Introduction to

Biopharmaceutics
Unit-I
Content:-
Absorption :
 Mechanisms of drug absorption through GIT.
 Factors influencing drug absorption though GIT.
 Absorption of drug from Non per oral extra-vascular routes.
Distribution :
 Tissue permeability of drugs.
 Binding of drugs.
 Volume of drug distribution
 plasma and tissue protein binding of drugs.
 Factors affecting protein-drug binding.
 Kinetics of protein binding.
 Clinical significance of protein binding of drugs.
 Biopharmaceutics
Pharmacokinetics
Bio - Pharmaco -
pharmaceutics kinetics

Movemen
Inside Related ts of
living to drug into
being dosage the body
form
Related to Physico- What body does to
chemical properties drug
A-Absorption
Solubility D-Distribution
Ionization M-Metabolism
Flow property etc. E-Elimination
Biopharmaceutics
It is defined as study of factors affecting rate and amount of drug reaches the
systemic circulation (blood)and the use of this information optimizes the therapeutic
efficacy of the drug.

Intake Absorption
Drug - mouth, stomach Blood systemic
circulation
Distribution

Degradation of drug in one to Targeted site

Metabolism
Another

Elimination
 Factors Affects ADME
Responses

Physicochemical properties-
Lipophilic-Good Absorption
Hydrophilic-Low absorption

 Factors-
 Solubility, flow property, external environment, disease /disorder etc.
Significance of biopharmaceutics-
 To decide the dosage form of the drug, their dose and route of
administration
 To study a ADME
 To understand bioavailability and bioequivalence of the drug.
Gastrointestinal Tract:
 The gastrointestinal tract is a muscular tube, approximately 6 m in length with
varying diameter.
 It stretches from the mouth to the anus and consists of four main anatomical areas;
the oesophagus, the stomach, the small intestine and the large intestine, or colon.
 The luminal surface of the tube is not smooth but very rough, thereby increasing
the surface area for absorption.
 The wall of the gastrointestinal tract is essentially similar in structure along its length,
consisting of four principal histological layers:
1. The serosa, which is an outer layer of epithelium with supporting connective tissues
which are continuous with the peritoneum.
2. The muscularis externa, which contains three layers of smooth muscle tissue, a thinner
outer layer, which is longitudinal in orientation, and two inner layers, whose fibres are
oriented in a circular pattern. Contractions of these muscles provide the forces for
movement of gastrointestinal tract contents and physical breakdown of food. (the
muscular wall of the gastrointestinal (GI) tract that surrounds the submucosa. It's
responsible for gut movement, such as peristalsis)
3. The submucosa, which is a connective tissue layer containing some secretory
tissue and which is richly supplied with blood and lymphatic vessels ( thin tubes that
carry lymph, a clear fluid, throughout the body and back to the bloodstream. They are part of the
lymphatic system, which is part of the immune system and helps protect the body from infection) .
 A network of nerve cells, known as the submucous plexus, is also located in
this layer.
4. The mucosa, which is essentially composed of three layers: the muscularis
mucosae, which can alter the local conformation of the mucosa, a layer of
connective tissue known as the lamina propria, and the epithelium.
5. The majority of the gastrointestinal epithelium is covered by a layer or layers
of mucus.
 This is a viscoelastic translucent aqueous gel that is secreted throughout the
gastrointestinal tract, acting as a protective layer and a mechanical barrier.
 Mucus is a constantly changing mix of many secretions and exfoliated
epithelial cells. It has a large water component (~95%).
 Its other primary components, which are responsible for its physical and
functional properties, are large glycosylated proteins called mucins.
 Mucins consist of a protein backbone approximately 800 amino acids long and
oligosaccharide side chains that are typically up to 18 residues in length.
 The mucous layer ranges in thickness from 5 μm to 500 μm along the length of
the gastrointestinal tract, with average values of approximately 80 μm.
 Mucus is constantly being removed from the luminal surface of the
gastrointestinal tract through abrasion and acidic and/or enzymatic breakdown,
and it is continually replaced from beneath.
 The turnover time has been estimated at 4 to 5 hours, but this may well be an
underestimate and is liable to vary along the length of the tract.
 The gastrointestinal tract includes the mouth, pharynx, esophagus, stomach,
small intestine, large intestine, and anus.
 The extent of drug absorption in a segment of the gastrointestinal tract depends
generally on the rate of absorption as well as on the exposed surface area and
time available for drug absorption.
 Mechanism of drug absorption:
The three broad categories of drug transport mechanisms involved in
absorption are –
A. Transcellular/intracellular transport
B. Paracellular/intercellular transport
C. Vesicular transport
A. Transcellular/Intracellular Transport
 It is defined as the passage of drugs across the GI epithelium.
 It is the most common pathway for drug transport.
 The 3 steps involved in transcellular transport of drugs are –

1. Permeation of GI epithelial cell membrane, a lipoidal barrier – this is the major

obstacle to drug absorption
2. Movement across the intracellular space (cytosol-fluid present inside the cell
membrane).
3. Permeation of the lateral or basolateral membrane- this is of secondary
importance.

The various transcellular transport processes involved in drug absorption are –

1) Passive Transport Processes –
 These transport processes do not require energy other than that of molecular
motion (Brownian motion) to pass through the lipid bilayer. Passive transport
processes can be further classified into following types –
A. Passive diffusion.
B. Pore transport.
C. Ion-pair transport.
D. Facilitated- or Carrier mediated-diffusion.
2. Active Transport Processes –
 This transport process requires energy from ATP to move drug molecules
from extracellular to intracellular milieu. These are of two types –
a. Primary active transport.
b. Secondary active transport – this process is further subdivided into two –
I. Symport (co-transport).
II. Antiport (counter-transport).
B. Paracellular/Intercellular Transport
 Paracellular/Intercellular Transport – is defined as the transport of drugs through the
junctions between the GI epithelial cells.
 This pathway is of minor importance in drug absorption.
 The two paracellular transport mechanisms involved in drug absorption are –
1. Permeation through tight junctions of epithelial cells – this process basically occurs
through openings which are little bigger than the aqueous pores.
 Compounds such as insulin and cardiac glycosides are taken up this mechanism.
2. Persorption – is permeation of drug through temporary openings formed by shedding
of two neighboring epithelial cells into the lumen.
Paracellular transport differs from pore transport in that the former involves transfer of
drug across epithelium and through the cellular junctions whereas in the case of latter,
the molecules are transferred from outside of the epithelial cell into the cell through
pores present in the cell membrane.
C. Vesicular or Corpuscular Transport (Endocytosis)
 Vesicular or Corpuscular Transport (Endocytosis) – Like active transport,
these are also energy dependent processes but involve transport of
substances within vesicles into a cell.
 Since the mechanism involves transport across the cell membrane, the
process can also be classified as transcellular.
 Vesicular transport of drugs can be classed into two categories –
1. Pinocytosis.
2. Phagocytosis.
 Passive Transport Processes
1) Passive Diffusion-
 Also called non-ionic diffusion, it is the major process for absorption of more than
90% of the drugs.
 The driving force for this process is the concentration or electrochemical gradient.
 It is defined as the difference in the drug concentration on either side of the
membrane.
 Drug movement is a result of the kinetic energy of molecules.
 Since no energy source is required, the process is called as passive diffusion.
 During passive diffusion, the drug present in the aqueous solution at the absorption
site partitions and dissolves in the lipid material of the membrane and finally leaves
it by dissolving again in an aqueous medium, this time at the inside of the membrane.
 Passive diffusion is best expressed by Fick’s first law of diffusion, which states that
the drug molecules diffuse from a region of higher concentration to one of lower
concentration until equilibrium is attained and that the rate of diffusion is directly
proportional to the concentration gradient across the membrane.
 It can be mathematically expressed by the following equation:

 dQ/dt = rate of drug diffusion (amount/time). It also represents the rate of

appearance of drug in blood
 D = diffusion coefficient of the drug through the membrane (area/time)
 A = surface area of the absorbing membrane for drug diffusion (area)
 Km/w = partition coefficient of the drug between the lipoidal membrane
and the aqueous GI fluids (no units) (CGIT – C) = difference in the
concentration of drug in the GI fluids and the plasma, called as the
concentration gradient (amount/volume)
 h = thickness of the membrane (length)
 Based on the above equation, certain characteristics of passive diffusion can
be generalized –
1. The drug moves down the concentration gradient indicating downhill
transport. The process is energy-independent.
2. The rate of drug transfer is directly proportional to the concentration
gradient between GI fluids and the blood compartment.
3. Greater the area and lesser the thickness of the membrane, faster the
diffusion; thus, more rapid is the rate of drug absorption from the intestine
than from the stomach.
4. The process is rapid over short distances and slower over long distance.
B. Pore Transport
 It is also called as convective transport, bulk flow or filtration.
 This mechanism is responsible for transport of molecules into the cell through the protein
channels present in the cell membrane.
 Following are the characteristics of pore transport –
1. The driving force is constituted by the hydrostatic pressure or the osmotic differences
across the membrane due to which bulk flow of water along with small solid molecules
occurs through such aqueous channels.
 Water flux that promotes such a transport is called as solvent drag.
2. The process is important in the absorption of low molecular weight (less than 100), low
molecular size (smaller than the diameter of the pore) and generally water-soluble drugs
through narrow, aqueous-filled channels or pores in the membrane structure—for example,
urea, water and sugars.
3. Chain-like or linear compounds of molecular weight up to 400 Daltons can be absorbed by
filtration.
 Drug permeation through water-filled channels is of particular importance in renal
excretion, removal of drug from the cerebrospinal fluid and entry of drugs into the
liver.
C. Ion-Pair Transport
 Yet another mechanism that explains the absorption of drugs like quaternary
ammonium compounds and sulphonic acids, which ionizes under all pH
conditions, is ion-pair transport.
 Despite their low o/w partition coefficient values, such agents penetrate the
membrane by forming reversible neutral complexes with endogenous ions
of the GIT like mucin.
 Such neutral complexes have both the required lipophilicity as well as
aqueous solubility for passive diffusion. Such a phenomenon is called as
ion-pair transport (Fig. 2.5).
 Propranolol, a basic drug that forms an ion pair with oleic acid, is absorbed
by this mechanism.
C. Carrier-Mediated Transport
 Some polar drugs cross the membrane more readily than can be predicted from
their concentration gradient and partition coefficient values.
 This suggests presence of specialized transport mechanisms without which many
essential water-soluble nutrients like monosaccharides, amino acids and vitamins
will be poorly absorbed.
 The mechanism is thought to involve a component of the membrane called as
the carrier that binds reversibly or non- covalently with the solute molecules to
be transported.
 This carrier-solute complex traverses across the membrane to the other side
where it dissociates and discharges the solute molecule.
 The carrier then returns to its original site to complete the cycle by accepting a
fresh molecule of solute.
 Carriers in membranes are proteins (transport proteins) and may be an enzyme
or some other component of the membrane.
 They are numerous in all biological membranes and are found dissolved in the
lipid bilayer of the membrane.
 Important characteristics of carrier-mediated transport are:
1. A carrier protein always has an uncharged (non-polar) outer surface which allows it
to be soluble within the lipid of the membrane.
2. The carriers have no directionality; they work with same efficiency in both directions.
3. The transport process is structure-specific i.e. the carriers have special affinity for and
transfer a drug of specific chemical structure only (i.e. lock and key arrangement);
generally the carriers have special affinity for essential nutrients.
4. Since the system is structure-specific, drugs having structure similar to essential
nutrients, called as false nutrients, are absorbed by the same carrier system. This
mechanism is of particular importance in the absorption of several antineoplastic agents
like 5-fluorouracil and 5- bromouracil which serve as false nutrients.
5. As the number of carriers is limited, the transport system is subject to competition
between agents having similar structure.
Two types of carrier-mediated transport systems have been identified.
They are—
a. facilitated diffusion and
b. active transport.
a. Facilitated Diffusion
 It is a carrier-mediated transport system that operates down the concentration
gradient (downhill transport) but at a much a faster rate than can be accounted by
simple passive diffusion.
 The driving force is concentration gradient (hence a passive process).
 Since no energy expenditure is involved, the process is not inhibited by metabolic
poisons that interfere with energy production.
 Facilitated diffusion is of limited importance in the absorption of drugs.
 Examples of such a transport system include entry of glucose into RBCs and
intestinal absorption of vitamins B1 and B2.
 A classic example of passive facilitated diffusion is the GI absorption of vitamin
B12.
 An intrinsic factor (IF), a glycoprotein produced by the gastric parietal
cells(epithelial cell in the stomach), forms a complex with vitamin B12 which is
then transported across the intestinal membrane by a carrier system (Fig. 2.7)
b. Active Transport
This transport mechanism requires energy in the form ATP.
Active transport mechanisms are further subdivided into –
1.Primary active transport –
 In this process, there is direct ATP requirement. Moreover, the process transfers only one
ion or molecule and in only one direction, and hence called as uniporter e.g. absorption
of glucose.
 Carrier proteins involved in primary active transport are of two types –
i. Ion transporters –
 Ion transporters are responsible for transporting ions in or out of cells.
 A classic example of ATP-driven ion pump is proton pump which is implicated in
acidification of intracellular compartments.
 Two types of ion transporters which play important role in the intestinal absorption of
drugs have been identified –
a. Organic anion transporter – which aids absorption of drugs such as pravastatin and
atorvastatin.
b. Organic cation transporter – which aids absorption of drugs such as diphenhydramine.
ii. ABC (ATP-binding cassette) transporters –
 ABC (ATP-binding cassette) transporters are responsible for transporting small
foreign molecules (like drugs and toxins) especially out of cells (and thus
called as efflux pumps) which make them clinically important.
 A classic example of ABC transporter is P-glycoprotein (P-gp).
 The latter is responsible for pumping hydrophobic drugs especially anticancer
drugs across the cells.
 Presence of large quantity of this protein thus makes the cells resistant to a
variety of drugs used in cancer chemotherapy, a phenomenon called as multi-
drug resistance.
 It is for this reason that P-gp is called as multi-drug resistance (MDR) protein.
 ABC transporters present in brain capillaries pump a wide range of drugs out of
brain.
2. Secondary active transport –
 In these processes, there is no direct requirement of ATP i.e. it takes advantage
of previously existing concentration gradient.
 The energy required in transporting an ion aids transport of another ion or
molecule (co-transport or coupled transport) either in the same direction or in
the opposite direction.
 Accordingly this process is further subdivided into –
i. Symport (co-transport) –
 involves movement of both molecules in the same direction e.g. Na+- glucose
symporter uses the potential energy of the Na+ concentration gradient to move
glucose against its concentration gradient.
 A classic example of symporter is peptide transporter called as H+-coupled
peptide transporter (PEPT1) which is implicated in the intestinal absorption of
peptide-like drugs such as β-lactam antibiotics.
ii. Antiport (counter-transport) –
 involves movement of molecules in the opposite direction e.g. expulsion of
H+ ions using the Na+ gradient in the kidneys.
 Figure 2.8 illustrates active transport of a drug and figure 2.9 represents the
types of active transpor
 Active transport is a more important process than facilitated diffusion in the
absorption of nutrients and drugs and differs from it in several respects:
1. The drug is transported from a region of lower to one of higher concentration
i.e. against the concentration gradient (in the case of ions, against an electrochemical
gradient) or uphill transport, without any regard for equilibrium.
2. The process is faster than passive diffusion.
3. Since the process is uphill, energy is required in the work done by the carrier.
4. As the process requires expenditure of energy, it can be inhibited by metabolic
poisons that interfere with energy production like fluorides, cyanide and
dinitrophenol and lack of oxygen, etc.
 Endogenous substances that are transported actively include sodium, potassium,
calcium, iron, glucose, certain amino acids and vitamins like niacin, pyridoxin
and ascorbic acid.
 Drugs having structural similarity to such agents are absorbed actively,
particularly the agents useful in cancer chemotherapy.
 Examples include absorption of 5- fluorouracil and 5- bromouracil via the
pyrimidine transport system, absorption of methyldopa and levodopa via an L-
amino acid transport system and absorption of ACE inhibitor enalapril via the
small peptide carrier system.
 A good example of competitive inhibition of drug absorption via active
transport is the impaired absorption of levodopa when ingested with meals rich
in proteins.
 Active transport is also important in renal and biliary excretion of many drugs
and their metabolites and secretion of certain acids out of the CNS.
C. Vesicular-
 Endocytosis It is a minor transport mechanism which involves engulfing
extracellular materials within a segment of the cell membrane to form a saccule or a
vesicle (hence also called as corpuscular or vesicular transport) which is then
pinched-off intracellularly (Fig. 2.11).
 This is the only transport mechanism whereby a drug or compound does not have to
be in an aqueous solution in order to be absorbed.
 This phenomenon is responsible for the cellular uptake of macromolecular
nutrients like fats and starch, oil soluble vitamins like A, D, E and K,
water soluble vitamin like B12 and drugs such as insulin.
 Another significance of such a process is that the drug is absorbed into the
lymphatic circulation thereby bypassing first- pass hepatic metabolism.
 Endocytosis includes two types of processes:
1.Phagocytosis (cell eating): adsorptive uptake of solid particulates, and
2. Pinocytosis (cell drinking): uptake of fluid solute.
 Orally administered Sabin polio vaccine, large protein molecules and the
botulism toxin (that causes food poisoning) are thought to be absorbed by
pinocytosis.
 Sometimes, an endocytic vesicle is transferred from one extracellular
compartment to another. Such a phenomenon is called as transcytosis
 Factors affecting drug absorption:
 A Pharmaceutical factors:
1. Physicochemical properties of drug:
a. Drug solubility and dissolution rate
b. Particle size and effective surface area
c. Polymorphism and amorphism
d. Pseudopolymorphism(hydrates or solvates)
e. Salt form of the drug
f. Lipophilicity of the drug
g. Drug stability
h. Stereochemical nature of the drug
2. Formulation factors:
a. Disintegration time
b. Manufacturing variables
c. Nature and type of dosage form
d. Pharmaceutical ingredients (excepients)
e. Product age and storage conditions
B. Patient related factors:
a. Age
b. Gastric emptying time
c. Intestinal transit time
d. Gastrointestinal pH
e. Diseased states
f. Blood flow through the GIT
g. Gastrointestinal contents
A. PHARMACEUTICAL FACTORS
 In order to design a formulation that will deliver the drug in the most
bioavailable form, the pharmacist must consider –
1.Physicochemical properties of the drug, and
2. Type of formulation (e.g. solution, suspension, tablet, etc.), and
3. Nature of excipients in the formulation.
1. PHYSICOCHEMICAL FACTORS AFFECTING DRUG ABSORPTION
i. Drug Solubility and Dissolution Rate:
 Except in case of controlled-release formulations, disintegration and de-
aggregation occur rapidly if it is a well-formulated dosage form.
 Thus, the two critical slower rate-determining processes in the absorption of
orally administered drugs are:
1. Rate of dissolution, and
2. Rate of drug permeation through the bio-membrane.
 Dissolution is the Rate Determining Step for hydrophobic, poorly aqueous
soluble drugs like griseofulvin and spironolactone; absorption of such drugs
is often said to be dissolution rate-limited.
 If the drug is hydrophilic with high aqueous solubility—for example, cromolyn
sodium or neomycin, then dissolution is rapid and RDS in the absorption of
such drugs is rate of permeation through the biomembrane.
 In other words, absorption of such drugs is said to be permeation rate-limited
or transmembrane rate-limited.
ii. Factors Affecting Drug Dissolution and Dissolution Rate
 Factors of in vivo importance that can affect dissolution and hence
absorption can be categorized into 2 classes:
1. Physicochemical properties of the drug, and
2. Dosage form factors.
 The various physicochemical properties of drug that affect drug dissolution
and its rate are— solubility, particle size, polymorphism, salt form,
pseudopolymorphism, complexation, wettability, etc.
 Dosage form factors include several formulation factors and excipients
incorporated in the dosage form.
Particle Size and Effective Surface Area of the Drug
 Particle size and surface area of a solid drug are inversely related to each other.
Smaller the drug particle, greater the surface area. Two types of surface area of
interest can be defined:
1. Absolute surface area which is the total area of solid surface of any particle, and
2. Effective surface area which is the area of solid surface exposed to the
dissolution medium.
 Polymorphism and Amorphism Depending upon the internal structure, a solid
can exist either in a crystalline or amorphous form.
 When a substance exists in more than one crystalline form, the different forms are
designated as polymorphs and the phenomenon as polymorphism.
 Polymorphs are of two types:
 Enantiotropic polymorph is the one which can be reversibly changed into another
form by altering the temperature or pressure
 e.g. sulphur, and Monotropic polymorph is the one which is unstable at all
temperatures and pressures e.g. glyceryl stearates .
 Monotropic polymorph is the one which is unstable at all temperatures and
pressures e.g. glyceryl stearates.
 The polymorphs differ from each other with respect to their physical properties
such as solubility, melting point, density, hardness and compression
characteristics.
 They can be prepared by crystallizing the drug from different solvents under
diverse conditions.
 The existence of the polymorphs can be determined by using techniques such as
optical crystallography, X-ray diffraction, differential scanning calorimetry,
etc.
 Depending on their relative stability, one of the several polymorphic forms will
be physically more stable than the others.
 Such a stable polymorph represents the lowest energy state, has highest
melting point and least aqueous solubility.
 The remaining polymorphs are called as metastable forms which represent the
higher energy state, have lower melting points and higher aqueous
solubilities.
 Because of their higher energy state, the metastable forms have a
thermodynamic tendency to convert to the stable form.
 Some drugs can exist in amorphous form (i.e. having no internal crystal
structure).
 Such drugs represent the highest energy state and can be considered as
supercooled liquids.
 They have greater aqueous solubility than the crystalline forms
because the energy required to transfer a molecule from crystal lattice
is greater than that required for non-crystalline (amorphous) solid— for
example, the amorphous form of novobiocin is 10 times more soluble than
the crystalline form.
Amorphous > Metastable > Stable.
Hydrates/Solvates (Pseudopolymorphism)
 The crystalline form of a drug can either be a polymorph or a molecular adduct or
both.
 The stoichiometric type of adducts where the solvent molecules are incorporated in
the crystal lattice of the solid are called as the solvates, and the trapped solvent as
solvent of crystallization.
 The solvates can exist in different crystalline forms called as pseudopolymorphs.
 This phenomenon is called as pseudopolymorphism.
 When the solvent in association with the drug is water, the solvate is known as a
hydrate.
 Hydrates are most common solvate forms of drugs.
Salt Form of the Drug
 Most drugs are either weak acids or weak bases.
 One of the easiest approaches to enhance the solubility and dissolution rate of such
drugs is to convert them into their salt forms
Drug pKa and Lipophilicity and GI pH—pH Partition Hypothesis
 The pH partition theory (Brodie et al) explains in simple terms, the process of drug absorption from
the GIT and its distribution across all biological membranes.
 The theory states that for drug compounds of molecular weight greater than 100, which are primarily
transported across the biomembrane by passive diffusion, the process of absorption is governed by:
1. The dissociation constant (pKa) of the drug.
2. The lipid solubility of the unionised drug (a function of drug Ko/w).
3. The pH at the absorption site.
 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.
 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:
 For, Acidic drugs
pH = pKa + log [ionized form]/ [Unionized form]
 For, Basic drugs
 pH = pKa + log [unionized form]/ [Ionized form]
 Lipophilicity and Drug Absorption,As mentioned earlier, it is the pKa of a
drug that determines the degree of ionisation at a particular pH and that
only the unionised drug, if sufficiently lipid soluble, is absorbed into the
systemic circulation.
 The drug will be poorly absorbed if it has poor lipid solubility (or low
Ko/w).
 In other words, a perfect hydrophilic-lipophilic balance (HLB) should be
there in the structure of the drug for optimum bioavailability.
DOSAGE FORM (PHARMACO-TECHNICAL) FACTORS
 Disintegration Time- Disintegration time (DT) is of particular importance in case of solid
dosage forms like tablets and capsules.
 In vitro disintegration test is by no means a guarantee of drug’s bioavailability because if
the disintegrated drug particles do not dissolve, absorption is not possible.
 However, if a solid dosage form does not conform to the DT, it portends bioavailability
problems because the subsequent process of dissolution will be much slower and
absorption may be insufficient.
 Coated tablets, especially sugar coated ones have long DT.
 Rapid disintegration is thus important in the therapeutic success of a solid dosage
form.
 DT of a tablet is directly related to the amount of binder present and the compression
force (hardness) of a tablet.
 A harder tablet with large amount of binder has a long DT.
 Disintegration can be aided by incorporating disintegrants in suitable amounts during
formulation.
 After disintegration of a solid dosage form into granules, the granules must de-
aggregate into fine particles, as dissolution from such tiny particles is faster than that
from granules.
Manufacturing/Processing Variables-
 Drug dissolution is the single most important factor in the absorption of
drugs, especially from the most widely used conventional solid dosage forms,
tablets and capsules.
 The dosage form related factors that influence dissolution and hence absorption
of a drug from such formulations are:
 Excipients (formulation ingredients apart from the active principles), and
Manufacturing processes.
 Pharmaceutical Ingredients/Excipients (Formulation factors) A drug is rarely
administered in its original form.
 Almost always, a convenient dosage form to be administered by a suitable
route is prepared. Such a formulation contains a number of excipients (non-drug
components of a formulation).
 Excipients are added to ensure acceptability, physicochemical stability during
the shelf-life, uniformity of composition and dosage, and optimum
bioavailability and functionality of the drug product.
 Despite their inertness and utility in the dosage form, excipients can influence
absorption of drugs.
 The more the number of excipients in a dosage form, the more complex it is
and greater the potential for absorption and bioavailability problems.
 As a general rule, the bioavailability of a drug from various dosage forms
decreases in the following order:
 Solutions > Emulsions > Suspensions > Capsules > Tablets > Coated Tablets
> Enteric Coated Tablets > Sustained Release Products.
 PATIENT RELATED FACTORS AFFECTING DRUG ABSORPTION
 Age
 In infants, the gastric pH is high and intestinal surface and blood flow to the GIT is low
resulting in altered absorption pattern in comparison to adults.
 In elderly persons, causes of impaired drug absorption include altered gastric emptying,
decreased intestinal surface area and GI blood flow, higher incidents of achlorhydria and
bacterial overgrowth in small intestine.
 Gastric Emptying Apart from dissolution of a drug and its permeation through the biomembrane,
the passage from stomach to the small intestine, called as gastric emptying, can also be a rate-
limiting step in drug absorption because the major site of drug absorption is intestine.
 Thus, generally speaking, rapid gastric emptying increases bioavailability of a drug. Rapid
gastric emptying is advisable where:
1. A rapid onset of action is desired e.g. sedatives.
2. Dissolution of drug occurs in the intestine e.g. enteric-coated dosage forms.
3. The drugs are not stable in the gastric fluids e.g. penicillin G and erythromycin.
4. The drug is best absorbed from the distal part of the small intestine e.g. vitamin B12.
 Intestinal Transit
 Since small intestine is the major site for absorption of most drugs, long
intestinal transit time is desirable for complete drug absorption.
 Gastrointestinal pH, A tremendous 107 fold difference in the hydrogen ion
concentration is observed between the gastric and colon fluids.
 The GI pH generally increases gradually as one move down the stomach to
the colon and rectum (see Fig. 2.22).
 GI fluid pH influence drug absorption in several ways:
1.Disintegration:
 The disintegration of some dosage forms is pH sensitive.
 With enteric-coated formulations, the coat dissolves only in the intestine
followed by disintegration of the tablet.
2. Dissolution:
 A large number of drugs are either weak acids or weak bases whose
solubility is greatly affected by pH.
 A pH that favours formation of salt of the drug enhances the dissolution of that
drug.
 Since drug dissolution is one of the important rate-determining steps in drug
absorption, GI pH is of great significance in the oral bioavailability of drugs.
 Weakly acidic drugs dissolve rapidly in the alkaline pH of the intestine
whereas basic drugs dissolve in the acidic pH of the stomach.
 Since the primary site for absorption of most drugs is small intestine, the
poorly water-soluble basic drugs must first dissolve in the acidic pH of
stomach before moving into the intestine.
Absorption of drug from Non per oral extra-vascular routes:
 NON PER ORAL Means other than oral routes which by passes the GIT
and reaches to systemic circulation.
 One of the major advantages of administering drugs by non-invasive
transmucosal (& transdermal) routes such as nasal, buccal, rectal, etc. is
that greater systemic availability is attainable .
 Moreover, peptide and protein drugs can also be delivered by such routes.
Buccal/Sublingual Administration
 The two sites for oral mucosal delivery of drugs are:
Sublingual route:
 The drug is placed under the tongue and allowed to dissolve.
Buccal route:
 The medicament is placed between the cheek and the gum.
 The barrier to drug absorption from these routes is the epithelium of oral mucosa.
 Passive diffusion is the major mechanism for absorption of most drugs; nutrients may be
absorbed by carrier-mediated processes.
 .
Rectal Administration:
 Despite its diminished popularity, the rectal route of drug administration is
still an important route for children and old patients.
 The drugs may be administered as solutions (microenemas) or
suppositories.
 Absorption is more rapid from solutions than from suppositories but is
more variable in comparison to oral route.
 Irritating suppository bases such as PEG promotes defecation and drug
loss. Presence of faecal matter retards drug absorption.
 Though highly vascularised, absorption is slower because of limited
surface area.
Topical Administration:
 Excluding the respiratory tract’s contact with the inhaled air, the skin is
virtually the sole human surface directly interfacing the body with the external
environment.
 It is the largest organ of the body weighing approximately 2 Kg and 2 m2 in
area and receives about 1/3rd of total blood circulating through the body.
 Majority of drugs applied topically are meant to exert their effect locally.
 When topically applied drugs are meant to exert their effects systemically, the
mode of administration is called as percutaneous or transdermal delivery.
 Percutaneous absorption occurs only if the topically applied drug permeates
the dermal capillaries and enters the blood stream.
Intramuscular Administration:
 Absorption of drugs from i.m. sites is relatively rapid but much slower in
comparison to i.v. injections. Factors that determine rate of drug absorption from i.m.
sites are:
1. Vascularity of the injection site: the decreasing order of blood flow rate to muscular
tissues in which drugs are usually injected is:
Arm (deltoid) > Thigh (vastus lateralis) > Buttocks (gluteus maximus)
2. Since blood flow rate is often the rate-limiting step in absorption of drugs from i.m.
sites, most rapid absorption is from deltoid muscles and slowest from gluteal region.
 The absorption rate decreases in circulatory disorders such as hypotension.
3. Lipid solubility and ionisation of drug: highly lipophilic drugs are absorbed rapidly
by passive diffusion whereas hydrophilic and ionised drugs are slowly absorbed
through capillary pores.
4. Molecular size of the drug: small molecules and ions gain direct access into
capillaries through pores whereas macromolecules are taken up by the lymphatic system.
 There is some evidence that small peptides and fluids can cross the endothelial tissue of
blood capillaries and lymph vessels by transport in small vesicles that cross the
Subcutaneous Administration:
 All factors that influence i.m. drug absorption are also applicable to
absorption from subcutaneous site.
 Generally, absorption of drugs from a s.c. site is slower than that from i.m.
sites due to poor perfusion, but this fact is of particular importance for the
administration of drugs for which a rapid response is not desired and for
drugs that degrade when taken orally e.g. insulin and sodium heparin.
 The rate of absorption of a drug from subcutaneous site can be increased in 2
ways:
1.Enhancing blood flow to the injection site: by massage, application of heat,
co-administration of vasodilators locally, or by exercise, and
2. Increasing the drug-tissue contact area: by co-administering the enzyme
hyaluronidase that breaks down the connective tissue and permits spreading
of drug solution over a wide area.
Pulmonary Administration
 In principle, all drugs intended for systemic effects can be administered by
inhalation since the large surface area of the alveoli, high permeability of
the alveolar epithelium and rich perfusion permit extremely rapid
absorption just like exchange of gases between the blood and the inspired
air.
 However, the route has been limited for administering drugs that affect
pulmonary system such as bronchodilators (salbutamol), anti-
inflammatory steroids (beclomethasone) and antiallergics (cromolyn).
Intranasal Administration
 The nasal route is becoming increasingly popular for systemic delivery
especially of some peptide and protein drugs.
 Drug absorption from nasal mucosa is as rapid as observed after
parenteral administration because of its rich vasculature and high
permeability.
 The route is otherwise used for drugs to treat local symptoms like nasal
congestion, rhinitis, etc.
 Two mechanisms for drug transport across the nasal mucosa have been
suggested—
a. A faster rate that is dependent upon drug lipophilicity, and
b. A slower rate which is dependent upon drug molecular weight. In case of
lipophilic drugs, rapid absorption by diffusion is observed up to 400 Daltons and
satisfactory absorption up to 1000 Daltons.
Intraocular Administration
 Topical application of drugs to the eyes is mainly meant for local effects such as
mydriasis, miosis, anaesthesia or treatment of infections, glaucoma, etc.
 Sterile aqueous solutions of drugs are widely used ophthalmic formulations
and administered in the conjunctival sac.
 The barrier to intraocular penetration of drugs is the cornea which possesses both
hydrophilic and lipophilic characteristics.
 Thus, for optimum intraocular permeation, drugs should possess biphasic
solubility.
 The pH of lachrymal fluid influences absorption of weak electrolytes such as
pilocarpine.
 On the other hand, pH of the formulation influences lachrymal output—higher
pH decreases tear flow and promotes drug absorption whereas lower pH
solutions increase lachrymation and subsequent drug loss due to drainage.
 Rate of blinking also influences drainage loss.
DISTRIBUTION
Introduction
 After entry into the systemic circulation, either by intravascular injection
or by absorption from any of the various extravascular sites, the drug is
subjected to a number of processes called as disposition processes.
 Disposition is defined as the processes that tend to lower the plasma
concentration of drug.
 The two major drug disposition processes are –
1.Distribution which involves reversible transfer of a drug between
compartments.
2. Elimination which causes irreversible loss of drug from the body.
Elimination is further divided into two processes –
a.Biotransformation (metabolism)
b. Excretion.
Distribution
 Distribution is defined as the reversible transfer of a drug between one
compartment and another.
 Since the process is carried out by the circulation of blood, one of the compartments
is always the blood or the plasma and the other represents extravascular fluids and
other body tissues.
 In other words, distribution is reversible transfer of a drug between the blood and
the extravascular fluids and tissues.
 Distribution is a passive process, for which, the driving force is concentration
gradient between the blood and the extravascular tissues.
 The process occurs by diffusion of free drug only until equilibrium is achieved.
 As the pharmacological action of drug depends upon its concentration at the site of
action, distribution plays a significant role in the onset, intensity and sometimes
duration of drug action.
Steps in Drug Distribution
 Distribution of drug present in systemic circulation to extravascular tissues
involves following steps –
1. Permeation of free or unbound drug present in the blood through the
capillary wall (occurs rapidly) and entry into the interstitial/extracellular
fluid (ECF).
2. Permeation of drug present in the ECF through the membrane of tissue
cells and into the intracellular fluid.
 This step is rate-limiting and depends upon two major factors –
a. Rate of perfusion to the extracellular tissue
b. Membrane permeability of the drug.
TISSUE PERMEABILITY OF DRUGS:
 The two major rate-determining steps in the distribution of drugs are:
1. Rate of tissue permeation, and
2. Rate of blood perfusion.
 If the blood flow to the entire body tissues were rapid and uniform, differences
in the degree of distribution between tissues will be indicative of differences in
the tissue penetrability of the drug and the process will be tissue permeation
rate-limited.
 Tissue permeability of a drug depends upon the physicochemical properties of
the drug as well as the physiological barriers that restrict diffusion of drug into
tissues.
Physicochemical Properties of the Drug:
 Important physicochemical properties of drug that influence its distribution are
molecular size, degree of ionization, partition coefficient and stereo-chemical
nature.
 Almost all drugs having molecular weight less than 500 to 600 Daltons easily
cross the capillary membrane to diffuse into the extracellular interstitial fluids.
 However, penetration of drugs from the extracellular fluid into the cells is a
function of molecular size, ionization constant and lipophilicity of the drug.
 Only small, water-soluble molecules and ions of size below 50 Daltons enter the
cell through aqueous filled channels whereas those of larger size are restricted
unless a specialized transport system exists for them.
 The degree of ionization of a drug is an important determinant in its tissue
penetrability.
 The pH of the blood and the extravascular fluid also play a role in the ionization
and diffusion of drugs into cells.
 A drug that remains unionized at these pH values can permeate the cells
relatively more rapidly.
 Since the blood and the ECF pH normally remain constant at 7.4, they do not
have much of an influence on drug diffusion unless altered in conditions such
as systemic acidosis or alkalosis.
 Most drugs are either weak acids or weak bases and their degree of ionisation
at plasma or ECF pH depends upon their pKa.
 All drugs that ionise at plasma pH (i.e. polar, hydrophilic drugs), cannot
penetrate the lipoidal cell membrane and tissue permeability is the rate-
imiting step in the distribution of such drugs.
 Only unionised drugs which are generally lipophilic, rapidly cross the cell
membrane.
Physiological Barriers to Distribution of Drugs
 A membrane (or a barrier) with special structural features can be a
permeability restriction to distribution of drugs to some tissues.
 Some of the important simple and specialized physiological barriers are:
1.Simple capillary endothelial barrier
2. Simple cell membrane barrier
3. Blood-brain barrier
4. Blood-CSF barrier
5. Blood- placental barrier
6. Blood-testis barrier.
1. The simple capillary endothelial barrier:
 The membrane of capil-laries that supply blood to most tissues is, practically
speaking, not a barrier to moieties which we call drugs.
 Thus, all drugs, ionised or unionised, with a molecular size less than 600
Daltons, diffuse through the capillary endothelium and into the interstitial fluid.
 Only drugs bound to the blood components are restricted because of the large
molecular size of the complex.
2. The simple cell membrane barrier:
 Once a drug diffuses from the capillary wall into the extracellular fluid, its
further entry into cells of most tissues is limited by its permeability through the
membrane that lines such cells.
 Such a simple cell membrane is similar to the lipoidal barrier in the GI
absorption of drugs (discussed in chapter 2).
 The physicochemical properties that influence permeation of drugs across such
a barrier are summarized in Fig. 3.4.
3. Blood-brain barrier (BBB):
 Unlike the capillaries found in other parts of the body, the capillaries in the
brain are highly specialized and much less permeable to water-soluble drugs.
 The brain capillaries consist of endothelial cells which are joined to one
another by continuous tight intracellular junctions comprising what is called as
the brain barrier (Fig. 3.5).
 Moreover, the presence of special cells called as pericytes and astrocytes,
which are the elements of the supporting tissue found at the base of
endothelial membrane, form a solid envelope around the brain capillaries.
 As a result, the intercellular (paracellular is circulation into the brain, it has to
pass through the cells (transcellular) are specific sites in the where the BBB
does not exist, namely, the trigger area and the median hypothalamic
eminence.
 Moreover, drugs administered betranasally may diffuse directly into the CNS
because of the continuity between submu cosal areas of the nose and the
subarachnoid space of the olfactory lobe).
 There is also virtual absence of pinocytosis in brain.
 A solute may thus gain access to brain via only one of the two pathways:
1. Passive diffusion through the lipoidal barrier:
 which is restricted to small molecules (with a molecular weight less than a
thresh-old of approximately 700 Daltons) having high o/w partition coefficient.
2. Active transport of essential nutrients such as sugars and amino acids.
 Thus, structurally similar foreign molecules can also pen-etrate the BBB by the
same mechanism.
 The effective partition coefficient of thiopental, a highly lipid soluble drug is
50 times that of pentobarbital and crosses the BBB much more rapidly.
 Most antibiotics such as penicillin which are polar, water-soluble and ionised at
plasma pH, do not cross the BBB under normal circumstances.
 The selective permeability of lipid-soluble moieties through the BBB makes
appropriate choice of a drug to treat CNS disorders an essential part of therapy;
for example, Parkinsonism, a disease characterized by depletion of dopamine in
the brain, cannot be treated by administration of dopamine as it does not cross
the BBB.
 Hence, levodopa, which can penetrate the CNS where it is metabolised to
dopamine, is used in its treatment.
 Targeting of polar drugs to brain in certain conditions such as tumour had always
been a problem.
Three different approaches have been utilized successfully to promote crossing the
BBB by drugs:
(i) Use of permeation enhancers such as dimethyl sulphoxide (DMSO).
(ii) Osmotic disruption of the BBB by infusing internal carotid artery with mannitol.
(iii) Use of dihydropyridine redox system as drug carriers to the brain.
 NO In the latter case, the lipid soluble dihydropyridine is linked as a carrier to the
polar drug to form a prodrug that readily crosses the BBB.
 In the brain, the CNS enzymes oxidize the dihydropyridine moiety to the polar
pyridinium ion form that cannot diffuse back out of the brain.
 As a result, the drug gets trapped in the CNS.
 Such a redox system has been used to deliver steroidal drugs to the brain
4. Blood-cerebrospinal fluid barrier:
 The cerebrospinal fluid (CSF) is formed mainly by the choroid plexus of the
lateral, third and fourth ventricles and is similar in composition to the ECF of
brain.
 The capillary endothelium that lines the choroid plexus have open junctions
or gaps and drugs can flow freely into the extracellular space between the
capillary wall and the choroidal cells.
 However, the choroidal cells are joined to each other by tight junctions
forming the blood-CSF barrier which has permeability characteristics similar
to that of the BBB (Fig. 3.6).
 As in the case of BBB, only highly lipid soluble drugs can cross the blood-
CSF barrier with relative ease whereas moderately lipid soluble and partially
ionised drugs permeate slowly.
 A drug that enters the CSp slowly cannot achieve a high concentration as the
bulk flow of CSP continuously removes the drug.
 For any given drug, its concentration in the brain will always be higher than
in the CSF.
 Although the mechanisms for diffusion of drugs into the CNS and CSF are
similar, the degree of uptake may vary significantly.
 In some cases, CSF drug concentration may be higher than its cerebral
concentra-tion e.g. sulphamethoxazole and trimethoprim, and vice versa in
other cases, e.g. certain B-blockers.
5. Blood-placental barrier:
 The maternal and the foetal blood vessels are separated by a number of
tissue layers made of foetal trophoblast basement membrane and the
endothelium which together constitute the placental barrier.
 The flow of blood in the maternal and the foetal blood vessels is shown in
Fig. 3.7.
 The human placental barrier has a mean thickness of 25 microns in early
pregnancy that reduces to 2 microns at full term which however does not
reduce its effectiveness.
 Many drugs having molecular weight less than 1000 Daltons and moderate
to high lipid solubility e.g. ethanol, sulphonamides, barbiturates, gaseous
anaesthetics, steroids, narcotic an-algesics, anticonvulsants and some
antibiotics, cross the barrier by simple diffusion quite rapidly.
 This shows that the placental barrier is not as effective a barrier as BBB.
Nutrients essential for the foetal growth are transported by carrier-mediated
processes.
 Immunoglobulins are trans-ported by endocytosis. An agent that causes toxic
effects on foetus is called as teratogen.
 Teratogenecity is defined as foetal abnormalities caused by administration
of drugs during pregnancy.
 Drugs can affect the foetus at 3 stages as shown in Table 3.2.
• It is always better to restrict all drugs during pregnancy because of
the uncertainty of their hazardous effects.
6.Blood-testis barrier:
 This barrier is located not at the capillary endothelium level but at sertoli-
sertoli cell junction. It is the tight junctions between the neighbouring
sertoli cells that act as the blood-testis barrier.
 This barrier restricts the passage of drugs to spermatocytes and spermatids.
VOLUME OF 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 equilibrated with the
drug do not have equal concentration.
Apparent volume of distribution = Amount of drug in body/Plasma drug concentration
VOLUME OF DISTRIBUTION
 A drug in circulation distributes to various organs and tissues.
 When the process of distribution is complete (at distribution equilibrium),
different organs and tissues contain varying concentrations of drug which
can be determined by the volume of tissues in which the drug is present.
 Since different tissues have different concentrations of drug, the volume
of distribution cannot have a true physiological meaning.
 However, there exists a constant relationship between the concentration
of drug in plasma C, and the amount of drug in the body, X.
 X∝ C
 Or,
 X=Vd C
 Where,
 Vd=Proportionality constant having the unit of volume and popularly called as apparent
volume of 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 equilibrated with the drug do not
have equal concentration.
 Thus, from equation, Vd is given by the ratio:
 Apparent Volume of Distribution = Amount of drug in the body
Plasma drug concentration
 Vd = X
C
 The apparent volume of distribution bears no direct relationship with the real volume of
distribution.
 The real volume of distribution has direct physiological meaning and is related to the body
water.
 The body water is made up of 3 distinct compartments as shown in the Table.
 The volume of each of these real physiological compartments can be determined by use of specific
tracers or markers (Table 3.5).
 The plasma volume can be determined by use of substances of high molecular weight or substances
that are totally bound to plasma albumin, for e.g. high molecular weight dyes such as Evans blue,
indocyanine green and 1-131 albumin.
 When given i.v., these remain confined to the plasma. The total blood volume can also be
determined if the haematocrit is known.
 The extracellular fluid (ECF) volume can be determined by substances that easily penetrate the
capillary membrane and rapidly distribute throughout the ECF but do not cross the cellmembranes,
for e.g. the Na+ Cl- Br- SCN- and SO42- ions and inulin, mannitol and raffinose.
 However, none of these substances are completely kept out of the cells.
 The ECF volume, excluding plasma is approximately 15 liters.
 The total body water (TBW) volume can be determined by use of substances that distribute
equally in all water compartments of the body (both intra- and extracellular), for eg heavy water
(D₂O), tritiated water (HTO) and lipid soluble substances such as antipyrine.
 The intracellular fluid volume is determined as the difference between the TBW and ECF volume.
 The intracellular fluid volume including those of blood cells is approximately 27 liters
 Since the tracers are not bound or negligibly bound to plasma or tissue proteins,
their apparent volume of distribution is same as their true volume of distribution.
 The situation is different with most drugs which bind to plasma proteins or
extravascular tissues or both.
 Certain generalizations can be made regarding the apparent volume of distribution
of such drugs:
1.Drugs which bind selectively to plasma proteins or other blood components, e.g.
warfarin (i.e. those that are less bound to extravascular tissues), have apparent
volume of distribution smaller than their true volume of distribution.
 The vd of such drugs lies between blood volume and TBW volume (i.e. between
6 to 42 litres); for example, warfarin has a vd of about 10 liters.
2. Drugs which bind selectively to extravascular tissues, e.g. chloroquine (i.e. those
that are less bound to blood components),have apparent volume of distribution larger
than their real volume of distribution.
 The Vd of such drugs is always greater than 42 liters or TBW volume; for example,
chloroquine has a Vd of approximately 15,000 liters.
 Such drugs leave the body slowly and are generally more toxic than drugs that do
not distribute deeply into body tissues.
 Thus, factors that produce an alteration in binding of drug to blood
components, result in an increase in Vd and those that influence drug
binding to extravascular components result in a decrease in V d.
 Other factors that may influence Vd are changes in tissue perfusion and
perme-ability, changes in the physicochemical characteristics of the drug
e.g. ionisation, changes in the body weight and age and several disease
states.
 Apparent volume of distribution is expressed in litres and sometimes in
litres/Kg body weight.
 The Vd of various drugs ranges from as low as 3 litres (plasma volume) to
as high as 40,000 liters (much above the total body size).
 Many drugs have Vd greater than 30 liters.
 The Vd is a characteristic of each drug under normal conditions and is
altered under conditions that affect distribution pattern of the drug.
METHODS FOR STUDYING DRUG DISTRIBUTION PATTERN
 New drug characterization assays are central in providing evidence of the
specificity and selectivity of drugs.
 It is thus essential to understand the drug distribution pattern in drug discovery.
 Studies for determining drug distribution pattern includes micro-dialysis, mass
spectrometry and imaging methods such as whole body autoradiography and
positron emission tomography (PET).
1. Microdialysis:
 This technique involves insertion of very fine probes into the tissues of the
living body, mostly into fluid spaces, such as the CSF, and other extracellular
fluids where possible.
 The probes consist of at least two concentric tubes, and a semipermeable
membrane separating them, positioned such that an artificial extracellular dialysis
fluid can be slowly infused through the probe and pass the membrane.
 Unbound drug molecules in the tissues surrounding the probe diffuse into the
flowing dialysate, which is then collected for analysis.
 The limitation of this technique is its invasive nature.
2. Matrix-assisted laser desorption ionization-mass spectrometry imaging
(MALDI-MSI):
 It is used to study drug localization within microenvironmental tissue compartments.
 Thin sections of animal tissues are exposed to pharmaceutical drugs by either spotting or
submerging.
 MALDI-MSI is then performed on the tissue to localize drug-distribution patterns. (colour code)
3. Autoradiography:
 In this approach, the radioactively labelled drug substance under investigation is administered
to test animals, usually mice or rats, which are killed at suitable time intervals, and frozen
sections of tissues or of the whole body prepared.
 An image of the radiation is obtained by placing the sections next to a photographic
emulsion.
 Thus, if a whole body section is used, and the radioactivity is specifically localized in highly
perfused organs such as the lungs and liver, the image will reveal this.
 Comparison with a normal photograph of the slice is used to identify which tissues contain the
radioactivity.
 Densitometry can be used to quantify the relative amounts of radioactivity.
4. Positron emission tomography (PET):
 Synthetic radioactive isotopes (e.g. 11C, 13N, 15O and 18F) with atomic
masses less than the naturally occurring stable isotopes have half-life
values of 2-110 minutes and emit positrons that interact with electrons to
emit gamma radiation that can be detected outside the body.
 The isotopes are generated in a cyclotron and incorporated into the drug
molecules immediately before the administration of the drug.
 PET scanning permits the production of images of live organisms
including humans.
PROTEIN BINDING OF DRUGS
 A drug in the body can interact with several tissue components of which the
two major categories are-
1. Blood, and
2. Extravascular tissues.
 The interacting molecules are generally the macromolecules such as proteins,
DNA or adipose.
 The proteins are particularly responsible for such an interaction.
 The phenomenon of complex formation with proteins is called as protein
binding of drugs.
Protein binding may be divided into –
1. Intracellular binding –
 where the drug is bound to a cell protein which may be the drug receptor;
if so, binding elicits a pharmacological response.
 These receptors with which drug interact to show response are called as
primary receptors.
2. Extracellular binding –
 where the drug binds to an extracellular protein but the binding does not
usually elicit a pharmacological response.
 These receptors are called secondary or silent receptors.
Mechanisms of Protein-
 Drug Binding of drugs to proteins is generally reversible which suggests that
it generally involves weak chemical bonds such as –
1.Hydrogen bonds
2. Hydrophobic bonds
3. Ionic bonds, or
4. Van der Waal’s forces.
 Irreversible drug binding, though rare, arises as a result of covalent binding
and is often a reason for the carcinogenicity or tissue toxicity of the drug;
 for example, covalent binding of chloroform and paracetamol metabolites to
liver results in hepatotoxicity
 Binding of drugs falls into 2 classes:
1. Binding of drugs to blood components like—
a. Plasma proteins
b. Blood cells Binding of drugs to extravascular tissue proteins, fats, bones,
etc.
 BINDING OF DRUGS TO BLOOD COMPONENTS:-
 Plasma Protein-Drug Binding Following entry of a drug into the systemic
circulation, the first things with which it can interact are blood
components like plasma proteins, blood cells and haemoglobin.
 The main interaction of drug in the blood compartment is with the plasma
proteins which are present in abundant amounts and in large variety.
 The binding of drugs to plasma proteins is reversible.
 The extent or order of binding of drugs to various plasma proteins is:
Albumin > α1-Acid Glycoprotein > Lipoproteins > Globulins
 TISSUE BINDING OF DRUGS (TISSUE LOCALIZATION OF DRUGS)
 The body tissues, comprise 40% of the body weight.
 Hence, tissue-drug binding is much more significant than thought to be. A drug can bind to one or
more of the several tissue components.
 Tissue-drug binding is important in distribution from two viewpoints:
1. It increases the apparent volume of distribution of drugs in contrast to plasma protein binding
which decreases it.
 This is because the parameter is related to the ratio of amount of drug in the body to the plasma
concentration of free drug and the latter is decreased under conditions of extensive tissue binding
of drugs.
2. 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 bromobenzene bind covalently to hepatic tissues.
 Factors influencing localization of drugs in tissues include lipophilicity
and structural features of the drug, perfusion rate, 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
 Several examples of extravascular tissue-drug binding are:
1.Liver: As stated earlier, epoxides of a number of halogenated hydrocarbons and
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.
 Lead is known to replace calcium from bones and cause their brittleness.
 Factors affecting protein-drug binding: Factors affecting protein-drug binding can be broadly
categorized as—
1. Drug related factors
a. Physicochemical characteristics of the drug
b. Concentration of drug in the body
c. Affinity of a drug for a particular binding component
2.Protein/tissue related factors .
3.Physicochemical characteristics of the protein or binding agent
a. Concentration of protein or binding component
b. Number of binding sites on the binding agent
4.Drug interactions .
5.Competition between drugs for the binding site (displacement interactions)
a. Competition between the drug and normal body constituents
b. Allosteric changes in protein molecule Patient related factors .
6.Age
a. a.Intersubject variations b. Disease states
1.DRUG RELATED FACTORS
a. Physicochemical Characteristics of the Drug
 As mentioned earlier, protein binding is directly related to the lipophilicity of
drug.
 An increase in lipophilicity increases the extent of binding, for example, the
slow absorption of cloxacillin in comparison to ampicillin after i.m. injection is
attributed to its higher lipophilicity and larger (95%) binding to proteins
while the latter is less lipophilic and just 20% bound to proteins.
 Highly lipophilic drugs such as thiopental tend to localize in adipose tissues.
 Anionic or acidic drugs such as penicillins and sulphonamides bind more to
HSA whereas cationic or basic drugs such as imipramine and alprenolol bind to
AAG.
 Neutral, unionised drugs bind more to lipoproteins.
b.Concentration of Drug in the Body
 The extent of protein-drug binding can change with both changes in drug
as well as protein concentration.
 The concentration of drugs that bind to HAS( Human serum albumin)
does not have much of an influence, as the therapeutic concentration of
any drug is insufficient to saturate it.
 However, therapeutic concentration of lidocaine can saturate AAG with
which it binds as the concentration of AAG (alpha acid glycoproptein)is
much less in comparison to that of HSA in blood.
c. Drug-Protein/Tissue Affinity
 Lidocaine has greater affinity for AAG than for HSA.
 Digoxin has more affinity for proteins of cardiac muscles than those of
skeletal muscles or plasma.
 Iophenoxic acid, a radio-opaque medium, has so great an affinity for
plasma proteins that it has a half-life of 2½ years.
2.PROTEIN/TISSUE RELATED FACTORS
a.Physicochemical Properties of Protein/Binding Component
 Lipoproteins and adipose tissue tend to bind lipophilic drugs by
dissolving them in their lipid core.
 The physiologic pH determines the presence of active anionic and cationic
groups on the albumin molecules to bind a variety of drugs.
b. concentration of Protein/Binding
 Component Among the plasma proteins, binding predominantly occurs with
albumin, as it is present in a higher concentration in comparison to other
plasma proteins.
 The amount of several proteins and tissue components available for
binding, changes during disease states.
c. Number of Binding Sites on the Protein
 Albumin has a large number of binding sites as compared to other proteins
and is a high capacity binding component.
 Several drugs are capable of binding at more than one site on albumin, e.g.
fluocloxacillin, flurbiprofen, ketoprofen, tamoxifen and dicoumarol bind to
both primary and secondary sites on albumin.
 Indomethacin is known to bind to 3 different sites.
 AAG is a protein with limited binding capacity because of its low
concentration and low molecular size.
 Though pure AAG has only one binding site for lidocaine, in presence of
HSA, two binding sites have been reported which was suggested to be due to
direct interaction between HSA and AAG.
3.DRUG INTERACTIONS
a.Competition Between Drugs for the Binding Sites (Displacement Interactions)
 When two or more drugs can bind to the same site, competition between them
for interaction with the binding site results.
 If one of the drugs (drug A) is bound to such a site, then administration of another
drug (drug B) having affinity for the same site results in displacement of drug A
from its binding site.
 Such a drug-drug interaction for the common binding site is called as
displacement interaction.
 The drug A here is called as the displaced drug and drug B as the displacer.
 Warfarin and phenylbutazone have same degree of affinity for HSA.
 Administration of phenylbutazone to a patient on warfarin therapy results in
displacement of latter from its binding site.
 The free warfarin may cause adverse hemorrhagic reactions which may be lethal.
 Phenylbutazone is also known to displace sulphonamides from their HSA
binding site
4. PATIENT RELATED FACTORS
a.Age-
 Modification in protein-drug binding as influenced by age of the patient is
mainly due to differences in the protein content in various age groups.
I. Neonates: Albumin content is low in newborn; as a result, the unbound
concentration of drug that primarily binds to albumin, for example phenytoin and
diazepam, is increased.
II. Young infants: An interesting example of differences in protein-drug binding
in infants is that of digoxin. Infants suffering from congestive cardiac failure are
given a digitalizing dose 4 to 6 times the adult dose on body weight basis.
This is contrary to one's belief that infants should be given low doses considering
their poorly developed drug eliminating system.
The reason attributed for use of a large digoxin dose is greater binding of the drug
in infants (the other reason is abnormally large renal clearance of digoxin in
infants).
III. Elderly:
 In old age, the albumin content is lowered and free concentration of
drugs that bind primarily to it is increased.
 Old age is also characterized by an increase in the levels of AAG and thus
decreased free concentration is observed for drugs that bind to it.
 The situation is complex and difficult to generalize for drugs that bind to
both HSA and AAG, e.g. lidocaine and propranolol.
Significance of protein or tissue binding of drugs:-
1) Absorption-
 The absorption equilibrium is attended by transfer of free drug from the site of
administration into the systemic circulation and When the concentration in
these two compartments becomes equal.
 Following equilibrium, the process may stop.
 However, binding of the absorbed drug to plasma proteins decreases free
drug concentration and disturbs such an equilibrium.
 Thus, sink conditions and concentration gradient are re-established which now
act as a driving force for further absorption.
 This is particularly useful in case of ionized drugs which are transported with
difficulty.
2) Systemic Solubility Drugs-
 Water insoluble drugs, neutral endogenous macromolecules such as heparin
and several steroids and oil soluble vitamins are circulated and distributed to
tissues by binding especially to lipoproteins which acts as vehicle for such
hydrophobic compounds.
3) DISTRIBUTION:-
 Plasma protein binding restricts the entry of drugs that have specific
affinity for certain tissues.
 This prevents accumulation of a large fraction of drug in such a tissues and
thus, subsequent toxic reactions.
 Plasma protein drug binding thus favors uniform distribution of drugs the
throughout the body by its buffer function (maintains equilibrium between the
free and bound drug).
 A protein bound drug in particular does not cross the BBB ,the placental
barrier and glomerulus.
Kinetics of protein binding-
 Consider protein P and Drug is D
 This theory applies the law of mass action
P+D PD
At equilibrium,
Ka = [PD]
[P] [D]
OR
PD= Ka [P] [D] …………………1
Where,
[P]=Concentration of free protein
[D]=Concentration of free Drug
[PD]= Concentration of protein drug complex
Ka=association state constant
Kd=Dissociation state constant
Kd= [P] [D]
[PD]
 If association constant [Ka] is greater than dissociation constant [Kd] it indicates
forward reaction
 PT=Total concentration of protein present
[PT]= [PD]+[P]……………2
 If r is a number of moles of drug bound to total protein then,
r= [PD] …………………………..3
[PT]
From equation 2 and 3
r= [PD]
[PD]+1…………………………………4
Putting the value of PD from equation 1 to equation 4
r = Ka [P] [D]
Ka [P] [D]+[P]
r= Ka [D] [P]
{Ka [D]+1} [P]
r= Ka[D]
Ka[D] +1……………………..5
 Equation 5 represents when there is only one binding site on the protein
 There is N number of binding sites are available per mole of protein then

r = N Ka [D]
Ka [D]+1
Value of association constant Ka and number of binding sites can be obtained by
plotting equation in four different ways
 It is made by plotting r Vs D
 When all the binding sites are occupied by the drugs.
 The protein is saturated and plateau is reached.
 A plateau r=N,
Where,
R=N/2
D=1/Ka (Concentration of drug)
r = N Ka [D]
Ka [D]+1

R= N/2= N Ka [D]
Ka [D]+1 ( cross multiplication)

N Ka [D]+N=2 N Ka [D]
 N Ka [D] +N= 2 N Ka [D] (Shift N to left side)
 N= 2 N Ka [D] - N Ka [D] [consider (D) as common]
 N= 2 N Ka- N Ka [D] [Consider Nka as common]
 N= N Ka [D] [Shift D to Left side]
 [D]= N
N Ka

D = 1
Ka
r = N Ka [D]
Ka [D]+1
r = N Ka [D]
Ka [D]+1
 r = N Ka [D]
Ka [D]+1
Rearrange the equation