The document discusses protein-drug binding interactions, detailing the mechanisms, types, and factors influencing binding between drugs and proteins, particularly within blood and extravascular tissues. It explains the significance of protein binding in drug absorption, distribution, elimination, and potential therapeutic applications. Additionally, it highlights various binding components such as plasma proteins and specific interactions that can impact drug efficacy and safety.

1. Protein Binding Interactions
Modern Bio-Analytical Techniques
M. Pharmacy II-Sem
Mehul H Jain
Pharmaceutical Analysis

2. A drug in a body can interact with several tissue components in which two major
categories are-
• Blood
• Extravascular tissue
The interacting molecules are generally the macromolecules such as proteins, DNA
or adipose.
The phenomenon of complex formation with protein is calls as protein drug binding.
It is mainly two types-
• Intracellular binding
where drugs binds to cell protein
• Extracellular binding
where drugs binds to extracellular protein
Introduction

3. 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. Vander 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.

4. Binding of drugs
TO BLOOD
COMPONENTS
Plasma
protein
Blood cell
TO
EXTRAVASCULAR
TISSUE
Liver
Kidney
Bones etc.

5. Binding of drugs to blood components
1. PLASMA PROTEIN-DRUG BINDING
• The main interaction of drug in the blood compartment is with the
plasma protein which are present in abundant amounts and in large
variety.
• The binding of drugs to plasma proteins is reversible.
• The extent or order of binding of drug to plasma proteins is:
Albumin › ὰ1-Acid glycoprotein › Lipoproteins › Globulins.

6. 2. Binding of drug to human serum Albumin (HAS)
• It is the most abundant plasma protein, having a M.W. of 65,000 with a large
binding capacity.
• Four different sites on HSA have been identified for drug-binding.
i. Site 1- Warfarin and azapropazone binding site
ii. Site 2- Diazepam binding site
iii. Site 3- Digitoxin binding site
iv. Site 4- Tamoxifen binding site
3. Binding of drug to ὰ1-acid glycoprotein
• 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.

7. 4. 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 :
1. Chylomicrons
2. Very low density lipoprotein (VLDL)
3. Low density lipoprotein (LDL)
4. High density lipoprotein (HDL)

8. 5. Binding Of Drug To Globulins
• It mainly binds to endogenous substances. In
plasma several globulins have been
identified.
• ἀ1-globulin:- (transcortin) corticosteroid
binding globulin.
• ἀ2-globulin:- (ceruloplasmin) it binds
vita. A, D, E, K & cupric ions.
• β1-globulin:- (transferrin) it binds to
ferrous ions.
• β2- globulin:- Binds to carotenoids
• γ-globulin:- Binds specifically to
antigens.
6. Binding Of Drugs 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. Thus, significant
RBC-drug binding is possible.
• The RBC comprises of 3 components
• Haemoglobin: It has a M.W. of
64,500. Drugs like phenytoin,
pentobarbital bind to haemoglobin.
• Carbonic anhydrase: Carbonic
anhydrase inhibitors drugs are bind to it
like acetazolamide
• Cell membrane: Imipramine &
chlorpromazine are reported to bind
with the RBC membrane.

9. Determination of protein-drug binding
1. Indirect techniques
• It based on separation of bound form from the free micro-molecule.
• Equilibrium Dialysis, Dynamic Dialysis, Ultra filtration, Ultracentrifugation, Gel
filtration are generally applied in biological samples.
2. Direct techniques
• Do not require the separation of bound form from micro-molecules.
• UV-Spectroscopy, Fluorimetry, HPLC are used.

10. Factors affecting protein-drug binding
1. Drug-related factors
• Physicochemical characteristics of drug
Protein binding is directly related to the lipophilicity & stereo selectivity of drug.
• Concentration of drug in the body
The extent of protein-drug binding can change with both changes in drug as well as
protein concentration.
• Affinity of a drug for a particular binding component
Drug having their own higher specific protein binding site.

11. 2. Protein/ tissue related factors
• 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 of drug to bind on the albumin.
• Concentration of protein or binding component
The amount of several proteins and tissue components available for binding,
changes during disease state
• Number of binding sites on the binding agent
Albumin has a large no. of binding sites as compared to other proteins and is a high
capacity binding component. AAG having low conc. & low molecular size therefore
it has limited binding capacity.

12. 3. Drug interactions
a. Competition between drugs for the binding sites [ Displacement interactions]
• When two or more drugs can bind to the same site, competition between them for
interaction with the binding site results.
• Such drug-drug interaction for the common binding site is called as displacement
interaction.
b. Competition between drug & normal body constituents
• The free fatty acids are known to interact with a no. of drugs that binds primarily
to HAS. The free fatty acid level increase in
• Physiological (fasting)
• Pathological (diabetes, myocardial infraction)
• Pharmacologically induced (after heparin or caffeine administration)

13. 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.
• The agent that produce such an effect is called as allosteric effector.
4. Patient-related factors
a. Age
b. Inter-subject variation
• These differences have been attributed to genetic and environmental factors.
c. Disease states
• Almost every serious chronic illness is characterized by decrease albumin content.

14. Significance of protein binding of drugs
1. Absorption
2. Systemic solubility of drugs
3. Distribution
4. Tissue binding, apparent volume of distribution and drug storage
5. Elimination
6. Displacement interaction and toxicity
7. Diagnosis
8. Therapy and drug targeting

15. ABSORPTION
• The absorption equilibrium is attained by transfer of free drug from the site of
administration into the systemic circulation and when the concentration in these two
compartments become equal.
• However, binding of the absorbed drug to plasma proteins decreases free drug
concentration and disturbs such an equilibrium.
• Thus, sink conditions and the concentration gradient are re-established which now act
as the driving force for further absorption.
• This is particularly useful incase of ionized drugs which are transported with difficulty.
SYSTEMIC SOLUBILITY OF DRUGS
• Water insoluble drugs, neutral endogenous macromolecules such as Heparin, Oil
soluble vitamins are circulated and distributed to tissues by binding especially to
lipoproteins which act as a vehicle for such hydrophobic compounds.

16. DIAGNOSIS
• The chlorine atom of chloroquine when replaced with radiolabeled I-131 can be used
to visualize melanomas of the eye since chloroquine has a tendency to interact with
the melanin of eyes.
DISTRIBUTION
• Plasma protein binding restricts the entry of drugs that have specific affinity for
certain tissues .
• This prevents accumulation of large fraction of drugs in such tissues and thus, reduce
toxic reactions.
• Plasma protein binding thus favors uniform distribution of drugs throughout the body
by its buffer function.
• A protein bound drug in particular does not cross the BBB, the placental barrier and
the glomerulus.

17. ELIMINATION
• Only the unbound drug is capable of being eliminated.
• This is because the drug protein complex cannot penetrate into the liver.
• The large molecular size of the complex also prevents it from getting filtered
through the glomerulus.
• Thus, drugs which are more than 95% bound are eliminated slowly i.e. they have
long elimination half lives.
THERAPY AND DRUG TARGETING
• The binding of drugs to lipoproteins can be used for site specific delivery of
hydrophilic moieties.
• This is particularly useful in certain cancer therapy's because certain tumor cells
have greater affinity to LDL than normal cells.
• Thus binding a suitable antineoplastic to it can be used as therapeutic tool.

18. Conclusion
• All pharmacokinetic parameters can be influenced by protein binding.
• Bound drug cannot penetrate through blood capillaries, so that the bound drug
pharmacologically inert.
• Plasma–protein bound drug have longer elimination half lives compare to the free
drug.
• Protein bound drug doesn’t cross BBB and placental barrier.

19. References
1. Brahmankar D.M., Jaiswal S.B., Biopharmaceutics and pharmacokinetics; A
Treatise, 2nd ed., Vallabh Prakashan, p. 116-136.
2. Tipnis H.P., Bajaj A., Principle and application of Biopharmaceutics and
pharmacokinetics, 1st ed., Carrier Publication, p. 73-84.
3. Shargel L., Wa-Pong S., Andrew B.C. Yu., Applied Biopharmaceutics and
pharmacokinetics, 5th ed., Mc Graw Hill company, p. 267-298.
4. Paradkar A., Bakliwal S., Biopharmaceutics and pharmacokinetics, 2nd ed.,
Nirali prakashan, p. 3.12-3.15.