Drug Distribution

By-
Prathamesh Suhas Patil
M. Pharm (Pharmaceutics)

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Once the drug enters systemic circulation, it is subjected to a number
of processes called drug disposition. Which includes-

1. Distribution- Reversible transfer of drug between compartments.

(blood and extravascular tissue)
2. Elimination- Irreversible loss of drug from the body comprises
metabolism and excretion.

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Like absorption through a passive diffusion mechanism,
distribution is also a passive process for which the driving force
is the concentration gradient between drug and extravascular
tissues.

Significance-
Pharmacological action depends on the concentration of a
drug at the site of action.

So, distribution plays an important role in-

1. Onset of action
2. Intensity of action/Therapeutic response/Toxic reaction Interrelation between different processes
of drug disposition
3. Duration of action

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Steps in Drug Distribution-
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
Schematic of the steps involved in drug distribution
(b) Membrane permeability of the drug.

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 FACTORS AFFECTING DISTRIBUTION OF DRUGS
1. Tissue permeability of drugs:
a. Physicochemical properties of the drug like molecular size, pKa, and o/w partition coefficient
b. Physiological barriers to diffusion of drugs
2. Organ/tissue size and perfusion rate
3. Binding of drugs to tissue components:
a. Binding of drugs to blood components
b. Binding of drugs to extravascular tissue proteins
4. Miscellaneous factors:
a. Age
b. Pregnancy
c. Obesity
d. Diet
e. Disease states
f. Drug interactions.

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1. Tissue permeability of drugs:
a. Physicochemical properties of the drug
- Molecular size, pKa, partition coefficient, and
stereochemical nature
- Almost all drugs having a molecular weight of less than 500 to 600 Daltons easily cross the capillary
membrane to diffuse into the extracellular interstitial fluids
- In the case of ionized molecules, only those below 50 Daltons are passed via pore transport while
others require a specialized transport system
- Drugs remaining unionized at the pH of blood (7.4) and are generally lipophilic can permeate
relatively faster
- E.g., pentobarbital and salicylic acid have almost the same Ko/w but the pentobarbital is more
unionized at blood pH and therefore distributes rapidly
- All drugs that ionize at plasma pH (i.e. polar, hydrophilic drugs), cannot penetrate the lipoidal cell
membrane and tissue permeability is the rate-limiting step in the distribution of such drugs.
- Stereochemical nature of the drug will also influence the distribution characteristics especially when
it has a tendency to interact with macromolecules like proteins.

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b. Physiological Barriers to the Distribution of Drugs

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.

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1. The Simple Capillary Endothelial Barrier
- Molecules above molecular size greater than 600
Dalton alone or in a bound state to blood
components are restricted to cross the capillary
endothelium

2. The Simple Cell Membrane Barrier

- Similar to the lipoidal barrier in the GI
absorption of drugs

Plasma membrane barrier and drug diffusion across it

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3. Blood-Brain Barrier (BBB)
- 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
- Astrocytes & Pericytes form a solid
envelope around the brain capillaries
- As the intercellular passage is blocked, a
drug has to pass through the cells
(transcellular) rather than between them
- Hence, only drugs having higher partition
coefficient values and low molecular
weight can cross the BBB.
- Essential nutrients such as sugars and
amino acids are actively transported.
- E.g., The effective partition coefficient of thiopental, a highly lipid soluble drug is 50 times that of
pentobarbital and crosses the BBB much more rapidly while antibiotics like penicillin are polar,
water-soluble, and ionized at plasma pH, do not cross the BBB.

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Different approaches to cross BBB
1. Use of permeation enhancers- dimethyl sulphoxide (DMSO)
2. Pro-drug approach- Levodopa Dopamine (Parkinsonism)
3. Osmotic disruption of the BBB - infusing the internal carotid artery with mannitol
4. Use of dihydropyridine redox system- as drug carriers to the brain
- Dihydropyridine (lipophilic) forms a complex with the drug which helps to cross 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. Hence, the drug gets trapped in the CNS
E.g., Steroidal drugs

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4. Blood-Cerebrospinal Fluid Barrier

- formed mainly by the choroid plexus of

the lateral, third, and fourth ventricles
- 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
- Highly lipophilic drugs can cross the
Blood CSF barrier more efficiently

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4. Blood Placental Barrier

- Barrier between maternal and foetal blood

vessels
- Both are separated by foetal trophoblast
basement membrane and endothelium
- Drugs having molecular weight less than 1000
Daltons and moderate to high lipid solubility can
cross by simple diffusion readily. This shows that
the placental barrier is not as effective as BBB.
- E.g., sulphonamides, barbiturates, etc.
- Nutrients are transported by carrier-mediated
transport while immunoglobulins by endocytosis.
- It is advised to avoid drugs during pregnancy that causes toxic effects to the foetus ( teratogenicity)
- E.g., phenytoin, methotrexate

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5. Blood Testis Barrier
- Located at the Sertoli-Sertoli cell junction
- Tight junction between neighbouring Sertoli cells that acts as a blood-testis
barrier
- restricts the passage of drugs to spermatocytes and spermatids

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2. Organ/Tissue size and Perfusion rate
- Perfusion rate is defined as the volume of blood that flows per unit time per unit volume of the
tissue.
- Distribution will be permeation rate-limited when-
a. The drug under consideration is ionic, polar, or water-soluble.
b. The highly selective physiologic barriers restrict the diffusion of drugs inside the cell.
- Distribution will be perfusion rate-limited when-
a. The drug is highly lipophilic
b. The membrane across which the drug is supposed to diffuse is highly permeable such as
those of the capillaries and the muscles
- Greater the blood flow, faster is the distribution
- Lungs > Kidneys > Liver > Heart > Brain > Muscles > Fat > Bones … (Order of perfusion rate)
- E.g., Thiopental – has high lipophilicity towards brain and adipose tissue but, it readily diffuses
first to the brain as it is a highly perfused organ.

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3. Binding of drugs to tissue compartments:
- Drug can bind to the components like plasma proteins, blood cells like haemoglobin and
extravascular proteins, and other tissues.

4. Miscellaneous Factors
A] Age
(a) Total body water – greater in infants
(b) Fat content – higher in infants and elderly
(c) Skeletal muscles – lesser in infants and elderly
(d) Organ composition – BBB is poorly developed & with high cerebral blood flow in infants
(e) Plasma protein content – Low albumin content in both infants & elderly
B] Pregnancy – Increase in volume of distribution due to growth of uterus, placenta, and foetus acts
as a separate compartment for drug distribution.
C] Obesity – High adipose tissue, so higher distribution of lipophilic drugs in fat.

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D] Diet – High-fat diet enhances the amount of fatty acids in the blood which increases the
binding of acidic drugs to albumin
E] Disease state – Mechanism of drug distribution altered due to:
(a) Altered albumin and other drug-binding protein concentration
(b) Reduced perfusion to organs or tissues
(c) Altered tissue pH
E.g., In meningitis and encephalitis, the BBB becomes more permeable and thus polar
antibiotics such as penicillin G and ampicillin which do not normally cross it, gain access to the
brain.
F] Drug Interactions – Drug interactions that affect distribution are mainly due to differences in
plasma protein or tissue binding of drugs.

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 VOLUME OF DISTRIBUTION
 The volume of distribution is the hypothetical volume of body fluid into which a drug is
dissolved or distributed.
 There is a constant relation between the concentration of drug in plasma(C), and the amount of
drug in the body (X).
X∝C
X = Vd C …. (Vd= proportionality constant i.e., Volume of Distribution)

 Vd is also called the apparent volume of distribution because all parts of the body equilibrated
with the drug do not have equal concentration.
Vd = X/C or
Apparent volm of distribution = Amount of drug in the body / Plasma drug concentration

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 The apparent volume of distribution bears no direct relationship with the real volume of
distribution.
 The real volume of distribution has direct physiologic meaning and is related to the body
water. The body water is made up of 3 distinct compartments.

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 Drugs that bind selectively to plasma proteins or other blood components, e.g. warfarin (i.e.
those that are less bound to extravascular tissues), have an 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 litres.

 Drugs that bind selectively to extravascular tissues, e.g. chloroquine (i.e. those that are less
bound to blood components), have an apparent volume of distribution larger than their real
volume of distribution. The Vd of such drugs is always greater than 42 litres or TBW volume;
for example, chloroquine has a Vd of approximately 15,000 litres. Such drugs leave the body
slowly and are generally more toxic than drugs that do not distribute deeply into body
tissues.

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 PROTEIN BINDING OF DRUGS
 A drug in the body can interact with several tissue components of which the two major
categories are –
1. Blood, (plasma proteins, blood cells) and
2. Extravascular tissues (tissue proteins, fats, bones, etc.)
 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 drugs
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.
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 Mechanisms of Protein-Drug Binding
 The 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, which is rare,
arises as a result of covalent binding and
is often a reason for the carcinogenicity
Protein-drug binding: Binding of drugs to various tissue components
or tissue toxicity of the drug; for
example, covalent binding of chloroform
and paracetamol metabolites to the liver
Note: results in hepatotoxicity
 Only the unbound drug moves reversibly between the compartments.

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 Binding of drugs to the Blood Components
• Plasma Protein-Dug Binding:
 The binding of drugs to plasma proteins is reversible.
 Albumin > α1-Acid Glycoprotein > Lipoproteins > Globulins

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• Binding of Drugs to Human Serum Albumin
 Human serum albumin (HSA) is the most abundant plasma protein (59% of
total plasma) having a molecular weight of 65000 with a large binding
capacity.
 There are 4 different sites on HAS for drug binding:
Site I: Also called as warfarin binding site, it represents the region to which a
large number of drugs are bound, e.g. several NSAIDs, sulphonamides,
phenytoin, sodium valproate, and bilirubin.
Site II: It is also called as the diazepam binding site. Drugs that bind to this
region include benzodiazepines, medium-chain fatty acids, ibuprofen,
ketoprofen, tryptophan, cloxacillin, probenecid, etc.
Site I and site II are responsible for the binding of most drugs.
Site III: is also called as digitoxin binding site.
Site IV: is also called as tamoxifen binding site.

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• Binding of drugs to α-1 Acid Glycoprotein (AAG):
 Mol. Wt. 44000, plasma concentration range – 0.04-0.1g%
 Binds to basic drugs like imipramine, amitriptyline, nortriptyline, etc.

• Binding of drugs to Lipoproteins:

 Hydrophobic bonding
 Lipoproteins are classified as
1. Chylomicrons (least dense and largest in size).
2. Very low-density lipoproteins (VLDL).
3. Low-density lipoproteins (LDL) (predominant in humans).
4. High-density lipoproteins (HDL) (most dense and smallest in size).
 Binding of drugs to lipoproteins is non-competitive i.e. there are no specific or nonspecific binding
sites and binding is not dependent on drug concentration.
 Basic, lipophilic drugs have relatively more affinity.

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• Binding of Drugs to Globulins:
 Several plasma globulins have been identified and are labelled as α1-, α2-, β1-, β2- and ϒ-
globulins.
1. α1-globulin: also called as transcortin or CBG (corticosteroid-binding globulin), it binds a
number of steroidal drugs such as cortisone and prednisone. It also binds to thyroxine and
cyanocobalamin.
2. α2-globulin: also called as ceruloplasmin, it binds vitamins A, D, E, and K and cupric ions.
3. β1-globulin: also called as transferrin, it binds to ferrous ions.
4. β2-globulin: binds to carotenoids.
5. ϒ-globulin: binds specifically to antigens.

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• Binding of Drugs to Blood Cells:
 More than 40% of the blood comprises of blood cells of which the major cell component is the
RBC. Of these, RBCs constitute 95%.
 RBCs are 500 times larger in diameter than the major plasma protein binding component,
albumin.
 It has 3 components:
1. Haemoglobin – Mol. Wt. 64000
- 6 to 7 times the concentration of albumin in the blood
- E.g., phenytoin, and pentobarbital binds to haemoglobin
2. Carbonic Anhydrase – E.g., Acetazolamide, chlorthalidone
3. Cell Membrane – Imipramine and chlorpromazine binds to the RBC membrane

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 Tissue Binding of Drugs
 The body tissues, apart from HSA, comprise 40% of the body weight which is 100 times that of
HSA.
 Tissue-drug binding results in the localization of a drug at a specific site in the body which
increases the biological half-life of the drug as well as can influence toxicity because-, a
number of drugs or their metabolites bind irreversibly to the tissue.
 E.g., Oxidation products of paracetamol, phenacetin, and chloroform binds covalently to the
hepatic tissue.
 Drugs that bind to both tissue and plasma components result in competition between drug-
binding sites.
 For the majority of drugs that bind to extravascular tissues, the order of binding is:
Liver > Kidney > Lung > Muscles

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 Examples of tissue-drug binding:
1. Liver – Halogenated hydrocarbons & paracetamol (Hepatotoxicity)
2. Lungs – Imipramine, Chlorpromazine, and Antihistamines accumulate in the lungs
3. Kidneys – Metallothionein, 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 by interacting with melanin
5. Eyes – Chloroquine and phenothiazines; retinal pigments also contain melanin
6. Hairs – Arsenicals, chloroquine, and phenothiazines deposit in hair shafts
7. Bones – Tetracycline binds to bones as well as teeth
8. Fats – Lipophilic drugs such as thiopental
9. Nucleic Acids – Chloroquine and quinacrine interact with DNA resulting in distortion of its
double helical structure.

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 FACTORS AFFECTING PROTEIN-DRUG BINDING
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
d. Physicochemical characteristics of the protein or binding agent
e. Concentration of protein or binding component
f. Number of binding sites on the binding agent
3. Drug interactions
g. Competition between drugs for the binding site (displacement
interactions)
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

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1. DRUG-RELATED FACTORS:
(a) Physicochemical Characteristics of the Drug –
 Protein binding is directly related to the lipophilicity of the drug.
 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, unionized drugs bind more to lipoproteins.
 Acidic drugs such as etodolac, flurbiprofen, and ibuprofen and basic drugs such as chloroquine,
disopyramide, and methadone shows stereoselective binding.

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(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 lidocaine can saturate AAG with which it binds as the
concentration of AAG is much less in comparison to that of HSA in blood.

(c) Drug-Protein/Tissue Affinity –

 Lidocaine has a greater affinity for AAG than for HSA.
 Digoxin has more affinity for proteins of cardiac muscles than those of skeletal muscles
or plasma.

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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

 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. ketoprofen
 AAG is a protein with limited binding capacity because of its low concentration and low molecular
size. E.g. Lidocaine

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3. DRUG INTERACTIONS:
(a) Competition Between Drugs for the Binding Sites
 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 the administration of another
drug (drug B) having affinity for the same site results in the displacement of drug A from
its binding site. Such a drug-drug interaction for the common binding site is called as
displacement interaction. Drug A here is called as the displaced drug and drug B as the
displacer.
 E.g., Warfarin and phenylbutazone have the same degree of affinity for HAS.
 Displacement interactions can result in an unexpected rise in the free concentration of the
displaced drug which may enhance clinical response or toxicity

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 For a drug that is 95% bound, a displacement of just 5%
of the bound drug results in a 100% rise in free drug
concentration.
 Clinically significant interactions will result when:
I. The displaced drug (e.g. warfarin) —
1. Is more than 95% bound.
2. Has a small volume of distribution (less than 0.15
L/Kg).
3. Shows a rapid onset of therapeutic or adverse effects.
4. Has a narrow therapeutic index.
II. The displacer drug (e.g. phenylbutazone) —
5. Has a high degree of affinity towards the drug to be
Fate of a drug after displacement interaction
displaced.
6. Competes for the same binding sites.
7. The drug/protein concentration ratio is high (above
0.10).
8. Shows a rapid and large increase in plasma drug
concentration.

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(b) Competition Between Drugs and Normal Body Constituents
 The free fatty acids interact with a number of drugs that bind primarily to HSA. It influences
binding of several benzodiazepines and propranolol (decreased binding) and warfarin (increased
binding).
 Acidic drugs such as sodium salicylate, sodium benzoate, and sulphonamides displace bilirubin
from its albumin-binding site.

(c) Allosteric Changes in Protein Molecules

 Alteration of the protein structure by the drug or its metabolite which modifies its binding
capacity.
 E.g. aspirin acetylates the lysine fraction of albumin thereby modifying its capacity to bind
NSAIDs like phenylbutazone (increased affinity) and flufenamic acid (decreased affinity).

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4. PATIENT-RELATED FACTORS:
(a) Age
 The difference in the protein-drug binding by age is mainly due to the differences in the protein
content in various age groups.
(i) Neonates: Albumin content is low; hence the amount of unbound drug is higher.
(ii) Young infants: Infants suffering from congestive cardiac failure are given a digitalizing dose 4 to
6 times the adult dose on body weight basis. The reason attributed for use of a large digoxin
dose is the 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. Also, an increase in the levels of AAG and thus decreased free
concentration is observed for drugs that bind to it. E.g., Lidocaine

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(b) Intersubject Variations
 The variation observed due to genetic and environmental factors.
 Few drugs showed a small difference. (Not more than 2 fold)

(c) Disease States

Hypoalbuminemia can severely impair protein-drug binding which can be caused by several
conditions like aging, CCF, trauma, burns, inflammatory states, renal and hepatic disorders,
pregnancy, surgery, cancer, etc.
Hyperlipoproteinemia, caused by hypothyroidism, obstructive liver disease, alcoholism, etc., affects
the binding of lipophilic drugs.

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In Summary, all factors, especially drug interactions and patient-related factors that affect
protein or tissue binding of drugs, influence:

1. Pharmacokinetics of drugs: A decrease in plasma protein—drug binding i.e. an increase

in unbound drug concentration, favors tissue redistribution and/or clearance of drugs
from the body (enhanced biotransformation and excretion).
2. Pharmacodynamics of drugs: An increase in the concentration of free or unbound drugs
results in increased intensity of action (therapeutic/toxic).

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 SIGNIFICANCE OF PROTEIN/TISSUE BINDING OF DRUGS
Absorption-
 The binding of the absorbed drug to plasma proteins decreases free drug concentration and
disturbs such equilibrium. Thus, sink conditions and the concentration gradient are re-
established which act as the driving force for further absorption.
Systemic Solubility of 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 act as a vehicle for such hydrophobic compounds.

Distribution
 Plasma protein binding restricts the entry of drugs that have specific affinity for certain
tissues.
 This prevents toxic reactions due to the accumulation of a large fraction of the drug.

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Tissue Binding, Apparent Volume of Distribution and Drug Storage
 A drug that is extensively bound to blood components remains confined to blood. So it has a
small volume of distribution.
 A drug that shows extravascular tissue binding has a large volume of distribution.
 A tissue or blood component that has a great affinity for a particular drug acts as a depot or
storage site for that drug.

Elimination
 Only the unbound or free drug is capable of being eliminated.
 Thus, drugs that are more than 95% bound are eliminated slowly i.e. they have long
elimination half-lives.
 However, penicillins have short elimination half-lives despite being extensively bound to
plasma proteins. This is because rapid equilibration occurs between the free and the bound
drug and the free drug is equally rapidly excreted by active secretion in renal tubules.
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Displacement Interactions and Toxicity
 Displacement interactions are significant in case of drugs which are more than 95% bound.
 A displacement of just 1% of a 99% bound drug results in doubling of the free drug
concentration i.e. a 100% rise.
 E.g,. Displacement interaction between warfarin & phenylbutazone

Diagnosis
 The thyroid gland has great affinity for iodine-containing compounds; hence any disorder of
the same can be detected by tagging such a compound with a radioisotope of iodine.

Therapy and Drug Targeting

 The binding of drugs to lipoproteins can be used for site-specific delivery of hydrophilic
moieties.
 Drug targeting in cancer treatment prevents normal cells from getting destroyed.

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 KINETICS OF PROTEIN-DRUG BINDING
If P represents proteins and D the drug, then applying law of mass action to reversible
protein-drug binding, we can write:
P + D  PD
At equilibrium,

Where,
[P] = concentration of free protein
[D] = concentration of free drug
[PD] = concentration of protein-drug complex
Ka = association rate constant
Kd = dissociation rate constant

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Ka > Kd indicates forward reaction i.e. protein-drug binding is favored. If PT is the total
concentration of protein present, bound and unbound, then:

If r is the number of moles of drug bound to total moles of protein, then,

Substituting the value of [PD] from the previous equation,

Above equation holds when there is only one binding site on the protein and the protein-drug
complex is a 1:1 complex. If more than one or N number of binding sites are available per
mole of the protein then:

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1. Direct Plot is made by plotting r versus [D] as shown
in Fig. Note that when all the binding sites are
occupied by the drug, the protein is saturated and a
plateau is reached. At the plateau, r = N. When r =
N/2, [D] = 1/Ka.
2. Scatchard Plot is made by transforming final
equation into a linear form. A plot of r/[D] versus r
yields a straight line (Fig. 4.4b). Slope of the line = –
Ka, y-intercept = NKa and x-intercept = N
3. Klotz Plot/Lineweaver-Burke Plot (Double Reciprocal
Plot): The reciprocal of final equation yields: A plot of
1/r versus 1/[D] yields a straight line with slope 1/NKa
and y-intercept 1/N

4. Hitchcock Plot is made by rewriting the above

equation as

Plots used for the study of protein-drug binding. (a) Direct plot;
(b) Scatchard plot; (c) Klotz plot; and (d) Hitchcock plot.
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