Protein binding interactions

introduction
mechanisms of protein drug binding
binding of drugs
binding of drugs to blood components
determination of protein drug binding
factors affecting
significance

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: Hydrogen bonds Hydrophobic bonds Ionic bonds 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

5. Binding of drugs to blood components 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 › ὰ 1 - Acid glycoprotein › Lipoproteins › Globulins.

6. 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. Site 1- Warfarin and azapropazone binding site Site 2- Diazepam binding site Site 3- Digitoxin binding site Site 4- Tamoxifen binding site Binding of drug to ὰ 1 - 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. 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 : Chylomicrons Very low density lipoprotein (VLDL) Low density lipoprotein (LDL) High density lipoprotein (HDL)

8. Binding Of Drug To Globulins It mainly binds to endogenous substances. In plasma several globulins have been identified. ἀ 1 - globulin:- ( transcortin ) corticosteroid binding globulin. ἀ 2 -globulin:- (ceruloplasmin) it binds vita. A, D, E, K & cupric ions. β 1 -globulin:- (transferrin) it binds to ferrous ions. β 2 - globulin:- Binds to carotenoids γ-globulin:- Binds specifically to antigens. 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 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. 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 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. 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. Drug interactions 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. 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. 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. Patient-related factors Age Inter-subject variation These differences have been attributed to genetic and environmental factors. Disease states Almost every serious chronic illness is characterized by decrease albumin content.

14. Significance of protein binding of drugs Absorption Systemic solubility of drugs Distribution Tissue binding, apparent volume of distribution and drug storage Elimination Displacement interaction and toxicity Diagnosis 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 Brahmankar D.M., Jaiswal S.B., Biopharmaceutics and pharmacokinetics; A Treatise, 2nd ed., Vallabh Prakashan , p. 116-136. Tipnis H.P., Bajaj A., Principle and application of Biopharmaceutics and pharmacokinetics, 1st ed., Carrier Publication, p. 73-84. Shargel L., Wa -Pong S., Andrew B.C. Yu., Applied Biopharmaceutics and pharmacokinetics, 5th ed., Mc Graw Hill company, p. 267-298. Paradkar A., Bakliwal S., Biopharmaceutics and pharmacokinetics , 2nd ed., Nirali prakashan, p. 3.12-3.15.

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