UNIT-I

Introduction to Biopharmaceutics
Absorption; Mechanisms of drug absorption through GIT, factors influencing drug
absorption though GIT, absorption of drug from Non per oral extra-vascular
routes, Distribution Tissue permeability of drugs, binding of drugs, apparent, volume of
drug distribution, plasma and tissue protein binding of drugs, factors affecting protein-
drug binding. Kinetics of protein binding, Clinical significance of protein binding of
drugs.

1. Introduction to Biopharmaceutics

Biopharmaceutics is defined as the study of factors influencing the rate and amount of
drug that reaches the systemic circulation and the use of this information to optimise the
therapeutic efficacy of the drug products. The process of movement of drug from its site of
administration to the systemic circulation is called as absorption. The concentration of drug
in plasma and hence the onset of action, and the intensity and duration of response depend
upon the bioavailability of drug from its dosage form. Bioavailability is defined as the rate
and extent (amount) of drug absorption. Any alteration in the drug’s bioavailability is
reflected in its pharmacological effects. Other processes that play a role in the therapeutic
activity of a drug are distribution and elimination. Together, they are known as drug
disposition. The movement of drug between one compartment and the other (generally
blood and the extravascular tissues) is referred to as drug distribution. Since the site of
action is usually located in the extravascular tissues, the onset, intensity and sometimes
duration of action depend upon the distribution behaviour of the drug. The magnitude
(intensity) and the duration of action depend largely upon the effective concentration and the
time period for which this concentration is maintained at the site of action which in turn
depend upon the elimination processes. Elimination is defined as the process that tends to
remove the drug from the body and terminate its action. Elimination occurs by two processes
— biotransformation (metabolism), which usually inactivates the drug, and excretion
which is responsible for the exit of drug/metabolites from the body.

In order to administer drugs optimally, knowledge is needed not only of the mechanisms of
drug absorption, distribution, metabolism and excretion (ADME) but also of the rate
(kinetics) at which they occur i.e. pharmacokinetics. Pharmacokinetics is defined as the
study of time course of drug ADME and their relationship with its therapeutic and toxic
effects of the drug. Simply speaking, pharmacokinetics is the kinetics of ADME or
KADME. The use of pharmacokinetic principles in optimising theuit individual patient
needs and achieving maximum therapeutic utility is called as clinical pharmacokinetics.
Figure 1 is a schematic representation of processes comprising the pharmacokinetics of a
drug.

Fig. 1. Schematic illustration of pharmacokinetic processes

The knowledge and concepts of biopharmaceutics and pharmacokinetics thus have an

integral role in the design and development of new drugs and their dosage forms and
improvement of therapeutic efficacy of existing drugs.

2. Absorption

Drug absorption is defined as the process of movement of unchanged drug from the site of
administration to systemic circulation. Following absorption, the effectiveness of a drug can
only be assessed by its concentration at the site of action. However, it is difficult to measure
the drug concentration at such a site. Instead, the concentration can be measured more
accurately in plasma. There always exist a correlation between the plasma concentration of a
drug and the therapeutic response and thus, absorption can also be defined as the process of
movement of unchanged drug from the site of administration to the site of measurement i.e.
plasma. This definition takes into account the loss of drug that occurs after oral
administration due to presystemic metabolism or first-pass effect.
 Fig. 2. Plots showing significance of rate and extent of absorption in drug therapy.

A drug that is completely but slowly absorbed may fail to show therapeutic response as the
plasma concentration for desired effect is never achieved. On the contrary, a rapidly absorbed
drug attains the therapeutic level easily to elicit pharmacological effect. Thus, both the rate
and the extent of drug absorption are important. Such an absorption pattern has several
advantages:

1. Lesser susceptibility of the drug for degradation or interaction due to rapid absorption.
2. Higher blood levels and rapid onset of action.
3. More uniform, greater and reproducible therapeutic response.
Drugs that have to enter the systemic circulation to exert their effect can be administered by
three major routes:
1. The Enteral Route: includes peroral i.e. gastrointestinal, sublingual/buccal and rectal
routes. The GI route is the most common for administration of majority of drugs.
2. The Parenteral Route: includes all routes of administration through or under one or
more layers of skin. While no absorption is required when the drug is administered i.v., it is
necessary for extravascular parenteral routes like the subcutaneous and the intramuscular
routes.
3. The Topical Route: includes skin, eyes or other specific membranes. The intranasal,
inhalation, intravaginal and transdermal routes may be considered enteral or topical according
to different definitions.

(a) Mechanism of drug absorption

The main mechanisms by which absorption occurs include:
 (a) Transcellular or intracellular transport
(b) Paracellular or
intercellular
transport
(c)Vescicular
transport or
endocytosis

Transcellular/Intracellular Transport is defined as the passage of drugs across the GI

epithelium. It is the most common pathway for drug 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.
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.

Mechanism of drug absorption

 Passive diffusion
 Pore transport
 Facilitated diffusion
 Active transport
 Ionic diffusion
 Ion pair transport
  Endocytosis

PASSIVE DIFFUSION
Drug molecule diffuse from a region of higher concentration to region of lower
concentration until equilibrium is attained.
• Major process for absorption of more than 90% of drugs
• Non ionic diffusion
• Driving force – concentration or electrochemical gradient
• Difference in the drug concentration on either side of the membrane
• Drug movement is a result of kinetic energy of molecules
Fick’s First law of diffusion

Where, dQ/dt = rate of drug

diffusion (amount/time)
D = diffusion coefficient
of the drug
A= surface area of the absorbing membrane for drug diffusion
Km/w = partition coefficient of drug between the lipoidal membrane & the aqueous
GI fluids
(CGIT – C) = difference in the concentration of drug in the GI fluids & the
plasma (Concentration Gradient)
h = thickness of the membrane

Characteristics of Passive diffusion:

 Energy independent
  Greater the area & lesser the thickness of the membrane, faster the diffusion
 The process rapid over for short distances
 Concentration equal on both the sides of the membrane - Equilibrium is attained
 Greater the PC of the drug faster the absorption
The passively absorbed drug enters blood, rapidly swept away & distributed into a larger
volume of body fluids
Hence, the concentration of drug at absorption site CGIT is maintained greater than the
concentration in the plasma. Such a condition is called as sink condition for drug
absorption.

PORE TRANSPORT
 It is also called as convective transport, bulk flow or filtration.
 Mechanism – through the protein channel present in the cell membrane.
 Drug permeation through pore transport – renal excretion, removal of drug from
CSF & entry of drug into the liver
 The driving force – hydrostatic or osmotic pressure differences across the
membrane. Thus, bulk flow of water along with the small solid molecules
through aqueous channels. Water flux that promotes such a transport is called as
solvent drag
 The process is important in the absorption of low molecular weight (<100D), low
molecular size (smaller than the diameter of the pore) & generally water soluble
drugs through narrow, aqueous filled channels or pores e.g. urea, water & sugars.
 Chain like or linear compounds (upto 400D)- filtration

IONIC OR ELECTROCHEMICAL DIFFUSION

 Charge on membrane influences the permeation of drugs.
 Molecular forms of solutes are unaffected by the membrane charge &
permeate faster than ionic forms.
 The permeation of anions & cations is also influenced by pH.
 Once inside the membrane, the cations are attached to negatively charge
intracellular membrane, thus giving rise to an electrical gradient.
 If the same drug is moving from a higher to lower concentration, i.e., moving
down the electrical gradient, the phenomenon is known as electrochemical
diffusion
 Thus, at a given pH, the rate of permeation may be as follows:
 Unionized molecule > anions > Cations

ION-PAIR TRANSPORT
 Responsible for absorption of compounds which ionizes at all pH values. e.g.
quaternary ammonium, sulphonic acids
 Ionized moieties forms neutral complexes with endogenous ions which have
both the required lipophilicity & aqueous solubility for passive diffusion.
 E.g. Propranolol, a basic drug that forms an ion pair with oleic acid & is absorbed
by this mechanism

CARRIER MEDIATED TRANSPORT

 Involves a carrier which reversibly binds to the solute molecules and forms
a solute-carrier complex.
 This molecule transverse across the membrane to the other side and dissociates,
yielding the solute molecule.
 The carrier then returns to the original site to accept a new molecule.
 There are two type of carrier mediated transport system
1) Facilitated diffusion
2) Active transport

FACILITATED DIFFUSION
 Facilitated diffusion is a form of carrier transport that does not require the
expenditure of cellular energy.
 Carriers are numerous in number & are found dissolved in cell membrane.
  The driving force is concentration gradient, particles move from a region of
high concentration to low conc.
 The transport is aided by integral membrane proteins.
 Facilitated diffusion mediates the absorption of some simple sugars, steroids,
amino acids and pyrimidines from the small intestine and their subsequent
transfer across cell membranes.

ACTIVE TRANSPORT
 It is characterized by the transport of drug against concentration gradient with
using energy. Requires energy, which is provided by hydrolysis of ATP for
transportation
 More commonly, metabolic energy is provided by the active transport of Na+, or
is dependent on the electrochemical gradient produced by the sodium pump,
Na+/K+ ATPase (secondary active transport).
 Primary Active Transport
 Direct ATP requirement
 The process transfers only one ion or molecule & only in one direction. Hence,
called as UNIPORT
• E.g. absorption of glucose
• ABC (ATP binding Cassette) transporters
Secondary Active Transport
 No direct requirement of ATP
 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
opposite direction.
2 types:
• Symport (co-transport)
• Antiport (counter transport)

ENDOCYTOSIS
It is a process in which cell absorbs molecules by engulfing them.
 Also termed as vesicular transport
 Minor transport mechanism involving engulfing extracellular materials within
segment of cell membrane to form a saccule or vesicle then pinched of
intracellularly.
 It occurs by 3 mechanisms:
o Phagocytosis
o Pinocytosis
 o Transcytosis

Phagocytosis (cell eating): adsorptive uptake of solid particulates

PINOCYTOSIS
 It is a form of endocytosis in which small particles are brought to the cell, forming
an invagination.
 These small particles are suspended in small vesicles.
 It requires energy in the form of ATP.
 It works as phagocytosis, the only difference being, it is non specific in the
substances it transports.
 This process is important in the absorption of oil soluble vitamins & in the uptake
of nutrients
TRANSCYTOSIS
 It is the process through which various macromolecules are transferred across the
cell membrane.
 They are captured in vesicles, on one side of the cell and the endocytic vesicle
is transferred from one extracellular compartment to another.
 Generally used for the transfer of IgA and insulin
ABSORPTION FACTORS

FACTORS INFLUENCING DRUG ABSORPTION

1. Physicochemical factors:
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. Pharmaceutical or Formulation or Dosage form related factors:

a. Disintegration time
b. Manufacturing variables
c. Nature and type of dosage form
d. Pharmaceutical ingredients (excepients)
e. Product age and storage conditions

3. Patient related or physiological or biological 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
h. Presystemic metabolism
A. Physicochemical properties of drug substances
1. Drug solubility and dissolution rate:
 The rate determining steps in absorption of orally administered drugs are:
 Rate of dissolution
 Rate of drug permeation through the biomembrane.

 Imp prerequisite for the absorption of a drug is that it must be present in aq

solution & this is depends on drug‘s aq solubility &its dissolution rate.

2. Particle size and effective surface area:

 Smaller the particle size (by micronization) greater is the effective surface area
more intimate contact b/w solid surface and aq solvent higher is the dissolution
rate increase in absorption efficiency
 E.g. poorly aqsoluble nonhydrophobic drugs like Griseofulvin, chloramphenicol
whose dissolution is rate limited.
 Particle size reduction has been used to increase the absorption of a large
number of poorly soluble drugs, such as bishydroxycoumarin, digoxin,
griseofulvin, nitrofurantoin,and tolbutamide.
 Griseofulvin has extremely low aqueous solubility, and material of normal
particle size gave rise to poor and erratic absorption.
 Microsize particles improve absorption, but it is improved even more when
it is formulated in ultramicrosize particles as a monomolecular dispersion in
polyethylene glycol.

3. Polymorphism and amorphism:

When sub exist in different crystalline form i.e. in polymorphic form then diff forms are
many compounds form crystals with different molecular arrangements, or polymorphs.
These polymorphs may have different physical properties, such as dissolution rate and
solubility.
Stable form
 Lowest energy state - Highest [Link].
 Least aq solubility
 Dis
solution
rate limited
Metastable
form
 Less stable form - Highest energy state - Lowest [Link].
 Higher aq solubility
 Better absorption and Bioavailability
 E.g The vitamin riboflavin exists in several polymorphic forms, and these have
a 20-fold range in aqueous solubility.
 Polymorphs that have no crystal structure, or amorphic forms, have different
physical properties from the crystalline forms.
 Absorption of many orally administered drugs is controlled by dissolution rate.
 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. E.g.
novobiocin
Amorphous form
 More soluble
 Rapidly dissolving
 Readily absorbed Crystalline form
 Less soluble
 Slower dissolving
 Not absorbed to significant extent

4. Solvates/hydrates:
 During their preparation, drug crystals may incorporate one or more solvent
molecules to form solvates.
  The most common solvate is water. If water molecules are already present in a
crystal structure, the tendency of the crystal to attract additional water to initiate
the dissolution process is reduced, and solvated (hydrated) crystals tend to
dissolve more slowly than anhydrous forms.
 Significant differences have been reported in the dissolution rate of hydrated and
anhydrous forms of ampicillin, caffeine, theophylline, glutethimide, and
mercaptopurine.
 The clinical significance of these differences has not been examined but is likely to
be slight.
 Solvates have greater solubility than their nonsolvates.e.g. chloroform solvates
of Griseofulvin, n- pentanol solvate of fludrocortisone.

5. 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 imp not the
pH of the bulk of the solution.
 E.g. of 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.

 Reverse in the case of salts of weak bases, it lowers the pH of diffusion layer and
the promoted the absorption of basic drugs.
 Other approach to enhance the dissolution and absorption rate of certain drugs is
by formation of in – situ salt formation i.e. increasing in pH of microenvironment
of drug by incorporating buffer agent.e.g. aspirin, penicillin
 But sometimes more soluble salt form of drug may result in poor absorption.e.g.
 sodium salt of phenobarbitone and phenobarbitone, tablet of salt of
phenobarbitone swelled, it did not get disintegrate thus dissolved slowly and
results in poor absorption.

6. Ionization state:
 Unionized state is imp for passive diffusion through membrane so imp for
absorption.
 Ionized state is imp for solubility.

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

 pH – partition theory states that for drug compounds of molecular weight more
than 100, which are primarily transported across the biomembrane by passive
diffusion, the process of absorption is governed by
 pKa of drug
 The lipid solubility of the unionized drug
 pH at the absorption site.
 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:

Lipophilicity and drug absorption:

 Ideally for optimum absorption, a drug should have sufficient aq solubility to
dissolve in fluids at absorption site and lipid solubility (Ko/w) high enough to
facilitate the partitioning of the rug in the lipoidal biomembrane i.e. drug should
have perfect HLB for optimum Bioavailability.
 And Ko/w = Distribution of drug in organic phase (octanol) / Distribution of drug
in aq phase
 As Ko/w i.e. lipid solubility i.e. partition coefficient increases percentage drug
absorbed increases.
 Pharmaceutical Factors

1. Disintegration
time: -
 Rapid disintegration is important to have a rapid absorption so lower D.T is
required.
 Now D.T of tablet is directly proportional to - amount o f binder
- Compression force.
And one thing should be remembered that in vitro disintegration test gives no means of a
guarantee of drugs B.A. because if the disintegrated drug particles do not dissolve then
absorption is not possible.
2. Manufacturing variables: - a). Method of granulation
 Wet granulation yields a tablet that dissolves faster than those made by other
granulating methods. But wet granulation has several limitations like formation
of crystal bridge or chemical degradation.
 Other superior recent method named APOC (agglomerative phase of
communition) that involves grinding of drug till spontaneous agglomeration and
granules are prepared with higher surface area. So tablet made up of this granules
have higher dissolution rate.
b) Compression force: -
 Higher compression force yields a tablet with greater hardness and reduced
wettability & hence have 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.

3. Nature and type of dosage form –

 Drug formulations are designed to provide an attractive, stable, and convenient method to use
products. Conventional dosage forms may be broadly characterized in order of decreasing
dissolution rate as solutions, solid solutions, suspensions, capsules and tablets, coated capsules and
tablets, and controlled release formulations.
A. Solutions
 Aqueous solutions, syrups, elixirs, and emulsions do not present a dissolution problem and generally
result in fast and often complete absorption as compared to solid dosage forms. Due to their
generally good systemic availability, solutions are frequently used as bioavailability standards
against which other dosage forms are compared.
B. Solid solutions
 The solid solution is a formulation in which drug is trapped as a solid solution or monomolecular
dispersion in a water-soluble matrix. Although the solid solution is an attractive approach to increase
drug absorption, only one drug, griseofulvin, is currently
marketed in this form.
C. Suspensions
 A 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. Liquid dosage forms, therefore, have several practical advantages besides simple
dissolution rate.
 However, they also have some disadvantages, including greater bulk, difficulty in handling, and
perhaps reduced stability.
D. Capsules and tablets
 These formulations differ from each other in that material in capsules is less impacted than in
compressed tablets. Once a capsule dissolves, the contents generally disperse quickly. The capsule
material,
 Although water soluble, can impede drug dissolution by interacting with the drug, but this is
uncommon.
 Tablets generally disintegrate in stages, first into granules and then into primary particles. As particle
size decreases, dissolution rate increases due to of increased surface area.
 Tablet disintegration was once considered a sufficient criterion to predict in vivo absorption.
 As a general rule, the bio-availability of a drug from various dosage forms decrease in the following
order: Solutions > Emulsions > Suspensions > Capsules > Tablets > Coated Tablets > Enteric
coateds Tablets > Sustained Release Products.
4. Pharmaceutical ingredients/Excipients: -
 More the no. of excepients in dosage form, more complex it is & greater the potential for absorption
and Bioavailability problems.
 Changing an excipient from calcium sulfate to lactose and increasing the proportion of magnesium
silicate, increases the activity of oral phenytoin.
 Systemic availability of thiamine and riboflavin is reduced by the presence of Fuller’s earth.
 Absorption of tetracycline from capsules is reduced by calcium phosphate due to complexation.
 Most of these types of interactions were reported some time ago and are unlikely to occur in the
current environment of rigorous testing of new dosage forms and formulations.
a) Vehicle-
 Rate of absorption – depends on its miscibility with biological fluid.
 Miscible vehicles (aq or water miscible vehicle) –rapid absorption e.g. propylene glycol.
 Immiscible vehicles - absorption –depends on its partitioning from oil phase to aq body fluid.
b) Diluents-
 Hydrophilic diluents-form the hydrophilic coat around hydrophobic drug particles –thus promotes
dissolution and absorption of poorly soluble hydrophobic drug.
c) Binders & granulating agent -
 Hydrophilic binders – imparts hydrophilic properties to granule surface – better dissolution of poorly
wettable drug. e.g. starch, gelatin, PVP.
 More amount of binder – increases hardness of tablet – decrease dissolution & disintegration rate.
d) Disintegrants -
 Mostly hydrophilic in nature.
 Decrease in amount of disintegrants – significantly lowers B.A.
e) Lubricants -
 Commonly hydrophobic in nature – therefore inhibits penetration of water into tablet and thus
dissolution and disintegration.
f) Suspending agents/viscosity agent –
 Stabilized the solid drug particles and thus affect drug absorption.
 Macromolecular gum forms unabsorbable complex with drug e.g. Na CMC.
 Viscosity imparters – act as a mechanical barrier to diffusion of drug from its dosage form and retard
GI transit of drug.
g) Surfactants –
 May enhance or retards drug absorption by interacting with drug or membrane or both.
 Surfactants have been considered as absorption enhancers, again mostly in animals. Polyoxyethylene
 ethers have been shown to enhance gastric or rectal absorption of lincomycin,penicillin,
cephalosporins, and fosfomycin in rats and rabbits.
 However, in humans, oral polyoxyethylene-20-oleyl ether resulted in poor and variable insulin
absorption.
 In general, unionic surfactants have little effect on membrane structure but cationic surfactants have
been associated with reversible cell loss and loss of goblet cells.
 Physiologic surfactants – bile salts – promotes absorption – e.g. Griseofulvin, steroids
 It may decrease absorption when it forms the unabsorbable complex with drug above CMC.
h) Bile salts-
 Bile contains conjugates of cholic acid and chenodeoxycholic acid, which emulsify dietary fat,
facilitate lipolysis, and transport lipid molecules through the unstirred layer of the intestinal mucosa
by micellar solubilization. The ability of bile salts to promote lipid absorption has prompted their
investigation as absorption enhancers for drugs, with modest success.
 Absorption of insulin can be increased by bile salts, both in experimental animals and in humans.
i) Colourants
 Even a low concentration of water soluble dye can have an inhibitory effect on dissolution rate of
several crystalline drugs.
 The dye molecules get absorbed onto the crystal faces and inhibit the drug dissolution. For example:
Brilliant blue retards dissolution of sulfathiazole.

5. Product age and storage conditions –

 Product aging and improper storage conditions adversely affect B.A.

PATIENT RELATED FACTORS AFFECTING DRUG ABSORPTION

Gastrointestinal tract
The gastrointestinal tract (GIT) comprises of a number of components, their primary function being
secretion, digestion and absorption. The mean length of the entire GIT is 450 cm. The major functional
components of the GIT are stomach, small intestine (duodenum, jejunum and ileum) and large intestine
(colon) which grossly differ from each other in terms of anatomy, function, secretions and pH.

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

1. Rapid onset of action is desired – Sedatives
2. Dissolution of drug occurs in intestine- Enteric coated tablets
3. Drugs not stable in gastric fluid – Penicillin G & erythromycin
4. Drugs absorbed from distal part of intestine- Vitamin B 12

Delay in gastric emptying is recommended

1. Food promotes drug dissolution & absorption – Griseofulvin
2. Disintegration & dissolution of dosage form is promoted by gastric fluids
3. Drugs dissolve slowly- Griseofulvin
4. Drugs irritate gastric mucosa- Aspirin, phenylbutazone & nitrofurantoin
5. Drugs absorbed from proximal part of small intestine – Vitamin B & C

Factors influencing gastric emptying

1. Volume of meal – Larger bulk longer gastric emptying time
2. Composition of meal – Rate of gastric emptying: Carbohydrates > Proteins > Fats
3. Physical state & viscosity – Liquid meals (hour to empty) > solid meals (6 to 7 hrs)
4. Temperature of meal - high or low temperature of ingested (in comparison with to body temperature)
reduce gastric emptying
5. Gastrointestinal pH- Less acidic pH of stomach promotes gastric emptying while more acidic pH retards
6. Electrolytes – Water isotonic solutions, solutions empty stomach rapidly whereas a higher electrolyte
concentration decreases gastric emptying
7. Body posture –Gastric emptying favoured while standing and lying on right side and vice versa
8. Emotional state – Stress & anxiety promotes while depression retards it
9. Exercise -Vigorous physical activity retards
10. Disease states: Diseases like gastroenteritis, gastric ulcer, pyloric stenosis, diabetes and hypothyroidism
retard gastric emptying. Partial or total gastrectomy, duodenal ulcer and hyperthyroidism promote gastric
emptying rate.
11. Drugs: Drugs that retard gastric emptying include poorly soluble antacids (aluminium hydroxide),
anticholinergics (atropine, propantheline), narcotic analgesics (morphine) and tricyclic antidepressants
(imipramine, amitriptyline). Metoclopramide, domperidone and cisapride (prokinetic agents) stimulate
gastric emptying.

Intestinal transit
 Small intestine is major site for drug absorption: Long intestinal transit time is desired for complete
drug absorption.
 Residence time depends upon intestinal motility or contraction.
 Peristaltic contraction promotes drug absorption by increasing the drug intestinal membrane contact,
by enhancing drug dissolution.
 Delayed intestinal transit is desirable for: Drugs that release slowly (sustained release)
 When the ratio of dose to solubility is high. (chlorthiazide) Drugs that dissolve only in intestine (enteric
coated)
 Drugs which are absorbed from specific site in the intestine (Lithium carbonate, Vitamin B) When drug
penetrate the intestinal mucosa very slowly (e.g. acyclovir)
 When absorption of drug from colon is minimal.

Gastro intestinal PH:

 A difference in PH is observed between gastric and colon fluids. The GI PH increases gradually from
stomach to the colon and rectum.
 The PH of GI fluids influence the drug absorption in several ways:
i) Disintegration: some dosage forms is Ph sensitive , with enteric coating the coat dissolves only in in
intestine.
ii) Dissolution: A large no. of drugs whose solubility is affected by pH are weak acidic and weak basic
drugs.
Weak acidic drugs dissolve rapidly in the alkaline medium whereas
Weak basic drugs dissolve in acidic medium.
iii) Absorption: Depending on drug pKa and whether it is acidic or basic , absorption depends on the
amount of unionized form at site of absorption.
iv) Stability: GI pH affects chemical stability of drug. Eg. Acidic pH of stomach degrades Penicillin G
and erythromycin. Hence they are administered as prodrugs namely carindacillin and erythromycin
estolate.

Blood flow to GIT

GIT is extensively supplied by blood capillary, about 28% of cardiac output is supplied to GIT portion, most
drug reach the systemic circulation via blood only.
Any factor which affects blood flow to GIT may also affect absorption.

Disease state
 Several disease states may influence the rate and extent of drug absorption.
  Three major classes of disease may influence bioavailability of drug.
GI diseases
Achlorhydria : Decreased gastric emptying and absorption of acidic drugs like aspirin
Malabsorption syndrome and celiac disease: decreased absorption
Gastrectomy may cause drug dumping in intestine, osmotic diarrhea and reduce intestinal transit time.
CVS disease
In CVS diseases blood flow to GIT decrease, causes decreased drug absorption.
Hepatic disease
Disorders like hepatic cirrhosis influences bioavailability of drugs which under goes first pass metabolism.
E.g. propranalol

Gastro intestinal contents

1. Food- drug interaction: In general presences of food delay, reduce, increase or may not affect
absorption.
2. Fluid volume: high vol better absorption e.g. erythromycin
3. Interaction of drug with normal GI contents:
Bile salts: increases :lipid soluble drugs e.g. gresiofulvin
Decreased: neomycin, kanamycin
4. Drug-Drug interaction in the GIT:
(A) Physico chemical drug- drug interaction:
Adsorption: Eg; anti diarrheal preparations contains adsorbents like kaolin, prevents a absorption of many
drugs co-administered with them.
Complexation: Eg; calcium, aluminium salts decreases tetracycline
pH changes: Basic drugs changes gastric pH
E.g.; tetracycline with antacids
(B) Physiological interaction:
Decreased GI transit: Anticholinergics like propanthelin decrease GI transit and increased absorption of
ranitidine and digoxin
Increase GI emptying: Metoclopramide increases GI motility and increased GI absorption of tetracycline,
levodopa etc.
Altered GI metabolism: Antibiotics decrease bacterial metabolism of drug e.g. erythromycin increases
efficacy of digoxin.

Presystemic or First pass metabolism

 The loss of drug as it passes through GIT membrane, liver for the first time during the absorption
process.
  The main reason for the decrease in bioavailability of a drug is decreased absorbtion or first pass
metabolism.
Four primary systems which affect pre systemic metabolism of a drugs
1. Luminal enzymes.
2. Gut wall enzymes or mucosal enzymes.
3. Bacterial enzymes.
4. Hepatic enzymes.

Luminal enzymes: These are enzymes present in gut fluids and include enzymes from intestinal and
pancreatic secretions. E.g. hydrolases
Gut wall enzymes: Also called mucosal enzymes they are present in gut and intestine, colon. E.g. alcohol
dehydrogenase
Bacterial enzymes: GI microfloras scantily present in stomach and small intestine and are rich in colon. e.g.
sulphasalazine sulphapyridine + 5 ASA
Hepatic enzyme: several drug undergo firstpass hepatic metabolism, highly extracted ones being
isoprenaline, nitroglycerin, morphine etc.

AGE:
 In children & Infants Gastric pH is high and intestinal surface and flow to git is low.
 while in adults altered gastric emptying, decrease intestinal surface area, decrease gastric blood flow
& higher incidence of achlorhydria cause impaired drug absorption.

ABSORPTION OF DRUG FROM NON-PER ORAL ROUTE

• Buccal/Sublingual Administration
• Rectal Administration
• Topical Administration
• Inhalation Administration
• Intramuscular Administration
• Subcutaneous Administration
• Intranasal Administration
• Intraocular Administration
• Vaginal Administration

Buccal/Sublingual Administration
In buccal route the medicament is placed between the cheek and the gum. In sublingual the drug is placed
under the tongue.
• Barrier to drug absorption from these route is epithelium of oral mucosa.
• Absorption of drug is by passive diffusion. Eg; lozenges, nitrates and nitrites,
Advantages-
Rapid absorption and higher blood levels No first pass metabolism
No degradation of drugs such as that encountered in the GIT Presence of saliva facilitates both drug
dissolution and permeation.
Disadvantages:-
Concern for taste and discomfort
Limited mucosal surface- small doses are administered.

Rectal Administration
• An important route for children and old patients.
• The drug may be administered as solution or suppositories.
• Irritating suppositories bases such as PEG promotes defecation and drug loss, and presence of fecal
matter retards drug absorption.

• By passes the presystemic hepatic metabolism.

• Drug administered by this route includes Aspirin, paracetamol, few barbiturates.
Advantages-
Alternative route for administration of unpleasant drugs Avoids nausea, vomiting
Can be used in case of unconscious patients
Bypasses presystemic hepatic metabolism from lower half of rectum.
Disadvantages-
Absorption is sometimes irregular and incomplete and many drugs cause irritation of rectal mucosa.

Topical Administration
• Skin is the largest organ in the body weighing around 2kg and 2mtsq in area and receives about 1/3rd
of total blood circulating through the body.
• Topical mode of administration is called as percutaneous or transdermal delivery.
• The drug act either locally or systemically.
• Drug that administered precutaneously include lidocaine, testosterone , estradiol, etc.

Transdermal route-
 This route of administration achieves systemic effects by application of drugs to the skin, usually via a
 transdermal medicated adhesive patch.
 The rate of absorption can vary markedly, depending on the physical characteristics of the drug (lipid
soluble) and skin at the site of application.
 This route is most often used for the sustained delivery of drugs, such as the antianginal drug
nitroglycerin, the antiemetic scopolamine.

INJECTIONS
Intravenous (IV) Injection.

Drug is directly goes into blood stream

Intramuscular (IM) Injection.
Absorption of drugs from I.M. sites is relatively rapid but much slower than I.V. injection.
Subcutaneous (SC) Injection.
Absorption is slower than I.M. site due to poor perfusion

Intraperitoneal (IP) Injection.

[Link] is rarely employed in human beings but most widely used in laboratory animals

Inhalations Administration
 All drugs intended for systemic effect can be administered by inhalation since the larger surface area of
alveoli, higher permeability to the alveolar epithelium & rapid absorption just exchange of gases in
blood.
 Route has been limited for drugs such as bronchodilators, anti-inflammatory steroids and
o antiallergics.
 Drug do not under go first pass metabolism.
 lipid soluble drugs absorption rapid by passive diffusion and polar drug by pore transport.
 Generally administered by inhalation either as gases or aerosols

Intranasal administration
 Drug absorption by this route is as rapid as parenteral administered because of its high permeability
and rich vasculature.
 Popular for administration of peptides and protein drugs.
 Route treat local symptoms like nasal congestion, rhinitis.
 Absorption depends upon drug lipophilicity and molecular weight.
 Rapid absorption by diffusion is observed up to 400 -1000 dalt.

Intraocular Administration
 Mainly for the treatment of local effects such as mydriasis, meiosis, anesthesia and glaucoma.
  The barrier in the occular membrane is called cornea which contains both hydrophilic and lipophilic
characters.
 Thus for optimum intra occular permeation drug should posses biphasic solubility.
 pH of formulation influences lacrimal output.
 Rate of blinking.
 Volume of fluid.
 The addition of viscosity increasing agents in the ophthalmic solution will increases occular bio
availability.
 Ex: pilocarpine, timmolol, atropine.

Vaginal Administration
 Available in various forms tablets, creams, ointments, douches and suppositories.
 Used for systemic delivery of contraceptive and other steroids.
 By passes first pass metabolism.
 Factors effecting drug absorption are
 -pH of the lumen fluid 4-5.
 -vaginal secretions.
 -microbes at vaginal lumen.
 Bio availability of vaginal product was about 20% more compared with oral.
 Ex: steroidal drugs and contraceptives.
 DRUG DISTRIBUTION

 Drug distribution: refers to the reversible transfer of a drug between the blood and the extra
vascular fluids and tissues of the body
 Drugs come into the circulation after absorption. From plasma, drugs have to cross the capillary
membrane to come to interstitial space. And then need cross the cell-membrane to enter into the
intracellular fluid.

FACTORS AFFECTING DISTRIBUTION OF DRUGS

 Tissue Permeability of the Drug
a. Physiochemical Properties of the drug like Molecular size, pKa and o/w Partition coefficient.
b. Physiological Barriers to Diffusion of Drugs.
 Organ / Tissue Size and Perfusion Rate
 Binding of Drugs to Tissue Components
binding of drug to blood components
binding of drug to extra cellular components
 Miscellaneous Factors
Age, Pregnancy, Obesity, Diet, Disease states, and Drug Interactions…

 Tissue Permeability of the Drugs depends upon:

1. Rate of Tissue Permeability, and
2. Rate of Blood Perfusion.
The Rate of Tissue Permeability depends upon Physiochemical Properties of the drug as well as
Physiological Barriers that restrict the diffusion of drug into tissues.

A. Physiochemical Properties that influence drug distribution are:

i. Molecular size,
ii. pKa, and
iii. o/w Partition coefficient.

I) Molecular Size:
 Mol wt less than 500 to 600 Dalton easily pass capillary membrane to extra cellular fluid.
  Penetration of drug from ECF to cells is function of Mol size, ionization constant & lipophilicity of
drug.
 From extra cellular fluid to cross cell membrane through aqueous filled channels need particle size
less than 50 Dalton (small) with hydrophilic property.
 Large mol size restricted or require specialized transport system.

ii) Degree of Ionization (pKa):

 The pH at which half of a drug is unionized is called pKa.
 Most of the drugs are weak acids or bases & their degree of ionization depends upon pKa.
 The PH of Blood plasma, extra cellular fluid and CSF is 7.4(constant) except in acidosis and
alkalosis.
 All the drugs ionize at plasma pH (i.e. Polar, Hydrophilic Drugs) Can not penetrate the Lipoidal cell
membrane hence the distribution is limited for these type of drugs.

iii) o/w permeability:

 Polar and hydrophilic drugs are less likely to cross the cell membrane.
 Nonpolar and hydrophobic drugs are more likely to cross the cell membrane.
 Only unionized drugs that are generally lipophillic can cross the cell membrane. Among the drugs
having same o/w partition coefficient but differ in extent of drug Ionization, the drug which is less
ionized is absorbed or have greater permeability than that of more ionized form.
 Ex- Salicylic acid & phenobarbitone have same o/w Partition coefficient but phenobarbitone is more
unionized and hence distributed rapidly.
B. PHYSIOLOGICAL BARRIERS
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

Capillary supply the blood to the most inner tissue
All drugs ionized or unionized molecular size less than 600dalton diffuse through the capillary endothelium
to interstitial fluid
Only drugs that bound to that blood component can’t pass through this barrier Because of larger size of
complex
2. Simple cell membrane barrier
Once the drug diffuse through capillary to extracellular fluid, its further entry in to cells of most tissue is
limited
Simple cell Membrane is similar to the lipoidal barrier (absorption)
Non polar & hydrophillic drugs will passes through it (passively).
Lipophilic drugs with 50-600 dalton mol size & Hydrophilic, Polar drugs with ‹50dalton will pass this
membrane
3. BLOOD-BRAIN BARRIER (BBB):
 Unlike the capillary found in other parts of body, the brain capillaries made of tight junctions of
capillary cells.
 The brain capillaries consist of endothelial cells which are joined to one another by continuous tight
intercellular junctions comprising what is called as the blood-brain barrier.
 As a result the intercellular passage is blocked and for a drug to enter from capillary it has to pass
THROUGH the cells rather BETWEEN them.
 Since BBB is lipoidal barrier, it allows only the drugs having high o/w partition coefficient to diffuse
passively.
 3 different approaches have been utilized successfully to promote crossing the BBB by drugs:
a. use of permeation enhancers (dimethyl sulfoxide)
b. osmotic disruption of the BBB by infusing internal carotid artery with mannitol
c. using dihydropyridine redox system as drug carrier (active transport).

4. Blood-cerebrospinal fluid barrier:

 The cerebrospinal fluid (c.s.f) is formed mainly by the choroid plexus of the LATERAL, THIRD,
AND FOURTH VENTRICLES and is similar in composition to the ECF of brain.
 Here the capillary endothelium that lines the c.s.f has open junctions, and drug can flow freely in to
the extracellular spaces between capillary wall and choroidal cells.
 But the choroidal cells are joined to each other by tight junctions forming the blood-csf barrier which
has permeability characters similar to that of BBB.
 Although the mechanism for diffusion of drugs in to CNS ans CSF is similar, the degree of uptake
may vary significantly.
 5. PLACENTAL BARRIER:
 The maternal and fetal blood vessels are separated by a number of tissue layers made of fetal
trophoblast basement membrane and the endothelium which together constitutes the placental
barrier.
 Many drugs having molecular weight of less than 1000 Daltons and moderate to high lipid solubility
cross the barrier by simple diffusion process.
 This shows that placental barrier is not as effective a barrier as BBB.

6. BLOOD-TESTIS BARRIER:
This barrier is located not at the capillary endothelium level but at sertoli-sertoli junction, it is the tight
junction between the neighboring sertoli cells that act as the blood-testis barrier. This barrier restricts the
passage of drugs to spermatocytes and spermatids.
 ORGAN/TISSUE SIZE AND PERFUSION RATE
Distribution is permeability rate-limited in the following cases:
a. When the drug under consideration is ionic, polar or water-soluble.
b. Where the highly selective physiologic barriers restrict the diffusion of such drugs to the inside of the cell.
In contrast, distribution will be perfusion rate-limited when:
i. The drug is highly lipophilic.
ii. The membrane across which the drug is supposed to diffuse is highly permeable such as those of the
capillaries and the muscles.
Perfusion rate is defined as the volume of blood that flows per unit time per unit volume of the tissue. It is
expressed in ml/min/ml of the tissue.

BINDING OF DRUGS TO TISSUE COMPONENTS

A drug in the body can bind to several components such as the plasma proteins, blood cells and
haemoglobin (i.e. blood components) and extravascular proteins and other tissues.
This topic is dealt comprehensively in Protein Binding of Drugs.

Miscellaneous Factors:
Age: Differences in distribution pattern of a drug in different age groups are mainly due to differences in:
a) Total body water-which is greater in infants.
b) Fat content-is also higher in infants and elderly.
c) Skeletal muscles-are lesser in infants and elderly.
d) Plasma protein content-low albumin content in both infants and elderly.
Diet: A Diet high in fats will increase the free fatty acid levels in circulation thereby affecting binding of
acidic drugs such as NSAIDS to Albumin.
Obesity: In Obese persons, high adipose tissue content can take up a large fraction of lipophilic drugs.
Pregnancy: During pregnancy the growth of the uterus, placenta and fetus increases the volume available
for distribution of drugs.
Disease States: Altered albumin or drug – binding protein conc.

Altered or reduced perfusion to organs /tissues

Altered Tissue pH
Drug Interactions: Drug interactions that affect distribution are mainly due to differences in plasma protein
or tissue binding of drugs.
  Volume of Distribution
Definition: A hypothetical volume of body fluid into which a drug is distributed
 The Volume of distribution (VD), also known as apparent volume of distribution, is used to
quantify the distribution of a drug between plasma and the rest of the body after oral or parenteral
dosing.
 It is called as Apparent Volume because all parts of the body equilibrated with the drug do not have
equal concentration.
 It is defined as the volume in which the amount of drug would be uniformly distributed to produce
the observed blood concentration.
 The apparent volume of distribution is a proportionality constant relating the plasma concentration to
the total amount of drug in the body.
X α C

X = Vd C

Vd = X / C

Apparent volume of distribution = amount of drug in the body/ plasma drug concentration
 It is expressed in liters or liters /kg body weight.
 Apparent volume of distribution is dependent on concentration of drug in plasma.
 Drugs with a large apparent volume are more concentrated in extra vascular tissues and less
concentrated intravascular.
 Applications of volume of distribution
1.
Vd provides the qualitative information
2.
Amount of drug in the body, DB can be determined
In case of target C1 is known, dosage regimen can be fixed for therapy
3.
Loading dose required to achieve steady stage C1 can be estimated
4.
Dose required for individual patients can be calculated
5.
Drug – drug interaction can be explained
6.
Total body clearance can be calculated

PROTEIN BINDING OF DRUGS

 A drug in the body can interact with several tissue components of which the two major categories are
blood and extravascular tissues. The interacting molecules are generally the macromolecules such as
proteins, DNS and adipose tissue.
 The phenomenon of complex formation of drug with protein is called as protein binding of drug.
 As a protein bound drug is neither metabolized nor excreted hence it is pharmacologically inactive
due to its pharmacokinetic and Pharmacodynamic inertness.
 It remains confined to a particular tissue for which it has greater affinity Binding of drugs to proteins
is generally of reversible & irreversible in nature.
Protein + drug ⇌ Protein-drug complex
Protein binding may be divided into:
– 1. Intracellular binding
– 2. Extracellular binding

MECHANISMS OF PROTEIN DRUG BINDING:

 Binding of drugs to proteins is generally of reversible & irreversible.
 Reversible generally involves weak chemical bond such as:
1. Hydrogen bonds
2. Hydrophobic bonds
3. Ionic bonds
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.
 BLOOD
Drug binding to
blood
Blood components Plasma
-D D-
cells proteins
TISSUE Tissue
Liver localisation
P-D D D-Free drug D P-D
Drug + in plasma
metabolite Enzymes
binding to liver D
tissues *
P-D D + Carrier Receptor
P-M Metabolite
s Drug binding
to renal D-C
tissues

Biliary Active
KIDNEY
excretion secretio Pharmacologic
n Response
Excretion
in urine

 Binding of drugs falls into two classes:

1. Binding of drugs to blood components like -
(a) Plasma proteins
(b) Blood cells
2. Binding of drugs to extravascular tissue proteins, fats, bones, etc.

 BINDING OF DRUG TO BLOOD COMPONENTS

A. Plasma protein-drug binding:-
 The binding of drugs to plasma proteins is reversible.
 The extent or order of binding of drug to plasma proteins is:
Albumin › 1-Acid gl ὰ ycoprotein › Lipoproteins › Globulins

1. Binding of drug to human serum Albumin

 It is the most abundant plasma protein (59%), having M.W. of 65,000 with large drug binding
capacity.
 Both endogenous compounds such as fatty acid, bilirubin as well as drug bind to HSA.
 Four diff. sites on HSA for drug binding.
Site I: warfarin & azapropazone binding site.
Site II: diazepam binding site.
Site III: digitoxin binding site.
Site IV: tamoxifen binding site.
2. Binding of drug to α1-Acid glycoprotein: (orosomucoid)
 It has a M.W. 44,000 and plasma conc. range of 0.04 to 0.1 g%. It binds to no. of basic drugs like
imipramine, lidocaine, propranolol, quinidine.

3. Binding of drug to Lipoproteins:

 Lipoproteins are amphiphilic in nature. It contains combination of lipid & apoproteins. The
lipophilic lipid consist of triglycerides & cholesteryl esters and hydrophilic apoprotein consists of
free cholesterol & proteins.
 The M.W. of lipoproteins from 2 lakhs to 34 lakhs depends on their chemical composition. They
are classify on the basis of their density:

e.g. Acidic: Diclofenac. Neutral: Cyclosporin A. Basic: Chlorpromazine.

4. Binding of drug to Globulins:
 It mainly binds to endogenous substances. In plasma several globulins have been identified.

Globulin Synonym Binds to:

1. α Globulin Transcortine /Corticosteroid Binding Steroidal drugs, Thyroxin &

1 globulin Cyanocobalamine.
2. α Globulin Ceruloplasmine Vitamin A,D,E,K.
2
3. β Globulin Transferine Ferrous ions
1
4. β Globulin --- Carotinoids
2

5. γ Globulin --- Antigens

B. BINDING OF DRUG TO BLOOD CELLS

 In blood 40% of blood cells of which major component is RBC (95%). The RBC is 500 times in
diameter as the albumin. The rate & extent of entry into RBC is more for lipophilic drugs.
 The RBC comprises of 3 components.
a) Haemoglobin: It has a M.W. of 64,500 Dal. Drugs like phenytoin, pentobarbital bind to
haemoglobin.
b) Carbonic anhydrase: Carbonic anhydrase inhibitors drugs are bind to it like acetazolamide &
chlorthalidone.
c) Cell membrane: Imipramine & chlorpromazine are reported to bind with the RBC membrane.

 BINDING OF DRUG TO EXTRAVASCULAR TISSUE PROTEIN

 The tissue-drug binding is much more significant because the body tissues comprise 40% of the body
wt which is 100 times that of HAS.
 A tissue can act as the storage site for drugs.
 Importance: 1. It increases apparent volume of distribution of drug.
2. Localization of a drug at a specific site in body.
 Factors that influence localization of drug in tissues are lipophillicity & structural features of the
drug, perfusion rate, pH differences etc.
 The order of binding of drug to extravascular tissue is: Liver › Kidney › Lung › Muscles
Organ Binding of:

[Link] Irreversible binding of Epoxides of Halogenated Hydrocarbon & Paracetamol.

[Link] Basic drugs : Imipramine, Chlorpromazine, & AntiHistaminics.

[Link] Metallothione protein binds to Heavy metals & results in Renal accumulation toxicity.

[Link] Chloroquine & Phenothiazine binds to Melanin.

[Link] Chloroquine & Phenothiazine also binds to Eye Melanin & results in Retinopathy.
[Link] Arsenicals, Chloroquine, & Phenothiazine.
Tetracycline (yellow discoloration of teeth),
[Link]
Lead (replaces Ca & cause brittleness)
Lipophilic drugs (thiopental),
[Link]
Pesticides (DDT)
[Link]
Chloroquine & Quinacrin.
Acid
 Several example of extravascular tissue-drug binding are: Liver, Lungs, Kidneys, skin, eyes, hairs,
etc.
 It also seen in hairs, bones, fats & nucleic acids etc.

Concentration
Protein [Link] Drug that bind
(g%)
Human Serum
65,000 3.5 – 5.0 Large variety of all type of drug
Albumin
α 1-Acid
44,000 0.04-0.1 Basic drug such as impiramine
glycoprotein
200,000 to
Lipoprotein variable Basic lipophilic drug like chlorpromazine
3,400,000

α 1- Globulin 59,000 0.003-0.007 Steroid like corticosterone, and thyroxine

α2 - Globulin 1,34,000 0.015-0.06 Vitamin A, D, E, and cupric ions

Hemoglobin 64,500 11-16 Phenytoin, phenobarbital, phenothiazines

  FACTORS AFFECTING PROTEIN DRUG BINDING
1. Drug - related factors
a) Physicochemical characteristics of the drugs
b) Concentration of drugs in the body
c) Affinity of drug for particular binding components
2. Protein / Tissue related factors
d) Physicochemical characteristics of the protein or binding agents
e) Concentration of protein or binding components
f) Number of binding sites on the binding agents
3. Drug interactions
g) Competition between drugs for the binding site
h) Competition between the drug and normal body constituents
i) Allosteric changes in protein molecule
4. Patient-related factors
j) Age
k) Intersubject variations
l) Disease states

1. Drug-related factors
a. Physicochemical characteristics of the drug:-
 Protein binding is directly related to the lipophillicity of drug. An increase in lipophillicity increases
the extent of binding.
b. Concentration of drug in the body:-
 Alteration in the concentration of drug substance as well as the protein molecules or surfaces
subsequently brings alteration in the protein binding process.
c. Affinity of a drug for a particular binding component:-
 This factor entirely depends upon the degree of attraction or affinity the protein molecule or tissues
have towards drug moieties.
 For Digoxin has more affinity for cardiac muscles proteins as compared to that of proteins of skeletal
muscles or those in the plasma like HSA.
2. Protein/ tissue related factors:
a. Physicochemical characteristics of protein or binding agent:
 Lipoproteins & adipose tissue tend to bind lipophilic drug by dissolving them in their lipid core.
 The physiological pH determines the presence of active anionic & cationic groups on the albumin to
bind a variety of drug.
b. Concentration of protein or binding component:
 Among the plasma protein, binding predominantly occurs with albumin, as it is present in high
concentration in comparison to other plasma protein.
 The amount of several proteins and tissue components available for binding, changes during disease
state.
c. Number of binding sites:
 Albumin has a large number of binding sites as compared to other proteins. Indomethacin binds to 3
sites on albumin.

3. Drug interactions
a. Competition between drugs for the binding sites [Displacement interactions]:-
 A drug-drug interaction for the common binding site is called as displacement interaction. D.I. can
result in unexpected rise in free conc. of the displaced drug which may enhance clinical response or
toxicity. Even a drug metabolite can affect D.I.
 If the drug is easily metabolisable or excretable, it’s displacement results in significant reduction in
elimination half-life.
 e.g. Administration of phenylbutazone to a patient on Warfarin therapy results in Hemorrhagic
reaction.
b. Competition between drug & normal body constituents:-
 The free fatty acids are known to interact with a no. of drugs that binds primarily to HSA. The free
fatty acid level increase in physiological, pathological condition.
c. Allosteric changes in protein molecule:-
 The process involves alteration of the protein structure by the drug or it’s metabolite thereby
modifying its binding capacity.
 e.g. aspirin acetylates lysine fraction of albumin thereby modifying its capacity to bind NSAIDs like
phenylbutazone.
4. Patient-related factors
a. Age:
1. Neonates: Low albumin content: More free drug.
2. Young infants: High dose of Digoxin due to large renal clearance.
3. Elderly: Low albumin: So more free drug.
b. Intersubject variability: Due to genetics & environmental factors.
c. Disease states:-

 SIGNIFICANCE OF PROTEIN/TISSUE BINDING OF DRUG

a. Absorption-
 As we know the conventional dosage form follow first order kinetics.
 So when there is more protein binding then it disturbs the absorption equilibrium.
b. Distribution-
 A protein bound drug in particular does not cross the BBB, the placental barrier, the glomerulus.
 Thus protein binding decreases the distribution of drugs.
c. Metabolism-
 Protein binding decreases the metabolism of drugs & enhances the biological half life.
 Only unbound fractions get metabolized.
 e.g. Phenylbutazone & Sulfonamide.
d. Elimination
 Only the unbound drug is capable of being eliminated.
 Protein binding prevent the entry of drug to the metabolizing organ (liver ) & to glomerulus
filtration.
 e.g. Tetracycline is eliminated mainly by glomerular filtration.
e. Systemic solubility of drug
 Lipoproteins act as vehicle for hydrophobic drugs like steroids, heparin, oil soluble vitamins.
f. Drug action-
 Protein binding inactivates the drugs because sufficient concentration of drug can not be build up in
the receptor site for action.
 e.g. Naphthoquinone
g. Sustain release-
 The complex of drug protein in the blood acts as a reservoir & continuously supplies the free drug.
 e.g. Suramin sodium-protein binding for antitrypanosomal action.
h. Diagnosis-
 The chlorine atom of chloroquine replaced with radiolabeled I- 131 can be used to visualize-
melanomas of eye & disorders of thyroid gland.

 Determination of Protein-drug Binding

1. Indirect technique: Based on separation of bound form.
. Equilibrium dialysis
. Dynamic dialysis
. Ultrafiltration
. Diafiltration
. Gel filtration
. Ultracentrifugation
2. Direct technique: Do not required separation of bound form.
. UV Spectroscopy
. Fluorimetry
. Ion selective electrodes
  Kinetics of protein binding
Assumptions: The drug-protein binding is reversible.

On the protein molecule one binding site is present

Under this condition the protein binding of drug may be described as follows:
Protein (P) + Drug (D) Drug-Protein complex (D-P )
or, P + D PD

From the law of mass

action
[PD] eqn (i)
Ka 
[P] [D]

Where, Ka is the association constant. Drugs strongly bound to protein have a very large Ka.

[ ] this symbol denotes molar concentration

To study the binding behavior of drugs, a ratio ‘r’ is defined as follows:

r  moles of drug bound

total moles of protein

hence, r  [PD]
eqn. (ii)
[PD]  [P]

Substituting, [PD] = Ka [P] [D] from eqn (i) into eqn (ii) we get:

Ka [P] [D]
r   Ka [D]
Ka [P][D]  [P] eq. (iii)
1  Ka [D]

Eqn. (iii) describes the situation where 1 mole of drug binds to one mole of protein in a 1 : 1 complex.

If drug molecules can bind independently to ‘n’ number of identical sites per protein molecule then the
following equation may be used:

nKa[D]
r
1  Ka [D]........................................
(1)

The value of association constant, Ka and the number of binding sites N can be obtained by plotting
equation 1 in four different ways as shown below.
 Direct Plot:
 It is made by plotting r versus [D] as shown in Fig. 1. Note that when all the binding sites are
occupied by the drug, the protein is saturated and plateau is reached. At the plateau, r = N. When r =
N/2, [D] = 1/Ka.

Scatchard Plot:
 It is made by transforming equation 1 into a linear form. Thus,
r + rKa[D] = N

Ka[D] r = N Ka[D] -

rKa[D]

r/[D] = N Ka – rKa......................................(2)

 A plot of r / [D] versus r yields a straight line (Fig. 2). Slope of the line = – Ka,

y intercept = NKa and x-intercept = N.

Klotz Plot/ Lineweaver-Burke Plot (Double Reciprocal Plot):
 The reciprocal of equation 1 yields:
1 / r = 1 / nka [D] + 1/n............................(3)

 A plot of 1/ r versus 1/ [D] yields a straight line with slope 1/ NKa and y-intercept
1/ N (Fig.3).
Hitchcock Plot:
 It is made by rewriting equation 2 as –
NKa [D] / r = 1 + Ka [D]
[D] / r = 1/ NKa + [D] / N....................................(4)

 Equation 4 is Hitchcock equation according to which a plot of [D] / r versus [D]

yields a straight line with slope 1/N and y-intercept 1/ NKa (see Fig. 4).