Protein Binding and Clinical Application

these slides cover the Protein Binding, Mechanisms of P-D Binding, Plasma P-D Binding, Extravascular Tissue P-D Binding, Apparent volume of Distribution, Kinetics of P-D Binding and Clinical Significance of Protein-Drug (P-D) Binding

1. Protein Binding Dr. Rameshwar Dass Guru Gobind Singh College of Pharmacy, Yamunanagar, Haryana

2. Contents Introduction Mechanisms of protein drug binding Classes of protein drug binding. Binding of drug to blood components. Plasma proteins Blood cells Binding of drug to extravascular tissue protein Factors affecting protein drug binding Significance of protein/tissue binding of drug

3. Introduction

4. Mechanisms of P-D Binding

5. Reversible P-D Binding

6. P-D Binding To Blood compone n ts Plasma Proteins Blood cells To Extra va s c u lar Tissues Proteins Fats Bones

7. Plasma P-D Binding

8. HSA P-D Binding 59% M.W. of 65,000 with large drug binding capacity

9. α1-Acid glycoprotein P-D Binding M.W. 44,000  Plasma  conc. 0.04 to 0.1 g%.  It  binds to no. of basic drugs like imipramine, lidocaine, propranolol,  quinidine

10. Lipoproteins P-D Binding : Binding by: Hydrophobic Bonds, Non- competative . M W. 2-34 Lacks Dalton. Lipid core composed of: Inside: triglyceride & cholesteryl esters. Outside: Apoprotein. Example: Acidic: Diclofenac. Neutral: Cyclosporin A. Basic: Chlorpromazine. L D L H D L V L D L Chylomicrons T y p e s

11. Globulin P-D Binding Globulin Synonym Binds to 1. α1 Globulin Transcortine / C o r t i c o s t e roid globulin Binding Steroidal drugs, Thyroxin & Cyanocobalamine. 2. α2 Globulin Ceruloplasmine Vitamin A,D,E,K. 3. β1 Globulin Transferin Ferrous ions 4. β2 Globulin --- Carotinoids 5. γ Globulin --- Antigens

12. Blood Cells -D Binding In blood 40% of blood cells Major component is RBC (95%). The RBC - 500 times in diameter as the albumin. RBC binding more for lipophilic drugs. The RBC comprises of 3 components. Hemoglobin: M .W. 64,500 Dal. Drugs binds phenytoin, pentobarbital. Carbonic anhydrase: Carbonic anhydrase inhibitors drugs are bind to it e.g. acetazolamide & chlorthalidone . Cell membrane: Imipramine & chlorpromazine bind with the RBC membrane.

13. Extravascular Tissue P-D Binding Binding order: Liver › Kidney › Lung › Muscles Tissue Drug examples L i ver Irreversible binding of Epoxides of Halogenated Hydrocarbon & Paracetamol L ungs Basic drugs: Imipramine, Chlorpromazine, & Anti- histaminics Kidney Metallothionine protein binds to Heavy metals & results in Renal accumulation and toxicity. Skin Chloroquine & Phenothiazine binds to Melanin Eye Chloroquine & Phenothiazine also binds to Eye Melanin & results in Retinopathy. Hairs Arsenicals, Chloroquine, & Phenothiazine. Bones Tetracycline(yellow discoloration of teeth), Lead(replaces Ca & cause brittleness) Fats Lipophilic drugs (thiopental), Pesticides (DDT) Nucleic Acid Chloroquine & Quinacrine.

14. Factors Affecting P-D Binding Drug related Factors and Effect Physicochemical characteristics Directly related to the lipophilicity of drug and increase in lipophilicity increases the extent of binding Directly related to the lipophilicity of drug and increase in lipophilicity increases the extent of binding Concentration The conc. of drug substance as well as the protein molecules or surfaces subsequently brings alteration in the P-binding process. The conc. of drug substance as well as the protein molecules or surfaces subsequently brings alteration in the P-binding process. Drug Affinity to protein The degree of attraction or affinity of the protein molecule or tissues have towards drug moieties. Digoxin has more affinity for cardiac muscles proteins than other protein. The degree of attraction or affinity of the protein molecule or tissues have towards drug moieties. Digoxin has more affinity for cardiac muscles proteins than other protein. Protein/ tissue related factors and Effect Physicochemical characteristics Lipoproteins & adipose tissue tend to bind lipophilic drug by dissolving them in their lipid core. pH determines the presence of active anionic & cationic groups on the albumin to bind a variety of drug. Concentration Binding predominantly depends on conc. Like albumin. Proteins and tissue components conc. May be changes during disease state

15. Factors Affecting P-D Binding Drug interactions and Effect Allosteric changes in P Directly related to the lipophilicity of drug and increase in lipophilicity increases the extent of binding Alteration of the protein structure by the drug or it’s metabolite changes its binding capacity Aspirin acetylates lysine fraction of albumin thereby modifying its capacity to bind NSAIDs like phenylbutazone Drug-drug competition for binding sites Administration of phenylbutazone to a patient on Warfarin therapy results in Haemorrhagic reaction Competition b/w D&F The free fatty acids are known to interact with a no. of drugs that binds primarily to HSA Patient-related factors Age Neonates: Low albumin content as compare to adults: More free drug Inter-subjects variability Due to genetics & environmental factors Disease states Renal and hepatic failure Decrease albumin conc. And less binding of acidic drugs; neutral and basic drugs are un affected Inflammatory states i.e , truama Increase AAG levels and inc. binding of basic drugs; neutral and acidic drugs are un affected

16. Apparent volume of Distribution Different tissue have diff. amount of drug and Vd cannot have a true physiologic meaning. But it depends on physiologic mechanism (primary parameter) The relationship between tissue-drug binding and volume of distribution (Amount of drug in body) α ( Conc. of drug in plasma) X α C X = V d .C V d =X/C Definition : Hypothetical Vol. of body fluid into which drug is dissolved or distributed. It is Apparent V d : Because all parts of body equilibrated with drug do not have equal amount. Extensively blood component bind drugs have low vol. of distribution Extra-vascular binding of drug show large vol. of distribution. Tissue binding of drug acts as depot/storage site.

17. Apparent volume of Distribution It depends on physiologic mechanism (primary parameter) V d =X/C Example: Vd 2 L/kg for lumefantrine Body fluid Volume (lit.) % of Body wt. % of TWB 1.Vascular fluid 6 9 15 (plasma) (3) (4.5) (7.5) 2.Extracellular fluid (excl uding plasma) 12 14 28 3.Intracellular fluid (excl uding blood cells) 24 34 57 Total Body 42 60 100

18. Kinetics of P-D Binding The bound drug is neither metabolized nor excreted due to its pharmacokinetic and pharmacodynamic inertness. Protein + Drug = Protein-Drug complex Note: applying law of mass action. P+D PD At equilibrium, Where, [P]=concentration of free protein [D]=concentration of free drug [PD]=concentration of protein-drug complex K a = association rate constant and K d =dissociation constant      

19. Kinetics of P-D Binding K a > K d indicates forward reaction i.e. protein-drug binding is favored. If P t is the total concentration of protein present , bound and unbound, then : P t =[PD]+[P]………….(2) If r is the number of moles of drug bound to total moles of protein, then, ……….( 3) Substituting the value of [PD] from equation (1) in equation (3) r = = ………(4) Equation (4) holds when there is only one binding site on the protein drug complex is 1:1 complex  

20. Direct Plot Kinetics It is made by plotting r vs [D] . Note that all binding sites are occupied by the drug. The protein is saturated and plateau is reached r = N where r = N/2 , [D]=1/k a

21. Scat-chard Plot It is made by transforming equation (5) into linear form r= r+r k a [D] r=N k a [D]-r k a [D] therefore, =N k a -r k a A plot of r/[D] vs r yields a straight line slope line=- k a y-intercept=N k a and x- intercept=N

22. Klotz Plot/Lineweaver- Burke Plot The reciprocal of equation = N A plot of 1/r vs 1/[D] yields a straight line S lope = - 1/N k a and y-intercept 1/N

23. Hitchcock Plot It is made by re-writing equation (5) as =1+ k a [D] Dividing both sides with N k a gives =

24. Clinical Significance P-D Binding Absorption The conventional dosage form follow first order kinetics . So when there is more protein binding then it disturbs the absorption equilibrium and 9ncrease the absorption. Distribution A protein bound drug in particular does not cross the BBB, the placental barrier, the glomerulus. It decreases the distribution of drugs Metabolism Protein binding decreases the metabolism of drugs & enhances the biological half life. Example: Phenylbutazone & Sulfonamide Elimination Only the unbound drug is capable of being eliminated. Pr e v en t th e dru g t o m e t a bol i ze (liver ) & glomerulus filtration ( Tetracycline ) The patients with acute hepatitis, protein binding of the glucocorticoids will be reduced and peak concentrations of administered glucocorticoids increased

25. Clinical Significance P-D Binding Drug action Protein binding inactivates the drugs because P-D not bind to the receptor site for action ( Naphthoquinone ) Aspirin reduces ketoprofen protein binding and increases its clearance Sustain release The complex of drug protein in the blood act as a reservoir & continuously supply the free drug. Diagnosis The Cl atom of chloroquine replaced with radiolabelled I 131 can be used to visualize-melanomas of eye & disorders of thyroid gland Systemic solubility of drug Lipoprotein act as vehicle for hydrophobic drugs like steroids, heparin, oil soluble vitamins