BIO- TRANSFORMATION

PHARMACOLOGY BIO TRANSFORMATION

1. BIOTRANSFORMATION DR MANISH MOHAN JR MD PHARMACOLOGY

2. BIOTRANSFORMATION Chemical alteration of the drug in the body. Conversion of nonpolar (lipid-soluble) compounds to polar (lipid- insoluble) 1 º site : liver Other sites- kidney, intestine, lungs, plasma.

3. CONSEQUENCES OF BIOTRANSFORMATION Active drug inactive metabolite E.g.: phenobarbitone, ibuprofen. Active drug active metabolite E.g.: morphine, allopurinol Inactive drug active metabolite E.g.: levodopa, enalapril

4. PHASES OF METABOLISM PHASE 1 (Non-synthetic /Functionalization ) -a functional group is generated or exposed -metabolite may be active or inactive. (b) PHASE 2 (Synthetic/Conjugation Phase ) - an endogenous radical is conjugated to the drug -metabolite is mostly inactive; except few drugs, e.g. glucuronide conjugate of morphine .

5. NONSYNTHETIC REACTIONS OXIDATION REDUCTION HYDROLYSIS CYCLIZATION DECYCLIZATION

6. SYNTHETIC REACTIONS GLUCURONIDE CONJUGATION ACETYLATION METHYLATION SULFATE CONJUGATION GLYCINE CONJUGATION GLUTATHIONE CONJUGATION RIBONUCLEOTIDE/RIBONUCLEOSIDE SYNTHESIS

7. DRUG METABOLISING ENZYMES 1. MICROSOMAL ENZYMES Location: smooth endoplasmic reticulum of liver Other sites: lungs, kidney, intestinal mucosa enzymes- cyp-450 ,UDPGT ,epoxide hydrolases, monooxygenases Catalyse oxidation ,reduction, hydrolysis and glucuronide conjugation 2. NON MICROSOMAL ENZYMES -location: cytoplasm and mitochondria of liver cells -esterase, amidases, flavoprotein oxidase, conjugases - catalyses - oxidation, reduction, hydrolysis, conjugations except glucuronidation

8. Metabolising enzymes PHASE 1 PHASE2 Other enzymes Cytochrome P450s (P450 or CYP) Sulfotransferases (SULT) Alcohol dehydrogenases Flavin-containing monooxygenases (FMOs) UDP-glucuronosyltransferases Aldehyde dehydrogenases Epoxide hydrolases (EHs) Glutathione-S-transferases (GSTs) NADPH-quinone oxidoreductase (NQO) N-Acetyltransferases (NATs) Methyltransferases (MTs)

9. CYTOCHROME P450 ENZYMES MONOXYGENASE ENZYME FAMILY Carryout oxidation in liver ,kidney, GI tract, skin and lungs. Oxidative reactions - CYP haeme protein, the reductase, NADPH, phosphtidyl choline and molecularO 2 . The reductase serves as the electron source for the oxidation

10. CYP450 FAMILY Most of the drug metabolizing enzymes are in CYP 1,2,3 families Families are designated by numbers 1,2,3,4 etc. Subfamilies - A,B,C,D etc. on the basis of amino acid sequence Eg: CYP2D6 family-2 subfamily-D gene number 6.

11. CYP ENZYMES CYP3A4 AND CYP3A5 ( 50% xenobiotic metabolism) Substrates: cyclosporine, dapsone, diazepam Location - liver, intestine, kidney Inducers: barbiturates, carbamazepine ↓(inhibitor) - erythromycin, clarithromycin

12. CYP ENZYMES…. CYP2D6 25-30% drug metabolism Inducer-unknown ↓ - quinidine, fluoxetine CYP2C8 Substrates: all trans retinoic acid Inducers: rifampicin and barbiturates ↓ :montelukast, pioglitazone

13. CYP ENZYMES…… CYP2C9 -substrates: diclofenac, losartan -inducer: carbamazepine, rifampicin - ↓ : fluconazole, fluvoxamine CYP2C19 12-15% drug metabolism Substrates: diazepam, propranolol Inducers: barbiturates ,rifampicin ↓ : fluconazole, ticlopidine

14. CYP ENZYMES…… CYP1A1 AND CYP1A2 substrates: theophylline, warfarin Inducers: rifampicin, smoking. ↓ : fluvoxamine CYP2E1 substrates: alcohol, halothane Inducers: c/c alcoholism ↓ : disulfiram,4-methylpyrazole

15. Flavin-Containing Monooxygenases Superfamily of phase1 enzyme Expressed high in liver 6 families of FMO Most abundant :FMO3 - cimetidine ,ranitidine. Genetic deficiency of FMO3- fish-odor syndrome

16. Hydrolytic Enzymes Hydrolysis of epoxides most of which produced by CYP Epoxides are highly reactive –cell toxicity 2 forms present - sEH(soluble epoxide hydrolases) - mEH (microsomal epoxide hydrolases) EHs participate in the deactivation of potentially toxic metabolites generated by CYPs.

17. PHASE 1 REACTIONS

18. OXIDATION Addition of oxygen/negatively charged radical or Removal of hydrogen /positively charged radical Reactions are mostly carried out by a group of monooxygenases in the liver. Final step : involves cytochrome p450 haemoprotein , NADPH, CYP450 reductase and O 2 .

19. OXIDATION TYPES 1.AROMATIC HYDROXYLATION(CYP dependent) R- R- -OH Eg : phenobarbitone ,phenytoin.

20. CYP dependent oxidations 2.Side chain hydroxylation R-CH 2 CH 3 RCH 2 CH 2 OH E.g : digitoxin phenylbutazone

21. 3. Oxidative dealkylation N-DEALKYLATION RNHCH 3 RNH 2 +HCHO e.g : morphine---normorphine amitriptyline----nortriptyline b. DEALKYLATION AT OXYGEN ATOM R-OCH 3 ROH+HCHO e.g : phenacetin to paracetamol

22. CYP dependent oxidations c. DEALKYLATION AT SULPHUR ATOM RSCH3 RSH+HCHO E.g:6 methyl thiopurine----mercaptopurine 4. DEAMINATION R-CH-NH 2- R RCOR+NH3 E.g : amphetamine-----phenylacetone derivatives

23. CYP dependent oxidations 5.Desulfurisation R1-P S  R1-P O | | R 2 R2 e.g : parathion to paraoxon 6. Oxidation at nitrogen atom RNH2 RNHOH e.g : chlorpheniramine, dapsone

24. CYP dependent oxidations 7.Oxidation at Sulphur atom R1-SH2-R2 R1-S=O | R2 eg : chlorpromazine to chlorpromazine sulfoxide cimetidine to cimetidine sulfoxide

25. CYP independent oxidations 8. Mitochondrial oxidation ( flavin monooxygenase) - require molecular oxygen, NADPH, flavin adenosine dinucleotide[FAD] - e.g : propylthiouracil, methimazole 9.DEHYDROGENATIONS -RCH2OH RCHO - e.g : ethanol metabolism

26. CYP independent oxidations 10.OXIDATIVE DEAMINATION Monoamine oxidase [MAO],diamine oxidase[DAO] RCH2NH2 RCHO+NH3 e.g : Phenylethylamine , epinephrine

27. REDUCTION addition of H2/positively charged radical or removal of O2/negatively charged radical Nitro reduction - RNO2 RNH2 e.g : chloramphenicol ,clonazepam

28. REDUCTION………… 2.AZO REDUCTION -R-N=N-R1 RNH2+R1NH2 -using hepatic AZO reductase - e.g : prontosil----to sulfanilamide. 3.KETOREDUCTION -R-C=O -R 1 R-CHOH-R1 - e.g : cortisone to hydrocortisone.

29. HYDROLYSIS -Drug is split combining with water -ESTER+WATER ALCOHOL+ACID esterase - amidases,peptidases -sites: liver, intestine, plasma -drugs: aspirin, lidocaine.

30. CYCLIZATION formation of ring structure from a straight chain compound E.g : Proguanil DECYCLIZATION opening up of ring structure of the cyclic drug molecule E.g : barbiturates, phenytoin

31. PHASE 2 REACTIONS

32. GLUCURONIDE CONJUGATION -conjugation to alpha –d-glucuronic acid -products are often excreted in the bile - requires enzyme UDP-GLUCURONYL TRANSFERASE - substrates: phenol,alcohol . - e.g : morphine, paracetamol

33. ACETYLATION requires co factor acetyl-CoA responsible enzyme is N-acetyl-transferase important in sulfonamide metabolism substrates: drugs with amino or hydralazine group e.g : INH, hydralazine.

34. SULFATE CONJUGATION -drug groups: alcohol, aromatic amines -catalyzed by cytoplasmic sulfotransferases -the essential co factor enzym-3’phosphoadenosine -5- phosphosulfate . - e.g : methyldopa, chloramphenicol GLYCINE CONJUGATION drug group: acetyl Co A derivative of carboxylic acid catalyzed by Acyl-CoA glycine transferase location: mitochondria E.g : salicylic acid, benzoic acid

35. CONJUGATION REACTIONS- METHYL CONJUGATION drug groups: amines, phenols catalyzing enzymes: trans methylases[cytoplasm] E.g : dopamine, epinephrine, histamine GLUTATHIONE CONJUGATION -drug groups: epoxides, nitro groups. -catalyzed by glutathione-s transferase - e.g : ethacrynic acid.

36. Conjugation reactions Ribonucleoside /ribonucleotide synthesis Activation of purine and pyrimidine antimetabolites E.g : 6-mercaptopurine

37. HOFMANN ELIMINATION Inactivation of the drug in the body fluids by spontaneous molecular rearrangement without the agency of any enzyme. Non enzymatic biotransformation. e.g : Atracurium

38. FIRST PASS METABOLISM -First pass effect/pre-systemic metabolism -Metabolism of a drug during its passage from the site of absorption into the systemic circulation. e.g : isosorbide dinitrate, xylocaine -Can result in bioavailability and therapeutic response Sites Gut wall , gut lumen Liver Lungs skin

39. ATTRIBUTES OF DRUGS WITH FIRST PASS METABOLISM Oral dose is higher than sublingual or parenteral Oral bioavailability is increased in patients with severe liver disease Marked individual variation in oral dose-difference in extent of first pass effect. Oral bioavailability if another drug competing with it in first pass effect if given concurrently e.g : CPZ and propranolol

40. MICROSOMAL ENZYME INDUCTION Certain drugs ,chemicals and carcinogens increase the synthesis of microsomal enzyme protein. E.g : phenobarbitone ,phenytoin induce CYP3A4 isoniazid, and chronic alcohol consumption induce CYP2E1 . Rate of metabolism of inducing drug/other drug increased. Induction takes 4-14 days to reach its peak and is maintained till the inducing agent is present

41. CONSEQUENCES OF INDUCTION 1.Decreased plasma level and decreased therapeutic effect of the co-administered drug 2.Decresed drug effect if the metabolite is inactive and vice versa 3.Tolerance-progressively reduced therapeutic effectiveness due to enhancement of their own metabolism. 4.Toxicity E.g : c/c alcoholics more prone to paracetamol toxicity 5.Therapuetic benefit E.g : treatment of neonatal jaundice

42. Possible uses of enzyme induction 1. Congenital nonhaemolytic jaundice 2. Cushing’s syndrome 3. Chronic poisonings

43. ENZYME INHIBITION One drug can competitively inhibit the metabolism of another drug. - e.g : allopurinol xanthine oxidase( azathioprine,6-mercaptopurine) A drug may inhibit one isoenzyme while being itself a substrate of another isoenzyme E.g : quinidine metabolized by CYP3A4 but inhibits CYP2D6 Inhibition occurs in a dose related manner and can precipitate toxicity of the object drug Blood flow limited metabolism e.g : propranolol reduce rate of lidocaine metabolism

44. SUMMARY Biotransformation- consequences Microsomal enzymes Phase 1 reaction Phase 2 reaction First pass metabolism Enzyme induction Enzyme inhibition

45. THANK YOU