorption rate of drugs from intramuscular
sites:
1) Vascularity of the Injection Site: The blood flow rate to muscular tissues in
which drugs are injected is arranged in its decreasing order as - Arms
(deltoid) > Thighs (vastus lateralis) > Buttocks (gluteus maximus). Blood
flow rate is the rate-limiting step in drug absorption from intramuscular sites.
The most rapid absorption occurs from deltoid muscles and slowest
absorption occurs from gluteal region.
2) Lipid Solubility and Drug Ionisation: Highly lipophilic drugs undergo
rapid absorption by passive diffusion, while the hydrophilic and ionised
drugs are slowly absorbed through the capillary pores.
3) Drug Molecular Size: Small molecules and ions directly enter the capillaries
through pores, while the macromolecules enter the lympha tic system. Small
peptides and fluids undergo cytopemphis, in which they cross the
endothelial tissue of blood capillaries and lymph vessels by getting
transported in small vesicles that cross the membrane.
4) Injection Volume and Drug Concentration: A drug i n concentrated form
and large volume injection undergoes faster absorption in comparison to the
drug given in dilute form and small volume injection.
5) pH, Composition and Viscosity of Injection Vehicle: When a drug solution
in acidic or alkaline pH ( e.g., phenytoin, pH 12) or in a non -aqueous solvent
such as propylene glycol or alcohol ( e.g., digoxin) is injected
intramuscularly, the drug precipitates at the injection site after a slow and
prolonged absorption. Viscous vehicles such as vegetable oils also sl ow
down the absorption of drug.
Merits of Drug Administration by Intramuscular (IM) Route
1) Uniform absorption occurs.
2) Onset of action is fast.
* *
3) Mild irritants can be given.

4) Prevent first pass metabolism.
5) No GIT-related factors.
Demerits of Drug Administration by Intramuscular (IM) Route

1) Only small quantities (10ml) of drug can be administered.
2) Local pain, abscess, and infection may occur.
3) Expensive.
1.4.5. Subcutaneous (SC) Route

Subcutaneous (SC) route of drug administration is similar to IM injections, but
the drug absorption in this route is slower than the IV route. The risk associated
with IV route can be reduced by using subcutaneous route. For example, solid
contraceptives (e.g., a single rod with etonogestrel) is implanted for prolonged
effect; Implanted programmable mechanical pumps are implanted to deliver
insulin in diabetic patients.
Merits of Drug Administration by Subcutaneous (SC) Route

1) Can be self-administered.
2) Complete but slow absorption.
3) Massage/ heat: vasoconstriction.
Demerits of Drug Administration by Subcutaneous (SC) Route

1) Painful.
2) Irritant drugs cause tissue damage.
3) Only small quantities of dose (2ml) can be injected.
1.4.6. Inhalational Route

This route delivers drug throughout the respiratory tract, mucous membranes and
pulmonary epithelium, as well as give fast effect as intravenous injections. Gases
or aerosol forms of drugs (like anaesthetics ) are administered through
inhalational route. This route is effective in treatment of patients with respiratory
complications such as asthma or chronic obstructive pulmonary disease.
Systemic side effects related to some drugs ( e.g., albuterol and corticosteroids,
and fluticasone) can be minimised in this route.
Merits of Drug Administration by Inhalational Route

1) Surface area of the respiratory endothelium is largeand cause rapid absorption.
2) Bronchodilators and inhaled steroids affect lungs with less systemic
absorption and minimum side effects.
3) Instant absorption of drug and rapid onset of action.
4) No hepatic first-pass metabolism of drug.
Demerits of Drug Administration by Inhalational Route
1) Specialised equipment required for drug delivery, e.g., inhalers.
2) Bioavailability of drug depends on the patient’s inhaler technique and drug
particle size.
3) Due to the use of inhaler, dose regulation is difficult.
* *
1.4.7. Transdermal Route

Transdermal patches are employed to deliver systemic effect of drug through
skin. The rate of absorption depends on physical characteristics of the skin and
application site. Transdermal patch es provide sustained de livery of drugs, e.g.,
anti-anginal drug (nitroglycerine), antiemetic (scopolamine), and contraceptives.
Merits of Drug Administration by Transdermal Route

1) Sustained effect.
2) No hepatic first-pass metabolism.
3) Convenient and good patient compliance.
Demerits of Drug Administration by Transdermal Route

1) Relatively slow onset of action.
2) Excessive absorption may cause inflamed, rough, abraded or burning effects
on skin.
3) Preferred for highly lipophilic drugs.
1.4.8. Topical Route

In topical route , the drug is applied on the skin surface (epidermis) or mucous
membrane by means of special formulations, e.g., creams, ointments, gels,
lotions, sprays, powders, and aerosols. By this route, local (affecting a small
area) to systematic (affecting the entire body) effects can be obtained. The drug is
absorbed through the pores in the skin ( e.g., sweat glands, hair follicles, etc.).
These dosage forms are used for treating skin infections, minim ising
inflammation, and protecting skin.
There are three pathways via which solutes can diffuse through the skin:
1) Transcellular (passive diffusion),
2) Intercellular (paracellular),
3) Transappendageal in which drug diffusion occurs through:
i) Hair follicles,
ii) Sweat glands, and
iii) Sebaceous glands.
The following factors influence passive percutaneous absorption of drugs:

1) Skin Conditions
i) Thickness of Stratum Corneum: Drug absorption is slow from foot and
palm as the skin in these regions has thick stratum corneum.
ii) Presence of Hair Follicles: Drug absorption is rapid from scalp as the
skin in this region has abundant hair follicles.
iii) Trauma: Drug absorption is facilitated when the stratum corneum is
destroyed by cuts, rashes, inflammation, mild burns, or other traumatic
conditions.
iv) Hydration of Skin: Drug absorption is facilitated when skin hydration is
promoted by soaking the skin in water or occluding it by using
*
emollients, plastic film, or dressing. *
v) Age: With the ageing of skin, gross histological changes occur. Aged
skin has hardened blood vessels and thus is more prone to allergic and
irritant effects of topically contacted chemicals. Infants (just like adults)
efficiently absorb drug through skin. Their ratio of surface area to body
weight is 3 times that of adults , hence, systemic toxicity of topically
applied drugs is of particular concern in infants.
vi) Skin Microflora: Skin surface holds a microbial population that
promote the biotransformation of topically applied therapeutic agents.
vii) Skin pH: The surface pH of nor mal human skin is 4 -6. A pH gradient
exists within the skin. Permeation of drugs prone to ionisation at skin pH
can be influenced.
viii) Skin Surface Lipids: Skin surface exhibits sebaceous glands that secrete
a mixture of lipids, which form an irregular film on the skin surface (0.4-
4.0mm thick) that can solubilise drug in it and influence its permeation.
ix) Anatomical Site: Different skin regions have differences in thickness of
stratum corneum, presence of appendages, blood circulation, and overall
skin thickness. All these factors have a direct influence on drug permeation.
2) Composition of Topical Vehicle
i) Vehicle or Base: The vehicle incorporating the drug influences drug
absorption. The vehicle in which the drug is dissolved promotes
absorption and not the one in which the drug is dispersed.
ii) Permeation Enhancers: Incorporation of certain chemicals such as
DMSO, propylene glycol, azone, etc., in topical formulations aid drug
penetration.
3) Application Conditions
i) Rubbing: On rubbing the area of drug application, blood circulation to
that area and thus drug penetration is influenced.
ii) Occlusion: Topical preparations produce an occlusive effect that prevents
moisture loss to the atmosphere from the skin and aqueous delivery vehicles.
This trapped endogenous or exogenous m oisture hydrates the stratum
corneum, makes it swell, and thus influences drug permeation across it.
iii) Loss of Vehicle: Loss of vehicle from the application site or
translocation of the applied dose from treated to untreated sites also
influences transdermal penetration. Evaporation of solvents decrease or
increase the drug solubility in the residual phase and thus the drug’s
thermodynamic activity and permeation, depending on the polarities of
the volatile solvents(s) and the drug.
4) External Factors
i) Environment Humidity and Temperature: Higher humidity and
temperature increase the hydration rate, local blood flow, and thus drug
absorption.
ii) Grooming: Frequency and vigour with which one bathes and the type of
soap one uses also contribute to variability in drug absorption.
* *
iii) Exposure to Chemicals: Occupational exposure to solvents accelerates

shedding of epidermal cells and thus enhances drug absorption.
iv) Chronic Use of Certain Drugs: Long-term use of cortisol or keratolytic
(like salicylic acid) enhances drug penetration.
Nitroglycerine, lidocaine, betamethasone, oestradiol, testosterone, etc. are
administered percutaneously. This route is useful for drugs having low oral
availability and short duration of action; effect of the latter category of drugs is
prolonged because percutaneous absorption is a slow process.
Merits of Drug Administration by Topical Route

1) Drug can be applied easily.
2) Less complication than oral delivery as drugs poorly absorb systemically.
3) Fast action on application site.
4) Creams and gels are less greasy and more convenient than ointments.
5) Lotions are best on hairy parts of the body.
Demerits of Drug Administration by Topical Route

1) Skin irritation occurs.
2) Improper absorption of certain drugs.
3) Ointments have longer duration of action due to sticky and oily texture.
1.4.9. Rectal Route

Suppositories are administered through rectal route. Drug is formulated with
waxy additives in wh ich the drug is dissolved or liquefie d on insertion into the
rectum. Drug absorbance occurs directly through thin, highly vascularised wall of
rectum. Around 50% of rectal drainage bypasses the portal circulation, i.e., less
biotransformation of drugs by liver. This route is used to avoid the destruction of
drug by intestinal enzymes or by low pH of stomach. Moreover, this rou te is
useful in preventing drug -induced vomiting due to oral administration as well as
in unconscious patient. The drug is administered in the form of suppositories,
through rectal route, when patient is not able to take drug orally (due to vomiting,
in consciousness) or have restrictions on eating (mostly after surgery).
Merits of Drug Administration by Rectal Route

1) Useful in patients suffering from nausea and vomiting.
2) Bypasses first pass metabolism, since absorption occurs from external
haemorrhoidal veins.
3) Gastric irritant drugs can be administered through this route.
Demerits of Drug Administration by Rectal Route

1) Rectal inflammation may occur.
2) Irregular absorption occurs.
1.4.10. Vaginal Route

Vagina is considered the best administration route for contraceptives, anti-fungals,
and antimicrobials for best achievement of local or systemic abs orption. Vaginal
wall contains a vast network of blood vessels, and thus is suitable for absorption of
* *
drugs for systemic use. Drug administration via vaginal route avoids the gut and
hepatic first pass metabolism, reduces gastrointestinal and hepatic sideeffects, and
enables local targeting of drugs to the reproductive organs.
Drug transport across vaginal membrane takes place in three major ways:
1) Transcellularly via concentration-dependent diffusion through cells,
2) Paracellularly mediated via tight junctions, and
3) Vesicular- or receptor-mediated transport.
Drug absorption from vaginal delivery system takes place in two main steps:
1) Drug dissolution in vaginal lumen, and
2) Membrane penetration.
Merits of Drug Administration by Vaginal Route

1) Enables prolonged drug release.
2) Minimises systemic side effects.
3) Increases bioavailability.
4) Utilises less amount of drug than utilised via oral route.
5) Avoids first-pass metabolism.
6) Eases self-medication.
7) Avoids contact with digestive fluid and minimises drug degradation.
8) Inhibits the occurrence of nausea, vomiting, and emesis , generally induced
after oral administration of drugs.
9) Enables a quick onset of action.
Demerits of Drug Administration by Vaginal Route
1) Gender specific.
2) Results in patient non-compliance.
3) Only a few drugs can be administered.
4) Drug absorption varies due to menstrual cycle, menopause, and pregnancy.
5) Affects the intercourse.
6) Demands maintenance of personal hygiene.
7) Some drugs are sensitive at vaginal pH.
1.4.11. Intraocular Administration

Drugs are topically applied to the eyes for local effects such as mydriasis,
meiosis, anaesthesia, treatment of infections, glaucoma, etc. Sterile aqueous
solutions of drugs are us ed as ophthalmic formulations and administered in the
conjunctival cul-de-sac. Cornea possessing hydrophilic and lipophilic
characteristics acts as a barrier to intraocular drug penetration. Thus, for optimum
intraocular permeation of drugs, they should po ssess biphasic solubility.
Lachrymal fluid pH influences absorption of weak electrolytes such as
pilocarpine. On the other hand, formulation pH influences lachrymal output;
ophthalmic solutions of higher pH decreases tear flow and promotes drug
absorption while ophthalmic solutions of lower pH increase lachrymation and
subsequent drug loss due to drainage. Rate of blinking also influences drainage
loss. Volume of fluid instilled into the eyes also affects drug bioavailability and
subsequent effectiveness.
* *
Human eye can usually hold around 10 l of fluid; hence, instilling concentrated
form of drug solution in small volume increases its effectiveness than when
administered in dilute form in large volume. Viscosity enhancers added in the
formulation prolong t he drug’s contact time with the eye, thus increase
bioavailability. Due to the same reason, oily solutions and ointments show
sustained drug action. Sometimes systemic absorption of a drug having low
therapeutic index (such as timolol) precipitates undesir able toxic effects.
Systemic entry of drugs occurs by the absorption r oute into lachrymal duct that
drains lachrymal fluid into the nasal cavity and GIT. This can be prevented by
simply closing the eyelid or by nasolacrimal occlusion in which the fingertip is
pressed on the inside corner of the eye after drug instillation.
1.4.12. Absorption of Drugs from Common Non per Oral

Routes of Drug Administration
Absorption, advantages and disadvantages of common routes of drug
administration are given in table 1.1:
Table 1.1: Absorption of Drugs from Common Routes of Drug Administration
Routes Bioavailability/ Advantages Disadvantages
Absorption
Intravenous Complete (100%) Drug is given for Increased chance for
(IV) systemic drug immediate or adverse reactions;
absorption. controlled effect; Possible anaphylaxis;
Can inject la rge Requires skill in insertion
fluid volumes; of infusion set; Tissue
Suitable for damage at site of
irritating drugs. injection (infiltration,
necrosis, or sterile
abscess).
Intramuscular Passive diffusion, Easier to in ject Irritating drugs can be
Injection (IM) endocytosis, pore than intravenous painful; Variable rates of
transport; Rapid injection; Larger absorption depending on
absorption from aqueous volumes can be muscle injected and
solutions; Slow used compared to blood flow.
absorption from non - subcutaneous
aqueous (oily) solutions. solutions.
Subcutaneous Passive diffusion; Rapid Generally used f or Rate of drug absorption
Injection (SC) absorption from aqueous vaccines and drugs depends on blood flow
solution; Slow not absorbed and injection volume.
absorption from depot orally, e.g.,
formulations. insulin.
Buccal or Passive diffusion, carrier No pre -systemic Some drug can be
Sublingual mediated transport; metabolism. swallowed; Not for most
(SL) Rapid absorption of drugs or drugs with high
lipid-soluble drugs. doses.
Rectal (PR) Passive diffusion; Useful when Absorption can be erratic;
Absorption may vary patient cannot Suppository can migrate
from suppository; More swallow to different position;
reliable absorption from medication; Used Some patient s may feel
enema (solution). for local and discomfort.
systemic effects.
* *
Vaginal Passive diffusion Increases Gender-specific; Patient

bioavailability; non-compliance.
Minimises
systemic side
effects; Quick
onset of action.
Transdermal Passive diffusion; Slow Transdermal Some irritation by patch
absorption, rate may delivery system or drug; Permeability of
vary; Increased (patch) is easy to skin variable with
absorption with use and withdraw; condition, anatomic site,
occlusive dressings. Continuous age and gender; Type of
release for a cream or ointment base
specified period; affects drug release and
Used for lipid - absorption.
soluble drugs with
low dose and low
MW; Low pre -
systemic
metabolism.
Inhalation Passive diffusion, pore Can be used for Particle size of drug
transport; Rapid local or systemic determines anatomic
absorption; Total dose effects. placement in respiratory
absorbed is variable. tract;
Can stimulate cough
reflex;
Some drugs can be
swallowed.
1.5. SUMMARY
The details given in the chapter can be summarised as follows:
1) Biopharmaceuticsis defined as, ―study of the interrelationship of
physicochemical properties of the drug, dosage form in which the rug d is given
and the administration route on the rate and extent of systemic drug absorption.‖
2) Biopharmaceutics is also defined as ―study of the factors influencing the
rate and amount of drug that reaches the systemic circulation and the use of
this information to optimise the therapeutic efficacy of drug products.‖
3) The GIT epithelium lining is the major cellular barrier to the absorption of
drugs from the GIT.
4) Passive diffusion (or non-ionic diffusion) is defined as the difference in the
drug concentrati on on either side of the membrane. Concentration or
electrochemical gradient is the driving force for this process.
5) The kinetic energy of drug molecules is responsible for the movement of drug.
6) Passive diffusion can be expressed by Fick’s first law of di ffusion,
according to which the drug molecules diffuse from a region of higher
concentration to lower concentration until equilibrium is achieved, and the
rate of diffusion is directly proportional to the concentration gradient across
the membrane.
* *
7) Carrier-mediated transport involves a membrane component, called the

carrier that reversibly or non -covalently binds to the solute molecules to be
transported.
8) Facilitated diffusion is a carrier -mediated transport system t hat works at a
much faster rate than the passive diffusion. Concentration gradient is the
driving force for this process that operates down the hill and thus is a passive
process.
9) Active transport involves movement of a substance from a region of low
concentration to high concentration, i.e., against its concentration gradient.
10) Active transport utilises energy, unlike passive transport that does not use
any type of ener gy. If it uses chemical energy from ATP, it is termed as
primary active transport. If it uses an electrochemical gradient, it is termed
as secondary active transport.
11) Pore transport (or connective transport, bulk flow or filtration) involves
the absorption of low molecular weight, low molecular size, and water -
soluble drugs ( e.g., urea, water, and sugar) through narrow, aqueous filled
channels or pores in the membrane structure.
12) Ionic or electrochemical diffusion involves the diffusion of ionic molecules
across the membrane as a function of potential di fference or electrical gradient.
13) Endocytosis is a minor transport mechanism in which the extracellular
materials are engulfed within a segment of the cell membrane to form a
saccule or a vesicle, which is then pinched-off intracellularly.
14) The process of endocytosis helps in the cellular uptake of macromolecular
nutrients (like fats and starch), oil soluble vitamins (like A, D, E, and K), and
drugs (like insulin).
15) Phagocytosis (cell eating) involves adsorptive uptake of solid particulates.
16) Pinocytosis (cell drinking) involves uptake of fluid solute.
17) At times, transcytosis occurs in which an endocytic vesicle is transferred
from one extracellular compartment to another.
18) Drug micronisation increases the drug solubility in small intestine (a better
absorption site), but also increases the drug degradation rate in stomach due
to increased surface area.
19) Dissolution rate is the amount of substance that goes into the solution per
unit time under standard conditions of temperature, pressure, and solvent
composition.
20) Noyes-Whitney equation describes the process of dissolution, and states that
the dissolution rate of drug (d c/dt) is directly proportional to the diffusion
coefficient of the drug in solution state in gastrointestinal fluid (D), effective
surface area of the drug particle available for drug dissolution (A), difference
in the saturation solubility of the drug in the diffusion layer (C s), and the
concentration of drug in solution state in the bulk of gastrointestinal fluid (C).
21) Danckwert’s model of dissolution does not consider the formation of
diffusion layer and the solid surface is con stantly exposed to fresh solvent
causing mass transfer.
* *
22) Limited solvation theory states that an intermediate concentration less than
saturation exist at the interface due to s olvation mechanism, which is the
function of solubility than that of the diffusion.
23) Good wettability results in particle size reduction.
24) The solubility of drugs can be easily enhanced by converting them into their
salt forms.
25) Brodie gave the pH partition theory to explain the absorption process of
drug from GIT and its distribution across the biological membranes.
26) The dissociation constant (pKa) of drug and the pH of body fluid at the
absorption site influence the amount of unionised drug.
27) Weakly acidic drugs with pKa > 8 are unionised at all pH values and hence
their absorption rate is rapid and independent of gastrointestinal pH.
28) Weakly acidic drugs with pKa ranging from 2.5 to 7.5 undergo pH -
dependent absorption. At acidic conditions of stomach (pH > pKa), they exist
in unionised form and undergo better absorption.
29) Strongly acidic drugs with pKa < 2.5 are ionised in the entire pH range of
GIT and hence are poorly absorbed.
30) Weakly basic drugs with pKa < 5.0 are ionised at all pH values and hence
undergo rapid and pH-independent absorption.
31) Weakly basic drugs with pKa ranging from 5 to 11 undergo pH-dependent
absorption. At alkaline conditions of the intestine, they exist in unionised
form and undergo better absorption.
32) Strongly basic drugs with pKa > 11 remain ionised in the entire pH range
of GIT and hence are poorly absorbed.
33) If a drug exists in unionised form, it will still undergo poor absorption,
provided its lipid solubility is low, i.e., it has a low value of Ko/w.
34) The lipid solubility of a drug can be determined from its oil/water partition
coefficient value (Ko/w).
35) A solid drug’s particle size and surface area are inversely proportional.
Smaller the drug particle, greater is its surface area.
36) Enantiotropic polymorphs can be reversibly changed into another form by
altering the temperature or pressure.
37) Monotropic polymorphs are unstable at all temperatures and pressures.
38) Stoichiometric type of adducts in which the solvent molecules are
incorporated in the crystal lattice of the solid are termed solvates, and the
trapped solvent is termed solvent of crystallisation.
39) Solvates can exist in different crystalline forms, which are known as
pseudopolymorphs, and the phenomenon is termed pseudopolymorphism.
40) If the solvent in association with the drug is water, the solvates are termed
hydrates, which are the most common solvate forms of drugs.
41) Aqueous solubility of the anhydrous form of a drug, i.e., anhydrates, is more
than that of the hydrates.
42) Non per Os or Oral indicates drug administration routes other than the oral
*
route, which bypasses the GIT and enter the systemic circulation. *
43) A major advantage of drug administration through non -invasive

transmucosal and transdermal routes (like nasal, buccal, rectal, etc.) is that a
greater systemic availability is attained.
44) Injections are preferred for orally unabsorbed drugs like atracuriu m
(neuromuscular blocker).
45) In intramuscular (IM) route , drug is delivered in the form of aqueous
solutions or depot preparations, i.e., drug suspension in non -aqueous vehicle
(polyethylene glycol).
46) Subcutaneous (SC) route of drug administration is simila r to IM injections,
but the drug absorption in this route is slower than the IV route.
47) In topical route , the drug is applied on the skin surface (epidermis) or
mucous membrane by means of special formulations.
48) Drug transport across vaginal membrane takes place transcellularly via
concentration-dependent diffusion through cells, paracellularly mediated via
tight junctions, and vesicular- or receptor-mediated transport.
1.6. EXERCISE
1.6.1. True or False

1) Endocytosis involves the absorption of low molecular weight dru gs through narrow,
aqueous filled channels or pores in the membrane structure.
2) If active transport utilises chemical energy from ATP, it is termed as primary active
transport.
3) Passive diffusion can be expressed by Fick’s first law of diffusion.
4) Passive diffusion is defined as the difference in the drug concentration on either side
of the membrane.
5) Pore transport is a minor transport mechanism in which the extracellular materials
are engulfed within a segment of the cell membrane.
6) Solubility is the amount of substance that goes into the solution per unit time under
standard conditions of temperature, pressure, and solvent composition.
7) Good wettability increases particle size.
8) Weakly basic drugs with pKa < 5.0 are io nised at all pH values and hence undergo
rapid and pH-independent absorption.
9) Enantiotropic polymorphs are unstable at all temperatures and pressures.
1.6.2. Fill in the Blanks

10) If active transport utilises _______, it is termed as secondary active transport.
11) _______ is the driving force for facilitated diffusion.
12) _______ involves uptake of fluid solute.
13) _______ states that an intermediate concentration less than saturation exist at the
interface due to salvation mechanism.
14) Weakly acidic drugs with pKa ______ _ are unionised at all pH values and hence
their absorption rate is rapid and independent of gastrointestinal pH.
15) The lipid solubility of a drug can be determined from its _______.
16) Smaller the _______, greater is its surface area.
* *
17) Aqueous solubility of the _______ is more than that of the _______ .
18) Drug transport across vaginal membrane takes place _______ , _______ , and
vesicular- or receptor-mediated transport.
Answers
1) False 2) True 3) True
4) True 5) False 6) False
7) False 8) True 9) False
10) Electrochemical gradient 11) Concentration gradient 12) Pinocytosis
13) Limited solvation theory 14) > 8
15) Oil/water partition coefficient value 16) Drug particle
17) Anhydrates and hydrates 18) Transcellularly and paracellularly
1.6.3. Very Short Answer Type Questions

1) Define biopharmaceutics.
2) Give the applications of biopharmaceutics.
3) Write about endocytosis.
4) What do you understand by active transport?
5) State the pH-partition theory.
6) Give the merits and demerits of sublingual route of drug administration.
1.6.4. Short Answer Type Questions

1) Enlist the different mechanisms of drug absorption and explain any two in detail.
2) Discuss how absorption of drug is affected by dissolution rate .
3) How pharmaceutical ingredients influence the absorption process?
4) Discuss the topical route of drug administration.
5) Write a note on intravenous and intramuscular routes of drug administration.
1.6.5. Long Answer Type Questions

1) Give a detailed review on mechanism of absorption through GIT .
2) Discuss the factors influencing drug absorption through GIT .
3) Write an exhaustive note on absorption of drugs from n on per oral extra -vascular
routes.
* *
Introduction to Distribution (Chapter 2) 45
CHAPTER Introduction to
2 Distribution
2.1. DISTRIBUTION
2.1.1. Introduction
The processes which lower the plasma drug concentration are termed
disposition. For the disposition of drugs, mainly two processes are involved:
1) Distribution: This process involves the reversible transfer of a drug between
ompartments. Drug distribution is also defined as the reversible transfer of a
drug between one compartment and another.
2) Elimination: This process involves irreversible loss of drug from the body.
Elimination is further divided into two processes, namely biotransformation
(metabolism) and excretion.
Tissue receptor or
site of action
Drug administration (oral, Plasma

parenteral, etc.) Protein or
tissue bound
Absorption Free drug Biotransformation

Metabolites (metabolism)
(from site of
administration)
Excretion
Figure 2.1: Inter-Relationships among Various Process of Drug Disposition

Distribution of drugs occurs through the circulatory system (by the circulation of
blood). Therefore, blood or plasma si gnifies one of the compartments, while
extravascular fluids and other body tissues signify the other compartments. In
other words, distribution of drug is a reversible transfer of drug between the
blood and the extravascular fluids and tissues.
Distribution is a passive transport process and the driving fo rce for this
process is obtained from the difference of concentration gradient between the
blood and extravascular tissues. By the diffusion process, the drug
concentration increases in tissues until it reaches equilibrium where the
amount of drug entering the tissues becomes equal to the amount of drug
draining out from the tissues. At equilibrium state, the drug concentration in
the tissues depends on the rate at which the drug is distributed in the tissues
and on the drug concentration in the plasma.
Plasma concentration Tissue concentration
* *
Drug distribution does not occur uniformly throughout the body because different
tissues get the drug from the plasma at different rates and in different
concentrations.
2.1.2. Tissue Permeability of Drugs

If the blood flows rapidly and uniformly to the entire body tissues, differences in
the degree of distribution between tissues will indicatethe differences in the tissue
penetrability of the drug. This process will be tissue permeability rate -limited. In
distribution of drugs, following are thetwo main rate-determining steps:
1) Rate of Tissue Permeability: Tissue permeability of a drug mainly depends
on two factors, i.e., the physicochemical properties of the drug and the
physiological barriers restricting diff usion of drug into tissues. Molecular
size, degree of ionisation, and partition coefficient are t he main
physicochemical properties influencing drug distribution.
Most of the drugs having molecular weight less than 500 -600 Daltons can
feasibly diffuse int o the extracellular interstitial fluids by crossing the
capillary membrane . But, penetration of drugs from the extracellular fluid
into the cells is determined by the molecular size, ionis ation constant , and
drug lipophilicity. Only small, water -soluble molecules and ions of size less
than 50 Daltons enter the cell via aqueous filled channels , while the larger -
sized particles can be passed by a specialised transport system.
The tissue permeability of a drug is mainly determined by its degree of
ionisation. The ionisation and diffusion of drugs into cells decide the pH of
blood and extravascular flu id. A drug which remains unionis ed at these pH
values permeates the cells with a faster rate.
The pH of blood and ECF generally remain constant at pH 7.4, therefore they
do not affect dr ug diffusion unless conditions like systemic acidosis or
alkalosis are static or remain unaltered.
Capillary wall Cell membrane
Intracellular
Blood ECF fluid
Unionised drug
(low Ka and large Ko/w)
Ionised drug + +
(large Ka and low Ko/w) Pore
Figure 2.2: Permeation of Unionised and Ionised Drugs across the

Capillary and Cell Membrane
Drugs are mostly weak acids or weak bases , and their degree of ionis ation at
plasma or ECF pH depends on their pK a value. All polar and hydrophilic
drugs get ionised at plasma pH, cannot penet rate the lipoidal cell membrane,
and tissue permeability is the rate -limiting step in the ir distribution. Only
lipophilic unionised drugs rapidly cross the cell membrane.
* *
In case of polar drugs in which permeability is the rate -limiting step in the ir
distribution, effective partition coefficient of drug is the driving force and it
is calculated as follows:
Fraction unionised K o/w of
Effective K o/w   …..(1)
at pH 7.4 unioniseddrug
The degree up to which effective partitio n coefficient influences rapid ity of
drug distribution can be explained by the following example (table 2.1):
Table 2.1: Distribution of Acidic Drugs in CSF
Drugs Relative Acidity Effective Ko/w at Relative Rate of
pH 7.4 Distribution
Thiopental Weaker acid 2.0 80
Salicylic acid Stronger acid 0.0005 1
Permeability is the rate-limiting step in drug distribution:

i) If the drug under consideration is ionic, polar, or water-soluble.
ii) If the highly selective physiological barriers restrict such drugs to diffuse
into the cell.
On the other hand, perfusion is the rate-limiting step in drug distribution:
i) If the drug is highly lipophilic.
ii) If the membrane across which the drug is to diffuse is highly permeable
(such as those of the capillaries and muscles).
Only highly lipophilic drugs (like thiopental) can cross the most selective
barriers such as the blood -brain barrier (BBB), while highly permeable
capillary wall allows almost all the drugs (except those bound to plasma
proteins) to pass. In both the cases, the rate of blood flow or perfusion to the
tissue is the rate -limiting step . Thus, greater the blood flow, faster the
distribution.
2) Rate of Blood Perfusion: Perfusion rate is the volume of blood that flows
per unit time per unit volume of the tissue
. Its unit is ml/min/ml of the tissue.
If K t/b represents tissue/bloo d partition coefficient of drug, the first -order
distribution rate constant (Kt) is expressed as:
Perfusion Rate
Kt  ….. (2)
Kt/b
The tissue distribution half-life is given as:

0.693 0.693Kt/b
Distribution on half  life   ….. (3)
Kt Perfusion Rate
The extent up to which a drug is distributed in a parti cular tissue or organ

depends on the size of tissue (i.e., tissue volume) and the tissue/blood
partition coefficient of the drug. This can be explained by taking the example
of thiopental (a lipophilic drug) , which has a high tissue/blood partition
coefficient towards the brain and higher for adipose tissue. Because brain
(site of action) is a highly perfused organ, thiopental diffuses very rapidly
*
into the brain and shows a rapid onset of action when given intravenously. *
Adipose tissue s are poorly perfused, therefore , distribution occurs very

slowly with the same drug. If thiopental concentration in the adipose tissue
shifts towards equilibrium, the drug rapidly diffuses out of the brain and
localises in the adipose tissue whose volume is 5 times more than brain and
has greater affinity for the drug. Such tissue redistribution results in rapid
termination of action of thiopental.
2.1.3. Physiological Barriers

Different types of barrier s are found in the body that restrict the distribution of
various compounds that enter the blood to different tissues. These barriers are
present in the body for protecting the sensitive tissues from the effect of various
chemicals that enter the blood via different routes.
The physiological barriers are discussed below:

1) Blood Capillary Membrane : Drug passes the capillary membrane t hrough
passive diffusion an d hydrostatic pressure. By passive diffusion , drug
molecules travel across the region of high concentration to low
concentration; it can be described by Fick’s law of diffusion.
dQ – DKA (Cp – C t )
Rate of Diffusion  
dt h
Where,
D = Drug diffusion coefficient in the membrane.
K = Lipid/water partition coefficient.
A = Membrane surface area.
Cp = Plasma drug concentration.
Ct = Tissue drug concentration.
h = Membrane thickness.
The negative sign indicates drug movement from inside the blood capillary
into the tissues.
2) Simple Capillary Endothelial Barrier : Capillaries supply blood to most of
the tissues. Capillary endothelium allows passage of drugs (ionised or
unionised of molecular size ˂ 600 Daltons) int o the interstitial fluid. Only
drugs bound to the blood components are restricted because of the large
molecular size of the complex.
3) Cell Membrane : Drug present in ECF is transported by passive diffusion
into the cell. Factors influencing the penetration of drugs into cells are same
as those observed in the gastrointestinal absorption of drugs.
For the transport of drugs, cell membrane acts like a lipid barrier.
Permeability of drugs through cell membrane chec ks the entry into the
cell.
The physicochemical properties that influence permeation of drugs across
such a barrier are illustrated in figure 2.3.
* *
Capillary wall Cell membrane

Intracellular
Blood ECF fluid
D Drugs of size less D D Bulk flow

P D
D
+ than 50 Daltons
–
D
D D
Lipophilic drugs D D Passive diffusion
P
50-600 Daltons
D
c
D + Polar/ionised drugs of + +
D D D Active transport
D size  50 Daltons
–
P D D
Figure 2.3: Cell Membrane Barrier and Drug Diffusion across it

4) Blood-Brain Barrier (BBB): Permeability of capillaries present in the brain
and spinal cord is different from that of the capillaries of rest of the body.
Endothelial cells of capillaries are covered by a layer of glial cells that have
tight intercellular junctions providing thicker lipid bar rier. This layer of glial
cells reduces diffusion and penetration of water -soluble and polar drugs into
the brain and spinal cord. Figure 2.4 represents this lipid barrier (BBB).
Brain
ECF of brain
Glial cells
Basement
Tight junctions membrane
Lipid-soluble drug
Blood Capillary
endothelium
Figure 2.4: Transport of Lipid-Soluble Drug across BBB
Normally, only lipid-soluble drugs can penetrate the interstitial fluid of the brain
and spinal cord, while water-soluble compounds need specific carriers to cross
the endothelial lining. In diffusion process , many transport mechanisms are
involved. Degree of ionisation in plasma and drug lipid solubility determines
the penetration rate of a drug into the brain. Highly lipid-soluble drugs (e.g.,
thiopental) cross BBB immediately, and reach the brain from plasma. Polar
drugs ( e.g., barbital) penetrate the CNS slowly. Weak organic acid s (e.g.,
penicillin G having pKa 2.6) are found in completely ionised form in plasma,
but penetrate the brain at a low rate due to poor lipid solubility.
Approaches to Promote Crossing the BBB
i) Permeation enhancers ( e.g., Dimethyl Sulfoxide or DMSO) are used for
*
increasing penetration. *
ii) Mannitol infused in internal carotid artery result in osmotic disruption of

BBB.
iii) Carriers (e.g., dihydropyridine redox system) are used to transport drug
into brain.
5) Blood-Cerebrospinal Fluid Barrier : Cerebrospinal fluid form s in choroid
plexus, found in the roof of the fourth ventricle and projects between the
cerebellum and pons on the lower bra in stem. At anterior brain stem in the
roof of the diencephalon, two extensive folds of choroid plex us originate and
extend through inter-ventricular foramina. Floors of the lateral ventricles are
covered by folds of the choroid plexus.
Drug diffuses easily through the blood -CSF barrier because the junctions
between the endothelial cells of blood capilla ries are open; so the drug
molecules easily reach the extracellular fluid (ECF) from the blood at the
barrier. But at the region of choroid plexus , tight junctions are present
between the cells that obstruct the penetration of polar drugs. So, only lipid -
soluble drugs can diffuse through the lipoidal barrier (figure 2.5).
Tight junction
CSF Choroid plexus cell
ECF
Capillary
endothelium
Open
junctions Lipid-soluble drug
Figure 2.5: Transport of Lipid-Soluble Drug across Blood Cerebrospinal

Fluid Barrier
As the CSF is almost devoid of protein, the CSF concentration of lipid -
soluble drugs represents the free drug concentration in plasma. Concentration
of drugs is greater i n brain tissues as compared to the CSF, e.g., in epileptic
patients concentration of p henytoin is 6 times higher in the temporal lobe
than in the CSF.
6) Placental Barrier: Maternal and the foetal blood vesse ls are separated by
placental barrier, which is made up of endothelium and many layers of tissue
of foetal trophoblast basement membrane.Figure 2.6 shows the blood flow in
maternal and foetal blood vessels. Placental barrier is less effective than BBB
because drugs having molecular weight less than 1000 Daltons and with
moderate to high lipid solubility ( e.g., ethanol, sulphonamides, barbiturates,
gaseous anaesthetics, steroids, narcotic analgesics, anticonvulsants , and
antibiotics) can cross the placental barrier through simple diffusion.
Immunoglobulins are transferred by endocytosis, whereas, nutrients essential
for foetal growth are transported bycarrier-mediated process.
* *
Drugs may give rise to lethal effects on the following two critical stages
during foetal development:
i) First Trimester: It is the duration when the foetal organs are
developing. At this stage , most of the drugs produce teratogenic effects
(congenital defects), e.g., thalidomide, phenytoin, isotretinoin, etc.
ii) Latter Stages: In the l atter stages of pregnancy, drugs may affect the
physiological functions of body, e.g., respiratory depression by
morphine.
Therefore, it is better to avoid using any drug during the pregnancy period
due to the uncertainty of harmful effects.
Foetal vein
Foetal artery
Foetal membrane
Drug movement (trophoblast + endothelium)
Maternal artery
Maternal vein
Figure 2.6: Placental Barrier and Blood Flow across Membrane
7) Blood-Testes Barrier: A layer is formed by the extensions of sustentacular

cells (sertoli cells) that surround the seminiferous tubule beneath the
spermatogonia. To maintain stable conditions, diffusion from interstitial fluid
to the seminiferous tubule is prevented by the tight junctions present between
the sustentacular cells.
2.1.4. Factors Affecting Drug Distribution

Drugs distribution is affected by the following factors:
1) Age: Distribution varies with difference in:
i) Total Body Content (Intracellular and Extracellular): It is maximum
in infants.
ii) Fat Content: It is greater in infants and in elderly people.
iii) Skeletal Muscles: It is less in infants and elderly people.
iv) Organic Composition: Poorly developed BBB, low myelin content, and
high cerebral blood flow in infants cause greater drug penetration in the
brain.
v) Plasma Protein Content: Albumin content is low in infants and elderly
people.
2) Pregnancy: Growth of uterus, placenta and foetus rais es volume for drug
distribution in pregnancy. Drug may also distribute in foetus which acts as a
separate compartment. Plasma and ECF volume also increases , but albumin
*
content is reduced. *
3) Obesity: High adipose ti ssue content results in low drug distribution and
perfusion. High fatty acid content alters the binding property of acidic drugs.
4) Diet: Fat-rich diet increases free fatty acid concentr ation in the blood that
affects the binding of acidic drugs, e.g., NSAIDs, albumin, etc.
5) Disease States: Drug distribution is severely affected in diseased conditions:
i) Alteration in albumin and other drug-protein concentration,
ii) Reduced or altered perfusion to organs and tissues, and
iii) Alteration in tissue pH.
In case of enceph alitis and meningitis, the BBB becomes more permeable,
therefore, concentration of ionic antibiotics (e.g., penicillin G and ampicillin)
increases in brain tissues.
6) Drug Interaction: Two or more drugs administered together compete for the
binding site, tend to replace each other, and the free drug may produce lethal
effects, e.g., phenylbutazone and warfarin.
2.1.5. Volume of Distribution

Varying concentrations of drug reaches different organs and tissues of the body.
The process o f distribution is considered to be complete at distribution
equilibrium. At this stage, different tissues and organs contain varying
concentrations of drug that can be determined by the volume of tissues in which
the drug is present. So, different body tis sues and organs have different
concentrations of drug. The physiological meaning of volume of distribution is
not clear. But, a constant relationship is seen between the amount of drug in body
(X) and the concentration of drug in plasma (C):
XC
Or, X  Vd
Where, Vd = Apparent volume of distribution, which is a proportionality constant

having the unit of volume.
Determination of Volume of Distribution

1) The drug dose is administered by a rapid intravenous bolus injection , and
then blood samples are taken at specific time intervals.
2) A suitable assay method is used to calculate the plasma concentration of each
drug sample.
3) The obtained data is plotted on a graph paper so that the plasma profile of the
drug can be obtained.
4) The drug level in plasma immediately after the administration of drug dose is
calculated by back-extrapolating the plasma concentration versus time profile
of the drug to time zero.
2.1.6. Apparent Volume of Distribution

Apparent volume of distribution is the hypothetical volume of body fluid into
which a drug is dissolved or distributed . It is named as apparent volume
because each part of the body equilibrated with the drug does not have equal
concentration.
* *
Thus, apparent volume of distribution can be represented as:

Amount of Drug in the Body
Apparent Volume of Distribution 
Plasma Drug Concentration
There is no direct relationship between the apparent volume of distribution and
true volume of distribution; while the real volume of distribution has direct
physiological meaning and is related to the body water. By using specific tracers
or markers, the volume of each of these real physiologic al compartments can be
determined. Hig h molecular weight substance s that can totally bind to plasma
albumin (e.g., high molecular weight dyes such as Evans blue, indocyanine green
and I -131 albumin) are used to determine the plasma protein. In case of
intravascular route, these remain intact to the plasma. If the concentration of
haematocrit is known, total blood volume can also be estimated.
The ECF volume can also be estimated by substances that can easily penetrate the
capillary membrane and rapidly distribute throughout the ECF butdo not cross the
cell membranes (e.g., the Na+, Cl  , Br, SCN and SO42 ions, insulin, mannitol, and
raffinose). The volume of ECF is approximately 15 litres,excluding the plasma.
Tracer elements either negligibly bound or do not bound to plasma or tissue
proteins, thus their apparent volume of distribution remain same to their true
volume of distribution. These conditions differ for most of drugs that bind to
extravascular tissues or plasma proteins or to both. A general concept can be
made in respect of apparent volume of distribution of these drugs:
1) Apparent Volume of Distribution Smaller than True Volume of
Distribution: Drugs that selectively bind to plas ma proteins or other blood
components (i.e., those that are less bound to extravascular tissues, e.g.,
warfarin) have apparent volume of distribution smaller than their true volume
of distribution. The V d of such drugs are found between blood volume and
Total Body Water ( TBW) volume (i.e., between 6 -42 litres ); for example,
warfarin has a Vd of about 10 litres.
2) Apparent Volume of Distribution Larger than True Volume of
Distribution: Drugs that selectively bind to extravascular tissues, (i.e., those
that are less bound to blood components, e.g., chloroquine) have apparent
volume of distribution larger than their true volume of distribution. The Vd of
such drugs is always greater than 42 litres or TBW volume. For example,
chloroquine has a V d of approximately 15,000 litres. Such drugs leave the
body slowly and are generally more toxic than the drugs that do not distribute
deeply into body tissues.
2.1.7. Binding of Drugs

In general, protein binding is defined as the binding of a drug to blood plasma
proteins. This binding can be between the drug and tissue membranes, RBCs,
and other blood components. The effectiveness of a drug on the body depends on
the amount of drug bound to protein. The bound drug remains in the blood and
the unbound drug metabolises into the active part of the drug. Therefore, if a drug
is 95% bound to a binding protein and 5% is free, it indicates that 5% of the drug
is active in the system and gives rise to pharmacological effects.
* *
Generally, protein binding is reversib le, and therefore a chemical equilibrium

develops, in which the chemical reaction occurs in backward and forward direction
without any net change in reactants and products. This indicatesthat while achieving
equilibrium, a cell which is effective at extra cting the unbound drug may extract
more drug as it disassociates. Reversible protein binding can be expressed as:
Protein + Drug ⇌ Protein-Drug Complex
Blood
Drug binding to
blood
components
Blood Plasma
D D proteins
cells
Tissue
Tissue
localisation
Liver
P-D D D (Free drug D P-D
Drug/metabolite in plasma)
+
binding to
Enzymes
liver tissues
D
P-D D + Carrier
Receptor
P-M Metabolites Drug binding Drug
to renal tissues D-C
Active
Secretion
Kidney
Biliary Excretion Pharmacologic

excretion in urine response
Figure 2.7: Protein-Drug Binding: Binding of Drugs to Various Tissue Components and its
Influence on Disposition and Clinical Response. Only the Unbound Drug Moves Reversibly
Between the Compartments
In the body, a drug interacts with several tissue components, of which blood and
extravascular tissues are the two major classes. Generally, macromolecules such
as proteins, DNA or adipose molecules, interact w ith body tissues. Protein
molecules interact by forming complex molecules, and this phenomenon of
complex formation is c alled protein binding of drugs . The importance of such
protein binding is that the bound drug is pharmacokinetically as well as
pharmacodynamically inert, thus indicating that a protein -bound drug is neither
metabolised nor excreted nor is pharmacologically active.
A bound drug is also restricted so that it remains confined to a particular tissue
for which it has greater affinity. Howeve r, a bound drug cannot undergo
membrane transport because of its enormous size and therefore its half -life is
enhanced. Binding of drugs falls into two classes:
1) Binding of drugs to blood components, like:
i) Plasma proteins, and
ii) Blood cells.
2) Binding of drugs to the extravascular tissues, proteins, fats, bones, etc.
2.1.7.1. Plasma Protein Binding of Drugs

When drug molecules reach systemic circulation, they interact with blood
components, i.e., plasma proteins, blood cells, and haemoglobin (table 2.2). But,
the drug molecules majorly interact with the plasma protein s present on cell
* *
membrane of various blood components in abundant amounts and large variety.

Binding of drugs to plasma proteins is a reversible proc ess and the extent of
binding of drugs to various plasma proteins occurs as albumin > 1-acid
glycoprotein > lipoproteins > globulins.
Table 2.2: Blood Proteins to which Drugs Bind
Proteins Molecular Concentration Drugs that Bind
Weight (g%)
Human Serum 65,000 3.5-5.0 Large variety of all types of drugs.
Albumin
α1-Acid 44,000 0.04-0.1 Basic drugs such as imipramine,
Glycoprotein lidocaine, quinidine, etc.
Lipoproteins 2,00,000 to Variable Basic, lipophilic drugs like
3,400,000 chlorpromazine.
α1-Globulin 59,000 0.003-0.007 Steroids like corticosterone and
thyroxine, and cyanocobalamin.
α2-Globulin 1,34,000 0.015-0.06 Vitamins A, D, E and K, and cupric ions.
Haemoglobin 64,500 11-16 Phenytoin, p entobarbital, and
phenothiazines.
Binding of drugs to different plasma proteins is discussed below:

1) Binding of Drugs to Human Serum Albumin (HAS): Human Serum
Albumin (molecular weight 65,000) is the most abundant plasma protein
(59% of total plasma and 3.5 -5.0%) having high drug binding capacity. The
therapeutic doses of most of the drugs are relatively much smaller and their
plasma concentration does not normally
achieve equimolar concentration with Site I Warfarin Binding Site
HSA. It has the property of binding to
several compounds of varied Site II Diazepam Binding Site
structures of endogenous origin ( e.g.,
fatty acids, bilirubin , and tryptophan) Site III Digitoxin Binding Site
as well as of exogenous origin ( e.g.,
Site IV Tamoxifen Binding Site
drugs, weak acids, neutral compounds
to weak bases, etc.). Drugs bind to the
Figure 2.8: Four Major Drug Binding
following four binding site s on the Sites on Human Serum Albumin
HSA (figure 2.8):
i) Site-I or Warfarin and Azathioprine Binding Site: Maximum number
of drugs bin d to this region, e.g., several NSAIDs (phenylbutazone,
naproxen, and indomethacin), sulfonamides ( sulfamethizole and
sulfadimethoxine), bilirubin sodium, phenytoin, and valproate.
ii) Site-II or Diazepam Binding Site: Benzodiazepines, medium chain
fatty acid s, ibuprofen, ketoprofen, tryptophan, cloxacillin, probenecid,
etc. bind to this region.
iii) Site-III: This site is also known as digitoxin binding site.
iv) Site-IV: This site is also known as tamoxifen binding site.
2) Binding of Drugs t o α1-Acid Glycoprotein (α1-AGP or AAG): The
molecular weight of α1-acid glycoprotein is about 44,000 and its plasma
* *
concentration ranges between 0.04 -0.1g%. Many basic drugs bind to it, e.g.,
propranolol, quinidine, disopyramide, imipramine, amitriptyline,nortriptyline,
and lidocaine.
3) Binding of Drugs to Lipoproteins: Depending on the chemical
composition, the molecular weight of lipoproteins varies between 0.2 to 3.4
million. On the basis of their density, lipoproteins are classified into:
i) Chylomicrons,
ii) Very Low Density Lipoproteins (VLDL),
iii) Low Density Lipoproteins (LDL, predominant in humans), and
iv) High Density Lipoproteins (HDL, most dense and smallest in size).
A drug binds to lipoproteins by dissolving the lipid core of the lipoproteins. The
binding capacity of drug depends on the lipid content of their lipid core. This
process of binding of drugs to lipoproteins is non-competitive.Many acidic (e.g.,
diclofenac), neutral e( .g., cyclosporine A) and basic drugs e.g.,
( chlorpromazine)
bind to lipoproteins, of which the basic lipophilic drugs have maximum affinity.
4) Binding of Drugs to Globulins: Many plasma globulins are recognised and
are labelled asfollows:
i) α1-Globulin or Transcortin or α1r CBG (Corticosteroid Binding
Globulin): It binds to  number of steroidal drugs, e.g., cortisone,
prednisone, thyroxin, and cyanocobalamin.
ii) α2-Globulin or Ceruloplasmin: It binds to vitamins A D, E and K and
cupric ions.
iii) β1-Globulin or Transferrin: It binds to ferrous ions.
iv) β2-Globulin: It binds to carotenoids.
v) γ-Globulin: It binds to antigens.
2.1.7.2. Binding of Drugs to Blood Cells

In blood, more than 40% are blood cells, in which RBC s constitute 95% of the
total blood cells. Therefore , binding of drug to RBCs is important. The diameter
of RBCs is 500 times more than the major p lasma protein binding component,
albumin. All the three components of RBC can bind to drugs:
1) Haemoglobin:Molecular weight of haemoglobin is 64,500 (almost equal to
HSA), but in blood its concentration is 7 -8 times of al bumin. Phenytoin,
pentobarbital, phenothiazines, etc. bind to haemoglobin.
2) Carbonic Anhydrase: Acetazolamide and chlorthalidone (ca rbonic
anhydrase inhibitors) bind to it.
3) Cell Membrane: Imipramine and chlorpromazine bind to RBC membrane.
Lipophilic drugs (e.g., phenytoin) have a greater rate and extent of entry into
RBCs. Hydrophilic drugs, e.g., ampicillin, do not enter RBCs.
2.1.7.3. Tissue Binding of Drugs

Almost all body tissues, except HSA, consist of 40% of the body weight which is
100 times that of HSA. Therefore, tissue-drug binding is very vital in the study of
drug distribution. A d rug has the capability to bind to one or more of the several
tissue components.
* *
The importance of tissue -drug binding in distribution can be explained by the

following two points:
1) Tissue-drug binding helps in increasing the apparent volume of distributi on
of drugs in contrast to plasma protein binding which decreases it. This
phenomenon occurs because the parameter is related to the ratio of drug
amount in the body to free drug plasma concentration, and the latter
decreases under extensive conditions of tissue-drug binding.
2) Due to tissue -drug binding, the drug gets distributed at some specific site in
the body (with a subsequent increase in biological half -life). This is so
because many drugs irreversibly bind to the tissues (opposite to the plasma
protein-drug binding ). Examples of tissue -drug binding are oxidation
products of paracetamol, phenacetin, chloroform, carbon tetrachloride, and
bromobenzene, which covalently bind to hepatic tissues.
Distribution of drugs in tissuesis affected by lipophilicity and structural features of
the drug, perfusion rate, pH differences, etc. Drugs bind to extravascular tissues in
the following order: Liver > Kidney > Lung > Muscle.
Several examples of extravascular tissue-drug binding are:
1) Liver: Epoxides of various h alogenated hydrocarbons and paracetamol bind
irreversibly to liver tissues and cause hepatotoxicity.
2) Lungs: Basic drugs like imipramine, chlorpromazine and antihistamines
accumulate in the lungs.
3) Kidneys: Metallothionein (a protein present in kidneys) bind s to heavy
metals (such as lead, mercury, and cadmium) and causes their renal
accumulation and thus toxicity.
4) Skin: Chloroquine and phenothiazines interact with melanin and accumulate
in the skin.
5) Eyes: Retinal pigments of the eye contain melanin that bind s with
chloroquine and phenothiazines, thus resulting in retinopathy.
6) Hairs: Arsenicals, chloroquine, and phenothiazines deposit in hair shafts.
7) Bones: Tetracycline binds to bones and teeth. If this antibiotic is
administered to infants or children during odontogenesis, they will have
permanent brown-yellow discolouration of teeth. Lead replaces calcium from
bones and makes them brittle.
8) Fats: Lipophilic drugs ( e.g., thiopental) and DDT (pesticide) accumulate in
adipose tissues by partitioning into it. Howe ver, high o/w partition
coefficient is not the only criteria for adipose distribution of drugs since more
lipophilic basic drugs (like imipramine and chlorpromazine) are not localised
in fats. Poor perfusion of adipose tissues could be the reason for such an
ambiguity. Adipose localisation of drugs occurs due to binding competition
between adipose and non -adipose tissues (lean tissues like muscles, skin and
viscera) and not due to partitioning.
9) Nucleic Acids:Molecular components of cells, like DNA, strongl y interact with
chloroquine and quinacrine, and result in distortion of its double helical structure.
* *
2.1.8. Factors Affecting Protein-Drug Binding

Factors affecting protein-drug binding are categorised as follows:
1) Drug-Related Factors
i) Physicochemical characteristics of drug,
ii) Concentration of drug in body, and
iii) Affinity of a drug for a particular binding component.
2) Protein/Tissue Binding-Related Factors
i) Physicochemical properties of the protein or binding agent,
ii) Concentration of protein or binding agent, and
iii) Number of binding sites on binding agent.
3) Drug Interactions
i) Competition between drugs fora binding site (displacement interactions),
ii) Competition between drugs and normal body constituents, and
iii) Allosteric changes in protein molecule.
4) Patient-Related Factors
i) Age,
ii) Intersubject variations, and
iii) Disease states.
2.1.8.1. Drug-Related Factors

The drug-related factors which affect protein binding are discussed below:
1) Physicochemical Characteristics of Drug: Protein binding directly depends
on the lipophilicity of drug; therefore an increase in lipophilicity increases
the extent of binding. Highly lipophilic drugs ( e.g., thiopental) localise in
adipose tissues, ani onic or acidic drugs ( e.g., penicillins and sulphonamides)
bind to HAS, cationic or basic drugs ( e.g., imipramine and alprenolol) bind
to AAG, and the neutral or unionised drugs bind to lipoproteins.
2) Concentration of Drug in Body: The extent of protein -drug binding can
change with the changes that occur in drug and protein concentration. The
concentration of drugs that bind to HSA shows a very little influence as the
therapeutic concentration of any drug is not sufficient to saturate it. But , the
therapeutic concentration of lidocaine can saturate AAG with which it binds
as the concentration of AAG is less than that of HSA in blood.
3) Drug-Protein/Tissue Affinity: The affinity of drug -protein/tissue binding
varies according to the drug molecule. For example, lidocaine shows more
affinity for AAG than for HAS; Digoxin has more affinity for proteins of
cardiac muscles than those of skeletal muscles or plasma; Iophenoxic acid (a
radio-opaque medium) has a great affinity for plasma proteins.
2.1.8.2. Protein/Tissue Binding-Related Factors

The protein/tissue binding-related factors affect ing the protein -drug binding are
discussed below:
1) Physicochemical Properties of Protein or Binding Agent: Lipophilic
drugs mainly bind to lipoproteins and adipose tissue s as they get easily
* *
dissolved in their lipid core. The presence of active anionic and cationic
groups on the albumin molecules to bind a variety of drugs depends on the
physiological pH (pH of blood, plasma, ECF, etc.).
2) Concentration of Protein or Binding Agent: Among the plasma proteins,
binding mainly occurs with albu min because its concentration is high er than
other plasma proteins. During diseased states, the amount of several protei ns
and tissue components available for binding gets changed.
3) Number of Binding Sites on Binding Agent: Albumin has numerous
binding sites as well as high capacity binding component as compared to
other proteins. Many drugs are capable of binding at more th an one site on
albumin. For example, flucloxacillin, flur biprofen, ketoprofen, tamoxifen,
and dicoumarol bind to both primary and secondary sites on albumin.
2.1.8.3. Drug Interactions

Drug-drug interaction affect drug-protein binding in the following ways:
1) Competition between Drugs for a Binding Site (Displacement
Interactions): A competition occurs between two or more drugs for
interacting with a binding site if the drugs can bind to the same site . If one
drug (drug A) binds to a specific site, administration of the other drug (drug
B) that has affinity for the same site causes dislocation or displacement of
drug A from its binding site. This type of drug -drug interaction for the
common binding site is known as displacement interaction.
In this interaction, drug A is called the displaced drug while the drug B is
called the displacer. In dis placement interactions , an unexpected rise is
observed in free concentration of the displaced drug , which ma y increase
clinical response or toxicity of the particular drug molecule. Displacement
interaction can be affected by a drug metabolite also. The clinical
importance of this type of interaction can be given as:
i) The displaced drug (e.g., warfarin) has a binding capacity of more than
95%, has a small volume of distribution (< 0.15 L/k g), shows a rapid
onset of therapeutic or adverse effects, and has a narrow therapeutic
index.
ii) The displacer drug (e.g., phenylbutazone) has a high affinity degree
because the drug to be displaced competes for the same binding site. The
drug/protein concentration ratio is high ( > 0.10) and shows a rapid and
large increase in plasma drug concentration.
The extent of displace ment depends on the concentration of displacer drug
and also on its affinity for the binding site with respect to that of the drug to
be displaced. If a drug has 95% binding capacity , a displacement of just 5%
of the bound drug results in a 100% rise in free drug concentration. If the
displaced drug has a small volume of distribution, it remains in the blood
compartment and gives rise to some toxic effects.
If the displaced drug has a large V d, it redistributes into a large volume of
body fluids and gives rise to negligible or insignificant clinical effects.
*
Increase in free drug concentration accompanying displacement also makes it *
more available for elimination by the liver and kidneys ( figure 2.9). If drug
gets metabolised or excreted easily, its displacement causes a significant
reduction in elimination half-life.
Enhanced
Site of action
pharmacologic
Receptor response
Blood
Tissue
Increase in free drug
Storage tissue redistribution
concentration
P-D D
Hepatic
(Displaced drug) Liver clearance
D1 (Displacer)
Renal
Kidney clearance
Figure 2.9: Fate of a Drug after Displacement Interaction
Displacement is almost insignificant if more selec tive, potent, and low dose
drugs are used.
2) Competition between Drugs and Normal Body Constituents: Among the
normal body constituents, the free fatty acids are the major ones that interact
with a number of drugs that bind primarily to HSA. The level of free fatty
acid increases in several physiologic al (fasting), pathologic al (diabetes,
myocardial infarction, and alcohol abstinence) and pharmacologically
induced conditions (after heparin and caffeine administration).
The fatty acids binding to albumin influence the binding of several
benzodiazepines and propranolol (decreased binding) and warfarin (increased
binding). Binding of b ilirubin to HSA can be compromised by some drugs,
and this is important for neonates who lack efficient BBB and bilirubin
metabolising capacity.
Acidic drugs ( e.g., sodium salicylate, sodium benzoate , and sulphonamides)
have the ability to displace bilirubin from its albumin binding site. T he free
bilirubin is not conjugated by the liver of neonates, and thus crosses the BBB
and precipitates the condition of kernicterus (degeneration of brain and
mental retardation).
3) Allosteric Changes in Protein Molecule: This mechanism is also used to
explain that how the drugs affect protein binding interactions. This process
includes alteration of protein structure by the drug or its metabolite to modify
its binding capacity. The agents producing such an effect are known as
allosteric effectors, e.g., aspirin acetylates the lysine fraction of albumin and
changes its capacity to bind NSAIDs , like phenylbutazone (increased
*
affinity) and flufenamic acid (decreased affinity). *
2.1.8.4. Patient-Related Factors

The patient-related factors that affect protein-drug binding are discussed below:
1) Age: A patient’s age modifies protein-drug binding due to the differences in
the protein content in various age groups:
i) Neonates: In new -borns, albumin content is very low , therefore the
unbound concentration of drug that primarily binds to albumin, e.g.,
phenytoin and diazepam, is increased.
ii) Young Infants: Binding of protein-drug in infants can be explained by an
example of digoxin. It has been observed that in fants suffering from
congestive cardiac failure are given a digitalising dose 4 to 6 times the adult
dose on body weight basis. It is opposite to the fact that infants should be
given low doses considering their poorly developed drug eliminating tem. sys
The reason behindusing large doses of digoxin is more binding of the drug in
infants (the other reason is large renal clearance of digoxin in infants).
iii) Elderly: In geriatric patient s, the albumin content is lowered and free
concentration of drugs that bind primarily to it is increased. It has been
also seen that the levels of AAG increases in old age, and therefore
decreased free concentration is observed for drugs that bind to it.
2) Intersubject Variations: Intersubject variability in drug binding is only
studied in few drugs which show that the difference is small and not more
than two times. These differences result in genetic and environmental factors.
3) Disease States: Protein-drug interaction gets altered in several pathologic
conditions due to the alterat ion in protein content. Be cause albumin is the
major drug -binding protein, hypoalbuminemia can severely impair protein -
drug binding. It may occur in almost all pathological conditions such as
ageing, CCF, trauma, burns, inflammatory states, renal and hepat ic disorders,
pregnancy, surgery, cancer, etc. Hyperlipoproteinemia may occur due to
hypothyroidism, obstructive liver disease, alcoholism, etc.,that affects binding
of lipophilic drugs. All factors, mainl y drug interactions and patient -related
factors, affecting protein or tissue binding of drugs influencethe following:
i) Pharmacokinetics of Drugs: A decrease in plasma protein -drug
binding, i.e., an increase in unbound drug concentration, favours tissue
redistribution and/or clearance of drugs from the bo dy (enhanced
biotransformation and excretion).
ii) Pharmacodynamics of Drugs: An i ncrease in concentration of free or
unbound drug increases the intensity of action (therapeutic/toxic).
2.1.9. Kinetics of Protein Binding

If P = pr oteins and D = the drug, then on applying law of mass action to
reversible protein drug binding:
…..(4)
At equilibrium:
[PD]
Ka  …..(5)
[P][D]
*
[PD] = Ka [P] [D] …..(6) *
Where, [P] = Concentration of free protein.

[D] = Concentration of free drug.
[PD] = Concentration of protein-drug complex.
Ka = Association rate constant.
Kd = Dissociation rate constant.
Ka > K d indicates forward reaction, i.e., protein-drug binding is favoured. If PT is
the total concentration of bound and unbound protein:
PT = [PD] + [P] …..(7)
If r = number of moles of drug bound to total moles of protein:
[PD] [PD]
r  …..(8)
[PT ] [PD]  [P]
On substituting the value of [PD] from equation (6) in equation (8):
K a [P][D] K a [D]
r  …..(9)
K a [P][D]  [P] K a [D]  1
Equation ( 9) holds when there is a single binding site on the protein and the
protein-drug complex is a 1:1 complex. If more than one or N number of binding
sites are available per mole of the protein:
NK a [D]
r …..(10)
K a [ D]  1
Plateau (saturation of binding sites
N at high drug concentration) NKa
r
N
r When all binding sites are occupied by [D]
2 the drug, protein is saturated and plateau Slope = –Ka
is reached. At plateau, r = N. When
1
r = N/2, [D] = 1/Ka
Ka N
r
[D] (b) Scatchard Plot
(a) Direct Plot
1 [D]
r r
1 1
Slope  Slope 
NKa N
1 1
N NK
a
1 [D
[D] ] Plot
(d) Hitchcock
(c) Klotz Plot
Figure 2.10: Plots Used for the Study of Protein-Drug Binding. (a) Direct
Plot, (b) Scatchard Plot, (c) Klotz Plot, and (d) Hitchcock Plot
The value of association constant (Ka) and the number of binding sites (N) can be
obtained by plotting equation (10) in four different ways (figure 2.10):
1) Direct Plot: It is made by plot ting r against (D) [ figure 2.10 (a)]. When all
the binding sites are occupied by the drug, the protein is saturated and
plateau is reached. At the plateau, r = N. When r = N/2, [D] = 1/Ka.
2) Scatchard Plot: It is made by transforming equation (10) into a linear form.
* *
NK a [D]
Thus, r …..(11)
K a [ D]  1
r + rKa [D] = NKa [D]
r = NKa [D] – rKa [D]
Therefore,
r
 NK a  rKa …..(12)
[D]
A plot of r/[D] versus r yields a straight line [figure 2.10 (b)] with slope as –
Ka, y-intercept as NKa, and x-intercept as N.
3) Klotz Plot/Lineweaver -Burke Plot (Double Reciprocal Plot): The
reciprocal of equation (10) yields:
1 1 1
  …..(13)
r NK a [D] N
A plot of 1/r versus 1/[D] yields a straight line with slope as 1/NK a and y -
intercept as 1/N [figure 2.10 (c)].
4) Hitchcock Plot: It is made by rewriting equation (13) as:
NKa [D]
 1  K a [D] …..(14)
r
On dividing both sides by NKa:
[D] 1 [D]
  …..(15)
r NK a N
Equation (15 ) is Hitchcock equation as per which a plot of [D]/r versus [D]
yields a straight line with slope 1/N and y-intercept 1/NKa [figure 2.10 (d)].
2.1.10. Clinical Significance of Protein Binding of Drugs

Protein-drug binding has the following clinical applications:
1) In a few cases, drug -protein interaction may cause a prolonged residence of
drug in the body. Plasma proteins are not a normal component of the
glomerular filtrate, and drugs bound to the proteins will not be filtered.
Therefore, a dr ug which gets eliminated by glomerular filtration will have a
long biological half-life if it were subjected to protein binding.
2) Due to protein binding, the fraction of diffusible drug reduces; drug
concentration is generally reduced at the sites of biotra nsformation when a
drug bounds in a greater extent. This reduces the rate of drug elimination.
3) Plasma proteins act as physiological solubilisers. For example,
bishydroxycoumarin is bound in the blood to the extent of 98%. It is because
the drug is much mor e soluble in blood than in simple aqueous solutions.
Bishydroxycoumarin remains insoluble at physiological pH without binding.
Therefore, a therapeutic dose of the drug cannot be given in travenously
without the precipitation of microcrystals in the blood vessels.
4) High blood levels and low volumes of distribution of the drugs results due to
excessive plasma protein binding. This indicates that the ratio of drug in the
*
blood to drug at the site of biological membrane may be long. *
5) Bacterial infections generally affect the organ cells. The efficiency of a drug
against a given pathogen can be evaluated by the drug’s intrinsic antibacterial
activity and its concentration in the extracellular spaces of the tissues, i.e.,
the site of infections. An antibiotic which is found in greater concentration in
the blood show extensive binding in its compartment and is found in low
concentration at the site of infection.
Another antibiotic with same potency, unbound to plasma proteins , and free
to be distributed may be present in high concentration at the site of infection.
This antibiotic is more clinically effective , despite the fact that it would
produce much lower blood levels than the first antibiotic.
6) The protein binding may also stimulate the probability of competitio n for the
binding sites on a protein molecule, and the amount of bound drug might be
decreased by another drug bound to plasma proteins. Protein binding may
influence the activity, distribution, and elimination of a drug. This
competitive phenomenon may have important clinical effects.
When two drugs are simultaneously given, displacement of drugs from
proteins occurs and increases the rate of biotransformation and elimination.
Due to this property, the compounds that are effective displacers may be used
potentially in case of drug intoxication to decrease the body drug content.
7) Another significant source of variation of free drug concentration in plasma
is the competitive binding between drugs and endogenous substances.
8) In diseased states, the electrolytic balance in the blood is changed, which
further changes the binding of drugs since the activity coefficient of drugs
also change. Some alterations are also observed in the 3D structures of
proteins when the electrolytic balance is disturbed. The effect of a ge on the
binding of drugs is also an important factor because the plasma volume and
its composition vary with age.
9) Physicochemical properties of drugs are also an important factor which plays
a significant role in the binding of drugs to blood components.
For example, tetracycline analogues show a correlation between their
physical properties and disposition characteristi cs. When the drug molecules
become more lipid-soluble, their interaction with proteins increases and their
elimination from the body decreases.
2.2. SUMMARY
1) The processes which lower the plasma drug concentration are termed
disposition.
2) Distribution involves the reversible transfer of a drug between compartme nts.
3) Drug distribution is also defined as the reversible transfer of a drug between
one compartment and another.
4) Elimination involves irreversible loss of drug from the body.
5) Distribution is a passive transport process and the driving force for this
process is obtained from the difference of concentration gradient between
the blood and extravascular tissues.
* *
6) Tissue permeability of a drug mainly depends on the physicochemical

properties of the drug and the physiological barriers restricting diffusion of
drug into tissues.
7) The tissue permeability of a drug is mainly determined by its degreeof ionisation.
8) Perfusion rate is the volume of blood that flows per unit time per unit
volume of the tissue. Its unit is ml/min/ml of the tissue.
9) Total body content (intracellular and extracellular) is maximum in infants.
10) Albumin content is low in infants and elderly people.
11) High adipose tissue content results in low drug distribution and perfusion.
12) Apparent volume of distribution is the hypothetical volume of body flu id
into which a drug is dissolved or distributed.
13) Drugs that selectively bind to plasma proteins or other blood components have
apparent volume of distribution smaller than their true volume of distribution.
14) Drugs that selectively bind to extravascular t issues have apparent volume of
distribution larger than their true volume of distribution.
15) Human Serum Albumin (HSA, molecular weight 65,000) is the most
abundant plasma protein (59% of total plasma and 3.5 -5.0%) having high
drug binding capacity.
16) Site-I binding site of HSA is known aswarfarin and azathioprine binding site.
17) Site-II binding site of HSA is known as diazepam binding site.
18) Site-III binding site of HSA is known as digitoxin binding site.
19) Site-IV binding site of HSA is known as tamoxifen binding site.
20) α1-Globulin or transcortin or α1r CBG (Corticosteroid Binding Globulin)
binds to  number of steroidal drugs.
21) α2-Globulin or ceruloplasminbinds to vitamins A D, E and K and cupric ions.
22) β1-Globulin or transferrin binds to ferrous ions.
23) β2-Globulin binds to carotenoids.
24) γ-Globulin binds to antigens.
25) Epoxides of various halogenated hydrocarbons and paracetamol bind
irreversibly to liver tissues and cause hepatotoxicity.
26) Basic drugs like imipramine, chlorpromazine and antihistamines accumulate
in the lungs.
27) Metallothionein binds to heavy metals (such as lead, mercury, and cadmium)
and causes their renal accumulation and thus toxicity.
28) Chloroquine& phenothiazines interact with melanin and accumulate in the skin.
29) Arsenicals, chloroquine and phenothiazines deposit in hair shafts.
30) Tetracycline binds to bones and teeth.
31) Lipophilic drugs and DDT accumulate in adipose tissues by partitioning into it.
32) Protein binding directly depends on the lipophilicity of drug; therefore an
increase in lipophilicity increases the extent of binding.
33) The displaced drug has a binding capacity of more than 95%, has a small
volume of distribution (< 0.15 L/kg), shows a rapid onset of therapeutic or
adverse effects, and has a narrow therapeutic index.
34) The displacer drug has a high affin ity degree because the drug to be
displaced competes for the same binding sites.
35) In new -borns, albumin content is very low, therefore the unbound
*
concentration of drug that primarily binds to albumin is increased. *
36) A decrease in plasma protein -drug binding, i.e., an increase in unbound drug
concentration, favours tissue redistribution and/or clearance of drugs from
the body (enhanced biotransformation and excretion).
37) An increase in concentration of free or unbound drug increases the intensity
of action (therapeutic/toxic).
2.3. EXERCISE
1) The processes which lower the plasma drug concentration are termed elimination.
2) Disposition involves reversible transfer of a drug between compartments.
3) Drugs that selectively bind to extravascular tissues have a pparent volume of distribution
larger than their true volume of distribution.
4) Site-I binding site of HSA is known as digitoxin binding site.
5) α2-Globulin binds to vitamins A D, E and K and cupric ions.
6) Basic drugs like imipramine, chlorpromazine and antihi stamines accumulate in the hair
shafts.
7) Metallothionein binds to heavy metals and causes their hepatic accumulation and thus
toxicity.

8) The tissue permeability of a drug is mainly determined by its _______.
9) The unit of perfusion rate is _______.
10) Site-IV binding site of HSA is known as _______.
11) β2-Globulin binds to _______.
12) _______ binds to antigens.
13) Chloroquine and phenothiazines interact with melanin and accumulate in the _______.
14) _______ directly depends on the lipophilicity of drug.
Answers
1) False 2) False 3) True
7) False 8) Degree of ionisation 9) ml/min/ml of the tissue
10) Tamoxifen binding site 11) Carotenoids 12) γ-Globulin
13) Skin 14) Protein binding

1) Define drug distribution.
2) Write about blood capillary membrane.
3) How drug distribution is affected by age?
4) What is volume of distribution?
5) Enlist the factors affecting protein-drug binding.

1) Discuss the factors affecting drug distribution.
2) Explain plasma protein binding of drugs.
3) How distribution is affected by patient- and drug-related factors?
4) Discuss the kinetics of protein binding.

1) Give a detailed review on different physiological barriers.
2) Discuss the factors affecting protein-drug binding.
3) Write an exhaustive note on tissue permeability of drugs.
* *
Introduction to Elimination (Chapter 3) 67
CHAPTER Introduction to
3 Elimination
3.1. DRUG METABOLISM
3.1.1. Introduction
Elimination is the major process for the removal of a drug from the body and the
termination of its action. It is defined as the irreversible loss of drug from the
body. Elimination occurs by t wo processes, i.e., metabolism and excretion. The
body metabolises some drugs chemically. Metabolism may either result in the
formation of inactive substances (metabolites) or substances that may resemble to
the original drug in terms of therapeutic activi ty or toxicity or substances that
may differ from the original drug.
Liver is the main site of metabolism for most drugs, and hence, almost every
drug passes through the liver. For the conversion of prodrug to active metabolites
or for the conversion of active drugs to inactive forms, the drugs should enter the
liver to be acted upon by many enzymes. A specific group of cytochrome P-450
enzymes involves in liver’s primary mechanism for metabolising drugs. The
metabolism rate of many drugs is controlled by the level of these cytochrome P -
450 enzymes. Since the enzymes possess a limited capacity of metabolism, they
are burdened, when the levels of drug present in the blood are high.
It is difficult for an infant to metabolise certain drugs , because at the t ime of

birth, the metabolic enzyme systems are not developed completely. Also, the
activity of the enzymes reduces with an increase in the age of an individual. This
is the reason why aged individuals and infants are unable to metabolise drugs as
efficiently as the younger adults and children. As a result, often smaller doses per
pound of body weight are dispensed for infants and aged individuals as compared
to young individuals or middle-aged adults.
The biochemical modification of a drug in the body is t ermed drug

metabolism or biotransformation . Once a drug enters the body it undergoes
absorption, distribution, metabolism, and finally the metabolites (resultant
products formed after metabolism) undergo excretion.
Active Drug
Metabolism
Active
metabolite Active
Inactive Active Inactivation (Duration of
metabolite Drug
Drug action gets
(Prodrug) prolonged)
Figure 3.1: Metabolism of Drug
* *
3.1.2. Organs Involved in Drug Metabolism

Though each biological tissue possesses a certain capability to metabolise drugs,
but the principal site of drug metabolism is the smooth endoplasmic reticulum of
the liver cell. Due to the given factors, liver functions as the major organ for
metabolism:
1) Size of the liver is quite large.
2) Chemicals absorbed by the gut perfuse the liver first.
3) Most of the drug -metabolising enzymes are present in very high
concentrations in the liver as compared to other organs.
After being swallowed, a drug is absorbed by the digestive system and the portal
vein carries it to the hepatic portal system, where it undergoes maximum
metabolism. Thus, the drug is said to exhibit first -pass effect. Epithelial cells of
the GIT, lungs, kidneys, and skin are the other possible sites where the drug can
undergo metabolism. However, localised side effects are often seen at these sites.
3.1.3. Enzymes Involved in Drug Metabolism

Metabolism of drugs involves many important enzymes and pathways. Based on
the type of reaction catalysed by them, they can be categorised as:
1) Enzymes involved in Phase I Metabolism include:
i) In Oxidation: Cytochrome P -450 monooxygenase system, flavin-
containing monooxygenase system, alcohol dehydrogenase, aldehyde
dehydrogenase, monoamine oxidase, and co-oxidation by peroxidase.
ii) In Reduction: NADPH-cytochrome P -450 reductase and reduced
(ferrous) cytochrome P -450. It is important to note that a chemical can
enter substrate cycle during reduction reactions. In this cycle, a free -
radical electron is gained by the chemical which is also quickly lost
towards oxygen to form a superoxide anion.
iii) In Hydrolysis: Esterase, amidase, and epoxide hydrolase.
2) Enzymes involved in Phase II Metabolism include:
i) In Methylation: Methyltransferase.
ii) In Sulphation: Glutathione S-transferases and sulfotransferases.
iii) In Acetylation: N-acetyltransferases and amino acid N-acyl transferases.
iv) In Glucuronidation: UDP-glucuronosyltransferases.
3.1.4. Factors Affecting Drug Metabolism

The following factors affect the biotransformation of a drug:
1) Inhibitors: Certain drugs, e.g., cimetidine, omeprazole, and ciprofloxacin,
can inhibit enzymes that metabolise a drug. Since the metabolising enzymes
are inhibited, metabolism of the administered drug decreases, which in turn
leads to an increase in the duration of its action.
2) Stimulators: Certain drugs like phenobarbitone and rifampic in can increase
the activity of enzymes that metabolise a drug. Hence, it proves
advantageous when drugs like phenytoin and warfarin are administered, as it
increases their metabolism.
* *
3) Age: Young children show poor drug metabolism as metabolic enzyme

systems are not developed completely. For example, grey baby syndrome is
seen in infants on administration of chloramphenicol as they lack glucuronyl
transferase required for the inactivation of chloramphenicol.
4) Sex: In comparison to males, the females possess lesser ability for drug
metabolism.
5) Species: Some enzymes may be species -specific. For example , rabbits
possess atropinase enzyme and hence are able to metabolise atropine
(therefore, atropine is non -toxic for rabbits); however, atropinase enzyme is
absent in humans and hence, atropine proves toxic for humans.
6) Genetics: Drug metabolising enzymes show hereditary patterns; deficiencies
of either of the enzymes belonging to the enzyme system can be inherited
from one generation to the other. For example, an individual in whom
Glucose-6-Phosphate Dehydrogenase (G-6-PD) enzyme is genetically
deficient shows haemolysis when primaquine is administered to them.
7) Body Temperature: Temperature of the body is directly proportional to
drug metabolism. Drug metabolism h as been found to increase with an
increase in body temperature and vice versa.
3.2. BASIC UNDERSTANDING OF METABOLIC

PATHWAYS
3.2.1. Introduction
Drug biotransformation involves enzymatic reactions that are divided into Phase
I and Phase II reactions ( table 3.1 ). Phase I reactions include hydrolysis,
reduction, and oxidation. These reactions slightly increase the hydrophilicity.
Phase II reactions include glucuronidation, sulfonation (or sulfation), acetylation,
methylation, conjugation with glutathione, and conjugat ion with amino acids.
These reactions increase the hydrophilicity by a greater extent.
Phase I reactions may or may not go before the Phase II reactions. For example,
heroin forms morphine -3-glucuronide by undergoing hydrolysis (Phase I) and
then conjuga tion with glucuronic acid (Phase II). However, morphine forms
morphine-3-glucuronide by direct conjugation with glucuronic acid (Phase II).
Table 3.1: Reactions Involved in Drug Metabolism and their Subcellular Location
Type of Phase-I
Reactions Enzymes Involved Subcellular Locations
Hydrolysis Esterase Microsomes, cytosol, lysosomes, and blood
Peptidase Blood lysosomes
Epoxidase and hydrolase Microsomes and cytosol
Reduction Azo- and nitro-reduction Microsomes, microflora, and cytosol
Carbonyl reduction Cytosol, microsomes, and blood
Disulphide reduction Cytosol
Sulphoxide reduction Cytosol
Quinone reduction Cytosol and microsomes
Reductive dehalogenation Microsomes
* *
Oxidation Alcohol dehydrogenase Cytosol

Aldehyde dehydrogenase Cytosol and mitochondria
Aldehyde oxidase Cytosol
Xanthine oxidase Cytosol
Monoamine oxidase Mitochondria
Diamine oxidase Cytosol
Prostaglandins H synthase Microsomes
Flavin monooxygenases Microsomes
Cytochrome P450 Microsomes
Glucuronide conjugation Microsomes
Sulphate conjugation Cytosol
Glutathione conjugation Cytosol and microsomes
Amino acid conjugation Mitochondria and microsomes
Acylation Mitochondria and cytosol
Methylation Cytosol, microsomes, and blood
3.2.2. Phase I Metabolic Pathways

A molecule of drug initially enters phase I metabolism, where it undergoes a
sequence of reactions, and at the end of this phase, the molecule shows the
following changes:
1) Forms a reactive site or a functional group, like OH, SH, or NH2.
During phase II, they successively conjugate with molecules, such as
glucuronic acid, acetyl CoA, etc.
2) Converts itself into forms that show reduced solubility in lipids as well as
in water so that its excretion is facilitated.
3.2.2.1. Hydrolysis Reaction

Drugs containing carboxylic acid (esterprocaine), amide (procainamide),
thioesters (spironolactone), phosphoric acid ester (paraoxon), and aci d anhydride
(diisopropylfluoro-phosphate) functional groups undergo hydrolysis. Hydrolysis
of carboxylic acid esters, amides, and thioesters is catalysed by carboxylesterases
located in various tissues and serum. Hydrolysis of phosphoric acid esters is
catalysed by paraoxonase (or organophosphatase), which is a serum enzyme.
Hydrolysis of phosphoric acid anhydrides is catalysed by
diisopropylflurophosphatase. Carboxylesterases catalyse the trans -esterification
of drugs in the presence of alcohol, e.g., conversion of cocaine to ethylcocaine.
1) Carboxylic Acid Esters
* *
2) Amides
3) Thioesters
4) Phosphoric Acid Esters
5) Acid Anhydride
6) Epoxides are Hydrolysed by Epoxide Hydrolase
* *
Another example of epoxide hydrolysis is conversion of leukotriene A 4 to

leukotriene B 4. Peptidases that cleave th e amide linkage between adjacent
amino acids act as amidases (hydrolysis reaction).
7) Organic Acid Esters
8) Inorganic Acid Esters
9) N-Hydroxylation
* *
N-hydroxylation of amides generates reactive intermediates that covalently bind

to macromolecules, e.g., paracetamol, and cause toxicity. Prolonge d usage or
overdosage of paracetamol causes liver damage. Hepatotoxicity is caused by N -
acetyl-p-benzoquinone metabolite, which is inactivated by glutathione
conjugation. Prolonged use or overdose of paracetamol causes depletion of
glutathione and the toxic metabolite of paracetamol causes liver damage.
3.2.2.2. Reduction Reaction

Drugs containing an aldehyde, ketone, disulphide, sulphoxide, quinine, N -oxide,
alkene, azo or nitro group undergo in vivo reduction, e.g., aldehyde can be
reduced to alcohol or sulphoxide can be reduced to sulphide. Aldehyde oxidase is
an enzyme involved in bioreductions.
1) Azo and Nitro Reduction: This reaction is catalysed by intestinal
microflora, cytochrome P-450 and NADPH-quinone oxidoreductase.
i) Azo Reduction
ii) Nitro Reduction
During azo reduction, N=N undergoes sequential reduction and gets cleaved
into two primary amines. Four reducing equivalents are required for this
reaction; whereas, six reducing equivalents are required for nitro reduction.
2) Carbonyl Reduction: This reaction is catalysed by carbonyl reductases
present in blood and cytosolic fraction of the liver, kidney, brain, and other
tissues. Reduction of aldehydes i nto primary alcohols and reduction of
ketones into secondary alcohols are examples of carbonyl reduction.
* *
Acetohexamide, daunorubicin, ethacrynic ac id, warfarin, and menadione are

other drugs that are reduced by carbonyl reductase.
3) Sulphoxide and N -Oxide Reduction: This reaction is catalysed by
thioredoxin-dependent enzymes present in liver and kidney cytosol.
4) Disulphide Reduction: Disulphides undergo reduction and get cleaved into

their respective sulphydryl components.
5) Reductive Halogenation: This reaction involves replacement of halogen

with hydrogen, and is catalysed by cytochrome P -450 and glutathione -S-
reductase. The C-F bond is resistant to reduction.
6) Quinone Reduction: Reduction of quinone into hydroxyquinones is

catalysed by DT-diaphorase (NADPH-quinone oxidoreductase).
* *
3.2.2.3. Oxidation Reaction

Liver cells (or hepatocytes) are the most common site for oxidation of a drug
molecule and microsomes form the site of oxidation within the hepatocytes.
Mixed Function Oxidase (MFO) is the enzyme system that causes oxidation of
drug. The following components make up the enzyme system:
1) Cytochrome Oxidase Enzyme:It is commonly known as Cytochrome P-450
(CYP-450) and is chemically a haemoprotein. It is the terminal enzyme of the
enzyme system that plays a role in drug oxidation. Oxidation of drug is caused
by reduction of CYP -450 (as it exhibits maximum absorption at 450nm),
hence, extra electrons are transferred to molecular oxygen, and so oxygen is
reduced (reduced form of oxygen is known as activated oxygen). Once the
electrons have been transferred, CYP -450 is free to again accept another
electron, i.e., it is recovered.
2) NADPH: A co -enzyme that forms one more significant member of this
system of oxidation.
3) Oxygen .
4) NADPH Cytochrome Reductase: It is chemically a flavoprotein, and
another enzyme involved in the process.
Steps of Oxidation
1) The drug along with oxidised cytochrome P -450 (Fe +++) enzyme forms a
binary complex.
2) The oxidised CYP-450 now receives one electron from the reduced flavopro tein,
and becomes reduced P-450 (Fe++) but still remains attached to the drug.
3) Subsequently, activation of an oxygen molecule is seen. Oxygen attaches to the
binary complex [obtained in step (1)], and forms a drug-reduced P-450 (Fe++)-
-Fe++-oxygen complex.
activated oxygen complex, generally, abbreviated as drug
4) Lastly, the oxidised drug splits and CYP -450 again converts into oxidised
(Fe+++) form; thus the enzyme is recovered.
DR —[Fe+++]
+++
Drug (DR) + [Fe ] (binary complex)
Reductase
[Fe++]
DR—[Fe++] O2 DR—[Fe++]
(reduced material)
Oxidised
DR+H2O
O2
Figure 3.2: Oxidation by MFO System
* *
Oxidation Reactions
1) Oxidation of Aromatic Carbon Atoms (Aromatic Hyd roxylation): In this
reaction, arene oxide (epoxide) is formed. It is a reactive intermediate that
undergoes rearrangement to yield arenols, and sometimes catechols and
glutathione conjugates.
The arene oxide intermediate is highly reactive and carcinogenic or cytotoxic

in some cases, e.g., epoxides of bromobenzene and benzo(a)pyrene.
Monosubstituted benzene derivatives undergo hydroxylation at ortho-, meta-
or para-positions, and most commonly the para-hydroxylated produc t is
formed, e.g., acetanilide is converted to paracetamol and phenylbutazone is
converted to oxyphenbutazone.
This reaction is favoured if the substituent is an activating or electron -rich

group, like amino group. Deact ivating or electron -withdrawing groups
(carboxyl and sulphonamide) retard or prevent aromatic hydroxylation, e.g.,
probenecid.
2) Oxidation of Olefins: Oxidation of non -aromatic carbon -carbon double
bonds is similar to aromatic hydroxylation, i.e., it forms epoxides to yield
1,2-dihydrodiols. Olefinic oxidation involving conversion of carbamazepine
to carbamazepine -10,11-epoxide that further converts into corresponding
trans-10,11-dihydrodiol is a well-known example.
* *
3) Oxidation of Benzylic Carbon Atoms: Carbon atoms attached to aromatic

rings (benzylic carbon atoms) undergo hydroxylation to yield corresponding
carbinols. If a primary carbinol is obtained, e.g., tolbutamide, it gets further
oxidised into aldehydes and then to c arboxylic acids. If a secondary carbinol
is obtained, it gets converted into ketone.
4) Oxidation of Allylic Carbon Atoms: Allylic carbon atoms lie adjacent to

olefinic double bonds. They undergo hydroxylation similar to benz ylic
carbons. Hydroxylation of hexobarbital into 3 -hydroxy hexobarbital is an
example of this reaction.
5) Oxidation of Carbon Atoms Alpha to Carbonyls and Imines: Several

benzodiazepines that contain a carbon atom (C -3) al pha to carbonyl (C=O)
and imino (C=N) functions readily undergo hydroxylation, e.g., diazepam.
* *
6) Oxidation of Aliphatic Carbon Atoms (Aliphatic Hydroxylation): Alkyl

or aliphatic carbon atoms undergo hydroxylation at the te rminal methyl
group (called as -oxidation) and the penultimate carbon atom (called as -1
oxidation). The latter oxidation reaction yields the major product, e.g.,
valproic acid.
7) Oxidation of Alicyclic Carbon Atoms (Alic yclic Hydroxylation):

Cyclohexane (alicyclic) and piperidine (non -aromatic heterocyclic) rings are
found in various molecules, e.g., acetohexamide and minoxidil, respectively.
Such rings undergo hydroxylation at C-3 or C-4 positions.
8) Oxidation of Carbon-Nitrogen Systems

i) N-Dealkylation: Alkyl groups attached to the nitrogen atom in nitrogen -
bearing compounds undergo N-dealkylation reactions, e.g., secondary and
tertiary aliphatic and aromatic amines, tertiary alicyclic amin es, and N -
substituted amides and hydrazines. Since N-dealkylation of amines yields
amines and that of amides yields amides, the reaction does not undergo
any change in the oxidation state. However, the removed alkyl group gets
oxidised. Mechanism of N-dealkylation involves oxidation of -carbon to
generate an intermediate, carbinolamine that undergoes rearrangement by
cleavage of C -N bond to yield the N -dealkylated product and the
corresponding carbonyl of the alkyl group (a primary alkyl and a
secondary alkyl are converted to aldehyde and ketone, respectively).
Tertiary nitrogen attached to different alkyl groups undergoes

dealkylation by removal of smaller alkyl group. A representative
example of each of the chemical cla sses of compounds capable of
undergoing N-dealkylation is given below.
* *
a) Secondary aliphatic amines, e.g., methamphetamine.
b) Tertiary aliphatic amines, e.g., imipramine.
ii) Oxidative Deaminat ion: This reaction, similar to N -dealkylation,

proceeds via carbinolamine pathway but the C-N bond cleavage occurs at
the bond linking the amino group to the larger portion of the drug
molecule. Thus, oxidative deamination is the reverse of N-dealkylation in
terms of the product formed, the carbonyl product retains a large portion of
the parent structure and the amines formed are simple,e.g., NH3.
iii) N-Oxide Formation: N-oxides are formed by the nitrogen atoms

exhibiting basic properties. Due to this reason, N -oxides can be formed
from amines but not from amides. The tertiary amines yield N -oxides.
The four categories of tertiary amines yielding N-oxides are:
a) Aliphatic amines, e.g., imipramine.
b) Alicyclic amines, e.g., nicotine.
c) Nitrogen atoms of aromatic heterocyclics, e.g., trimethoprim.
d) Amines attached to aromatic rings, e.g., N, N-dimethyl aniline.
* *
The N-oxide products are highly water -soluble and get excreted in urine.
But, they are prone to get reduced into the corresponding amine.
iv) N-Hydroxylation: Opposing the basic compounds that form N -oxides,
N-hydroxy formation is displayed by non -basic nitrogen atoms such as
amide nitrogen, e.g., lidocaine.
9) Oxidation of Carbon-Sulphur Systems

i) S-Dealkylation: Mechanism of S -dealkylation of thioethers (RSR ) is
similar to that of N -dealkylation, i.e., it proceeds via -carbon
hydroxylation. The C -S bond cleavage forms a thiol (RSH) and a
carbonyl product, e.g., 6-methyl mercaptopurine.
ii) Desulphuration: This reaction involves cleavage of carbon -sulphur

bond (C=S or thiono) and formation of a product with C=O bond. Such a
desulphuration reaction is ob served in thioamides (RCSNHR’), e.g.,
thiopental.
* *
iii) S-Oxidation: Thioethers also undergo S -oxidation reaction to yield

sulphoxides that further oxidise into sulphones (RSO 2R). Several
phenothiazines, e.g., chlorpromazine, also undergo S-oxidation.
10) Oxidation of Carbon -Oxygen Systems ( O-Dealkylation): This reaction,

similar to N -dealkylation, proceeds via -carbon hydroxylation and yield an
unstable hemiacetal or hemiketal intermediate that undergoes spontaneous C-
O bond cleavage to form alcohol (arenol or alkanol) and a carbonyl moiety.
Methyl ethers get rapidly dealkylated than the longer chain ethers, such as
the ones containing n -butyl group. The reaction results in active metabolites,
e.g., conversion of phenacetin to paracetamol and codeine to morphine.
11) Oxidation of Alcohol, Carbonyl and Carboxylic Acid: This reaction is

catalysed by non -microsomal dehydrogenase enzymes. Primary alcohols get
rapidly metabolised into aldehydes that further metabolise into carboxylic
acids; however, oxidation of secondary alcohols to ketones continues slowly.
In ethanol, reversible oxidation to acetaldehyd e occurs that further rapidly

oxidises into acetic acid since acetaldehyde is highly toxic and should not
accumulate in the body. With secondary alcohols, the oxidation rate
increases with an increase in alkyl chain length. Compounds with primary
*
and secondary alcohol groups are oxidised at the primary group. *
12) Miscellaneous Oxidative Reactions

i) Oxidative Aromatisation/Dehydrogenation: An example of metabolic
aromatisation of drugs is nifedipine.
ii) Oxidative Dehalogenation: This reaction commonly occurs in drugs

containing halogen, e.g., chloroform. Dehalogenation of this drug yields
phosgene that forms electrophiles capable of covalent binding to tissues.
3.2.3. Phase II Metabolic Pathways

In Phase II reactions, drugs (or their metabolites) are combined with hydrophilic
endogenous compounds to form complexes exhibiting adequate hydrophilic
character to allow rapid excretion.
These conjugation reactions include gl ucuronidation, sulphonation (or
sulphation), acetylation, methylation, conjugation with glutathione, and
conjugation with amino acids such as glycine, glutamic acid and taurine ( table
3.2). Phase II reactions are much faster than phase I reactions.
Table 3.2: Conjugation Reactions and Respective Functional Groups
Functional Groups Conjugation Reactions
–OH, –COOH, –NH2, –NH, –SH, and –CH Glucuronidation
Aromatic –OH, aromatic –NH2, and alcohols Sulphonation (sulphation)
Aromatic –NH2, aliphatic NH2, hydrazines, and Acetylation
–SO2NH2
Aromatic –OH, –NH2, –NH, and –SH Methylation
Epoxides and organic halides Glutathione conjugation
Aromatic –NH2 and –COOH Glycine conjugation
3.2.3.1. Glucuronidation
Glucuronidation is quantitatively the most significant phase II reaction.
Glucuronic acid conjugation occurs only when glucuronic acid gets activated.
The cofactor, Uridine Diphosphate -glucuronic acid (UDP -glucuronic acid) is
required during glucuro nidation, which is also catalysed by UDP -Glucuronyl
Transferases (UGTs, located in the endoplasmic reticulum of liver, kidney,
intestine, skin, brain, spleen, and nasal mucosa) (figure 3.3).
* *
UDP-glucuronic acid
Figure 3.3: Synthesis of UDP-Glucuronic Acid

An electron -rich nucleophilic heteroatom, i. e., O, N , or S, forms the site of
glucuronidation. Thus, aliphatic alcohols, phenols and carboxylic acids form O -
glucuronidation, primary and secondary amines form N -glucuronides, and free
sulphhydryl groups form S-glucuronides.
1) O-Glucuronides
Other examples include acetaminophen, codeine, morphine, naloxone,

oxazepam, propofol, propranolol, ketoprofen, naproxen, valproic acid,
fenoprofen, chloramphenicol, and trichloroethanol.
2) N-Glucuronides
Other examples include benzidine, cyproheptadine, imipramine, lamotrigine,

*
meprobamate, sulphisoxazole, and tripelennamine. *
3) S-Glucuronides
4) C-Glucuronides: Steroids, bilirubin, catechols, and phenylbutazone undergo

C-glucuronidation. Glucuronide conjugates are polar, water -soluble, and are
eliminated from the body through urine or bile. The size of aglycone (parent
compound) decides whether glucuronides will be eliminat ed through bile or
urine. Glucuronic acid is ionised at physiological pH and enhances
elimination because:
i) It increases the aqueous solubility of drug or its metabolites.
ii) It is recognised by the biliary and renal organic transport system, thus
glucuronides are secreted in urine and bile.
3.2.3.2. Sulphation
Sulphate conjugation forms a highly water -soluble sulphuric acid ester. This
reaction is catalysed by sulphotransferases (sulphokinases), which are cytosolic
enzymes found in liver, kidney, intestinal tract, lung, platelets, and brain. The
cofactor required for sulphation is 3 '-Phosphoadenosine-5'-Phosphosulphate
(PAPS). Many drugs undergoing O -glucuronidation also undergo sulphation. In
sulphation reaction, just as in glucuronidation, the sulphate is activated before the
reaction with substrate.
The sulphate is first converted into Adenosine -5'-Phosphosulphate (APS), which

is then metabolised to 3 '-Phosphoadenosine-5'-Phosphosulphate (PAPS). This
activated sulphate is then made to react with a substrate.
3.2.3.3. Acetylation
N-acetylation is a major biotransformation route for drugs with an aromatic
amine or a hydrazine group. This reaction requires acetyl coenzyme A (acetyl
CoA) cofactor and is catalysed by N-acetyltransferases.
* *
N-acetylation reaction involves the following two steps:

1) The acetyl group from acetyl -CoA is transferred to an active site cysteine
residue in an N-acetyltransferase with release of coenzyme A.
2) The acetyl group is transferred from the acylated enzyme to the amino group
of the substrate. The enzyme is regenerated.
N-acetyltransferases are cytosolic enzymes found in liver.
3.2.3.4. Methylation
The metabolites resulting from methylation are not more polar or water-soluble.
They possess equal or higher pharmacological activity than the parent drug. This
reaction is catalysed by methyl transferase.
Methylation reaction involves the synthesis of an activated coenzyme, i.e., S -

Adenosyl Methionine (SAM, donor of m ethyl group), from L -methionine and
ATP. The methyl group is then transferred to a substrate.
Examples of methyl transferases are Catechol -O-Methyl Transferase (COMT),

Phenyl Ethanolamine-N-Methyl Transferase (PNMT), etc.
3.2.3.5. Glutathione Conjugation

Glutathione (GSH) is a tripeptide comprised of glycine, cysteine, and glutamic
acid. Its formation is catalysed by -glutamylcysteine synthetase.
Glutathione conjugates are thioesters formed by nucleophilic attack of glutathione

thiolate anion (GS−) on electrophilic carbon atom or electrophilic heteroatoms (O,
N, or S). Glutathione conjugation reactions are of the following two types:
1) Displacement Reactions: In these reactions, glutathione displaces an
electron-withdrawing group.
2) Addition Reactions: In these reactions, glutathione is added to an activated
double bond.
* *
Displacement Reaction
Addition Reaction
Conjugation of Heteroatom
Electrophiles are very toxic as they have the ability of binding to macromolecules
(proteins and DNA) and causing cellular damage and mutations. Hence,
conjugation of electrophiles with glutathione is an important detoxification
reaction.
Glutathione conjugates are either eliminated in bile or first they are converted to
mercapturic acid in the kidney and then excreted in urine.
* *
3.2.3.6. Amino Acid Conjugation

Drugs are conjugated with amino acids by the following two pathways:
1) Conjugation of drugs containing a carboxylic acid group with the amino
group of amino acids, e.g., glycine, glutamate, and taurine. This pathwa y
involves initial activation of drugs with CoA. Thus, the acyl -CoA thioether
formed reacts with amino group of amino acids.
2) Conjugation of drugs containing aromatic hydroxylamine with carboxylic
acid groups of amino acids, e.g., serine and proline.
The acceptor amino acid used for conjugation depends on the species and drug.
Apart from glycine, glutamine and taurine, other amino acids, e.g., ornithine,
arginine, histidine, serine, and several dipeptides, e.g., glycylglycine,
glycyltaurine and glycylvaline, are also used for conjugation.
Conjugation with amino acids is generally a detoxification reaction.
3.3. DRUG EXCRETION
3.3.1. Introduction
Excretion removes drugs and/or their metabolites from the body. For cessation of
the pharmacological action of a drug, its excretion, in intact or unchanged form is
important. Kid neys are principal organs for excretion; and excretion through
kidneys is termed as renal excretion. Non -renal excretion is the term used to
describe excretion by other organs except the kid neys. The other organs include
lungs, biliary system, intestine, salivary glands, and sweat glands.
All lipid -soluble drugs are converted into water -soluble compounds by the
metabolic processes. This conversion enables their excretion (from the body). In
case, the drug or its metabolites are water -soluble, it is excreted unchanged.
Therefore, it can be said that for a drug to be eliminated from the body, both
metabolism and excretion are important.
3.3.2. Clearance and Renal Clearance

Clearance may be defined as the complete removal of a drug in a specified time
period from the hypothetical volume of body fluids containing the drug. A unit of
ml/min expresses clearance of a drug and its value remains constant for any
particular plasma drug concentration. Clearance describes the relationship
between plasma drug concentration and the rate of drug elimination.
Elimination Rate
Clearance(Cl)  ….. (1)
* *
Renal Clearance (ClR)

Blood volume or plasma volume, completely cleared of the drug in its unchanged
form, by the kidneys per unit time, defines the term renal clearance (Cl R).
Mathematically, it can be expressed as:
Rate of Urinary Excretion
Cl R  ….. (2)
Renal clearance can be physiologically expressed as the ratio of rate of

reabsorption subtrac ted from the sum total of the rate of filtration and rate of
secretion to plasma drug concentration (C):
Rate of Filtration  Rate of Secretion – Rate of Reabsorption
Cl R  ….. (3)
C
Clearance of a drug by an organ is not more than the blood flow to that organ
because the rate of overall clearance i s limited by the drug delivery to that organ
via blood. If the volume of distribution and elimination rate constants are known,
clearance can be calculated for a one-compartment model. Thus,
Cl = Vd . Kel …..(4)
Clearance is a measure of renal unction.
f Creatinine clearance is widely used for this
purpose. Creatinine is a by-product of muscle catabolism and is chemically amine.
Serum creatinine is measured in clinical practice for this purpose. As muscle
catabolism and creatinine levels differ with age, weight, and sex, different formulas
are used to calculate creatinine clearance from serum creatinine concentration.
For children between 1 to 20 years:
0.7
0.48  H  W 
Cl cr  .
Scr  70 
…..(5)
For adults above 20 years:

Males:
(140  Age)  W
Cl cr  …..(6)
72 Scr
Females:
(140  Age)  W
Cl cr  …..(7)
85 Scr
Where, Clcr = Creatinine clearance (ml/min).
Scr = Serum creatinine (mg%)
H = Height (cm).
W = Weight (kg).
Age = in years.
Normal creatinine clearance is 120-130ml/min. Creatinine clearance < 10ml/min
indicates severe renal impairment. Renal function can be expressed as follows:
Clcr of patient
Renal Function (RF)  …..(8)
Clcr of normal subject
Hence, dose in patients with renal impairment = (Normal dose)(RF)
* *
Given below are some drawbacks and precautions when c alculating Cl cr from
serum creatinine:
1) Liver dysfunction is associated with a significant over -prediction of Cl cr.
These equations should be used with caution in patients with liver disease.
2) Thin individuals have low serum creatinine concentrations secon dary to
decreased muscle mass, resulting in a significant over-prediction of Clcr.
3) Elderly patients have low serum creatinine concentrations secondary to
decreased muscle mass, leading to a possible over-prediction of Clcr.
3.3.3. Renal Excretion of Drugs

Kidneys are responsible for the excretion of nearly all drugs as well as their
metabolites, to a certain extent. Renal route exclusively is responsible for the
excretion of some drugs, like gentamicin. Water -soluble, non -volatile, small
molecular size (< 500 Daltons) agents that undergo slow metabolism, undergo
excretion via urine.
Blood
Glomerulus 1) Glomerular filtration of

water and unbound drugs
and metabolites
1
2) Active tub ular secretion of
acidic and basic drugs and
2
Proximal metabolites
Tubule
3 3) Active reabsorption of
Blood acidic and basic endogenous
compounds and passive
reabsorption of lipophilic
Loop of Henle drugs
4) Urinary excretion of drugs

and metabolites that are
Distal 4 filtered and/or actively
Tubule secreted and not reabsorbed.
Collecting Urine
Tubule
Figure 3.4: Simplified Diagram of the Processes involved in Urinary Excretion

of Drugs
Nephron is the basic functional unit of kidney, which is involved in excretion.
One million nephrons are present in each of t he kidney. Each nephron has the
following parts:
1) Glomerulus, 2) Proximal tubule, 3) Loop of Henle,
4) Distal tubule, and 5) Collecting tubule.
Drug excretion via urine is determined by the following processes namely

glomerular filtration, active tubular secretion, and active or passive tubular re -
absorption (figure 3.4).
* *
Concentration of drugs in the lumen tends to show an increase by the glomerular

filtration and active tubular secretion, thus they are said to assist the process of
excretion. However, tubular re -absorption reduces the rate of excretion besides
preventing the drug from moving out of the body. Accordingly, the following
equation can be used to denote the rate of excretion:
3.3.3.1. Glomerular Filtration

Glomerular filtratio n is a process involving the filtration of most compounds
irrespective of whether they are ionised or unionised, with an exception of
macromolecules (bounded to plasma proteins or blood cells). It is a non -selective
process which occurs only in one directi on. Glomerulus also behaves as a
negatively charged selective barrier, which promotes the retention of negatively
charged compounds. The hydrostatic pressure of blood flowing in the capillaries
acts as the driving force for filtration by the glomerulus. Only 10% (or 120 -
130ml/min) of cardiac output is filtered through the glomeruli, out of the 25% (or
1.2 litres) of blood/min that reaches the kidneys through the renal artery. The rate
of filtration is known as Glomerular Filtration Rate (GFR) . Although, aro und
180 litres of protein and free ultra -filtrate of cells pass through the glomeruli
every day, yet, nearly 1.5 litres only is excreted as urine. The remaining filtrate
undergoes reabsorption from the tubules.
An agent, excreted solely by filtration without being either secreted or reabsorbed

in the tubules helps to determine the GFR. The rate of excretion of such an agent
ranges from 120 -130ml/min. Estimation of GFR employs the use of creatinine,
inulin, mannitol, and sodium thiosulfate. Also, creatinine and inulin are
extensively used for the estimation of renal function.
3.3.3.2. Active Tubular Secretion

Active tubular secretion is a carrier-mediated process. Compounds are transported
in a direction opposite to that of the concentration gradient during active t ubular
secretion by utilising energy. The capacity of this system is limited and it gets
saturated. Two mechanisms of active tubular secretion have been identified:
1) A system that secretes organic acids or anions ( e.g., penicillin, salicylates,
glucuronides, sulphates, etc.) This system is similar to the one which secretes
endogenous acids, like uric acid.
2) A system that secretes organic bases or cations ( e.g., morphine,
mecamylamine, hexamethonium, endogenous amines like catecholamine,
choline, histamine, etc.).
Both the mechanisms are comparatively non-selective and do not depend on each
other. However, both of them can be bidirectional, i.e., the substances can
undergo active secretion as well as reabsorption, e.g., uric acid.
Active secretion remains un changed by alterations in pH and protein binding,
because the bound drug rapidly dissociates at the moment when unbound drug
gets excreted. However, it is influenced by renal blood flow opposite to the
* *
process of glomerular filtration. The values of rate o f excretion of drugs

undergoing active secretion are greater than the normal GFR value of 130ml/min,
e.g., penicillin exhibits a renal clearance value of 500ml/min. Both glomerular
filtration and tubular secretion are indicated by such a high value.
3.3.3.3. Tubular Re-Absorption

Tubular re-absorption of drugs follows the process of glomerular filtration. The
entire length of the renal tubule shows tubular reabsorption. When the values
obtained for the rate of excretion of a drug do not exceed that of GFR of
130ml/min, re-absorption of that drug is indicated. Agents that are completely re-
absorbed after filtration, ( e.g., glucose) possess a clearance value of zero. Re -
absorption increases the half-life of a drug, in contrast to tubular secretion.
Tubular re-absorption can take place in either of the two ways:
1) Active Tubular Re -Absorption: High threshold endogenous substances or
nutrients like electrolytes, glucose, vitamins, amino acids, etc., which need to
be conserved by the body, usually undergo active tubular r e-absorption.
Active re-absorption is shown by very few drugs, e.g., oxopurinol.
2) Passive Tubular Re-Absorption: Numerous exogenous substances, including
drugs, commonly exhibit passive tubular re-absorption. The back diffusion or
re-absorption of water along with sodium and other inorganic ions establishes
the concentration gradient, i.e., the driving force for such a process. Less than
1% of glomerular filtrate is excreted as urine; hence, if a drug does not undergo
either secretion or re -absorption, its c oncentration in the urine will be 100
times more than that of free drug in plasma owing to water re -absorption.
3.3.4. Factors Affecting Renal Excretion of Drugs

Factors affecting drug excretion are:
1) Physicochemical Properties of Drugs: Various factors affect the
physicochemical properties of drugs:
i) Molecular Weight (MW): The excretion of drugs with large molecular
weights is difficult, compared to that of drugs with smaller molecular
weights. This is especially seen in case of glomerular filtration.
ii) Lipid Solubility: The solubility of a drug in lipids is inversely
proportional to its urinary excretion. For example , if drug A is more
soluble in lipids compared to drug B, then drug A will show increased
volume of distribution and a decreased renal excretion as compared to
that exhibited by drug B (increased lipid solubility of a drug increases its
volume of distribution and decreases renal excretion).
iii) Volume of Distribution: The volume of distribution of drugs (V d) is
inversely proportional to their clearance. A drug that possesses a large Vd
shows poor excretion in urine, while drugs possessing a smaller V d
(restricted to blood) show sufficient excretion (higher rate of excretion).
2) Renal Blood Flow: Drugs that show increased perfusion also show an
increase in their excretion; this is especially significant for drugs that
*
undergo excretion by glomerular filtration. *
3) Binding Characteristics of Drugs: Drugs that are bounded to plasma

proteins act as macromolecules, and their filtration by the glomerulus is not
possible. Glomerular filtrate contains only those drugs that are unbound or
free. Drugs that are protein-bound possess extended half-lives.
4) Drug Renal Clearance: It is the rate at which a drug is excreted by the
kidney into urine comparative to the plasma drug concentration. Adequate
renal function determines the renal clearance of several drugs and their
metabolites.
In cases of impaired renal clearance, t ½ of a drug may increase and the body
may show the presenc e of toxic levels of the drug. Renal clearance is
significant particularly for certain drugs which are:
i) Excreted mainly by the kidneys, and
ii) Possess limited therapeutic index (e.g. lithium, digoxin, and warfarin).
Diseases decrease renal clearance in the following two patterns:
i) Diseases that Decrease Renal Blood Flow: For example, congestive
heart failure, haemorrhage, and cardiogenic shock.
ii) Diseases that Decrease Renal Excretion: For example, renal disease
(e.g., glomerulonephritis) that may increase the half-life (t ½) of drugs.
5) Plasma Drug Concentration: Glomerular filtration and renal reabsorption
are affected by the concentration of drug in plasma. If the drug is not protein
bound, its glomerular filtration is directly proportional to its plasma
concentration. In drugs showing renal reabsorption, excretion occurs only
when concentration in glomerular filtrate is higher than reabsorption capacity.
6) Urine pH: For weakly acidic (pKa 3 -7) or weakly basic (pKa 6 -12) drugs,
the degree of ionisation in tubular fluid depends on pH, for example:
i) Methamphetamine (Weak Base, pKa 10): Renal excretion is 4 times
faster in acidic urine than in alkaline urine because at lower pH, the
ionisation is lower, reabsorption is less, and excretion of
methamphetamine is more.
ii) Phenobarbital (Weak Acid, pKa 7.4): Renal excretion is 7 times faster
in alkaline urine. However, renal clearance is only a small fraction of
total clearance.
iii) Salicylic Acid (Weak Acid, pKa 3.5): This acid is mainly ionised at
physiological pH. At pH 5.0, the amount of non-ionised form is 25 times
of that present at pH 7.4. In over dosage of aspirin, systemic alkali and
tubular acidosis occurs that enhances tubular reabsorption and prolongs
half-life of elimination. This can be reversed by giving systemi c alkali
and fluids and producing an alkaline urine with high flow rate.
7) Biological Factors: Various biological factors, like age, sex and species,
influence the drug excretion. Circadian rhythm also plays an important role.
Females show 10% lower renal e xcretion than males; in neonates, renal
function is only 60 -70% in comparison to adults; in elderly, renal function
and hence renal excretion is reduced.
* *
8) Pregnancy: The volume and composition of body fluids undergo significant

changes in pregnant women. The plasma volume, the red cell mass, and the
albumin mass increases. The plasma volume shows greatest increase (up to
40%), and the hematocrit and plasma albumin concentration also increase.
Total body volume also increases between 6-8 lines.
Pregnancy significantly increases renal blood flow and GFR in very early
pregnancy. GFR increases up to 50% by the end of first trimester. Hence,
plasma concentration of components handled by filtration falls; e.g., normal
serum creatinine in pregnancy is about 40-50mmol/l.
9) Drug Interactions: Drug excretion is influenced by many drug interactions
and involve the following effects:
i) Forced diuresis,
ii) Alteration in urine pH,
iii) Alteration in intrinsic clearance,
iv) Alteration in renal blood flow,
v) Alteration in binding characteristics, and
vi) Alteration in active secretion.
If the drug is highly protein bound and if any other drug displaces it,
excretion of such highly protein bound drug increases. For example,
furosemide enhances the excretion of gentamicin by displacing it from its
protein binding sites; the excretion of basic drugs is enhanced by the
acidification of urine, ( e.g., by drugs like ammonium chloride) and the
excretion of acidic drugs is enhanced by the alkalinisation of urine ( e.g., by
drugs like bicarbonates).
10) Disease States: Renal excretion is an important route of excretion, and hence
the excretion of drugs will be affected by renal impairment or renal
dysfunction. Renal impairment can be caused by hypertension, diabetes,
nephrotoxicity (by aminoglycosides, lead, or mercury), polynephritis
(inflammation of kidneys), and hypovolemia.
For example, in uremia associated with decreased glomerular filtration and
accumulation of fluids, renal excretion of drugs is reduced. Thus, half -life of
the drug increases, thereby increasing its accumulation in body.
3.3.5. Non-Renal Routes of Drug Excretion

Non-renal or extra -renal routes for drug excretion are the terms used to describe
excretion of drugs and their metabolites by all other routes except the renal route.
The several routes of non-renal excretion are as follows:
1) Biliary Excretion of Drugs - Enterohepatic Cycling: Bile is produced by the
hepatic cells present along the bile canaliculi. The production and secretion of
bile is an active process. Bile is secreted in the liver, stored in the gall bladder,
and released in the duodenum (as and when required). Bile flows at a steady
rate of 0.5 -1ml/mm in humans. Bile significantly aids the digestion and
absorption of fats. Re-absorption of nearly 90% of the secreted bile acids takes
place in the intestine, and they are carried back to the liver for re -secretion.
*
Faeces form the mode of excretion for the rest (10%) of the secreted bile acids. *
The secretion of bile is a capacity -limited process and gets saturated, since it
is an active process. The process of bile secretion is similar to that of active
renal secretion. The secretion of organic anions, cations, and neutral polar
compounds are linked to the existence of varying transpo rt mechanisms for
the same. If a drug concentration in bile pigments is less than drug
concentration in plasma, the drug is said to have a small biliary clearance and
vice versa. The ratio of concentration of bile to the concentration of plasma
in some cases can give a value approximating 1000. The biliary clearance in
such cases can be 500ml/min or more in value.
Based on their bile/plasma concentration ratios, the compounds excreted in
bile can be categorised into the following three groups:
i) Group A: Compounds with bile/plasma concentration ratio of about 1
are categorised in Group A, e.g., sodium, potassium and chloride ions
and glucose.
ii) Group B: Compounds with bile/plasma concentration ratio of more than 1
(generally ranging from 10 -1000) are categorise d in Group B, e.g., bile
salts, bilirubin glucuronide, creatinine, sulfobromophthalein conju
gates, etc.
iii) Group C: Compounds with bile/plasma concentration ratio of less than 1
are categorised in Group C, e.g., sucrose, inulin, phosphates,
phospholipids, and mucoproteins.
2) Pulmonary Excretion: The process of simple diffusion through lungs aids
in the absorption of gaseous and volatile substances (like general
anaesthetics, e.g., halothane). In the same way, diffusion can also help in
their excretion (they can be excreted into the expired air by diffusion).
Pulmonary blood flow, rate of respiration, solubility of the volatile substance,
etc., constitute the factors that influence the pulmonary excretion of a drug.
Those gaseous anaesthetics that are not very soluble in blood, e.g., nitrous
oxide, show rapid excretion. It is usually seen that gaseous drugs which are
intact undergo excretion; however, their metabolites do not undergo
excretion. Lungs are the organ of excretion for those compounds that are
highly soluble in blood and tissues like alcohol.
3) Salivary Excretion: It involves excretion of drugs through saliva by the
process of passive diffusion. The pH-partition hypothesis forms the basis for
the prediction of salivary excretion of drugs. Though salivary pH ranges from
5.8-8.4, its mean value in human is 6.4. Drugs that exist in unionised forms and
that are soluble in lipids, at the mean pH undergo passive salivary excretion.
For weak acids,
S 1  10( pHsalivapKa ) f plasma
Ra    ….. (9)
P 1  10( pHplasmapKa ) f saliva
For weak bases,

S 110( pKa  pHsaliva) f plasma
Rb    ….. (10)
P 110( pKa pHplasma) f saliva
* *
Where,
fplasma and fsaliva = Free drug fractions in plasma and in saliva, respectively.
For weak acids, the S/P ratios are found to be < 1, while for weak bases they
are found to be > 1. This means that basic drugs are excreted i n saliva in
more amounts as compared to drugs that are acidic in nature. Some drugs
show a high (as high as 0.1%) salivary concentration value. Several drugs
show a fairly constant S/P ratio. Therefore, by detecting their quantity in
salivary excretion, th eir concentration in blood can be determined, e.g.,
caffeine, theophylline, phenytoin, carbamazepine, etc. Some drugs ( e.g.,
lithium) are secreted in saliva actively, and sometimes, their concentration in
saliva is found to be 2 -3 times more than that in t he plasma. Saliva also
actively secretes penicillin and phenytoin.
Salivary
Oral glands
administration
Distribution and
GIT elimination
Faecal Blood
excretion
Figure 3.5: Salivary Cycling of Drugs
The unpleasant after taste in the mouth of a patient, who is on drug treatment,
indicates salivary excretion of the drug. A few cases have been reported
where this process resu lts in adverse effects like black hairy tongue in
patients on antibiotic therapy, gingival hyperplasia in patients on phenytoin,
etc. Salivary secretion is inhibited by some basic drugs, and hence results in
dryness of mouth. A cycling process ( figure 3.5), resembling enterohepatic
cycling can be seen in drugs undergoing salivary excretion, e.g.,
sulphonamides, antibiotics, clonidine, etc.
4) Mammary Excretion: A drug that is excreted in milk can enter in infants
feeding on breast, and therefore, it is significant. Milk contains lactic secretions
synthesised in the extracellular fluid. It also contains rich amounts of fats and
proteins. Lactating mothers secrete nearly 0.5-1 litres of milk per day.
Drug excretion in milk is not a passive process. It depends on:
i) pH-partition behaviour,
ii) Molecular weight,
iii) Lipid solubility, and
iv) Degree of ionisation.
The pH of milk ranges from 6.4 -7.6 and possesses a mean pH of 7.0. Free,
unionised, lipid-soluble drugs show passive diffusion in the alveolar cells of
the mammary gland. The ratio of concentration of drug in Milk to that in
Plasma (M/P) determines the amount of drug excreted in milk. Since milk
bears an acidic nature as compared to plasma, drugs that are weakly basic in
* *
nature show a greater concentration in milk , just like in case of saliva, and
they possess an M/P ratio of more than 1. For drugs weakly acidic in nature,
the reverse is true. Studies have revealed that the excretion of acidic drugs in
milk is inversely correlated with its molecular weight and par tition
coefficient; while in basic drugs, it is inversely correlated to the degree of
ionisation and partition coefficient.
Drugs that show extensive binding with plasma proteins ( e.g., diazepam) are
secreted in milk in lesser amounts. Drugs that undergo excretion via milk can
bind to the proteins that are present in milk. Excretion of drugs in milk is
usually in amounts less than 1% and the portion consumed by infants are in
amounts that are too less to accomplish either beneficial or toxic levels.
However, certain powerful drugs like barbiturates, morphine, and ergotamine
may induce toxic effects in infants.Interaction of bilirubin with sulphonamides
resulting in jaundice and discoloration of teeth with tetracycline are some
instances showing that excretion of a drug in milk may result in adverse effects.
5) Skin Excretion: The pH-partition hypothesis also regulates the excretion of
drugs through skin via sweat. Drugs and their metabolites that are passively
excreted through the skin are accountable to some extent for urticaria and
dermatitis in addition to other hypersensitivity reactions. Sweat shows the
excretion of compounds like benzoic acid, salicylic acid, alcohol and
antipyrine along with heavy metals such as lead, mercury, and arsenic.
6) Gastrointestinal Excretion: Generally, drugs are seen to be excreted in the
GIT when they have been administered through parenteral route and their
concentration gradient favours passive diffusion. The process of GI excretion
of drugs is opposite to that of GI absorpti on. In GIT, stomach is a specific
site that shows the excretion of water-soluble and ionised forms of drugs that
are either weak acids or bases, e.g., nicotine and quinine. GIT also shows the
absorption and excretion of drugs that are administered orally. Excretions of
drugs in GIT are followed by their re -absorption (into the systemic
circulation) and hence are recycled.
7) Genital Excretion: Drugs may also be excreted in the reproductive tract and
genital secretions. Semen has shown the presence of some drugs.
3.4. SUMMARY
1) Elimination is defined as the irreversible loss of drug from the body.
2) Liver is the main site of metabolism for most drugs.
3) A specific group of cytochrome P-450 enzymes involves in liver’s primary
mechanism for metabolising drugs.
4) The biochemical modification of a drug in the body is termed drug
metabolism or biotransformation.
5) Young children show poor drug metabolism as metabolic enzyme systems
are not developed completely.
6) In comparison to males, the females possess lesser ability for drug metabolism.
* *
7) Temperature of the body is directly proportional to drug metabolism.

8) Drugs containing carboxylic acid (esterprocaine), amide (procainamide),
thioesters (spironolactone), phosphor ic acid ester (paraoxon), and acid
anhydride (diisopropylfluoro -phosphate) functional groups undergo
hydrolysis.
9) Drugs containing an aldehyde, ketone, disulphide, sulphoxide, quinine, N -
oxide, alkene, azo or nitro group undergo in vivo reduction.
10) Carbonyl reduction is catalysed by carbonyl reductases present in blood
and cytosolic fraction of the liver, kidney, brain, and other tissues.
11) Sulphoxide and N -oxide reduction is catalysed by thioredoxin -dependent
enzymes present in liver and kidney cytosol.
12) Reductive halogenation involves replacement of halogen with hydrogen,
and is catalysed by cytochrome P450 and glutathione-S-reductase.
13) Quinone reduction of quinone into hydroxyquinones is catalysed by DT -
diaphorase (NADPH-quinone oxidoreductase).
14) Liver cells (or hepatocytes) are the most common site for oxidation of a drug
molecule and microsomes form the site of oxidation within the hepatocytes.
15) Mixed Function Oxidase (MFO) is the enzyme system that causes oxidation
of drug.
16) Cytochrome oxidase enzyme , commonly known as Cytochrome P-450
(CYP-450) and is chemically a haemoprotein.
17) Alkyl groups attached to the nitrogen atom in nitrogen -bearing compounds
undergo N-dealkylation reactions.
18) Oxidative deamination proceeds via carbinolamine pathway but the C -N
bond cleavage occurs at the bond linking the amino group to the larger
portion of the drug molecule.
19) Desulphuration reaction involves cleavage of carbon -sulphur bond (C=S or
thiono) and formation of a product with C=O bond.
20) Thioethers undergo S-oxidation reaction to yield sulphoxides that further
oxidise into sulphones (RSO2R).
21) Oxidative dehalogenation reaction occurs in drugs containing halogen, e.g.,
chloroform.
22) In Phase II reactions , drugs (or their metabolites) are combined with
hydrophilic endogenous co mpounds to form complexes exhibiting adequate
hydrophilic character to allow rapid excretion.
23) Glucuronic acid conjugation occurs only when glucuronic acid gets
activated.
24) The cofactor, Uridine Diphosphate -glucuronic acid (UDP -glucuronic
acid) is required during glucuronidation, which is also catalysed by UDP -
Glucuronyl Transferases.
25) An electron -rich nucleophilic heteroatom, i.e., O, N or S, forms the site of
glucuronidation.
26) Steroids, bilirubin, catechols, and phenylbutazone undergo C-
glucuronidation.
27) Sulphate conjugation reaction is catalysed by sulphotransferases
(sulphokinases), which are cytosolic enzymes found in liver, kidney,
*
intestinal tract, lung, platelets, and brain. *
28) N-acetylation reaction requires acetyl coenzyme A (acetyl CoA) cofactor

and is catalysed by N-acetyltransferases.
29) Methylation reaction involves the synthesis of an activated coenzyme, i.e.,
S-Adenosyl Methionine (SAM, donor of methyl group), from L -methionine
and ATP.
30) Glutathione (GSH) is a tripeptide comprised of glycine, cystei ne, and
glutamic acid. Its formation is catalysed by -glutamylcysteine synthetase.
31) Glutathione conjugates are thioesters formed by nucleophilic attack of
glutathione thiolate anion (GS−) on electrophilic carbon atom or electrophilic
heteroatoms (O, N, orS).
32) Clearance may be defined as the complete removal of a drug in a specified
time period from the hypothetical volume of body fluids containing the drug.
33) Renal clearance can be physiologically expressed as the ratio of rate of
reabsorption subtracted from the sum total of the rate of filtration and that of
secretion to plasma drug concentration (C).
34) Normal creatinine clearance is 120-130ml/min.
35) Glomerular filtration is a process involving the filtration of most
compounds irrespective of whether they are ionised or unionised, with an
exception of macromolecules (bounded to plasma proteins or blood cells).
36) The rate of filtration is known as Glomerular Filtration Rate (GFR).
37) Active tubular secretion is a carrier-mediated process.
38) High threshold endogenous substances or nutrients like electrolytes, glucose,
vitamins, amino acids, etc., which need to be conserved by the body, usually
undergo active tubular re-absorption.
39) Numerous exogenous substances, including drugs, commonly exhibit passive
tubular re-absorption.
40) The excretion of drugs with large molecular weights is difficult, compared to
that of drugs with smaller molecular weights.
41) The solubility of a drug in lipids is inversely proportional to its urinary
excretion.
42) The volume of distribution of dru gs (V d) is inversely proportional to their
clearance.
43) Drugs that show increased perfusion also show an increase in their excretion.
44) Drugs that are bounded to plasma proteins act as macromolecules, and their
filtration by the glomerulus is not possible.
3.5. EXERCISE
1) Liver is the main site of metabolism for most drugs.
2) Temperature of the body is inversely proportional to drug metabolism.
3) Drugs containing an aldehyde, ketone, disulphide, sulphoxide, quinine, N -oxide,
alkene, azo or nitro group undergo hydrolysis.
4) S-methylation is catalysed by cytochrome P450 and glutathione-S-reductase.
5) Cytochrome oxidase enzyme, commonly known as Cytochrome P-450 (CYP-450) and
is chemically a protein.
6) Desulphuration reaction involves cleavage of carbon -sulphur bond and formation of
*
a product with C=O bond. *
7) Oxidative dehalogenation reaction occurs in drugs containing oxygen.

8) N-acetylation reaction requires acetyl coenzyme A cofactor and is catalysed by N -
acetyltransferases.
9) Glutathione is a tripeptide comprised of glycine, cysteine, and glutamic acid.
10) Numerous exogenous substances exhibit active tubular re-absorption.

11) The biochemical modification of a drug in the body is termed drug _______.
12) Carbonyl reduction is catalysed by _______ pres ent in blood and cytosolic fraction
of the liver, kidney, brain, and other tissues.
13) _______ are the most common site for oxidation of a drug molecule.
14) _______ is the enzyme system that causes oxidation of drug.
15) Uridine Diphosphate -glucuronic acid is requ ired during glucuronidation, which is
also catalysed by _______.
16) Normal creatinine clearance is _______.
17) The rate of filtration is known as _______.
18) The solubility of a drug in lipids is inversely proportional to its _______.
19) The volume of distribution of drugs is _______ proportional to their clearance.
20) Drugs that show increased perfusion also show an increase in their _______.
Answers
10) False 11) Biotransformation 12) Carbonyl reductases
13) Liver cells 14) Mixed function oxidase 15) UDP-Glucuronyl Transferases
16) 120-130ml/min 17) Glomerular filtration rate 18) Urinary excretion
19) Inversely 20) Excretion

1) Define drug metabolism.
2) Enlist the enzymes involved in drug metabolism.
3) How drug metabolism is affected by genetics?
4) Give two examples of hydrolysis reaction.
5) What is glucuronidation?
6) Define clearance.
7) How renal excretion of drugs is affected by their physicochemical properties?

1) Discuss the factors affecting drug metabolism.
2) Write about oxidation reaction of phase I metabolic pathway.
3) Discuss the glutathione conjugation reaction.
4) Discuss the factors affecting renal excretion of drugs.
5) Explain any two non-renal routes of drug excretion.

1) Give a detailed review on phase II metabolic pathways.
2) Discuss the non-renal routes of drug excretion.
3) Write an exhaustive note on renal excretion of drugs.
* *
CHAPTER Bioavailability and

4 Bioequivalence
4.1. BIOAVAILABILITY
4.1.1. Introduction and Definition

Bioavailability is the rate and extent of an administered dose of drug that
reaches the systemic circulation in unchanged form . It is a major
pharmacokinetic property of drug. Bi oavailability of a drug administered
intravenously is 100%. A drug’s therapeutic effectiveness depends on its ability to
provide the medicament at a sufficient rate and in sufficient amount to its site of
action in order to achieve the desired pharmacological response. This effectiveness
of a drug dosage form is known as bioavailability or physiologic availability or
biologic availability. The pharmacological response of many drugs can be directly
related to their plasma levels; therefore, bioavailability can also be defined as the
rate and extent of absorption of unchanged drug from its dosage form .
Bioavailability of a drug from its dosage form depends on:
1) The administration route,
2) Patient-related factors,
3) Physicochemical properties of the drug, and
4) Dosage form characteristics.
The drug dose given to the patient is the administered dose. The dose available
to the patient is the bioavailable dose (usually less than the administered dose).
The effect of administration route on bioavailable dose is in the following order:
Parenteral > Oral > Rectal > Topical
4.1.2. Objectives of Bioavailability Studies

The important objectives of bioavailability studies are:
1) It involves the primary development stages for a suitable dosage fo rm of a
new drug entity.
2) It helps in determining how efficiency of absorption is influenced by the
excipients, patient-related factors, and possible interactions with other drugs.
3) It helps in the development of new formulations of the existing drugs.
4) It controls the quality of a drug product during the early stages of marketing
to determine how drug absorption is affected by processing fac tors, storage,
and stability conditions.
4.1.3. Types of Bioavailability

Bioavailability of drugs is of two types:
1) Absolute bioavailability, and
2) Relative bioavailability.
* *
Bioavailability and Bioequivalence (Chapter 4) 101
4.1.3.1. Absolute Bioavailability

The absolute bioavailability of a given drug from a dosage form is the fraction
(or percentage) of the administered dose absorbed int o the systemic
circulation in unchanged form . It is calculated by comparing the total amount
of unchanged drug reaching the systemic circulation after administering a known
dose of the dosage form via any route with the total amount of unchanged drug
reaching the systemic circulation after administering an equivalent dose of the
drug in the form of an intravenous bolus injection.
When a drug is administered via intravenous route, the administered dose is
introduced directly into the systemic circulation, i.e., it does not have to pass
through any absorption barriers, and therefore gives 100% bioavailability. Due to
this reason, an intravenous bolus injection serves as a reference to compare the
systemic availability of the drug administered via different routes.
Absolute bioavailability of a drug can
be calculated from plasma data by Concentration of drug in
comparing the total areas under the Aqueous solution
by intravenous
plasma concentration -time curves bolus injection
plasma
obtained after administering equivalent

doses of the drug via absorption site and
via intravenous route i n the same Aqueous
subject at different times. Figure 4.1 solution by
preoral route
shows the plasma concentration -time
Time following administration
curves obtained after administering of a single dose
equivalent doses of the drug via Figure 4.1: Typical Plasma Concentration-Time
intravenous bolus injection and Curves obtained by Administering Equivalent
Doses of the Same Drug by Intravenous Bolus
gastrointestinal route. Injection and by the Oral Route
For equivalent doses of administered drug:

(AUCT ) abs
Absolute bioavailability 
bioavailability
Absolute …. (1)
AUCT iv
Where, (AUC T)abs = Total area under the plasma con centration-time curve after
administering a single dose via absorption site.
(AUCT)iv = Total area under the plasma concentration -time curve after
administering a single dose by rapid intravenous injection.
If different drug doses are administered by both the routes, the dose sizes can be
corrected as follows:
(AUCT ) abs /D abs
Absolute bioavailability 
bioavailability
Absolute …. (2)
AUCT iv /D iv
Where, Dabs = Size of the single dose of drug administered via absorption site.
Div = Size of the drug dose administered as an intravenous bolus injection.
Sometimes, different dosages of drugs should be administered via different

routes. The dose administered via intravenous route is often kept low to avoid
toxic si de effects and also for ease of formulation. Bioavailability data for
* *
different dosages should be calculated carefully as sometimes the drug

pharmacokinetics are non -linear; due to which different doses will result in an
incorrect figure for absolute bioav ailability if calculated using a simple ratio, as
in equation (2) . Absolute bioavailability can also be determined using urinary
excretion data by comparing the total cumulative amounts of unchanged drug
excreted in the urine after administering the drug v ia absorption site and
intravenous route (bolus injection) to the same subject at different times.
For equivalent doses of administered drug:
(X )
Absolute bioavailability  u abs
Absolutebioavailability ….. (3)
X u iv
Where, (X u)abs and (X u)iv = Total cumulative amounts of unchanged dru g
excreted in the urine after administering equivalent single doses of drug via
absorption site and intravenous bolus injection, respectively.
If different doses of drug are administered:
(X ) /D
Absolute bioavailability  u abs abs
Absolutebioavailability ….. (4)
X u iv /D iv
Absolute bioavailability of a drug from a par ticular type of dosage form may be
expressed as a fraction or a percentage.
Factors related to oral route of drug administration may affect bioavailability, and
these effects can be studied by measuring absolute bioavailability obta ined by
administering a drug as a simple aqueous solution (that does not precipitate on
contact with or dilution by gastrointestinal fluids) by oral and intravenous routes,
e.g., pre -systemic metabolism by the intes tine or liver, formation of complexes
between the drug and endogenous substances ( e.g., mucin) at the site of
absorption, and drug stability in the gastrointestinal fluids.
4.1.3.2. Relative Bioavailability

For drugs that cannot be administered by intra venous bolus injection, their
relative (or comparative) bioavailability is determined instead of the absolute
bioavailability. Relative bioavailability can be determined by comparing the
bioavailability of a drug from a test dosage form with the same drug administered
in a standard dosage form. The latter is either an orally administered solution
(from which the drug is known to be well -absorbed) or an established
commercial preparation of proven clinical effectiveness.
Hence, relative bioavailability is the fraction (or percentage) of a drug that is
absorbed in unchanged form into the systemic circulation from a dosage form of the
relative to a recognised (or clinically proven) standard dosage form of the same drug.
Plasma concentration-time curves are used for calculating relative bioavailability of a
drug administered as equal doses of a test dosage form and a recognised standard
dosage form by the same administration route to the same subject at different times:
(AUCT ) test
Relative bioavailab
Relative ility 
bioavailability …..(5)
AUCT standard
* *
Where, (AUC T)test and (AUC T)standard = Total areas under the plasma
concentration-time curves after administering a single dose of the test dosage
form and of the standard dosage form, respectively.
If test and standard dosage forms are administe red in different doses, the dose
size can be corrected as follows:
(AUCT ) abs /D test
Relative
Re lative bioavailab ility 
bioavailability ….. (6)
AUCT s tan dard /D s tan dard
Where, D test and D standard = Sizes of the single doses of test and standard dosage
forms, respectively.
Relative bioavailability (alike absolute bioa vailability) can be expressed as a
fraction or a percentage, and can also be determined from urinary excretion data
as follows:
(X u ) test
Relative bioavailability 
bioavailability
Re lative ….. (7)
X u s tan dard
Where, (X u)test and (X u)standard = Total cumulative amounts of unchanged drug
excreted in the urine after administering single doses of the test dosage form and
standard dosage form, respectively.
If the test and standard dosage forms are administered in different doses at
different times, the total amount of unchanged drug excreted in th e urine per unit
dose of drug should be used in this equation.
Relative bioavailability is measured to determine the effects of dosage form
differences on systemic bioavailability of a drug. Many dosage form factors can
influence the drug bioavailability , like the type of dosage form ( e.g., tablet,
solution, suspension, and hard gelatin capsule), differences in the formulation of
a particular type of dosage form, and manufacturing variables employed in the
production of a particular type of dosage form.
4.1.4. Measurement of Bioavailability

The methods employed in the quantitative evaluation of bioavailability are
broadly divided into:
1) Pharmacokinetic Methods: These are indirect methods as they are based on
the assumption that the pharmacokinetic properties of a drug reveal its
therapeutic efficacy. The two major pharmacokinetic methods are:
i) Plasma level-time studies, and
ii) Urinary excretion studies.
2) Pharmacodynamic Methods: These methods are complementary to
pharmacokinetic approaches and involve direct measurement of drug effect
on a pathophysiologic process as a function of time. The two pharmacodynamic
methods for determination of bioavailability from:
i) Acute pharmacologic response, and
ii) Therapeutic response.
* *
4.1.4.1. Plasma Level-Time Studies

The method of determining plasma drug concentration is based on the assumption
that two dosage forms exhibiting su perimposable plasma level-time profiles in a
group of subjects should show identical therapeutic activity.In a single dose study,
serial blood samples are collected for 2 -3 biological half -lives after drug
administration. Their drug concentration is analy sed and is plotted against the
corresponding time of sample collection to obtain the plasma level -time profile.
Sampling should be started within 5 minutes of drug administration via intravenous
route, and is continued at every 15 minutes. The disposition phase can be properly
described only if not less than 3 sample points are taken for the drug following
one-compartment model and 5 to 6 points are taken for the drug following two -
compartment model. For oral dose, 3 points should be taken on the ascending part
of the curve to accurately determine K a. The points for disposition or descending
phase of the curve should be taken in a manner similar to that for intravenous dose.
Given below are the three parameters of plasma level -time studies that are
significant in the determination of bioavailability:
1) Cmax: It is the peak that indicates the point at which the drug concentration in
plasma is maximum. Peak plasma concentration (or peak height
concentration or maximum drug concentration ) is the drug concentratio n
at peak. Cmax is usually expressed in mcg/ml.
Peak level depends on the administered dose and the absorption and
elimination rate of drug. Peak is the point of time when the absorption rate and
the elimination rate of drug becomes equal. The portion of ht e curve to the left
of peak indicates the absorption phase where the drug’s absorption rate is
greater than its elimination rate. The portion of the curve to the right of peak
indicates the elimination phase where the drug’s elimination rate is greater anth
its absorption rate. Peak concentration is related to pharmacological response
and should be more than the Minimum Effective Concentration (MEC) and
less than the Maximum Safe Concentration (MSC).
2) tmax: It is the time the drug requires to reach peak con centration in plasma
after extravascular administration. t max is expressed in hours. Time of peak
concentration is used in estimating the absorption rate of drug. It influences
the onset time and onset of action. t max is used for evaluating the efficacy of
drugs employed in treating acute conditions (pain and insomnia) that can be
treated with a single dose of a drug.
3) AUC: It is the area under the plasma level-time curve that gives a measure
of the extent of absorption or the amount of drug that reaches the systemic
circulation. The extent of bioavailability can be determined by following
equations:
F
AUCoral Div ….. (8)
AUCiv Doral
Fr 
AUCtest Dstd ….. (9)
AUCstd D test
* *
Where, D = Dose administered.

Subscripts iv and oral = Administration route.
Subscripts test and standard = Test a nd standard doses of the same drug
to determine relative availability.
With multiple dose study, the method involves drug administration for 5
biological half-lives with a dosing interval either equal to or greater than the
biological half-life (i.e., administration of at least 5 doses) to reach the steady-
state. A blood sample should be collected at the end of previous dosing interval
and 8 to 10 samples should be collected after administering the next dose.
The extent of bioavailability is given as:
Fr 
AUCtest Dstd test ….. (10)
AUCstd D testl std
Where, [AUC] values = Area under the plasma level-time curve of one dosing
interval in a multiple dosage regimen, after reaching the steady
-state (figure 4.2).
 = Dosing interval.
Bioavailability can also be determined from the peak plasma concentration at
steady-state (Css,max) as follows:
Css,max test Dstd test
Fr  ….. (11)
Css,max std Dtestl std
The absorption rate is not important in multiple dosing methods.
Dose Dose Dose Dose Dose Dose
     
CCss,max
ss,max
Plasma Concentration C
Steady-state
level
C
Css,min
ss,min
Area under the

curve for one
dosing interval
at steady-state
Dosing interval 
Figure 4.2: Determination of AUC and on Multiple Dosing upto Steady-States
4.1.4.2. Urinary Excretion Studies

Urinary excretion studies, performed to assess bioavailability, rely on the
principle that the urinary excretion of unchanged drug and the plasma
concentration of drug are directly proportional. Thus, even if 10 -20% of a drug
gets excreted in the urine, its bioavailability can be determined. Urinary excretion
* *
study is used for drugs that get excreted in urine in unchanged form ( e.g., certain
thiazide diuretics and sulphonamides) and for drugs that have urine as the site of
action ( e.g., urinary antiseptics such as nitrofurantoin and hexamine). The
concentration of drug metabolites that get excreted in urine is never considered
for calculations, because a drug before getting absorbed may undergo pre -
systemic metabolism at different stages.
The urinary excretion method involves collecting urine samples at regular
intervals for 7 biological half-lives, analysing the unchanged drug in the collected
sample, and determining the amount of drug excreted at each interval and
cumulative amount ex creted. Each time of sampling, the subject should
completely empty the bladder to avoid errors in the next urine sample that may
result from the residual amount. In the beginning of the study, frequent sampling
is essential to correctly calculate the absorption rate.
Given below are the major parameters that are examined in urinary excretion data
obtained with a single dose study:
1) (dXu/dt)max: It is the maximum urinary excretion rate, obtained from the
peak of plot between excretion rate and midpoint time of urine collection
period. It is analogous to the C max obtained from plasma level studies since
the rate of appearance of drug in urine and its concentration in systemic
circulation are proportional. The value of (dX u/dt)max increases with the
increase in rate of and/or extent of absorption.
2) (tu)max: It is the time for maximum excretion rate. It is analogous to t max of
plasma level data. The value of u(t)max decreases with increase in absorption rate.
(dXu/dt)max
Rate of excretion
(tu)max
Midpoint time of urine collection period
Figure 4.3: Plot of Excretion Rate versus Time. The curve is
analogous to a typical plasma level-time profile obtained after
oral administration of a single dose of drug
3) Xu: It is the cumulative amount of dru
g excreted in urine. It is related to the AUC
of plasma level data. The value of Xu increases with the increase in extent of
absorption.The extent of bioavailability is calculated from
the following equations:
F
X  D

u oral iv
X  D

..… (12)
u iv oral
F 
X  D

u test std ….. (13)
r
X  D

u std testl
* *
With multiple dose study to steady -state, the equation for computing
bioavailability is:
X u ,ss test Dstd test
Fr 
X u,ss std D testl test
….. (14)
Where, (X u,ss) = Amount of drug excreted unchanged during a single dosing

interval at steady-state.
4.1.4.3. Acute Pharmacologic Response

When pharmacokinetic methods cannot be used successfully and easily to
measure bioavailability, an inaccurate, non -reproducible, or an acute
pharmacologic effect such as change in ECG or E EG readings, pupil diameter,
etc., is related to the time course of a given drug. Then bioavailability can be
determined by making of pharmacologic effect -time curve and dose -response
graphs. For this method, responses of the drug for not less than 3 biolo gical half-
lives should be measured to obtain a good estimate of AUC.
The method of acute pharmacologic response has a disadvantage that the

pharmacologic response may vary and thus an accurate correlation between the
measured response and drug available from the formulation cannot be
established. Also, the observed response may be due to an active metabolite
whose concen tration is not proportional to the concentration of parent drug
inducing the pharmacologic effect.
4.1.4.4. Therapeutic Response

The therapeutic response method involves observing the clinical response of a drug
formulation in patients who are suffering from the disease for which th e drug is
intended to be used. The therapeutic responsemethod has several disadvantages:
1) Quantitation of observed response is so inappropriate that reasonable
assessment of relative bioavailability between two dosage forms of the same
drug cannot be done.
2) Bioequivalence studies are conducted using a crossover design in which each
subject is given each test dosage form, and it is anticipated that the subject’s
physiological status does not change during the entire study.
3) If multiple -dose protocols for a drug are not employed, a patient who
requires the drug for a disease will be administered only a single dose of the
drug in every few days or each week.
4) Many patients receive more than one drug, and the bioavailability study
results obtained could be compromised because of a drug-drug interaction.
4.1.5. Significance of Bioavailability Studies

Bioavailability studies have the following significant features:
1) They help in estimating the fraction of the orally administered dose that is
absorbed into the systemic circulation when compa red to the bioavailability
for a solution, suspension, or intravenous dosage form that is completely
available.
* *
2) They provide useful information that is required for establishing dosage

regimen and for drug label ling, such as distribution and elimination
characteristics of the drug.
3) They provide information regarding the performance of a formulation.
4) They help in the determination of influence of excipient on absorption.
5) They help in the development of new formulations of existing drug.
6) They control the qua lity of drug product by determining the influence of
processing factors, storage and stability on absorption.
7) They help in making comparison of drug in different dosage form or same
dosage form of different manufacturer.
4.2. METHODS TO ENHANCE THE

DISSOLUTION RATES AND BIOAVAILABILITY
OF POORLY SOLUBLE DRUGS
4.2.1. Introduction
Various techniques are available for enhancing the solubility of poorly soluble
drugs. Some of these approaches are as follows:
1) Chemical Modifications:
i) Use of salt forms ii) Co-crystallisation
iii) Co-solvency iv) Hydrotrophy
v) Solubilising agents vi) Nanotechnology
2) Physical Modifications:
i) Particle size reduction
a) Micronisation b) Nanosuspension
c) Sonocrystallisation d) Supercritical fluid process
ii) Modification of the crystal habit
a) Polymorphs
b) Pseudopolymorphs
iii) Complexation - Use of complexing agents
iv) Solubilisation by surfactants
a) Microemulsions
b) Self microemulsifying drug delivery system
v) Drug dispersion in carriers
4.2.2. Use of Salt Forms

Salts have improved solubility and dissolution characteristics than the original
drug. A minimum difference of 3 units between the pKa value of the group and
that of its counter -ion is required to form stable salts. The solubility of alkali
metal salts of acidic drugs (like penicillins) and strong acid salts of basic drugs
(like atropine) in water is more than that of the parent drug. Factors influencing
salt selection are physical and chemical properties of the salt, safety of counter -
ion, therapeutic indications, and administration route.
* *
Salt formation has the following limitations:

1) Salts of neutral compounds cannot be formed.
2) Salts of very weak bases or acids can be formed with much difficulty.
3) The salt may be hygroscopic, exhibit polymorphism, or has poor processing
characteristics.
4) Conversion of salt to free acid or base form of the drug on surface of solid
dosage form prevents or retards drug release.
5) Precipitation of unionised drug in the gastrointestinal milieu that has poor
solubility.
4.2.3. Co-Crystallisation
With the application of co -crystals or molecular complexes , drug solubility can
be improved. If the solvent forms an integral part of the network structure and at
least two component crystal, it is termed as co-crystal. If the solvent do es not
directly involve itself in the network (as in open framework structures), it is
termed as clathrate (or inclusion complex). A co-crystal is a crystalline material
consisting of two or more molecular and electrically neutral species bound by
non-covalent forces.
Co-crystals are more stable as the co -crystallising agents are solids at room
temperature. Only three co -crystallising agents, i.e., saccharin, nicotinamide and
acetic acid, are recognised as safe (GRAS), thus limiting their pharmaceutical
applications. Co -crystallisation may also occur between two active
pharmaceutical ingredients by using sub -therapeutic amounts of drug substances
(such as aspirin or acetaminophen). Co-crystals can be prepared by evaporating a
heteromeric solution or by grin ding the components together. Another technique
of preparing co -crystals includes sublimation, growth from the melt, and slurry
preparation. Formation of molecular complexes and co -crystals and their
importance is increasing day -by-day as an alternative to salt formation, mainly
for neutral compounds or those having weakly ionisable groups.
4.2.4. Cosolvency
Solubilising the drugs in co -solvents is another technique of solubility
enhancement of poorly soluble drugs. Adding an organic cosolvent to water can
change the drug solubility. Water solubility of weak electrolytes and non -polar
molecules is poor and this can be improved by adding another solvent that alters
the solvent polarity. This process is termed cosolvency, and the solvent used for
increasing solubility is termed a cosolvent.
The system of cosolvent involves solvent blending in which the interfacial

tension between the aqueous solution and hydrophobic solute is reduced.
Cosolvents mostly have hydrogen bond donor and/or acceptor groups and small
hydrocarbon regions. Their hydrophilic hydrogen bonding groups ensure water
miscibility, and their hydrophobic hydrocarbon regions interfere with water’s
hydrogen bonding network, thus reducing the intermolecular attraction of water.
Cosolvents disrupt the wate r’s self-association, reduce water’s ability to squeeze
out non-polar, hydrophobic compounds, and thus increase solubility. A different
* *
perception is that cosolvents facilitate solubilisation by making the polar water

environment more non -polar like the so lute. Solubility enhancement as high as
500-fold is achieved by using 20% of 2-pyrrolidone.
4.2.5. Hydrotrophy
Hydrotrophy is the increase in water solubility due to the presence of large
amounts of additives. Its mechanism of improving solubility is closely rel ated to
complexation that involves a weak interaction between the hydrotrophic agents
(e.g., sodium benzoate, sodium acetate, sodium alginate, and urea) and the solute.
An example of hydrotrophy is solubilisation of theophylline with sodium acetate
and sodium alginate.
4.2.6. Solubilising Agents

Solubility of poorly soluble drugs can also be improved by using solubilising
materials. For example , PEG 400 is used for improving the solubility of
hydrochlorothiazide; Modified Gum Karaya (MGK) was evaluated as a carri er
for dissolution enhancement of nimodipine; addition of caffeine and
nicotinamide for improving the aqueous solubility of halofantrine (antimalarial).
4.2.7. Nanotechnology Approaches

Nanotechnology is the study and use of materials and structures at the nanos cale
level of approximately 100 nanometers (nm) or less. For many new chemical
entities of low solubility, enhancement of oral bioavailability by micronisation is
not sufficient because the micronised product may undergo agglomeration, which
decreases the effective surface area for dissolution.
4.2.8. Particle Size Reduction

Particle size reduction techniques by various milling processes are well -
established and are a standard part of formulation development. Particle size of
the drug is reduced by micronisation, nanosuspension, sonocrystallisation, etc.
techniques. As particle size decreases, surface area of particle increases, and this
increases the solubility.
Micronisation
Drug solubility is often intrinsically related to drug particle size. By reducing the
particle size of the drug, its surface area is increased and its dissolution properties
are improved. The conventional methods of particle size reduction (such as
comminution and spray drying) rely on mechanical stress to disaggregate the
active compound. M icronisation is used to increase the surface area for
dissolution.
Nanosuspension
Nanosuspensions are sub -micron colloidal dispersion of pure drug particles,
which are stabilised by adding surfactants. They offer the advantages of
increased dissolution ra te due to larger surface area exposed, and absence of
Ostwald ripening due to the uniform and narrow particle size range obtained
that eliminates the concentration gradient factor.
* *
Sonocrystallisation
Recrystallisation of poorly soluble materials with liquid solvents and anti-solvents
has been successfully employed for particle size reduction. Sonocrystallisation is a
novel approach for particle size reduction that involves inducing crystallisation
using ultrasound waves at a frequency range of 20 -100kHz. T his technique
enhances the nucleation rate, is an effective means of size reduction, and also
controls size distribution of the active pharmaceutical ingredients. In most cases,
ultrasound waves are used in the range of 20 kHz-5 MHz.
Supercritical Fluid Process

Application of novel nanosizing and solubilisation technology has increased the
use of Supercritical Fluid (SCF) processes for particle size reduction. SCF is a
dense non -condensable fluid with temperature and pressure greater than its
critical temperature (Tc) and critical pressure (Tp). By manipulating the pressure
of SCFs, the favourable properties of gases, like high diffusivity, low viscosity,
and low surface tension, may be imparted upon liquids to control drug
solubilisation with a supercritical fluid. SCFs are highly compressible, and allow
moderate changes in pressure to alter the density and mass transport
characteristics of fluid, which determine its solvent power.
Once the drug particles solubilise in SFs, they may be recrystallised at r educed
particle sizes. SCF process allows micronisation of drug particles within a narrow
range of particle size, often to sub-micron levels. The current SCF processes have
the ability to create nanoparticulate suspensions of particles 5 -2,000nm in
diameter. The most widely employed SCF processing methods for micronised
particles are Rapid Expansion of Supercritical Soluti ons (RESS) and gas anti -
solvent recrystallisation (GAS). These methods are used by the pharmaceutical
industries using carbon dioxide (CO2) as the SCF due to its favourable processing
characteristics like low critical temperature (Tc = 31.10°C) and pressure (Pc =
73.8 bar).
In the method of RESS, a drug or a mixture of drug and polymer is solubilised in

SCF and this solution is sprayed thr ough a conventional nozzle or capillary tube
into a lower pressure environment. Rapid expansion the solution undergoes
reduces CO 2 density and its solvent power, thus supersaturating the lower
pressure solution. This supersaturation results in the recrysta llisation and
precipitation of pure drug or drug -polymer particles of greatly reduced size,
narrow size distribution, and high purity. For example , solubility of nifedipine
has been improved by using RESS. In the GAS process, the drug or drug -
polymer mixtu re is solubilised into a solvent via conventional means and then
sprayed into a SCF; the drug should be insoluble in the SCF, and the SCF should
be miscible with the organic solvent. The SCF diffuses into the spray droplets,
causing expansion of the present solvent, and precipitation of the drug particles.
4.2.9. Modification of the Crystal Habit

Polymorphism is the element’s or compound’s ability to crystallise in more than
one crystalline form. Different drug polymorphs are chemically same, but differ
in their physicochemical properties like solubility, melting point, density, texture,
* *
and stability. In the same way, amorphous form of drug is more suitable than the
crystalline form as higher energy is associated with the former and increase in
surface area. The dissolution of different solid forms of drug follows the given
order: Amorphous >Metastable polymorph >Stable polymorph
4.2.10. Complexation
Complexation involves association between two or more molecules to form a
non-bonded entity with a well -defined stoichiome try. Complexation relies on
weak forces such as London forces, hydrogen bonding, and hydrophobic
interactions. The complexing agents commonly used in improving bioavailability
of poorly soluble drugs are given in the table below:
Table 4.1: List of Complexing Agents
Types Examples
1) Inorganic lB
2) Coordination Hexamine cobalt(III) chloride
3) Chelates EDTA and EGTA
4) Metal-Olefin Ferrocene
5) Inclusion Cyclodextrins and Cholic acid
6) Molecular Complexes Polymers
4.2.11. Solubilisation by Surfactants

Surfactant molecule s have separate polar and non -polar regions. They mostly
consist of a hydrocarbon segment that is linked to an anionic, cationic, Zwitter
ionic or non -ionic polar group. When small polar molecules are added they
accumulate in the hydrophobic core of the micelles. This solubilisation process is
important in industrial and biological field. Presence of surfactants decreases the
surface tension but increases the solubility of drug in an organic solvent.
Microemulsion
Jack H. Shulman first used the term microemulsion in 1959. It is a four -
component system comprising of an external phase, an internal phase, a
surfactant, and a co -surfactant. On adding a surfactant that is soluble in the
internal phase (unlike the co -surfactant), an optically clear, isotropic,
thermodynamically stable emulsion is formed, which is termed microemulsion
because the internal or dispersed phase is < 0.1μ droplet diameter.
Microemulsion is formed spontaneously and does not involve the input of
external energy as in case of coarse emulsions.
The surfactant and co -surfactant alternate each other and a mixed film i s formed
at the interface, which contributes to the stability of microemulsion. Non -ionic
surfactants, such as Tweens (polysorbates) and Labrafil (polyoxyethylated oleic
glycerides), having high HLB values are used so that o/w droplets are
immediately form ed during production. Microemulsion is advantageous over
coarse emulsion due to its ease of preparation involving spontaneous formation,
thermodynamic stability, transparent and elegant appearance, increased drug
loading, enhanced penetration through biolo gical membranes, increased
bioavailability, and less inter - and intra -individual variability in drug
pharmacokinetics.
* *
4.2.12. Drug Dispersion in Carriers

The approach of solid dispersion for particle size reduction and dissolution and
absorption rate enhancement of drugs was first introduced in 1961. The term
solid dispersion refers to the dispersion of one or more active ingredients in an
inert carrier in a solid state, prepared by the melting (fusion) method, solvent
method, or fusion solvent method.
The novel preparation techniques include rapid precipitation by freeze drying,

spray drying in the presence of amorphous hydrophilic polymers, supercritical
fluid technology, and melt extrusion method. Polyvinyl pyrrolidone,
polyethylene glycols, Plasdone -S63020, a nd surfactants (Tween -80, docusate
sodium, Myrj -52, Pluronic -F68, and SLS) are the most commonly used
hydrophilic carriers for solid dispersions.
With the approach of solid dispersion using suitable hydrophilic carriers, the
solubility of etoposide, glyb uride, itraconazole, ampelopsin, valdecoxib,
celecoxib, and halofantrine can be improved. The eutectic combination of
chloramphenicol/urea and sulphathiazole/urea are the examples for the
preparation of a poorly soluble drug in a highly water soluble carrier.
Poorly Water Soluble
Drug
Tablet/capsule Dosage form Solid dispersion/Solution
Disintegration Disintegration
Large solid particle Colloidal particles/

Drug in GIT
(usually 5-100) Fine oily globule (<1)
Lower dissolution rate Higher dissolution rate

Absorption into
Body System
Figure 4.4: Schematic Representation of Bioavailability
Enhancement of a Poorly Water-Soluble Drug by Solid Dispersion
Simple Eutectic Mixture

A eutectic mixture of a sparingly water -soluble drug and a highly water -soluble
carrier is thermodynamically regarded as an intimately blended physical mixture of
two crystalline components. Increased surface a rea increases the dissolution rate,
thus concluding that the increase in dissolution was due to decreased particle size.
Solid Solutions
Solid solution is a binary system comprising of a solid solute molecularly dispersed
in a solid solvent. Solid solutions are also termed molecular dispersions or mixed
crystals as the two components crystallise together in a homogeneous one phase
system. Reduction of particle size up to the molecular level increases the aqueous
solubility of solid solutions, thus they undergo faster dissolution than eutectics and
* *
solid dispersions. Solid solutions are prepared by fusion method in which a

physical mixture of solute and solvent are melted together and then rapidly
solidified. Such systems, prepared by fusion, are termedmelts.
Solid solutions are classified on the following two bases:

1) Miscibility between the Drug and the Carrier: Solid solutions are divided
into two categories on this basis:
i) Continuous Solid Solution: In this, the drug and the carrier are miscible
in all proportions.
ii) Discontinuous Solid Solution: In this, solubility of each com ponent in
the other is limited.
2) Distribution of Drug in Carrier Structure: Solid solutions are divided into
two categories on this basis:
i) Substitutional Crystalline Solid Solution: In this, if the drug and
carrier molecules are almost of same size, the drug molecule substitutes
for the carrier molecules in its crystal lattice.
ii) Interstitial Crystalline Solid Solution: In this, if the size of drug
molecule is 40% or less than the size of carrier molecules, the drug
molecules occupy the interstitial spaces in the crystal lattice of carrier
molecules (figure 4.5).
Crystal lattice of carrier
molecules
Drug molecule
Substitutional solid solution Interstitial solid solution
Figure 4.5: Types of Crystalline Solid Solution
Glass Solution of Suspension

A glass solution is a homogenous system in which a glassy or a vitreous of the
carrier solubilises drug molecules in its matrix. PVP dissolved in organic solvents
undergoes a transition to a glassy state on solvent evaporation.
Compound or Complex Formation

This system is characterised by complexation of two components in a binary
system during solid dispersion preparation. Availability of the drug from the
complex depends on the solubility dissociation constant and the intrinsic
absorption rate of the complex.
Amorphous Precipitation
When drug precipitates as an amorphous form in the ine rt carrier, amorphous
precipitation occurs. Higher energy state of the drug in this system produces
much greater dissolution rates than the corresponding crystalline forms of the
drug.
* *
4.3. IN VITRO-IN VIVO CORRELATIONS (IVIVC)

Development of a drug and optimisation of a formulation are very tedious, time
taking and expensive process, and during these processes in vitro -in vivo
correlations (IVIVC) play an important role. Optimisation of a formul ation
includes alteration in formulation, composition, equipment, batch sizes , and
manufacturing processes. If any such types of one or more alterations are made to
the formulation, the new formulation demands carrying out bioequivalence
studies to prove i ts similarity with the previous formulation and thus increases
the cost of optimisation process. As a result of these alterations , the market cost
of the new formulation increases. These problems can be resolved by developing
in vitro tests that reflect bi oavailability data. IVIVC is the technique which can
be used to develop new pharmaceuticals so that the number of human studies
during the development of formulation can be reduced. So IVIVC acts as a
surrogate for in vivo bioavailability and supports bio waivers.
United State Pharmacopoeia (USP) defined IVIVC as “the establishment of a

rational relationship between a biological property or a parameter derived
from a biological property produced by a dosage form, and a
physicochemical property or characteristic of the same dosage form”.
Food and Drug Administration (FDA) defined IVIVC as “ a predictive

mathematical model describing the relationship between an in-vitro property
of a dosage form and an in-vivo response”.
The in vitro studies are performed to analyse the rate or extent of drug dissolution
or release, while the in vivo studies help to study the plasma drug concentration
or amount of drug absorbed. IVIVC is performed to obtain drug dissolution
results from two or more products so that the simila rity or dissimilarity of
expected plasma drug concentration profiles can be determined. It is important to
know how to establish similarity or dissimilarity of in vivo response, i.e., plasma
drug concentration profiles b efore deducing a relationship betwee n the results
obtained from in vitro and in vivo studies. The method used to establish similarity
or dissimilarity of plasma drug concentration profile s is called bioequivalence
testing. There are well -defined guidelines and standards for establishing
bioequivalence between drug profiles and products.
4.3.2. Purpose of IVIVC

The purposes or objectives of IVIVC can be given as:
1) To Reduce Regulatory Burden: Under some specific circumstances, IVIVC
serves as a substitute for additional in vivo experiments.
2) In Optimisation of Formulation: The optimisation of formulations includes
alterations in composition, manufacturing process, equipment, and batch
sizes. Therefore, an exhaustive study of bioequivalence (BE)/bioavailab ility
(BA) is necessary to prove th e validity of a new formulation that is
*
bioequivalent with the target formulation. *
3) To Justify the Product’s Therapeutic Quality: IVIVC technique is used to

prove the therapeutic efficiency of the formulation.
4) To Scale -up Post-Approval Changes (Time and Cost Saving during
Product Development): Validated IVIVC process is used as approval tool
for biowaivers during the filings of a Level 3 (or Type II in Europe)
variation, either during scale-up or post-approval, and also for line extensions
(e.g., different dosage strengths).
5) Used as Surrogate for in vivo Bioequivalence and Support Biowaivers
(Time and Cost Saving): The main aim of an IVIVC process is to utilise in
vitro dissolution profiles as a surrogate for in vivo bioequivalence studies and
to support biowaivers.
4.3.3. Levels of IVIVC

As per the FDA guidelines, there are four levels of IVIVC, which include levels
A, B, C, and multiple C. The correlation level concept is based on the ability of
the corre lation to reflect the entire plasma drug level -time profile which is the
result of administration of the given dosage form.
4.3.3.1. Level A Correlation

The level A c orrelation of IVIVC has regulatory relevance and cor relates the
entire in vitro and in vivo profiles. It is the highest category of correlation and
represents a point -to-point relationship between in vitro dissolution rate and in
vivo input rate of the drug from dosage form.
It is important to achieve l evel A co rrelation because it allows bio waiver for
changes in manufacturing site, raw material suppliers, and minor changes in
formulation. Level A correlation defines a direct relationship between in vivo
data such as the measurement of in vitro dissolution rate is required to determine
the biopharmaceutical rate of the dosage form.
4.3.3.2. Level B Correlation

The level B correlation of IVIVC relies on the principles of statistical moment
analysis. This level includes comparison of in vitro dissolution time (MDT vitro)
of the product to either in vivo residence time (MRT) or in vivo dissolution time
(MDT vivo). Level B correlation is not so useful for regulatory purposes because
it does not reflect the actual in vivo plasma level curves, and also in vitro data
from such a correlation cannot be used to justify the extremes of quality control
standards.
4.3.3.3. Level C Correlation

The level C correlation relates one dissolution time p oint (t 50%, t 90%, etc.) to one
mean pharmacokinetic parameter (AUC, t max, or C max). It is the weakest level of
correlation as partial relationship between absorption and dissolution is
established because it does not represent the complete shape of plasm a drug
concentration-time curve (the critical factor that defines the performance of a
drug product). Therefore, the usefulness of level C correlation is restricted during
the prediction of in vivo drug performance.
* *
In the initial phase of formulation deve lopment, l evel C correlations can be

useful when pilot formulations are being selected while waiver of an in vivo
bioequivalence study (biowaiver) is generally not possible.
4.3.3.4. Multiple Level C Correlations

Mutliple level represents the relationship between one or more desirable
pharmacokinetic parameters (C max, AUC, etc.) and concentration of drug
dissolved at different time point s of dissolution profile. If the correlation has
been established over the entire dissolution profile with one or more
pharmacokinetic parameters of interest, the m ultiple level C correlation can be
used to justify biowaivers.
It is important that a multiple l evel C correlation should be based on minimum
three dissolution time points including the early, middle, and late stages of the
dissolution profile. Also the development of a level A correlation occurs, when
multiple level C correlation is achieved at each time point at the same parameter
so that the effect on in vivo performance of any change in dissolution can be
evaluated.
4.3.3.5. Level D Correlation

The level D correlation is a semi -quantitative (qualitative analysis) and rank
order correlation. It is not a formal correlation and is not useful for regulatory
purpose, but can be used during the development or processing of a formulation.
4.3.4. In vitro Drug Dissolution Models

An in vitro test can be useful if it predicts the in vivo behaviour to such an extent
that there is no need to conduct an in vivo bioavailability test. Despite attempts to
standardise the test performance, the in vitro dissolution technique is still by no
means a perfect approach. The efforts are mainly aimed at mimicking the
environment offered by the biological system.
An ideal dissolution apparatus should have the following features:
1) It should be simple in design, easy to operate, and functional under variable
conditions.
2) The fabrication dimensions and positioning of all components should be
precisely specified and reproducible.
3) It should provide an easy way of introducing the dosage form into the
dissolution medium and once immersed, it should hold it in a regular and
reliable manner.
4) It should permit controlled variab le intensity of mild, uniform, and non -
turbulent liquid agitation.
5) It should provide minimum mechanical abrasion to the dosage form during
the test period so that the microenvironment surrounding the dissolving form
does not get disrupted.
6) It should maintain perfect sink conditions.
7) It should prevent or eliminate the evaporation of dissolution medium and
maintain it at a fixed temperature within a specified narrow range. The
*
dissolution apparatuses are mainly thermostatically controlled at 37C. *
8) It should provide ease of drawing samples for automatic or manual analysis

without interrupting the flow characteristics of the liquid.
9) It should facilitate good inter-laboratory agreement.
10) It should be sensitive enough to reveal the process changes and formul ation
differences, but still should yield repeatable results under identical conditions.
11) It should permit evaluation of disintegrating, non -disintegrating, dense or
floating tablets or capsules, and finely powdered drugs.
The dissolution apparatus has g radually evolved from a simple beaker type to a
highly versatile and fully automated instrument. The dissolution apparatuses can
be classified into two principal types based on the absence or presence of sink
conditions:
1) Closed-Compartment Apparatus: It i s a limited -volume apparatus that
operate under non -sink conditions. The dissolution fluid is restrained to the
container size. Example, beaker type apparatuses such as the rotating basket
and the rotating paddle apparatus.
2) Open-Compartment (Continuous Flow-Through) Apparatus: In this, the
dosage form is contained in a column which is brought in continuous contact
with fresh, flowing dissolution medium (perfect sink condition).
A third type of apparatus, called dialysis system , is used for very poorly
aqueous soluble drugs that require large volume of dissolution fluid for
maintenance of sink conditions.
3) Rotating Basket Apparatus (Apparatus 1): It was first described by
Pernarowski et al. It is a closed -compartment, beaker type apparatus
containing a cyl indrical glass vessel (one litre capacity) with hemispherical
bottom. This vessel is partially immersed in a water bath to maintain 37 C
temperature. A cylindrical basket of 22 mesh (meant to hold the dosage form)
is centrally placed in the vessel such tha t it is 2cm above the bottom of the
vessel. The basket is rotated by a variable speed motor through a shaft (figure
4.6a). While withdrawing samples, the basket should remain in motion.
4) Rotating Paddle Apparatus (Apparatus 2): It was first described by Levy
and Hayes . Its assembly is same as that for Apparatus 1, with the only
difference that instead of the rotating basket , a paddle is used as a stirrer
(figure 4.6b). The dosage form is sunk to the bottom of the vessel. Sinkers
prevent the floating of ca psules and other floatable forms. Such preparations
are attached with a small, loose, wire helix to prevent them from floating.
5) Reciprocating Cylinder Apparatus (Apparatus 3): It comprises of a set of
cylindrical flat -bottomed glass vessels fitted with rec iprocating cylinders
(figure 4.6c). It is used for dissolution testing of controlled-release bead-type
(pellet) formulations.
6) Flow-Through Cell Apparatus (Apparatus 4): It comprises of a reservoir
for the dissolution medium and a pump that forces the diss olution medium
through the cell holding the test sample (figure 4.6d). It is used in either:
i) Closed-mode where the fluid is re-circulated and is of fixed volume, or
ii) Open-mode where the fluids are continuously replenished.
* *
The test sample (table, capsule s, or granules) is placed in the vertically

mounted dissolution cell that allows the fresh solvent to be pumped in
(between 240 and 960ml/h) from the bottom ( figure 4.6d). This apparatus
has the following benefits:
i) It provides ease of maintaining sink cond itions during dissolution which
is required for drugs of limited aqueous solubility,
ii) It allows using large volume of dissolution fluid, and
iii) It provides feasibility for automation of apparatus.
7) Paddle Over Disc Apparatus (Apparatus 5): It comprises of a s ample
holder or disc that holds the product. The disc is placed at the bottom of
Apparatus 2 (rotating paddle apparatus; figure 4.6e) and the apparatus is
operated in the usual way. It is used for evaluating transdermal products.
(e) Apparatus 5 (f) Apparatus 6

(a) Apparatus 1 (b) Apparatus 2
Acrylic Spring Angled disc

Reciprocating
rod Teflon holder disc
cylinder
(c) Apparatus 3 (d) Apparatus 4 (g) Apparatus 7 – Reciprocating Holders
Figure 4.6: Official USP Dissolution Apparatus – (a) Apparatus 1 – Rotating Basket
Apparatus, (b) Apparatus 2 – Rotating Paddle Apparatus, (c) Apparatus 3 – Reciprocating
Cylinder Apparatus, (d) Apparatus 4 – Flow Through Cell Apparatus, (e) Apparatus 5 –
Paddle Over Disc Apparatus, (f) Apparatus 6 – Cylinder Apparatus, and (g) Apparatus 7 –
Reciprocating Disc Apparatus
8) Cylinder Apparatus (Apparatus 6): It is similar to Apparatus 1 ( figure

4.6f) with the only difference that the basket is replaced with a stainless steel
cylinder to hold the sample. The sample is mounted on an inert porous
cellulosic material and adhered to the c ylinder. It is used for evaluating
transdermal products.
9) Reciprocating Disc Apparatus (Apparatus 7):In this, the samples are placed
on disc-shaped holders (figure 4.6g) using inert porous cellulosic support that
vertically reciprocates via drive inside a glass container containing dissolution
medium. The test is carried out at 32 C temperature and at 30 cycles/min
reciprocating frequency. It is used for evaluating transdermal products and
non-disintegrating controlled-release oral preparations.
Table 4.2 lists the various types of dissolution apparatus and their applications:
Table 4.2: Compendial Dissolution Apparatus Types and Their Applications
Apparatus Name Drug Formulation Tested
Apparatus 1 Rotating basket Conventional tablets, chewable tablet s,
controlled-release formulations.
Apparatus 2 Rotating paddle Tablets, orally disintegrating tablets, chewable
tablets, capsules, controlled -release products,
suspensions.
* *
Apparatus 3 Reciprocating Controlled-release formulations, chewable

cylinder tablets.
Apparatus 4 Flow-through cell Formulations containing poorly soluble drugs,
powders and granules, microparticles, implants.
Apparatus 5 Paddle over disc Transdermal formulations.
Apparatus 6 Cylinder Transdermal formulations.
Apparatus 7 Reciprocating disc Controlled-release formulations (non -
disintegrating oral formulations and transdermal
formulations).
4.3.5. Applications of an IVIVC

IVIVC has the following applications:
1) In Drug Delivery Sys tem: Many rate controlling technologies ( e.g.,
diffusion-dissolution, matrix retardation, osmosis, etc.) are used as modified
release dosage forms. Such technologies are used for control ling and
prolonging the release of drugs when the drug is administered either orally or
parenterally. As a result , novel drug delivery systems (such as OROS,
liposomes, niosomes, pharmacosomes, microspheres, nanoparticles,
implants, in situ gelling system, organogels, transdermal drug delivery
systems, parenteral depots, etc .) have been developed as a substitute to
conventional dosage forms. The primary objective of these dosage forms is
to achieve zero-order, long term, pulsatile, or “on demand” delivery.
2) In Early Stages of Drug Delivery Technology Development: During drug
development, the most important stage is drug candidate selection, which is
based on the drug developability criteria that include physicochemical
properties of drug, results obtained from preformulation, and the p reliminary
studies (that involve several in vitro systems and in vivo animal models )
addressing efficacy and toxicity.
Since IVIVC explores the relationship between in vitro and in vivo
properties, IVIVC studies of the drug in animal models during drug
candidate selection represents the feasibili ty of drug delivery system for
given drug candidates. Such correlations should incorporate study designs
that include the study of several formulations of the modified-release dosage
forms and a rank order of release (fast/slow) of the formulations. At thi s
stage, better design and development efforts are expected in the future, even
though the formulations and methods used are not up to the mark.
3) In F ormulation Assessment – In vitro Dissolution: In product
development, a suitable dissolution method that can distinguish the
performance of formulations with different release rates in vitro and in vivo
is an important tool. On the basis of the nature of correlation, the dissolution
method can be changed . If the discriminatory in vitro method is validated,
further development in formulation relies on in vitro dissolution only.
4) Dissolution Specifications: IVIVC plays an important role in setting
specifications like modified-release dosage forms require dissolution testing
over multiple time points. Specificationtime points are generally selected in the
early, middle, and late stages of dissolution profiles. The range of dissolution
* *
specification in very rare cases exceeds 10% of the dissolution of the pivotal
clinical batch, if IVIV correlations are not present. But in the presence of
IVIVC, a range of specifications are applicable based on the prediction of
concentration-time profiles of test batches bioequivalent to the reference batch.
The dissolution specifications can be established in the presence of an IVIVC
that starts after obtaining the reference (pivotal clinical batch) dissolution
profile. Dissolution of batches with different dissolution properties (including
slowest and fastest batches) are used along with the IVIVC model, and the
concentration-time profiles should be deduced using a suitable convolution
method. Specifications should be established in such a way that all batches
with dissolution profiles between the fastest and slowest batches are
bioequivalent and less optimally bioequivalent to the reference batch.
5) Future Biowaivers: During drug development, various changes are made in
the formulations due to various reasons, for example, unexpected problems
in stability, development, availability of better materials, better processing
results, etc. If an established IVIVC is present, bioequivalence studies can be
avoided by using the dissolution profile from the changed formulation, and
predicting the in vivo concentration-time profile. Such a type of established
profile can be used as a surrogate for the in vivo bioequivalence study. This is
a cost-saving approach as it reduces drug development spending and speedy
implementation of post -approval changes. The post -approval changes may
range from minor (e.g., change in non-release-controlling excipient) to major
(e.g., site change, equipment change, change in manufacture method, etc.).
6) IVIVC in Parenteral Drug Delivery: IVIVC is also applicable to parenteral
dosage forms that are either injected or implanted (such as controlled -release
particulate syst ems, depot system, implants, etc.); but, the success rate of
such development of IVIVC for parenteral dosage forms is very low due to
multiple reasons. For establishing a correlation between the in vitro and in
vivo data, sophisticated modelling techniques are needed which are
unpredictable and unavoidable.
7) Biowaivers: A validated IVIVC procedure can be used for justification of a
biowaiver in filings of a Level 3 (or Type II in Europe) variation, either
during scale -up or during post -approval, and also for line extensions ( e.g.,
different dosage strengths). The primary condition before granting a
biowaiver is that the prediction of in vivo performance of the product with
the modified in vitro release rate should remain bioequivalent with the
originally test ed product (i.e., the new dissolution rate remains within the
IVIVC based biorelevant corridor). The FDA guidance puts forward the
following categories of biowaivers:
i) Biowaivers without an IVIVC,
ii) Biowaivers using an IVIVC: Non-narrow therapeutic index drugs,
iii) Biowaivers using an IVIVC: Narrow therapeutic index drugs,
iv) Biowaivers when in vitro dissolution is independent of dissolution test
conditions, and
v) Situations for which an IVIVC is not recommended for biowaivers.
* *
Biowaivers is granted for changes in manufacturing site, equipment,

manufacturing process, and formulation composition according to a
predictive and reliable IVIVC. These changes range from minor ones ( that
are not important to change product performance ) to major ones where an
IVIVC fails to validate the change for regulatory judgment.
8) Mapping: This process establishes a relation between Critic al
Manufacturing Variables (CMV, including formulation processes) and
equipment variables that can affect drug release from the product. The
process of ma pping specifies boundaries of in vitro dissolution profiles
according to the acceptable bioequivalent criteria.
The main goal is to develop such product specifications , which ensure
bioequivalence of future batches prepared within the limits of acceptabl e
dissolution specifications. Mapping-based dissolution specifications increase
the credibility of dissolution as a bioequivalent surrogate marker as well as
provide continuous assurance and predictability of product performance.
4.3.6. Limitations in the IVIVCArising from In Vivo Data

Following are the limitations of IVIVC:
1) In IVIVC, more than one dosage form is essential and sometimes intravenous
or solution becomes compulsory to calculate deconvolution.
2) In IVIVC, pharmacokinetics and absorption of drug should be linear . If the
pharmacokinetic processes depend on the fraction of dose reaching the
systemic circulation (or of the dose administered) or on the rate of
absorption, the formulation and simulation cannot be compared . Such non -
linearity is due to saturable absorption processes (active absorption),
induction or inhibition of metabolism, the rate/absorption dependent first past
effect, etc. These points should be analysed before establishing an IVIVC.
3) The limiting factor for IVIVC should not be absorption, because it is
convenient to attempt an IVIVC if solubility is not the limiting factor in
comparison to drug release. Drug release should depend on the formulation,
and should be slower than dissolution and absorption.
4.4. BIOEQUIVALENCE STUDIES

Several formulations of the same drug are designed in the same dose, in a similar
dosage form, and to be given by the same route. Substitution of one product for
another can be done if their therapeutic efficacy is same as the standard accepted.
The clinical performance of such drug products can be ensured by performing
bioequivalence studies.
Some important terms applicable to bioequivalence studies are defined below:
1) Equivalence: It is a relative term that compares drug products with respect
to a specific characteristic or function or to a defined set of standards.
2) Chemical Equivalence: It indicates that two or more drug products contain
*
the same amount of the same labelled chemical subst ance as an active ingredient.*
3) Pharmaceutical Equivalence: It indicates that two or more drug products

are identical in strength, quality, purity, content uniformity, and
disintegration and dissolution characteristics; however, they differ in their
excipients.
4) Bioequivalence: It is a relative term indicating that the drug substance in two
or more identical dosage forms, reaches the systemic circulation at the same
relative rate and extent, i.e., their plasma concentration -time profile s are
identical without any signifi cant statistical differences. Bioequivalence is
indicated if any statistically significant differences are observed in the
bioavailability of two or more drug products.
5) Therapeutic Equivalence: It indicates that two or m ore drug products
containing the same therapeutically active ingredient produce identical
pharmacological effects and can control the disease to the same extent.
4.4.2. Objectives for Bioequivalence Studies

If a new product is designed as a pharmaceutical equivalent or alternative to be a
substitute for an approved medicinal product, the equivalence with this product
should be shown or justified. Bioequivalence stud ies should be performed to
ensure the clinical performance of such drug products. Bioequivalence studies
are conducted if there is:
1) A risk of bio-inequivalence, and/or
2) A risk of pharmacotherapeutic failure or diminished clinical safety.
4.4.3. Types of Bioequivalence Studies

The two types of bioequivalence studies are:
1) In vivo Bioequivalence Studies: The sequence of criteria used for evaluating
the need for in vivo studies is as follows:
i) Oral immediate release products with systemic action:
a) These are indicated for serious conditions requiring a ssured
response,
b) They have a narrow therapeutic margin,
c) Pharmacokinetics complicated by absorption <70% or absorp tion
window, non-linear kinetics, pre-systemic elimination >70%,
d) They have unfavourable physi cochemical properties, e.g., low
solubility, metastable modifications, instability, etc.,
e) They have documented evidence for bioavailability problems, and
f) No relevant data is available, unless the applicant justifies that in
vivo study is not necessary.
ii) Non-oral immediate release products.
iii) Modified release products with systemic action.
In vivo bioequivalence studies are conducted as the bioavailability studies,
i.e., by the pharmacokinetic and pharmacodynamic methods.
2) In vitro Bioequivalence Studies: If the above criteria are not applicable,
*
comparative in vitro dissolution studies are sufficient. In vitro dissolution *
studies can be used instead of in vivo bioequivalence studies under the

following circumstances, which are termed biowaivers (or exemptions):
i) The drug product and the active substance it con tains differs only in
strength, provided that all the following conditions hold:
a) Pharmacokinetics are linear,
b) Qualitative composition is same,
c) Ratio between the active substance and the excipients is same, or (in
case of small strengths) the ratio between the excipients is same,
d) Both the products are produced by the same manufacturer at the
same production site,
e) A bioavailability or bioequivalence study has been conducted with
the original product, and
f) The in vitro dissolution rate is same under the same test conditions.
ii) The original manufacturer of the drug product has slightly reformulated
or has slightly changed the manufac turing method in ways that can be
argued to be inappropriate for bioavailability.
iii) The drug product meets all the following requirements:
a) The product is in the form of a solution or solubilised form (elixir,
syrup, tincture, etc.),
b) The product contains active ingredient in the same concentration as
the approved drug product, and
c) The product contains no such excipients that affect the ab sorption of
active ingredient.
iv) An acceptable IVIVC and the in vitro dissolution rate of the new product
is equivalent to that of the already approved medicinal product. Furthermore :
a) The product is intended for topical administration (cream, oint ment,
gel, etc.) to exert local effect,
b) The product is intended to be for oral administration but not to be
absorbed (antacid or radio-opaque medium), and
c) The product is inhaled as a gas or vapour.
It can be concluded from the above -mentioned criteria for drug prod ucts that
bioavailability and bioequivalence are self-evident.
4.4.4. Bioequivalence Study Parameters

If two products show similar rate and extent of drug release, they are said to be
bioequivalent, i.e., they will release the drug molecules in the same amount and
rate (speed). These characteristics of two drug products inside the body ( in vivo
situation) can be studied by the following parameters:
1) AUC: It is the area under plasma drug concentration -time curve. It provides
information on the amount of drug in plasma, i.e., extent of release.
2) Cmax: It is the maximum plasma drug concentration. It partially depends on
the rate of drug release from the formulation.
3) tmax: It is the time required to reach maximum plasma drug conce ntration. It
also depends on the rate of drug release from the formulation.
4) t1/2: It is the elimination half -life. It provides information on the elimination
*
of the drug from the body. *
Given below are some other parameters that are used in the bioequivalence study:
1) Normalised C max: Cmax and t max show significant intra -subject variability,
and hence normalised C max is used in some cases. It is calculated by the
following equation:
C
Normalised Cmax = max …..(15)
AUC
Studies indicate that normalised C max in comparison to C max shows less intra-
subject variability.
2) Mean Residence Time (MRT): It is the time a drug molecule spends in the
body before getting excreted. MRT can be calculated by first determining the
Area Under the Moment Curve (AUMC) by the following equation:
t
 t C  t C 
AUMC0-t    i i i-1 i-1 t i  t i-1  ..... (16)
i 1  2 
Equation (16) indicates that AUMC can be calculated in the same manner as
AUC, with the only difference that in the latter drug plasma concentration is
multiplied by time. AUMC 0-t is calculated from AUMC 0-, and then MRT is
calculated by the following equation:
AUMC0-
MRT  ..... (17)
AUC0-
3) Plasma Trough Fluctuation (%): It is used in the bioequivalence study of
sustained release formulations, which are designed such that they maintain
steady-state plasma drug concentration for extended time periods. Hence, in
bioequivalence study of sustained release formulations, the steady -state
plasma drug concentrations obtained from two drug products are compared.
Cmin is the lowest plasma drug concentration just before the next dose, and %
PTF is the % change in plasma drug concentration between two administered
doses. It is calculated by the following equation:
C max  C min
%PTF  ….. (18)
C average
Where, Caverage = Average plasma drug concentration during the dosingeriod.
p
4.4.5. Evaluation of Bioequivalence Data

The bioequivalence data obtained is evaluated by the following methods:
1) Analytical Method: This method for drug measurement should be validated
for accuracy, precision, sen sitivity, and specificity. Using multiple analytical
methods during a bioequivalence study does not give valid results as
different methods yield different values. Data should be presented in
tabulated as well as graphical form to ease evaluation. The plas ma drug
concentration-time curve should be available for each drug product and each
subject.
2) Pharmacokinetic Evaluation of the Data: For single -dose studies,
*
including a fasting study or a food intervention study, the pharmacokinetic *
analyses include calcu lating AUC to the last quantifiable concentration and
to infinity, tmax, Cmax, elimination rate constant ( k), elimination half-life (t1/2),
and other parameters for each subject. For multiple -dose studies,
pharmacokinetic analyses include calculating the s teady-state AUC, t max,
Cmin, Cmax, and % fluctuation for each subject. Proper statistical evaluation
should be performed on the estimated pharmacokinetic parameters.
3) Statistical Evaluation of the Data: Bioequivalence is determined by
comparing population averages of a bioequivalence metric, such as AUC and
Cmax. This approach is termed average bioequivalence , and involves
calculating a 90% confidence interval for the ratio of averages (population
geometric means) of the bioequivalence metrics for the test a nd reference
drug products. Bioequivalence can be established if the calculated confidence
interval falls within a prescribed bioequivalence limit (usually 80 -125% for
the ratio of the product averages).
Another approach is termed individual bioequivalenc e (proposed by the
FDA) that requires a replicate crossover design for estimating within -subject
variability for the test and reference drug products, and subject -by
formulation interaction.
Bioequivalence can be proved only if there is no statistical dif ference
between the bioavailability of the test product and the reference product.
There are many statistical approaches (or parametric tests) that are used for
comparing the bioavailability of drug from the test dosage form and from the
reference dosage f orm. Many of these approaches assume that the data are
distributed according to a normal distribution or “bell-shaped curve”.
The distribution of many biological parameters ( Cmax and AUC) has a longer
right tail than observed in a normal distribution. The true distribution of these
biological parameters may also be difficult to establish because small number
of subjects are used in a bioequivalence study. The distribution of data that
has been transformed to log values resembles a normal distribution compared
to the distribution of non -log-transformed data. Therefore, for determining
bioequivalence, log transformation of the bioavailability data ( e.g., Cmax,
AUC) is performed before statistical data evaluation.
The obtained bioequivalence data is statisti cally evaluated by the following
two methods:
i) Analysis of Variance (ANOVA): This is a statistical procedure in which
the data is tested for differences within and between treatment and control
groups. A bioequivalent product should not produce any signific ant
differences in the tested pharmacokinetic parameters.
AUC(0-t), AUC (0-∞), tmax, and Cmax obtained for each treatment or dosage
form are usually tested. Other metrics of bioavailability have also been
used for comparing the bioequivalence of two or more formulations. The
ANOVA may evaluate variability in subjects, treatmen t groups, study
period, formulation, and other variables, depending on the study design. If
the data shows large variability, the mean difference for each
* *
pharmacokinetic parameter (such as AUC) may be masked, and the

investigator might mistakenly conclude the two drug products to be
bioequivalent.
A statistical difference between the pharmacokinetic parameters obtained
from two or more drug products is statistically significant if there is a
probability of less than 1 in 20 times or 0.05 probability ( p>0.05) that
these results would have happened on the basis of chance alone.
Probability indicates the level of statistical significance. If p<0.05, the
differences between the two drug products are not considered statistically
significant.
ii) Two One -Sided Test s Procedure: This method is also termed as
confidence interval approach . This statistical method validates if the
bioavailability of the drug from the test formulation is too low or high
than that of the reference product. This approach aims to determine i f
there are large differences (i.e., > 20%) between the mean parameters.
The 90% confidence limits are estimated for the sample means.
The interval estimate is based on a Student’s t distribution of the data,
in which a 90% confidence interval about the ra tio of means of the two
drug products should be within ±20% for measuring the rate and extent
of drug bioavailability. For most drugs, a 20% difference in AUC or Cmax
between the two formulations would have no clinical significance. The
lower 90% confidence interval for the ratio of means cannot be less than
0.80, and the upper 90% confidence interval for the ratio of the means
cannot be more than 1.20. The 90% confidence interval is set at 80-125%
when log-transformed data are used. These confidence limits are termed
as the bioequivalence interval.
The 90% confidence interval is a function of sample size and study of
inter- and intra -subject variability. For analysing a single -dose, fasting
study, ANOVA is performed on the log -transformed AUC and Cmax
values. No statistical differences should be present between the mean
AUC and Cmax parameters for the test (generic) and reference drug
products. The 90% confidence intervals about the ratio of the means for
AUC and Cmax values of the test drug product should neither be less than
0.80 (80%) nor greater than 1.25 (125%) of that of the reference product
based on log-transformed data.
4.4.6. Significance of Bioequivalence Studies

Bioequivalence studies have the following significant features:
1) They provide a link between the pivotal and early clinical trial formulation.
2) They assist in the determination of therapeutic equivalence between the
pharmaceutical equivalence generic drug product and a corresponding
reference listed drug.
3) They provide information on product quality and performance when there are
changes in components, composition, and manufacture method after approval
*
of the drug product. *
4) They evaluate the absolute bioavailability of dosage form compared with

reference dosage forms.
5) They perform the dose proportionality study to determine if bioavailability
parameters are linear over the proposed dosage range.
6) They involve an intervention study to examine the effect of food and
concomitant medication.
7) They involve a dosage form p roportionality study to determine if equipotent
drug treatments administered at different dose strength of the market form
produce equivalent drug bioavailability.
8) They are needed as a result of changes in the formulation or manufacturing
processes.
4.5. SUMMARY
1) Bioavailability is the rate and extent of an administered dose of drug that
reaches the systemic circulation in unchanged form.
2) The drug dose given to the patient i s the administered dose . The dose
available to the patient is the bioavailable dose.
3) The absolute bioavailability of a given drug from a dosage form is the
fraction (or percentage) of the administered dose absorbed into the systemic
circulation in unchanged form.
4) Relative bioavailability can be determined by comparing the bioavailability
of a drug from a test dosage form with the same drug administered in a
standard dosage form.
5) Cmax is the peak that indicates the point at which the drug concentration in
plasma is maximum. It is usually expressed in mcg/ml.
6) tmax is the time the drug requires to reach peak concentration in plasma after
extravascular administration. It is expressed in hours.
7) AUC is the area under the plasma level -time curve that gives a me asure of
the extent of absorption or the amount of drug that reaches the systemic
circulation.
8) Urinary excretion studies , performed to assess bioavailability, rely on the
principle that the urinary excretion of unchanged drug and the plasma
concentration of drug are directly proportional.
9) (dXu/dt)max is the maximum urinary excretion rate, obtained from the peak of
plot between excretion rate and midpoint time of urine collection period.
10) (tu)max is the time for maximum excretion rate.
11) Xu is the cumulative amount of drug excreted in urine.
12) The method of acute pharmacologic response has a disadvantage that the
pharmacologic response may vary and thus an accurate correlation between
the measured response and drug available from the formulation cannot be
established.
* *
13) The therapeutic response method involves observing the clinical response
of a drug formulation in patients who are suffering from the disease for
which the drug is intended to be used.
14) If the solvent forms an integral part of the network struct ure and at least two
component crystal, it is termed as co-crystal.
15) If the solvent does not directly involve itself in the network (as in open
framework structures), it is termed as clathrate (or inclusion complex).
16) Water solubility of weak electrolytes a nd non -polar molecules is poor and
this can be improved by adding another solvent that alters the solvent
polarity. This process is termed cosolvency, and the solvent used for
increasing solubility is termed a cosolvent.
17) The system of cosolvent involves solvent blending in which the interfacial
tension between the aqueous solution and hydrophobic solute is reduced.
18) Hydrotrophy is the increase in water solubility due to the presence of large
amounts of additives.
19) Jack H. Shulman first used the term microemulsion in 1959.
20) The term solid dispersion refers to the dispersion of one or more active
ingredients in an inert carrier in a solid state, prepared by the melting (fusion)
method, solvent method, or fusion solvent method.
21) A eutectic mixture of a sparing ly water -soluble drug and a highly water -
soluble carrier is thermodynamically regarded as an intimately blended
physical mixture of two crystalline components.
22) Solid solution is a binary system comprising of a solid solute molecularly
dispersed in a solid solvent. Solid solutions are also termed molecular
dispersions or mixed crystals as the two components crystallise together in
a homogeneous one phase system.
23) In continuous solid solution , the drug and the carrier are miscible in all
proportions.
24) In discontinuous solid solution, solubility of each component in the other is
limited.
25) In substitutional crystalline solid solution, if the drug and carrier molecules
are almost of same size, the drug molecule substitutes for the carrier
molecules in its crystal lattice.
26) In interstitial crystalline solid solution, if the size of drug molecule is 40%
or less than the size of carrier molecules, the drug molecules occupy the
interstitial spaces in the crystal lattice of carrier molecules.
27) A glass solution is a hom ogenous system in which a glassy or a vitreous of
the carrier solubilises drug molecules in its matrix.
28) United State Pharmacopoeia (USP) defined IVIVC as “the establishment of
a rational relationship between a biological property or a parameter derived
from a biological property produced by a dosage form, and a
physicochemical property or characteristic of the same dosage form”.
* *
29) Food and Drug Administration (FDA) defined IVIVC as “a predictive

mathematical model describing the relationship between an in-vitro property
of a dosage form and an in-vivo response”.
30) The level A correlation of IVIVC has regulatory relevance and correlates
the entire in vitro and in vivo profiles.
31) The level B correlation of IVIVC relies on the principles of statistical
moment analysis.
32) The level C correlation relates one dissolution time point (t 50%, t 90%, etc.) to
one mean pharmacokinetic parameter (AUC, tmax, or Cmax).
33) Multiple l evel C correlations represent the relations hip between one or
more desirable pharmacokinetic parameters (C max, AUC, etc.) and
concentration of drug dissolved at different time points of dissolution profile.
34) The level D correlation is a semi-quantitative (qualitative analysis) and rank
order correlation.
35) Equivalence is a relative term that compares drug products with respect to a
specific characteristic or function or to a defined set of standards.
36) Chemical equivalence indicates that two or more drug products contain the
same amount of the same labelled chemical substance as an active ingredient.
37) Pharmaceutical equivalence indicates that two or more drug products are
identical in strength, quality, purity, content uniformity, and disintegration
and dissolution characteristics; however, they differ in their excipients.
38) Bioequivalence is a relative term indicating that the drug substance in two or
more identical dosage forms, reaches the systemic circulation at the same
relative rate and extent, i.e., their plasma concentration -time profiles are
identical without any significant statistical differences.
39) Therapeutic e quivalence indicates that two or more drug products
containing the same therapeutically active ingredient produce identical
pharmacological effects and can control the disease to the same extent.
40) Mean Residence Time (MRT) is the time a drug molecule spends in the
body before getting excreted.
41) Plasma Trough Fluctuation (%) is used in the bioequivalence study of
sustained release formulations, which are designed such that they maintain
steady-state plasma drug concentration for extended time periods.
42) Analytical method for drug measurement should be validated for accuracy,
precision, sensitivity, and specificity.
43) Average bioequivalence involves calculating a 90% confi dence interval for
the ratio of averages (population geometric means) of the bioequivalence
metrics for the test and reference drug products.
44) Individual bioequivalence requires a replicate crossover design for
estimating within-subject variability for the test and reference drug products,
and subject-by formulation interaction.
45) Analysis of Variance (ANOVA) is a statistical procedure in which the data
is tested for differences within and between treatment and control groups.
46) Two one-sided tests procedureis also termed asconfidence interval approach .
* *
4.6. EXERCISE

1) The relative bioavailability of a given drug from a dosage form is the fraction of the
administered dose absorbed into the systemic circulation in unchanged form.
2) (dXu/dt)max is the m aximum urinary excretion rate, obtained from the peak of plot
between excretion rate and midpoint time of urine collection period.
3) The acute pharmacological response method involves observing the clinical response
of a drug formulation in patients who are suffering from the disease for which the
drug is intended to be used.
4) In continuous solid solution, solubility of each component in the other is limited.
5) In substitutional crystalline solid solution , if the drug and carrier molecules are
almost of same size, the drug molecule substitutes for the carrier molecules in its
crystal lattice.
6) A glass solution is a homogenous system in which a glassy or a vitreous of the
carrier solubilises drug molecules in its matrix.
7) The level C correlation of IVIVC relie s on the principles of statistical moment
analysis.
8) Chemical equivalence indicates that two or more drug products contain the same
amount of the same labelled chemical substance as an active ingredient.
9) Statistical method for drug measurement should be va lidated for accuracy, precision,
sensitivity, and specificity.

10) The drug dose given to the patient is the _______ and the dose available to the
patient is the _______.
11) Cmax is usually expressed in _______.
12) _______ is the cumulative amount of drug excreted in urine.
13) Solid solutions are also termed _______ or _______ .
14) The level _______ correlation is a semi -quantitative (qualitative analysis) and rank
order correlation.
15) _______ indicates that two or more drug products containing the s ame
therapeutically active ingredient produce identical pharmacological effects and can
control the disease to the same extent.
16) _______ is the time a drug molecule spends in the body before getting excreted.
17) _______ requires a replicate crossover design f or estimating within -subject
variability for the test and reference drug products, and subject -by formulation
interaction.
18) _______ is a statistical procedure in which the data is tested for differences within
and between treatment and control groups.
Answers
10) Administered dose and bioavailable dose 11) mcg/ml
12) Xu 13) Molecular dispersions & mixed crystal
14) D 15) Therapeutic equivalence
16) Mean residence time 17) Individual bioequivalence
18) ANOVA
* *

1) Define bioavailability.
2) What is relative bioavailability?
3) Give the objectives of bioavailability studies.
4) Define tmax and Cmax.
5) How bioavailability can be enhanced by using salt form of drugs?
6) Give the level D correlation of In Vitro-In Vivo Correlation.
7) Enlist the dissolution models.
8) Give any one limitation of In Vitro-In Vivo Correlation.
9) Define bioequivalence.
10) What is MRT?

1) Discuss in detail about absolute bioavailability.
2) Give the significance of bioavailability studies.
3) Write about urinary excretion studies.
4) Explain the levels of In Vitro-In Vivo Correlation.
5) Give the applications of In Vitro-In Vivo Correlation.
6) Write in detail the objectives and types of bioequivalence studies.
7) Give the significance of bioequivalence studies.

1) Discuss about the measurement of bioavailability in detail.
2) Explain briefly the in vitro drug dissolution models.
3) Write an exhaustive note on evaluation of bioequivalence data.
* *
Pharmacokinetics (Chapter 5) 133
CHAPTER Pharmacokinetics
5
5.1. PHARMACOKINETICS
5.1.1. Definition and Introduction

Pharmacokinetics is the study of rate processes involved in absorption,
distribution, metabolism , and excretion of a drug . It includes the study of
biological, physiological and physicochemical factors that influence the transfer
processes of drugs in the body, and also influence the rate and extent of ADME
of those drugs in the body. In most of the cases, both pharmacologic al and
toxicological actions are considered with respect to the plasma concentration of
drugs. The study of pharmacokinetics helps the pharmacists in individualising
therapy for the patients.
The word pharmacokinetic has been originated from the Greek word pharmakon
which means drug and kinesis which means motion or change of rate. Thus ,
pharmacokinetics can also be defined as “ the kinetics of drug absorption,
distribution, metabolism and excretion (KADME) and their relationship
with the pharmacological, therapeutic or toxicological response in humans”.
Absorption is the process of movement of unchanged drug from the site of
administration to systemic circulation or to the site of measurement (i.e.,
plasma).
Distribution is the reversible transfer of a drug between the blood and the
extravascular fluids and tissues.
Elimination is the major process of drug removal from the body and termination
of its action. It is defined asthe irreversible loss of drug from the body
. Elimination
is a two-step process which involves biotransformation and excretion.
Metabolism (or biotransformation) of drugs is the chemical conversion of one
form to another.
Excretion is the process by which drugs and/or their metabolites are
irreversibly transferred from internal to external environment.
5.1.2. Objectives
The objectives of pharmacokinetics are as follows:
1) The main objective of pharmacokinetics is to measure the extent of drug
absorption, distribution, biotransformation, and excretion in intact, living
animals or humans. The information obtained from such studies is used to
determine the effect of alterations in dose, dosage regimen, administration
*
route, and physiologic state on drug accumulation and disposition. *
2) Pharmacokinetic profile of a drug can be inferred by analysing the changes in

concentration of a drug or its metabolite in body fluids in the given time
period.
3) At any time a fter drug administration in known dose or dosage regimen , its
plasma or urine concentration is the net result of its absorption, distribution,
metabolism, and exc retion. Therefore, the main purpose is to resolve the
observed kinetic profiles into their components.
4) By using appropriate experimental design, various types of models , and
kinetic data analysis, the contribution of absorption, distribution, metabolism,
and excretion can be individually isolated. After this , quantisation can be
achieved by maintaining material balance at all times.
5) It helps to understand the basics of a therapeutic drug monitoring service.
6) It is used to describe and understand how physiological changes affect the
pharmacokinetic profile of a drug in very young and elderly individuals.
7) In drug disposition, subsequent absorption of drugsoccurs, while in elimination,
an irreversible loss of drugoccurs from the body by metabolism and excreti on.
5.1.3. Applications
Following are the applications of pharmacokinetics:
1) Equations of pharmacokinetics are used to determine the drug bioavailability.
2) Using the principles of pharmacokinetics the dosing pattern of any d rug can
be fixed.
3) The principles of pharmacokinetics are also used to determine the dosing
pattern of controlled release dosage forms.
4) In case of kidney failure, the drug dose that should be given can be measured
by using pharmacokinetic principles.
5.2. PHARMACOKINETIC MODELS
5.2.1. Introduction
Many mathematical models are used to describe the rate processes of drug
absorption, distribution, metabolism, and excretion. These models are also used
to develop various equations which analyse drug concentrations in bod y with
respect to time. Since drug concentrations depend on time, the drug concentration
and time variables are termed as dependent and independent variables,
respectively. Direct measurement of pharmacokinetic parameters is not possible ;
thus, can be dete rmined through in vitro experiments using the given set of
dependent and i ndependent variables, which are collectively termed as data.
These data are used to design a pharmacokinetic model and test its validity.
Selection of pharmacokinetic model for data analysis relies on a hypothesis and

set of assumptions that describe the biological events mathematical ly. The
pharmacokineticist should be careful while depending on a pharmacokinetic
model for determining drug action. For data analysis, often a simple st
pharmacokinetic model and statistical methods are used to select the best model
that fits the data.
* *
If it is found that the model does not appropriate ly fit all the experimental
observations, a new and a more complex model (hypothesis) is devised and tested
for validity. It should always be known that the pharmacokinetic data should not
replace the clinical observations in patient and sound judg
ement by the clinician.
5.2.2. Compartment Models

The most traditional and common appro ach used for pharmacokinetic
characterisation of a drug is compartment analysis. In this approach, the body is
considered to be composed of various compartments that reversibly communicate
with each other. If e very organ, tissue or body fluid that can be e quilibrated with
the drug is considered as a compartment, a body can have numerous
compartments and mathematical description of such a model will be highly
complex. Therefore, tissues with similar drug distribution properties are pooled
to form a hypothetical compartment that is kinetically homogeneous.
These compartments are not real physiological or anatomic region , but are
hypothetical or virtual. These compartments represent the bio system and are
assumed to comprise of tissues with similar blood flow and affinity for drug. A
hypothetical model comprising of one, two or maximum three functional
compartments (arranged serially or parallelly to each other) is used to analyse the
kinetics of most of the drugs. It is also considered that the rate of drug movement
between compartments (i.e., entry and exit) follow first-order kinetics.
5.2.2.1. Types
Compartment models are divided into the following two categories based on their
arrangement, i.e., whether parallel to each other or in a series:
1) Mammillary Model: This is the most common compartment modelto be used
in pharmacokinetic studies. In this model , one or more peripheral
compartments are connected to a central compartment , consisting of plasma
and highly perfused tissues in which the drug undergoes rapid distribution. The
administered drug firstly reaches the central compartment and then distributes
to all the other compartments connected to the central compartment. Since the
major organs involved in drug elimination (i.e., kidney and liver) are found in
the central compartment, drug eliminationis assumed to occur from there.
Several types of compartment models have been shown in figure 5.1.
Compartment 1 is the plasma or central compartment, and compartments
2, 3 and 4 are the tissue or peripheral compartments . The letter K
indicates pharmacokinetic rate constants, and the numbers describe the
movement direction of drug between the compartments. For example, K12
indicates the rate constant with respect to drug move ment from compartment
1 to compartment 2.
Parameters of Mammillary Model
Model 1 (figure 5.1) can be described by two parameters, i.e., the volume of
compartment and the elimination rate constant (K). In model 4, the
pharmacokinetic parameters include the volume of compartment 1 and 2 and
*
the rate constants Ka, K13, K12 and K21 for total six parameters. *
During the study of these models, it is necessary to know whether drug

concentration data can be directly obtained from each compartment. For
models 3 and 4 (figure 5.1) the data obtained from compartment 2 cannot be
obtained easily since tissue sampling is a tedious task and drug concentration
may not be homogenous in different tissues.
The amount of drug in the tissue compartments can be mathematically
calculated if the data regarding the amount of drug absorbed and eliminated
per unit time is obtained by the sampling of compartment 1. Various
mathematical equations are used to describe these models and evaluate
various pharmacokinetic parameters.
Model 1: One-compartment open model I.V. injection
Model 2: One-compartment open model with first-order absorption
Ka K
1
Model 3: Two-compartment open model I.V. injection

K12
1 2
K21
K13
Model 4: Two-compartment open model with first order absorption
K12
Ka
1 2
K21
K13
Figure 5.1: Various Compartment Models

2) Catenary Model: In this model, the compartments are joined to each other
in a chain or series ( figure 5.2). However, this model is rarely preferred
owing to the drawback that it is not physiologically/anatomically comparable
because the various organs are directly linked to the blood compartment.
K01 K12 K23
1 2 3
K21 K32
Figure 5.2: Catenary Model
5.2.2.2. Advantages
Following are the advantages of compartment models:
1) They give a visual representation of variou s rate processes involved in drug
disposition.
2) They provide various rate constants required to describe these processes.
3) They help the pharmacokineticist to derive differential equations for various
rate processes so that changes in drug concentration in various compartments
can be described.
* *
4) They help in determining drug concentration -time profile under normal

physiological as well as pathological conditions.
5) They are also used in the development of dosage regimens.
5.2.2.3. Disadvantages
Following are the disadvantages of compartment models:
1) All the physiologic al functions or the anatomic structure of the species
cannot relate to the compartment model. Therefore, many assumptions have
to be made to interpret the data obtained from compartment models.
2) Exhaustive effort is required to find an exact model that can be used to
predict and describe the ADME of a drug under consideration.
3) Compartment modelling is based on curve fitting of plasma concentration
with multi-exponential mathematical equations.
4) This approach is not a common procedure and is applicable only for some
specific drugs.
5) The behaviour of drug in body can fit more than one c ompartmental model
depending on the drug administration route.
6) Sometimes, it becomes difficult to explain the differences between results
obtained from human and animal experiments using compartment models.
7) A variation in the study model can be observed.
5.2.3. Non-Compartmental Models

In non-compartmental analysis, assumption of specific compartment model is not
required; therefore it is also called as themodel-independent method. This method
of analysis can be used for any compartment model in which the drugs or
metabolites follow linear kinetics. This modelis based on statistical moment theory
in which experimental data is collected after the administration of a single dose of
drug.
AUC 
t 2  t 1 C1  C2 
Concentration × Time
2
AUM
C
Concentration
AUC 
t 4  t 3  C3
C1
2
AUC
Trapezoid
C2
C3
Time t1 t2 t3 t4
Figure 5.3: AUC and AUMC Plots
If the time course of drug concentration in plasma is considered as a statistical
distribution curve:
AUMC
MRT ..... (1)
AUC
* *
Where MRT = Mean residence time.

AUMC = Area under the first-moment curve.
AUC = Area under the zero-moment curve.
After plotting a graph between the product of plasma drug concentration and time
(C.t) versus time (t) from zero to infinity ( figure 5.3), the value of AUMC is
obtained, that can be mathematically represented as:

AUMC   C t dt ..... (2)
0
From a plot of plasma drug concentration versus time from zero to infinity, the
value of AUC is obtained, which is mathematically represented as:

AUC   C dt ……(3)
0
The values of AUMC and AUC can be calculated from their respective graphs
using the trapezoidal rule in which the curve is divided by a series of vertical
lines int o a number of trapezoids, followed by calculating the area of each
trapezoid separately and then adding them together.
MRT is the average amount of time a drug spends in the body before being
eliminated. Thus, it can be said that MRT is the statistical mo ment analogy of
half-life (t 1/2). Practically, MRT defines the time for the elimination of 63.2% of
the intravenous bolus dose. The values will always be greater when the drug is
administered in any form or through any route other than the intravenous bolus.
The non -compartmental model is mostly used to analyse the important

pharmacokinetic parameters , such as bioavailability, clearance, and apparent
volume of distribution. This method is also used to determine half -life, rate of
absorption, and first-order absorption rate constant of the drug.
5.2.3.1. Statistical Moment Theory

Compartment models can be used in pharmacokinetic analysis of drug
concentration in blood versus time data with the application of various
mathematical equ ations devised on the basis of certain assumptions. In non -
compartmental methods , such assumptions and mathematical equations are not
required because the non-compartmental models used for predicting absorption,
distribution, and elimination parameters rely on the statistical moment theory.
The statistical moment theory helps in studying the changes in drug

concentration in plasma and/or tissues with time . The total area under the
curve from time zero to infinity ( AUC0) is the zero momen t of a drug
concentration in plasma versus time curve.
The total area under the curve obtained from a plot of drug concentration
versus time is the zero moment of a drug concentration in a plasma -time profile.
The total area under the curve obtained from a plot of product of drug
concentration and time versus time (AUMC0) is the first moment of a plasma
concentration-time profile.
* *
Concentration
Time (hrs.)
Figure 5.4: Plots of Drug Concentration (g/ml) ( ) and Drug
Concentration-Time (g-hr/ml) (X) versus Time, during and after an
Hour Constant Rate Intravenous Infusion. The Area under the Drug
Conc. versus Time Plot to Infinity is AUC0, the Area under the Drug
Conc. Time versus Time Plot to Infinity is AUMC0.
During the calculation of various pharmacokinetic parameters, the plasma
concentration versus time data is plotted ( figure 5.4) and the AUC from t = 0 to
the last sampling time (t*) is calculated using the trapezoidal rule. The values of
(C) × (t) (i.e., product of concentration of drug and time) are plotted against time
(figure 5.4). The area under the (C) × (t) versus t plot from t = 0 to the last
sampling time (t*) is called the first moment of drug concentration with
respect to time (AUMC).
The area under the curve from t* to  for both the curves can be calculated using
 
appropriate equations to obtain AUC t* and AUMC t* . By adding these areas to
t*
AUC 0 t* and AUMC0 , the total areas under zero moment and first moment
curves, i.e., AUC  
0 and AUMC0 , respectively are obtained. The above obt ained
values are used to calculate various pharmacokinetic parameters.
5.2.3.2. Advantages
Following are the advantages of non-compartmental analysis:
1) The simple algebraic equations are used for easy derivation of the
pharmacokinetic parameters.
2) Similar mathematical treatment can be used for any drug or metabolite,
provided that they follow first-order kinetics.
3) A detailed description of drug disposition is not needed.
The main drawback of non-compartmental analysis is that it offers limited
information about the plasma drug concentration -time profile and most of the
time it deals with averages.
5.2.4. Physiological Models

Physiological pharmacokinetic models (or blood flow or perfusion models) rely
on known anatomic and physiological data. These models describe the data
kinetically considering that the blood flow distributes a drug to various body
* *
parts. Drug uptake into organs is de termined by binding the drug in these tissues.
In contradiction of the tissue volume of distribution, the actual tissue volume is
used. There are many tissue organs in the body, and each tissue volume and its
drug concentration should be estimated. The phy siological model potentially
predicts the accurate tissue drug concentration, which the compartment model
fails to do.
A physiological pharmacokinetic model, despite the limitation, provides a better
understanding of the changes in drug distribution from one animal species to
another by the physiological factors. Other major differences are:
1) Data fitting is not required in perfusion model. Drug concentrations in
various tissues depend on the organ tissue size, blood flow, and
experimentally determined dru g tissue-blood ratios (i.e., drug partition
between tissue and blood).
2) Due to some pathophysiological conditions, blood flow, tissue size, and the
drug tissue -blood ratio may vary. Effect of these variations on drug
distribution should be considered in physiological pharmacokinetic models.
3) Physiological pharmacokinetic models can be applied to several species,
and human data can be extrapolated with some drugs. In compartment
models, the volume of distribution is a mathematical concept with no
relation to t he blood volume and blood flow, thus extrapolation is not
possible. So far, many drugs (like digoxin, lidocaine, methotrexate, and
thiopental) have been described using perfusion models. It is not possible to
estimate the tissue levels of these drugs using compartment models;
however, they describe blood levels well. A perfusion model is shown in
figure 5.5.
QH
Heart
QM
Muscle
QS
SET
QR
RET
Ke QK
Kidney
Km QL
Liver
Figure 5.5: Pharmacokinetic Model of Drug Perfusion

In a perfusion model, the number of tissue compartments varies with drug-to-
drug. The tissues or organs in which the drugs have little or no penetration
(brain, bones, and other parts of CNS) are not considered. If each organ is
described separately with a differential equation, the model will turn out to
be very complex and mathematically difficult. A simpler and equally good
approach is that the tissues with similar blood perfusion properties are
* *
grouped into a single compartment. For example, distribution of lidocaine in

blood and various organs have been successfully described using perfusion
model; organs like lungs, liver, brain, and muscles were individually
described by differential equations, while other tissues were grouped as RET
(Rapidly Equilibrating Tissue) and SET (Slowly Equilibrating Tissue).
5.2.4.1. Types
The physiological models are categorised into the following two types:
1) Blood Flow Rate -Limited Models: These models are more popular and
common in use. They rely on the assumption that drug movement in a body
region is more rapid than its delivery rate to that region by the perfusing
blood. Ho wever, this assumption is applicable to low molecular weighed,
poorly-ionised and highly lipophilic drugs ( e.g., thiopental, lidocaine, etc.)
having high membrane permeability.
2) Membrane Permeation Rate -Limited Models: These models are more
complex. They are used for highly polar, ionised and charged drugs. In these
models, the cell membrane acts as a barrier for the drug that gradually
permeates by diffusion; thus, these models are also called diffusion-limited
models. Equations for these models are very co mplicated due to the time lag
in equilibration between the blood and the tissue.
5.2.4.2. Advantages
The physiological pharmacokinetic models have the following advantages over
the conventional compartment models:
1) Their mathematical treatment is direct.
2) They do not require data fitting; drug concentration in various body regions
can be estimated based on the organ or tissue volume, perfusion rate and
experimentally determined tissue-to-plasma partition coefficient.
3) They provide accurate description of drug concentration -time profile in any
organ or tissue, and thus, provide a better insight of drug distribution
characteristics in the body.
4) They can easily predict the effect of altered physiology or pathology on drug
distribution from the changes in various pharmacokinetic parameters that
correspond to actual physiologic and anatomic measures.
5) They can correlate data in several animal species and with some drugs, and
then can extrapolate to humans.
The physiological pharmacokinetic models have the following disadvantages:
1) Obtaining the experimental data using these models is a very exhaustive
process.
2) Most of them assume an average blood flow for individual subjects, h ence
making the prediction of individualised dosing is difficult.
3) The number of data points in these models is less than the pharmacokinetic
parameters to be assessed.
4) They can monitor drug concentration in body with much difficulty since
*
exhaustive data is required. *
5.3. ONE COMPARTMENT OPEN MODEL
5.3.1. Introduction
One-compartment open model is the simplest model that describes the body as a
single, kinetically homogeneous unit having no barriers to drug movement. Also
in this model, final distribution equilibrium between the drug in plasma and other
body fluids is rapidly attained and maintained all the times. This model applies
only to those drugs that rapidly distribute throughout the body.
The anatomical reference compartment is the plasma and the drug concentration
in plasma represents the drug concentration in all body tissues, i.e., any change in
plasma drug concentration indicates a proportional change in drug concentration
throughout the body. However, the model does not assume that the plasma drug
concentration is equal to drug concentration in other body tissues. The term open
indicates that the input ( absorption) and output (elimination) are unidirectional
and the drug can be eliminated from the body. Figure 5.6 shows s uch a one -
compartment model.
Ka Blood and KE Metabolism
Drug Other Body
Input Tissues Output Excretion
(Absorption) (Elimination)
Figure 5.6 Representation of One-Compartment Open Model showing
Input and Output Processes
The following assumptions help in deriving mathematical equations for one -
compartment open model:
1) Drug absorption from the absorption sit e may be explained by the first -order
kinetics.
Explanation: Most drugs are absorbe d by passive diffusion governed by the
first-order process, i.e., the drug absorption rate and the drug concentration at
absorption site are proportional. Drugs which are absorbed by active
transport, facilitated diffusion, etc., do not follow this assumption.
2) A drug on entering the systemic circulation rapidly distributes to other body
fluids and tissue, thus a dynamic equilibrium is rapidly achieved between the
drug in the blood and the drug in other tissues.
Explanation: The drug distributing to highly p erfused tissues, like heart,
lungs, liver, kidney, etc., can distribute instantaneously and attain
equilibrium between the drug levels in plasma and other body fluids and
tissues. If the drug is distributed to poorly perfused tissues, the time required
for its distribution and equilibrium is considered. Hence, this simple model is
not used for pharmacokinetic analysis of the data obtained with such drugs.
3) Any changes that occur in the drug plasma levels indicate proportional
changes in the tissue drug levels.
Explanation: A dynamic equilibrium exists between the drug concentration
in plasma and drug concentration in tissues. Thus, a change in plasma drug
level reflects a proportional change in the drug levels in tissues.
* *
4) Drug elimination from the body follo ws apparent first -order kinetics and its
rate constant (K) is known as an apparent first-order rate constant.
Explanation: Drug elimination from the body occurs by different processes,
like renal, biliary, biotransformation, excretion in the expired air, etc.
5.3.2. Intravenous Injection (Bolus)

If a drug that rapidly distributes in the body is given as a rapid intravenous
injection (i.e., I.V. bolus or slug), it takes 1 -3 minutes for complete circulation ,
and therefore the absorption rate is neglected in calculations. This model can be
represented as follows:
Blood and other KE
Body Tissues
The general expression for rate of drug presentation to the body is as follows:
dX
 Rate in (absorption)  Rate out (elimination) ..... (4)
dt
Since the rate in or absorption is absent, equation (4) becomes:
dX
  Rate out ..... (5)
dt
If the rate out or elimination follows first-order kinetics:
dX
  KE X ..... (6)
dt
Where, KE = First-order elimination rate constant.
X = Amount of drug in the body remaining to be eliminated at time (t).
The negative sign indicates that the drug is lost from the body. Now, the various
related pharmacokinetic parameters can be estimated.
Determination of Pharmacokinetic Parameters from Plasma Data after

Intravenous Injection (Bolus)
The different pharmacokinetic parameters that can be determined after
intravenous bolus administration are:
1) Elimination rate constant,
2) Elimination half-life,
3) Apparent volume of distribution,
4) Clearance,
5) Total clearance, and
6) Area under curve.
5.3.2.1. Elimination Rate Constant (KE)

A drug that follows one-compartment kinetics and is administered as a rapid I.V.
injection experiences decline in plasma drug concentration due to elimination of
drug from the body (and not due to distribution); this phase is called as
elimination phase.
* *
On integrating equation 6):

lnX = lnXo – KE t ..... (7)
Where, Xo = Amount of drug at time(t) = 0, i.e., the initial amount of druginjected.
Equation (7) can also be written in the exponential form as:
X  X 0 e KE t ..... (8)
Equation ( 8) indicates that disposition of a drug following one -compartment

kinetics is mono-exponential.
On transforming equation (8) into common logarithms (log base 10):
KE t
log X  log Xo  ..... (9)
2.303
Direct determination of the amount of drug in the body (X) is difficult, thus
advantage is taken of the fact that a constant relationship exists between the
plasma drug concentration (C, easily measurable) and X; thus:
X = Vd C ..... (10)
Where, V d = Apparent volume of distribution (a proportionality constant). It is a
pharmacokinetic parameter that allows the use of plasma drug concentration
instead of amount of drug in the body. Therefore, equation (6) becomes:
K t
log C  log C o   E ..... (11)
2.303
Where, Co = Plasma drug concentration immediately after I.V. injection.
Equation ( 11) is of a straight line and indicates that a semi -logarithmic plot of
log C versus t will be linear with y -intercept log C o. Elimination rate constant is
directly obtained from the slope of the line (figure 5.7b). It has units of min –1.
Thus, a linear plot is easily handled mathematically than a cur ve which will be
obtained from C versus t plot on a regular (Cartesian) graph paper
(figure 5.7a).
log Co
Log Concentration (log scale)
a) Regular Plot b) Semi-log Plot
log C2
K log C  log C
1 2
Concentration
Slope  E
2.303 2.303( t  t )
log C1
1 2
t1/2 t1/2
t1 t2
Time
Figure 5.7 (a) Cartesian Plot of a Drug that follows One-Compartment Kinetics and
given by Rapid I.V. Injection, and (b) Semi-logarithmic Plot for the Rate of
Elimination in a One-Compartment Model
* *
Thus, C o, K E, and t 1/2 can be readily obtained from log C versus t graph.
Elimination of drug from the body is the sum of urinary excretion, metabolism,
biliary excretion, pu lmonary excretion, and other involved mechanisms. Thus,
KE is an additive property of rate constants for each of these processes and is
called the overall elimination rate constant.
KE = Ke + Km + Kb + Kl + …… ..... (12)
If the number of rate constants involved and their values are known, evaluating
the fraction of drug eliminated by a particular route becomes easier. For
example, if a drug is eliminated by urinary excretion and metabolism, the
fraction of drug excreted unchanged in urine (F e) and the fraction of drug
metabolised (Fm) is given as:
Ke
Fe  ..... (13)
KE
K
Fm  m ..... (14)
KE
5.3.2.2. Elimination Half-Life (t1/2)

Elimination half -life (or biological half -life) is the oldest, best known
pharmacokinetic parameter, and was once considered the most important drug
characteristic. It is the time taken for the amount of drug in the body and the
concentration of drug in plasma to decline by one -half or 50% of its ini tial
value. It is expressed in hours or minutes. Half-life is related to elimination rate
constant as follows:
0.693
t1/ 2  ..... (15)
KE
Increased physiologic understanding of pharmacokinetics signifies that half -life

is a secondary parameter that depends on the primary parameters, like clearance
and apparent volume of distribution as follows:
0.693Vd
t1/ 2  ..... (16)
Cl T
5.3.2.3. Apparent Volume of Distribution (Vd)

Apparent volume of distribution is an independent pharmacokinetic drug
parameter. It is closely related to the physiological mechanisms of the body, thus
is considered a primary parameter.
On modifying equation (7):
Amountof drug in the body X
Vd   ..... (17)
Plasma drug concentration C
Vd is the measure of the extent of drug distribution and is expressed in litres.

For evaluating the V d of a drug in a best and simplest way, it is administered by
rapid I.V. injection and the following equation is used:
X o I.V.bolus dose
Vd   ..... (18)
Co Co
* *
Equation (1 8) can be used for drugs following one -compartment kinetics

because V d can be estimated when distribution equilibrium is achieved between
drug in plasma and drug in tissues. Such equilibrium is established for a drug that
follows one -compartment k inetics. A more useful non -compartmental method
that can be applied to many compartment models for estimating Vd is:
For drugs given as I.V. bolus:
Xo
Vd ( area )  ..... (19)
K E .AUC
For drugs administered extravascularly (e.v.):
F Xo
Vd ( area )  ..... (20)
K E .AUC
Where, X o = Dose administered and F = Fraction of drug absorbed in the

systemic circulation. F = 1, i.e., the drug achieves complete availability when the
drug is administered via intravenous route.
5.3.2.4. Clearance (ClR)

Many difficulties occur when elimination rate constant and half -life are applied
as pharmacokinetic parameters in an anatomical physiological context and as a
measure of drug elimination mechanisms. Clearance is the most impor tant
parameter in clinical drug applications and is useful in evaluating the mechanism
by which a drug is eliminated by the whole organism or by a particular organ.
Rate of elimination
Clearance  ..... (21)
dX/dt
Or, Cl  ...... (22)
C
Clearance is the theoretical volume of body fluid contain ing the drug (i.e.,
that fraction of apparent volume of distribution) from which the drug has
been completely removed in a given period of time.
5.3.2.5. Total Body Clearance (ClT)

Drug elimination from the body involves several processes that occur in kidneys,
liver, lungs, and other eliminating organs. Clearance at an individual organ level
is termed organ clearance. It can be estimated by dividing the elimination rate
by each organ with the drug concentration presented to it. Thus,
Rate of elimination by kidney
Renal Clearance Cl R  ..... (23)
C
Rate of elimination by liver
Hepatic Clearance Cl H  ......(24)
C
Rate of elimination by other organs
Other Organ Clearance Cl others  ..... (25)
C
Total body clearance (Cl T or total systemic clearance) is an additive property of
individual organ clearances. Hence,
ClT = ClR + ClH + Clothers ..... (26)
* *
5.3.2.6. Area Under Curve (AUC)

AUC can be determined by various methods. It can be accurately determined by
collecting adequate num ber of blood samples. For first -order kinetics, blood
collection up to 6 half -lives provides information about 98% of the process or
reaction. The following methods are used for determining AUC:
1) Planimeter: It is an instrument used for me chanically measuring the area of
plane figures drawn on rectilinear graph paper.
2) Cut and Weigh Method: In this method, the total area under the curve on
rectilinear graph paper is cut and accurately weighed on an analytical
balance. The obtained total weight is divided by the weight of unit area of the
same paper.
3) Trapezoidal Rule: In this method, the plasma drug concentration-time curve
is expressed as a series of straight lines, and thus AUC is divided into
trapezoids. Area of each trapezoid can be easi ly calculated, and sum of all
trapezoidal areas yield the true area under the curve (figure 5.8).
concentration
Plasma drug
Time
Figure 5.8: Graphical Representation for
Linear Trapezoidal Method to Estimate
Let us assume that f(t) is a function thatAUC
describes plasm a
drug concentration -
time curve, and (t) is a function that is linear between two su ccessive plasma
drug level-time points.
The AUC expressed by (t)   ( t )  dt  will be approximately t ( t )  dt
tn

tn
 0
t  0
tn
The integral t (t ) is expressed as the sum of total number of trapezoids into
0
which the curve is divided (n).

tn t1 t2 tn
t 0
(t )  dt   (t )  dt   (t )  dt ...   (t )  dt
t0 t0 t n 1
…..(27)
Since it is an area of trapezoid:

t1 t t
t 0 (t )  dt  1 2 0 (C0  C1 ) …..(28)
Where, C0 and C1 = Plasma drug concentration at time, t0 and t1.

Similarly,
tn t n  t n 1
t n 1
( t )  dt 
2
(Cn 1  Cn ) …..(29)
* *
Based on equation s (28) and (29), equation (27) can be expressed as:
tn t t t t t t
t 0 (t )  dt  1 2 0 (C0  C1 )  2 2 1 (C1  C2 ) ...  n 2 n 1 (Cn 1  Cn ) …..(30)
n 1
t11  t i
( t )  dt  
tn
Thus, t 0
i 0 2
(Ci  Ci 1 ) …..(31)
The true representation of AUC depends on t he number of blood samples

collected, i.e., more the number of blood samples collected better is the accuracy
in determining AUC.
5.3.3. Intravenous Infusion

Rapid I.V. injection is not suitable if the dr ug precipitates toxicity or if
maintenance of a stable concentration or amount of drug in the body is required.
In such a case, the drug ( e.g., several antibiotics, theophylline, procainamide,
etc.) is administered as I.V. infusion at a constant rate (zero -order). The duration
of constant rate infusion is much longer than the half -life of the drug; this is
contrary to the short duration of infusion of an I.V. bolus (few seconds).
Advantages of such a zero-order infusion of drugs are:

1) The rate of infusion can be easily controlled to fit individual patient’s needs.
2) Fluctuation in maxima and minima (peak and valley) plasma level is
prevented; this is desired when the drug has a narrow therapeutic index.
3) Other drugs, electrolytes and nutrients can be admin istered simultaneously
by the same infusion line in critically ill patients.
The model can be represented as follows:
Ro Blood and KE
Drug Other Body Elimination
Zero-order Tissues
infusion rate
The rate of change in the amount of drug in the body (dX/dt) at any time during
infusion, is the difference between the zero -order rate of drug infusion (R 0) and
the first-order rate of elimination (–KEX):
dX
 R o  KEX ..... (32)
dt
On integrating and rearranging equation (32):

X  R o (1  e K E t ) ..... (33)
KE
Since X = V dC, equation (3 3) can be trans formed into concen tration terms as
follows:
Ro
C (1  e K E t ) ..... (34)
K E Vd
C  R o (1  e K E t ) ..... (35)
Cl T
* *
Since the drug amount in body is zero at the beginning of constant rate infusion,
no elimination occurs. With time, the amount of drug in body gradually increases
(elimination rate < infusion rate) up to a point after which the plasma drug
concentration reaches a constant value, i.e., steady-state, plateau or infusion
equilibrium (elimination rate = infusion rate) (figure 5.9).
Infusion rate
(2RO)
CSS Steady-state
Infusion stopped
Infusion rate
(RO) When plotted on a semi-log
graph yields a straight line with
Infusion time (T) slope = –KE/2.303
Time
Figure 5.9: Plasma Concentration -Time Profile for a Drug given by Constant Rate I.V.
Infusion (the two curves indicate different infusion rates Ro and 2Ro for the same drug)
At steady -state, the rate of change of amount of drug in the body is zero, so
equation (38) becomes:
Zero = Ro – KE Xss
Or KE Xss = Ro ..... (36)
On transforming and rearranging equation (36) to concentration terms:
Ro R Infusion rate
Css =  o , i.e., ..... (37)
K E Vd ClT Clearance
Where, XSS and CSS = Amount of drug in the body and plasma drug concentration
at steady-state, respectively.
The value of K E (and t1/2) can be obtained from the slope of straight line obtained
from a semi-log plot (log C versus t) of plasma concentration-time data collected
at the time when the drug is no more infused. Alternatively, K E can be calculated
from the data collected during infusion to steady-state.
On substituting R0/ClT = CSS from equation (37) in equation (35):
C = CSS (1 – e–KEt) ..... (38)
On rearranging equation (38):
 CSS  C  K E t
 e ..... (39)
 CSS 
On transforming equation (39) into log form:
C  C  KEt
Log  SS  ..... (40)
 CSS  2.303
A semi -log plot of (C SS – C)/CSS versus t yields a straight line with slope of
–KE/2.303 (figure 5.10).
* *
C  C
log  ss 
 Css  K E
Slope 
2.303
t
Figure 5.10: Semi-log Plot to Compute KE from
Infusion Data Up to Steady-State
The time to reach steady-state concentration depends on the elimination half -life.
An increase in infusion rate will increase the plasma concentration attained at
steady-state. If since the beginning of infusion (t 1/2), n is the number of half -lives
passed, equation (38) can be written as:
C = CSS [1 − (t1/2)n] ..... (41)
The percent of C SS achieved at the end of each t 1/2 is the sum of C SS at previous
t1/2 and the concentration of drug remaining after a given t1/2.
Determination of Pharmacokinetic Parameters from Plasma Data after

Intravenous Infusion
The first-order elimination rate constant and elimination half-life can be obtained
from a semi-log plot of post-infusion concentration-time data. Equation (40) can
also be used for this purpose. Apparent volume of distribution and total systemic
clearance can be computed from steady -state concentrat ion and infusion rate
[equation (36)].
These two parameters can also be computed from the total area under the curve
till the end of infusion:
R oT R T
AUC   o  CssT ….. (42)
K E Vd ClT
Where, T = Infusion time
5.3.4. Extravascular Administration

On administering a drug via extravascular route (oral, intramuscular, rectal, etc.),
it should get absorbed to exert its therapeutic activity. Absorption rate can be
mathematically described as a zero -order or first -order process. A large number
of plasma concentration -time profiles can be described by one -compartment
model with first -order absorption and elimination. Under certain conditions,
absorption of some drugs is also described by zero -order (constant rate) kinet ics.
Differences between zero-order and first-order kinetics are shown in figure 5.11.
Zero-order absorption process has a constant absorp tion rate, and is independent
of Amount Remaining to be Absorbed (ARA). Its regular ARA versus t plot is
linear with slope equal to absorp tion rate while its semi -log plot shows an ever -
increasing gradient with time. On the other hand, first -order absorption process
shows a decline in the rate with ARA, i.e., absorption rate depend on ARA; its
regular plot is curviline ar and semi -log plot is a straight line with slope equal to
absorption rate constant.
* *
Log ARA
Zero-order
ARA
Zero-order
First-
order
First-order
Time Time
a) Cartesian Plot b) Semi-log Plot
Figure 5.11: Distinction between Zero-Order and First-Order Absorption
Processes. (a) Regular Plot and (b) Semi-log Plot of Amount of Drug
Remaining to be Absorbed (ARA) versus Time (t)
After extravascular administration, the rate of change in the drug amount in body
(dX/dt) is the difference between the rate of input (absorption, dX ev/dt) and rate
of output (elimination, dXE/dt).
dX/dt = Rate of absorption – Rate of elimination
dX dX ev dX E
  ..... (43)
dt dt dt
The plasma concentration -time profile of a drug following one -compartment
kinetics shows the absorption phase, post -absorption phase, and elimination
phase (figure 5.12).
Cmax
Absorption rate = Elimination rate
Post-absorption phase
Absorption
phase
Elimination phase
Time
Figure 5.12: Absorption and Elimination Phases of Plasma
Concentration-Time Profile Obtained After Extravascular
Administration of a Single Dose of Drug
During the absorption phase, the absorption rate > the elimination rate.
dX ev dX E
 ..... (44)
dt dt
At peak plasma concentration, the absorption rate = the elimination r ate, and
the change in amount of drug in the body is zero.
dX ev dX E
 ..... (45)
dt dt
During the post-absorption phase, some drug still remains to be absorbed at the
extravascular site, and the elimination rate > the absorption rate.
dX ev dX E
 ..... (46)
dt dt
After the completion of drug absorption , the absorption rate be comes zero and
the plasma level-time curve shows only the elimination phase.
* *
Zero-Order Absorption Model

This model is similar to the model for constant rate infusion.
Blood and
Drug at R0 KE
Other Body Elimination
e.v. site Zero-order Tissues
absorption
As in the case of several controlled drug delivery systems, the drug absorption
rate is constant and continues till the amount of drug at the absorption site ( e.g.,
GIT) gets depleted. The equations explaining the plasma concen tration-time
profile for constant rate intravenous infusion are also applicable to this model.
First-Order Absorption Model

A drug that enters the body by first -order absorption process gets distributed
according to one-compartment kinetics and gets eliminated by first-order process.
This model is depicted as follows:
Blood and
Drug at Ka KE
other Body Elimination
e.v. site First-order Tissues
absorption
The differential form of the equation (43) is:

dX
 Ka Xa  K EX ..... (47)
dt
Where,
Ka = First-order absorption rate constant
Xa = Amount of drug at the absorption sit
e remaining to be absorbed(i.e., ARA).
On integrating equation (47):
X
K a F Xo
(K a  K E )

e K E t  e K a t  ..... (48)
On transforming equation (48) into concentration terms:

C
K a F Xo
Vd (K a  K E )

e K E t  e K a t  ..... (49)
Where, F = Fraction of drug absorbed systemically after e.v. administration.
5.3.4.1. Determination of Pharmacokinetic Parameters- Cmax and tmax

At peak plasma concentration, th e absorption rate becomes equal to the
elimination rate, i.e., K aXa = K EX, and the rate of change in plasma drug
concentration (dC/dt) becomes zero. This rate can be obtained by differentiating
equation (49).
dC

K a F X0
dt Vd (K a  K E )
 
 K E e  K E t  K a e  K a t = zero ..... (50)
On simplifying equation (50):

Ka t
K E e K E t  K a e t ..... (51)
* *
On converting equation (51) to logarithmic form:

K t K t
log K E  E  log K a  a ..... (52)
2.303 2.303
Where, t = tmax
On rearranging equation (52):
2.303log (Ka /K E )
t max  ..... (53)
Ka  KE
Equation (53) shows that as K a becomes greater than K E, t max becomes smaller
since (Ka – KE) increases faster than log Ka/KE.
By substituting equation ( 53) in equation (4 9), the value of Cmax can be
obtained. However, this can be expressed in a simpler way as follows:
F X o K E t max
C max  e ..... (54)
Vd
It is shown that at Cmax, when Ka = KE, tmax = 1/KE. Hence, equation (54) reduces to:
F X o 1 0.37 F X o
C max  e  ..... (55)
Vd Vd
Since, FXo/Vd represents Co after I.V. bolus, the maximum plasma concentration
that can be attained after extravascular administration is 37% of the maximum
level that can be attained w ith I.V. bolus in the same dose. If bioavailability is
<100%, lower concentration will be attained.
5.3.4.2. Elimination Rate Constant

Elimination rate constant can be obtained from the elimination phase of the
plasma level-time profile. For the drugs administere d via extravascular route, the
absorption rate is much greater than the elimination rate (K at >> K Et). Hence,
e–Kat approaches zero faster than e –KEt. At this stage absorption completes, and
the change in plasma drug concentration depends only on eliminat ion rate and
equation (49) reduces to:
K a FX0
C e– K E t …..(56)
Vd (K a – K E )
On transforming equation (56) into log form:

K a FX0 K t
log C  – E …..(57)
Vd (K a – K E ) 2.303
A plot of log C versus t yields a straight line with slope –KE/2.303 (half-life can
be computed from KE). Elimination rate constant can also be deduced from
urinary excretion data.
5.3.4.3. Absorption Rate Constant (Ka)

Absorption rate constant can be computed by the method of residuals (or
feathering , peeling, and stripping ). This method is used in pharmacokine tics
to resolve a multi -exponential curve into its individual components. For a drug
* *
that follows one -compartment kinetics and is administered via extravascular

route, the plasma drug concentration is expressed by a bi -exponential
equation ( 58).
C
K a FX 0
Vd (K a – K E )

e –KEt – e –Ka t  …..(58)
If KaFX0/Vd(Ka–KE) = A, a hybrid constant:

C  A e –KE t – A e –Ka t …..(59)
During the elimination phase, when absorption is almost over, K a >> K E and the
value of second exponential e – K a t , approaches zero; whereas the first exponential
e – Ka t , approaches a finite value. Hence, equation (59) reduces to:

C  A e–KEt …..(60)
On taking logarithm of equation (60):

 K t
log C  logA – E …..(61)
2.303

Where, C = the back extrapolated plasma concentration value. A plot of log C
versus t yields a bi -exponential curve having a terminal linear phase with slope
–KE/2.303 ( figure 5.13). Back extrapolation of the straight line to time zero
yields y-intercept log A.
log K a FX0
Log A =
Vd (K a  K E )
Back extrapolated terminal

portion of curve (log C values)
Log C
True plasma concentration

values (log C values)
Residual curve (log Cr values)
K E
Slope = 2.303
K a
Lag time Slope = 2.303
t0
t
Figure 5.13: Plasma Concentration-time Profile after Oral Administration of a
Single Dose of a Drug. The Biexponential Curve has been Resolved into its Two
Components, i.e., Absorption and Elimination
By deducting true plasma concentration values, i.e., equation ( 59) from the
extrapolated plasma concentration values, i.e., equation (60), a series of residual
concentration values (C) is obtained:

(C – C)  Cτ  A e – Kat …..(62)
Ka t
log C  log A – …..(63)
*
2.303 *
A plot of log C  versus t yields a straight line with slope –Ka/2.303 and y -
intercept log A (figure 5.13). Absorption half-life can be obtained from Ka using
the relation 0.693/K a. Thus, the method of residuals allows resolution of the bi -
exponential plasma level -time curve into its two exponential components. This
method gives best results if the difference between K a and K E is large (K a/KE 
3). In some cases, K E obtained after I.V. bolus of the same drug is much larger
than the K a obtained by the method of residuals ( e.g., isoprenaline). If KE/Ka  3,
the terminal slope gives the value of K a (and not K E), while the slope of residual
line gives the v alue of K E (and not K a). This is called flip-flop phenomenon,
since the slopes of the two lines exchange their meanings.
Ideally, the extrapolated and the residual lines intersect each other on y -axis, i.e.,
at time t = zero, and there is no lag in absorp tion. However, if such an
intersection occurs at time > zero, it indicates time lag , which is the time
difference between drug administration and start of absorption. It is denoted by t 0
and represents the beginning of absorption.
The above method for Ka estimation is a curve-fitting method that is suitable for
drugs that get rapidly and completely absorbed and follow one -compartment
kinetics even when given intravenously. However, if the drug absorption is
affected by gastrointestinal motility or enzymati c degradation, and if the drug
shows multi-compartment characteristics after intravenous administration (which
is true for virtually all drugs), the value of K a computed by curve-fitting method
is invalid even if the drug has undergone absorption by first -order kinetics. The
so obtained K a is the best estimate of first -order disappearance of drug from the
GIT instead of the first-order appearance in the systemic circulation.
Wagner-Nelson Method for Estimation of Ka

In the method of residuals, absorption process is assumed to be of the first -order
kinetics. This assumption is valid for dosage forms in which absorption process
is the rate -determining step, i.e., in solutions and rapidly dissolving dosage
forms. In cases where drug release from the dosage form is the rate-limiting step,
the absorption process follows zero-order, mixed zero, and first-order kinetics, or
even more complex processes.
Assumptions
The Wagner-Nelson method of calculation does not require a model assump tion
concerning the absorption process. It does require the assumption that:
1) The body behaves as a single homogeneous compartment, and
2) The elimination process of drug follows first-order kinetics.
Wagner-Nelson method is a better alternative to curve -fitting method in the

estimation of Ka. It involves determination of Ka from % unabsorbed-time plots and
does not require the assumption of zero- or first-order absorption kinetics. When a
single dose of drug is administered via oral route, at any given time,the amount of
drug absorbed into the systemic circulation (XA) is the sum of the amount of drug in
body (X) and the amount of drug eliminated from the body (X E). Thus:
X A = X + XE ..... (64)
* *
The amount of drug i n the body is X = V dC. The amount of drug eliminated at
any time (t) can be calculated as:
XE = KE Vd [AUC]ot ..... (65)
On substituting the values of X and XE in equation (64):
XA = Vd C + KE Vd [AUC]ot ..... (66)
The total amount of drug absorbed into the systemic circulation from time zero to
infinity (XA) can be given as:
XA = Vd C + KE Vd [AUC]o ..... (67)
Since at t = , C = 0, equation (67) reduces to:
XA = KE Vd [AUC]o ..... (68)
Log % ARA
Slope = Ka /2.303
t
Figure 5.14: Semi-log Plot of %ARA versus t
according to Wagner-Nelson Method
The fraction of drug absorbed at time t is given as:
X A VdC  K E Vd [A  C]o C  K E [A  C]o
t t

 
 
..... (69)
XA K E Vd [A  C]o K E [A  C]o
% drug unabsorbed at any time is:

 X   C  K E A  Co t 
% ARA  1 A 100 1  
100 ..... (70)
 X A   K E [A  C]o 
The Wagner-Nelson method involves collecting blood samples after a single oral
dose at regular intervals of time till the entire amount of drug is elimi nated from
the body. The value of K E is obtained from a plot of log C versus t and [AUC] ot
and [AUC] o are obtained from a plot of C versus t. A semi -log plot of %
unabsorbed (i.e., %ARA) versus t yields a straight line with slope Ka/2.303
(figure 5.14). If a regular plot of the same is a straight line, the absorption is
assumed to be following zero-order kinetics.
Advantages of Wagner-Nelson Method

1) Absorption and elimination processes give accurate determinations of Ka.
2) Absorption process does not have to follow first-order kinetics.
3) The method can be used for investigating the absorption process.
Disadvantages of Wagner-Nelson Method

1) The method is only applicable to drugs having one-compartment characteristics.
2) Problem arises when a drug that obeys one-compartment model after extravascular
*
administration shows multi
-compartment characteristics after intravenous injection. *
Loo–Riegelman Method for Estimation of Ka

Loo-Reigelman method is used for determining the absorption rate constant (K a)
from plasma concentration -time profile of a drug that obeys two -compartment
model. This method utilises the % drug unabsorbed versus time plot. The amount
of drug (that obeys two-compartment model) absorbed after oral administration is
the sum of the amount of drug in the central compartment (X c), tissue
compartment (Xt), and the amount of drug eliminated:
Ab = Xc + Xt + X3 ..... (71)
The terms in equation (71) can also be expressed as:
Xc = Vc.C ..... (72)
Xt = Vt.Ct ..... (73)
t
X3  Vc .K13 C.dt  Vc K13AUC0 t
 ..... (74)
0
On substituting the values of Xc and X3 in equation (71):

Ab = Vc.C + Xt + VcK13[AUC]0t ..... (75)
On dividing equation (75) by Vc:
 C  t  K 13 AUC0
Ab X t
..... (76)
Vc Vc
On putting t = , equation (76) becomes:
Ab α
 0  0  K 13 AUC0  K 13 AUC0
t α
..... (77)
Vc
Where, Ab = Amount of drug absorbed from the dosage form. The r atio of Ab 
to the dose is the fraction of the dose absorbed (F).
F = Ab/X0 ..... (78)

The fraction of dose absorbed at any time in comparison to Ab can be obtained
by dividing equation (76) by equation (77):
Ab C  X t /Vc  K13AUC 0
t

K13AUC 0
..... (79)
Abα α
Ab C  X t /Vc  K13AUC 0
t

K13AUC 0
Or, ..... (80)
Abα α
Where, Ct = Xt/Vc = Apparent tissue concentration.

On plotting the fraction of dose unabsorbed [1 – (Ab/Ab)] against time, a
straight line with slope –K/2.303 is obtained. The value of abso rption rate
constant can be obtained from the slope ( figure 5.15). The C and K[AUC] 0t
values can be obtained from the plot of plasma drug level versus time. The values
for Ct can be computed by the Loo-Riegelman method using equation:
(Ct)tn = K12Ct  K12 C t n 1 1  e  K 21t  + (Ct)tn-1 e
 K 21t
..... (81)
K 21
* *
Where,
Ct = Apparent tissue concentration
tn = Time of sampling for sample n
tn-1 = Time of sampling for the sampling point proceeding sample n
(C)tn–1 = Concentration of drug at central compartment for sample n –1
C = Concentration difference at central compartment between two sampling
times
t = Time difference between two sampling times
Fraction of drug unabsorbed
[1 − (Ab/Abα)]
Slope = –Ka/2.303
Time (hours)
Figure 5.15: Plot of the Fraction of Drug Unabsorbed [1 – (Ab/Ab)]
versus Time used to determine the Absorption Rate Constant by Loo-
Riegelman Method for a Drug that Follows a Two-Compartment Model
For example, plasma is sampled at times t = 0 and 0.5 hours, and corresponding
concentrations of the drug in the central compartment are 0 and 2g/ml.
Then, (Ct)tn = 2g/ml and (C)tn –1 = 0, C = 2g/ml, t = 0.5 hours
If the drug is administered via intravenous route for estimating the distribution
and elimination rate constants, only then the absorption rate can be estimated by
Loo-Riegelman method. For drugs that cannot be given intravenously, K a cannot
be calculated by the Loo-Riegelman method.
Advantage of Loo Riegelman

The method has no limitation on the order of the absorption process.
Disadvantage of Loo Riegelman

The method requires the plasma concentration -time data after intravenous bolus
and oral administration to obtain all the necessary kinetic constants.
5.3.5. Methods of Elimination

If plasma level-time data is not availabl e, necessary information can be obtained
from urine data regarding elimination kinetics of a drug. The urine excretion
method has some advantages over other methods in the analysis of a
pharmacokinetic system:
1) This method is used when no accurate analytica l techniques are available for
accurate measurement of plasma drug concentration.
2) Urine samples can be conveniently collected while withdrawing blood
samples periodically cannot be conveniently collected.
* *
3) Urine drug concentration can be determined using a less sensitive analytical

method, while plasma drug concentration is determined using a highly
sensitive analytical technique. If urine drug concentrations are low, large
urine drug samples are readily assayed.
4) First order elimination, excretion and absor ption rate constants and fraction
excreted unchanged are calculated from the data obtained from urine drug
analysis. This data also helps in determining the first -order metabolism or
extra-renal excretion rate constant from the difference, KE – Ke = KM.
5) Urine drug data can be used for measuring absolute and relative
bioavailability without fitting the data to a complex mathematical model.
6) This method is non-invasive, therefore, assures better compliance.
7) When urine drug -time data is linked with plasma level -time data, renal
clearance of unchanged drug can be deduced from the following relation:
Total amount of drug which gets excreted unchanged
ClR 
Area under the plasma level - time curve
Total systemic clearance and non -renal clearance can also be estimated if V d
is known.
Disadvantages of Urine Method
1) Vd and ClT cannot be calculated only using urine data.
2) The method is less accurate than the plasma drug method because it gives
only a rough estimate of pharmacokinetic parameters.
3) If the drug release is very slow, the resulting urinary drug concentration will
be too low to be assessed with accuracy.
4) If the drug has a very long biological half -life, urine samples should be
collected for several days till the entire drug has excreted.
Criteria for Getting Valid Urinary Excretion Data

1) At least 10% of the drug administered should b e excreted unchanged in the
urine.
2) A specific analytic method should be used for determining unchanged drugs.
Further, metabolites should not interfere in this method.
3) After fasting overnight, the patient should administer 400ml water to
promote diuresis so that sufficient urine samples can be collected.
4) After administering 400ml water, the patient’s bladder should be completely
emptied after an hour and the urine sample is collected as a blank. The drug
should be first administered with 200ml water, follow ed by 200ml at hourly
intervals for the next 4 hours.
5) While collecting urine samples, the patients are advised to empty their
bladders completely.
6) Frequent sample should be collected to get a good curve.
7) While sampling, the exact time and volume of urine xcreted
e should be recorded.
8) The individual collection period should be less than the biological half-life of
the drug.
9) To collect more than 99% of the excreted drug, urine samples should be
collected for at least 7 half-lives of the drug.
10) Urine excretion ar te is also influenced by changes in urine pH and urine volume.
* *
5.3.5.1. Determination of Pharmacokinetic Parameters from Urine

Data after Intravenous Bolus Administration
The urine data is shown in table 5.1:
Table 5.1: Urinary Excretion Data Following I.V. Bolus of 100mg of a Drug
Observation Treatment of Data
Concentration Urine Mid- Amount Excretion Cumulative Amount
Urine Collected
Time of Urine
Collection (t)
of Unchanged Collection Point of Excreted Rate Amount Remaining

Volume of
(Hours)
Sample
Drug in Urine Interval Urine in Time (mg/H) Excreted to be

(ml)
(mcg/ml) dt Collection Interval dXu/dt (mg) Excreted

(or t) (t*) (mg) Xut Xu
dXu –Xut
(or Xu)
0 0 – – – – – – 0 66.7
1 0-2 140 250 2 1 35.0 17.5 35.0 31.7
2 2-4 150 100 2 3 15.0 7.5 50.0 16.7
3 4-6 90 80 2 5 7.2 3.6 57.2 9.5
4 6-8 200 20 2 7 4.0 2.0 61.2 5.5
5 8-12 310 10 4 10 3.1 0.8 64.3 2.4
6 12-24 600 04 12 18 2.4 0.2 –
66.7

Xu 
From ur inary excretion data, the first -order elimination (and excretion) rate
constants can be determined by using the following two methods:
Rate of Excretion Method
In the rate of excretion metho d, the rate of urinary drug excretion (dX u/dt) is
directly proportional to the amount of drug in the body (X):
dX u
X
dt
dX u
Or,  Ke X ...... (82)
dt
Where, K e = first -order urinary excretion rate constant (a proportionality
constant). According to the first-order disposition kinetics:
X = XO e-KEt ..... (83)
On substituting equation (83) in equation (82):
dX u
 K e X Oe  K E t ..... (84)
dt
Where, XO = Dose administered (intravenous bolus).
dX u
log  log K e X O  K E t log e
dt
1
But, log e = log 2.718 = 0.4343 =
2.303
dX u K t
 log  log K e XO  E ..... (85)
*
dt 2.303 *
Equation (85) is used to draw a semi -log plot of rate of excretion (log dX u/dt)
versus time (t) that gives a straight line with slope –KE/2.303 (figure 5.16). The
slope of such an excretion rate versus time plot is re lated to elimination rate
constant (KE), and not to excretion rate constant (Ke). However, the excretion rate
constant can be obtained from the intercept on y -axis (log K e Xo). From K E and
Ke, the elimination half -life and non -renal elimination rate consta nts can be
computed.
log Ke Xo
dX u
log slope = –KE/2.303
dt
Figure 5.16: Semi-log Plot of Excretion Rate versus Time of

Urine Collection Period for Computing Elimination Rate
Constant after I.V. Bolus Administration
This method has an advantage that it can be used for drugs with long half -lives.
For such drugs, urine samples are collected only for 3 -4 half -lives. Another
advantage is that it is not essential to collect all the urine samples because
collection of any two urine samples at two times yield points on the rate plot
through which a straight line can be constructed.
The disadvantage of this method is that while estimating K E a high degree of

fluctuation occurs in the drug e limination rate and the data obtained are so
scattered that estimation of drug half -life becomes difficult. These problems,
however, can be overcome by sigma-minus method.
Sigma-Minus Method
In sigma-minus method, from equation (84):
dX u KEt
 K e XO e
dt ……(86)
dX u KEt
Or,  K e XO e dt
dt
 dX u  K e XO  e
 KEt
dt
KEt
e
X u  K e XO I ..... (87)
 KE
Where, I = Integration constant whose value is obtained from initial conditions ,
i.e., t = 0, Xu = 0.
* *
Thus, equation (87) becomes:

eo
0  K e XO I
 KE

Or, I  K e X O e0 1
KE
 ...... (88)

e KEt K e X O
Xu KeXO 
 KE KE
K X
X u  e O (1 e K E t ) ..... (89)
KE
In equation (89), Xu = cumulative amount of drug excreted unchanged in urine at
any time (t). The time, after 6 -7 half -lives, approaches infinity and e KEt
becomes e  K E that further becomes zero, and hence the cumulative amou nt
excreted at infinity time ( X u ) can be given by the equation:
K eXO
Xu  ..... (90)
KE
X u  X u (1  e  K E t )
X u  X u  X u e  K E t
Or, X u  X u  X u e  K E t ..... (91)
K t
log (Xu  Xu )  log Xu  E ..... (92)
2.303
Where, X u – Xu = ARE at any given time = Amount remaining to be excreted at
the given time.
By using equation (92), KE can be calculated from the urinary excretion data; but
this data is applicable to a drug that follows one -compartment model and is
administered as I.V. bolus.

Data after Intravenous Infusion
The data obta ined during constant rate I.V. infusion is used for determining the
elimination rate constant. The equation describing the urinary excretion rate of
unchanged drug, when administered as I.V. bolus, is also applicable when it is
administered at constant rate as I.V. infusion. Thus:
dX u
 KeX ..... (93)
dt
If a drug is given as I.V. infusion, the amount of drug in the body (X) is given as:
R
X  0 (1  e  K E t ) ..... (94)
KE
* *

dX u K e R 0
 (1  e K E t ) ..... (95)
dt KE

KeR 0 t KeR 0
Xu   2 (1  e K E t ) ..... (96)
KE KE
If a drug is infused for a long period to attain steady -state in the plasma, the term
e  K E t approaches zero and equation (96) becomes:
K R t K R
Xu  e 0  e 2 0 ..... (97)
KE KE
On plotting the cumulative amount of drug excreted (X u) against time (t), a

curvilinear plot is obtained (figure 5.17). The linear portion of this plot has a
slope K e R0/KE. On extrapolatin g the linear portion to time axis, x -intercept is
obtained that is equal to 1/KE because when Xu = 0, t = 1/KE.
Xu
KeR 0
Slope
KE
1/KE
t
Figure 5.17: Regular Plot of Xu versus t during Constant
Rate I.V. Infusion

Data after Extravascular Administration
The equation for rate of excretion when the drug is administered via
extravascular route can be written as:
dX u
 KeX ..... (98)
dt
If a drug is administered intr avenously and absorbed by first -order process, X is

given as:
K FX

X  a O e K E t  e K a t
(Ka  K E )
 ..... (99)
dt

( Ka  KE )
e 
dX u K e K a FX O K E t
 e K a t  …..(100)
* *

K K FX O  1 eK E t K E eK a t 
Xu  e a     ..... (101)
KE  K a ( K E  K a ) K a (KE  K a ) 
At t = , equation (101) becomes:

K FX
X u  e O ..... (102)
KE

ARE  (Xu  XO ) 
Xu
(Ka  K E )

 K a e K E t  K E e K a t  ..... (103)
A semi-log graph of Xu  XO versus time (t) yields a bi -exponential curve. If K a >
KE, the slope of the terminal linear portion of the curve gives the valu e of K E for
the drug. Equation ( 104) is also used to estimate the absorption rate constant
(Ka) by the method of residuals.
The urinary excretion data obtained after administering a drug orally can be
analysed by the Wagner-Nelson method to evaluate K a by drawing %ARA plots.
In this method, urine samples are collected for sufficient time intervals to ensure
accurate estimation of K E (but should not be collected to time infinity). The
equation obtained relating % ARA with urine excretion rate is as follows:
 X   dX u / dt  K E X u 
% ARA  1  A  100  1   100 ..... (104)
 Xu   K E X u 
A semi-log plot of %ARA versus t yields a straight line with slope –Ka/2.303.
Accurate values of K a are obtained from urine excretion data of drugs having
slow rate of absorption. Collecti on of urine samples at very short intervals of
time is difficult for drugs having rapid absorption rate.
5.4. SUMMARY
1) Pharmacokinetics is the study of rate processes invol ved in absorption,
distribution, metabolism, and excretion of a drug.
2) The word pharmacokinetic has been originated from the Greek word pharmakon
which means drug and kinesis which means motion or change of rate.
3) Absorption is the process of movement of u nchanged drug from the site of
administration to systemic circulation or to the site of measurement (i.e.,
plasma).
4) Distribution is the reversible transfer of a drug between the blood and the
extravascular fluids and tissues.
5) Elimination is the major proce ss of drug removal from the body and
termination of its action. It is defined as the irreversible loss of drug from the
body.
* *
6) Metabolism (or biotransformation) of drugs is the chemical conversion of

one form to another.
7) Excretion is the process by which drugs and/or their metabolites are
irreversibly transferred from internal to external environment.
8) The most traditional and common approach used for pharmacokinetic
characterisation of a drug is compartment analysis.
9) In mammillary model, one or more peripheral compartments are connected
to a central compartment, consisting of plasma and highly perfused tissues in
which the drug undergoes rapid distribution.
10) In catenary model, the compartments are joined to each other in a chain or
series
11) In non-compartmental analysis, assumption of specific compartment model
is not required; therefore it is also called as the model-independent method.
12) MRT is the average amount of ti me a drug spends in the body before being
eliminated.
13) The values of AUMC and AUC can be calculated from their respective
graphs using the trapezoidal rule in which the curve is divided by a series of
vertical lines into a number of trapezoids, followed by calculating the area of
each trapezoid separately and then adding them together.
14) The total area under the curve from time zero to infinity (AUC 0) is the zero
moment of a drug concentration in plasma versus time curve.
15) The total area under the curve obtai ned from a plot of drug concentration
versus time is the zero moment of a drug concentration in a plasma -time
profile.
16) The total area under the curve obtained from a plot of product of drug
concentration and time versus time (AUMC 0) is the first moment of a
plasma concentration-time profile.
17) Physiological pharmacokinetic models (or blood flow or perfusion models)
rely on known anatomic and physiological data.
18) Blood flow rate-limited models rely on the assumption that drug movement
in a body region is more rapid than its delivery rate to that region by the
perfusing blood.
19) Membrane permeation rate -limited models are used for highly polar,
ionised and charged drugs.
20) One-compartment open model is the simplest model that de scribes the
body as a single, kinetically homogeneous unit having no barriers to drug
movement.
21) A drug that follows one -compartment kinetics and is administered as a rapid
I.V. injection experiences decline in plasma drug concentration due to
elimination of drug from the body (and not due to distribution); this phase is
called as elimination phase.
22) Elimination half-life (or biological half-life) is the time taken for the amount
of drug in the body and the concentration of drug in plasma to decline by
one-half or 50% of its initial value. It is expressed in hours or minutes.
* *
23) Vd is the measure of the extent of drug distribution and is expressed in litres.
24) Clearance is the theoretical volume of body fluid contain ing the drug (i.e.,
that fraction of appare nt volume of distribution) from which the drug has
been completely removed in a given period of time.
25) Clearance at an individual organ level is termed organ clearance.
26) Planimeter is an instrument used for mechanically measuring the area of
plane figures drawn on rectilinear graph paper.
27) Absorption rate constant can be computed by the method of residuals (or
feathering, peeling, and stripping).
28) Wagner-Nelson method involves determination of K a from % unabsorbed -
time plots and does not require the assumpti on of zero - or first -order
absorption kinetics.
29) Loo-Reigelman method is used for determining the absorption rate constant
(Ka) from plasma concentration -time profile of a drug that obeys two -
compartment model.
30) In the rate of excretion method , the rate of urinary drug excretion (dX u/dt)
is directly proportional to the amount of drug in the body (X).
5.5. EXERCISE
1) The most traditional and common approach used for pharmacokinetic
characterisation of a drug is non-compartment analysis.
2) In mammillary model, the compartments are joined to each other in a chain or series
3) The total area under the curve from time zero to infinity is the zero moment of a drug
concentration in plasma versus time curve.
4) The total area under the curve obtained from a plot of product of drug concentration
and time versus time is the first moment of a plasma concentration-time profile.
5) Membrane permeation models rely on known anatomic and physiological data.
6) Vd is the measure of the extent of drug distribution and is expressed in hours.

7) The word pharmacokinetic has been originated from the Greek word _______ and
_______ .
8) Non-compartmental analysis is also called as the _______ .
9) _______ are used for highly polar, ionised and charged drugs.
10) _______ is an instrument used for mechanically measuring the area of plane figures
drawn on rectilinear graph paper.
11) _______ can be computed by the method of residuals.
12) In the _______, the rate of urinary drug excretion is directly proportional to the
amount of drug in the body.
Answers
7) pharmakon and kinesis 8) Model-independent method
9) Membrane permeation rate-limited models 10) Planimeter
11)
*
Absorption rate constant 12) Rate of excretion method *

1) Define pharmacokinetics.
2) What are the applications of pharmacokinetics?
3) Give the advantages of pharmacokinetic models.
4) What are catenary model?
5) What is trapezoidal rule?
6) Give the advantages and disadvantages of non-compartment models.
7) Give the types of physiological models.
8) What is elimination half-life?
9) Give the advantages and disadvantages of Wagner Nelson method .
10) Define clearance.
11) What is flip-flop phenomenon?
12) Give the criteria for getting valid urinary excretion data.

1) Discuss the applications of pharmacokinetics.
2) Write about the types of compartment models.
3) Give the statistical moment theory.
4) Explain the physiological models.
5) Write in detail the calculation of elimination rate constant, elimin ation half -life,
AUC, and clearance for one compartment open model for IV bolus .
6) Give the method of residuals.
7) Briefly explain the sigma minus method.
8) Write a note on extravascular administration of one compartment open model.

1) Discuss about compartment models.
2) Explain briefly about non-compartment models.
3) Write an exhaustive note on methods of elimination.
4) How pharmacokinetic parameters can be evaluated from one compartment open
model for IV bolus?
* *
CHAPTER Multicompartment
6 Models
6.1. MULTI-COMPARTMENT MODELS
6.1.1. Introduction
One-compartment model describes the pharmacokinetic profile of many drugs. In
such case, instantaneous dist ribution equilibrium is assumed, and decline in the
amount of drug in the body with time is expressed as elimination by an equation
with a mono -exponential term. Instantaneous distribution, however, is not
possible for larger number of drugs and drug disposition is not mono -exponential
but bi- or multi-exponential. The reason for this is that the body is composed of a
heterogeneous group of tissues, each with different degree of blood flow, drug
affinity, and therefore different equilibration rates.
An ideal pharmacokinetic model should have a rate constant for each tissue
undergoing equilibrium (which is difficult mathematically). The best approach is
to combine the tissues together based on the similarity in their distribution
characteristics. Just like one -compartment models, drug disposition in multi -
compartment models is also assumed to follow first-order kinetics.
Multi-compartment drug characteristics can be understood by giving it as

intravenous bolus and observing the decline in plasma drug concentration with
time. The number of exponentials required to describe such a plasma level -time
profile d etermines the number of kinetically homogeneous compartments into
which a drug will distribute.
Multi-compartment models are based on the following assumptions:

1) Blood/plasma and the highly perfused tissues (like brain, hea rt, lung, liver,
and kidneys) form the central compartment.
2) Other tissues with similar distribution characteristics are combined together
to constitute peripheral compartment tissues based on the similarity in their
distribution characteristics.
3) Drugs admi nistered via intravenous route are directly introduced into the
central compartment.
4) Irreversible drug elimination by hepatic biotransformation or renal excretion
occurs from the central compartment.
5) Reversible distribution occurs between central and perip heral compartments,
and a finite time is required to attain distribution equilibrium.
6) After drug equilibration between central and peripheral compartments, drug
elimination follows first-order kinetics.
7) The rate processes involving drug passage in and out of individual
compartments follow first -order kinetics, and the plasma level -time curve is
* *
Multicompartment Models (Chapter 6) 169
described by the sum of series of exponential terms , each of which

corresponds to the first -order rate processes associated with a given
compartment.
8) Peripheral compartment cannot be accessed for direct measurement, and it is
not a site of drug elimination or clearance.
6.1.2. Two Compartment Open Model

Two-compartment models are the most common multi -compartment model. In
these models, the body tissues are classified into the following two categories:
1) Central Compartment or Compartment 1: This comprises of blood and
highly perfused tissues (like liver, lungs, kid neys, etc.) that rapidly attain
equilibrium with the drug. Elimination occurs from this compartment.
2) Peripheral or Tissue Compartment or Compartment 2: This comprises of
poorly perfused and slow equilibrating tissues (like muscles, skin, adipose,
etc.), and is a hybrid of several functional physiologic units.
Compartment in Series
Plasma Intracellular fluid
Ka VP K12 Vc
D C1 D1 C2 D2
Input K21
Output Kel
Metabolism Elimination
Compartment in Parallel
D1
VP C1 Metabolism
Ka Kel
D Output
Input
Vc C2 Elimination
D2
Figure 6.1: Open Two-Compartment Pharmacokinetic Model. (C1 -Concentration
in Compartment 1; D1 - Amount of Drug in Compartment 1; C2 -Concentration in
Compartment 2; D2 - Amount of Drug in Compartment 2).
Classification of a particular tissue (like brain) into central or peripheral
compartment depends on the drug ’s physicochemical properties. A drug that is
highly lipophilic can cross the BBB, and in this case brain will be included in the
central compartment. On the other hand, a polar drug ca nnot cross the BBB and
in this case brain in spite of being a highly perfused organ will be included in the
peripheral compartment.
The two-compartment model, depending on the compartment from whic h drug

elimination has occurred, is categorised into the following three types:
1) Two-compartment model with elimination from central compartment,
2) Two-compartment model with elimination from peripheral compartment, and
3) Two-compartment model with elimination from both the compartments.
If suitable information is no t present, elimination occurs from the central
compartment.
* *
Through one - and two -compartment models, the processes of pharmacokinetics

can be understood. But, these models are crude approximation of complex body
processes, and for being more precise and accurate, the body is sub-divided into a
larger number of compartments.
Dose
Blood
Brain
Carcass
Liver
Bile
Gut Tissue
Gut
Lumen
Faecal Matter
Kidney
Skin
Hair
Urine
Figure 6.2: Nine-Compartment Model of Mercury
In figure 6.2, a nine-compartment research model for mercury is shown. Separate
compartments are included for major organ systems. Input to the model is oral
(into the gut) and excretion occurs through hair, urine, and faeces. The model
studied had 18 compartments, nine for each of two chemical forms of mercury.
With the help of computers, the mathematical equations for 9 and 18
compartment models can be easily so lved. However, these large models behave
in a more complex manner than the one- or two-compartment models.
6.1.3. Two Compartment Open Model – I.V. Bolus

Two-compartment open model - I.V. bolus is shown below with elimination from
the central compartment:
1 K12 2
Central Peripheral
Compartment K21 Compartment
KE
After administering a drug (that follows two -compartment kinetics) as

intravenous bolus, the decline in plasma concentration is bi -exponential. This
indicates that there are two d isposition processes, i.e., distribution and
elimination, which are not obvious in a regular arithmetic plot but can be
identified in a semi-log plot of C versus t (figure 6.3).
* *
Log drug concentration

Initial rapid decline
(Distributive phase)
Slow terminal decline

(Elimination phase)
Central compartment
Peripheral compartment
Time
Figure 6.3: Changes in Drug Concentration in the Central (Plasma)
and the Peripheral Compartment after I.V. Bolus of a Drug that Fits
Two-Compartment Model
Drug concentration in the central compartment initially undergoes a rapid decline
due to drug distribution from the central to the peripheral compartment. This
phase is therefore termed the distributive phase . After a while, pseudo -
distribution equilibrium is attained between the two compartments, and then the
subsequent loss of drug from the central compartment becomes slow due to
elimination. This phase of slower rate process is termed the post-distributive or
elimination phase . Contrary to the central compartment, drug concentration in
the peripheral compartment f irst increases and reaches maximum in the
distribution phase. Thereafter, the drug concentration declines in the post -
distributive phase (figure 6.3).
Let K 12 and K 21 be the first -order distribution rate constants representing
reversible drug transfer bet ween the central and peripheral compartments; they
are termed microconstants or transfer constants. Let subscript c and p define
central and peripheral compartments, respectively. The rate of change in drug
concentration in the central compartment is expressed as:
dC c
 K 21Cp  K12Cc  K ECc …..(1)
dt
On extending the relationship X = VdC to equation (1):
dC c K 21X p K12Xc K E Xc
   ….. (2)
dt Vp Vc Vc
Where,
Xc and Xp = Amountsof drug in the central and peripheral
compartments, respectively.
Vc and Vp = Apparent volumes of the central and peripheral compartments, respectively.
The rate of change in drug concentration in the peripheral compartment is
expressed as:
dC p
 K12Cc  K 21Cp …..(3)
dt
K X K 21X p
 12 c  …..(4)
Vc Vp
* *
On integratin g equations ( 2) and ( 4), an equation forms that represent drug

concentration in the central and peripheral compartments at any given time (t):
X  K    t  K 21    t 
C c  0  21 e   e  …..(5)
Vc          
X  K   K  
C p  0  12 e t   12 e t  …..(6)
Vp          
Where, X0 = Intravenous bolus dose.
 and  = Hybrid first-order constants for the rapid distribution phase and the
slow elimination phase, respectively.
The hybrid first -order constant values ( and ) depend on the first -order ra te
constant values (K12, K 21 and K E). The mathematical relation ships between the
former and latter are given as:
 +  = K12 + K21 + KE …..(7)
 = K21KE …..(8)
Equation (6) can be simplified as:
Cc = Ae–t + Be–t …..(9)
Cc = Distribution exponent + Elimination exponent
Where, A and B = Hybrid constants for the two exponents (i.e., distribution and
elimination), and can be resolved graphically by the method of residuals.
X K    K 21   
A  0  21   C0   …..(10)
Vc        
X0  K 21     K 21   
B    C0   …..(11)
Vc         
Where, C0 = Plasma drug concentration after intravenous injection.
Determination of Pharmacokinetic Parameters after I.V. Bolus

Method of residuals can be used to determine all the parameters of equation (9).
Other parameters of the model, i.e., K 12, K 21, K E, etc., can be derived by proper
substitution of these values.
C0 = A + B …..(12)
C 0
KE  …..(13)
A  B
AB(  ) 2
K12  …..(14)
C 0 (A  B)
A  B
K 21  …..(15)
C0
For two -compartment model, K E = rate constant for drug elimination from the
central compartment and  = rate constant for drug elimination from the body.
Overall elimination t1/2 should be calculated from the value of .
* *
Area under the plasma concentration-time curve (AUC) can be obtained as:
A B
AUC   …..(16)
 
The apparent volume of central compartment (Vc) is given as:
X X0
Vd  0  …..(17)
C 0 K E AUC
The apparent volume of peripheral compartment (Vp) is given as:
VK
Vp  c 12 …..(18)
K 21
The apparent volume of distribution at steady
-state or equilibrium(Vd,ss) is given as:
Vd,ss = Vc + Vp …..(19)
It is also given as:
X0
Vd ,area  …..(20)
AUC
Total systemic clearance is given as:
ClT = Vd …..(21)
The pharmacokinetic parameters can be calculated from the urinary excretion
data as follows:
dX u
 K e Vc …..(22)
dt
An equation identical to equation ( 6) can be derived for rate of excretion of
unchanged drug in urine:
dX u
 K e Aet  K e Be t …..(23)
dt
Equation ( 23) can be resolved into individual exponents by the method of
residuals as described for plasma concentration-time data.
Renal clearance is given as:
ClR = KeVc …..(24)
Methods of Residuals
This method aids in resolving the bi-exponential disposition curve, obtained after
intravenous bolus of a drug that follows two -compartment model, into its
individual exponents. On rewriting equation (9):
Cc  Ae – t  Be –t …..(25)
The bi -exponential curve in figure 6.4 shows that the initial decline due to
distribution is more rapid than the terminal decline due to elimination, i.e., the
rate constant  >> , and hence the term e –t approaches zero more rapidly than
the does e–t. Thus, equation (25) reduces to:

C – Be –βt …..(26)
* *

 t
log C  log B – …..(27)
2.303

Where, C  Back extrapolated plasma concentration values.
Log C0
Log A
Log C
Log B
Back extrapolated terminal portion of elimination

phase (log C values)
True plasma concentration curve (log C values)
Residual line (log Cr values)
Slope = – /2.303
Slope = – /2.303
Time
Figure 6.4: Resolution of Bi-exponential Plasma Concentration-Time Curve
by the Method of Residuals for a Drug that Follows Two-Compartment
Kinetics on I.V. Bolus Administration
A semi-log plot of C versus t yields the terminal linear phase of the curve with
slope –/2.303. On back extrapolating to time zero, y -intercept log B is yielded
(figure 6.4). The t ½ for the elimination phase can be obtained from t ½ = 0.693/.
On subtracting extrapolated plasma concentration values of the elimination phase
[equation ( 26)] from the corresponding true plasma concentration values
[equation (25)], a series of residual concentration values (Cr) are obtained.

Cr  C – C  Ae– t …..(28)

at
log Cr  log A  …..(29)
2.303
A semi-log plot of C r versus t yields a straight line with slope –/2.303 and y -
intercept log A (figure 6.4).
6.1.4. Two Compartment Open Model – I.V. Infusion

Two-compartment open model – I.V. infusion is shown below with elimination
from the central compartment:
1 K1 2
RO
Central 2 Peripheral
1
KE
The plasma or central compartment concentration of a drug that follows two -

compartment model w hen administered at a constant rate (zero -order)
intravenous infusion is expressed as:
R 0   K E     t  K E    t 
C 1   e   e  .....(30)
Vc K E           
* *
The second and the third term in the bracket becomes zero at steady -state (i.e., at
time infinity), and equation (30) reduces to:
R0
Css  ..... (31)
Vc K E
On substituting VCKE = Vd in equation (31):

R R
Css  0  0 ..... (32)
Vdβ Cl T
The loading dose (X0,L) to obtain Css immediately at the start of infusion can be
calculated from equation (33):
R
X0, L  CssVc  0 ..... (33)
KE
6.1.5. Two Compartment Open Model - Extravascular

Administration
The two-compartment open model - extravascular administration is shown below
with elimination from the central compartment:
Ka 1 K12 2
Central Peripheral
KE
The rate of change in concentration of a drug that enters the body by first -order
absorption process and distributes according to the two-compartment model in
the central compartment is described by an absorption exponent, and the two
usual exponents describing drug disposition. The plasma concentration at any
time (t) is given by equation (34):
C = Ne–Kat + Le–t + Me–t ..... (34)
C = Absorption exponent + Distribution exponent + Elimination exponent
Where, Ka,  and  have usual meanings; while L, M and N are coefficients.
C oP
A
B 
Slope   ( 0.434  )
2.303

Slope   ( 0.434  )
2.303
Ka
Slope  
2.303
Time
Figure 6.5: Semi-log Plot of Plasma Drug Concentration versus Time for
Two Compartment Model after Extravascular Administration
* *
The above multi-exponentials can be determined by the method of residuals. In the


first step, elimination line ( figure 6.5 solid line, Slope   ) is subtracted
2.303
from the curve to result in a two -compartment residual plot having a post -

absorptive linear segment (figure 6.5, Slope   ) . On subtracting the
2.303
K
residuals from this plot, a line with lsope  a is produced (figure 6.5).
2.303
o
The intercepts A and B are same. C P is the y-axis intercept of absorption residual
line and is theoretically equals to A + B.
6.1.6. Kinetics of Multiple Dosing

A single dose of drug is not sufficient for treating certain diseases. So drugs are
repeated at specific time intervals for maintaining the therapeutic plasma drug
concentration throughout the treatment period. The objective of drug treatment is
to achieve and maintain plasma drug concentration within the therapeutic range
with minimum fluctuations. Thus, the objective for antibiotics is to maintain
plasma drug concentration above minimum effective concentration; the objective
for drugs with narrow therapeutic range is to maintain the plasma drug
concentrations below toxic levels.
When the first dose is administered in an oral multiple dosing regimen, the
plasma drug concentration increases and reaches the peak and then declines.
When the second d ose is administered, plasma drug concentration again
increases to reach a level higher than the first dose, and continues to increase till
a steady state plasma drug concentration is achieved (figure 6.6).
C
Time
Figure 6.6: Plasma Drug Concentration-Time Profile after Multiple
Dosing Regimen (C – Average Steady State Plasma Drug Concentration)
At steady state, the drug input and o utput will be equal. The amount of drug that
accumulates in the body relative to the first dose is calculated as follows:
1
R ( 0.693 ) / t 1
…..(35)
1 e /2
Where, R = Accumulation factor (depends on dosing interval and half-life).

 = Dosing interval.
Thus, smaller the /t1/2 ratio, greater will be the accumulation.
* *
From first-order kinetics, approximately 90% of steady state will be reached within
four half-lives. However, the time required to reach the steady state depends on the
) depends on:
drug’s half-life. Average steady state plasma drug concentration (C
1) Maintenance dos (X0),
2) Fraction of the dose absorbed (F),
3) Dosing interval (), and
4) Clearance (Cl).
F.X 0
C'  …..(36)
Cl  
1.44  F  X 0  t1/2
C' 
Vd  τ
AUC
 …..(37)

Where, Vd = Volume of distribution.
ACU = Area under the plasma drug concentration-time curve after single
maintenance dose.
From equations (36) and (37) maintenance dose can be calculated as:
C' Cl  
X0 
F
C' V  
 …..(38)
1.44  F  t1 / 2
6.1.7. Steady State Drug Levels

The time required to reach steady state depends on the drug’s half -life. If K a >>
KE, the drug reaches the plateau in approximately five half-lives; this is called the
plateau principle , which also means that K E determines the rate at which the
multiple dose steady state is reached. The time taken to reach steady state is not
influenced by the dose size, dosing interval, and number of doses.
Maximum and Minimum Concentration During Multiple Dosing

If n is the number of doses administered, C max and C min obtained o n multiple
dosing after the nth dose is given as:
1  e  nKe  
C n , max  C 0  K E  
….(39)
 1 e 
1  e  nKe    K E 
C n , min  C 0  K E  
e  C n , max e  K E  ….(40)
 1 e 
The maximum and minimum concentrations of drug in plasma at steady -state are
given by the following equations:
C0
Css, max  ….(41)
1  e K n 
C eK E
Css, min  0 K  Css, max e K E  ….(42)
1 e E
* *
Where, C 0 = concentration attained from instantaneous absorption and

distribution (obtained by extrapolating the elimination curve to time zero).
Equations (39) to ( 42) can be written in terms of amount of drug in the body,
and fraction of dose absorbed (F) should be considered.
Fluctuation is the ratio of C max to C min. Greater the r atio, greater is the
fluctuation. Like accumulation, fluctuation depends on dosing frequency, half -
life, and the absorption rate of drug. Greatest fluctuation is observed when the
drug is given as intravenous bolus; while small fluctuations are observed wh en
the drug is given extravascularly and it undergoes continuous absorption. The
average drug concentration at steady-state (Css,av) depends on:
1) Maintenance dose (Xo),
2) Fraction of dose absorbed (F),
3) Dosing interval (), and
4) Clearance (ClT) (or Vd and KE or t1/2) of the drug.
Average concentration and drug content on multiple dosing to steady state:
FX 0
Css, av  ….(43)
Cl T 
1.44FX0 AUC (single dose)
  ….(44)
Vd τ τ
Where, the coefficient 1.44 is the reciprocal of 0.693, while AUC is the are a
under the curve after a single maintenance dose. Equation ( 44) is used for
determining the maintenance dose of a drug to achieve a desired concentration.
Since X = VdC, the body content at steady state is given as:
1.44FX 0 t1 / 2
Xss, av  ….(45)

These average values are not arithmetic mean of C ss,max and C ss,min as the plasma
drug concentration undergoes exponential decline.
6.1.8. Calculation of Loading and Maintenance Doses and

their Significance in Clinical Settings
A drug is therapeutically active when it attains the desired steady state in five
half-lives. However if the drug is having a long half -life, it will take a longer
time. The plateau can be reached immediately after administering a dose t hat
yields the desired steady state before starting the maintenance doses. Such an
initial dose or first dose intended to be therapeutic is termed priming or
loading dose (X0,L). The equation used for evaluating loading dose is given as:
Css, av Vd
X 0, L  ….. (46)
F
The value of Cmax after extravascular administration is always smaller than that after
intravenous administration. Therefore, the loading dose is proportionally small. If
drugs have low therapeutic indices, the loading dose can be divided into smaller
doses which are to be given at various intervals before the first maintenance dose.
* *
If Vd is not known, the loading dose can be determined as:

X 0, L 1

X0 1 e 
K a 
1  e K E   ….. (47)
Equation (47) is true when Ka >> K E and the drug is distributed rapidly. If the
drug is given in travenously or if drug is absorbed rapidly, the absorption phase
can be neglected and equation (47) becomes:
X 0, L 1
  R ac
X0  
1  e K E 
….. (48)
Loading Dose
The ratio of loading dose to maintenance dose (X 0,L/X0) is termed dose ratio. As
a rule, when
1)  = t1/2, dose ratio = 2.0.
2)  > t1/2, dose ratio < 2.0.
3)  < t1/2, dose ratio > 2.0.
Figure 6.7 shows that if loading dose is not optimum, and is either too low or too
high, the steady state is attained in five half -lives just as when no loading dose is
given. Loading dose X0,L Maintenance doses X0
X0 X0 X0 X0
   
Dose ratio > 2
MSC
Dose ratio = 2
Dose ratio < 2
MEC
Dosing interval in hours

Figure 6.7: Schematic Representation of Plasma Concentration-Time Profiles that
Result when dose Ratio is Greater than 2.0, Equal to 2.0 and Smaller than 2.0
Maintenance of Drug within Therapeutic Range

Maintenance of drug concentration within the therapeutic window depends on:
1) Drug therapeutic index,
2) Drug half-life, and
3) Drug convenience.
In drugs with short half -life (< 2.5 hours) and narrow therapeutic i ndex, e.g.,
heparin, it is not easy to maintain such a level as the dosing frequency must
be less than the half -life. However, some drugs like penicillin with half -life =
0.9 hours may be given less frequently (in every 4 -6 hours) but the
maintenance dose should be larger so that the plasma concentration is
maintained above the minimum inhibitory level.
* *
A drug with intermediate half-life (3-8 hours) but low therapeutic index should
be given at intervals less than or equal to half -life. A drug having high
therapeutic index should be given at intervals between 1 to 3 half -lives. A drug
with long half -life (> 8 hours) should be giv en once every half -life. Steady state
in such a case can be rapidly attained by administering a loading dose.
A drug with very lon g half-life (> 24 hours), e.g., amlodipine, should be given
once in 24 hours.
6.2. SUMMARY
1) In multi-compartment models, blood/plasma and the highly perfused tissues
(like brain, heart, lung, liver, and kidneys) form the central compartment.
2) In multi -compartment models, drugs administered via intravenous route are
directly introduced into the central compartment.
3) In multi -compartment models, irreversible drug elimination by hepatic
biotransformation or renal excretion occurs from the central compartment.
4) In multi -compartment models, after drug equilibration between central and
peripheral compartments, drug elimination follows first-order kinetics.
5) In multi -compartment models, peripheral compartment c annot be accessed
for direct measurement, and it is not a site of drug elimination or clearance.
6) Central compartment or compartment 1 comprises of blood and highly
perfused tissues (like liver, lungs, kidneys, etc.) that rapidly attain
equilibrium with the drug. Elimination occurs from this compartment.
7) Peripheral or tissue compartment or compartment 2 comprises of poorly
perfused and slow equilibrating tissues (like muscles, skin, adipose, etc.), and
is a hybrid of several functional physiologic units.
8) Methods of residuals aids in resolving the bi -exponential disposition curve,
obtained after intravenous bolus of a drug that follows two -compartment
model, into its individual exponents.
9) The time required to reach steady state depends on the drug’s half-life.
10) If K a >> K E, the drug reaches the plateau in approximately five half -lives;
this is called the plateau principle, which also means that K E determines the
rate at which the multiple dose steady state is reached.
11) Fluctuation is the ratio of C max to C min. Greater the ratio, greater is the
fluctuation.
12) The ratio of loading dose to maintenance dose (X0,L/X0) is termed dose ratio.
13) A drug with intermediate half-life (3-8 hours) but low therapeutic index
should be given at intervals less than or equal to half-life.
14) A drug having high therapeutic index should be given at intervals between 1
to 3 half-lives.
15) A drug with long half-life (> 8 hours) should be given once every half-life.
16) A drug with very long half -life (> 24 hours), e.g., amlodipine, should be
given once in 24 hours.
* *
6.3. EXERCISE

1) In multi-compartment models, blood/plasma and the highly perfused tissues form the
peripheral compartment.
2) In multi -compartment models, after drug equilibration between central and
peripheral compartments, drug elimination follows zero-order kinetics.
3) Fluctuation is the ratio of C max to Cmin.
4) A drug with intermediate half-life (3-8 hours) but low therapeutic index should be
given once every half-life.
5) A drug with very long half-life (> 24 hours) should be given once in 24 hours.

6) In multi -compartment models, irreversible drug elimination by hepatic
biotransformation or renal excretion occurs from the _______.
7) _______ comprises of poorly perfused and slow equilibrating tissues, and is a hybrid
of several functional physiologic units.
8) The time required to reach steady state depends on the drug’s _______.
9) The ratio of loading dose to maintenance dose is termed _______.
10) A drug with long half-life, i.e., _______, should be given once every half-life.
Answers
4) False 5) True 6) Central compartment
7) Peripheral compartment 8) Half-life 9) Dose ratio
10) > 8 hours

1) What are loading and maintenance doses?
2) Give the advantages of pharmacokinetic models.
3) Give any two assumptions of multi-compartment models.
4) Draw the graph for method of residuals.

1) Discuss the steady state drug levels.
2) Write a short note on multi-compartment models.
3) Write about two compartment open model.

1) Discuss about kinetics of multiple dosing.
2) Write an exhaustive note on two compartment open model for IV bolus.
* *
CHAPTER Non-Linear
7 Pharmacokinetics
7.1. NON-LINEAR PHARMACOKINETICS

7.1.1. Introduction
At therapeutic doses, the change in amount of drug in the body or the change in
plasma drug concentration due to absorption, distribution, binding, metabolism,
or excret ion is proportional to the administered dose (whether a single dose or
multiple doses). In such a case, the rate processes follow first -order or linear
kinetics and the semi -log plots of C versus t for different doses are super -
imposable when corrected for dose administered. This is the principle of
superposition. Pharmacokinetic parameters, like F, K a, K E, V d, Cl R and Cl H
describe that the time -course of a drug in the body remains unaffected by the
dose, i.e., their pharmacokinetic is dose-independent.
In some cases, the rate process of a drug’s ADME depends on carrier or enzymes
that are substrate -specific, have definite capacities, and are susceptible to
saturation at high drug concentration. In these cases, first -order kinetics
transform into a mixture of first -order and zero -order rate processes and the
pharmacokinetic parameters alter with the administered dose. Pharmacokinetics
of such drugs is dose -dependent. Other synonymous terms are mixed -order, non-
linear, and capacity -limited kinetics. Drugs wit h such a pharmacokinetic profile
give rise to variability in pharmacological response.
7.1.2. Factors Causing Non-Linearity

Non-linearity can occur in drug absorption, distribution, metabolismand excretion.
Drug Absorption
Non-linearity in drug absorption occurs due to the following reasons:
1) When Absorption is Solubility or Dissolution Rate -Limited: When a
drug, e.g., griseofulvin, is administered at higher doses, a saturated solution
of the drug is formed in the GIT or at any other extravascular site and the
absorption rate attains a constant value.
2) When Absorption Involves Carrier -Mediated Transport Systems:
Saturation of the transport system at higher doses of vitamins, e.g.,
riboflavin, ascorbic acid, cyanocobalamin, etc., causes non-linearity.
3) When Pre -Systemic Gut Wall or Hepatic Metabolism Attains
Saturation: Saturation of pre -systemic metabolism of drugs, e.g.,
propranolol, hydralazine and verapamil, at high doses increases
bioavailability.
* *
Non-Linear Pharmacokinetics (Chapter 7) 183
The parameters, F, K a, C max and AUC, undergo a decrease in the first two cases
and undergo an increase in the last case. Changes in gastric emptying and
gastrointestinal blood flow and other physiological factors are some other causes
of non -linearity in drug absorption. Non -linearity in drug absorption is of little
consequence unless availability is significantly affected.
Drug Distribution
Non-linearity in distribution of drugs administered at high doses occurs due to
the following reasons:
1) Saturation of Binding Sites on Plasma Proteins: A fixed number of
binding sites are present on plasma proteins for a particular drug, e.g.,
phenylbutazone and naproxen; therefore, with increase in drug concentration,
the fraction of unbound drug also increases.
2) Saturation of Tissue Binding Sites: With large single bolus doses or
multiple dosing of drugs, e.g., thiopental and fentanyl, the tissue storage sites
undergo saturation.
In both the cases, the free plasma drug concentration increases; however, Vd
increases in the first case and decreases in the last one. Clearance also alters
depending on the extraction ratio of the drug. Clearance of a drug with high
Extraction Ratio ( ER) is increased due to saturation of binding sites. Unbound
clearance of dru gs with low ER is unaffected and the pharmacological response
increases.
Drug Metabolism
Non-linear kinetics of clinical importance is capacity -limited metabolism as
small changes in dose administered produce large variations in plasma drug
concentration at steady state. It is a major source of large inter -subject variability
in pharmacological response.
Non-linearity in drug metabolism occurs due to the following reasons:

1) Capacity-Limited Metabolism due to Enzyme and/or Cofactor
Saturation: This occurs in phenytoin, alcohol, theophylline, etc.
2) Enzyme Induction: The peak plasma concentration of carbamazepine
decreases on repetitive administration over a time period. Auto -induction
characterised in this case is also dose-dependent. Thus, enzyme induction is a
common cause of dose- and time-dependent kinetics.
Saturation of enzyme decreases Cl H and increases C ss; while, the reverse is true
for enzyme induction. Other causes of non -linearity in metabolism are saturation
of binding sites, inhibitory e ffect of the metabolite on enzyme, and pathological
situations such as hepatotoxicity and changes in hepatic blood flow.
Drug Excretion
The two active processes in renal excretion of a saturable drug are:
1) Active Tubular Secretion: After the saturation of carrier system, a decrease
*
in renal clearance of drug, e.g., penicillin G, occurs. *
2) Active Tubular Reabsorption:After the saturation of carriersystem, an increase

in renal clearance of drug, e.g., water-soluble vitamins and glucose, occurs.
Other sources of non -linearity in renal excretion are forced diuresis, changes in
urine pH, nephrotoxicity, and saturation of binding sites.
7.1.3. Michaelis-Menten Method of Estimating Parameters

Michaelis-Menten equation describes the kinetics of capacity-limited or saturable
processes:
dC Vmax C
  ….. (1)
dt K m  C
Where, –dC/dt = Rate of decline of drug concentration with time.
Vmax = Theoretical maximum rate of the process.
Km = Michaelis constant.
Three situations can be considered depending on the values of Km and C:
1) When Km = C: Under this situation, equation (1) reduces to:
dC Vmax
  ….. (2)
dt 2
The rate of process is one-half of its maximum rate (figure 7.1):
Vmax
Zero-order rate at high doses

dC Vmax
dt 2 Mixed-order rate at intermediate doses
First-order rate at low doses
Km
C
Figure 7.1: Plot of Michaelis-Menten Equation [Elimination Rate (dC/dt) versus
Concentration (C)]. Initially, the rate increases linearly (first-order) with con-
centration, becomes mixed-order at higher concentration and then reaches
maximum (Vmax) beyond which it proceeds at a constant rate (zero-order)
2) When Km >> C: Under this condition,Km + C = Km and equation (1) reduce to:
dC Vmax C
  ….. (3)
dt Km
Equation (3) is similar to the one describing first -order elimination of a
drug, where V max/Km = KE. This indicates that the drug concentration in body
resulting from usual dosage regimens of most drugs is below the K m value of
elimination process (however, phenytoin and alcohol are certain exceptions).
3) When Km << C: Under this condition, Km + C = C andequation (1) reduces to:
dC
  Vmax ….. (4)
dt
* *
Equation (4) is similar to the one describing a zero -order process, i.e., the
rate-process occurs at a constant rate (V max) and is independent of drug
concentration, e.g., metabolism of ethanol.
Estimation of Km and Vmax

The parame ters of capacity -limited processes, like metabolism, renal tubular
secretion, and biliary excretion, can be defined by assuming one -compartment
kinetics for the drug and that elimination involves a single capacity -limited
process. The K m and V max parameters can be evaluated from the plasma
concentration-time data obtained after intravenous bolus administration of a drug
with non-linear elimination characteristics. On re-writing equation (1):
dC Vmax C
  ….. (5)
dt K m  C
On integrating equation (5) followed by conversion to log base 10:
(C  C ) Vmax
log C  log C 0  0  ….. (6)
2.303K m 2.303 K m

Log C o
Log C
Log Co
Terminal linear portion of the curve

 Vmax
with slope
2.303 K m
t
Figure 7.2: Semi-log Plot of a Drug given as I.V. Bolus with Non-
Linear Elimination and that Fits One-Compartment Kinetics
A semi-log plot of C versus t yields a curve with a terminal linear portion having
slope –Vmax/2.303Km. Back extrapolation to time zero gives y -intercept of log

C 0 (figure 7.2). This line can be described by the following equation:
 Vmax
log C  log C 0  ….. (7)
2.303 K m
Equations (6) and (7) are similar at low plasma concentrations. On equating and
simplifying both the equations:

(C 0  C) C0
 log ….. (8)
2.303K m C0
The value of Km can be obtained from equation (8). V max can be calculated by
substituting the value of Km in the slope value.
Vmax and Km can also be obtained by determining the rate of change of plasma drug
concentration at different times and by using the reciprocal equation
of (1). Thus:
1 Km 1
  ….. (9)
dC dt Vmax C m Vmax
* *
Where, Cm = plasma concentration at midpoint of the sampling interval. A double

reciprocal plot or the Lineweaver-Burke plot of l/(dC/dt ) versus 1/Cm of
equation (9) yields a straight line with slope Km/Vmax and y-intercept 1/Vmax.
Lineweaver-Burke plot has a disadvantage that its points are clustered. Thus,
Hanes-Woolf plot (equation 10 ) and Woolf-Augustinsson-Hofstee plot
(equation 11) are used that give more reliable plots and uniformly scat
tered points.
Cm Km Cm
  ….. (10)
dC dt Vmax Vmax
dC dC dt K m
 Vmax  ….. (11)
dt Cm
Equations (10) and (11) are rearrangements of equation (9) . Equation (10) is
used to pl ot C m/(dC/dt) versus Cm. Equation (11) is used to plot dC/dt versus
(dC/dt)/Cm. The K m and V max parameters can be determined from the slopes and
y-intercepts of the two plots.
Km and Vmax from Steady State Concentration

When a drug is administered as a co nstant rate intravenous infusion or as a
multiple dose regimen, the steady state concentration (C ss ) is given in terms of
Dosing Rate (DR):
DR = CSSC1T …..(12)
Where, DR = R o when the drug is administered as zero -order intravenous
infusion; and DR = FX o/ when the drug is administered as multiple oral dosage
regimen (F = fraction bioavailable, Xo = oral dose, and  = dosing interval).
At steady state, the dosing rate and the decline rate of plasma drug concentration
are the same. If the decl ine (elimination) is due to a single capacity -limited
process (e.g., metabolism):
V C
DR  max ss …..(13)
K m  C ss
A plot of Css versus DR yields a hockey-stick shaped curve (figure 7.3):
Css
Km
Vmax
2 Vmax
DR
Figure 7.3: Curve for a Drug with Non-Linear Kinetics Obtained by
Plotting the Steady State Concentration versus Dosing Rates
Several measurements should be made at s teady state during dosage with
different doses in order to accurately define the characteristics of a curve.
* *
The parameters Km and Vmax can be graphically determined in the following ways:
1) Lineweaver-Burke Plot/Klotz Plot: On taking reciprocal of equation (13):
1 Km 1
  ….. (14)
DR Vmax C ss Vmax
Equation (14) is similar to equation (9). A plot of 1/DR versus 1/Css yields a
straight line with slope Km/Vmax and y-intercept 1/Vmax (figure 7.4):
1
DR Km
slope 
Vmax
1
Vmax
1
C ss
Figure 7.4: Lineweaver-Burke/Klotz Plot for Estimation of Km
and Vmax at Steady State Concentration of Drug
2) Direct Linear Plot : A pair of C ss, viz., Css,1 and C ss,2 obtained with two
different dosing rates (DR1 and DR 2) is plotted (figure 7.5). The points C ss,1
and DR 1 are joined to form a line, and the points C ss,2 and DR 2 are joined to
form a second line. The intersection point of these two lines is extrapolated
on DR axis to obtain Vmax and on x-axis to obtain Km.
DR
Vmax
DR1
DR2
Css,1 Css,2 Km
Css 0 Km
Figure 7.5: Direct Linear Plot for Estimation of Km and Vmax at
Steady-State Concentrations of a Drug Given at Different Dosing Rates
3) Graphical Method : This method of estimating K m and V max involves re -
arranging equation (13) to yield:
K DR
DR  Vmax  m ….. (15)
Css
A plot of DR versus DR/Css yields a straight line with slope –Km and y -
intercept V max. The pa rameters K m and V max can also be determined
numerically by setting-up simultaneous equations:
Vmax C ss,1
DR 1  ….. (16)
K m  C ss , 1
* *
Vmax C ss, 2
DR 2  ….. (17)
K m  C ss , 2
On combining equations (16) and (17):

DR 2  DR 1
Km  ….. (18)
DR 1 DR 2

C ss ,1 C ss, 2
After computing K m, it is substituted in any one of the two simultaneous

equations (i.e., 16 or 17) to obtain Vmax.
Km is much less variable than V max. Hence, if mean K m for a drug is known
from an earlier study, V max can be determined from a single measurement of
Css at any given dosing rate.
The parameters K m and V max estimated by assuming one -compartment system

and a single capacity -limited process have several limitations. More complex
equations result and the computed Km and Vmax will be larger when:
1) The drug is eliminated by more than one capacity-limited process.
2) The drug exhibits parallel capacity -limited and first -order elimination
processes.
3) The drug follows multi-compartment kinetics.
However, K m and V max obtained under su ch situations are little practically

applicable in dosage calculations.
Significance of Michaelis-Menten Constant

1) If the value of Km of a particular enzyme-substrate system is known, it can be
predicted whether the cell needs more enzymes or more substrate to speed up
the enzymatic reaction.
2) If a reaction with two similar substrates ( e.g., glucose and fructose) can be
catalysed by an enzyme in the cell, it will prefer the substrate for which the
enzyme has lower Km value.
3) The value of K m is an approximate measure of the concentration of substrate
of the enzyme in the cell where reaction is taking place. The enzymes
catalysing reactions with more concentrated substrates ( e.g., sucrose) have
relatively high K m value. In contrast, the enzymes that react with substrates
present in very low concentrations ( e.g., hormones) have comparatively
lower Km values for the substrates.
The Time Required to Attain 90% of the True Steady State Plasma
Concentration for Phenytoin
The time required to attain 90% of the true steady state plasma concentration for
phenytoin administered at different rates, where V = 50 L, V max = 500mg/day,
and K m = 4μg/ml ( = 4mg/l) is to be determined. From equation ( 19), the time
required to attain 90% of the steady state concentration can be determined for
various daily doses.
* *
K mV
(2.303Vmax – 0.9R )  t 0.9. …..(19)
Vmax – R 2
For 100mg dose:
(4mg L–1 )(50L)
–1 2
(2.303[500mg day –1 ] – 0.9[100mg day–1]) = 1.33 days.
(400mg day )
For 200mg dose:

(4mg L–1 )(50L)
(2.303[500mg day–1 ] – 0.9[200mg day–1]) = 2.16 days.
(300mg day–1 ) 2
For 300mg dose:

(4mg L–1 )(50L)
–1 2
(2.303[500mg day –1 ] – 0.9[300mg day–1]) = 4.41 days.
(200mg day )
For 400mg dose:

(4mg L–1 )(50L)
(2.303[500mg day–1 ] – 0.9[400mg day–1]) = 15.8 days.
(100mg day–1 ) 2
At times and doses above plasma phenytoin concentrations can be achieved by

making slight modifications in the steady state phenytoin concentration as
follows:
0.9(Cp)ss = 0.9KmR/(Vmax–R). …..(20)
From this data table 7.1 can be constructed.
If the value of K m was 5.7mg/l (and not 4mg/l), it can be said that since K m is the
numerator of equation ( 19), K m and the time to reach 90% of the steady state
phenytoin concentration are directly proportional. Similarly, 0.9(C p)ss is also
directly proportional to K m. Therefore, each value can be multiplied by the factor
(5.7/4.0) to obtain the figures given in table 7.2.
Table 7.1: Time (t0.9) to 90% of Steady State Plasma Concentration (C p)ss Level
as a Function of Daily Dose (R); K m = 4.0mg L–1
–1
R (mg day ) t0.9 (days) 0.9(Cp)ss (mg L–1)
100 1.33 0.90
200 2.16 2.40
300 4.41 5.10
400 15.8 14.4
Table 7.2: Time (t0.9) to 90% of Steady State Plasma Concentration (C p)ss Level
as a Function of Daily Dose (R); K m = 5.7 mg L–1
–1
R (mg day ) t0.9 (days) 0.9(Cp)ss (mg L–1)
100 1.90 1.28
200 3.08 3.42
300 6.28 7.27
400 22.5 20.5
* *
7.2. SUMMARY
1) After the saturation of carrier system, a decrease in renal clearance of drug,
e.g., penicillin G, occurs.
2) After the saturation of carrier system, an increase in renal clearance of drug,
e.g., water-soluble vitamins and glucose, occurs.
3) Michaelis-Menten equation describes the kinetics of capacity -limited or
saturable processes.
4) A double reciprocal plot or the Lineweaver-Burke plot of l/(dC/dt) versus
1/Cm yields a straight line with slope Km/Vmax and y-intercept 1/Vmax.
5) Lineweaver-Burke plot has a disadvantage that its points are clustered.
Thus, Hanes-Woolf plot and Woolf-Augustinsson-Hofstee plot are used
that give more reliable plots and uniformly scattered points.
7.3. EXERCISE
1) After the saturation of carrier system, a decrease in renal clearance of drug occurs.
2) After the saturation of carrier system, a decrease in renal clearance of drug occurs.
3) Michaelis-Menten equation describes the kinetics of capacity -limited or saturable
processes.

4) A double reciprocal plot or the Lineweaver -Burke plot of l/(dC/dt) versus 1/Cm
yields a straight line with slope _______and y-intercept _______.
5) _______ has a disadvantage that its points are clustered.
6) _______and ______are used that give more reliable plots and uniformly scat
tered points.
Answers
1) True 2) False 3) True 4) Km/Vmax and 1/Vmax
5) Lineweaver-Burke plot 6) Hanes-Woolf plot and Woolf-Augustinsson-Hofstee plot

1) What is the principle of superposition?
2) Write the Michaelis-Menten equation.
3) What is the equation when K m >> C?
4) Give the significance of Michaelis-Menten equation.

1) Give the graphical determination of parameters K m and Vmax.
2) What are the factors causing non-linearity?

1) Discuss Michaelis-Menten equation.
2) Discuss briefly about non-linear pharmacokinetics.
* *
Index 191
Index
A L
Absorption of Drugs from Non per Oral Extra- Levels of IVIVC, 116
Vascular Routes, 31 Level A Correlation, 116
Apparent Volume of Distribution, 52, 145 Level B Correlation, 116
Area Under Curve, 147 Level C Correlation, 116
Level D Correlation, 117
B Loading and Maintenance Doses, 178
Binding of Drugs, 53 Loo –Riegelman Method, 157
Binding of Drugs to Blood Cells, 56
Bioavailability, 100, 128 M
Bioequivalence, 123, 130 Measurement of Bioavailability, 103
Bioequivalence Study Parameters, 124 Mechanisms of Drug Absorption, 10, 11, 12
Biopharmaceutics, 9, 40 Methods of Residuals, 173, 180
Applications, 9 Methods to Enhance the Dissolution Rates and
Bioavailability of Poorly Soluble Drugs,
C 108
Cell Drinking, 16, 41 Michaelis Menten Equation, 184
Clearance, 146 Multi-compartment models, 168
Clearance and Renal Clearance, 87
Clinical Significance of Protein Binding of N
Drugs, 64 Non-Compartmental Models, 137
Compartment Models, 135 Non-Linear Pharmacokinetics, 182
Catenary Model, 136, 165, 166 Non-Renal Routes of Drug Excretion, 93
D O
Distribution, 45, 65 One Compartment Model after IV Drug
Factors Affecting Drug Distribution, 51 Administration, 190
Drug Metabolism, 67 One-compartment open model, 142, 165
E P
Elimination Half-Life, 145 Pharmacokinetics, 133, 164
Elimination Rate Constant, 143 Phase I Metabolic Pathways, 70
Evaluation of Bioequivalence Data, 125 Phase II Metabolic Pathways, 82
Physiological Barriers, 48
F Placental Barrier, 50
Factors Affecting Protein-Drug Binding, 59 Physiological Models, 139
Factors Affecting Renal Excretion of Drugs, Advantages, 141
91 Disadvantages, 141
Factors Influencing Drug Absorption, 16 Types, 141
Pharmaceutical Factors, 26
Physicochemical Factors, 16 R
Fick’s first law of diffusion, 12, 40, 43 Rate of Excretion Method, 160
Regulatory Requirements for Bioequivalence
I Studies
In vitro Drug Dissolution Models, 117 Review, 132, 167, 181
In Vitro-In Vivo Correlation, 115 Renal Excretion of Drugs, 89
K S
Kinetics of Multiple Dosing, 176 Sigma-Minus Method, 161
Kinetics of Protein Binding, 62 Statistical Moment Theory, 138
Steady State Drug Levels, 177
* *
Bibliography
 Chatwal G.R., Biopharmaceutics and Pharmacokinetics, Himalaya Pub.
 Brahmankar D.M., Jaiswal Sunil B. , Biopharmaceutics and
Pharmacokinetics A Treatise, Vallabh Prakashan.
 Venkateswarlu V., Khar Roop K., Biopharmaceutics and Pharmacokinetics,
PharmaMed Press.
 Kulkarni J.S., Pawar A.P., Shedbalkar V.P., Biopharmaceutics and
Pharmacokinetics, CBS Publishers.
 Madan P.L., Biopharmaceutics and Pharmacokinetics, Jaypee Brothers.
 Gibaldi Milo, Biopharmaceutics and Clinical Pharmacokinetics , 4 th Edition,
PharmaMed Press.
 Notari Robert E., Biopharmaceutics and Clinical Pharmacokinetics: An
Introduction, 4th Edition, Marcel Dekker Inc.
 Kar Ashutosh, Essentials of Biopharmaceutics And Pharmacokinetics ,
Elseveir.
 Shargel Leon, Yu Andrew, Wu-Pong Susanna, Applied Biopharmaceutics &
Pharmacokinetics, 6th Edition, McGraw Hill Professional.
* *

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Prof. (Dr.) N.B. Santha Sheela

Mr. Sunil Kumar

Books are Available for Online Purchase at: tppl.org.in

THAKUR PUBLICATION PVT. LTD., LUCKNOW

Biopharmaceutics & Pharmacokinetics

Copyright © All Rights Reserved

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Please e-mail us at, thakurpublication@gmail.com

I am extremel y thankful to Dr. R. Sundhararajan , M.Pharm, Ph.D., Principal,

- Prof. (Dr.) N.B. Santha Sheela

I am very thankful to my teachers, parents and family members for encouraging

-Mr. Sunil Kumar

Bioavailability and Bioequivalence

3.2.2.2. Reduction Reaction 73 4.2.2. Use of Salt Forms 108

5.2. Pharmacokinetic Models 134 Intravenous Infusion

CHAPTER Introduction to Biopharmaceutics

Biopharmaceutics is defined as, ―study of the interrelationship of

Biopharmaceutics is also defined as ―study of the factors influencing the rate

1.1.2. Applications of Biopharmaceutics

Solid dosage Dissolution Non-ionic Non-ionic

1.2.2. Mechanisms of Drug Absorption Through GIT

The following mechanisms are involved in the transport of drug molecules

1.2.2.1. Passive Diffusion

Passive diffusion can be expressed by Fick’s first law of diffusion, according to

1.2.2.2. Carrier-Mediated Transport

Carrier-mediated transport system is

1.2.2.3. Facilitated Diffusion

Intestinal Membrane Blood

Figure 1.4: Facilitated Diffusion of Vitamin B12

1.2.2.4. Active Transport

GI lumen Membrane Blood

Drug-carrier Dissociation Free

Figure 1.5: Active Absorption of a Drug

An example of competitive inhibition of drug absorption through active transport

1.2.2.5. Pore Transport

1.2.2.6. Ionic or Electrochemical Diffusion

1.2.2.7. Ion-Pair Transport

Figure 1.7: Endocytic Uptake of Macromolecules

1.3. FACTORS INFLUENCING DRUG

1.3.2. Physicochemical Factors

Salt Form of the Drug

An orally administered drug undergoes degradation due to the acidic or

Due to degradation of drug, the following consequences may occur:

The compromise between solubility and s tability of drug in gastrointestinal

The following theories explain the process of drug dissolution:

Since this dissolution process involves the contact of fresh pockets of

Agglomerate with PEG

When phenytoin is agglomerated by spherical crystallisation, the formed

proportional to their absorption from colon; doxycycline is better

Limitations of pH-Partition Theory

concept, e.g., derivatives of barbituric acid h ave almost same pK a, but

For Weak Bases

For Weak Acids

For Basic Drugs

However in hydrophobic drugs (like aspirin, phenacetin , and phenobarbital),

1.3.3. Pharmaceutical Factors

In granulation, many steps are involved that influence drug dissolution:

greater amount, it may increase hardness and decrease disintegration or

b) Membrane permeability is enhanced by increasing lipophilicity (e.g.,