CAREWELL PHARMACY

M Pharmacy Notes
Pharmaceutics
Advanced Biopharmaceutics & Pharmacokinetics

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Syllabus:-
Unit 1. Drug Absorption From The Gastrointestinal Tract:
Gastrointestinal tract, Mechanism of drug absorption, Factors
affecting, pH–partition theory, Formulation and physicochemical
factors: Dissolution rate, Dissolution process, Noyes–Whitney equation
and drug dissolution, Factors affecting the dissolution rate.
Gastrointestinal absorption: role of the dosage form: Solution (elixir,
syrup and solution) as a dosage form ,Suspension as a dosage form,
Capsule as a dosage form, Tablet as a dosage form ,Dissolution
methods ,Formulation and processing factors, Correlation of in vivo
data with in vitro dissolution data. Transport model: Permeability-
Solubility-Charge State and the pH Partition Hypothesis, Properties of
the Gastrointestinal Tract (GIT), pH Microclimate Intracellular pH
Environment, Tight-Junction Complex. Solubility: Experimental
methods. Permeability: In-vitro, in-situ and In-vivo methods.

Unit 2 - Biopharmaceutic Considerations in Drug Product Design and

In Vitro Drug Product Performance: Introduction, Biopharmaceutic
Factors Affecting Drug Bioavailability, Rate- Limiting Steps in Drug
Absorption, Physicochemical Nature of the Drug Formulation Factors
Affecting Drug Product Performance, In Vitro: Dissolution and Drug
Release Testing, Compendial Methods of Dissolution, Alternative
Methods of Dissolution Testing, Meeting Dissolution Requirements,
Problems of Variable Control in Dissolution Testing Performance of
Drug Products: In Vitro–In Vivo Correlation, Dissolution Profile
Comparisons, Drug Product Stability, Considerations in the Design of a
Drug Product.

Unit 3 - Pharmacokinetics: Basic considerations, Pharmacokinetic

models, Compartment modeling: One compartment model- IV bolus, IV
infusion, Extra-vascular; Multi Compartment model: Two compartment -

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model in brief, Non-Linear Pharmacokinetics: Cause of non-linearity,

Michaelis – Menten equation, Estimation Kmax and Vmax. Drug
interactions: Introduction, The effect of protein-binding interactions,
The effect of tissue-binding interactions, Cytochrome P450-based
drug interactions, Drug interactions linked to transporters.

Unit 4 - Drug Product Performance, In Vivo: Bioavailability and

Bioequivalence: Drug Product Performance, Purpose of Bioavailability
Studies, Relative and Absolute Availability, , Methods for Assessing
Bioavailability, Bioequivalence Studies, Design and Evaluation of
Bioequivalence Studies, Study Designs, Crossover Study Designs,
Evaluation of the Data, Bioequivalence Example, Study Submission and
Drug Review Process, The Biopharmaceutics Classification System,
Generic Biologics (Biosimilar Drug Products),Clinical Significance of
Bioequivalence Studies, Special Concerns in Bioavailability and
Bioequivalence Studies, Generic Substitution.

Unit 5 - Application of Pharmacokinetics: Modified-Release Drug

Products, Targeted Drug Delivery Systems and Biotechnological
Products. Relationship between Pharmacokinetics including
Pharmacodynamics: Generation of a pharmacokinetic– pharmacodynamic
(PKPD) equation, Pharmacokinetic and pharmacodynamic, interactions.
Pharmacokinetics and pharmacodynamics of biotechnology drugs:
Introduction, Proteins and peptides, Monoclonal antibodies,
Oligonucleotides, Vaccines (immunotherapy),Gene therapies.

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Unit 1 - Drug Absorption from the Gastrointestinal

Tract
Absorption is the process of movement of drug from its site of
administration to the systemic circulation.

Drug Absorption: is defined as the process of movement of unwanted

drug from the site of administra
administration
tion to systemic circulation.

Plots showing significance of rate and exten

extentt of absorption in drug
therapy

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Gastrointestinal Tract
Basic structure

 The gastrointestinal tract is a muscular tube lined by a special layer

of cells, called epithelium.
 Although
though each section of the tract has specialized functions, the
entire tract has a similar basic structure with regional variations.

The wall is divided into four layers as follows:

1. Mucosa - The innermost layer of the digestive tract has specialized

epithelial
helial cells supported by an underlying connective tissue layer
called the lamina propria. The lamina propria contains blood vessels,
nerves, lymphoid tissue and glands that support the mucosa.
Depending on its function, the epithelium may be simple (a single
sin
layer) or stratified (multiple layers).
2. Submucosa - The submucosa surrounds the muscularis mucosa and
consists of fat, fibrous connective tissue and larger vessels and
nerves. At its outer margin there is a specialized nerve plexus called
the submucosall plexus or Meissner plexus. This supplies the mucosa
and submucosa.

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3. Muscularis external - This smooth muscle layer has inner circular

and outer longitudinal layers of muscle fibers separated by the
myenteric plexus or Auerbach plexus. Neural innervations control
the contraction of these muscles and hence the mechanical
breakdown and peristalsis of the food within the lumen.
4. Serosa/mesentery - The outer layer of the GIT is formed by fat
and another layer of epithelial cells called mesothelium.

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Mechanisms of Drug Absorption

The three board categories of drug transport mechanisms involved in
absorption are:-

1. Transcellular/intracellular transport
2. Paracellular/intercellular transport
3. Vesicular transport

1. Transcellular/intracellular transport

It is defined as the passage of drugs across the Gl epithelium. It is the

most common pathway for drug transport.

3 steps involved in transcellular transport of drugs are -

a. Permeation of Gl epithelial cell membrane

b. Movement across the intracellular space
c. Permeation of the lateral or baso lateral membrane

Two types of transcellular transport are:

i. Passive transport processes

ii. Active transport processes

i. Passive Transport Processes: These transport processes do not

require energy other than that of molecular motion (Brownian
motion) to pass through the lipid bilayer.
They are classified into:
a) Passive diffusion
b) Pore transport
c) Ion-pair transport
d) Facilitated- or mediated-diffusion

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ii. Active Transport Processes: These transport processes require

energy from ATP to move drug molecule from extracellular to
intracellular milieu.
They are of two types:
a) Primary active transport
b) Secondary active transport - they are
I. Symport (Co-transport)
II. Antiport (counter-transport)

2. Paracellular/Intercellular Transport

It is defined as the transport of drugs through the junction between

the GI epithelial cells. This pathway is of minor importance in drug
absorption.

The two mechanisms involved in drug absorption are:

1. Permeation through tight junctions of epithelial cell

2. Persorption

3. Vesicular or Corpuscular Transport (Endocytosis)

Like active transport, these are also energy dependent processes but
involve transport pf substances within vesicles into a cell. Since the
mechanism involves transport across the cell membrane.

They are classified into two categories:

a. Pinocytosis
b. Phagocytosis

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Important transport processes and drugs absorbed through them

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Factors affecting Drug Absorption

The chain of events that occur following administration of a solid
dosage form such as a tablet or a capsule until its absorption into
systemic circulation.

The process consists of four steps:

1. Disintegration of the drug product.

2. Disaggregation and subsequent release of the drug.
3. Dissolution of the drug in the fluids at the absorption site.
4. Absorption
orption i.e. movement of dissolved drug through the GI
membrane into the systemic circulation and away from the
absorption.

In a series of kinetic or rate processes, the rate at which the drug

reaches the systemic circulation is determined by the slowest of the
various steps involved in the sequence.

Such step is called as the rate determining or rate

rate-limiting
limiting step (RDS).

The rate and extent of drug absorption from its dosage form can be
influenced by a number of factors in all these steps. The various

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factors that influence drug absorption called as biopharmaceutic

factors in dosage for design.

PH–Partition Theory of Drug Absorption

Introduction

 A drug injected Intravascular directly enters the systemic

circulation and exerts its pharmacological effects.
 But, Majority of drugs are administered orally which is intended to
act systemically.
 Such drugs can exert their pharmacological actions only when they
enter into systemic circulation by the process of absorption.
 Drug absorption is defined as the process of movement of
unchanged drug from the site of administration to systemic
circulation.

pH- PARTITION THEORY

 Understanding of the interrelationships between the rate of drug

absorption, the dissociation constant (pKa) and the pH of the
absorption site is known as the pH-partition theory.
 The theory states that for drug compounds of molecular weight
greater than 100, which are primarily transported across the bio
membrane by passive diffusion.

The process of absorption is governed by:

1. The dissociation constant (pKa) of the drug.

2. The lipid solubility of the unionized drug.
3. The pH at the absorption site.

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Brodie proposed the partition theory to explain the influence of GI pH

and drug pKa on the extent of drug transfer or drug absorption.

 pH partition theory of drug absorption is based on the GIT is a

simple lipid barrier to the transport of drugs and chemicals.
 Accordingly the unionized form of an acid or basic drug, if
sufficient lipid soluble, is absorbed but the ionized form is not.
 The larger the fraction of drug is in the unionized form at a specific
absorption site, the faster is the absorption.

DRUG pKa and GI pH

 The fraction of drug in solution that exist in the unionized form is a

function of both dissociation constant of the drug and the pH of the
solution.
 The dissociation constant is often expressed for both acids and
bases as pKa (the basic logarithm of the acidic dissociation
constant).
 It is customary to express the dissociation constants of both acidic
and basic drugs by pKa values.
 The lower the pKa of an acidic drug, the stronger the acid i.e.,
greater the proportion of ionized form at a particular pH. The
higher the pKa of a basic drug, the stronger the base.
 Thus from the knowledge of pKa of the drug and pH at the
absorption site (or biological fluid), the relative amount of ionized
and unionized drug in solution at a particular pH and the percent of
drug in solution at this pH can be determined by Henderson-
Hasselbach equation,

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When the concentration of ionized drug becomes equal, (since log1=0)

and thus pH = pka. The pka is the characteristic of the drug.

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A membrane barrier that separates the aqueous solutions of different

pH such as GIT and plasma then the theoretical ratio R of drug
concentration on either side of the mem
membrane
brane can be given by the
equation,

pH Range In GIT

 The pH range in GIT from 11-8

 stomach is from 1--3
 Intestine (from duodenum to colon) 5
5-8,
 Then certain generalization regarding ionization and absorption of
drugs can be made, as predicted from pH partit
partition
ion hypothesis.

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Influence of Drug pKa and GI pH on Drug Absorption

Lipophilicity (Ko/w) and Drug Absorption

 The unionised drug ->> if sufficiently lipid soluble

soluble->> is absorbed into
the systemic circulation.
 Iff it has poor lipid solubility -> it will be poorly absorbed.
 Therefore for optimum absorption ->> a perfect HLB should be
required.
 The lipid solubility of a drug is determined from its oil/water
oi
partition coefficient (Ko/w) value.

Limitations of pH-partition
partition Hypothesis:

1. Presence of virtual membran

membrane pH
2. Absorption of ionized drug
3. Influence of GI surface area and residence time of drug.
drug
4. Presence of aqueous unstirred diffusion layer.

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1. Presence of virtual membrane pH:

PH-absorption
absorption curve for Acidic and basic drugs.

Dotted lines - Curves predicted b

by pH-partition
partition hypothesis (only
unionized drug absorbed)

Bold lines - the practical curves (less sleeps and shifted)

The Virtual pH also called as the microclimate pH, is different from

the luminal pH exists at the membrane surface.

2. Absorption of ionized
ed drugs:

 As per hypothesis - only unionized form of drug absorbed and

a
ionized drug is negligible ((3to4 times less) - Principle of non-ionic
non
diffusion.
 pH absorption curve shift suggested that ionized forms of some
drugs also get absorbed to a some extent.
 If such drugs have large lipophilic group in their structure, despite
their ionization, they will be absorbed passively or by active
transport, ion pair transport and convective flow.

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3. Influence of GI surface area and residence time of drug

 According to theory - (condition of unionized form in large extent)

 Acidic drugs are best absorbed from stomach
 Basic drugs are best absorbed from intestine
 But Surface area of Stomach and intestine is different.
 Once a Acidic drug reaches the intestine, the remaining fraction will
be poorly absorbed.
 It may not be attain its therapeutic level.
 But irrespective of GI pH and degree of ionization both acidic and
basic drugs are more rapidly absorbed from intestine, because of its
large surface area, long residence time.

4. Presence of aqueous unstirred diffusion layer

 The bulk of the luminal fluid is not in direct contact with the
membrane but a barrier called as aqueous unstirred diffusion layer
present in between them.
 Such layer has thickness and it acts as barrier to absorption of
drugs.
 As per pH- partition theory the rate limiting step in the absorption
was partitioning in the lipid barrier.
 Presence of aqueous unstirred diffusion layer, a drug must diffuse
first through this barrier and then through the lipoidal barrier.
 Thus, drugs having large Partition coefficient can rapidly penetrate
the lipid membrane but diffusion through aqueous unstirred layer is
the rate limiting step.

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Formulation and Physicochemical Factors:

Pharmaceutical Factors: include factors relating to the physiochemical
properties of the drug and dosage form characteristics and
pharmaceutical ingredients.

Physicochemical properties of drug substances:

1. Drug solubility and dissolution rate

2. Particle size and effective surface area
3. Polymorphism and amorphism
4. Pseudo polymorphism (hydrates/solvates)
5. Salt form of the drug
6. Lipophilicity of the drug
7. pKa of the drug and gastrointestinal pH
8. Drug stability
9. Stereo chemical nature of the drug

Dosage form characteristics and a Pharmaceutical Ingredients

(Pharmaco-technical factors):

1. Disintegration time (tablets/capsules)

2. Dissolution time
3. Manufacturing variables
4. Pharmaceutical Ingredients (excipients/ adjuvants)
5. Nature and type of dosage form
6. Product age and storage conditions

Patient-Related Factors: include factors relating to the anatomical,

physiological and pathological characteristics of the patient.

1. Age
2. Gastric emptying time
3. Intestinal transit time

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4. Gastrointestinal pH
5. Disease state
6. Blood flow through the GIT
7. Gastrointestinal contents
a. Other drugs
b. Food
c. Fluids
d. Other normal GI contents
8. Contact time with gastrointestinal mucosa
9. Presystemic metabolism by
a. Luminal enzymes
b. Gut wall enzymes
c. Bacterial enzymes
d. Hepatic enzymes

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Dissolution
Definition:

 Dissolution is a process in which a solid substanc

substancee solubilizes in a
given solvent i.e. mass transfer from the solid surface to the
liquid phase.
 Dissolution is the rate determining step for hydrophobic, poorly
aqueous soluble drugs.
E.g. Griseofulvin, spironolactone

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Mechanism of Dissolution

a. Diffusion
sion layer model
b. Danckwert's model
c. Interfacial barrier model

Dissolution mechanisms - 2 steps:

1. Interfacial reaction >>>> cause liberation of solid particles into

boundary layer (C).
2. Migration of solute from boundary layer into bulk of solution (C) by
diffusion
sion & convection.
 Overall rate of dissolution depends on the slowest step.
 Usually Step (2) is the RDS.

Fick's law:

Where, k = rate constant

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Noyes and Whitney Equation

The rate of change in concentration of dissolved material with time it
directly
y proportional to the concentration difference between the two
sides of diffusion layer:

Where,

 dc/dt - Dissolution rate of drug.

 k - Rate constant.
 Cs - Concentration of solution at solid surface
surface.
 Сь - Bulk of the solution
solution.

Modified Noyes-Whitney's
Whitney's Equation:-

 Brunner incorporated surface area 'A' in Noyes & Whitney equation.

dc/dt = kA (Cs - Cb)

 Afterwards Brunner, incorporated Fick's law of diffusion &

expanded his given eq
equation to include diffusion coefficient 'D',
thickness of stagnant diffusion lay
layer
er ‘h' & volume of dissolution
medium 'v'.

Where,

 D = diffusion coefficient of drug.

 A = surface area of dissolving solid.
 Kw/o = water/oil partition coefficient of drug.

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 V = volume of dissolution medium.

 h = thickness of stagnant layer.
 Cs - Cb = conc. gradient for diffusion of drug.

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Factors Affecting Dissolution Rate

1. Factors related to Physicochemical Properties of Drug.
Drug
2. Factors related to Drug Product Formulation
Formulation.
3. Processing Factor.
4. Factors Relating Dissolution Apparatus
Apparatus.
5. Factors Relating
lating Dissolution Test Parameters
Parameters.

1. Physicochemical Properties of Drug

1. Particle Size of Drug

 Surface
urface area increases with decrease in particle size, higher
dissolution rates may be achieved through reduction of particle size.
E.g. Micronisation of n
non-hydrophobic
hydrophobic drug like griseofulvin leads to
increase in dissolution rate.
 Micronisation of hydrophobic powders can lead to aggregation and
floatation, when powder is dispersed into dissolution medium.
E.g. hydrophobic drugs like aspirin, phenacetin and phenobarbital.

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2. Drug Solubility

 Minimum aqueous solubility of 1% is required to avoid potential

solubility limited absorption problems.
 Studies on several compound of different chemical classes and a
wide range of solubility revealed that initial di
dissolution
ssolution rate of
these substances is directly proportional to their respective
solubility.
E.g. Poorly soluble drug: griseofulvin, spironolactone.
Hydrophilic Drug: neomycin
neomycin.

3. Solid State Characteristics

 Anhydrous forms dissolve faster than hydrated for form

m because they
are thermodynamically more active than hydrates.
E.g. Ampicillin anhydrate faster dissolution rate than trihydrate.
 Amorphous forms of drug tend to dissolve faster than crystalline
materials.
E.g. Novobiocin, Griseofulvin.
 Where in the dissolution
olution rate of amorphous erythromycin estolate is
markedly lower than the crystalline form of erythromycin estolate.

Metastable (high activation energy) polymorphic form has better

dissolution than stable form. Eg. Methyl prednisone.

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4. Salt Formation

 It is one of the common approaches used to increase drug solubility

and dissolution rate.
 It has always been assumed that sodium salts dissolve faster than
their corresponding insoluble acids.
E.g. sodium and potassium salts of Penicillin G, phenytoin,
barbiturates, tolbutamide etc.
 While in case of Phenobarbital dissolution of sodium salt was slower
than that of weak acid. Due to decreased disintegration of sodium
salt.
 Hydrochlorides and sulphates of weak bases are commonly used due
to high solubility.
E.g. epinephrine, tetracycline.

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Gastrointestinal Absorption: Role of the Dosage

Form
Introduction

 A drug injected intra vascularly directly enters the systemic

circulation and exerts its pharmacological effects. But Majority of
drugs administered extra vascularly (orally).
 If intended to act systemically, such drugs can exert their
pharmacological actions only when they come into blood circulation
from their site of application. So, absorption is an important step.
 Absorption: Movement of active drug (or prodrug) from the site of
administration to the systemic circulation.
 Drug formulations are designed to provide an attractive, stable, and
convenient method to use products.
 Conventional dosage forms may be broadly characterized as;
a. Solutions
b. Suspension
c. Capsules
d. Tablets

The bioavailability of a drug to decrease in the following order:

Solution > suspension > capsule > tablet > coated tablet

 One drug can routinely produce a 2 to 5-fold difference in the rate

or extent of gastro-intestinal absorption depending on the dosage
form of the formulation.
 In some cases, even greater difference observed. A difference of
more than 60-fold- has been found in the absorption rate of spirono
lactone from the worst formulation to the best formulation.

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Nature and Type

ype of Dosage

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Solutions
 Solutions such as syrups and elixirs show fast and often complete
absorption of drug because they do not have dissolution problem.
 However, dilution of the drug solution with gastric fluid may result
in precipitation that may re-disperse rapidly due to extremely fine
nature of precipitate.
 The factors that affect drug absorption from solution include
viscosity, reversible complexation, chemical stability and micellar
solubilization.
 The vehicle used in syrups, elixirs and emulsions may be aqueous or
non- aqueous (e.g., PEG, PG, alcohol) or non-water miscible (e.g.,
vegetable oils).
 The rate of drug absorption from non-aqueous or non-water miscible
vehicle based solution is less than the rate of drug absorption from
water based solution.
 The selection of vehicle for solution dosage form depends on the
physiochemical properties of the drug.
Ex. Paracetamol drop is prepared with PEG 400 as it is sparingly
soluble in water.
 Certain materials such as sorbitol or hydrophilic polymers are added
to a solution dosage form, to improve pour ability and palatability by
increasing the viscosity of the preparation.
 Due to good systemic availability, solutions are frequently used as
bioavailability standards against which other dosage forms are
compared.
 Rapid and complete absorption may be observed in some instances,
particularly if the oil is administered in emulsified form.
 Administration of indoxole dissolved in the oil phase of Lipomul -
Oral (o/w).

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 Resulted in a threefold improvement in the extent of absorption

compared to that observed after administration of an aqueous
suspension and a nine fold improvement compared to a hard gelatin
capsule.

Suspensions
 Drug in a suspension is in solid form, but is finely divided and has a
large surface area. Drug particles can diffuse readily between the
stomach and small intestine so that absorption is relatively
insensitive to stomach emptying rate.
 Adjusting the dose to a patient's needs is easier with solutions and
suspensions than with solid dosage forms.
 Several studies have demonstrated the superior bioavailability
characteristics of suspensions compared to those of solid dosage
forms.
 Ex. the blood levels of trimethoprim and sulfa methoxazole were
compared in 24 healthy subjects following oral administration of
3 forms, the absorption rate of each drug was significantly
greater with the suspension than with the tablet or capsule.
 Penicillin blood conc. following oral administration of various
dosage forms show higher level with suspension of Phenoxymethyl
penicillin.
 Finally divided solid particles in suspension are stabilized with
suspending agents.
 Suspending agents retard the rate sedimentation of dispersed
particles.
 Absorption of drug in suspension form is not greatly affected by
stomach emptying rate.
 But suspending agent may increase the viscosity of drug vehicle and
thereby may diminish rate of drug dissolution.

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 Other critical factors that affect drug absorption include particle

size, crystal forms and formation of non-absorbable complexes
 Suspending agent may form non-absorbable complexes with drug eg.,
divalent metals form in suspension of multivitamins and essentials
elements form complex with sodium carboxymethylcellulose that
poorly get absorb in body.
 As dissolution is taking place at the surface of solute smaller
particle having larger surface area may dissolve rapidly.
 Bioavailability studies with drugs suspended in oi1-in-waier emulsions
have yielded some promising results.
 One study compared the absorption of micronized griseofulvin after
its administration to healthy subjects in a corn oil-in-water emulsion,
an aqueous suspension, and two different commercial tablets.
 The extent of absorption of the drug after administration of the
emulsion was about twice that observed after administration of the
aqueous suspension or tablets.
 MOA; based on the ability of fatty acids, liberated during the
digestion of corn oil, to inhibit gastrointestinal motility (which would
increase the residence time of the drug in the small intestine) and
to stimulate gallbladder evacuation

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Capsules
 In capsule on disruption of the shell, the encapsulated powder mass
should disperse rapidly to expose a large surface area to the
gastrointestinal fluids.
 Diluents added to capsules dosage form may affect the dissolution
of filled drug in capsule shell.
 Hydrophilic diluents are added in the capsule of a poorly water
soluble drugas they enhance the dispersion rate of the aqueous fluid
to the contents of the shell.
 This results in better dissolution of the drug in the biological fluid.
 Sometimes wetting agents are also added to improve dispersion
rate.
 Further, drug absorption from capsule may also be affected by
particle size and chemical and physical incompatibility of the drug
with a filler and other ingredients.
 Certain drugs are formulated in soft gelatin capsule as a solution
from which drug disperses and dissolves more rapidly as compared
to hard gelatin capsule.
 Moreover, soft gelatin capsule leaves less residual drug in gut and
hence causes minimal irritation.
 This approach is more useful for the drugs that causes local
irritation.
 The use of dicalcium phosphate as a diluent in tetracycline capsules
has been found to significantly impair absorption because a poorly
soluble calcium- tetracycline complex is formed in the powder mass
or during dissolution.
 Factors that influence drug absorption from capsule dosage forms
include - particle size and crystal form of the drug, and selection of
diluents and fillers.

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 A soft elastic capsule containing 0.4 mg of digoxin is about

equivalent to a tablet containing 0.5 mg of the drug i.e; mean
absorption was 75% of the dose from the tablet and 97% from the
capsule.

Tablets

 Many factors related to the formulation or production of tablets

may affect drug dissoluti
dissolution and absorption.
 Most formulations require the incorporation of hydrophobic
hydrophobi
lubricants, such as magnesium stearate, to produce an acceptable
tablet. In general, the larger the quantity of lubricant in a
formulation the slower is the dissolution rate.
 Compression
pression force may also be an important factor in drug
bioavailability from compressed tablets.
 The in vitro disintegration time of tablets has been shown to be
directly proportional to compression force and tablet hardness.
 High compression forces may als also
o increase the strength of the
internal structure of the granules and retard dissolution of drug
from the granules and disintegration of the granules.

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 A novel approach to enhance the availability of poorly water-soluble

drugs from tablets has been used in a marketed griseofulvin
product.
 A molecular dispersion of the drug in (Polyethylene glycol 6000, A
water-soluble waxy polymer that congeals at about 60C) is prepared
and suitably modified for incorporated into a tablet dosage form.
 The absorption of griseofulvin from this product appears to be
complete and about twice that observed from commercial tablets
containing micronized drug.

Coated Tablet

 The coating must dissolve or disrupt before tablet disintegration

and drug dissolution can occur.
 The disintegration of certain coated tablets appears to be the rate-
limiting process in drug absorption.
 Film-coated tablets are compressed tablets that are coated with a
thin layer or film of a material that is usually water soluble or
dispersible.
 A number of polymeric substances with film forming properties may
be used including hydroxy propyl methylcellulose and
carboxymethylcellulose.
 The film coat should disrupt quickly in the fluids of the
gastrointestinal tract, independent of pH.
 Sugar coating may affect the bioavailability of a drug. Alternatives
include the film-coated tablet and the press coated tablet.

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Enteric coated Tablets

 Enteric coated is special film coated tablet which are used to bypass
gastric fluid so that the drug gets dissolve in intestine.
 They show a delayed absorption and therefore a delayed onset of
action.
 They also show high inter and intra subject variability due to
difference in gastric emptying rate.
 The modern approach to enteric-coating makes use of polymer like
cellulose acetate phthalate that are "insoluble' at pH I to 3 but
'soluble" at pH5 to 7.
 The thickness of the coating may also affect bioavailability.
 Studies with quinine tablets coated with cellulose acetate phthalate
show a decrease in both rate and extent absorption with increasing
thickness of the coating.

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Dissolution Methods
Two Methods:-

1. Official methods
2. Non official methods

1. Official Methods:

Apparatus are used according to standards specified. The USP includes

seven apparatus design for drug release and dissolution testing of
immediate release and for oral dosage form, for extended release,
enteric coated, Transdermal drug delivery system.

Methods are listed below:

1. Rotating basket method

2. Paddle method
3. Flow-through method
4. Reciprocating cylinder method
5. Paddle over disk method
6. Rotating cylinder method
7. Reciprocating disk method

Apparatus 1 - Basket

Useful for

 Capsules
 Beads
 delayed release / enteric coated dosage forms
 floating dosage forms
 surfactants in media

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Standard volume

 900/1000 mL
 1, 2, 4 L vessels

Dissolution Apparatus-3
3 (Reciprocating Cylinder)

Design:

 Vessel:
 Set of cylindrical flat bottom glass vessels
 Set of reciprocating cylinders
 Stainless
tainless steel fittings.
 Agitation type: -Reciprocating
Reciprocating
 Volume of dissolution medium:
medium:-200-250ml
 Water bath:- Maintain at 37±0.5°C
 Dosage form is placed in cylinder
 Use: Tablets, beads, controlled and extended
release formulations.

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Apparatus 4 Flow-Through
Through Cell

Advantages:

a. Infinite sink condition can be achieved hence low soluble drugs

studies can be done.
b. It is easy to change pH of media during test, avoiding hot spots
as seen in basket method.
c. Minimum dwell time, avoiding problems of degradation
degradati of drug
during process
d. Ease of sampling and automation of data reduction.
e. Adaptability to current USP calibrator
calibrators.

Disadvantages:

a. Large volume of media is required.

b. Control of constant flow rate is difficulty.
c. Clogging results in damage to equipment.
d. Pump should produce pulse free flow.

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e. Pressure may build up due to clogging hence pressure transducer

should used to regulate pressure and to maintain constant flow
rate.

USP Apparatus 5 Paddle over Disk

Standard Paddle Used

 Typical Volume 900 mL

 Temperature 32 °C

Useful for

 Transdermal Patches
 Ointments
 Floaters
 Emulsions
 Bolus

Modifications

 Disk Design
 Volume

Apparatus - 6 (Rotating Cylinder)

Design:

 Vessel: same as apparatus 1.

 Shaft: Stainless steel 316, basket is replaced with cylinder is
used.
 Sample
 Mounted to inner porous cellulosic material and adheres to
cylinder.
 Dosage unit is placed in cyli
cylinder and release
ase from side out.
 Water-bath: maintained at 32+0.5°C
32+0.5°C.

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Use:

 Transdermal patches cannot be cut into small size.

 Solid dosage forms.

USP Apparatus 7 - Reciprocating Holder

Reciprocating Holder

 Sample Holder.
 Temperature 32 °C.

Sequential Media Tubes

 Typical Volumes 50 to 200 mL

Useful for

 Transdermal Patches
 Solid Dosage Forms
 PH Profile
 Small Volumes

Modifications

1. Dosage Form Holder

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Non-Official Methods

Tracking of dissolution from different dosage form is sometimes is not

possible with the existing official methods. So several non-official
methods are designed for dissolution testing.

These includes,

1. Percutaneous absorption technique.

2. Rotating bottle method for sustained release dosage form.
3. Dialysis system.

1. Percutaneous Absorption Technique:

 Percutaneous absorption is related to the absorption across the

skin. Percutaneous absorption methods are currently used to study
transfer kinetics through membranes.
 These are useful in testing membrane characteristics and studying
absorption through skin.
 These are generally use for patches, the patches generally mounted
in same position as the simulated skin membrane and serves as the
donor side of the system.
 Some of the tech. that are used in percutaneous absorption
measurement includes side-by-side, the Franz cell, and flow through
cell design.

2. Rotating Bottle Method:

 This is probably the oldest method used for sustained release

dosage forms.
 The system consists of 12 small bottle attached to a horizontal
shaft that rotates at a slow rate of 6-50 rpm.
 The whole assembly is placed in a constant water bath.

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 Each bottle consists of 60 ml of dissolution fluid that is decant

through a 40-mesh screen after each sampling period and is
replaced by fresh fluid.

Dialysis System

 In this case very poorly soluble drugs, where perfect sink condition
would necessitate a huge volume of solvent with conventional
methods, a different approaches utilizing dialysis membranes, was
tried as a selective barrier between the fresh solvent and the cell
compartment containing the dosage form.
 It has been used with some success in case of other dosage form
such as suspensions, creams and ointments.

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Formulation and Processing Factors

Introduction

 A drug injected intravascularly directly enters systemic circulation

& exerts its pharmacological effects.
 If intended to act systemically, such drugs can exert their
pharmacological actions only when they come into blood circulation
from their site of application.
 Majority of drugs are administered extravascular only.
 Absorption is an important step.

Processing Factors

Manufacturing Processes - Manufacturing processes that influence

drug dissolution are:

1. Compression force

 Density
 Porosity
 Hardness
 Disintegration time
 Dissolution of tablets

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Method of Granulation

Pharmaceutical
ceutical Ingredients/Excipients

 Despite their inertness & utility in the dosage form, excipients can
influence absorption of drug.
 More number of excipients in a dosage form, the more complex it is
& greater potential for absorption & bioavailability problems
probl

Excipients are added to ensure,

1. Stability
2. Uniformity
3. Function ability
4. Bioavailability
5. Acceptability

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Vehicle

Absorption depends to a large extent on its miscibility with biological

fluids.

Diluents

 Diluents are commonly added to tablet (and capsule) fformulations

ormulations if
the required dose is inadequate to produce the necessary bulk.
 Eg of drug-diluent
diluent interaction resulting in poor bioavailability is that
of tetracycline & DCP

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Binders & Granulating Agents

 Promote cohesive compacts before & after compression.

 Eg polymeric materials like starch, cellulose derivatives, acacia,
PVP, etc.

Colorants

 Very low concentration of water-soluble dye inhibitory effect on

dissolution.
 Dye molecules get adsorbed onto the crystal faces & inhibit drug
dissolution.
E.g. brilliant blue retards dissolution of sulfathiazole.

Lubricants

 To aid flow of granules.

 To reduce inter particle friction.
 Sticking of particles to dies & punches.
 Eg: Hydrophobic in nature (several metallic stearates & waxes)

Coatings

 Deleterious effect of various coatings on drug dissolution from a

tablet dosage form is enteric coat > sugar coat > nonenteric film
coat.
 E.g. Shellac coated tablets, on prolonged storage, dissolve more
slowly in the GIT

Disintegrants

 Agents overcome cohesive strength of tablet & break them up on

contact with water which is an important prerequisite to tablet
dissolution.
 These are hydrophilic in nature. Eg: MCC.

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Mechanism:

Buffers

 They create right atmosphere for dissolution. e.g. buffered aspirin

tablets.
 Buffer containing potassium cations inhibit the drug absorption. e.g.
vitamin B2 & sulphanilamide.

Surfactants

They may enhance or retard drug absorption.

1. Promotion of wetting & dissolution of drugs.

2. Better membrane contact of drug for absorption.
3. Enhanced membrane permeability of the drug.

Suspending Agents/Viscosity Imparters

 Hydrophilic polymers like vegetable gums, semisynthetic gums &

synthetic gums.
 Stabilize drug particles by reducing their rate of settling & by
increasing viscosity of the medium they also affect palatability &

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pourability of solution dosage forms. Eg Na-CMC complex

amphetamine increases drug absorption

Crystal Growth Inhibitors

 Eg crystal growth inhibitors like PVP & PEG inhibit conversion of a

high energy metastable polymorph into stable

Complexing Agents

 These agent alters stability, solubility, molecular size, partition

coefficient & diffusion coefficient
 Pharmacologically inert & must dissociate either at the absorption
site or following absorption into the systemic circulation
 Complexation has been used to enhance drug absorption are:
 Enhanced dissolution through formation of a soluble complex
e.g. ergotamine tartarate-caffeine complex.
 Enhanced lipophilicity for better membrane permeability e.g.
caffeine-PABA complex.
 Enhanced membrane permeability e.g. enhanced GI absorption
of heparin.

Solutions

 Solutions is most rapidly absorbed.

 Drug dissolution is absent

Factors influencing absorption of solution are:

1. Viscosity
2. Surfactants
3. Solubilizer
4. Stabilizer
5. Stability

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Emulsions

Superior to suspensions in administering poorly aqueous soluble

lipophilic drugs.

Absorption increases 3 fold over its aqueous suspension

Factors influencing drug absorption of emulsion are:

1. Surface area
2. Interfacial tension
3. Droplet size
4. Surfactants
5. Lipophilicity

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Suspension

Drug dissolution which is generally rapid due to the large surface area
of the particles.

Factors affecting absorption

a. Particle size polymorphism

b. Wetting agents
c. Viscosity of the medium
d. Suspending agent

Powders

 Though powders are superior to tablets & capsules

 They are not in use nowadays due to handling & palatability
problems.
Factors to be considered in the absorption of drug from powders are:
a. Particle size
b. Polymorphism
c. Wettability

Capsules

Powders & granules are administered in hard gelatin capsules whereas

viscous fluids & oils in soft elastic shells.

Factors of importance in case of hard gel:

 Drug particle size

 Density
 Polymorphism
 Intensity of packing
 Influence of diluents & excipients

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Factors of importance in case of soft gel:

 Between the drug & the diluents

 Between drug & gelatin shell

Tablets

Compressed Tablets > Film Coated Tablets > Sugar Coated Tablets >
Enteric Coated Tablets>Sustained Release Products.

Factors to be considered in the absorption of drug from tablets

are:

 Effective surface area

 Area dissolution
 Deaggregation
 Permeability
 Excipients/API

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In-Vitro In-Vivo Correlation

 According to Food & Drug administration: IVIVC is a predictive
mathematical model describing the relationship between an in-vitro
property of a dosage form and a relevant in-vivo response.
 According to FDA 1977 on bioavailability & bioequivalence stated
that dissolution test the preferred in-vitro test should be
correlated with in-vivo data.
 The in-vitro property is the rate or extent of drug dissolution or
release while the in-vivo response is the plasma drug concentration
or amount of drug absorbed.
 It establishes relationship between biological property (pharmaco-
dynamic effect & plasma drug concentration) and physicochemical
property of drug substance such as dissolution rate.
 The in-vitro dissolution characteristics are dependent on the
physical properties of active pharmaceutical ingredients, the drug
dissolution, hydrodynamics of dissolution apparatus & dissolution
media.
 Unless the in-vitro dissolution reflects the in-vivo performance the
results obtained will have no relevance.

Objectives:

1. To ensure batch to batch uniformity in physiological performance by

utilizing in-vitro experimental data.
2. To serve as a tool in developing new, efficacious and safe dosage
form.
3. It assumes great imp. Especially for such formulations which
contains drugs having narrow therapeutic window (Eg: Diltiazem,
carbamazepine & Nifedipine) or variable therapeutic response (Eg:
Theophylline, digoxin etc.,)

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IVIVC can be achieved using:

1. Pharmacological correlations based on clinical observations.

2. Semi-quantitative correlation based on drug blood level or urinary
excretion data.
3. Quantitative correlation arising from absorption kinetics &
calculation of an in- vivo absorption rate constants.

Different Methods of IVIVC:

a. Simple point type

b. Comparison of Profiles

a. Simple Point Type: The percentage of drug dissolved in a given time

is correlated with the bioavailability of the Drug product.

b. Comparison of Profiles:

In vivo Data

 Plasma concentration time profile.

 Pharmacokinetic parameters.
 Percent drug absorbed time profile.
 Statistical movement analysis.

In vitro Data

 Percent drug dissolution profile.

 Kinetic parameters.
 Percent drug dissolved time profile.
 Statistical movement analysis.

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Percent of drug dissolved versus percent of drug absorbed

 If a drug is absorbed completely after dissolution, a linear

correlation may be obtained and by comparing the percent drug
absorbed to the percent drug dissolved.
 In choosing the dissolution medium and use a slow dissolution
stirring rate so that invivo dissolution is approximated.
Eg: The drug Aspirin is absorbed rapidly and completely from GIT.
Therefore a change in the dissolution rate from a dosage form may
be reflected in a change in the amount and rate if drug absorption
during the period of observation.
 Differences in the dissolution rates of dosage forms will be
reflected in the rate and extent of the drug only if the drug
absorption is dissolution rate limited.

Dissolution rate versus absorption rate:

 If dissolution of the drug is rate limiting, a faster dissolution rate

may result in a faster rate of appearance of drug in the plasma. It
may be possible to establish a correlation between rate of
dissolution and rate of absorption of the drug.
 The absorption is more difficult to determine then peak absorption
time. Therefore, the absorption time may be used in correlating
dissolution data to absorption data.
 In the analysis of invitro invivo drug correlation rapid drug
absorption may be distinguished from the slower drug absorption by
observation of the absorption time for the preparation.
Eg: In study involving three sustained released Aspirin products,
the dissolution time for the preparations were linearly correlated to
the absorptions times for various amounts aspirin absorbed.

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Different Stages in IVIVC:

Stage 1 IVIVC:

 Represents a point-to-point relationship between in vitro dissolution

rate and in vivo rate of the drug from the dosage form.
 Super imposable absorption rate curves and mathematical
descriptions are similar Alternative to in vivo method.
 Manufacturing method changes, formula modification performed
without in vivo data.

Stage 2 IVIVC

 Level B IVIVC utilizes the principles of statistical moment analysis.

 In this level of correlation, the mean in vitro dissolution time (MDT
vitro) of the product is compared to either mean in vivo residence
time (MRT).
 Not point to point correlation.
 This level is not reliable to justify changes in manufacturing or
formula changes.

Stage 3 IVIVC:

 Single point correlation

 Relates dissolution time with pharmacokinetic property like AUC
(AREA UNDER CURVE)
 Helps to develop formulations

Stage 4 IVIVC: (multiple stages 4)

 Amount of drug dissolved at different time intervals can be

correlated with many pharmacokinetic parameters.
 Scale up and post approval changes (SUPAC) can be made to the
formulation.

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Properties of GI Tract
1. Gastric Emptying:

 Few drugs are absorbed from the stomach.

 Most of the drugs are retained in stomach temporarily, largely in
solution, and are progressively delivered to the small intestine
where they are absorbed.
 For this reason gastric emptying is a critical factor in drug
absorption.
 The emptying time of stomach through the pylorus is proportional
to the volume remaining in the stomach.
 This constitutes a first order process which can be characterized
by a single half-life value.
 The different type of food shows different values of gastric
emptying time. The order ofgastric emptying can be summarized
as follows.

Carbohydrates < Proteins < Water << Lipids

Example: Paracetamol absorption is rate limited due to gastric

emptying.

2. Intestinal transit time:

 The intestinal transit rate also has a significant influence on the

drug absorption, since it determines the residence time of the drug
in the absorption site.
 Increasing the rate of gastric emptying and gastro-intestinal
motility increases the rate of absorption of a drug but, for digoxin
and riboflavin, increased gastrointestinal motility is associated with
a decrease in the rate of absorption.

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 Since, intestine is the major site of absorption of most of the

drugs; Long intestinal transit time is desirable for the complete
absorption of drugs.
Delayed intestinal transit is desirable for:
 Drugs that dissolve only in intestine (enteric coated).
 Drugs absorbed from specific sites in the intestine.

Other factors:

a. GI motility
b. Gl pH
c. Drug stability in GIT
d. Surface area
e. Blood flow to GIT

3. Water fluxes in the GI tract:

 In addition to the transport of material through the GI tract, water

fluxes due to secretion and reabsorption in the different segments
may significantly influence drug absorption.
 Over a 24 hour period approximately the flow rate into the
duodenum reaches between 6-10 liter of fluid per day.
 Most of this fluid is reabsorbed in the duodenum so that at its
distal end the flow rate is reduced to 3-5 liter per 24 hour. It is
further reduced to 1.5-2 liter/24 hour by the end of the jejunum,
0.7-1.2 liter/24 hour by the end of the ileum, and is only 0.1 liter/24
hour at the end of the colon, i.e. in the faeces.
 Water crosses the mucosal membrane through pores that are too
small for transfer of drug molecules across.
 Water fluxes in the gut wall therefore are unlikely to have a direct
effect on drug absorption. However, the presence of large volumes
of water in the duodenum, and to a lesser extent in the jejunum and

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ileum, may influence the dissolution of sparingly soluble drugs. The

considerable water flux in these GI segments may also facilitate
intra luminal transport of dissolved drug molecules towards the
absorption sites on the mucosa.
 The secretion and reabsorption of water also modifies the luminal
concentration of drug and therefore its rate of absorption.

Permeability-Solubility-Charge State:
 According to Ficks first law, passive diffusion of a solute is the
product of diffusivity and concentration gradient of the solute
inside the membrane.
 The membrane/water apparent partition coefficient relates the
latter internal gradient to the external bulk- water concentration
difference between the two solutions separated by the membrane.
 For an ionizable molecule to permeate by passive diffusion most
efficiently, the molecule needs to be in its uncharged form at the
membrane surface.
 The amount of the uncharged form present at a given pH, which
directly contributes to the flux, depends on several important
factors, such as pH, binding to protein and bile acids, self-binding,
and solubility.

PH Partition Hypothesis
 pH -partition theory explains the process of drug absorption from
the GIT and its distribution across all biological membranes.
 pH-partition theory states that for drug compounds of molecular
weight more than 100, which are primarily transported across the
bio membrane by passive diffusion, the process of absorption is
governed by
 Dissociation constant pKa of drug.

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 The lipid solubility of the unionized drug

 pH at the absorption site

As most of the drugs are weak electrolytes, their degree of ionization

depends upon the pH of the biological fluid.

This theory depends on some assumptions are as follows:-

1. The GIT is simple lipoidal barrier for transport of drug.

2. Larger the fraction of unionized drug, faster the absorption
3. Greater the lipophilicity of unionized drug, better the absorption.

a) Pka of drug:-

Amount of drug that exist in unionized form and in ionized form is a

function of pKa of drug & pH of the fluid at the absorption site and it
can be determined by Henderson-hesselbach equation:

 pH = pKa + log [lionized form/ unionised form ] ..........For, weakly

acidic drugs
 pH= pKa + (log unionized form/ionized form)........

For, Weakly basic drugs.

a. When conditions reach to the equilibrium the second term of both

equations become zero (as log1 is equal to zero). Now pH becomes
equal to pKa which is the characteristic feature of the drug.
b. The GIT has range of pH from 1 to 8, i.e. stomach has pH around 1-3
and intestine from 5-8. So on the basis of pH partition hypothesis
some generalizations can be made for absorption of drugs.

For Weak acidic drug:-

a. Very weak acids (pKa> 8) such as phenytoin, ethosuximide are remain

unionized at all pH values and therefore their absorption is rapid
and independent of GI pH.

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b. Acids in the pKa range 2.5 to 7 .5 are greatly affected by changes in

pH and therefore their absorption is pH-dependent; e.g. several
NSAIDs like aspirin, ibuprofen, phenylbutazone, and a number of
penicillin analogs. Such drugs are better absorbed from acidic
conditions of stomach.
c. Stronger acids with pKa < 2.5 such as cromolyn sodium are ionized in
the entire pH range of GIT and therefore remain poorly absorbed.

For Basic drugs:

a. Very weak basesi (pKa< 5) such as caffeine, diazepam etc are remain
non ionized at all the ph values therefore their absorption is rapid
and pH independent.
b. Bases in the pKa range 5 to 11.0 are greatly affected by changes in
pH and hence their absorption is pH-dependent; e.g. Several
morphine analogs, chloroquine, imipramine and amitriptyline. Such
drugs are better absorbed from the relatively alkaline conditions of
the intestine

Lipophilicity and Drug Absorption

 As mentioned earlier, it is the pKa of a drug that determines the

degree of ionization at a particular pH and that only the unionized
drug, if sufficiently lipid soluble, is absorbed into the systemic
circulation.
 Thus, even if the drug exists in the unionized form, it will be poorly
absorbed if it has poor lipid solubility (or low Ko/w).
 Ideally, for optimum absorption, drug should have sufficient aqueous
solubility to dissolve in the fluids at the absorption site and lipid
solubility (Ko/w) high enough to facilitate the partitioning of the
drug in the lipoidal biomembrane and into the systemic circulation.

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 In other words, a perfect hydrophilic -lipophilic balance (HLB)

should be there in the structure of the drug for optimum
bioavailability.
 The lipid solubility of a drug is determined from its oil/water
partition coefficient (KO/W) value.
 This value is a measure of the degree of distribution of drug
between one of the several organic, water immiscible, lipophilic
solvents such as n-octanol, etc. and an aqueous phase.
 In general, the octanol/pH 7.4, buffer partition coefficient value in
the range of I to 2 of a drug is sufficient for passive absorption
across lipoidal membranes.
 In yet another study by Schanker on a series of barbituric acid
derivatives having .same pKa, the percent absorbed increased with
an increase in the partition coefficient of the drug.
 Thus, to enhance the bioavailability of a drug, not only its dissolution
rate but also its rate of permeability should be considered

Limitations of pH partition hypothesis

a. The pH-partition theory provides a basic frame work for

understanding drug absorption, but it is an over simplification of a
more complex process
b. The theory indicates that the relationship between pH and
permeation or absorption rate is described by an S- shaped curve
corresponding to the dissociation curve of the drug.
c. For a simple acid or base, the inflection point of the pH- absorption
curve should occur at a pH equal to the pka of the drug.

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PH-absorption curve for acidic and basic drugs. Dotted lines

indicate curves predicted by pH-partition hypothesis And bold lines
indicate that practical curves.

d. In general pH absorption curves are less steep then expected and

are shifted to higher pH values for acids and to lower pH values for
bases.
1. Presence of virtual membrane pH.
2. Absorption of ionized drug.
3. Influence of GI surface area and residence time of drug.
4. Presence of aqueous unstirred diffusion layer.

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PH Microclimate
The experimental pH-absorption curves are less steep and shift to the
left (lower pH values) for a basic drug and to the right (higher pH
values) for an acidic drug. This led to the suggestion that a virtual pH,
also called as the microclimate pH, different from the lumenal pH
exists at the membrane surface.

 The absorption of short-chain weak acids in the rat intestine, as a

function of pH, appears not to confirm to the pH-partition
hypothesis.
 Similar anomalies were found with weak bases.
 The apparent pKa values observed in the absorption pH curve were
shifted to higher values for acid sand to lower values for bases,
compared with the true pKa values.
 Such deviations could be explained by the effect of an acid layer on
the apical side of cells, the so-called acid Ph microclimate.
 Shiau et al directly measured the microclimate pH, pHm, to be 5.2
6.7 in different sections of the intestine (very reproducible values
in a given segment) covered with the normal mucus layer, as the
luminal (bulk) pH, pHb, was kept at 7.2.
 Good controls ruled out pH electrode artifacts. With the mucus
layer washed off, pHm rise from 5.4 to 7.2. Values of pHb as low as
3 and as high as 10 remarkably did not affect values of pHm.
 Glucose did not affect pHm when the microclimate was established.
 However, when the mucus layer had been washed off and pHm Was
allowed to rise to pHb, the addition of 28 mM glucose caused the
original low pHm to be reestablished after 5 min.
 Shiau et al hypothesized that the mucus layer was an ampholyte (of
considerable pH buffer capacity) which created the pH acid
microclimate.

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 Said et al measured pHm in rat intestine under in vitro and in vivo

conditions. As pHb was kept constant at 7.4, pHm values varied 6.4-
6.3 (proximal to distal duodenum), 6.0 -6.4 (proximal to distal
Jejunum), 6.6- 6.9 (proximal to distal ileum), and was 6.9 in the
colon.
 Serosal surface had normal ph. When glucose or sodium was removed
from the bathing solutions, the pHm values began to rise. Metabolic
inhibitors (1 m Miodoacetate or 2,4- dinitrophenol) also caused the
pHm values to rise.
 Said et al hypothesized that a Na+ /H+ antiporter mechanism,
dependent metabolism, was responsible for the acid pH
microclimate.
 The tips of villi have the lowest phm valuesthe secretion, whereas
the crypt regions have pHm>8 values.
 Most remarkable was that microclimate (pHm 8) was observed in the
human stomach, whose bulk phb is generally about 1.7.
 In the stomach and duodenum, the near- neutral microclimate pH
Was attritbuted to the secretio of HCO3 from the epithelium.

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Tight Junction Complex

 Many structural components of the tight junctions (TJ) have been
defined in the last ten years. Lutz and Siahaan reviewed the protein
structural components of the (TJ). The occluding protein complex
that makes the water pores so restrictive.
 Freeze-fracture electron micrographs of the constrictive region of
the TJ show netlike arrays of strands (made partly out the
cytoskeleton.) Circumscribing the cell, forming a division between
the apical and the baso lateral sides.
 A region ten strands wide forms junctions that have very small pore
openings; fewer strands produce leakier junctions. The actual cell-
cell adhesions occur in the adhesions junctions, located further away
from the apical side.
 Apparently three calcium continuously link 10-residue portions
cadherin proteins spanning from two adjoining cell walls. Calcium-
binding agents can open the junctions by interactions with the
cadherin complex.

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Permeability: In-Vitro, In-Situ and In-Vivo

Methods
Different methods of determining drug absorption

1. In vitro method
2. In Vivo Method
3. In situ method

1. In vitro Method

 In vitro method are carried out outside of the body and are used to
determine the permeability of drug using live animal tissues.
 In vitro models have been introduced to assess the major factors
involved in the absorption process and predict the rate and extent
of drug absorption.
 Here, the intestine of lower experimental animals such as rats,
guinea pigs, rabbits are taken for the study.

The different in vitro methods are: Physico chemical methods:

1. Partition coefficient
2. Artificial membrasnes
3. Chromatographic retention indices
4. Brush border membrane vesicles (BBMV)
5. Isolated intestinal cells
6. Tissues techniques
a. Everted small intestinal sac technique
b. Everted sac modification
c. Circulation techniques
d. Everted intestinal ring or slice techniques
7. Diffusion cell method
8. Cell culture techniques

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1. Partition Coefficient

 Partition coefficient between an oil and water phase, log P, is one of

the easiest property of a drug molecule that can be determined.
 It provides a measure of the Lipophilicity of a molecule and can be
used to predict to what extent it will cross the biological membrane.
Eg. Octanol is selected as an oil phase as it has similar properties to
biological membranes.
 It's important to note that Log P does not take the degree of
ionization into consideration and hence log D is used.

2. Artificial Membrane

 An artificial membrane, or synthetic membrane, is a synthetically

created membrane which is usually intended for separation purposes
in laboratory or in industry.
 Synthetic membranes have been successfully used for small and
large-scale industrial processes since the middle of twentieth
century.
 A wide variety of synthetic membranes is known.
 They can be produced from organic materials such as polymers and
liquids, as well as inorganic materials. The most of commercially
utilized synthetic membranes in separation industry are made of
polymeric structures. They can be classified based on their surface
chemistry, bulk structure, morphology, and production method. The
chemical and physical properties of synthetic membranes and
separated particles as well as a choice of driving force define a
particular membrane separation process.
 The most commonly used driving forces of a membrane process in
industry are pressure and concentration gradients. The respective
membrane process is therefore known as filtration. Synthetic
membranes utilized in a separation process can be of different

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geometry and of respective flow configuration. They can also be

categorized based on their application and separation regime.
 The best known synthetic membrane separation processes include
water purification, reverse osmosis, dehydrogenation of natural gas,
and removal of cell particles by microfiltration and ultra filtration,
removal of microorganisms from dairy products, and Dialysis.

3. Chromatographic Retention Indicies

 In gas chromatography, Kovats retention index (shorter Kovats

index, retention index; plural retention indices) is used to convert
retention times into system- independent constants.
 The index is named after the Hungarian- born Swiss chemist Ervin
Kováts, who outlined this concept during the 1950s while performing
research into the composition of the essential oils.
 The retention index of a certain chemical compound is its retention
time normalised to the retention times of adjacently eluting n-
alkanes. While retention times vary with the individual
chromatographic system (e.g. with regards to column length, film
thickness, diameter, carrier gas velocity and pressure, and void
time), the derived retention indices are quite independent of these
parameters and allow comparing values measured by different
analytical laboratories under varying conditions.
 Tables of retention indices can help identify components by
comparing experimentally found retention indices with known values

4. Brush Border Membrane Vesicles

 A brush border (striated border or brush border membrane) is the

microvilli-covered surface of simple cuboidal epithelium and simple
columnar epithelium cells found in certain locations of the body.

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 Microvilli are approximately 100 nanometers in diameter and their

length varies from approximately 100 to 2,000 nanometers in length.
 Because individual microvilli are so small and are tightly packed in
the brush border, individual microvilli can only be resolved using
electron microscopes

5. Using Isolated Intestinal Cells

 Here, the small intestine is perfused with enzyme solutions that

release the cells are treated with chelating agents or enzymes ▸
The freshly isolated cells are suspended in buffer solution
 At the time of experiment, the cells are separated, resuspended in
buffer containing the drug under 02/CO2 and shaken well. ▸ After
a specific period of time, the cells are separated by filtration,
extracted and drug absorbed is determined.

6. Tissue Techniques

a. Everted small intestinal sac technique:

 This method involves isolating a small segment of the intestine of a

laboratory animal such as rat, inverting the intestine and filling sac
with a small volume of drug free buffer solution.
 Both the segments are tied off and the sac is immersed in an
Erlenmeyer Flask containing a large volume of buffer solution that
contains the drug.
 The flask and its contents are they oxygenated and the whole
preparation is maintained at 37 degree celsius and shaken mildly.
 At predetermined time intervals, the sac is removed and the
concentration of drug in the serosal fluid is determined/assayed for
drug content.

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b. Everted Sac Modification

 In this method, the test animal is fasted for a period of 20-24 hr

and water is allowed.
 The animal is killed and the entire small intestine is everted.
Segments,5-15 cm in length are cut from a specific region of the
intestine.
 The distal end of the segment is tied and the proximal end is
attached to the cannula. The segment is suspendedin a mucosal
solution which contains the drug.
 A drug free buffer is then placed in the serosal compartment.

c. Circulation Techniques

 In this method, small intestine may or may not be everted.

 This involves isolating either the entire small intestine of small lab
animal or a segment and circulating oxygenated buffer containing
the drug through the lumen.
 Drug free buffer is circulated on the serosal side of the intestinal
membrane and oxygenated
 Absorption rate from the lumen to the outer solution are
determined by sampling both the fluid circulating through the lumen
and outside

d. Everted Intestinal Ring or Slice Technique

 In this technique, the entire small intestine is isolated from the

fasted experimental animal and washed with saline solution and dried
by blotting with filter paper.
 The segment is tied at one end and by placing on glass rod it is
carefully everted and cut into small rings.
 The everted intestinal rings are then incubated in drug containing
buffer maintained at 37 degree celsius with constant oxygenation.

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 Under optimal conditions, rings remain viable for up to 2 hrs and the
transport of drug is stopped by rinsing the rings with ice cold
buffer and drying them.

7. Diffusion Cell Method

 In this method, small segments of small intestine are mounted

between two glass chambers filled with buffer at 37 degree celsius.
 Diffusion cell consist of two compartments:
a. Donor compartment - which contains the drug solution and the
lower end of which contains the synthetic or natural GI
membrane that interface with the receptor compartment.
b. Receptor compartment - which contain the buffer solution.

8. Cell Culture Techniques

 Cell culture is the complex process by which cells are grown under
controlled conditions, generally outside their natural environment.
 In this technique, differentiated cells of the intestine, originating
from CaCo2 cells (cells or carcinoma of colon )are placed on
synthetic polycarbonate membrane previously treated with an
appropriate material such as collagen which on incubation aids
reproduction of cells while not retarding drug permeation
characteristics
 These models are based on the assumption that passage of the
drugs across the intestinal epithelium is the main barrier for drugs
to reachthe circulation.

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2. In Vivo Methods

In vitro and In situ techniques gives us an idea about absorption, but in

vivo method gives us an idea about some important factor that
influence motility and the effects of drugs on the GIT Can be
determined.

The In Vivo method can be classified into:

1. Direct method
2. Indirect Method

1. Direct Method

 The drug levels in blood or urine is determined as a function of time.

For this, a suitable sensitive reproducible analytical procedure
should be developed to determine the drug in the biological fluid.
 In this method, blank urine or blood sample is taken from the test
animal before the experiment.
 The test dosage form is administered to the animal and at
appropriate intervals of time the blood or urine samples are
collected and assayed for the drug content.
 From the data, we can determine the rate and extent of drug
absorption.
 In this method, the experimental animal chosen should bear some
resembles to man.
 It is reported that pigs most closely resemble to man but are not
used due to the handling problems.
 The other animals that can be used are dogs, rabbits and rats.

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2. Indirect Method

 When the measurement of drug concentration in the blood or urine

is difficult or not possible, but a sensitive method is available to
test the activity, then absorption studies can be done by this
indirect method.
 In this method, pharmacological response of the drug is related to
the amount of the drug in the body.
 The response is determined after the administration of a test
dosage form; LD 50 appears to be dependent on the rate of the
absorbing of drug.

3. In Situ Method

 It stimulates the in vivo conditions for drug absorption and are

based on perfusion of a segment of GIT by drug solution and
determination of amount of drug diffused through it.
 In situ refers to those method in which the animals blood supply
remains intact in which the rate of absorption determined from
these methods may be more realistic than those determined from in
vitro techniques
 These models are powerful tools to study the mechanistic aspects
of this important process.
 Acts as a bridge between in vivo and in vitro methods.

I - Absorption of drug from small intestine

Absorption of drug from small intestine is described in two methods:

1. Perfusion technique:
 Doluisio method
 Single pass perfusion method

2. Intestinal loop techniques

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II- Absorption from the stomach

 Fasted adult male rats are anaesthetized, stomach is exposed and

the cardiac end is ligated.
 An incision is made in the pylorus in which the cannula is introduced
and ligated.
 The lumen is washed several times with saline and subsequently with
0.1 N HCL solutions containing 0.15M NaCl.
 The drug solution of known concentration is introduced into the
stomach and after 1 hour, the solution is removed from the gatric
pouch and assayed for drug content.

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Unit 2 - Biopharmaceutic Considerations in Drug

Product Design and In Vitro Drug Product
Performance
Introduction

 Biopharmaceutics studies the in vitro impact of physicochemical

properties of drugs and drug products on delivery to body under
normal or pathologic conditions.
 Biopharmaceutics links the physical and chemical properties of drug
and drug product to their performance, in vivo.
 The aim of biopharmaceutics is to adjust the delivery of drug from
drug products in such a manner as to provide: optimal therapeutic
activity and safety for the patient.
 Biopharmaceutic considerations often determine the ultimate dose
and dosage form of a drug product. For example, the dosage for a
drug intended for local activity, such as a topical dosage form, is
often expressed in concentration or as % of the active drug in the
formulation.
 Drug products include the active drug substance combined with
special additional ingredients (excipients) that make up the dosage
form.
 Although excipients are considered inert with respect to
pharmacodynamics activity, excipients are important in the
manufacture of the drug product and provide functionality to the
drug product with respect to drug release and dissolution.

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Each route of drug application presents special Biopharmaceutic

considerations in drug product design.

By carefully choosing the route of administration and properly

designing the drug product, the bioavailability of the active drug can
be varied from rapid and complete absorption to a slow, sustained rate
of absorption or even virtually no absorption, depending on the
therapeutic objective.

Example: An eye medication may require special Biopharmaceutic

considerations including appropriate pH, isotonicity, local irritation to
the cornea, draining by tears, and concern for systemic drug
absorption.

Scope of Biopharmaceutics

 Encompass all the possible physiological factors which may effect

the drug in various dosage forms.
 Encompass all the possible effects of various dosage forms on
biological region.
 A primary concern in biopharmaceutics is the bioavailability of
drugs.
 Bioavailability is the assessment of the rate and extent at which the
active drug becomes available at the site of action.

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Biopharmaceutics Considerations in Drug Product

Design
1. Pharmacodynamics considerations
2. Drug considerations
3. Drug product considerations
4. Patient considerations
5. Manufacturing considerations

1. Pharmacodynamics Considerations - Therapeutic objective

 Toxic effects
 Adverse reactions

2. Drug Considerations

 Chemical & physical properties of drug

 pka & pH profile
 Particle size
 Polymorphism
 Hygroscopicity
 Partition coefficient
 Excipients interaction
 pH stability profile
 Solubility

3. Drug Product Considerations

 Pharmacokinetics of drug
 Bioavailability of drug
 Desired dose of drug
 Dosing frequency

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4. Patient Consideration

 Compliance & acceptability of drug product

 Cost

5. Manufacturing Considerations

 Cost
 Availability of raw materials
 Stability
 QC

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Biopharmaceutic
pharmaceutic Factors Affecting Drug
Bioavailability
There are various factors which affect the bioavailability of drug-
drug

1. Pharmaceutical Factors
2. Patient Related Factors
3. Route of Administration

1. Pharmaceutical Factors:

It is expected that, bioavailability of drugs to be in this decreasing

order-

Solutions > Suspension > Capsule > Tablet > Coated Tablet

Pharmaceutical Factors: Formulation factors

1. Disintegration time
2. Manufacturing variables
a. Method of granulation
b. Compression force
3. Nature & type of dosage form
4. Pharmaceutical ingredients
5. Product age & storage conditions

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1. Disintegration time (DT)

 It is defined as the time taken by the solid dosage form to

breakdown into smaller particles in the body after their ingestion.
 Order of disintegration of the solid dosage forms:

Capsules > Tablets > Coated tablets > Enteric coated tablets >
sustained release tablets

 Harder the tablet, greater is its disintegration time.

 Disintegration of solid dosage forms can be enhanced by
incorporating appropriate amounts of disintegrants in the
formulation.

2. Manufacturing variables

a. Method of granulation

 Wet granulation: By selecting a suitable granulating liquid, the

dissolution rate of insoluble drugs can be enhanced.
 Direct compression: dissolution rate of tablets prepared by this
method are higher than the wet granulation method.

b. Compression force

 Higher compression force yields a tablet with greater hardness

and reduced wettability & hence has a long D.T. but on other hand
higher compression force cause crushing of drug particles into
smaller ones with higher effective surface area which in decrease
in D.T.
 So effect of compression force should be thoroughly studied on
each formulation.

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3. Nature and type of dosage form

Depending upon the nature and type of dosage form, the absorption
pattern of a drug decreases in the following order;

Solutions > Emulsions > Suspensions > Capsules > Tablets > Coated
tablets > Enteric coated tablets > Sustained release tablets

4. Pharmaceutical Ingredients

More the no. of excipients in dosage form, more complex it is & greater
the potential for absorption and Bioavailability problems.

a. Vehicle: Vehicles are used in Parenteral and oral liquids

preparations.
 Rate of absorption - depends on its miscibility with biological
fluid.
 Miscible solvents-rapid
rapid absorption of drug.
 Immiscible solvent
solvent-slow absorption of drug.

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 Non-Aqueous water immiscible E.g.: Vegetable oil, Sesame oil,

Peanut oil.
 Aqueous - E.g.: Water, Syrup
 Non-Aqueous water miscible E.g.: Propylene glycol, Glycerol,
Sorbital.

b. Diluents: Diluents are added to increase the bulk of the dosage

form, especially in tablets and capsules.

 Hydrophilic diluents - Form the hydrophilic coat around

hydrophobic drug particles-thus promotes dissolution and
absorption of poorly soluble hydrophobic drug.
 Inorganic diluents - E.g.: Dibasic calcium phosphate, Calcium
carbonate.
 Organic diluents - E.g.: Dextrose, Sorbitol, Mannitol. Diluents

c. Binding Agents

 Although binders are incorporated to produce cohesive bonding

between granules during the process of compaction of tablets.
 Hydrophilic binders are for enhancing the dissolution rate of
poorly soluble drug. E.g. starch, gelatin, PVP.
 More amount of binder increases hardness of tablet and
decreases dissolution & disintegration rate.

d. Disintegrating Agents

 They are added to the tablet to disrupts the cohesive forces

between the granules, thereby causing the breakdown of the
tablet to attain faster dissolution.
 Mostly hydrophilic in nature, increase in disintegration increases
the bioavailability.
E.g.: Guar gum, Starch, Microcrystalline cellulose

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e. Lubricating Agents

 These agents when added to a tablet formulation decrease the

friction between the granules and die wall of the tablet press.
 Commonly hydrophobic in nature - therefore inhibits penetration
of water into tablet and thus dissolution and disintegration.
 Insoluble Lubricants, E.g.: Mineral oil, Talc
 Soluble Lubricants, E.g.: PEG 4000, PEG 6000.

f. Surfactants

 They are commonly used in the formulations as solubilizers,

emulsifiers, wetting agents etc.
 At lower concentrations, they increase the rate of absorption of
poorly water soluble drugs.
 Physiologic surfactants like bile salts they promotes absorption
E.g.: Griseofulvin, steroids

g. Complexing Agents

They increase the absorption rate of other drugs due to ;

 Formation of soluble complexes which enhances the dissolution.

 Increased Lipophilicity which enhances membrane permeability.

h. Colorants

 Water-soluble dyes even in least concentrations get adsorbed on

the crystal faces and delay their dissolution rate.
 E.g.: Brilliant blue retards dissolution of sulfathiazole.

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5. Product age and storage Conditions: Alterations in storage

conditions and prolonged duration of storage of drug products may
modify their physicochemical properties resulting in altered drug
absorption patterns.

Patient Related Factors:

1. Age
2. Gastric Emptying
3. Intestinal Transit
4. Diseases
5. Effect of Food
6. Blood Flow to GIT
7. First pass metabolism

1. Age
 Absorption pattern of drugs may vary among different age
groups.
 Infant have less acidic G.I fluids, smaller intestinal surface area
and comparatively less blood flow than adults.
 Intestinal surface area and blood flow, bacterial overgrowth in
small intestine, altered gastric emptying, which retards the drug
absorption

2. Gastric Emptying

 Gastric emptying is the entry of gastric content into the small

intestine.
 Gastric emptying rate: It is the rate at which gastric contents
empty into the small intestine.
 Gastric emptying time: It is the time required for gastric content
to empty into the small intestine.

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 Rapid Gastric Emptying is required when the consumed drugs

 Are unstable in gastric pH (Penicillin G)
 Are better absorbed from the small intestine (Vit B12)
 Delayed Gastric Emptying is required when
 The drug (Griseofulvin) dissolves slowly.
 Food enhances the dissolution and absorption of drugs.

3. Intestinal Transit

 The residence time of food or drug substance in intestine is

known as intestinal transit time.
 As small intestine is major site of absorption, longer or delayed
transit time is required for the complete absorption of drugs.
 Delayed intestinal transit is recommended for those drugs which
 Exhibits sustained release action. (Diclofenac sodium)
 Are enteric coated and hence dissolves only in the intestine.

4. Diseases

a. GI Diseases and Infections: Drug absorption may be influenced by

several pathophysiological conditions of GIT. Malabsorption
syndrome like celiac disease and Chrons disease.
b. Gastrointestinal Surgery: Gastrointestinal surgery especially
gastrectomy may cause drug dumping in the intestine.

5. Effect of Food

 The presence of food in the GI tract can affect the bioavailability

of the drug from an oral drug product.
 Food contains amino acids, fatty acids, and many nutrients that may
affect intestinal pH and solubility of drugs.
 The effects of food are not always predictable and can have
consequences.

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Some effects are:

 Delay in gastric emptying

 Stimulation of bile flow
 A change in the pH of the GI tract
 A change luminal metabolism of the drug substance
 Physical or chemical iinteraction
nteraction of the meal with the drug
product or drug substance.
 The absorption of some antibiotics, such as penicillin and
tetracycline, is decreased with food; whereas other drugs,
particularly lipid-soluble
soluble drugs such as griseofulvin and metazalone,
are better absorbed when given with food containing a high fat
content.

6. Blood Flow to GIT: Increase in the blood flow to the site of

absorption (GIT), increases the drug absorption as rapid removal of
drug from its absorption site helps to maintain sink con
conditions.
ditions.

7. First pass metabolism: A drug administered orally, passes through

the GIT and liver where it undergoes extensive metabolism before
reaching the systemic circulation, thereby leading to decreased
bioavailability, this phenomenon is called as fir
first
st pass metabolism.

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Rate-Limiting
Limiting Steps in Drug Absorption
 Systemic drug absorption from a drug product consists of a
succession of rate processes.
 For solid oral, immediate
immediate- release drug products (e.g., tablets,
capsules), the rate processes include:
include:-
1. Disintegration of the drug product and subsequent release of the
drug,
2. Dissolution of the drug in an aqueous environment, and
3. Absorption across cell membranes into the systemic circulation.
 In the process of drug disintegration, dissolution, and absorption,
absorptio
the rate at which drug reaches the circulatory system is determined
by the slowest step in the sequence. The slowest step in a series of
kinetic processes is called the rate
rate-limiting step.
 Except for controlled release products, disintegration of a solid
soli oral
drug product is usually more rapid than drug dissolution and drug
absorption.
 For drugs that have very poor aqueous solubility, the rate at which
the drug dissolves (dissolution) is often the slowest step and
therefore exerts a rate
rate-limiting effect on drug bioavailability.
 In contrast, for a drug that has a high aqueous solubility, the
dissolution rate is rapid, and the rate at which the drug crosses or
permeates cell membranes (absorption) is the slowest or rate- rate
limiting step.

Rate processes of dr
drug bioavailability

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Physicochemical Nature of the Drug Formulation

Physicochemical properties of drug substances: A detailed study of
physicochemical properties of drug molecules may help to enhance the
rate as well as the extent of drug absorption. It also serves to
formulate the drug in most suitable dosage form.

1. Drug dissolution & solubility rate.

2. Particles size & effective surface area.
3. Salt form of drug.
4. Polymorphism & amorphism.
5. Solvates & hydrates.
6. Ionization state.
7. Drug pKa & lipophilicity & GI pH - pH partition hypothesis.

1. Drug dissolution & solubility rate

 Dissolution is the process of solubilization of a substance in a

given solvent.
 Drug dissolution rate is the amount of drug that goes into
solution per unit time under the standard conditions of o
temperature, pH, solvent composition and constant solid surface
area.
 Dissolution plays a significant role as it is regarded as the rate
determining step in the process of absorption.
 Solutions > Suspensions > Capsules > Tablets > Coated tablets.

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Solubility: The quantity of drug that dissolves in the gastrointestinal

fluid is indicative of vivo drug absorption.

 A drug is said to undergo appreciable bio absorption if it exhibits

aqueous solubility greater than 10mg/ml at 37°C and pH ranging
between 1-7.
 When the solubility is less than 1mg/ml, it undergoes undesirable GI
absorption.
 Thus with the aid of solubility and dissolution data, potential
problems related to bioavailability and therapeutic activity of the
drug can be recognized.
 Both solubility and absorption can be correlated by the concept of
maximum absorbable dose (MAD).

2. Particle Size & effective surface area

Types of surface area:

1. Absolute surface area: it is the total solid surface area

2. Effective surface area: it is the solid surface area of particle
exposed to the dissolution medium.
 In order to convert the absolute surface area to effective surface
area, surfactants like polysorbate 80 or diluents like PEG, dextrose
are used.
 Griseofulvin, chloramphenicol on micronization show increased
absorption and decrease in therapeutic dose.

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3. Salt form of drug

 At given pH, the solubility of drug, whether acidic/basic or its salt,

is a constant. While considering the salt form of drug, pH of the
diffusion layer is important not the pH of the bulk o
off the solution.
 E.g. salt of weak acid. Which increases the pH of the diffusion
layer, which promotes the solubility and dissolution of a weak acid
and absorption is bound to be rapid.

4. Amorphism and Polymorphism

 Amorphous forms generally dissolve faster than crystalline forms

because no energy is needed to break up the crystal lattice.
 For this reason, the amorphous form is often preferred over the
crystalline form and several drugs, including hydrocortisone and
prednisolone, are marketed in the amorphic form.

 The crystalline form of drugs may exist as polymorphs or molecular

adducts or both.

5. Solvates & hydrates:

 During their preparation, drug crystals may incorporate one or more

solvent molecules to form solvates.
 The solvent trapped is known as sol
solvent
vent of crystallization.
 The solvates may exists in varying crystalline forms known as pseudo
polymorphs and the phenomenon is known as pseudo polymorphism.

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 The molecular complex is referred as hydrates if water molecules

has been reported as solvent.
 Anhydrous - Drug is not associated with water, monohydrate and
dehydrated - drug is associated with one and more water molecules
respectively.
 The anhydrous form have higher energy states, higher aq.
solubilities, dissolves at faster rate and hence exhibit higher
bioavailability.
Ex: anhydrous ampicillin is more soluble than their hydrous form.

6. Ionization state:

 Unionized state is important for passive diffusion through

membrane, hence important for absorption.
 Ionized state is important for solubility.

7. Drug pKa & lipophilicity & GI pH - pH partition hypothesis :

 pH - Partition theory states that drug compounds of molecular

weight more than 100 Daltons, which are primarily transported
across the biological membrane by passive diffusion, their process
of absorption is governed by
 Pka of drug (Dissociation constant)
 The lipid solubility of unionized drug
 pH at the absorption site.

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Drug Product Performance

 Defined as the release of the drug substance from the drug product
leading to bioavailability of the drug substance.
 The assessment of drug product performance is important since
bioavailability is related both to the pharmacodynamics response and
to adverse events.
 Thus, performance tests relate the quality of a drug product to
clinical safety and effic
efficacy.

Bioequivalence Studies in New Drug Development (NDA):

Bioequivalence Studies in Generic Drug Development (ANDA):

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Factors Affecting Drug Product Performance

1. Physiochemical nature of the drug

The physical and chemical properties of the drug substance as well as

the excipients are important considerations in the design of a drug
product. For example,

 Intravenous solutions are difficult to prepare with drugs that

have poor aqueous solubility.
 Drugs that are physically or chemically unstable may require
special excipients, coatings, or manufacturing processes to
protect the drug from degradation.
 The potent pharmacodynamic activity of drugs such as estrogens
and other hormones, penicillin antibiotics, cancer
chemotherapeutic agents, and others, may cause adverse
reactions to personnel who are exposed to these drugs during
manufacturing.

2. The nature of the excipients in the drug product

 Excipients in the drug product affect the dissolution kinetics of

the drug, either by altering the medium in which the drug is
dissolving or by reacting with the drug itself.
 Suspending agents increase the viscosity of the drug vehicle and
thereby diminish the rate of drug dissolution from suspensions.
 Excessive quantity of magnesium stearate (a hydrophobic
lubricant) in the tablet formulation may repel water and retard
drug dissolution and slow the rate of drug absorption.
 Coatings, particularly shellac, will crosslink upon aging and
decrease the dissolution rate.
 Low concentrations of surfactants decrease the surface tension
and increase the rate of drug dissolution, whereas higher

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surfactants concentrations tend to form micelles with the drug

and thus decrease the dissolution rate.
 Excipients, such as sodium bicarbonate, may change the pH of the
medium surrounding the active drug substance.
 Aspirin, a weak acid when formulated with sodium bicarbonate,
will form a water-soluble salt in an alkaline medium, in which the
drug rapidly dissolves.
 Excipients in a formulation may interact directly with the drug to
form a water-soluble or water-insoluble complex.
 For example, if tetracycline is formulated with calcium carbonate,
an insoluble complex of calcium tetracycline is formed that has a
slow rate of dissolution and poor absorption.

3. The method of manufacturing

 High compression of tablets without sufficient Disintegrants may

cause poor disintegration of a compressed tablet.
 In some cases, a drug product is designed so that it may be used
in conjunction with a specialized medical device.
 For example, a drug solution or suspension may be formulated to
work with a nebulizer or metered-dose inhaler for administration
into the lungs.
 Both the physical characteristics of the nebulizer and the
formulation of the drug product can influence the droplet
particles and the spray pattern that the patient receives upon
inhalation of the drug product.
 Bioavailability of a drug from different dosage forms would
decrease in the following order;

Solution > suspension > capsule > tablet > coated tablet

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Dissolution and Drug Release Tests

 Dissolution and drug release tests are in-vitro test s that measure
the rate and extent of dissolution or release of the drug substance
from a drug product, usually in an aqueous medium under specified
conditions
 The dissolution test is an important quality control procedure for
the drug product and is often linked to product performance in vivo.
 In-vitro drug dissolution studies are most often used for monitoring
drug product stability and manufacturing process

Conditions that May Affect Drug Dissolution and Release: Drug and
formulation related

1. Drug substance
 Particle size
 Polymorph
 Surface area
 Chemical stability in dissolution media

2. Formulation of drug product

 Excipients (lubricants, suspending agents, etc)

Conditions that May Affect Drug Dissolution and Release:

methodology related

1. Medium
 Volume
 pH
 Molarity
 Co-solvents, added enzymes/surfactants
2. Temperature of medium
3. Apparatus

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4. Hydrodynamics
 Agitation rate
 Shape of dissolution vessel
 Placement of tablet in vessel
 Sinkers (for floating products and products th at stick to side of
vessel)

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Compendial Methods of Dissolution

USP apparatus type I (Basket)

Common problems associated with the rotating basket test

 Clogging
 In homogeneity in the agitation
 Wire basket corrode
corrodes
 Low Media Agitatio
Agitation
 Dissolution being accelerated due to
abrasion

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USP apparatus type II (Paddle)

 A paddle replaces the basket as the source of

agitation
 The metallic blade and shaft comprise a single
entity that may be coated with a suitable inert
coating to prevent corrosion.
rrosion.
 For hard-gelatin
gelatin capsules and other floating
dosage forms, a "sinker" is required to weight
the sample down until it disintegrates and
releases its contents at the bottom of the
vessel.

USP apparatus type III (Reciprocating Cylinder)

 This type iss used for modified release

dosage forms.
 It is necessary to obtain a correlation
between in vitro dissolution results and
the bioavailability of these products (in
vitro-in
in vivo correlation).
 So it is essential for the pH,
composition, ionic strength, vis
viscosity
and agitation speed of the medium to
be altered during the dissolution test,
thus simulating passage of the product through the gastrointestinal
tract.
 For this purpose the reciprocating cylinder method was developed.
 The internal cylinders remain iin
n each line of vessels, in reciprocal
movement, for pre- programmed times and intensities (dips per
minute or "dpm") in the apparatus.

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 During emersion, the agitation system rises until the screen in the
lower cover touches the dosage form, which separates from the
screen and floats freely in the dissolution medium when the stirring
system activates.
 After the programmed period, the rods rise until the internal
cylinders are positioned over the vessels, where they remain for a
pre- established timeframe so that the dissolution medium can
drain.

USP apparatus type IV (Flow through Cell)

Cells: Different types of cells are available for testing tablets,

powders, suppositories, hard- and soft- gelatin capsules, implants,
semisolids, suppositories, and drug-eluting stent

Pump and Flow Patterns:

 Peristaltic and pulsating piston pumps can be used. Usually a

sinusoidal pulse rate of 120 ± 10 pulses per minute is used. The pulse
rate remains constant independent of the selected flow rate
 The need for further stirring is eliminated due to the pulsating
pattern of the pump
 The open system is selected for samples that require high volume of
media (i.e., low solubility compounds), and the closed system is
selected when a low volume of medium is required.

Filtration:

 A filter is at the inner top of the cell to retain undissolved material.

Usually glass fiber filters are used.
 Appropriate selection of the filter is required for efficient
filtration and to avoid backpressure created by filter resistance.

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Sampling:

 The collected samples can be analyzed directly by a UV vis

spectrophotometer or they can be collected in fractions and
analyzed by HPLC

USP apparatus type V (Paddle over Disc)

This uses the paddle apparatus (USP 2) with the sample, usually a
Transdermal delivery system, being attached to a stainless-steel disk,
which is then placed on the bottom of the vessel, directly under the
paddle.

USP apparatus type VI (Rotating Cylinder)

This is a modification of the basket apparatus (USP Apparatus 1) with

the basket being replaced by a stainless steel cylinder. The sample is
again usually a Transdermal delivery system attached to the outside of
the cylinder.

USP apparatus type VII (Reciprocating Holder)

 This variant is based on a sample holder that oscillates up and down

in the medium vessel.
 The sample holder may take the form of a disk, cylinder, or a spring
on the end of a stainless steel or acrylic rod, or it may simply be the
rod alone.
 The sample is attached to the outside of the sample holder either
by virtue of being self-adhesive (e.g., transdermal delivery system)
or is glued in place using a suitable adhesive.

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Alternative Methods for Dissolution

Non official methods

a. Natural convection non sink methods

1. Klein solvmeter method

2. Nelson hanging pellet method
3. Levy static disk method

b. Forced convection non sink methods

1. Tumbling method
2. Levy or Beaker method
3. Rotating disk method

a. Natural Convection Non Sink Method

 'In this method the density difference is utilized for replacing the
surrounding dissolution medium'

Klein Solvmeter method

 Carrier device surrounded by flat and is immersed in dissolution

medium
 When dosage form is placed in the boat the bar moves and as dosage
form dissolves it moves upwards
 Amount of dosage form dissolved Amount of dosage form dissolved
height of bar movement

Nelson hanging pellet method - Aluminum strip having provision for

holding dosage form which is in turn connected perfectly maintained
balance arm of strip.

Dosage form is mounted on aluminum strip with help of wax. This

method can be employed to know intrinsic dissolution rate.

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To prevent disintegration further high pressures can be applied and

also constant surface.

Levy static Disk method

 Acrylic holder containing dosage form is inserted into a known

volume of medium through rubber stopper.
 Acrylic holder containing dosage form is inserted into a known
volume of medium through rubber stopper

Disadvantages: effect of conc. On dissolution medium is ignored and

the surface area of dosage form while dissolving is assumed constant
which is not impractical.

b. Forced Convection Non Sink Methods

Tumbling Method:

 The Drug/ Dosage form with the dissolution medium is placed in test
tube that is in turn clamped to the revolving drum which is rotated
at the speed of 6- 12rpm in water bath at 37 C
 The test tubes are removed and the medium is assayed at regular
time points for the dissolved drug amount

Beaker method

 Dissolution medium, 250ml of 0.1N HCl at 37°C placed in a 400ml

beaker.
 Agitation by three blade polyethylene stirrer, 5cm diameter and
rotates at 60 rpm.
 Stirrer immersed to a depth of 2.7 cm in medium and in the center.
 Tablets are placed in a beaker and test was carried out.
 Samples are removed and assayed for the content.

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Rotating disk method

 Developed by late Eino nelson and described by Levy and Sahli.

 In this method, the drug is compressed in a non-disintegrating disc
without excipients.
 The disc is mounted in a holder so that only one face of the disc is
exposed to the dissolution medium.
 The holder and disc are immersed in medium and held in a fixed
position as in static disc method and rotated at a given speed in
rotating disc method.
 Samples are collected at predetermined times.
 Surface area of the drug through which dissolution occurs is kept
constant –intrinsic dissolution Rate.

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Meeting Dissolution Requirements

According to the Code of Federal Regulations (CFR), a drug product
application should include the following specifications necessary to
ensure the

 Identity,
 Strength,
 Quality,
 Purity,
 Potency,
 Bioavailability of the drug product,
 Including, and acceptance criteria relating to, dissolution rate in
the case of solid dosage forms.

Dissolution acceptance criteria

For the selection of the dissolution acceptance criteria, the following

points should be considered:

1. The dissolution profile data from the pivotal clinical batches and
primary (registration) stability batches should be used for the
setting of the dissolution acceptance criteria of your product (ie,
specification- sampling time point and specification value).
 A significant trend in the change in dissolution profile during
stability should be justified with dissolution profile comparisons
and in vivo data in those instances where the similarity testing
fails
2. Specifications should be established based on average in vitro
dissolution data for each lot under study, equivalent to USP Stage 2
testing (n = 12).
3. For immediate-release formulations, the last time point should be
the time point where at least 80% of drug has been released.

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 If the maximum amount released is less than 80%, the last time
point should be the time when the plateau of the release profile
has been reached. Percent release of less than 80% should be
justified with data (eg, sink conditions information)
4. For extended-release formulations, a minimum of three time points
is recommended to set the specifications. These time points should
cover the early, middle, and late stages of the release profile.
 The last time point should be the time point where at least 80%
of drug has been released. If the maximum amount released is
less than 80%, the last time point should be the time when the
plateau of the release profile has been reached.
5. The dissolution acceptance criterion should be set in a way to ensure
consistent performance from lot to lot, and this criterion should not
allow the release of any lots with dissolution profiles outside those
that were studied clinically.
 The term Q means the amount of drug dissolved within a given
time period established in the drug product specification table
and is expressed as a percentage of label content.
 For example, a value of Q = 80% at 30 minutes means that the
mean percent dissolved of 12 units individually tested is at least
80% at the selected time point of 30 minutes.
 When implementing dissolution as a quality control tool for batch
release and stability analysis, the testing should follow the
recommendations listed in the USP method <711> for immediate-
release dosage forms and <724> for modified-release dosage
forms.
 For example, for Stage 1, which considers the testing of 6 units,
each unit must meet the criterion of not less than 85% at 30
minutes for a drug product whose acceptance criterion was set to
Q = 80% at 30 minutes.

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 Testing should continue through the three stages (S1, S2, S3)
unless the results conform at either Stage 1 or Stage 2 (Table
15-9)
 The USP-NF monographs may have multiple dissolution tests for
generic drug products that are approved by the FDA as
therapeutic equivalents.
 Although both the brand and approved generic drug products are
bioequivalent, there in vitro dissolution profiles may be different.
 Ideally, both methods should have very similar discriminating
ability; however, this can only be determined when an IVIVR or an
IVIVC has been established for the drug products rending the
method not only discriminating but also predictive of in vivo
performance.

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Problems of Variable Control in Dissolution Testing

The source of deviation from accurate results in dissolution test can
be broadly classified into following categories:

1. Equipment Related Factors

2. Process Related Factors
3. Drug Substance Properties Related Factors
4. Drug Product Properties Related Factors
5. Miscellaneous Factors

1. Equipment Related Factors

Dissolution equipment is a machine. The initial quality of the device and

its subsequent care and maintenance will influence both operational
reliability and product dissolution rate results. A discussion of the
dissolution equipment is important as the dissolution rate is generated
by the stirring mechanism interacting with the dosage form in the
media. The environment in which it operates will affect performance,
and it needs to be running properly at all times

Some of the parameters associated:

a. Dissolution Test Vessel

b. Paddle/ Basket Shaft
c. Vibrations
d. Use of Filters
e. Calibration of Dissolution Vessel

a. Dissolution Test Vessel:

 Dissolution testing is an attempt to create a perfectly controlled

space, with a hydrodynamically consistent environment. "Ideally the
upper portion of each vessel would be perfectly cylindrical, and its
bottom would be a perfect hemisphere.

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 They are made one at time by manually blowing a molten mass of

glass into molds. As a result, the vessels are not uniform with
respect to weight, height, cylindrical shape, hemispherical
curvature, and inner diameter. The inside surface of each should be
inspected for abnormalities."
 Some have concluded that a major source of dissolution result
variability appears to be the geometric parameters of the
dissolution vessel and stirring mechanism.
 A precision vessel (Takao, Japan) was manufactured by a new glass
processing technology and displayed an almost ideal inner shape
consisting of a cylinder and hemisphere. In contrast, two
conventional vessels, vessel A (manufacturer A, Japan) and vessel B
(manufacturer B, USA), exhibited distortion and unevenness in
various places. The vessel bottoms in particular deviated from the
ideal hemispherical shape, and curvature among vessels differed
widely. The inside of the cylinder of these two manufacturers'
vessels also deviated from the ideal circular shape.
 The dissolution results of USP Prednisone Calibrator tablets were
compared for the precision vessel and vessel A. For vessel A,
however, test results varied widely between vessels used and
between positions in the dissolution tester. In addition, the mean
values of prednisone dissolution percentages obtained from six
positions differed significantly (p < 0.05) among vessels.
 These results suggest that the shape of a glass vessel is critical to
obtaining unvarying and reproducible dissolution test results.

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b. Paddle/ Basket Shaft:

Close inspection of USP apparatus I & II before use can help identify
sources of error. In both cases, shafts must be straight and true. The
paddles are sometimes partially coated with Teflon. This coating can
peel and partially shed from paddle, causing flow disturbance of
hydrodynamics within the vessel. Paddles can rust and become nicked
and dented; this can adversely affect dissolution hydrodynamics and be
a source of contamination.

Two types of basket shafts are commercially available to the analyst.

One type has an O-ring inset in the disk at the end of the shaft with
the basket fitting around the O-ring. The other has three clips
attached to the disk at the end of the shaft. The basket is attached
by fitting between the clips and the disk. There was no difference
between the two basket shaft types for the three development
products.

The difference showed a higher dissolution rate using the clipped

basket shaft design. The clipped basket shaft is the official USP
design; however there are some drawbacks to this design. The clips
protrude and disturb the fluid flow in the vessel.

c. Vibrations:

Vibration is a complicated concept that can result in the addition of

energy to a system. The addition of energy from an external source
can alter the results of a dissolution evaluation. Such an alteration is
an unacceptable source of error that must be minimized. As far as USP
is concerned it has been noted that there is brief mention of
vibrations requirements consisting of statement that "No part of the
assembly, including the environment in which the assembly is placed,

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contributes significant motion, agitation, or vibration beyond that due

to the smoothly rotating stirring element".

d. Use of Filters

Most current dissolution procedures require samples to be withdrawn

from the vessel, the exception being when the concentration is
determined in situ with fiber optics. No matter whether the sample is
withdrawn manually or automatically, effective filters are necessary to
prepare the sample for analysis; otherwise, undissolved material from
the medium could influence the results. All of the filter materials
available on the market for dissolution testing may not be equally
suitable for this task.

For example, it is important that filter materials should have little or

no tendency to absorb the drug, since adsorption to the filter will
result in out-of-specification results.

e. Calibration of Dissolution Apparatus

The process by which a test apparatus is determined to meet the

compendial (dimensional and operational) specifications has been
termed mechanical calibration, while the use of reference standard
tablets is given the designation chemical calibration. Chemical
calibration allows a final and summative confirmation of the suitable
operation of the integrated dissolution assembly, beyond the evaluation
of its separate component attributes (e.g., stirring element dimensions,
control of rotation speed, dissolution medium volume).

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2. Process Related Factors

The dissolution test should not be considered as absolute analytical

procedure. It is always comparative, whether as a bioequivalence test
assessing different formulations, as a stability test comparing stored
products with the original one, or as a quality control test comparing
different batches against previously established limits. As a
comparative test procedure, the consistency and reproducibly of both
the analytical equipment itself and the techniques used are essential.

Various factors associated with dissolution test procedure that may

lead to errors:

1. Use of water as dissolution media

2. Sample introduction
3. Single point Vs multiple point sampling
4. De-aeration of media
5. Controlled release dosage forms

1. Use of Water as Dissolution Media:

Many compendial dissolution tests specify water as the dissolution

medium. When dissolution procedures were first being added to the
monographs, the default "First Case" was usually specified. This
consisted of 900 mL of water as dissolution medium with USP
Apparatus 1 (Basket Method) at 100 rpm, or later, USP Apparatus 2
(Paddle Method) at 50 rpm. Products not meeting the "First Case"
tolerance specification of NLT 75% "Q" in 45 minutes using this
method was asked to submit data for a new procedure.

2. Sample Introduction:

Sample introduction can be tricky and unfortunately at times, not easy

to perform reproducibly. Products can have a dissolution rate that is

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"position dependent". For example, if the tablet is offcenter, the

dissolution rate is high due to shear forces or if it is in the centre,
coning may occur and dissolution rate will go down. Film coated tablets
can be sticky and pose problems related to tablet position in the vessel.
Suspensions can be introduced in a variety of ways. Some examples are
to manually use syringes or pipettes, pour from beaker, or automate
delivery using calibrated pipettes. Each method has its own set of
limitations. Mixing of the sample will generate air bubbles; therefore,
the mixing time of suspension samples must be strictly uniform to
reduce biased results.

3. Single Point Vs Multiple Point Sampling

A seemingly robust and discriminating method can present unexpected

results based on the number of sampling time points and sample
withdrawals.

As in a "profile" experiment (typically used in the R&D or stability

testing environments) versus a single- pull (typically used in the QC
environment), resulted in different dissolution rates. It is believed
that this discrepancy is a result of a greater disturbance of the fluid
hydrodynamics in the dissolution vessel caused by the additional
insertions and residence of the sampling probe in the vessel during
multi-point sampling. This may be due to hydrodynamic effects that can
potentially influence in vitro dissolution results. Additional examples in
the literature suggest that the hydrodynamic conditions, drug release
pattern, or mechanical forces of anin vitro dissolution are crucial to
the drug release rate.

4. Deaeration of Media:

The level of dissolved gases is related to the presence of bubbles.

Bubbles are common and will cause problems in non-deaerated medium.

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It is stated that bubbles can interfere with dissolution test results

and should be avoided. Dissolved air can slow down dissolution by
creating a barrier; either adhering to tablet surface or to basket
screens or particles can cling to bubbles on the glass surface of the
vessel or shafts.

5. Controlled Release Dosage Forms

The current trend is to evaluate each and every sustained or controlled

release dosage form on individual basis. The formulation scientists and
regulatory authorities face an enormous challenge of generalising the
test conditions for dissolution testing because most individual drug
candidates for sustained or controlled release dosage forms and their
delivery design possess diverse physicochemical and pharmacokinetic
properties requiring specific considerations. The difficulties are also in
simulating in vivo conditions in in vitro. Since most sustained or
controlled release preparations are designed for prolonged release and
therapeutic effect, variabilities in in vivo conditions (such as presence
and nature of food in the gastrointestinal tract, time of the day the
dosage form is administered) which can substantially affect the
release profile of the drug are bound to happen.

6. Automation:

While automation of dissolution sampling is very convenient and labor

saving, errors often occur with these devices because the analysts
tend to overlook problem area. Sample lines are often a source of error
for a number of reasons: unequal lengths, crimping, wear beyond limits,
disconnection, carry-over, mix-ups or crossing and inadequate cleaning.

Pumping tubes may wear out through normal use or repeated organic
solvent rinsing and may necessitate replacement.

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3. Drug Substance Properties Related Factors

Knowledge of drug properties like solubility of drug, effect of pH

change, crystalline structure is important. One could anticipate
precipitation of the drug as the pH changes in solution, or if release
from the dosage form leads to super saturation of the test media e.g.
preparation of a standard solution may is an important step. It is
customary to use a small amount of alcohol to dissolve the standard
completely.

A history of the typical absorptivity range of the standard can be very

useful to determine if the standard has been prepared properly.

4. Drug Product Properties Related Factors

Dissolution profile may help in identifying trends and effects of

formulation changes. When the results are highly variable, it indicates
that the method is not robust. Two major casual factors influence
variability: Mechanical and Formulation.

Mechanical causes can arise from the dissolution conditions chosen.

An apparatus or speed change may change the results. The formulation

can have poor content uniformity, additionally, reactions and/ or
degradation may be occurring in situ.

The film coating may cause sticking to the vessel walls. Upon aging,
capsule shells are known for the pellicle formation and tablets may
become harder or softer, depending upon the excipients and drug
interaction with moisture, which in turn may affect the dissolution and
disintegration rate.

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5. Miscellaneous Factors

1. Personal Errors:

Anomalous dissolution usually involves some of following observations:

floating chunks of tablets, spinning, coning, mounding, gumming,
swelling, capping, off-center position, sticking, particles adhering to
apparatus or vessel walls, sacs, swollen/rubbery mass. A well trained
analyst can pinpoint many of such problems.

Along with good documentation, familiarity with the dissolution

behavior of the product is essential in quickly identifying changes in
stability or changes associated with a modification of the formulation.

2. Cleaning:

The analyst should take special care to examine this aspect when
validating the method. In many laboratories, where different products
are tested on the same equipment, this is a critical issue that, if
inadequately monitored, may be a cause of inspection failures.

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Dissolution Profile Comparison

Definition:

It is graphical representation [in terms of concentration vs. time] of

complete release of drug from a dosage form in an appropriate
selected dissolution medium.

I.e. in short it is the measure of the release of A.P.I from a dosage

form with respect to time.

Importance of dissolution profile Comparison :

 Dissolution profile of an A.P.I. reflects its release pattern under the

selected condition sets. i.e. either sustained release or immediate
release of the formulated formulas.
 For optimizing the dosage formula by comparing the dissolution
profiles of various formulas of the same A.P.I
 FDA has placed more emphasis on dissolution profile comparison in
the field of post approval changes.
 The most important application of the dissolution profile is that by
knowing the dissolution profile of particular product of the BRAND
LEADER, we can make appropriate necessary change in our
formulation to achieve the same profile of the BRAND LEADER.

Objective of dissolution profile Comparison:

 To develop in-vitro in-vivo correlation which can help to reduce

costs, speed-up product development and reduce the need to
perform costly bioavailability human volunteer studies.
 Establish the similarity of pharmaceutical dosage forms, for which
composition, manufacture site, scale of manufacture, manufacture
process and/or equipment may have changed within defined limits.

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Methods to Compare Dissolution Profile

Graphical Method

 In this method we plot graph of Time V/S concentration of solute

(drug) in the dissolution
lution medium or biological fluid.
 The shape of two curves is compared for comparison of dissolution
pattern and the concentration of drug at each point is compared for
extent of dissolution.
 If two or more curves are overlapping then the dissolution profile
profi is
comparable.
 If difference is small then it is acceptable but higher differences
indicate that the dissolution profile is not comparable.

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Statistical Analysis:

1. Student's t-Test: Following tests are commonly used

used:

a. Paired t-test
b. Unpaired t-test

Calculated 't' value is compared with tabulated value of 't' if the

calculated value exceeds the tabulated value, then the null hypothesis
should be rejected and vice versa.

2. ANOVA Method (Analysis of Varience)

This test is generally applied to differen

differentt groups of data. Here we
compare the variance of different groups of data and predict whether
the data are comparable or not.

Minimum three sets of data are required. Here first we have to find
the variance within each individual group and then compare them
th with
each other.

ANOVA table:

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Compare the calculated F

F- value with the tabulated value at particular
degrees of freedom and level of significance. If the calculated value is
less than the tabulated value, then degrees of variance is insignificant.

Model
el dependent methods:

Zero order kinetics:

Qt = Q0 + K0t

Where,

 Qt is the amount of drug dissolved in time t

 Qo is the initial amount of drug in the solution
 Ko is the zero order release constant expressed in units of
concentration/time.

Plot: Cumulative amount

mount of drug released versus time.

Applications: Transdermal systems, as well as matrix tablets with low

solubility drugs in coated forms, osmotic systems, etc.

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First order model:

log C = log C0 - Kt/2.303

Where,

 Co is the initial concentration of drug,

 K is the first order rate constant, and
 t is the time.

Plot: log cumulative percentage of drug remaining vs. time which would
yield a straight line with a slope of -K/2.303.

Application: This relationship can be used to describe the drug

dissolution in pharmaceutical
armaceutical dosage forms such as those containing
water soluble drugs porous matrices.

First order plot:

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Higuchi model (Diffusion matrix formulation)

This is given by Higuchi.

Q = K√T

Where,

 Q is the amount of drug released in time't' per unit area,

 K is higuchi constant
 T is time in hr.

Plot: The data obtained is to be plotted as cumulative percentage drug

release versus Square root of time.

Application:

Modified release pharma

pharmaceutical
ceutical dosage forms, transdermal systems
and matrix tablets with water soluble drugs.

Higuchi Plot:

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Hixson-Crowell model:

Hixson and Crowell described this

Wo1/3 - Wt1/3 = Kt

Where,

 W0 is the initial amount of drug

 Wt is the remaining amount of drug at time t .

Plot: Data is to be plotted as cube root of drug percentage remaining

in matrix versus time.

Application: This expression applies to pharmaceutical dosage form

such as tablets, where the dissolution occurs in planes that are parallel
to the drug surface if the tablet dimensions diminish proportionally in
such a manner that the initial geometrical form keeps constant all the
time.

Korsmeyer - Peppas model:

 The Korsemeyer and Peppas described this method..

 It is given by the equation:

Mt/Ma = Ktn

Where,

 Mt / Ma is fraction of drug released

 t = time
 K=constant includes structural and geometrical characteristics of
the dosage form
 n= release component which is indicative of drug release
mechanism where, n is diffusion exponent.
 If n= 1, the release is zero order.
 n = 0.5 the release is best described by the Fickian diffusion.

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 0.5 < n < 1 then release is through anomalous diffusion or case two
diffusion. In this model a plot of percent drug release versus
time is linear.

Some models with linear equation for graphical presentation:

Model Independent Approach Using a Similarity Factor:

The difference factor (fl) calculates the percent (%) difference

between the two curves at each time point and is a measurement of the
relative error between the two curve
curves:

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Where n is the number of time points, R is the dissolution value of the

reference (pre change) batch at time t, and T is the dissolution value
of the test (post change) batch at time t.

The similarity factor (2) is a logarithmic reciprocal square root

transformation of the sum of squared error and is a measurement of
the similarity in the percent (%) dissolution between the two curves.

Limits for similarity and difference factors

Advantages:

1. They are easy to compute

2. They provide a single number to describe the comparison of
dissolution profile data.

Disadvantages

1. The values of f1 and f2 are sensitive to the number of dissolution

time point used.

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2. If the test and reference formulation are inter changed f2 is

unchanged but f1 is not yet differences between the two mean
profiles remain the same.
3. The basis of the criteria for deciding the difference or similarity
between dissolution profiles is unclear.

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Unit 3 - Pharmacokinetics - Pharmacokinetic Models

Compartmental Model
A compartment is a group of tissues with similar blood flow and drug
affinity.

A compartment is physiologic and anatomic region.

Compartment is the traditional and most widely used approach to

pharmacokinetic characterization of drug. These models simply
interpolate the instrumental data and allow on empirical formula to
estimate drug concentration with time.

Assumptions of Compartmental Models

 The body is represented as a series of compartment arranged in

series or parallel to each other.
 The rate of drug movement between compartments is described by
first order kinetics.
 Rate constants are used to represent rate of entry into and exit
from compartment.
 A statistical analysis of plasma concentration time data is another
method used to find out no of compartments.

Application of Compartment Modeling

a. It is simple and flexible approach and widely used.

b. It gives a visual representation of various rate process involved in
drug disposition.
c. It is useful in predicting drug concentration time profile in both
normal and pathological composition.
d. It is useful in relating plasma drug levels in therapeutic and toxic
levels.

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One Compartment Model (Instantaneous Distribution

Model)
 The time course of drug concentration determined after the
administration can satisfactorily explained by assuming the body as
a single well mixed compartment with first order disposition
process.
 The body is constituted as a single kinetically homogeneous unit with
no barriers to movement of drug.
 Elimination is a first order process with first order rate constant.
 Drug moves dynamically in and out of the compartment then rate of
input will be greater than the rate of output.

One Compartment "Open Model"

The term open indicates that the input (availability) and output
(elimination) are unidirectional and that the drug can be eliminated
from the body.

Classification of One Compartment Open Model

Depending upon rate of input several one compartment models are

defined:

a. One compartment open model - intravenous bolus administration.

b. One compartment open model - continuous intravenous infusion.
c. One compartment open model - extra vascular zero - order
absorption.
d. One compartment open model - extra vascular first - order
absorption.

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Intravenous Bolus Administration

When a drug that distributes in body is given in the form of a lipid
intravenous injections, its takes about one or three minutes, for
complete circulation.

The model can be diagrammatically depicted as

as:

It can be mathematically represented as: The general expression

for rate of drug presentation to the bo
body is:

dX/dt = Rate in (availability) - Rate out (elimination)

Since rate in or absorption is absent, the equation becomes:

dX/dt = Rate out

If the rate out or elimination follows first

first-order
order kinetics, then:

dX/dt = - KEX

Where,

 KE = first - order elimina

elimination rate constant, and
 X = amount of drug in the body at any time "t" remaining to be
eliminated.
 Negative sign indicates that the drug is being lost from the body.

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Estimation of Pharmacokinetic Parameters

For a drug that follows one

one-compartment kinetics
tics and administered as
rapid i.v. injection, the decline in plasma drug concentration is only due
to elimination of drug from the body (and not due to distribution), the
phase being called as elimination phase.

Elimination phase can be characterized by 3 parameters-

-

1. Elimination rate constant

2. Elimination half-life
life
3. Clearance

(a) Cartesian plot of a drug that follows one

one-compartment
compartment kinetics and
given by rapid i.v. injection, and

(b) Semi logarithmic plot for the rate of elimination in a one-

one
compartment model.

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Elimination Half-Life:

It is defined as the time taken for the amount of drug in the body as
well as plasma concentration to decline by one
one-half
half or 50% its initial
value.

t1/2 = 0.693/KE

1. Apparent volume of distribution, and

2. Clearance.

Apparent Volume
olume of Distribution:

These parameters are closely related with the physiologic mechanisms

in the body, they are called as primary parameters.

Clearance

Clearance is defined as the theoretical volume of body fluid containing

drug (i.e. that fraction of apparent volume of distribution) from which
the drug is completely removed in a given period of time. It is
expressed in ml/min or liters/hour.

ClR = Rate of elimination by kidney

kidney/C

The total body clearance, CIT, also called as total systemic clearance,
is an additive property of individual organ clearances.

ClT = СlR + ClH + Clothers

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Intravenous Infusion
Rapid i.v. injection is unsuitable when the drug has potential to
precipitate toxicity or when maintenance of a stable concentration or
amount of drug in the body is desired. In such a situation, the drug
(for example,
xample, several antibiotics, theophylline, procainamide, etc.) is
administered at a constant rate (zero
(zero-order)
order) by i.v. infusion.

In contrast to the short duration of infusion of an i.v. bolus (few

seconds), the duration of constant rate infusion is usually much longer
than the half-life
life of the drug.

Advantages of zero-order
order infusion of drugs include:

a. Ease of control of rate of infusion to fit individual patient needs.

b. Prevents fluctuating maxima and minima (peak and valley) plasma
level, desired especially when the drug has a narrow therapeutic
index.
c. Other drugs, electrolytes and nutrients can be conveniently
administered simultaneously by the same infusion line in critically ill
patients.

The model can be represented as follows:

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Plasma concentration-time
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)

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Extra Vascular Administration

 When a drug is administered by extra vascular route (e.g. oral, i.m.,
rectal, etc.), absorption is a prerequisite for its therapeutic activity.
 The rate of absorption may be described mathematically as a zero- zero
order or first-order
order process. A large number of plasma
concentration- time profiles can be described by a one-
one
compartment
tment model with first
first-order
order absorption and elimination.
However, under certain conditions, the absorption of some drugs
may be better described by assuming zero
zero-order
order (constant rate)
kinetics.

Distinction between zero

zero-order and first-order
order absorption processes.
"Figure a" is regular plot, and "Figure b a" semi log plot of amount of
drug remaining to be absorbed (ARA) versus time t.

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Zero-order
order absorption is characterized by a constant rate of
absorption:

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Multi Compartment Models

Introduction

 One compartment is described by mono- exponential term i.e.

elimination.
 For large class of drugs this terms is not sufficient to describe its
disposition.
 It needs a bi- or multi- exponential terms
 The body is composed of a heterogeneous group of tissues each with
different degree of blood flow and affinity for drug and therefore
different rates of elimination.
 Multi-compartment characteristics are best described by
administration as i.v. bolus.

Two Compartment Model

The simplest and commonest is the two compartment model which

classifies the body tissues in two categories:

1. Central compartment or Compartment 1.

2. Peripheral or Tissue Compartment or Compartment 2.
 Compartment 1 comprises of blood and highly perfused tissues like
liver, lungs, kidneys, etc.
 Elimination usually occurs from this compartment.
 Compartment 2 comprises of poorly perfused and slow equilibriating
tissues such as muscles, skin, adipose, etc.

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General Grouping of Tissue According to Blood Supply:

Depending upon the compartmen

compartmentt from which the drug is eliminated,
the 2 compartment model can be further categorized into:

 With elimination from Central compartment

compartment.
 With elimination from peripheral compartment.
 With elimination from both the compartments.

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Two compartment Open model - I.V. bolus

administration
 Elimination from central compartment
compartment.

 After the iv bolus of a drug the decline in the plasma conc. is bi-
bi
exponential.
 Two disposition processes
processes- distribution and elimination.
 These two processes are only evident when a semi log plot of C vs t
is made.
 Model Structure for Two Compartment Model
Model.

Where,

 X0 = IV bolus dose administered

administered.
 Cc = X1 = amount of drug in the central compartment
compartment.
 Cp = X2 = amount of drug in the peripheral compartment.
compartment
 k12 = rate constant for transfer of drug from the central to the
peripheral compartment
compartment.

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 k21 = rate constant for transfer of drug from the peripheral to the
central compartment..
 kE = first order elimination rate constant.
 Elimination of drug out of the central compartment
 The second, slowerer rate process, is called as the post
post- distributive
or elimination phase.
 In contrast to central compartment, the conc. of drug in the
peripheral compartment first increases and reaches its max.

The Rate change in drug conc. in the central compartment is given

by,

Extending the relationship

relationship, X = V d C

Where, Vc and V p = apparent volumes of C1 and C2

The rate of change in drug conc. in the peripheral component is

given by:

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On integration equation gives conc. of drug in central and

peripheral compartments
rtments at given time t:

Two Compartment Open Model - Intravenous

Infusion

The plasma or central compartment conc. of a drug when administered

as constant rate (0 order
order) i.v. infusion is given as:

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At steady state the second and the third term in the bracket
becomes zero and the equation reduces to: Now, The loading dose

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Two-Compartment
Compartment Open Model - Extra vascular
administration
 First Order Absorption
 The model can be depicted as follows:

 The rate of change in drug conc. in the ce

central
ntral compartment is
described by three exponents: An absorption exponent, and
 Two usual exponents that describe drug disposition.
 The plasma conc. at time t is,

Where, L, M & N are coefficients

coefficients.

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Nonlinear Pharmacokinetics
 The rate process of drug's ADME is depending upon carrier or
enzymes that are substrate specific, have definite capacities and
are susceptible to saturation at a high drug concentration.
 In such cases, an essentially first-order kinetics transform into a
mixture of first-order and zero-order rate processes and the
pharmacokinetic parameters are changed with the size of the
administered dose.
 Pharmacokinetics of such drugs are said to be dose- dependent.
Terms synonymous with it are mixed-order, nonlinear and capacity-
limited kinetics.

Detection of Non-Linearity in Pharmacokinetics

There are several tests to detect non-linearity in pharmacokinetics but

the simplest ones are:

1. First test: Determination of steady state plasma concentration at

different doses.
2. Second test: Determination of some important pharmacokinetic
parameters such as fraction bioavailability, elimination half life or
total systemic clearance at different doses of drug. Any change in
these parameters is indicative to non-linearity which are usually
constant.

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Causes of Non-Linearity

1. Drug Absorption

Three causes:

1. Solubility/dissolution of drug is rate-limited; Griseofulvin - at high

concentration in intestine.
2. Carrier - mediated transport system; Ascorbic acid - saturation of
transport system.
3. Presystemic gut wall / hepatic metabolism attains saturation;
Propranolol.
 These parameters affected F, Ka, Cmax and AUC.
 A decrease in these parameters is observed in former two causes
and an increase in latter cause.

2. Drug distribution

At high doses non-linearity due to two causes:

1. Binding sites on plasma proteins get saturated; Phenylbutazone.

2. Tissue binding sites get saturated.
 In both cases there is increase in plasma drug concentration.
 Increase in Vd only in (1)
 Clearance with high ER gets increased due to saturation of binding
sites.

3. Drug metabolism

Non-linearity occurs due to capacity limited metabolism, small changes

in dose administration - large variations in plasma concentration at
steady state - large intersubject variability.

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Two imp causes:

1. Capacity - limited metabolism - enzyme & cofactor saturation;

Phenytoin, Alcohol.
2. Enzyme induction - decrease in plasma concentration;
Carbamazepine.
 Auto induction in dose dependent concentration.
 Saturation of enzymes - decrease in Cl- increase in C.
 In case of enzyme induction reverse condition.
 Other reasons include saturation of binding sites, inhibitory effects
of the metabolites on the action of enzymes.

4. Drug excretion - Two active processes which are saturable:

1. Active tubular secretion - Penicillin G

2. Active tubular reabsorption - Water soluble vitamins & Glucose.
 Saturation of carrier systems - decrease in renal clearance in case
of I & increase in II. Half life also increases.
 Other reasons like forced diuresis, change in urine pH,
nephrotoxicity & saturation of binding sites.
 In case of biliary excretion non-linearity due to saturation -
Tetracycline & Indomethacin.

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Examples of drugs showing nonlinear pharmacokinetics

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Michaelis – Menten Equation

The kinetics of capacity limited or saturable
urable processes are best
described by Michaelis - Menten equation.

Where,

 -dC/dt
dC/dt = rate of decline of drug conc. with time
 Vmax = Theoretical maximum rate of the process
 KM = Michaelis constant

Three situations can now be considered depending upon the value

v of
Km and C.

1. When KM = C:

Under this situation, eq I reduces to,

-dC/dt = Vmax/2............II
II

 The rate of process is equal to half of its maximum rate.

 This process is represented in the plot of dc/dt vs. C. shown in fig.

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2. If a drug at low conc. undergoes a saturable biotransformation

then KM >> C:

here, K+C=K and eq. I reduces to,

-dC/dt =VmaxC/KM..............III

Above eq. is identical to the one that describe first order elimination
of drug, where VMax/KM=KE.

3. When K<<C:

Under this condition, KM + C = C and eq. I will become,

-dC/dt =Vmax ………………..IV

Above eq. is identical to the one that describe a zero order process i.e.
the rate process occurs at constant rate V and is independent of drug
conc.

E.g. metabolism of ethanol

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Estimation of Vmax & Km

In enzymatic kinetic work, the classic Michaelis - Menten equation:

V = Vmax.C / KM + C…………….1

Where,

 V = reaction rate,
 C = substrate conc. both are used to determine Vmax & Km.

The velocity of the reaction (V) at various concentration levels

lev of drug
(C) is determined either by in
in-vitro experiments or in-vivo
vivo experiments
at constant enzyme levels.

Method 1

By reciprocating equation (1), we get:

When 1/V is plotted against 1/C, a straight line is obtained with a slope
of Km/Vmax and an Intercept
ercept of 1/Vmax.

E.g. A plot of 1/VV vs 1/ C (shown in the figfig.) gave an intercept of

0.33μmol and a slope of 1.65, Now, calculate V max and KM.

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Now,

 Intercept= 1/Vmax 0.33 μ mol.

 Vmax = 3 μ mol/ml min
 Slope = Km/ Vmax So, 1.65 = Km/ Vmax
 Km = 1.65 X 3 = 4.95 μ mol/ ml
 X-axis intercept = -1/
1/ Km

Method 2

Multiplying eq. 2 by C, we get:

A plot of C/V vs C gives a straight line with 1 / V max as the slope and
Km/Vmax as the
e intercept (shown in the fig.)

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Method 3

The equation can also be written as:

V = -K
Km V/C + Vmax …………………… (4)

A plot of V vs V/C gives a straight line with a slope of -Km & an

Intercept of Vmax. (Shown in the fig.
fig.)

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Drug Interactions
When the pharmacological activity of a drug is altered by the
concomitant use of another drug or by the presence of some other
substance.

The drug whose activity is affected by such an interaction is called as

the object drug.

The agent which precipitates such an interaction is referred to as the

precipitant.

Drug interactions include:

1. Drug-drug interactions.
2. Food-drug interactions, for example, inhibition of metabolism of
several drugs by grapefruit juice.
3. Chemical-drug interactions, for example, interaction of a drug with
alcohol, tobacco or environmental chemicals.
4. Drug-laboratory test interaction, for example, alteration of
diagnostic laboratory test results by the presence of drug.
5. Drug-disease interactions, for example, worsening of disease
condition by the drug.

Factors Contributing to Drug Interactions - Some of the more

important risk factors that lead to drug interactions include:

1. Multiple drug therapy

2. Multiple prescribers
3. Multiple pharmacological effects of drug
4. Multiple diseases/Predisposing illness
5. Poor patient compliance
6. Advancing age of patient
7. Drug related factors

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Protein Binding of
f Drug
 The phenomenon of complex formation with proteins is called as
protein binding of drugs.
 The formation of a drugdrug-protein
protein complex is often named drug-
drug
protein binding.

The drug protein binding ma

may be of two types:

1. Reversible - When the drug bind the protein with weaker chemical
bonds such as hydrogen bond or vander
vanderwaal's forces.
2. Irreversible - Irreversible drug protein binding is usually a result
of chemical activation of the drugs, which then attaches strongly to
the protein or macromolecules by cova
covalent
lent chemical bonding.

Binding off Drugs to Blood Components

a. Albumin
b. Alpha 1- Acid Glycoprotein
c. Lipoprotein
d. Erythrocytes (RBC)
e. Immunoglobulins

The extent or order of binding of drugs to various plasma proteins is:

Abumin > alpha1 - Acid Glycoprotein > Lipo
Lipoproteins
proteins > Globulins.
Globulins

Effect of protein binding interactions

a. Apparent volume of distribution

distribution: It is defined as the hypothetical
volume of body fluid into which a drug is dissolved or distributed. It
is called as apparent volume because all parts of the body bod
equilibrated with the drug do not have equal concentration.

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b. Effect of Protein Binding on Apparent Volume of Distribution:

 The extent of drug protein binding affects VD.

 Drugs that are highly bound to plasma proteins have a low fraction
of free drug (fu= unbound or free drug fraction).
 The plasma protein-bound drug does not diffuse easily and is
therefore less extensively distributed to tissues
 Drugs with low plasma protein binding have larger fu, generally
diffuse more easily into tissues, and have a greater volume of
distribution.
 Drugs such as furosemide, sulfisoxazole, tolbutamide, and warfarin
are bound greater than 90% to plasma proteins and have a VD value
ranging from 7.7 to 11.2 L per 70-kg body weight.
 Basic drugs such as imipramine, nortriptyline, and propranolol are
extensively bound to both tissue and plasma proteins and have very
large VD values.

Displacement of drugs from plasma proteins can affect the

pharmacokinetics of a drug in several ways: directly increase the
free (unbound) drug concentration as a result -

1. Reduced binding in the blood;

2. Reaches the receptor sites directly, causing a more intense
pharmacodynamic (or toxic) response;
3. Causing a transient increase in VD and decreasing partly some of the
increase in free plasma drug concentration;
4. More drug diffusion into tissues of eliminating organs, particularly
the liver and kidney, resulting in a transient increase in drug
elimination.

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c. Effect of Protein Binding on Elimination of Drug

 Protein binding decreases the renal excretion of the drugs and

enhance the biological half-life. Only the free drugs excreted
through filtration for example tetracycline.
 The binding of drugs in extra vascular organs decreases their
concentration in blood and thereby shows their elimination by liver,
kidney and lungs.
 However, the binding of drug to plasma protein may retard the
elimination of drugs depending on the mechanism of elimination, for
example the binding of drug to the plasma protein lowers their
unbound concentration in blood and thereby decreases their rate of
elimination by glomerular filtration and by inefficient transport
system in the kidney.
 Binding to plasma protein would decreases the rate of elimination of
lipid soluble drugs that diffuse rapidly from the glomerular
filtration back into the blood, even though they are rapidly
transported by active transport system.
 In addition, the binding of drug to plasma protein would also
decreases the rate of drug metabolism by relatively inactive enzyme
system in liver.

d. Effect of Protein Binding on Patient with Kidney Disease

 Albumin is primarily responsible for the binding of acidic drugs

whereas basic drugs appear to bind preferentially to AAG (alpha-
amino glycoprotein).
 The low concentration of albumin and AAG in patients with liver
disease indicates the inability of the liver to synthesize these drug-
binding proteins.
 Impaired binding of acidic and basic drugs is well documented in
patients with liver disease.

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 The albumin concentrations in the transplant patients were low

despite stable biochemical liver tests. Because albumin is primarily
responsible for binding of acidic drugs, liver transplant patients
would not be able to bind acidic drugs as efficiently as the normal
subjects.
 However, the AAG concentrations were elevated in all the transplant
patients studied. AAG is an acute-phase reactant and is primarily
synthesized in the liver.
 AAG concentrations are known to increase in plasma.

e. Effect of Protein Binding on Patients with Hepatic Disease

 Protein binding affects distribution.

 The impaired liver is unable to synthesize plasma proteins (albumin)
adequately.
 Liver impairment causes accumulation of substances (bilirubin) that
displace drugs from protein-binding sites.
 A statistics on this case shows that liver disease patients with
normal concentration of albumin and bilirubin have unbound
percentages of frusemide similar to those in healthy subjects.

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Effect of Tissue Binding Interactions

1. Tissue binding of drugs (tissue localization of drugs)

A drug can bind to one or more of the several tissue components.

Tissue-drug binding is important in distribution from two viewpoints:

 It increases the apparent volume of distribution of drugs in

contrast to plasma protein binding which decreases it.
 Tissue-drug binding results in localization of a drug at a specific site
in the body (with a subsequent increase in biological half-life). This
is more so because a number of drugs bind irreversibly with the
tissues (contrast to plasma protein-drug binding); for example,
oxidation products of paracetamol, phenacetin, chloroform, carbon
tetrachloride and bromo benzene bind covalently to hepatic tissues.

Factors influencing localization of drugs in tissues include:

1. Lipophilicity and structural features of the drug,

2. Perfusion rate,
3. pH differences, etc.

Extensive tissue-drug binding suggests that a tissue can act as the

storage site for drugs. Drugs that bind to both tissue and plasma
components result in competition between drug binding sites.

For majority of drugs that bind to extravascular tissues, the order of

binding is:

Liver > Kidney > Lung > Muscles

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Several Examples of Extra vascular Tissue-drug Binding are:

1. Liver: Paracetamol bind irreversibly to liver tissues resulting in

hepatotoxicity.
2. Lungs: Basic drugs like imipramine, chlorpromazine and
antihistamines accumulate in lungs.
3. Kidneys: Metallothionin, a protein present in kidneys, binds to heavy
metals such as lead, mercury, and cadmium and results in their renal
accumulation and toxicity.
4. Skin: Chloroquine and phenothiazines accumulate in skin by
interacting with melanin.
5. Eyes: The retinal pigments of the eye also contain melanin. Binding
of chloroquine and phenothiazines to it is responsible for
retinopathy.
6. Hairs: Arsenicals, chloroquine and phenothiazines are reported to
deposit in hair shafts.
7. Bones: Tetracycline is a well-known example of a drug that binds to
bones and teeth. Administration of this antibiotic to infants or
children during odontogenesis results in permanent brown-yellow
discoloration of teeth.
8. Fats: Lipophilic drugs such as thiopental and the pesticide DDT
accumulate in adipose tissues by partitioning into it.
9. Nucleic Acids: Molecular components of cells such as DNA interact
strongly with drugs like chloroquine and quinacrine resulting in
distortion of its double helical structure.

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Role of Cytochrome P450 in Drug Interactions

Introduction

 The cytochrome P450 (P450 or CYP) isoenzymes are a group of

heme- containing enzymes embedded primarily in the lipid bilayer of
the endoplasmic reticulum of hepatocytes.
 Isoenzyme : Enzymes that differ in amino acid sequence but
catalyze the same chemical reaction.
 The endoplasmic reticulum is a network of tubules and flattened
sacs that produce and process lipids and proteins in plant and animal
cells.
 A hepatocyte is a cell of the main parenchymal tissue of the liver.
 It takes part in the metabolism of many drugs, steroids and
carcinogens.
 The most intensively studied route of drug metabolism is the P450-
catalysed mixed function oxidation reaction which conforms to the
following stoichiometry.

NADPH + H+ + O2 + RH® NADP+ + H2O + ROH

 RH represents an oxidisable drug substrate.

 ROH is the hydroxylated metabolite more than 30 human CYP
isozymes have been identified to date.
 It has been estimated that 90% of human drug oxidation can be
attributed to six main enzymes (CYP1A2, 2C9, 2C19, 2D6, 2E1 and
3A4/5).

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Clinical Example of P450-Based Interactions

1. Terfenadine

 Terfenadine is the first non-sedating H1- antihistamine drug.

 It is rapidly oxidized by CYP3A4 to two metabolites, acyclinol and an
alcohol derived from the oxidation of a t-butyl methyl group.
 The alcohol is further oxidized to a carboxylic acid by either
CYP3A4 or dehydrogenase.
 This carboxylic acid then binds to the H1 histamine receptor and
should relieve allergy symptoms.
 The oxidation of terfenadine by CYP3A4 can be inhibited strongly
by azole antifungal or antimicrobial agents such as ketoconazole and
erythromycin.
 Therefore, the blood concentration of terfenadine increased.
 High blood levels of terfenadine have been associated with cardiac
problems including dysrhythmias, torsade de pointes, and abnormal
ventricular rhythms.
 For this reason, very carefully controlled co-administration of
terfenadine is advised.

2. Cimetidine

 Cimetidine inhibits antihistamine H2-receptor binding and is used in

the treatment of gastric ulcers.
 The mechanism of inhibition appears to involve the imidazole ring of
cimetidine with competitive binding, which is not present in
ranitidine.
 It also exhibits selective inhibition of reactions catalysed by
CYP2D6 and 3A4.

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 Unlike ranitidine, cimetidine significantly increased the maximum

plasma concentration (Cmax), AUC and the total amount of
disopyramide excreted unchanged in the urine.
 But the serum profile of mono-N- dealkyldisopyramide, a metabolite
of disopyramide, was not affected significantly.
 Ranitidine had no significant effect on the pharmacokinetics of
disopyramide and mono- N-dealkyldisopyramide.

3. Grapefruit juice

 Grapefruit juice could markedly increase the oral bioavailability of a

number of medications.
 Co-administration of grapefruit juice with the calcium channel
antagonist felodipine resulted in a large increase in serum felodipine
concentrations, as well as an enhancement of the pharmacodynamic
effects of the drug
 Flavonoids (e.g. quercetin, naringenin, kaempferol) found in large
amounts in oranges, grapefruit and their juices are known to alter
the activity of P450 enzymes (P450 isoenzyme).
 The major grapefruit-specific flavonoid is naringin.
 It is believed that this naringin mainly inhibits the enzyme (CYP3A)
that metabolizes calcium antagonists.
 Grape fruit juice is well known as potent inhibitors of cytochrome
P450 3A4.
 It increases bioavailability of several drugs known to be metabolized
by CYP3A4.

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4. Omeprazole

 Omeprazole is a proton-pump inhibitor used widely for the

treatment of gastric ulcers.
 Omeprazole is converted to hydroxyomeprazole and omeprazole
sulphone primarily by CYP2C19 and CYP3A4 respectively.
 Omeprazole reduced the plasma clearance and prolonged the half
life of phenytoin and diazepam but did not affect the apparent
volume of distribution and plasma protein binding of either diazepam
or phenytoin.
 Omeprazole appears to be a competitive inhibitor of CYP2C19, and
involved in its metabolism.

5. Erythromycin

 Erythromycin, an antimicrobial agent, is known to inhibit a number of

drug oxidation reactions catalysed by CYP3A4.
 It inhibits the oxidation of terfenadine, cyclosporin and numerous
other drugs both in vivo and in vitro.
 Erythromycin N-demethylation itself is catalysed by CYP3A4/5.

6. Cyclosporin

 Cyclosporin is the most popular immunosupressant used in organ

transplantation.
 The major pathway of cyclosporin metabolism is via CYP3A4.
 Cyclosporin is mainly used as immunosuppressant for organ
transplantation, the CYP3A4 level in the donor's liver as well as the
recipient's liver, small intestine and other tissues must always be
taken into consideration.

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7. Rifampicin

 Rifampicin and isoniazid are key drugs used in the treatment of

tuberculosis, while rifampicin is highly effective in inducing hepatic,
drug metabolic P450 enzyme.
 When enzyme induction is achieved, the pharmacological effects of
a specific drug may be reduced, since not only the metabolism of
rifampicin itself, but also the metabolism of the other drug is
accelerated.
 Rifampicin is also known to induce CYP3A4 and CYP2C9.

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Drug Interactions Linked to Transporters

Introduction

 Drug transporters will affect the nature of the drug and its
effects.
 Uptake - The action of taking up
 Efflux-the flowing out of a particular particle.
 Uptake and efflux transporters determine plasma and tissue
concentrations of a broad variety of drugs.

Transporters expressed in the:

a. Small intestine
b. Liver
c. Kidney
d. Blood brain barrier.
 Blood-brain barrier or the maternal-fetal barrier, have been shown
to protect sensitive tissues from potentially toxic compounds.
 Inhibition or induction of drug transporters by coadministered
drugs can alter pharmacokinetics and pharmacodynamics of the
victim drugs.
 Each transporter has a specific pattern of tissue expression.

Classification

 Transporters mediating the uptake of drugs into cells.

 Transporters mediating the export of drugs or drug metabolites out
of cells.
 Efflux transporters such as P-glycoprotein, the breast cancer
resistance protein, and the multidrug resistance protein are
localized to the apical membrane of entrecotes [a cell of the

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intestinal lining 1, thereby limiting], bioavailability of orally

administered substrates.
 After absorption and passage through the portal vein, drugs reach
the basolateral [situated below and toward the side] membrane of
hepatocytes.
 Inhibition transporters of this efflux by concomitantly administered
drugs results in an increased bioavailability of the victim drug.
 Whereas Induction of these intestinal efflux transporters reduces
bioavailability of drug substrates.
 Drug transporters also play a major role for drug secretion from the
proximal tubular cells of the kidney into urine.
 Uptake and efflux transporters for organic anions are also
expressed in the kidney. Inhibition of these processes by
concomitantly administered drugs leads to a reduced renal clearance
of the victim drug.

A. Uptake Transporters

1. Intestinal lumen

 The human OATP family consists of 11 members grouped into six

subfamilies based on their amino acid sequence similarities.
 From these OATP proteins, to this day four have been identified as
being important for drug therapy.
 OATP1A2 reported to be expressed in the intestinal epithelium, the
renal epithelium, and highly expressed in brain capillary endothelial
cells.

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Transporters expressed in enterocytes of the human intestinal

epithelium. Uptake transporters discussed in this review are colored
in red, export pumps in blue.

2. Hepatocytes

 OATP1B1 and OATP1B3 exclusively expressed in human hepatocytes.

 OATP2B1 the OATP family member being expressed in almost all
tissues.
 OATP1B3 seems to be expressed in colon polyps, colon cancer and in
pancreatic adeno carcinoma.
 These play an important role for drug disposition.
 An important drug class with compounds being transported by
OATP1A2, OATP1B1, OATP1B3, and OATP2B1 are HMG-CoA
reductase inhibitors (statins).
 Pitavastatin has been identified as substrate for all four OATPs.

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 Several other drugs have been identified as substrates for these

OATP family members, including antibiotics, chemotherapeutic
agents, antihistaminic drugs, and diuretics.
 The OAT/OCT Family of organocation/anion/zwitterion
transporters consists of 23 human members, including the cation
transporters OCTs, the anion transporters OATS.
 The OCT1 protein exhibits a broad tissue distribution with a high
expression in liver and weaker expression in some other tissues such
as spleen and lung.
 Drugs transported by or inhibiting all three OCTs in vitro include
anesthetic drugs -ketamine and cocaine, b-blockers -propranolol
antidepressants-Citalopram and oral antidiabetic drugs metformin.

Transporters expressed in human hepatocytes. Uptake transporters

discussed in this review are colored in red, export proteins in blue.

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3. Brain intestinal space

 The anion transporters OAT1 is expressed in different tissues with

the highest expression in kidney and located there in the basolateral
membrane of proximal tubule cells.
 In addition, mRNA tr
transcripts
anscripts of OAT1 have been detected in the
brain [cerebellum, hippocampus, and hypothalamus and the choroid
plexus. OAT2 is highly expressed in human liver and to a lower
extent also in the kidney with the same localization as the OAT1
protein.

Transporters expressed in human brain capillary endothelial cells.

Uptake transporters discussed in this review are colored in red,
export proteins in blue.

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B. Efflux Transporters

 The human multidrug resistance/transporters of antigen

presentation (MDR/TAP) family consist of 11 different ABC
transporters including the peptide transporters.
 TAP1 and TAP2 are the phospholipid translocator.
 The drug export pump, and the bile salt export pump are important
for drug disposition.
 P-gp is the most studied export pump mediating the transport of
drugs and drug conjugates from the intracellular to the
extracellular space.
 It is expressed in several tissues including intestine, kidney, liver,
brain and placenta.
 P-gp is expressed in the apical membrane of the entire intestine
from duodenum to rectum
 P-gp is also important for the blood-brain barrier as a defense
mechanism against the penetration of toxins and drugs into the
central nervous system.
 P-gp has a very wide substrate spectrum mediating the export of a
variety of drugs from different drug classes. Substrates include
anticancer drugs (e.g., docetaxel, etoposide, vincristine),
immunosuppressants [e.g. cyclosporine (CSA), tacrolimus], antibiotics
(e.g., macrolides), statins (e.g., atorvastatin, lovastatin), cardiac
drugs (e.g., digoxin, digitoxin), and b- blockers e.g., carvedilol;
 P-gp is an important determinant of pharmacokinetics and an
important mediator of transporter-mediated drug-drug interactions.
 The second MDR1/TAP family member, which is in particular
important for drug side effects via inhibition of this transporter, is
the bile salt export pump BSEP.
 BSEP shows the highest expression in liver and is located in the
canalicular membrane of hepatocytes.

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 The apically localized export pump MRP2 seems to be the most

important member of this family for drug disposition.
 MRP2 represents an apically localized export pump of the MRP
family and is highly expressed in liver, kidney, small intestine, gall
bladder, and placenta.
 The exclusively apical localization is in line with its role for the
excretion of many phase II metabolites of drugs and endogenous
compounds into bile, urine, and the intestinal lumen.
 Breast cancer resistance protein (BCRP) is a member of the ABCG
family of ABC transporters. This is so-called half-size ABC
transporter is expressed in different tissues including intestine,
liver, kidney, brain, in addition in testis and placenta.
 Substrates include antivirals e.g., acyclovir, statins e.g., pravastatin
and rosuvastatin, anticancer drugs e.g., topotecan, antibiotics e.g.,
ciprofloxacin and ofloxacin, as well as diclofenac, sulfasalazine, and
cimetidine.
 Furthermore, endogenous substances and metabolites such as
vitamin K3 estrone-3-sulfate, dehydroepiandrosterone sulfate, and
uric acid have been shown to be transported by BCRP.
 Substances that inhibit BCRP-mediated export in vitro without being
transported itself include cyclosporine, Omeprazole, pantoprazole,
saquinavir, and tacrolimus.
 MATE1 is expressed in both liver and kidney, MATE2-K is highly
expressed in kidney and to lower levels also in colon and testis.

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Unit 4 - Drug Product Performance In-Vivo

Drug Product Performance

 Drug product performance, in vivo, may be defined as the release of

the drug substance from the drug product leading to bioavailability
of the drug substance.
 The assessment of drug product performance is important since
bioavailability is related both to the pharmacodynamics response and
to adverse events.
 Thus, performance tests relate the quality of a drug product to
clinical safety and efficacy.

Bioavailability

 Bioavailability studies are drug product performance studies used to

define the effect of changes in the physicochemical properties of
the drug substance, the formulation of the drug, and the
manufacture process of the drug product (dosage form).
 Drug product performance studies are used in the development of
new and generic drug products.
 Bioavailability is one aspect of drug product quality that links the in
vivo performance of a new drug product to the original formulation
that was used in clinical safety and efficacy studies.

Bioequivalence

Bioequivalence studies are drug product performance tests that

compare the bioavailability of the same active pharmaceutical
ingredient from one drug product (test) to a second drug product
(reference).

Bioavailability and bioequivalence can be considered as measures of the

drug product performance in vivo.

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Bioequivalence Studies in New Drug Development (NDA)

During drug development, bioequivalence studies are used to

compare:

a. Early and late clinical trial formulations.

b. Formulations used in clinical trials and stability studies, if
different.
c. Clinical trial formulations and to-be-marketed drug products, if
different.
d. Product strength equivalence, as appropriate.

Bioequivalence study designs are used to support new formulations of

previously approved products, such as a new fixed-dose combination
version of two products approved for co administration, or modified-
release versions of immediate-release products.

Post Approval, in vivo bioequivalence studies may be needed to support

regulatory approval of major changes in formulation, manufacturing, or
site, in comparison to reference formulation (usually the pre change
formulation)

Drug product performance and new drug product development for NDAs.

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 Equivalent drug product performance is generally demonstrated by

an in vivo bioequivalence study in normal healthy volunteers.
 Equivalent drug product performance may be demonstrated in vitro
using comparative dissolution profiles.
 After the drug product is approved by the FDA and marketed, the
manufacturer may perform changes to the formulation. These
changes to the marketed drug product are known as post approval
changes. It is termed as SUPAC (scale-up and post approval change
based on several FDA guidance documents), could include a change in
the supplier of the active ingredient, a change in the formulation, a
change in the manufacturing process, and/or a change in the
manufacturing site. In each case, the manufacturer must
demonstrate that drug product performance did not change and is
the same for the drug product manufactured before and after the
SUPAC change.

Bioequivalence Studies in Generic Drug Development (ANDA)

 A generic drug product is a multisource drug product that has been

approved by the FDA as a therapeutic equivalent to the reference
listed drug product (usually the brand or innovator drug product)
and has proven equivalent drug product performance.
 Clinical safety and efficacy studies are not generally performed on
generic drug products. Since the formulation and method of
manufacture of a drug product can affect the bioavailability and
stability of the drug, the generic drug manufacturer must
demonstrate that the generic drug product is pharmaceutically
equivalent, bioequivalent, and therapeutically equivalent to the
comparator brand-name drug product.

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 Drug product performance comparison for oral generic drug

products is usually measured by in vivo bioequivalence studies in
normal healthy adult subjects under fasted and fed conditions.

Purpose of Bioavailability and Bioequivalence Studies

 Bioavailability and bioequivalence studies are important in the
process of approving pharmaceutical products for marketing.
 Bioavailability is defined as the rate and extent to which the active
ingredient or active moiety is absorbed from a drug product and
becomes available at the site of action.
 Bioavailability data provide an estimate of the fraction of drug
absorbed from the formulation, and provide information about the
pharmacokinetics of the drug.
 Relative bioavailability studies compare two drug product
formulations.
 A bioequivalence study is a specialized type of relative
bioavailability study.
 Bioequivalence is defined as the absence of a significant difference
in the rate and extent to which the active ingredient or active
moiety becomes available at the site of drug action when
administered at the same molar dose under similar conditions in an
appropriately designed study.
 Bioavailability and bioequivalence data play pivotal roles in regulatory
submissions for marketing approval of new and generic drugs
throughout the world.
 Clinical studies are used to determine the safety and efficacy of
drug products.
 Bioavailability studies are drug product performance studies used to
define the effect of changes in the physicochemical properties of

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the drug substance, the formulation of the drug, and manufacture

process of the drug product (dosage form).
 Bioequivalence studies are used to compare the bioavailability of the
same drug (same salt or ester) from various drug products.
 Bioavailability and bioequivalence can be considered as performance
measures of the drug product in vivo.
 If the drug products are pharmaceutically equivalent, bioequivalent,
and therapeutically equivalent (as defined by the regulatory agency
such as the FDA), then the clinical efficacy and the safety profile
of these drug products are assumed to be similar and may be
substituted for each other.

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Relative and Absolute Availability

 A drug product's
roduct's bioavailability provides an estimate of the relative
fraction of the administered dose that is absorbed into the
systemic circulation
 Determining the fraction (f) of administered dose absorbed involves
comparing the drug product's systemic exposur
exposuree (represented by
the concentration-versus
versus-time
time or pharmacokinetic profile) with that
of a suitable reference product.
 For systemically available drug products
products,, bioavailability is most
often assessed by determining the area under the drug plasma
concentration-versus--time profile (AUC).
 The AUC is considered the most reliable measure of a drug's
bioavailability, as it is directly proportional to the total amount of
unchanged drug that reaches the systemic circulation.

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Absolute Bioavailability
 Absolute bioavailability
ioavailability compares the bioavailability of the active
drug in the systemic circulation following extra vascular
administration with the bioavailability of the same drug following
intravenous administration.
 Intravenous drug administration is considered 100% absorbed.
 The route of extravascular administration can be inhaled,
intramuscular, oral, rectal, subcutaneous, sublingual, topical,
transdermal, etc.
 The absolute bioavailability is the dose
dose-corrected
corrected AUC of the
extravascularly administered drug prod
product
uct divided by the AUC of
the drug product given intravenously.

Thus, for an oral formulation, the absolute bioavailability is

calculated as follows:

 Fabs is the fraction of the dose absorbed, expressed as a

percentage.
 AUCpo is the AUC following oral ad
administration.
 Div is the dose administered intravenously
intravenously.
 AUCiv is the AUC following intravenous administration
administration.
 Dpo is the dose administered orally
orally.

Absolute availability, Fabs may be expressed as a fraction or as a

percent by multiplying F abs × 100. A drug
ug given by the intravenous route
will have an absolute bioavailability of 100% (f = 1). A drug given by an
extra vascular route may have an Fabs = 0 (no systemic absorption) and
Fabs = 1.0 (100% systemic absorption).

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Relative Bioavailability
In a relative
e bioavailability study, the systemic exposure of a drug in a
designated formulation (generally referred to as treatment A or
reference formulation) is compared with that of the same drug
administered in a reference formulation (generally referred to as
treatment
eatment B or test formulation).

In a relative bioavailability study, the AUCS of the two formulations

are compared as follows:

 Frel is the relative bioavailability of treatment (formulation) A,

expressed as a percentage
percentage.
 AUCA is the AUC following adminis
administration
tration of treatment
(formulation) A.
 DA is the dose of formulation A
A.
 AUCB is the AUC of formulation B; and
 DB is the dose of formulation B
B.

Relative bioavailability studies used in drug development include studies

to characterize food effects and drug
drug-drug interactions.

In a food-effect
effect bioavailability study, oral bioavailability of the drug
product given with food (usually a high
high-fat, high-calorie
calorie meal) is
compared to oral bioavailability of the drug product given under fasting
conditions. The drug produ
product
ct given under fasting conditions is treated
as the reference treatment.

The goal of a drug-drug

drug interaction study is to determine whether
there is an increase or decrease in bioavailability in the presence of

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the interacting drug. The general drug-drug interaction study design

compares drug relative bioavailability with and without (reference
treatment) the interacting drug.

Relative bioavailability studies are used in developing new formulations

of existing immediate- release drug products, such as new modified-
release versions or new fixed-dose combination formulations.

In the case of a new modified-release version, the reference product

is the approved immediate-release product.

In the case of a new fixed-dose combination, the reference product

can be the single-entity drug products administered either separately
(ie, three treatments for a fixed-dose combination doublet) or
concurrently according to an approved combination regimen (ie, two
treatments).

Relative bioavailability study designs are also commonly used for

bridging formulations during drug development, for example, to
evaluate how drug systemic availability from a new premarket
formulation compares with that from an existing premarket
formulation.

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Methods of Assessing Bioavailability

1. Pharmacokinetic Methods
a. Plasma Level - Time Studies
b. Urinary Excretion Studies

2. Pharmacodynamics Methods

a. Acute Pharmacological Response

b. Therapeutic Response

1. Pharmacokinetic Methods - Widely used and based on assumption

that Pharmacokinetic profile reflects the therapeutic effectiveness of
a drug.

a. Plasma Level - Time Studies

 Most common type of human bioavailability studies.

 Based on the assumption that there is a direct relationship between
the concentration of drug in blood or plasma and the concentration
of drug at the site of action.
 Following the administration of a single dose of a medication, blood
samples are drawn at specific time intervals and analyzed for drug
content.
 A profile is constructed showing the concentration of drug in blood
at the specific times the samples were taken.
 Bioavailability (the rate and extent of drug absorption) is generally
assessed by the determination of following three parameters.

They are:

a. Cmax (Peak plasma concentration)

b. tmax (time of peak)
c. Area under curve

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Plasma
sma Drug Concentration - Time Profile

a. Cmax (Peak plasma drug concentration)

 Maximum concentration of the drug obtained after the

administration of single dose of the drug.
 Expressed in terms of μg/ml or mg/ml.

b. tmax : (Time of peak plasma conc.)

 Time required achieving peak concentration of the drug after

administration.
 Gives indication of the rate of absorption.
 Expressed in terms of hours or minutes.

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c. AUC: Is the measurement of the extent of the drug bioavailability

It is the area under the dr
drug plasma level-time
time curve from t =0 & t =
∞, and is equal to the amount of unchanged drug reaching the general
circulation divided by the clearance.

Measurement of AUC

1. Trapezoidal method

 Most common method of estimating AUC.

 Divide the plasma conc - time curve into several trapezoids. -
Count the trapezoids & find the area.
 Total area of the trapezoids will approximate the area under the
curve.
 More number of trapezoids formed more accurate will be the
result.

The area of one trapezoid between time t 1 and t2 is:

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2. Cut & Weigh Method:

 Preparing
eparing calibrated plot by cutting squares of graph &weights
are recorded & plotted against weight V, area.
 Sample curve is cut & weight is recorded.
 By interpolation method area of sample graph is found.

3. Planimeter:

 Instrument for mechanically measurin

measuring g the area under the curve.
 Measures area by tracing outline of curve.
 Disadvantage: Degree of error is high due to instrumental &
human error.

4. Counting the Square

Square:

 Total no. of squares enclosed in the curve is counted.

 Area of each square determined using relationship:
Area = (height) (width)

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The extent of bioavailability can be determined by the following

equations:

For single dose study:

For multiple dose study:

Where [AUC] values are area under the plasma level time curve of one
dosing interval
rval in a multiple dosage regimen, after reaching the steady-
steady
state.

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b. Urinary Excretion Studies:

 Urinary excretion of unchanged drug is directly proportional to

plasma concentration of drug.
 Thus, even if a drug is excreted to some extent (at least 10 to 20%)
in the urine, bioavailability can be determined.
Eg: Thiazide diuretics, Sulphonamides.
 Method is useful when there is lack of sufficiently sensitive
analytical technique to measure drug concentration.
 Noninvasive method, so better patient compliance.

Three Important Parameters in urine excretion data for single dose

study:

a. (dxu/dt)max
b. (tu)max
c. Xu∞

a. (dx/dt)max: (Maximum urinary excretion rate)

 Its value increases as rate and/or extent of absorption increases.

 Obtained from peak of plot between rates of excretion versus
midpoint time of urine collection period.

b. (t)max:

 Time for maximum excretion rate.

 Its value decreases as absorption rate increases.
 Analogues of tmax of plasma level data.

c. Xu∞
 Cumulative amount of drug excreted in urine.
 Related to AUC of plasma level data.
 It increases as the extent of absorption increases.

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The extent of bioavailabil

bioavailability is calculated from equation::

For single dose study:

For multiple dose study:

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2. Pharmacodynamics methods

a. Acute Pharmacologic Response Method

 When bioavailability measurement by pharmacokinetic method is

difficult, an acute pharmacologic effect such as effect on pupil
diameter, EEG & ECG readings related to time course of drug.
 Bioavailability can then be determined by construction of
pharmacological effect- time curve as well as dose response graphs.

Disadvantage:

 It tends to be more variable.

 Observed response may be due to an active metabolite whose
concentration is not proportional to concentration of parent drug.

b. Therapeutic Response Method

This method based on observing the clinical response to a drug

formulation given to patient suffering from disease.

Drawbacks: The major drawbacks of this method are that quantization

of observed response is too improper to allow for reasonable
assessment of relative bioavailability between two dosage forms of the
same drug.

E.g.: Anti-inflammatory drugs.

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Bioequivalence Studies
It is defined as "the absence of a significant difference in the rate
and extent to which the active ingredient or active moiety in
pharmaceutical equivalents or pharmaceutical alternatives becomes
available at the site of drug action when administered at the same dose
under similar conditions in an appropriately designed study".

Understanding the terms:

1. Pharmaceutical equivalent

a. It refers to drug products, which contain the same active ingredient

in the same strength (concentration) and dosage form, and is
intended for the same route of administration. In general, it has the
same labeling and meets compendial and other standards of
strength, quality, purity, and identity.
b. Pharmaceutical equivalent does not necessarily imply therapeutic
equivalence as differences in the excipients and/or the
manufacturing process can lead to differences in product
performance.

2. Pharmaceutical Alternatives

a. Drug products are considered pharmaceutical alternatives if they

contain the same therapeutic moiety, but are different salt, esters,
or complexes of that moiety, or are different dosage forms or
strengths. Different dosage forms and strengths within a product
line by a single manufacturer are thus pharmaceutical alternatives,
as are extended-release products when compared with immediate or
standard-release formulations of the same active ingredients.

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Equivalence Studies Needed for Marketing

Pharmaceutically equivalent multi-source pharmaceutical products must
be shown to be therapeutically equivalent to one another in order to be
considered interchangeable.

Several test methods are available to predict bio-equivalence,

including:

a. Pharmacokinetic studies in humans in which the active drug

substance or one or more metabolites are measured in an accessible
biologic fluid such as plasma, blood or urine.
b. Comparative pharmacodynamics studies in humans.
c. Comparative clinical trials.
d. In-Vitro Studies.

Bioequivalence study designs

1. Pilot Study

 A pilot study in a small number of subjects can be carried out

before proceeding with a full bioequivalence study.
 The study can be used to validate analytical methodology, assess
variability, optimize sample collection time intervals, and provide
other information. For example, for conventional immediate-release
products, careful timing of initial samples may avoid a subsequent
finding in a full- scale study that the first sample collection occurs
after the plasma concentration peak.
 For modified-release products, a pilot study can help determine the
sampling schedule to assess lag time and dose dumping. A pilot study
that documents bioequivalence may be acceptable, provided that its
design and execution are suitable and a sufficient number of
subjects have completed the study.

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2. Replicate Study Designs

 Replicate study designs are recommended for bioequivalence studies

of modified-release dosage forms and highly variable drug products
(within- subject coefficient of variation ≥ 30%), including those that
are immediate release, modified-release, and other orally
administered drug products.
 Replicate study designs offer several scientific advantages
compared to non- replicate designs.

The advantages of replicate study designs are that they:

a. Allow comparisons of within-subject variance for the test and

reference products.
b. Indicate whether a test product exhibits higher or lower within-
subject variability in the bioavailability measures when compared to
the reference product.
c. Suggest whether a subject-by-formulation interaction may be
present.
d. Provide more information about factors underlying formulation
performance.
e. Reduce the number of subjects needed in the bioequivalence study.

3. Non-replicate Study Designs - Non-replicate study designs are

recommended for bioequivalence studies of most orally administered,
immediate-release dosage forms.

4. Food-Effect Studies

 Food-effect bioequivalence studies focus on demonstrating

comparable bioavailability between test and reference products
when administered with meals. Usually, a single-dose, two- period,
two- treatment, two-sequence crossover study is recommended for
food-effect bioequivalence study.

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 Food-effect bioequivalence studies are generally recommended for

modified release products. Food- effect bioequivalence studies are
also recommended for certain conventional release drug products.
 Selection of conventional release drug products that require food
studies is based upon certain considerations, such as:
a. Documented evidence of effect of food on drug absorption (e.g.,
cefaclor);
b. The drug is recommended to be administered with food;
c. The drug may produce gastric irritation under fasting conditions,
thus may be taken with food (e.g., NSAIDs)

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Evaluation of Bioequivalence
a. Comparative pharmacokinetic studies
b. Comparative pharmacodynamics studies
c. Comparative clinical trials
d. Comparative in vitro tests
e. Any other approach deemed adequate by FDA

a. Pharmacokinetic studies:

 BE between a test (T) and reference (R) product can be achieved by

the conduct of comparative pharmacokinetic studies. These studies
are generally performed with a limited number of healthy
volunteers, e.g., 24-36 subjects.
 Most studies have a two-sequence, two-period, crossover design
where each subject is randomly assigned to either sequence TR or
RT with an adequate washout interval between the two treatment
periods.
 Derived from the plasma or serum concentration-time profile, the
rate of drug absorption is commonly expressed by maximum
concentration (Cmax) and time to maximum concentration (Tmax)
whereas the extent of absorption is expressed by the area-under-
the-curve from time zero after drug administration to time infinity
(AUCt) and/or to the last quantifiable drug concentration (AUCt).
 AUCt may be calculated using the simple trapezoidal rule while AUC t
can be estimated by summing up AUC, and Ct/λz where Ct is the last
quantifiable concentration and λz is the terminal rate constant.
 Both AUCs and Cmax are statistically analyzed using the two one-
sided tests procedure to determine if the average values between
the T and R products are comparable.
 These comparisons require the calculation of a 90% confidence
interval for the geometric mean ratios of the T and R products. BE

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is generally declared if the 90 % confidence interval is within the BE

limit of 80.00- 125.00 %.
 However, the BE limits for highly variable drugs and narrow
therapeutic index drugs have been scaled to the intrasubject
variability of the reference product in the study.
 To obtain geometric means, the data of AUCs and Cmax are log-
log
transformed prior to conducting an analysis of variance (ANOVA),
then back-transformed
transformed before calculating the T/R ratio.

b. Pharmacodynamics Studies

1. Dose-Response
Response Relationship: Pharmacodynamics endpoints selected
for BE studies are required to have the capacity of detecting potential
differences between the test and reference products.

 The basic pharmacodynamics study design for BE determination may

include two doses of the reference product.
 This can be ascertained by a pilot study that demonstrates the
existence of a clear dose
dose-response
response relationship, which should be
done before the conduct
onduct of pivotal BE studies.
 Depending on the drugs, the dosedose-response curve
urve may be linear,
nonlinear, steep, or shallow. A shallow dose
dose-response
response curve may not
allow for detection of potential formulation differences between
products. Linearity may be obtained in some cases when the dose is
expressed on logarithmic scale.
 For many drugs, however, the dose
dose-response
response relationship based on a
pharmacodynamics endpoint is nonlinear and can be fitted to a
hyperbolic Emax model as follows
follows:

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Where,

 E is the estimated (fitted) value of pharmacodynamic response,

 E0 is the baseline pharmacodynamic effect,
 Emax is the maximum pharmacodynamic effect, and
 ED50 is the dose where the pharmacodynamic effect is half-maximal.

c. Comparative Clinical Trials

 Clinical responses are often located near or at the plateau of the

dose-response curve, thus insensitive to distinguish the therapeutic
difference between a test and reference formulation.
 As a result, conduct of these studies for BE assessment requires a
large number of patients to detect formulation differences. •
Demonstration of dose-response relationships is not required for
clinical BE studies since they are intended only to confirm the lack
of important clinical differences between products in comparison.
 Because of all the reasons mentioned above, BE studies using clinical
endpoints will be considered only when both pharmacokinetic and
pharmacodynamics approaches are impossible for BE determination.
 Several FDA guidance documents for industry are available on the
application of clinical approaches to document BE for topical drug
products.
 Typically, a randomized, double-blind, placebo-controlled, parallel
group study is required. However, placebo treatments are not
needed for drugs treating infectious diseases.
 BE is established if the T product is equivalent to the R product and
superior to the placebo treatment. In the case of nasal sprays for
local action, the USFDA may waive the in vivo BE studies and also
for solution-based products as BA/BE is self- evident for these
products. However, such testing is required for suspension based

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nasal sprays due to the lack of a suitable method for particle size
determination in suspension formulations.
 Moreover, in vivo BE testing cannot be exempted for nasal solutions
in metered dose devices because they are drug device combination
products.
 Ex- For establishment of equivalence in local delivery of suspension-
based nasal sprays, the US FDA has recommended clinical trials in
seasonal allergic rhinitis patients. The study design is a randomized,
double-blind, placebo-controlled, parallel group of 14-day duration.
The clinical endpoints for equivalence and efficacy analyses are
patient self-rated mean total nasal symptom scores.
 In general, for drug products that BE determination is made on the
basis of pharmacodynamics or clinical endpoints, measurement of
the active ingredients, or active moieties in an accessible biological
fluid (i.e., pharmacokinetic approach) is necessary to ensure
comparable systemic exposure (albeit minimal) between the T and R
product.
 However, for some locally acting drug products, such
pharmacokinetic studies may be limited by the labeled maximum
dose, drug bioavailability, and sensitivity of the bioassay used.
 In such circumstances, pharmacodynamics or clinical studies could
be used to document comparable systemic effects of these drug
products.

d. In-vitro dissolution testing:

 Dissolution/release testing is the most commonly used in vitro

method for BE assessment.
 Although in vitro dissolution/release testing has seldom been used
alone as a tool for BE demonstration, dissolution/release information
along with the in vivo study data is routinely submitted by drug

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sponsors for BE documentation of orally administered drug

products.
 Dissolution/release data have often been employed to substantiate
BE when there is a minor change to formulation or manufacturing. In
addition, in vitro dissolution/release data are utilized to support
waiver of BA/BE studies for lower strengths of a drug product,
provided that an acceptable in vivo study has been conducted for a
higher strength and compositions of these strengths are
proportionally similar.
 Together with the use of BCS, in vitro dissolution/release testing
has played an increasingly important role in the regulatory
determination as to whether the waiver off in vivo BE studies can be
granted for an immediate-release drug product (FDA 2000).
 To serve as an indicator for BE, an in vitro dissolution/ release test
should be correlated with a predicative of in vivo BA (FDA 1995,
2003).
 In this setting, the in vitro dissolution/release methodology should
be optimized to closely mimic the physiological environment in vivo.
 For a drug product, proper in vitro dissolution/release behavior in
the presence of different formulations with defined in vivo
absorption characteristics will be useful to facilitate the
establishment of an in vitro-in vivo correlation (IVIVC).
 The in vitro dissolution/release method developed in such a manner
may be utilized as a surrogate for BA/BE studies when a change
occurs in manufacturing or formulation.

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Cross-over
over designs

Replicated crossover design: Sequence studies

studies- 3-way
way and 4-way
4
design

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Sampling

 In a typical BE study, the T and R product are generally

administered with 8 oz (i.e., 240 mL) of water to each participating
subject under fasting conditions, unless the study is to be
conducted under fed conditions where a high- fat meal will be given.
 For fasting studies, subjects are usually fasted overnight before
drug administration in the following day and standardized meals will
be provided to subjects no less than 4 h after dosing.
 For BE studies with pharmacokinetic measures, under normal
circumstances, a series of blood samples (rather than urine or tissue
samples) will be collected after dosing and parent drug (and major
metabolites) concentrations in serum or plasma will be measured.
 However, depending on the drug kinetics, whole blood may be more
appropriate for analysis of some drugs, e.g., tacrolimus.

Criteria for comparisons:

 It focuses on the degree of certainty needed in the analysis of

relative BA or BE studies. An equivalence approach is generally
recommended.

The approach usually relies on:

a. A criterion to allow the comparison.

b. A confidence interval for the criterion.
c. A BE limit (also called the goalpost).
 Log- transformation of exposure measures is generally
recommended.
 To compare measures in these studies, data are analyzed by using an
average BE criterion with other criteria allowed more recently.

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Drug review processes: INDs- NDAS:

BE documentation may be useful during the IND-NDA period to
establish links between:

a. Early and late clinical trial formulations;

b. Formulations used in clinical studies and stability studies, if
different; and
c. Clinical trial formulations and the to-be-marketed drug product.
 In each comparison, the new formulation or new method of
manufacture is the test product, and the prior formulation or
method of manufacture is the reference product. It may not be
possible to conclude BE because the test product produces higher or
lower measures of rate and extent of absorption or because the
performance of the test or reference is more variable.
 In some cases, "bioinequivalence" is observed because of inadequate
numbers of subjects entered into the BE study.

ANDAs: Sponsors of ANDAS are required to establish BE between a

pharmaceutically equivalent generic drug product and the
corresponding listed drug.

Post approval Changes:

 Information on the types of in vivo BE studies and in vitro

dissolution needed for post-approval changes to drug products
approved as either NDAS or ANDAs are provided in FDA guidance.
 In the presence of certain major changes in components and
composition, and/or method of manufacture after approval, in vivo
BE between pre- and post change product may need to be re-
established.
 Under such circumstances, for approved NDAs, the drug product
after change should be compared with the drug product before

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change, whereas for approved ANDAS, the drug product after

change should be compared with the reference listed drug.

Biopharmaceutical Classification System [BCS]

The Biopharmaceutical Classification System was first developed by in
1995, by Amidon et al & his colleagues.

Definition: "The Biopharmaceutical Classification System is a a

scientific framework for classifying a drug substance based on its
aqueous solubility & intestinal permeability & dissolution rate”.

To saved time fast screening is required so drug substances are

classified on basis of solubility and permeability. This classification is
called Biopharmaceutical Classification System.

Factor Affecting on BCS

The Biopharmaceutical Classification System has been developed to

provide a scientific approach to allow for to prediction in vivo
pharmacokinetics of oral immediate release (IR) drug product by
classifying drug compound based on their,

a. Solubility
b. Permeability
c. Dissolution

a. Solubility

 The Maximum Amount of solute dissolved in a given solvent under

standard conditions of temperature, pressure and pH.
 Solubility is the ability of the drug to be solution after dissolution
 The higher single unit dose is completely soluble in 250 ml at pH 1-
6.8 (37°C).

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b. Permeability

 Permeability of the drug to pass the biological membrane which is

the lipophilic.
 Permeability is indirectly based on the extent of absorption of a
drug substance.
 Drug substance is con
considered
sidered to be highly permeable, when the
extent of absorption in human determined to be 90% or more of
administered drug or compare to in vivo reference dose.

c. Dissolution

 It is process in which solid substance solubilises in given solvent i.e

mass transfer
fer from solid surface to liquid phase.
 Using USP apparatus I at 100 rpm or USP apparatus II at 50 rpm .
 Dissolution Media [900 ml],
1. N HCl or simulated gastric fl
fluid
uid (pH 1.2) without enzyme.
2. pH 4.5 buffer & pH 6.8 buffer.
3. Simulated intestinal fluid withou
without enzyme.

Biopharmaceutical Classification System for Drug

Drug:

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Class - I

 Ideal for oral route administration.

 Drug absorbed rapidly.
 Drug dissolved rapidly.
 Rapid therapeutic action.
 Bioavailability problem not expected for immediate release drug
product.
E.g. Metoprolol, Propranolol, Diltiazem.

Class - II

 Oral route for administration.

 Drug absorb rapidly.
 Drug dissolves slowly.
 Bioavailability is controlled by dosage form and rate of release of
the drug substance
E.g. Nifedipine, naproxen.

Class - III

 Oral route for administration.

 Drug absorbance is limited.
 Drug dissolves rapidly.
 Bioavailability is incomplete if drug is not release or dissolve in
absorption window.
E. g. Cimitidine, Metformin, Insulin.

Class - IV

 Poorly absorbed by orally administration.

 Both solubility & permeability limitation.
 Low dissolution rate.
 Slow or low therapeutic action.

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 An alternate route of administration may be needed.

E.g. Taxol, Chlorthiazole, Cefexime Trihydrate.

Application

a. To predict in vivo performance of drug product using solubility and

permeability measurements.
b. Aid in earliest stages of drug discovery research.
c. To use in biowaiver considerations.
d. For research scientist to decide upon which drug delivery
technology to follow or develop.
e. Also for the regulation of bioequivalence of the drug product during
scale up and post approval.

Class Boundaries

Highly Soluble

 The highest dose strength is soluble in ≤ 250 ml water over a pH

range of 1 to 7.5.
 The volume estimate - a glassful (8 ounce)

Highly Permeable - When the extent of absorption in humans is

determined to be > 90% of an administered dose.

Rapidly Dissolving - When > 85% of the labeled amount of drug

substance dissolves within 30 minutes using USP apparatus I or II in a
volume of ≤ 900 ml buffer solutions.

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Generic Biologics (Biosimilar Drug Products)

Introduction

Generic - A generic drug is a pharmaceutical drug that contains the

same chemical substance as drug that was originally protected by
chemical patents

Biologics - A therapeutic agent manufactured in a living system such

as a microorganism, plant or animal cell using recombinant DNA
technology,

Biological products

 Biological products are a diverse category of products and are

generally large, complex molecules.
 These products may be produced through biotechnology in a living
system, such as a microorganism, plant cell, or animal cell
 And that are often more difficult to characterize than small
molecules drugs

Biosimilar - A Biosimilar drug that is very much like another biological

drug (called the reference drug) that has already been approved by
the U.S. Food and Drug Administration (FDA) and has no clinically
meaningful differences in terms of safety and effectiveness from the
reference product

A Biosimilar drug is a safe and effective, high-quality treatment that is

"highly similar with no meaningful clinical differences" to a brand name
biologic drug that's used to treat a certain disease.

 Almost identical copy of an original biological product.

 They are large molecules with complex structures.
 They are prepared from engineered organism.
E.g.: Insulin produced from E-Coli.

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According WHO

 The term is Similar Biologic Product.

 Similar to an already licensed reference bio therapeutic product
in terms of quality, safety & efficacy.

According US-FDA

 The term is Follow-on Biologic.

 Highly similar to reference biologic product without clinically
meaningful differences in safety, purity and potency.

History

 In late 1960's, the FDA developed the Abbreviated New Drug

Application (ANDA) for approval of generic drug.
 The 1st ever "similar biologic product manufactured by India was a
Hep-B vaccine named BIOVAC B in 2000.
 The 1st biosimilar approved by EMEA (Europe, Middle East, Africa)
was OMNITROPE which is a biosimilar Recombinant Human Growth
Hormone in Year 2006.
 US-FDA manufactured 1st follow up biologic Zarxio (Filgrastim) in
2015.

Biosimilars Include

1. Blood and Plasma products

2. Non recombinant products
3. Recombinant products
4. Monoclonal antibodies
5. Vaccines

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Requirements for Biosimilars

 It should be biologic product

product.
 Reference product should be an already licensed biologic product.
product
 The biosimilar should demonstrate a high similarity of safety,
quality and efficacy to that of the reference product.
product
 Similarity should be determined by comparing the Biosimilar
product with the reference product based on quality, non clinical
and clinical studies
studies.

Difference between biolo

biologic and biosimilars

 Biologic drugs are large, complex proteins made from living cells
through highly complex manufacturing processes.
 Unlike generic drugs, which are copies of chemical drugs.
drugs
 A Biosimilar is a copy of a biologic medicine that is similar, b
but not
identical, to the original medicine
medicine.
 It enters the market subsequent to a previously authorized version
whose patent has expired and is approved only after showing that it
is "highly similar" to an approved biological product
product.

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 Compared to a biologic, it can be easier to get a Biosimilar Barshi

Shahu approved by regulatory agencies
 Because the structural difference between biologics and biosimilars
is so minimal, it becomes very easy to make the mistake of assuming
they are identical. However, this does not make them
interchangeable
 Biosimilars are often given the same indications as the originator
drugs in order to reduce the development cost, hence the market
price, making it more accessible and affordable to patients

Difference between biosimilars and generic drug

 Biosimilar drugs and generic drugs are two different things

 Generic drugs contain identical medicinal ingredients to their
reference products. They are composed of small molecules that are
chemically synthesized.
 Biosimilar drugs and their reference biologics are very similar but
not identical.

Source

 Living organism (Biosimilar)

 Chemical synthesis (Generic)

Size

 Large molecule (Biosimilar)

 Small molecule (Generic)

Structure

 Complex, heterogeneous (Biosimilar)

 Well defined (Generic)

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Manufacturing process

 Difficult (Biosimilars)
 Relative simple (Generic)

Stability

 Unstable, sensitive to external conditions (Biosimilars)

 Stable (Generic)

Immunogenicity

 Immunogenic (Biosimilars)
 Mostly non immunogenic (Generic)

Interchangeable with reference product

 No (Biosimilars)
 Yes (Generic)

Cost

 High (Biosimilars)
 Low (Generic)

Biosimilar Industries around the World

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Biosimilar Industries in India

 Biosimilars approved and used in India mainly consist of the

vaccines, monoclonal antibodies, insulin, and recombinant proteins.
 India has achieved the distinction of being the second largest
supplier of vaccines in the world:
1. Cipla
2. Lupin
3. Biocon ltd
4. Cadila Healthcare (Zydus)
5. Dr. Reddy's laboratories

Who approves biosimilars in India?

 The regulations stem from biosimilar guidelines originally Drafted in

2012 by 2 Indian governmental agencies, the Central Drugs
Standard Control Organization (CDSCO) and the Department Of
Biotechnology (DBT), meant to address and improve the development
and approval process for Biosimilars.
 India has over 95 approved biosimilars in the domestic market more
than any other country and market penetration.
 Biosimilars emerging as one of the fastest-growing sectors in the
market.
 Rules regarding clinical trials are much more stringent for
biosimilars than those applied to generics.

Biosimilar are also regulated for various processes (e.g, clinical

trials, import and manufacture) and are governed primarily by:

 Drugs and Cosmetics Act (1940; and Rules 1945);

 New Drugs and Clinical Trial Rules (2019);
 Guidelines on Similar Biologics;

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 Regulatory Requirements for Marketing Authorization in India

(2016);
 Rules for Manufacture, Use, Import, Export and Storage of
Hazardous Microorganisms and Genetically Engineered Organisms
or Cells (1989);
 Environment
ment (Protection) Act (1986);
 Regulations and Guidelines on Biosafety of Recombinant DNA
Research and Biocontainment (2017);
 CDSCO Guidance for the Industry (2008);
 Guidelines for Generating Pre
Pre-clinical
clinical and Clinical Data for rDNA
Vaccines, Diagnostics and other Biological (1999);

Some Biosimilar Approved In India

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Are biosimilar drugs safe?

 There are strict rules about how a biosimilar drug is tested for its
Safety
 Just like any drug, a biosimilar drug is tested in clinical trials to
make sure it is safe to use in people
 The data from the clinical trials are carefully reviewed by the (FDA)
to be sure the biosimilar drug is just as safe and effective as its
brand name biologic drug this means it has met the strict standards
for being safe

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Clinical Significance of Bioequivalence Studies

1. Products Requiring Be Studies

Products requiring be studies are mainly

1. Drugs with narrow margin of safety,

E.g. digoxin, antiarrhythmics, anticoagulants, cytostatics, lithium,
phenytoin, cyclosporine, sulphonylureas, theo-phylline
2. Critical use drugs, i.e. drugs indicated for serious conditions
requiring assured therapeutic response,
E.g. antiinfectives, cardiovascular drugs, antiepileptics,
antiasthmatics; and
3. Sustained or modified release products, due to the difficult
formulation.

2. Drug Interchangeability

 In practice, bioequivalence in drug absorption has been interpreted

that the confidence interval for the ratio of means (of drug
absorption) is within bioequivalence limits.
 An alternative would be to show that the tolerance intervals (or a
distribution free model) overlap sufficiently.
 Many practitioners interpret that generic drug products and the
innovative drug product can be used interchangeably because they
are therapeutically equivalent.
 The FDA, however, does not indicate that approved generic drug
products and the innovative drug products can be used
interchangeably.
 The FDA only indicates that an approved generic drug product can
be used as a substitute to the innovative drug product.
 Basically, drug interchangeability can be classified either as
 Drug prescribability or

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 Drug switchability
 Drug prescribability is defined as the physician's choice for
prescribing an appropriate drug product for his/her new patients
between a brand-name drug product and a number of generic drug
products that have been shown to be bioequivalent to the brand-
name drug product.
 Drug switchability, on the other hand, is related to the switch from
a drug product (e.g., a brand-name drug product) to an alternative
drug product (e.g., a generic copy of the brand-name drug product)
within the same subject, whose concentration of the drug product
has been titrated to a steady, efficacious, and safe level.
 As a result, drug switchability is considered more critical than drug
prescribability in the study of drug interchangeability for patients
who have been on medication for a while.
 Drug switchability, therefore, is exchangeability within the same
subject.

3. Comparing Generic and Innovator Drugs:

Bioequivalence (BE) study is required to show whether a generic copy

product can be interchangeable with the brand innovator product.

E.g.: BE studies of clopidogrel generics :

 Clopidogrel is a prodrug that must undergo hepatic metabolism to

become the active metabolite.
 The active metabolite is highly unstable and thus difficult to
measure.
 Therefore, the BE study of clopidogrel may be a pharmacokinetic
study or a pharmacodynamic study.
 The pharmacokinetic BE study of clopidogrel is based on the
measurement of clopidogrel parent compound

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 The pharmacodynamic BE study of clopidogrel is based on the

inhibition of ADP binding to its platelet membrane receptors, P2Y12,
which causes platelet aggregation.

4. Drug Formulations

 Bioequivalence for most of oral tablets or capsules is demonstrated

in vivo by comparing the rate and extent of absorption that is
bioavailability of the generic product with that of the innovator
product.
 This is done by measuring the active ingredient concentration in
blood, plasma, serum or other biological fluids over a certain period
of time for both the generic and innovator products, also called test
and reference drugs respectively.
 By doing so the bioequivalence studies frequently rely on
pharmacokinetic measures such as area under the concentration-
time curve (AUC) and peak drug concentration (Cmax)
 Use of Alternate routes:Drugs with high hepatic first pass
metabolism should be given by routes other than oral. ie., sublingual,
transdermal eg., Nitroglycerine
 High oral doses: Some drugs have high hepatic extraction ratio.
 Less dose in hepatic Disease: In severe hepatic cirrhosis/portal
systemic shunts, the dose of the drugs with large extraction ration
and hepatic first pass effect should be reduced otherwise toxicity

5. Drug Development

 Bioequivalence studies are very important for the development of a

pharmaceutical preparation in the pharmaceutical industry.
 Their rationale is the monitoring of pharmacokinetic and
pharmacodynamics parameters after the administration of tested
drugs.

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 The target of such study is to evaluate the therapeutic

compatibility of tested drugs (pharmaceutical equivalents or
pharmaceutical alternatives).

6. Clinical Interpretation

 Clinical interpretation is important in evaluating the results of a

bioequivalence study.
 Bioequivalence studies should be conducted for the comparison of
two medicinal products containing the same active substance.
 Two products marketed by different licensees, containing same
active ingredient(s), must be shown to be therapeutically equivalent
to one another in order to be considered interchangeable.
 Differences of less than 20% in AUC & Cmax between drug products
are unlikely to be clinically significant in patients.
 Minimize product to product variability by different manufactures &
lot to lot variability with a single manufacture.

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Special Concerns in Bioavailability and Bioequivalence

Design and Conduct of Studies

Pharmacokinetic studies

Basic Study Design

a. What are the scientific questions to be answered.

b. The nature of the reference material and the dosage form to be
tested
c. The availability of analytical methods
d. Benefit risk ratio considerations in regard to testing in humans

Study Population

Selection of the number of subjects

The number of subjects required for a study should be statically

significant and is determined by the following considerations:

1. The error variance with associated from a pilot experiment, from

previous studies or from published data.
2. The significance level desired usually 0.05
3. The expected deviation from the reference product compatible with
bioequivalence
4. The required power, normally >80% to detect the maximum allowable
difference (usually 20%) in primary characteristics to be studied

Selection Criteria for Subjects

 To minimize intra and inter individual variation subjects.

 Healthy adult volunteers with the aim to minimize variability and
permit detection of difference between the study drugs.
 Women taking contraceptive drugs should normally avoided.

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 If the drug product is intended for use in both sexes attempt

should not be included in the studies.
 For a drug representative a potential hazard in one of users, the
choice of subjects may be narrowed. example: studies on
teratogenic drugs

Genetic Phenotyping - If a drug is known to be subject to major

genetic polymorphism, studies could be performed in panels of
subjects of known phenotype or genotype for the polymorphism in
questions.

Study Conditions

 Selection of blood sampling points

 Fasting and fed state conditions
 Steady state conditions

In following cases - an additional "steady state study" is considered

appropriate:

1. Where the drug has a long terminal elimination half life and blood
concentrate after a single dose cannot be followed for a sufficient
time.
2. Where assay sensitivity is inadequate to follow the terminal
elimination phase for an adequate period of time
3. For drugs, which are a necessary part of therapy, but where multiple
dose therapy is required, e.g. cytotoxics
4. For modified-release products where it is necessary to assess the
fluctuation in plasma concentration in plasma concentration over a
dosage interval at steady state.
5. For those drugs which induce their own metabolism or show large
intra individual variability.
6. For enteric-coated preparations where the coating is innovative.

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7. For combination products where the ratio of plasma concentration

of the individual drug is important.
8. For drugs that exhibit non-linear (i.e. dose or time dependant)
pharmacokinetics.
9. Where the drug is likely to accumulate in the body

Characteristics to be investigated during bioavailability/

bioequivalence studies

 Measured concentrations of the active in the biological matrix

 Where the concentrations of the drug may be too low to accurately
measure in the biological matrix
 Limitations of the analytical method
 Unstable drugs
 Drug with a very short half life in case of prodrugs

Bioanalytical Methods

The bio analytical methods methods used to determine the drug and its
metabolites in plasma, serum, blood or urine or any other suitable
matrix must be well characterised, standardised, fully validated and
documented to yield reliable results that can satisfactorily interpreted

The validation of the analytical method can be envisaged to consist

of two distinct phases:

1. The pre-study phase which comes before the actual start of the
study and involves the validation of the method on biological matrix
human plasma samples and spiked plasma samples.
2. The study phase in which the validated bio analytical method is
applied to the actual analysis of samples from bioavailability and
bioequivalence studies mainly to confirm the stability, accuracy and
precision

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Pre study phase

1. Stability of the drug/ metabolite in the biological matrix

2. Specificity
3. Sensitivity
4. Precision and accuracy
5. Recovery
6. Range and intensity
7. Analytical system stability

Study phase

 In general, with acceptable as defined by validated data, the

analysis of biological sample can be done by single determination
without a need for a duplicate or replicate analysis.
 The need for duplicate analysis should be assessed on a case-by case
basis. A procedure should be developed that documents the reason
for re-analysis.

Quality Control sample

 Quality control samples are samples with known concentration

prepared by spiking drug free biological fluid with drug.
 These samples are prepared in low, medium and high concentration.
 To avoid possible confusion between quality control samples and
standard solutions during review process, preparation of quality
control samples at concentration different from those used for the
calibration id recommended.

Repeat analysis

 In most studies some samples will require re-analysis because of

aberrant results due to processing errors, equipment's failure or
poor chromatography.

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 The criteria for re-analysis of such samples should be stated.

 The criteria for repeat analyses should be determined prior to
running the study and recorded in the protocol/ laboratory
operating procedures.

Statiscal Evaluation

1. Data analysis - Reducing consumers risk as well as manufacture's

risk.
2. Statiscal analysis. Example - ANOVA, chi square test
3. Criteria for bioequivalence - To establish bioequivalence, the
calculated 90% confidence interval for AUC and Cmax should fall
within the bioequivalence range, usually 80-125%. This is equvilaent
to the rejection of two one sided t-tests with the full hypothesis of
non- bioequivalence at 5% level of significance. The non-parametric
90% interval for Tmax within clinically acceptable range

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Generic Substitution
 Drug product selection &generic drug product substitution are
major responsibilities for physicians, pharmacists and others who
prescribes dispense or purchase drugs.
 To facilitate that FDA published, Approved Drug Products with
Therapeutic Equivalence Evaluations
 Orange book which identifies drug products approved on the basis
of safety and effectiveness.
 They serve as public information and advice to health agencies,
prescribers and pharmacists to promote public education in the area
of drug product selection.
 To contain drug costs, most state have adopted generic substitution
laws to allow pharmacist to dispense a generic drug product for a
brand- name drug product that has been prescribed.
 Some states have adopted positive formulary which lists
therapeutically equivalent or interchangeable drug product that
pharmacist may dispense.
 Others use a negative formulary, which lists drug products that are
not therapeutically equivalent, or interchange of which is prohibited.
 And if the drug is not negative formulary, the unlisted generic drug
products are assumed to be therapeutically equivalent and may not
be interchanged.

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Approved Drug Products with Therapeutic Equivalence Evaluation

 Orange book contains therapeutic equivalence evaluations for

approved drug products made by various manufacturers.
 These marketed products are evaluated according to specific
criteria, the evaluation codes used for these drugs are listed

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 The concept of therapeutic equivalence as used to develop the

Orange Book applies only to drug products containing the same
active ingredient.
 And does not encompass a comparison of different therapeutic
agents used for same condition.
Eg: propoxyphene HCL versus pentazocine HCL for treatment of
pain.

Generic Substitution Of Modified - Release Drug Products

 Generic extended- release drug products may have different

release mechanism compared to brand drug product.
 The modified drug release drug products have different
pharmacokinetic profiles.
 And even different clinical efficacy compared to conventional form
of the drug given in the same daily dose.
 Since the pharmacokinetic profile may differ, the practitioner
needs to consult the FDA publication, approved Drug Products with
Therapeutic Equivalence Evaluation (Orange Book),to determine
which of these drugs can be substituted.

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Unit 5 - Application of Pharmacokinetics

Modified-Release Drug Products:

Modified release drug product is those that alter the timing and/or
the rate of release of drug substance.

Types of modified release drug products are:

1. Delayed release
2. Extended release
3. Targeted release
4. Orally disintegrating tablet

1. Delayed Release Drug Products - A dosage form that releases a

discrete portion/portions of drug at a time other than the promptly
release after administration. An initial portion may be released
promptly after administration.

E.g., Enteric-coated dosage forms are common delayed-release

products (enteric-coated aspirin and other NSAID products).

2. Extended Release Drug Products

 A dosage form that allows at least a twofold reduction in dosage

frequency as compared to that drug presented as an immediate-
release (conventional) dosage form.
 It includes controlled-release, sustained-release, and long-acting
drug products.

3. Targeted Release Drug Products

 A dosage form that releases drug at or near the intended

physiologic site of action.
 Targeted-release dosage forms may have either immediate- or
extended-release characteristics.

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4. Orally Disintegrating Tablets - ODTs have been developed to

disintegrate rapidly in the saliva after oral administration. It may be
used without the addition of water. The drug is dispersed in saliva and
swallowed with little or no water.

Biopharmaceutic Factors:

 The ER oral drug products remain in the gastro-intestinal (GI) tract

longer than conventional, immediate release, drug products. Thus,
drug release from an ER drug product is more subject to be
affected by the anatomy and physiology of the GI tract, GI transit,
pH, and its contents such as food compared to an immediate-release
oral drug product.
 In some cases, there may be a specific absorption site or location
within the GI tract in which the extended-release drug product
should release the drug.
 This specific drug absorption site or location within the GI tract is
referred to as an "absorption window". The absorption window is the
optimum site for drug absorption.

1. Stomach

 The stomach receives food or liquids from the oesophagus. It is a

"mixing and secreting" organ.
 In the presence of food, the stomach is in the "digestive phase"; in
the absence of food, the stomach is in the "interdigestive phase".
 If the drug is administered during the digestive phase; Fatty
material, nutrients, and osmolality may further extend the time of
the drug staying in the stomach. When the drug is administered
during the interdigestive phase, the drug may be swept along rapidly
into the small intestine. The drug release rates from some

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extended-release drug products are affected by mechanism of drug

release.
 The rate of drug release of various ER formulations can be affected
by the composition of the co- administered meal.

2. Small Intestine and Transit Time

 The small intestine provides an enormous surface area for drug

absorption because of the presence of microvilli.
 Its transit time of a solid preparation has been concluded to be
about 3 hours or less in 95% of the population.

3. Large Intestine

 Here, drug transit time is slow. The rectum has a pH of about 6.8-
7.0 and contains more fluid compared to the colon. Drugs are
absorbed rapidly when administered as rectal preparations.
 However, the transit rate through the rectum is affected by the
rate of defecation. Presumably, drugs formulated for 24-hour
duration must remain in this region to be absorbed.

pH Values against Transit Time at Different Segments of GI

Tract:

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Dosage form selection for MRDP

The properties of the drug and the size of the required dosage are
important in formulating an extended-release product. These
properties will also influence the selection of appropriate dissolution
media, apparatus, and test parameters to obtain in vitro drug release
data that will reflect in vivo drug absorption.

E.g.

 Drug with low aqueous solubility generally should not be formulated

into a non- disintegrating tablet, because risk of incomplete drug
dissolution is high.
 Drug with low solubility at neutral pH should be formulated as an
erodible tablet, so that most of drug is released before it reaches
the colon.
 A drug with high water solubility in acidic pH in stomach but very
insoluble at intestine pH may be difficult to formulate into ER drug
product. The osmotic type of controlled drug release system may be
more suitable for this type of drug.
 With too much coating, bioavailability gets reduced.

Kinetics of extended-release dosage forms

 The amount of drug required in an extended-release dosage form to

provide a sustained drug level in the body is determined by the
pharmacokinetics of the drug, the desired therapeutic level of the
drug, and the intended duration of action.
 In general, the total dose required (Dtot) is the sum of the
maintenance dose (Dm) and the initial dose (D) released immediately
to provide a therapeutic blood level.

Dtot = D1+Dm

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 In practice, Dm (mg) is released over a period of time a

and
nd is equal to
the product of td (the duration of drug release) and the zero-order
rate kr0 (mg/h). Therefore, can be expressed as:

Dtot = DI +krotd

 Ideally, the maintenance dose (Dm) is released after D, has

produced a blood level equal to the therapeutic drug level (C).
However, due to the limits of formulations, Dm actually starts to
release at t = 0. Therefore, D, may be reduced from the calculated
amount to avoid "topping."

 It describes the total dose of drug needed, with t, representing the

time needed to reach peak drug concentration after the initial dose.
 For a drug that follows a one
one-compartment
compartment open model, the rate of
elimination (R) needed to main
maintain
tain the drug at a therapeutic level
(Cp) is:

R = kVDCP

 Where kr0 must be equal to R in order to provide a stable blood level

of the drug. it provides an esestimation
timation of the release rate (kr0)
required in the formulation. The above equation may also be written
writt
as

R = CPClT

 Where ClT is the clearance of the drug. In designing an extended-

extended
release product, D, would be the loading dose that would raise the
drug concentration in the body to Cp, and the total dose needed to
maintain therapeutic concentration in the body would be simply

Dtot = DI+CpClT

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Modified Drug Del

Delivery Products
Modified-release
release drug products are designed for different routes of
administration based on the physicochemical, pharmacodynamics (PD),
and pharmacokinetic (PK) properties of the drug an
andd on the properties
of the materials used in the dosage form.

Characteristics of Extended
ended Release Oral Dosage Forms

The drugs best suited for incorporation into an extended release

product have the following characteristics:

 They exhibit neither very slo

sloww nor very fast rates of absorption and
excretion.
 They should uniformly absorb from the gastrointestinal tract.
 They are administered in relatively small doses.
 Possess a good margin of safety.
 They are used in the treatment of chronic rather than acute
conditions.

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Types of Extended-Release Products:

1. Drug Release from Matrix - A matrix is an inert solid vehicle in

which a drug is uniformly suspended. A variety of excipients based on
wax, lipid, as well as natural and synthetic polymers have been used as
carrier material in the preparation of such matrix type of drug
delivery systems. The drug release from such matrix systems is mainly
controlled by the diffusion process, concomitant swelling, and/or
erosion process.

Classification of Matrix Tablets

Based on the retarded materials used, matrix tablets can be

divided into five types:

a. Hydrophobic matrix (plastic matrix)

b. Lipid matrix
c. Hydrophilic matrix
d. Biodegradable matrix
e. Mineral matrix.

Embedding drug in inert plastic matrix

 By this method, the drug is granulated with an inert plastic material

such as polyethylene, polyvinyl acetate, or polymethacrylate, and the
granulation is compressed into tablets.
 The drug is slowly released from the inert plastic matrix by
diffusion. The inert tablet matrix, expended of drug, is excreted
with the fecus.

Embedding drug in slowly eroding or hydrophilic matrix system

 By this process, the drug substance is combined and made into

granules with an excipient material that slowly erodes in body fluids,
progressively releasing the drug for absorption.

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 When these granules are mixed with granules of drug prepared without
the excipient, the uncombined granules provide the immediate drug
effect whereas the drug-excipient granules provide extended drug
action.
 Matrix system can also be classified according to their porosity
situation, including microporous, and nonporous system.
Gum type matrix tablets
 Some excipients have a remarkable ability to swell in the presence
of water and form a substance with a gel-like consistency. When
this happens, the gel provides a natural barrier to drug diffusion
from the tablet.
 Gelatin dissolves rapidly after the gel is formed. Drug excipients
such as methylcellulose, gum tragacanth, Veegum, and alginic acid
form a viscous mass and provide a useful matrix for controlling drug
release and dissolution.

Polymeric Type Matrix Tablets

 The most important characteristic of this type of preparation is

that the prolonged release may last for days or weeks rather than
for a shorter duration (as with other techniques).
 An early example of an oral polymeric matrix tablet was Gradumet
(Abbott Laboratories), which was marketed as an iron preparation.
The non-biodegradable plastic matrix provides a rigid geometric
surface for drug diffusion, so that a relatively constant rate of drug
release is obtained.

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2. Slow release coated beads, granules or microspheres

 In these systems, the drug is distributed onto beads, pellets,
granules, or other particulate systems. The size of these beads can
be very small (microencapsulation) for injections or larger for oral
drug delivery. Several approaches have been used to manufacture
beaded formulations including pan coating, spray drying, fluid-bed
drying, and extrusion-spheronization.
 Pan coating is a modified method adopted from candy manufacturing.
Cores or nonpareil seeds of a given mesh size are slowly added to
known amount of fine drug powder and coating solution and rounded
for hours to become coated drug beads. The drug-coated beads are
then coated with a polymeric layer, which regulates drug release
rate by changing either the thickness of the film or the composition
of the polymeric material.

3. Microencapsulated Drug

 Microencapsulation is a process of encapsulating microscopic drug

particles with a special coating material, therefore making the drug
particles more desirable in terms of physical and chemical
characteristics.
 It is a process by which solids, liquids, or even gases may be
enclosed in microscopic particles by formation of thin coatings of
wall material around the substance.
 The typical encapsulation process usually begins with dissolving the
wall material, say gelatin, in water. The material to be encapsulated
is added and the two-phase mixture thoroughly stirred. With the
material to be encapsulated broken up to the desired particle size, a
solution of a second material, usually acacia, is added. This additive
material concentrates the gelatin into tiny liquid droplets.

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 One of the advantages of microencapsulation is that the

administered dose of a drug is subdivided into small units that are
spread over a large area of the gastrointestinal tract, which may
enhance absorption by diminishing localized drug concentration.
 A common drug that has been encapsulated is aspirin. Aspirin has
been microencapsulated with ethyl cellulose, making the drug
superior in its flow.

4. Ion-Exchange Products:

Ion-exchange technique has been popularly applied in water purification

and chemical extraction. Ion-exchange preparations usually involve an
insoluble resin capable of reacting with either an anionic or a cationic
drug. An anionic resin is negatively charged so that a positively charged
cationic drug may attach the resin to form an insoluble non-absorbable
resin-drug complex.

It provides protection for very bitter or irritating drugs. Ion exchange

has been combining with a coating to obtain a more effective sustained
release product.

Process

 A solution of a cationic drug may be passed through a column

containing an ion-exchange resin, forming a complex by the
replacement of hydrogen atoms.
 The resin-drug complex is then washed and may be tableted,
encapsulated, or suspended in an aqueous vehicle. The release of the
drug is dependent upon the pH and the electrolyte concentration in
the gastrointestinal tract.
 Release is greater in the acidity of the stomach than in the less
acidic environment of the small intestine. E.g., Tussionex
pennkinetic, an an oral suspension containing Hydrocodone polistirex

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and chlorpheniramine polistirex suspension and phentermine resin

capsules.
 The insoluble drug complex containing the resin and drug dissociates
in the GIT in the presence of the appropriate counter ions. The
released drug dissolves in the fluids and is rapidly absorbed.

5. Osmotic drug delivery systems: Osmotic drug delivery systems

have been developed for both oral extended-release products known as
gastrointestinal therapeutic systems (GITS) and for parenteral drug
delivery as an implantable drug delivery (e.g., osmotic minipump). Drug
delivery is controlled by the use of an osmotically controlled device in
which a constant amount of water flows into the system causing the
dissolving and releasing of a constant amount of drug per unit time.

Process in osmotic mini pump

 The pioneer oral osmotic pump drug delivery system is the Oros
system, developed by Alza.
 The system is composed of a core tablet surrounded by a semi
permeable membrane coating have a 0.4 mm diameter hole produced
by laser beam.
 The system is designed such that only a few drops of water are
drawn into the tablet each hour.
 The rate of inflow of water and the function of the tablet depends
upon the existence of an osmotic gradient between the contents of
the bi-layer core and the fluid in the GI tract.
 Drug delivery is essentially constant as long as the osmotic gradient
remains constant.
 Here, The drug release rate may be altered by;
 Changing the surface area,
 The thickness or composition of the membrane,
 Changing the diameter of the drug release orifice.

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6. Gastro retentive Systems:

 The extended-release
release drug product should release the drug
completely within the region in the GI tract in which the drug is
optimally absorbed. Due to GI transit, the extended
extended-release
release drug
product continuously moves distally down the GI tract. In some
cases, the extended--release drug product
ct containing residual drug
may exit from the body. Pharmaceutical formulation developers have
used various approaches to retain the dosage form in the desired
area of the gastrointestinal tract.
 One such approach is a gastro
gastro-retentive
retentive system that can remain
rema in
the gastric region for several hours and prolong the gastric
residence time of drugs.
 Usually, the gastro-retentive
retentive systems can be classified into several
types based on the mechanism applied such as
a. High-density systems
b. floating systems
c. Expandable systems
d. Super porous hydrogels
e. Mucoadhesive
ucoadhesive or bioadhesive systems
f. Magnetic systems
g. Dual working systems

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7. Transdermal Drug Delivery Systems:

Skin represents the largest and most easily accessible organ of the
body. A Transdermal drug delivery sys
sys-tem (patch)
tch) is a dosage form
intended for delivering drug across the skin for systemic drug
absorption.

Transdermal drug absorption also avoids presystemic metabolism or

"first-pass"
pass" effects. It delivers the drug through the skin in a
controlled rate over an exte
extended period of time.

8. Core Tablets

 A core tablet is a tablet within a tablet, the inner core is usually

used for the slow-drug
drug-release
release component, and the outside shell
contains a rapid-release
release dose of drug.
 Formulation of a core tablet requires two gragranulations.
nulations. The core
granulation is usually compressed lightly to form a loose core and
then transferred to a second die cavity, where a second granulation
containing additional ingredients is compressed further to form the
final tablet.
 The core material may
ay be surrounded by hydro
hydro-phobic
phobic excipients so
that the drug leaches out over a prolonged period of time. This type
of preparation is sometimes called a slowslow-erosion
erosion core tablet,
because the core generally contains either no disintegrant or
insufficient disintegrant
sintegrant to fragment the tablet.

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9. Prolong Action Tablets

 An alternate approach to prolong the action of a drug is to reduce

the aqueous solubility of the drug, so that the drug dissolves slowly
over a period of several hours. The solubility of a drug is dependent
on the salt.
 Another application of prolong-action tablets is also called as
pulsatile drug delivery system. This chrono pharmaceutical
formulation is usually used in the treatment of circadian rhythm
dysfunction disease.

10. Repeat Action Tablets

 Repeat action tablets are prepared so that an initial dose of drug is

released immediately followed later by a second dose. The tablets
may be prepared with the immediate release dose in the tablets
outer shell or coating with the second dose in the tablets inner core,
separated by a slowly permeable barrier coating.
 Repeat action dosage forms are best suited for the treatment of
chronic conditions requiring repeated dosing.
 The drugs utilized should have low dosage and fairly rapid rates of
absorption and excretion.

11. Multitablet System

 Small spheroid compressed tablets 3 to 4 mm in diameter may be

prepared to have varying drug release characteristics.
 They may be placed in gelatin capsule shells to provide the desired
pattern of drug release.
 Each capsule may contain 8 to 10 minitablets, some uncoated for
immediate release and others coated for extended drug release.

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12. Complex Formation: Certain drug substances when chemically

combined with certain other chemical agents form chemical complexes
that may be only slowly soluble in body fluids, depending upon the pH of
the environment. This slow dissolution rate provides the extended
release of the drug.

13. Combination Product: A product comprised of two or more

regulated components, that is, drug/device, biologic/device,
drug/biologic, or drug/device/biologic, that are physically, chemically,
or otherwise combined or mixed and produced as a single entity. e.g.,
Monoclonal antibody combined with a therapeutic drug

14. Modified Release Parenteral Dosage Forms:

 It include microspheres, liposomes, drug implants, inserts, drug-

eluting stents, and nanoparticles.
 These formulations are designed by entrapment or
microencapsulation of the drug into inert polymeric or lipophilic
matrices that slowly release the drug, in vivo, for the duration of
several days or up to several years. Modified-release parenteral
dosage forms may be biodegradable or nonbiodegradable.
Nonbiodegradable implants need to be surgically removed at the end
of therapy.

Evaluation of Modified-Release Drug Products

In vitro/In vivo correlations (IVIVCS)

 IVIVCS is critical to the development of oral extended-release

products. Assessing IVIVCS is important throughout the periods of
product development, clinical evaluation, and submission of an
application for FDA- approval for marketing, and during post
approval for any formulation or manufacturing changes.

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Three categories of IVIVCS are included;

Level A - A predictive mathematical model for the relationship

between the entire in vitro dissolution/release time course.

E.g., the time course of plasma drug concentration or amount of drug

absorbed.

Level B - A predictive mathematical model of the relationship

between summary parameters that characterize the in vitro and in
vivo, time courses.

Level C - A predictive mathematical model of the relationship

between the amount dissolved in vitro at a particular time (or T50%)
and a summary parameter that characterizes the in vivo time course
(e.g. Cmax or AUC).

Dissolution Studies

 Reproducibility of the method

 Proper choice of the medium
 Maintenance of sink condition
 Control of solution hydrodynamics
 Dissolution rate as function of pH, ranging from 1-8

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Evaluation of In-Vivo Bioavailability Data

1. Pharmacokinetic Profile

 Plasma drug conc.-time curve should adequately define bioavailability

of drug from dosage form.
 The bioavailability data should demonstrate the extended release
characteristics of the dosage form compared to
reference/immediate release product.

2. Steady State Plasma Drug Concentration

 Fluctuation = C∞ max - C∞ min /C∞ avθ

 Where C∞ av is equal to [AUC]/T

3. Rate of Drug Absorption - For a extended release drug product to

claim zero- order absorption, Wagner nelson method is used.

4. Occupancy Time

 For drugs whose therapeutic window are known, plasma drug conc.
Maintained above the minimum effective drug concentration.
 The time required to obtain plasma drug levels within therapeutic
window is known as occupancy time.

Advantages of MRDP:

a. Reduction in drug blood level fluctuation.

b. Frequency reduction in dosing.
c. Patient compliance
d. Reduced adverse side effect.
e. Reduction in health care cost.

Disadvantages of MRDP:

a. Dose-dumping
b. Less flexibility in accurate dose adjustment.

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Targeted Drug Delivery Systems

 Targeted drug delivery system is a special form of drug delivery
system where the medicament is selectively targeted or delivered
only to its site of action or absorption and not to the non-target
organs or tissues or cells.
 It seeks to concentrate the medication in the tissues of interest
while reducing the relative concentration of the medication in the
remaining tissues.
 This improves efficacy and reduce side effects.

Ideal Characteristics:

 It should be nontoxic, biocompatible, biodegradable, and

physicochemical stable in vivo and in vitro.
 Restrict drug distribution to target cells or tissues or organs and
should have uniform capillary distribution.
 Controllable and predicate rate of drug release.
 Drug release does not effect the drug action.
 Therapeutic amount of drug release.
 Minimal drug leakage during transit.
 Carriers used must be bio-degradable or readily eliminated from the
body without any problem and no carrier induced modulation of
diseased state.
 The preparation of the delivery system should be easy or reasonably
simple, reproductive and cost effective.

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Reasons for Drug Targeting:

General considerations in targeted drug delivery

Considerations in the development of site

site-specific
specific or targeted drug
delivery systems include:

a. The anatomic and physiologic characteristics of the target

t site,
including capillary permeability to macromolecules and cellular
uptake of the drug.
b. The physicochemical characteristics of the therapeutically active
drug.
c. Physical and chemical characteristics of the carrier.
d. Selectivity of the drug
drug-carrier complex.
e. Any impurities introduced during the conjugation reaction linking the
drug and the carrier that may be immunogenic, be toxic, or produce
other adverse reactions.

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Target Site:

 The accessibility of the drug-carrier complex to the target site may

present bioavailability and pharmacokinetic problems, which also
include anatomic and/or physiologic considerations.
E.g., Targeting a drug into a brain tumor requires a different route
of drug administration (intrathecal injection) than targeting a drug
into the liver or spleen.
 Moreover, the permeability of the blood vessels or biologic
membranes to macromolecules or drug- carrier complex may be a
barrier preventing delivery and intracellular uptake of these drugs.
Site-Specific Carriers:
 To target a drug to an active site, one must consider whether there
is a unique property of the active site that makes the target site
differ from other organs or tissue systems in the body.
 The next consideration is to take advantage of this unique
difference so that the drug goes specifically to the site of action
and not to other tissues in which adverse toxicity may occur.
 In many cases the drug is complexed with a carrier that targets the
drug to the site of action.

e.g., one of the first approved drugs developed using

pharmacogenomic principles is Herceptin (trastu-zumab), a monoclonal
antibody designed to bind to the human epidermal growth factor
receptor. This receptor is over expressed on HER-2 positive breast
cancer cells. Therefore, the drug will preferentially bind HER-2
positive breast cancer cells, though other noncancerous cells may also
express the receptor

 Similarly, trastuzumab has also been used as targeting agents for

anticancer drug- encapsulated nanoparticles in clinical studies.

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 The successful application of these delivery systems requires the

drug-carrier complex to have both affinity for the tar-get site and
favourable pharmacokinetics for delivery to the organ, cells, and sub
cellular target sites.
 An additional problem, particularly in the use of protein carriers, is
the occurrence of adverse immunological reactions-an occurrence
that is partially overcome by designing less immunoreactive proteins.
 Humanized mAbs are an example of a therapeutic protein
engineered to be less immunoreactive.

Drugs

 Most of the drugs used for targeted drug delivery are highly
reactive drugs that have potent pharmacodynamic activities with a
narrow therapeutic range.
 These drugs are often used in cancer chemotherapy. Many of these
drugs may be derived from biologic sources, made by a semi
synthetic process using a biologic source as a precursor, or produced
by recombinant DNA techniques.
 The drugs may also be large macromolecules, such as proteins, and
are prone to instability and inactivation problems during processing,
chemical manipulation, and storage.

Targeting Agents

 Properly applied, drug targeting can improve the therapeutic index

of many toxic drugs. However, monoclonal antibodies are not the
"magic bullet" for drug targeting that many people had hoped. One
difficulty encountered is that the large molecule reduces the total
amount of active drug that can be easily dosed (i.e., the ratio of
drug to carrier).

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 In contrast, conventional carriers or targeting agents that are not

specific are often many orders of magnitude smaller in size, and a
larger effective drug dose may be given more efficiently. Antibody
fragments comprised of either the double- or single-chain variable
regions are also being tested as smaller drug targeting agents.
 In addition to employing monoclonal antibodies in liposomes and
other delivery systems. The resulting conjugate can theoretically
deliver the drug directly to a cell that expresses a unique surface
marker.
E.g., a tumor cell may over express the interleukin-2 receptor. In
this case, a cytotoxic molecule such as recombinant diphtheria toxin
is coupled to an mAb specific for the interleukin-2 receptor (Ontak).
The conjugate delivers the toxin preferentially to these tumor cells.
An overall tumor response rate for Ontak is 38%, with side effects
including acute hypersensitivity reaction (69%) and vascular leak
syndrome (27%).
 Myoscint is an 111In-labeled mAb targeted to myosin that is used to
image myocardial injury in patients with suspected myocardial
infarction. An immune response to mAb drugs may develop, since
mAbs are produced in mouse cells. "Humanized" mAbs are
genetically engineered to produce molecules that are less
immunogenic.

Oral Immunization:

 Antigens or fragmented antigenic protein may be delivered orally

and stimulate gut-associated lymphoid tissue (GALT) in the
gastrointestinal tract. This represents a promising approach for
protecting many secretory surfaces against a variety of infectious
pathogens, but products have not yet reached clinical trials.

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 Immunization against salmonella and Escherichia coli in chickens was

investigated for agricultural purposes. Particulate antigen delivery
systems, including several types of microspheres, have been shown
to be effective orally inducing various types of immune response.
Encapsulation of antigens with mucosal adjuvants can protect both
the antigen and the adjuvant against gastric degradation and
increase the likelihood that they will reach the site of absorption.

Advantages:

a. Drug administration protocols may be simplified.

b. Toxicity is reduced by delivering a drug to its target site, thereby
reducing harmful systemic effects.
c. Drug can be administered in a smaller dose to produce the desire
effect.
d. Avoidance of hepatic first pass metabolism.
e. Enhancement of the absorption of target molecules such as peptides
and particulates.
f. Dose is less compared to conventional drug delivery system.
g. No peak and valley plasma concentration.
h. Selective targeting to infections cells that compare to normal cells.

Disadvantages:

a. Rapid clearance of targeted systems.

b. Immune reactions against intravenous administered carrier systems.
c. Insufficient localization of targeted systems into tumour cells.
d. Diffusion and redistribution of released drugs.
e. Requires highly sophisticated technology for the formulation.
f. Requires skill for manufacturing storage, administration.
g. Drug deposition at the target site may produce toxicity symptoms.
h. Difficult to maintain stability of dosage form.

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Biotechnological Products
Many diseases occur as a result of variability in the genes involved in
producing essential enzymes or proteins in the body. The genes are
coded in deoxyribonucleic acid (DNA), helical double- stranded
molecules folded into chromosomes in the nucleus of cells. The Human
Genome Project was created more than a decade ago to sequence the
human genome, This national effort is continuing to yield information
on the role of genetics in congenital defects, cancer, disorders
involving the immune system, and other diseases that have a genetic
link.

As a result, biotechnology, or the use of biological materials to create

a specific product, in this case pharmaceuticals, has become an
important sector of the pharmaceutical industry and accounts for the
fastest growing class of new drugs in the market. Nucleic acid, protein
and peptide drugs, and diagnostics are the main drug products
emerging from the biopharmaceutical industry.

Biotechnological Drugs: Pharmaceutical biotechnology consist of the

combination of two branch which are "Pharmaceutical science" and
"Biotechnology".

Pharmaceutical Science: It can be simply define as the branch of

science that deals with the formulation compounding and dispensing of
drugs.

Biotechnology: Biotechnology drug differ from Pharmaceutical drugs in

that they use biotechnology as a means for manufacturing, which
involves the manipulation of microorganism, such as bacteria, or
biological substance, like enzymes, to perform a specific process.

Ex:- Antibiotics, vaccines etc.

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Biotechnological Products: Biotechnology can be defined as application

of technology using the living organisms to obtain useful products. The
products made by the biotechnology process include, pharmaceuticals
(medicine), food, and water purification, genetic known as
biotechnological products.

Types of Biotechnological Products:

a. Industrial and Environmental biotechnology.

b. Medical/pharmaceutical biotechnology
c. Agricultural biotechnology
d. Diagnostic research biotechnology

Examples of biotechnological products:

1. Proteins and peptides

2. Monoclonal antibodies
3. Oligonucleotides
4. Vaccines (immunotherapy)
5. Gene therapies

1. Proteins and Peptides:

Proteins: Proteins are the large organic compounds made of amino

acids arranged in linear chain and joined together by peptide bonds.

 Protein > 50 amino acids

 Molecular weight above 5000

Peptides: These are short polymer formed from the linking in a

defined order of amino acids.

 Peptide < 50 amino acids

 Molecular weight less than 5000

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Scientific advances in molecular and cell biology have resulted in the

development of two new biotechnologies.

 The first utilizes Recombinant Dna to produce protein product.

 The second technology is HYBRIDOMA TECHNOLOGY. Various
protein and peptide drug are epidermal growth factor, tissue
plasminogen activator.

2. Monoclonal Antibodies:

 Antibody or immunoglobulin's are protein m

molecules
olecules produced by a
specialized group of cells called B
B-lymphocytes
lymphocytes in mammals.
 An antibody is a protein produced by white blood cells and used by
the immune system to identify and neutralize foreign objects like
bacteria, viruses and foreign substances
substances.. Each antibody recognizes a
specific antigen unique to its target.
 Monoclonal antibodies (mAb) are antibodies that are identical
because they were produced by one type of immune cell, all clones
of a single parent cell.
 Polyclonal antibodies are antibodie
antibodiess that are derived from
different cell lines.
 The power of mAb lies in their highly specific binding of only one
antigenic determinant. As a result, mAb drugs, targeting agents, and
diagnostic are creating new ways to treat and diagnose.
 Monoclonal antibodies
dies can also target and deliver toxin specifically
to cancer cells and destroy them while sparing normal cells and
important detectors used in laboratory diagnostics.

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3. Oligonucleotides

 Antisense drugs consist of nucleotides linked together in short DNA

or RNA sequence known as oligonucleotides.
 Antisense oligonucleotides drugs, are drugs that seek to block DNA
transcription or RNA translation in order to moderate many disease
processes.
 Oligonucleotides are chemically synthesized by using
phosphoramidite. The oligonucleotide chain proceeds in the direction
of 3' to 5' terminus.
 These are the molecules made of synthetic genetic material, which
interact with the natural genetic material that codes the
information for production of proteins.
 Antisense RNA prevents protein translation of certain mRNA
strands by binding to them.
 Antisense DNA can used to target a specific complementary RNA.
E.g.,Mipomersen for high cholesterol.

4. Vaccines (Immunotherapy)

 A vaccine is a biological preparations that improves immunity to a

particular disease.
 A vaccine typically contains an agent that resembles a disease
causing microorganism and is often made from weakened or killed
forms of the microbe, its toxins or one of its surface proteins.
 Vaccines are dead or inactivated organisms or purified product
derived from them.

The different types of vaccines are:

A) Traditional Vaccines:

1. Killed 2. Live, attenuated 3. Toxoid 4. Subunit

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B) Innovative Vaccines:

1. Conjugate vaccines 2. Recombinant vector vaccine 3. T-cell receptor

peptide vaccine 4.Valence 5. Heterotypic

5. Gene Therapy

 A technique for correcting defective genes that are responsible for

disease development.
 In this use of DNA as a pharmaceutical agent to treat disease.
 The most common form of gene therapy involves using DNA that
encoded a functional, therapeutic gene to replace a mutated gene.

Approaches for gene therapy:

1. Gene modification
a. Replacement therapy
b. Corrective gene therapy

2. Gene transfer

a. Physical (Microinjection, Gene gun, naked DNA, Electroporation)

b. Chemical (Liposomes, Cationic liposomes, Oligonucleotides etc.)
c. Biological (Viral vector, mammalian artificial chromosomes)

3. Gene transfer in specific cell lines

a. Somatic gene therapy

b. Germ line gene therapy

4. Eugenic approach (gene insertion)

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Introduction to Pharmacokinetic & Pharmacodynamics

The purpose of studying pharmacokinetics and pharmacodynamics is to
understand the drug action, therapy, design, development and
evaluation.

Pharmacokinetic Study:

 It is a branch of Pharmacology which deals with the study of

Absorption, Distribution, Metabolism, Excreation/Elimination.
 Pharmacokinetics is the study of "What the body does to the drug"

Pharmacodynamics Study

 In Greek, Pharmcon - Drug Dynamics - Action.

 Pharmacodynamics is the study of biochemical and physiologic
effect of drug. It is the study of "What the drug does to the body"

Pharmacokinetics: It involves Four Processes:

1. Absorption
2. Drug distribution
3. Metabolism
4. Drug elimination

1. Absorption

 It is the process of entry of drug from site of administration into

systemic circulation.
 The bioavailability of the drug depends on the extent of the
absorption.
 Bioavailability is the percentage of drug that reaches the systemic
circulation in an unchanged form and becomes available for biological
effect following administration by any route.

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 Bioequivalence occurs when two formulations of the same compound

have the same bioavailability and the same rate of absorption.

Factors influencing absorption:

1. Factors Related To Drug:

a. Physicochemical properties
 Degree of ionization,
 Degree of solubility,
 Chemical nature,
 valence.
 High lipid / water partition coefficient increases absorption

b. Pharmaceutical form of drug - e.g., Absorption of solutions is

better than suspensions or tablets.

2. Factors Related To Patient:

a. Route of administration

Absorption is faster from i.v. > inhaled > i.m. > oral > dermal
Administration.

b. Area and vascularity of absorbing surface

 Absorption is directly proportional to both area and vascularity.

Thus absorption of the drug across the intestine is more
efficient than across the stomach, as Intestine has more blood
flow and much bigger surface area than those of the Stomach.

c. State of absorbing surface - e.g. atrophic gastritis and mal-

absorption syndrome decrease rate of absorption of drugs.

d. Rate of general circulation - E.g. in shock, peripheral circulation is

reduced and I.V. route is used.

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e. Presence of other drugs and other Specific factor - E.g. intrinsic

factor of the stomach is essential for vitamin B12 absorption from
lower ileum and adrenaline induces vasoconstriction so delay absorption
of local anaesthetics.

3. First-Pass Effect (Pre-Systemic Metabolism):

Where drugs must pass through gut mucosa and liver before reaching
systemic circulation.

a. Gut first-pass effect: E.g. benzyl penicillin is destroyed by gastric

acidity, insulin by digestive enzymes.
b. Hepatic first-pass effect: E.g. lidocaine (complete destruction so
not effective orally) and propranolol (extensive destruction)

2. Distribution

Distribution is the movement of drug from the central compartment

(blood) to peripheral compartments. Here the concentration gradient is
being the driving force for the movement from plasma to tissues.

It depends on;

 Ionization
 Molecular size
 Binding to plasma proteins
 Differences in regional blood flow Presence of tissue-specific
transporters.

Volume Of Distribution (Vd):

It is defined as the volume of fluid required to contain the total

amount of drug Q in the body at the same concentration as that
present in the plasma, Cp

Vd = Q/Cp

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Importance of Vd:

 It helps in estimating the total amount of drug in body at any time.

 Amount of drug = Vd x plasma concentration of drug at certain time
 Vd is important to determine the loading dose.
 Loading dose = Vd x desired concentration

3. Metabolism (Biotransformation):

 Biotransformation means chemical alteration of the drug in the

body.
 It is needed to render non-polar (lipid-soluble) compounds polar
(lipid insoluble) so that they are not reabsorbed in the renal tubules
and are excreted.
 The primary site for drug metabolism is liver; others are-kidney,
intestine, lungs and plasma.
Phases of biotransformation:
 Phase I (Non-synthetic) reactions - A functional group is
generated or exposed-metabolite may be active or inactive.
 Phase II (Synthetic) reactions - Mostly a conjugation reaction -
Metabolite is mostly inactive (except few drugs).

Phase I (Non-synthetic) Reactions

Introduction or unmasking of functional group by oxidation, reduction

hydrolysis, Cyclization, Decyclization.
These reactions may result in;
a. Drug inactivation (most of drugs)
b. Conversion of inactive drug into active metabolite (cortisone→
cortisol)
c. Conversion of active drug into active metabolite (phenacetin→
paracetamol)
d. Conversion to toxic metabolite (methanol → formaldehyde)

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Phase II (Synthetic) Reactions

 Functional group or metabolite formed by phase I is masked by

conjugation with natural endogenous constituent as glucuronic acid,
glutathione, sulphate, acetic acid, glycine or methyl group.
 These reactions usually result in drug inactivation with few
exceptions.
E.g. morphine-6-conjugate is active
 Most of drugs pass through phase I only or phase II only or phase I
then phase II.
 Some drugs as isoniazid passes first through phase II then phase I
(acetylated then hydrolyzed to isonicotinic acid).

Factors Affecting Drug Metabolism

a. Drugs - One drug can competitively inhibit the metabolism of

another if it utilizes the same enzyme or cofactors either by Enzyme
induction or by Enzyme inhibition.

b. Genetic Variation - The most important factor is genetically

determined polymorphisms.

E.g., Isoniazid is metabolized in the liver via acetylation. There are

two forms (slow and fast) of the enzyme responsible for acetylation
(N-acetyl transferase ), thus some patients metabolize the drug
quicker than others. Slow acetylators are prone to peripheral neuritis
while fast acetylators are prone to hepatic toxicity.

c. Nutritional State - Conjugating agents are sensitive to body

nutrient level.

E.g., low protein diet can decrease glycine.

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d. Dosage - High dose can saturate metabolic enzyme leading to drug

accumulation. If metabolic pathway is saturated due to high dose or
depletion of endogenous conjugate, an alternative pathway may appear.

e. Age - Drug metabolism is reduced in extremes of age (old patients

and infants)

4. Elimination or Excrection:

Elimination -Termination of Drug Action by which a drug or metabolite

is eliminated from the body. Drugs and their metabolites are excreted
in Urine, Faeces, Exhaled air, Saliva and sweat.

 Two-stage kidney process (filter, absorption)

 Metabolites that are poorly reabsorbed by kidney are excreted in
urine.
 Some drugs have active (lipid soluble) metabolites that are
reabsorbed into circulation (e.g., pro- drugs).
 Other routes of elimination: lungs, bile, skin.

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Pharmacodynamics

 Pharmacodynamics refers to the relationship between drug

concentration at the site of action and the resulting effect,
including the time course and intensity of therapeutic and adverse
effects.
 The effect of a drug present at the site of action is determined by
that drug's binding with a receptor.
 The concentration at the site of the receptor determines the
intensity of a drug's effect.

Drug Action: Four major types of bio- macromolecular targets of drug

action is there,

a. Enzyme
b. Transmembrane ion channel
c. Membrane bound transporter
d. Receptor

Factors Affecting Drug Response:

 Density of receptors on the cell surface.

 The mechanism by which a signal is transmitted into the cell by
second messengers.
 Regulatory factors that control gene translation and protein
production may influence drug effect.
Dose-Response Curves

Individual responses to varying doses;

 Threshold: Dose that produces a just-noticeable effect.

 ED50: Dose that produces a 50% of maximum response.
 Ceiling: Lowest dose that produces a maximal effect.

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Dose-Response Functions:

 Efficacy ED50= median effective dose.

 Lethality LD50= median lethal dose.
 Therapeutic Index = LD 50 /ED 50 = toxic dose/effective dose.
 This is a measure of a drug's safety.
 A large number = a wide margin of safety.
 A small number = a small margin of safety.

Duration of Effect

Duration of effect is determined by a complex set of factors,

including;

 The time that a drug is engaged on the receptor

 Intracellular signaling
 Gene regulation.

Time Course Studies important for;

 Predicting dosages/dosing intervals

 Maintaining therapeutic levels
 Determining time to elimination

Tolerance

The effectiveness can decrease with continued use is referred to as

tolerance.

Tolerance may be caused by;

 The pharmacokinetic factors, such as increased drug metabolism,

that decrease the concentrations achieved with a given dose.
 The pharmacodynamics factors like when the same concentration
at the receptor site results in a reduced effect with repeated
exposure.

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Drug Interaction:
Drug interaction may be defined as an alteration in the effects of one
drug by prior or concurrent administration of another drug.

 Drug that precipitates the interaction - Precipitant drug

 Drug whose action is affected - Object drug

Types of Drug Interactions:

1. Drug-drug interactions.
2. Drug-food interactions.
3. Chemical-drug interactions.
4. Drug-laboratory test interactions.
5. Drug-disease interactions.

Factors contributing to drug interactions:

1. Multiple drug therapy.

2. Multiple prescribers.
3. Multiple pharmacological effects of drug.
4. Multiple diseases
5. Poor patient compliance.
6. Drug-related factors.
7. Advancing age of patient

Mechanism of drug interactions:

The three mechanisms by which an interaction can develop are;

1. Pharmaceutical interactions.
2. Pharmacokinetic interactions.
3. Pharmacodynamic interactions.

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1. Pharmaceutical Interactions:

Also called as incompatibility. It is a physicochemical interaction that

occurs when drugs are mixed in i.v. Infusions causing precipitation or
inactivation of active principles.

E.g., Ampicillin, chlorpromazine & barbiturates interact with dextran

in solutions and are broken down or from chemical compounds.

2. Pharmacokinetic Interactions:

"These interactions are those in which ADME properties of the object

drug are altered by the precipitant and hence such interactions are
also called as ADME interactions".

The resultant effect is altered plasma concentration of the object

drug. These are classified as:

1. Absorption interactions
2. Distribution interactions
3. Metabolism interactions
4. Excretion interactions

1. Absorption Interactions: Are those where the absorption of the

object drug is altered. The net effect of such an interaction is:

 Faster or slower drug absorption.

 More, or, less complete drug absorption.

Major mechanisms of absorption interactions are:

a. Complexation and adsorption.

b. Alteration in GI pH
c. Alteration in gut motility.
d. Inhibition of GI enzymes.
e. Alteration of GI micro flora.

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2. Distribution Interactions - Are those where the distribution

pattern of the object drug is altered.

The major mechanism for distribution interaction is alteration in

protein-drug binding.

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3. Metabolism Interactions: Are those where the metabolism of the

object drug is altered.

Mechanisms of metabolism interactions include;

a. Enzyme induction - Increased rate of metabolism.

b. Enzyme inhibition - Decreased rate of metabolism. It is the most
significant interaction in comparison to other interactions and can
be fatal.

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4. Excretion Interactions: Are those where the excretion pattern of

the object drug is altered. Major mechanisms of excretion interactions
are;

 Alteration in renal blood flow.

 Alteration of urine PH
PH.
 Competition for active secretions -Forced dieresis.

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3. Pharmacodynamics Interactions - Are those in which the activity

of the object drug at its site of action is altered by the precipitant.
Such interactions may be direct or indirect.

These are of two types:

1. Direct pharmacodynamics interactions.

2. Indirect pharmacodynamics interactions.

1. Direct Pharmacodynamics Interactions:

In which drugs having similar or opposing pharmacological effects are

used concurrently.

The three consequences of direct interactions are:

A. Antagonism.
B. Addition or summation.
C. Synergism or potentiation

A. Synergism

 When the therapeutic or toxic effects of two drugs are greater

than the sum of effects of individual drugs.
 It is an enhancement of action of one drug by another.
 E.g.: Combination of sulfamethoxazole and trimethoprim is used as
antimicrobial agent.
 Alcohol enhances the of analgesics activity aspirin.

B. Additive Effect

 Net effect of two drugs used together is equal to the sum of the
individual drug effects.
 E.g.: Combination of thiazide diuretic and beta adrenergic blocking
drug is used for the treatment of hypertension.

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C. Antagonism:

 The effects of one drug can be reduced or abolished by the

presence of another drug.
 The interacting drugs have opposing actions
 E.g.: Blockade of antiparkinsonian action of levodopa by neuroleptics
and metoclopramide having anti dopaminergic action.
 Acetylcholine and noradrenaline have opposing effects on heart rate

2. Indirect Pharmacodynamics Interaction:

 In which both the object and the precipitant drugs have

unrelated effects but latter in some way alerts the effects of
the former.
 Example: morphine and nalorphine.

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Drug-Food Interactions

Garlic

 When combined with diabetes medication could cause dangerous

decrease in blood sugar level.
 Some garlic sensitive individuals may experience heart burn and
flatulence. It also has anti- clotting properties (interaction with
anticoagulants).

Orange Juice

 It must not be consumed with antacids containing aluminium.

 The juice increases the absorption of aluminium and leads to severe
constipation.

Milk - It contains elements like Mg and Ca which chelate antibiotics

like tetracycline and hence decrease its absorption and effect.

Grapefruit Juice - It inhibits CYP3A4; increasing levels of

antidepressants (sertraline), benzodiazepines, verapamil.

Vitamin K - Vit.k rich foods reduce the effectiveness of

anticoagulants (such as warfarin), increasing the risk of clotting.

Fiber in OATMEAL and other cereals - When consumed in large

amounts, can interfere with the absorption of digoxin.

Alcoholic Beverages - It tend to increase the depressive effect of

medications such as benzodiazepines, antihistamines, antidepressants,
antipsychotics, muscle relaxants and narcotics.

 Disulfiram like reaction with metronidazole.

 Increase metabolism of warfarin and phenytoin.

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Smoking

 It increases activity of drug metabolizing enzymes in the liver.

 Diazepam, Theophylline, Olanzapine are metabolized rapidly and
their effect is decreased.

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Pharmaceutical Biotechnology
 Pharmaceutical biotechnology consist of the combination of two
branch which are "Pharmaceutical science" and "Biotechnology"
 Pharmaceutical science: It can be simply define as the branch of
science that deals with the formulation compounding and dispensing
of drugs.
 Biotechnology: Biotechnology drug differ from Pharmaceutical drugs
in that they use biotechnology as a means for manufacturing, which
involves the manipulation of microorganism, such as bacteria , or
biological substance, like enzymes, to perform a specific process.

Biotechnological Products
 Biotechnology can be defined as application of technology using the
living organisms to obtain useful products.
 The products made by the biotechnology process include,
pharmaceuticals (medicine), food, and water purification, genetic
known as Biotechnological products.

Types of biotechnology products:

a. Industrial and Environmental Biotechnology

b. Medical/Pharmaceutical Biotechnology
c. Agricultural Biotechnology
d. Diagnostic Research Biotechnology.

Examples of Biotechnological products

1. Proteins and Peptides

2. Monoclonal antibodies
3. Oligonucleotides
4. Vaccines (immunotherapy)
5. Gene therapies

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1. Protein and Peptides

Protein - Protein are the large organic compound made of amino acids
arranged in linear chain and joined together by peptide bonds.

 Protein > 50 amino acids

 Molecular weight above 5000

Peptide - These are short polymer formed from the linking in a

defined order of amino acids.

 Peptide
eptide < 50 amino acids
 Molecular weight less than 5000

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Pharmacokinetics

1. Absorption:-

Enteral Administration

 Peptides and proteins, unlike conventional small-molecule drugs, are

generally not therapeutically active upon oral administration.
 The lack of systemic bioavailability is mainly caused by two factors:
 high gastrointestinal enzyme activity, and
 low permeability through the gastrointestinal mucosa.
 Thus, although various factors such as permeability, stability and
gastrointestinal transit time can affect the rate and extent of
absorption of orally administrated proteins, molecular size is
generally considered the ultimate obstacle.

Advantages of oral administration is still desired route of delivery

for protein drugs due to:

a. Its convenience
b. Cost-effectiveness
c. Painlessness

Strategies to overcome the obstacles associated with oral delivery

of proteins:

 Suggested approaches to increase the oral bioavailability of protein

drugs include encapsulation into micro- or nanoparticles thereby
protecting proteins from intestinal degradation.
 Other strategies are chemical modifications such as amino acid
backbone modifications and chemical conjugations to improve the
resistance to degradation and the permeability of protein drug.
 Co-administration of protease inhibitors for the inhibition of
enzymatic degradation.

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 The substantial peptidase and protease activity in the

gastrointestinal tract makes it the most efficient body
compartment for peptide and protein metabolism & gastrointestinal
mucosa presents a major absorption barrier for water soluble
macromolecules such as peptides and proteins.
 Due to the lack of activity after oral administration for most
peptides and proteins, administration by injection or infusion - that
is, by intravenous (IV), subcutaneous (SC), or intramuscular (IM)
administration – is frequently the preferred route of delivery for
these drug products.

Parenteral Administration:

 Most peptide and protein drugs are currently formulated as

parenteral formulations because of their poor oral bioavailability.
 Major routes of a administration
dministration include intravenous (IV),
subcutaneous (SC), and intramuscular (IM) administration.
 In addition, other non
non-oral
oral administration pathways are utilized,
including nasal, buccal, rectal, vaginal, transdermal, ocular and
pulmonary drug delivery

 Exception: IM or SC injections may be more appropriate on

achieving biologic activity of the product
 Since IV administration as either a bolus dose or constant rate
infusion, however, may not always provide the desired
concentration-time
time profile.

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For example,

1. Luteinizing hormone-releasing hormone (LH-RH) in bursts stimulates

the release of follicle-stimulating hormone (FSH) and luteinizing
hormone (LH), whereas a continuous baseline level will suppress the
release of these hormones.
2. To avoid the high peaks from an IV administration of leuprorelin, an
LH-RH agonist, a long acting monthly depot injection of the drug is
approved for the treatment of prostate cancer.

Inhalational Administration:-

 Inhalational delivery of peptides and proteins offers the advantage

of ease of administration, the presence of a large surface area (75
m2) available for absorption, high vascularity of the administration
site, and bypass of hepatic first pass metabolism.
 Disadvantages of inhalation delivery include the presence of certain
proteases in the lung, potential local side effects of the inhaled
agents on the lung tissues (i. e., growth factors and cytokines), and
molecular weight limitations.
 The success of inhaled peptide and protein drugs can be exemplified
by inhaled recombinant human insulin products, with Exubera being
the first approved product (2006), and several others in clinical
development.
 Inhaled insulin offers the advantages of ease of administration and
rapid onset with a shorter duration of action for tighter
postprandial glucose control as compared to subcutaneously
administered regular insulin.
 Dornase-a, which is indicated for the treatment of cystic fibrosis, is
another example of a protein drug successfully administered
through the inhalation route.

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Transdermal Administration :-

 Transdermal drug delivery offers the advantages of bypassing

metabolic and chemical degradation in the gastrointestinal tract, as
well as first-pass metabolism by the liver.
 Methods frequently used to facilitate transdermal delivery include
sonophoration and iontophoresis. Both methodologies increase skin
permeability to ionic compounds.
 Therapeutic doses of insulin, interferon-y, and epoetin-a have all
been successfully delivered transdermally via sonophoresis

2. Distribution

 The rate and extent of protein distribution is largely determined by

the molecule size and molecular weight, physiochemical properties
(e.g., charge, lipophilicity), binding to structural or transport
proteins, and their dependency on active transport processes to
cross biomembranes.
 Since most therapeutic proteins have high molecular weights and are
thus large in size, their apparent volume of distribution is usually
small and limited to the volume of the extracellular space due to
their limited mobility secondary to impaired passage through
biomembranes.
 After IV application, peptides and proteins usually follow a
biexponential plasma concentration-time profile that can best be
described by a two- compartment pharmacokinetic model.
 The central compartment in this model represents primarily the
vascular space and the interstitial space of well-perfused organs
with permeable capillary walls, especially liver and kidneys, while the
peripheral compartment comprises the interstitial space of poorly
perfused tissues such as skin and (inactive) muscle.

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 Active tissue uptake can substantially increase the volume of

distribution of peptide and protein drugs, as for example observed
with atrial natriuretic peptide (ANP).
 Another factor that can influence the distribution of therapeutic
peptides and proteins is binding to endogenous protein structures.
Physiologically active endogenous peptides and proteins frequently
interact with specific binding proteins involved in their transport
and regulation. Ex :- growth hormone
 Protein binding not only affects whether the peptide or protein drug
will exert any pharmacological activity, but on many occasions it may
also have an inhibitory or stimulatory effect on the biological
activity of the agent. Eg :- Recombinant cytokines.

3. Metabolism & Elimination:-

 Proteolysis: - Proteolytic enzymes such as proteases and peptidases

are ubiquitous throughout the body. As proteases and peptidases
are also located within cells, intracellular uptake is seen more an
elimination rather than a distribution process.
 Gastrointestinal: - For orally administered peptides and proteins,
the gastrointestinal tract is the major site of metabolism.
Presystemic metabolism is the primary reason. Parenterally
administered peptides and proteins may also be metabolized in the
intestinal mucosa following intestinal secretion.
 Hepatic :- the liver may also contribute substantially to the
metabolism of peptide and protein drugs. Proteolysis usually starts
with endopeptidases that attack in the middle part of the protein,
and the resulting oligopeptides are then further degraded by
exopeptidases.
 The ultimate metabolites of proteins, amino acids and dipeptides,
are finally reutilized in the endogenous amino acid pool. The rate of

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hepatic metabolism is largely dependent on specific amino acid

sequences in the protein.

Pathways of renal metabolism of peptides and proteins: glomerular filtration

followed by either (1) intraluminal metabo
metabo- lism or (II) tubular reabsorption
with intracellular lysosomal metabolism and (III) per
peritubular
itubular extraction with
intracellular lysosomal metabolism (Modified from Maack et al. (1985)).

Renal: - Renal metabolism of peptides and small proteins is mediated

through three highly effective processes. Consequently, only minuscule
amounts of intact protein
rotein are detectable in the urine.

1. The first mechanism involves the glomerular filtration of larger,

complex peptides and proteins, followed by reabsorption into
endocytic vesicles in the proximal tubule and subsequent hydrolysis
into small peptide fragme
fragments and AA.
2. The second mechanism entails glomerular filtration followed by intra
luminal metabolism, predominantly by exopeptidases in the luminal
brush border membrane of the proximal tubules.

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3. The third mechanism is peritubular extraction of peptides and

proteins from post glomerular capillaries and intracellular
metabolism.
 The determining factors for clearance of protein and peptide
include molecular weight as well as a molecule's physico-chemical
physico
properties, including size, overall charge, lipophilicity,
lipophilicity functional
groups, secondary and tertiary structure.

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Pharmacodynamics

 Protein therapeutics are usually highly potent compounds with steep

dose- effect curves as they are targeted therapies towards a
specific, well-described pharmacologic structure or mechanism.
 Thus, a careful characterization of the concentration-effect
relationship, i.e., the pharmacodynamics, is especially desirable for
protein therapeutics.
 In Protein therapeutics only too often most emphasis is laid on the
pharmacokinetic performance of the system, i.e. the plasma level
versus time profile of the drug to be accommodated.
 However, drug effects (pharmacodynamics) also exhibit their own
rate and time profiles, although they are dependent on drug
concentrations in plasma.
 It is very important that pharmacokinetics and pharmacodynamics
are studied simultaneously, so that their relationship is clearly
established.
 This will make it possible to predict the drug effect profile from
pharmacokinetic data, including the rate of input from the delivery
system.
 Such approaches will make it possible to better define the optimal
rate and time profiles of drug delivery.

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Application:

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2. Monoclonal Antibodies
 Antibody or immunoglobulin's are protein molecules produced by a
specialized
cialized group of cells called B
B-lymphocytes
lymphocytes in mammals.
 An antibody is a protein produced by white blood cells and used by
the immune system to identify and neutralize foreign objects like
bacteria, viruses and foreign substances. Each antibody recognizes a
specific antigen unique to its target.
 Monoclonal antibodies (mAb) are antibodies that are identical
because they were produced by one type of immune cell, all clones of
a single parent cell.
 An antigen can be a foreign molecule that interacts with the cells of
the immune system, triggering an immune response.
 The molecules on the antigens to which the antibodies attach
themselves are called Epitopes.
 The region of the antibody which binds to the Epitope is called a
Paratope.

 The power of mAb lies in ttheir

heir highly specific binding of only one
antigenic determinant. As a result, mAb drugs, targeting agents, and
diagnostic are creating new ways to treat and diagnose.
 Monoclonal antibodies can also target and deliver toxin specifically
to cancer cells and ddestroy
estroy them while sparing normal cells and
important detectors used in laboratory diagnostics.

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Pharmacokinetic of mAbs:

1. Absorption:

 Due to their high mol. Mass,The majority of mAbs that have been
approved or are currently in clinical development are administered
by intravenous (IV) infusion.
 Consequently, extra vascular routes have been chosen as
alternatives, including subcutaneous administration and
intramuscular administration.
 The mAbs enter the lymphatic system by convective flow of
interstitial fluid into the porous lymphatic vessels. The molecular
mass cut-off of these pores is >100-fold the molecular mass of
mAbs. From the lymphatic vessels, the mAbs are transported uni
directionally into the venous system.
 It has been shown that antibodies can reach the systemic
circulation after oral administration, but only to a very small extent.
The antibodies pass the intestinal epithelium not by passive
transcellular but by receptor-mediated transcellular or paracellular
transport.

2. Distribution

In general, the distribution of classical mAbs in the body is poor.

Limiting factors are, in particular, the high molecular mass and the
hydrophilicity/polarity of the molecules.

Transport

 Permeation of mAbs across the cells or tissues is accomplished by

transcellular or paracellular transport, involving the processes of
diffusion, convection, and cellular uptake. Due to their physico-
chemical properties, the extent of passive diffusion of classical
mAbs across cell membranes in transcellular transport is minimal.

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 Endocytosis is an absorptive process of large and polar molecules

such as mAbs, and involves the formation of intracellular vesicles
from parts of the cell membrane.
 The mAbs initially distribute into a restricted central volume (Vc) of
3-5 L, which in humans approximates the serum volume.

3. Elimination

Clearence

 As glomerular filtration has an approximate molecular size limit of

20–30 kDa, mAbs do not undergo filtration in the kidneys due to
their relatively large size.
 The situation is different, however, for low molecular-mass antibody
fragments, which can be filtered.
 Tubular secretion has not been reported to occur to any significant
extent for mAbs, and peptides/small proteins are readily
reabsorbed in the proximal or distal tubule of the nephron or are
even metabolized.
 Thus, renal elimination in total is uncommon or low for mAbs. Biliary
excretion of mAbs has been reported only for IgA molecules, and
only to a very small extent. Therefore, total clearance (CL) does
usually not comprise renal or biliary clearance.

Binding to Antigen - Binding of mAbs not only affects distribution but

also reflects another means of elimination. Binding of the Fab region to
the antigen with high affinity must be regarded as almost irreversible.
The antigen-antibody complex, if located on the surface of a cell, will
be internalized and subsequently degraded.

Binding to Anti-Idiotype Antibodies: A third elimination pathway

occurs if anti-idiotype antibodies are formed as an immune response of
the human body to the administration of mAbs. Following repeated

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administration, anti-idiotype antibodies are usually observed after one

to two weeks, with the extent of the adverse reaction strongly
depending on several

Pharmacodynamics

 mAbs have been marketed for use in the treatment of a wide range
of conditions, including cancer, autoimmunity and inflammatory
disease.
 It is convenient to discuss antibody P'dynamic relating to 4 main
categories of applications.
1. Immunotoxicotherapy, where Ab is employed to alter the
P'kinetic & P'dynamic of soluble ligands(eg. Drugs, cytokines,
xenobiotics)
2. Elimination of target cells.
3. Alteration of cellular function.(eg. Receptor blockade)
4. Targeted drug delivery.

Application of monoclonal antibodies.

The application of monoclonal antibodies can be broadly categorized as:

1. Diagnostic Applications

 Biochemical analysis
 Diagnostic Imaging

2. Therapeutic Applications

Direct use of MAbs as therapeutic agents

 In the treatment of cancer

 In the treatment of AIDS

MAbs as targeting agents.

3. Protein Purification

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Oligonucleotides
 Antisense drugs consist of nucleotides linked together in short DNA
or RNA sequence known as oligonucleotides.
 Antisense oligonucleotides drugs, are drugs that seek to block DNA
transcription or RNA translation in order to moderate many disease
processes.
 Oligonucleotide's are chemically synthesized by using
phosphoramidite.
 The oligonucleotide chain proceeds in the direction of 3' to 5'
terminus.

 Antisense oligonucleotide's are the molecules made of synthetic

genetic material, which iinteract
nteract with the natural genetic material
that codes the information for production of proteins.
 Antisense RNA prevents protein translation of certain mRNA
strands by binding to them.
 Antisense DNA can used to target a specific complementary RNA.
 Antisense oligo nucleotides are defined as the oligonucleotides with
8 to 50 nucleotides in length that can bind to RNA through Watson-
Watson
Crick base pairing and thereafter modulate its function.

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 Based on this definition, ASOs include classic ASOS as well as

siRNA and microRNA (miRNA) oligonucleotides, which are double-
double
stranded oligonucleotides.
 All of the antisense pharmacological actions involve three common
processes: (i) access of ASO to their action sites in the cells, (ii)
binding of their target RNA, and (iii) po
post-binding
binding events, such as
degradation of the RNA through endogenous enzymes.

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Pharmacokinetics:

1. Absorption:

 Antisense oligonucleotides cannot be delivered by oral

administration because their gastrointestinal absorption is low as a
consequence of their high molecular weight, their hydrophilicity, and
their charge.
 Therefore, ASOs are generally administered by parenterally.
Phosphorothioate antisense oligonucleotides (PTOs) are mostly
applied intravenously.
 They are rarely administered by s.c. because of an insufficient
stability toward degrading nucleases and their propensity to cause
inflammatory reactions at the injection site after continuous or
repeated dosing.

2. Distribution:

 The tissue distribution properties of second-generation ASOS are

generally similar to that observed for phosphorothioate antisense
oligonucleotide (PTO).
 First, ASOS are readily and almost completely distributed from the
plasma to the tissues.
 The plasma concentration versus time profile of ASOS has been
shown in many studies to be multiphasic with a fast decline in ASO
concentration in the first 24 h after administration and an initial
half-life reflecting tissue distribution following i.v. delivery in the
range of 30-90 min or even shorter depending on the chemical
modification and the specific ASO
 The highest concentrations of oligonucleotides in all species studied
were found in kidney, liver, spleen, and lymph nodes, but

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oligonucleotides can be measured in almost every tissue, except

brain, at 24 h after IV administration.
 Consistent with the pattern of distribution, liver and kidney were
monitored closely for evidence of toxicity in mouse and monkey
toxicology studies.

3. Metabolism & Elimination:-

 Oligonucleotides are metabolized by nucleases ubiquitously

expressed by cells in most tissues.
 PTOs lacking ribose modifications at their 3′ and 5′ ends are
primarily degraded by exonucleases generating 3' or 5' shortened
fragments but also by endonucleases in tissues. These fragmented
oligonucleotides may still possess antisense activity.
 On the contrary, second-generation ASOS protected at the 3' and
5' from exonuclease degradation by chemical modifications are
initially metabolized by endonucleases in tissues, leading to short
fragments, which may be further degraded by exonucleases.
 In contrast to PTOS, the metabolites of second-generation ASOS
resulting from the initial endonuclease cleavage are too short to still
possess antisense activity.
 The elimination half-life of oligonucleotides in plasma reflects their
metabolism in tissues, the equilibration of full-length ASOS and
metabolites between tissues and blood, and their excretion by the
kidneys.

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Pharmacodynamics

 The mechanism of action for antisense compounds is to inhibit gene

expression sequence--specifically
specifically by hybridization to mRNA through
Watson-Crickck base pair interactions.
 This is followed by degradation of the target mRNA through an
RNase Hdependent terminating mechanism.
 Consequently, the ASO prevents translation of the encoded protein
product, or the disease
disease-causing
causing factor in a highly sequence-specific
sequence
manner.

Examples:

1. Mipomersen for high cholesterol

2. Affinitak and a Genasense against cancer
3. AV 1-6002 and AV 1-6003
6003 for the treatment of Hemorrhagic fever.

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Application of Oligonucleotides

Antisense drugs are being researched to treat a variety of diseases

such as:

a. Lung cancer
b. Colorectal carcinoma
c. Pancreatic carcinoma
d. Malignant melanoma
e. Diabetes
f. Amyotrophic lateral sclerosis (ALS)
g. Asthma

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Vaccines (Immunotherapy)
 A vaccine is a biological preparation that improves immunity to a
particular disease.
 A vaccine typically contains an agent that resembles a disease
causing microorganism and is often made from weakened or killed
forms of the microbe, its toxins or one of its surface proteins.
 Vaccines are dead or inactivated organisms or purified product
derived from them.

The different types of vaccines are:

a. Traditional vaccines
b. Innovative vaccines

a. Traditional vaccines

1. Killed
2. Live, attenuated
3. Toxoid
4. Subunit.

1. Killed: some vaccines contain killed, but previously virulent,

microorganism that have been destroyed with chemicals,heat,
radioactivity or antibiotics.

Examples: are influenza, cholera, polio, hepatitis A, and rabies.

2. Live attenuated: some vaccines contain live, attenuated

microorganisms, many of these are active viruses that have been
cultivated under conditions that disable their virulent properties or
that use closely related but less dangerous organisms to produce a
broad immune response.

Examples: yellow fever, measles, mumps.

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3. Toxoid: Toxoid vaccines are made from inactivated toxic compound

that cause illness rather than the microorganism.

Examples: are Tetanus and Diphtheria.

4. Subunit: Protein subunit-rather than introducing an inactivated or

attenuated microorganism to an immune system(which would constitute
a whole agent vaccine),a fragment of it can create an immune response.

Examples: meningococcal disease and pneumococcal disease.

b. Innovative vaccines

1. Conjugate vaccines
2. Recombinant vector vaccine
3. t-cell receptor peptide vaccine
4. Valence
5. Heterotypic.

1. Conjugate vaccines: certain bacteria have polysaccharide outer

coats that are poorly immunogenic .By linking these outer coats to
protein (ex., toxin),the immune system can be led to recognize the
polysaccharide as if it were a protein antigen.

Example: Hib (haemophilus influenza type b) disease.

2. Recombinant vector vaccine: by combining the physiology of one

microorganism and the DNA of the other, immunity can be created
against diseases that have complex infection process.

Example: Hepatitis B

3. T-cell receptor peptide vaccine: they show the modulation of

cytokine production and improve cell mediated immunity and are under
development. Use to stimulate anti tumour T cell.

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4. Valence:

a. Monovalent: use to immunize against single antigen.

b. Multivalent: used to immunize against two or more microorganism.

Use: Hepatitis A, Hepatitis B, mumps,rubella,diphtheria, chickenpox

etc.

5. Heterotypic: vaccines that are pathogens of other animals that

either do not cause disease or cause mild disease in the organism being
treated. Use: Diphtheria

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Pharmacokinetics

1. Absorption & distribution:

Intranasal: Intranasal vaccine administration is optimal for antigens

(Ag) distribution into the nasal-associated lymphoid tissue (NALT),
which contains high levels of Dendritic Cell (DC) and T-cells. NALT is
especially relevant for immune response against airborne pathogens and
to a lesser extent to mucosal infections, due to its predominant
polarization to humoral response.

 This route is characterized by a rapid and direct systemic

absorption.
 Intranasal administration has shown to produce greater Ag Cmax
(maximum concentration) and AUC (area under curve of the
pathogen or molecule administered as vaccine) compared to IM
administration.

Intradermal or Transcutaneous: DNA vaccination using intradermal

administration is also associated with a higher number of Ag at the
injection site compared to IM route, prolonging the Ag exposure time.

Intravenous: IV administration of DNA vaccines as naked DNA

plasmids normally leads to a rapid blood and tissue degradation of the
vectors, while after IM administration their persistence in muscle
tissues has been shown to vary depending on the DNA vaccine dose,
vector, and use of adjuvants

2. Metabolism and Excretion:

 The metabolism and excretion processes are not well studied for
vaccines because PK studies are not required for vaccine approval,
and also because they are assumed to be irrelevant regarding
vaccine efficacy or interaction with other drugs.

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 Considering the low and expected few doses administered in

vaccination, these assumptions seem reasonable. However, as chronic
Ag exposure is associated with tolerance development, complete Ag
elimination should be guaranteed in order to avoid chronic exposure
leading to a decrease in vaccine efficacy.
 Regarding DNA vaccines, one of the main concerns is the plasmid
integration of vaccine into host DNA. This integration depends
mainly on the nature of the foreign plasmid and DNA, but it must be
considered that a very low elimination (which can take years)
increases the chances of plasmid integration.
 Therefore, demonstration of complete elimination of these vaccines
may become relevant to assure safety or to avoid interaction of
vaccine DNA with other pathogens or microorganisms.

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Gene Therapy
Gene therapy can be defined as an experimental technique for

 Correcting
orrecting defective genes
 Inserting a normal gene to replace an abnormal gene

Approaches in gene therapy

A. Types of gene therapy

1. Somatic gene therapy

2. Germ line gene therapy

B. Gene modification

1. Gene replacement
2. Gene correction
3. Gene augmentation

C. Gene transfer methods

1. Viral gene transfer (biological)

2. Non
on viral gene transfer :
a. Physical
hysical method
b. Chemical
hemical method

A. Types of Gene Therapy

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Types of Somatic Gene Therapy

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B. Gene Modification

1. Gene Replacement: Removal of a Mutant Gene sequence from the

Host Genome and its replacement with a normal Functional Gene.
2. Gene Correction: Involves only defective portion of a mutant gene
which is altered to provide the Functional Gene.
3. Gene Augmentation: Defective gene is modified by introducing a
normal genetic sequence into Host Genome without altering the
defective one.

C. Gene Transfer Methods - To transfer the desired gene into a

target cell, a carrier is required Such vehicles of gene delivery are
known as vectors

Two main classes

1. Viral vectors
2. Non viral vectors

1. Viral gene transfer (biological)

a. Retrovirus vector system

b. Adenovirus vector system
c. Adeno associated virus vector
d. Herpex simplex virus vector

2. Non Viral Vectors

a. Physical Methods:

i. Electroporation
ii. Microinjection
iii. Gene Gun

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b. CHEMICAL METHOD:

i. Calcium phosphate mediated DNA transfer

ii. Liposome mediated gene transfer

Systemic and Organ Pharmacokinetics

Naked Plasmid DNA (pDNA):

 Administration of naked DNA into the body is the simplest means of

gene therapy. A conventional intravenous injection of pDNA results
in low or undetectable transgene expression in major organs.
 The reason for this low efficiency can be found in the
physicochemical and biological properties of the DNA. DNA is a big
molecule with a molecular weight over 2000 kDa and strong anionic
charge and is easily degraded by the existing DNases in the blood.
Therefore, its permanence and distribution in the body are limited.
 Understanding the in vivo fate of DNA itself is a prerequisite to
develop safe and efficient gene delivery systems.

Formulated pDNA:

 Tissue distribution of the gene therapy system is essential, since

transgene expression only occurs in those cells transfected with the
genetic material.
 In vivo, tissue distribution is determined by the physicochemical and
biological properties of the vector employed.
 Therefore, formulation, along with the route of administration, is
crucial to achieve the therapeutic objectives.
 Cationic lipid/ DNA complexes have been proved to be rapidly
cleared from the bloodstream after intravenous (iv) injection in
general terms, accumulating primarily in lung and liver, and to a less
extent in spleen.

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 Nevertheless, there is redistribution between these two first

organs, with an initial accumulation in lung, followed by a gradual
increase in liver.
 This observation has been explained by a first
first-pass
pass effect in the
presence of serum com
components,
ponents, lipoplexes could form aggregates
being passively targeted to pulmonary microvasculature, the first
capillary bed encountered after iv injection.
 The hepatic redistribution would be due to complex dissociation and
small complex carried away by blood flow from the lung.
 The use of different co
co-helpers
helpers lipids, lipid: DNA charge ratio and
size have been proved to influence tissue distribution.
 Tissue distribution of polyplexes is more easily controlled since
cationic polymers interact less with blood components.
 Thus, targeted delivery can be achieved by controlling the
physicochemical and biological properties of the complex.

Barriers in gene therapy after in vivo administration

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Pharmacodynamics

 The DNA can integrate into host's genome (main characteristic of

retroviruses) or maintain an extrachromosomal location. Upon
integration, vector genes appear to be expressed for a long period;
however, it may induce carcinogenesis.
 On the other hand, extrachromosomal DNA is progressively reduced
in the number of copies by cellular division and loss by degradation
generating a transient expression .
 Regardless of the disposition, DNA has to be transcribed to mRNA,
which will then be exported to the cytoplasm and traduced into its
encoding protein.
 At this level, gene expression is going to be regulated by different
factors, such as the disposition, the plasmid stability in the nucleus
or the DNA expression cassette used.

Application

a. Gene therapy used to treat type I diabetes

b. Gene therapy for cancer treatment
c. Parkinson's disease
d. Severe Combined Immune Deficiency(ADA-SCID)
e. Cystic fibrosis
f. Hemophilia
g. Blindness

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