BIOTRANSFORMATION OF XENOBIOTICS

General pharmacology- Biotransformation of xenobiotics

1. BIOTRANSFORMATION of XENOBIOTICS BIOTRANSFORMATION OF XENOBIOTICS DR. JERIN JAMES IInd YEAR MD PHARMACOLOGY SRM MEDICAL COLLEGE ,CHENNAI

2. overview What are xenobiotics What is biotransformation Sites of biotransformation Drug metabolising enzymes Phase I reactions Phase II reactions Factors affecting biotransformation reaction Role of biotransformation in new drug development Conclusion Dr. Jerin James 2

3. xenobiotics Xenobiotics are substances foreign to the body Xenos – foreign , bios – life Can be from: Natural sources –e.g. plant products, alkaloids, poisons Artificially manufactured – e.g.drugs , chemicals , pesticides Dr. Jerin James 3

4. Definition : BIOTRANSFORMATION Enzyme catalysed Biochemical transformation of drug Within living organism Lipid soluble drug/metabolite → water soluble form Excreted through the kidney Mainly Liver Also kidney,intestine,adrenal cortex,lungs,placenta,skin Dr. Jerin James 4

5. BIOTRANSFORMATION Dr. Jerin James 5 Absorption & Distribution Water insoluble xenobiotic Biotransformation Water soluble xenobiotic Excreted by urine or bile

6. SITES OF BIOTRANSFORMATION Dr. Jerin James 6

7. Outcomes of biotransformation 1. Formation of active metabolite from inactive/prodrug., Telampicillin → Ampicillin 2. Formation of inactive drug from pharmacologically active drug., Phenobarbitone → hydroxyphenobarbitone 3. Formation of an active drug from an equally active drug., Diazepam → Oxazepam 4. Formation of toxic metabolites 5. Change in pharmacological action/new action ., Iproniazid → Isoniazid (antidepressant) (antitubercular) Dr. Jerin James 7

8. PHASES of biotransformation reactions Drug oxdn / redn / congugtn prdt hydrolysis Dr. Jerin James 8 PHASE I PHASE II Some drugs directly enter Phase II metabolism Following Phase I the drug may be activated, unchanged or inactivated Conjugated drug is usually inactive DRUG

9. PHASE I reactions Degradative reactions Drug is converted to a smaller polar/non polar metabolite By introduction of a new functional group Mainly microsomal reactions Few are non-microsomal Oxidation, Reduction , Hydrolysis Metabolites formed may be active/inactive Dr. Jerin James 9

10. Phase ii reactions =Synthetic reactions = conjugation reactions Makes molecule more polar Reactions are catalysed by microsomal/ mitochondrial/cytoplasmic enzymes Metabolite formed is polar, water soluble , inactive Dr. Jerin James 10

11. First pass metabolism/ pre-systemic elimination/ first pass effect Metabolism of a drug That occur before drug entering systemic circulation Occur for drugs that are taken orally Significant amount of drug is inactivated before reaching systemic circulation Certain amount of drug is absorbed as it passes through GIT wall and portal circulation ↓d bioavailability of the drug Diminished therapeutic effect Imipramine,morphine,propranolol,buprenorphine,lignocaine,testosterone Bypassed by parenteral administration of drug Dr. Jerin James 11

12. Xenobiotic metabolising enzymes Dr. Jerin James 12 Goodman & Gilmans ,The pharmacological basis of therapeutics .13 th ed. P-87

13. Microsomal enzymes Smooth endoplasmic reticulum of liver mainly & intestinal mucosa, lung and kidney , between the phospholipid bilayer Principal enzymes- Mixed function Oxidases ( MFO) or Cytochrome P-450 Most important pathway of drug metabolism They are a superfamily of enzymes,all of which contain an iron containing protein – Heme Hemoproteins Heme contains one atom of iron in hydrocarbon cage, that functions to bind O2 in the CYP active site Enzyme in reduced f give a product whose absorption peak is at 450 cm-1 e.g. glucoronyl trsnsferase Non specific action Can be induced/activated Can metabolise only lipid soluble drugs Cytochrome P450 = CYP Dr. Jerin James 13

14. Location of cyp Goodman & Gilman, The pharmacological basis of therapeutics, 13 th ed ,page 88 Dr. Jerin James 14

15. Cytochrome p 450 Classified into families designated by no.’s 1,2,3 Subfamilies designated by letters A,B,C,D Amino acid sequence cDNA cloning studies Another number is added to indicate specific isoenzyme eg CYP 2D6 Dr. Jerin James 15

16. Cytochrome p 450 Important CYPs for drug metabolism : CYP 3A CYP 2D CYP 2C Exhibit genetic polymorphism Result in interindividual variation in drug response Dr. Jerin James 16

17. Cyp 3a 4 & cyp 3a5 Substrates Steroids Macrolides CCB Hormones Antihistamines Induced by Barbiturates Carbamazepine Phenytoin Rifampicin Inhibitted by (‘ zole ’ drugs,mycin drugs,CCBs,antihistaminics ) Erythromycin , Claruthromycin Ketoconazole, Fluconazole Verapamil,Diltiazem Ritonavir Grapefruit juice Dr. Jerin James 17

18. Cyp 2d 6 Substrates TCAs Propafenone Sertraline Propranolol Codeine metoprolol Inducers Rifampicin Dexamethasone Inhibitors Quinindine fluoxetine Dr. Jerin James 18

19. Cyp 2c8 ,CYP 2C9 Substrates Phenytoin warfarin Inducers Barbiturates Rifampicin Inhibitors fluconazole Dr. Jerin James 19

20. Cyp 2c 19 Substrate Diazepam Proton pump inhibitors TCA phenytoin Inducers Barbiturates Phenytoin Inhibitors Fluoxamine Ticlopidine fluoxetine Dr. Jerin James 20

21. NON-MICROSOMAL ENZYMES Present in cytoplasm, Mitochondria of hepatocytes & other tissues, Plasma eg. MAO, Esterases, Amidases, Transferases & Conjugages Catalyse Phase II reactions (except glucuronide conjugation), certain oxidations, reductions & hydrolytic reactions Non- inducible Can be inhibited Shows genetic variations eg pseudocholine esterase ,Acetyl transferase Dr. Jerin James 21

22. Phase I reactions/ NON-SYNTHETIC REACTIONS Oxidation, Reduction, Hydrolysis Makes molecule more susceptible to Phase II reactions Involve addition/uncovering of a reactive group This functional group can be acted upon by phase II/ conjugating enzymes Dr. Jerin James 22

23. Phase I reactions/ NON-SYNTHETIC REACTIONS OXIDATIONS MICROSOMAL OXIDATION( CYP Dependent) A romatic hydroxylations eg. Phenobarbitone →para hydroxy phenobarbitone Phenytoin, propranolol,warfarin Aliphatic hydroxylations eg pentobarbitone → hydroxy pentobarbitone Digoxin, ibuprofen Dr. Jerin James 23

24. Phase I reactions/ NON-SYNTHETIC REACTIONS N-O-S- dealkylation N-dealkylation : removal of one alkyl group from amino nitrogen eg. Morphine → normomorphine Mephobarbitone → phenobarbitone O-dealkylation : removal of one alkyl group from eg. phenacetin → paracetamol S-dealkylation : removal of one alkyl group from eg. 6 methyl thiopurine → mercaptopurine N- & S- oxidation : Eg. Chlorpromazine → chlorpromazine sulfoxide Dr. Jerin James 24

25. Phase I reactions/ NON-SYNTHETIC REACTIONS Deamination eg.amphetamine → phenyl acetone derivative Desulfurisation eg.parathion → paraoxon Dr. Jerin James 25

26. Phase I reactions/ NON-SYNTHETIC REACTIONS 2. NON-MICROSOMAL OXIDATION (CYP Independent) Mitochondrial oxidation eg. epinephrine → VMA Cytoplasmic oxidation eg. alcohol → acetaldehyde → acetic acid Plasma oxidative process eg.histamine → imidazole acetic acid Dr. Jerin James 26

27. Phase I reactions/ NON-SYNTHETIC REACTIONS CYP P 450 independent oxidation enzymes: Flavin Mono Oxygenases (FMO) Alcohol Dehydrogenase(ADH) Aldehyde oxidase Xanthine oxidase Peroxidase Prostaglandin synthase myeloperoxidase Dr. Jerin James 27

28. Phase I reactions/ NON-SYNTHETIC REACTIONS REDUCTIONS Microsomal reductions Nitro reduction eg. R-No2 → R-NH2 +FADH (chloramphenicol) Azo reduction eg.R -N═N-R → R-NH2 +R1 NH2+FADH Hepatic azoreductase (e.g. sulfasalazine) Keto reduction R-C=O-R → R-CH-OH-R eg. Cortisone → hydrocortisone Dr. Jerin James 28

29. Phase I reactions/ NON-SYNTHETIC REACTIONS 2. Non microsomal reduction Chloral hydrate → trichlorethanol Dr. Jerin James 29

30. Phase I reactions/ NON-SYNTHETIC REACTIONS HYDROLYSIS Microsomal hydrolysis Pethidine → pethidinic acid Hepatic membrane bound esterase Non microsomal hydrolysis by estrases and amides Procaine → PABA Atropine → Tropic acid Dr. Jerin James 30

31. PHASE II REACTIONS/ SYNTHETIC REACTIONS MICROSOMAL CONJUGATION GLUCORONIDE CONJUGATION Parent drug/Phase I metabolite that contain phenolic, alcoholic, carboxylic, amino/ mercapto groups Undergo conjugation reaction with UDP glucuronic acid Catalysed by UDP glucoronyl transferase enzyme → drug-glucuronide conjugate, polar, readily excreted →inactive products ,( except morphine glucuronide which is active) Drug + UDPGA → Drug-glucuronide + UDP Glucoronyl transferase Eg. Morphine, paracetamol, aspirin Dr. Jerin James 31

32. PHASE II REACTIONS/ SYNTHETIC REACTIONS NON MICROSOMAL CONJUGATION N- Acetylconjugation (cytosol) N-acetyl transferase Acetyl CoA – co factor R-NH2 → R-NH.CO.CH3 N-acetyl transferase Acetyl CoA Isoniazid, PAS , Dapsone, Sulfonamides Dr. Jerin James 32

33. PHASE II REACTIONS/ SYNTHETIC REACTIONS 2 . Sulfate conjugation ( cytosol) Sulfotransferases 3’phospho adenosine 5-Phospho sulfate (PAPS) – cofactor Sulfate conjugates are highly polar →excreted in urine eg. Aspirin, methyl dopa, paracetamol, corticosteroids Dr. Jerin James 33

34. PHASE II REACTIONS/ SYNTHETIC REACTIONS 3 . Amino acid conjugation (Mitochondria) Coupling with glycine/glutamine Glycine transferase Acetyl CoA – cofactor Eg. Aspirin , Benzoic acid, Nicotinic acid Dr. Jerin James 34

35. 4 . Methyl conjugation (cytosol ) Transmethylase Cofactor(methyl donor) - S-adenosine methionine Eg. O-methylation : Dopamine , epinephrine N- methylation : Histamine Dr. Jerin James 35

36. PHASE II REACTIONS/ SYNTHETIC REACTIONS 5 . Glutathione conjugation (cytoplasm/microsomes ) Glutathione –S-transferase enzyme Eg. Epoxides, No2 group containing drugs Dr. Jerin James 36

37. PHASE II REACTIONS/ SYNTHETIC REACTIONS 6. Ribosides & Riboside phosphates Fo rmation of ribonucleosides & ribonucleotides by purines and antimetabolites used in cancer chemotherapy Dr. Jerin James 37

38. Non enzymatic biotransformation (Hoffmann e4limination) Metabolism in the plasma spontaneously Molecular rearrangement Without enzyme action Eg. Atracurium Dr. Jerin James 38

39. Some peculiarities in biotransformation Phase II reaction before phase I Same drug can be metabolised by different drugs simultaneously e.g. Amitryptiline metabolised by CYP 2D6, 2C9 AND 2C19 Same CYP can metabolise different drugs simultaneously e.g. CYP3A4 can bind and metabolize diazepam and testosterone simultaneously Dr. Jerin James 39 ISONIAZID N-ACETYL CONJUGATE (phase II ) HYDROLYSIS (Phase I )

40. Factors affecting biotransformation Physico chemical properties of the drug Molecular size Acidity/basicity Pka Lipophilicity Interaction with drug metabolising enzymes 2. Chemical properties of the drug Enzyme induction Enzyme inhibition Environmental chemicals 3. Biological factors – age ,sex, diet.. Dr. Jerin James 40

41. Factors affecting biotransformation Dr. Jerin James 41 Enzyme induction Xenobiotics induce metabolism of its own (auto metabolism) or other drugs by binding to nuclear receptor and activating expression of target genes by transcription Goodman & Gilman, The pharmacological basis of therapeutics, 13 th ed ,page 88 Goodman & Gilmans , The pharmacological basis of therapeutics, 13 th Ed p-

42. Factors affecting biotransformation Enzyme inducers induce CYPs ,increase metabolism of many other drugs , resulting in therapeutic failure Eg. Barbiturates, Carbamazepine, Glutethimide, Griseofulvin, Phenytoin ,Primidone, Rifabutin, Rifampicin etc Goodman & Gilmans , The Pharmacological basis of therapeutics, 13 th Ed p- Dr. Jerin James 42

43. Factors affecting biotransformation Enzyme inhibition Enzyme inhibitors decrease drug metabolising capacity of CYPs Inhibitors compete for active site of CYPs – drug cannot bind Result in increase in drug level – that lead to drug toxicity The potency of the inhibitor is determined by lipophilicity and strength of bond between inhibitor and active site of CYP Eg. Amiodarone, Clarithromycin, Clotrimazole, Erythromycin, Ketoconazole, Metronidazole, Chloroquine, Ritonavir, Grape fruit juice etc Dr. Jerin James 43

44. Factors affecting biotransformation Genetic variation Drugs can behave diffferntly in different individuals due to genetic variations Eg. People lacking Pseudo-choline esterase due to genetic variation , prolonged apnoea can occur when Succinyl choline is administered Goodman & gilmans . The pharmacoliogicla basis of therapeutics, 13 th Ed Dr. Jerin James 44

45. ROLE OF BIOTRANSFORMATION IN DRUG DEVELOPMENT PROCESS Two key elements of new drug development, Efficacy and Safety are directly related to drug biotransformation. The capacity to metabolise xenobiotics has made development of drugs more time consuming and costly ,partly due to Species difference in expression of enzymes that metabolise drugs & thereby limit the utility of animal models to predict drug effects in humans Interindividual variations in the capacity of humans to metabolise drugs Drug-drug interactions involving xenobiotic metabolising enzymes Metabolic activation of chemicals to toxic and carcinogenic derivatives Dr. Jerin James 45

46. ROLE OF BIOTRANSFORMATION IN DRUG DEVELOPMENT PROCESS COMPUTER BASED (IN SILICO) SYSTEMS COMPACT Camitro META MetabolExpert METEOR IN-VITRO SYSTEMS Human liver S9 fractions Human liver microsomes Huan liver cytosol fractions Hep G2 cell line BC2 cell line Dr. Jerin James 46

47. ROLE OF BIOTRANSFORMATION IN DRUG DEVELOPMENT PROCESS Human liver S9 fractions Most widely used In-Vitro system for metabolic screening in new drug development Contain both Phase I and Phase II metabolic enzymes The microsomes component of the S9 fraction contain cytochrome P450 isoforms (phase I metabolism) and other enzyme activities. The cytosolic portion contains the major part of the activities of transferases(phase II metabolism). Relatively inexpensive Easy to use Can be automated comprehensive and high quality data at reasonable expense for drug discovery programs Dr. Jerin James 47

48. conclusion Xenobiotics are any substances foreign to the body Biotransformation aims to convert water insoluble drugs/substances to water soluble form and is excreted via kidney/bile. In phase I reaction, addition of functional group dramatically changes the biological property of the xenobiotic CYP 450 is the principal enzyme of phase I reactions In phase II reaction, the phase I metabolite is conjugated to increase the water solubility Biotransformation can determine the efficacy and toxicity of a drug by controlling its biological t ½ Dr. Jerin James 48

49. conclusion Different factors that influence xenobiotic metabolism CYP inducers and inhibitors are the most important cause for drug-drug interactions Prediction of metabolism and ADRs by knowledge of biotransformation with modern in vitro and in silico methods are emerging as the most important part in new drug development process Dr. Jerin James 49

50. References K.D. Tripathi, Essentials of medical pharmacology, 7th Ed Goodmann & Gilmann , Pharmacological Basis of Therapeutics,13 th Ed H.P. Rang, M.M.Dale,J.M . Ritter,P.KMoore Pharmacology, 5 th Ed Sharma & Sharma Dr. Jerin James 50

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