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Biotransformation of Xenobiotics Barbara M. Davit, PhD, DABT Division of Bioequivalence, Office of Generic Drugs, CDER, FDA Introduction to the Theory.

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Presentation on theme: "Biotransformation of Xenobiotics Barbara M. Davit, PhD, DABT Division of Bioequivalence, Office of Generic Drugs, CDER, FDA Introduction to the Theory."— Presentation transcript:

1 Biotransformation of Xenobiotics Barbara M. Davit, PhD, DABT Division of Bioequivalence, Office of Generic Drugs, CDER, FDA Introduction to the Theory and Methods in Toxicology Sept. 17, 2001

2 2 Overview Major Phase I and Phase II enzymes Reaction mechanisms, substrates Enzyme inhibitors and inducers Genetic polymorphism Detoxification Metabolic activation FDA guidances related to biotransformation

3 3 Introduction Purpose –Converts lipophilic to hydrophilic compounds –Facilitates excretion Consequences –Changes in PK characteristics –Detoxification –Metabolic activation

4 4 Comparing Phase I & Phase II

5 5 Biotransformation by liver or gut enzymes before compound reaches systemic circulation Results in lower systemic bioavailbility of parent compound Examples: propafenone, isoniazid, propanolol First Pass Effect

6 6 Phase I: Hydrolysis Carboxyesterases & peptidases –hydrolysis of esters –eg: valacyclovir, midodrine –hydrolysis of peptide bonds –e.g.: insulin (peptide) Epoxide hydrolase –H 2 O added to expoxides –eg: carbamazepine

7 7 Phase I: Reductions Azo reduction –N=N to 2 -NH 2 groups –eg: prontosil to sulfanilamide Nitro reduction –N=O to one -NH 2 group –eg: 2,6-dinitrotoluene activation N-glucuronide conjugate hydrolyzed by gut microflora Hepatotoxic compound reabsorbed

8 8 Carbonyl reduction –Alcohol dehydrogenase (ADH) Chloral hydrate is reduced to trichlorothanol Disulfide reduction –First step in disulfiram metabolism Sulfoxide reduction –NSAID prodrug Sulindac converted to active sulfide moiety Reductions

9 9 Quinone reduction –Cytosolic flavoprotein NAD(P)H quinone oxidoreductase two-electron reduction, no oxidative stress high in tumor cells; activates diaziquone to more potent form –Flavoprotein P450-reductase one-electron reduction, produces superoxide ions metabolic activation of paraquat, doxorubicin Reductions

10 10 Dehalogenation –Reductive (H replaces X) Enhances CCl 4 toxicity by forming free radicals –Oxidative (X and H replaced with =O) Causes halothane hepatitis via reactive acylhalide intermediates –Dehydrodechlorination (2 Xs removed, form C=C) DDT to DDE Reductions

11 11 Alcohol dehydrogenase –Alcohols to aldehydes –Genetic polymorphism; Asians metabolize alcohol rapidly –Inhibited by ranitidine, cimetidine, aspirin Aldehyde dehydrogenase –Aldehydes to carboxylic acids –Inhibited by disulfiram Phase I: Oxidation-Reduction

12 12 Monoamine oxidase –Primaquine, haloperidol, tryptophan are substrates –Activates 1-methyl-4-phenyl-1,2,5,6- tetrahydropyridine (MPTP) to neurotoxic toxic metabolite in nerve tissue, resulting in Parkinsonian-like symptoms Phase I: Monooxygenases

13 13 Peroxidases couple oxidation to reduction of H 2 O 2 & lipid hydroperoxidase –Prostaglandin H synthetase (prostaglandin metabolism) Causes nephrotoxicity by activating aflatoxin B1, acetaminophen to DNA-binding compounds –Lactoperoxidase (mammary gland) –Myleoperoxidase (bone marrow) Causes bone marrow suppression by activating benzene to DNA-reactive compound Monooxygenases

14 14 Flavin-containing mono-oxygenases –Generally results in detoxification –Microsomal enzymes –Substrates: nicotine, cimetidine, chlopromazine, imipramine –Repressed rather than induced by phenobarbital, 3-methylcholanthrene Monooxygenases

15 15 Microsomal enzyme ranking first among Phase I enzymes with respect to catalytic versatility Heme-containing proteins –Complex formed between Fe 2+ and CO absorbs light maximally at 450 ( ) nm Overall reaction proceeds by catalytic cycle: RH+O 2 +H + +NADPH ROH+H 2 O+NADP + Phase I: Cytochrome P450

16 16 Cytochrome P450 catalytic cycle

17 17 Hydroxylation of aliphatic or aromatic carbon –(S)-mephenytoin to 4-hydroxy-(S)- mephenytoin (CYP2C19) –Testosterone to 6 -hydroxytestosterone (CYP3A4) Cytochrome P450 reactions

18 18 Cytochrome P450 reactions Expoxidation of double bonds –Carbamazepine to 10,11-epoxide Heteroatom oxygenation, N-hydroxylation –Amines to hydroxylamines –Omeprazole to sulfone (CYP3A4)

19 19 Heteroatom dealkylation –O-dealkylation (e.g., dextromethorphan to dextrophan by CYP2D6) –N-demethylation of caffeine to: theobromine (CYP2E1) paraxanthine (CYP1A2) theophylline (CYP2E1) Cytochrome P450 reactions

20 20 Cytochrome P450 reactions Oxidative group transfer –N, S, X replaced with O –Parathion to paroxon (S by O) –Activation of halothane to trifluoroacetylchloride (immune hepatitis)

21 21 Cytochrome P450 reactions Cleavage of esters –Cleavage of functional group, with O incorporated into leaving group –Loratadine to Desacetylated loratadine (CYP3A4, 2D6)

22 22 Cytochrome P450 reactions Dehydrogenation –Abstraction of 2 Hs with formation of C=C –Activation of Acetaminophen to hepatotoxic metabolite N-acetylbenzoquinoneimine

23 23 Gene family, subfamily names based on amino acid sequences At least 15 P450 enzymes identified in human liver microsomes Cytochrome P450 expression

24 24 Cytochrome P450 expression Variation in levels, activity due to: –Genetic polymorphism –Environmental factors: inducers, inhibitors, disease –Multiple P450s can catalyze same reaction (lowest K m is predominant) –A single P450 can catalyze multiple pathways

25 25 Major P450 Enzymes in Humans

26 26 Major P450 Enzymes in Humans

27 27 Major P450 Enzymes in Humans

28 28 Major P450 Enzymes in Humans

29 29 Major P450 Enzymes in Humans

30 30 Major P450 Enzymes in Humans

31 31 Major P450 Enzymes in Humans

32 32 Major P450 Enzymes in Humans

33 33 Metabolic activation by P450 Formation of toxic species –Dechlorination of chloroform to phosgene –Dehydrogenation and subsequent epoxidation of urethane (CYP2E1) Formation of pharmacologically active species –Cyclophosphamide to electrophilic aziridinum species (CYP3A4, CYP2B6)

34 34 Drug-drug interactions due to reduced rate of biotransformation Competitive –S and I compete for active site –e.g., rifabutin & ritonavir; dextromethorphan & quinidine Mechanism-based –Irreversible; covalent binding to active site Inhibition of P450

35 35 Induction and P450 Increased rate of biotransformation due to new protein synthesis –Must give inducers for several days for effect Drug-drug interactions –Possible subtherapeutic plasma concentrations –eg, co-administration of rifampin and oral contraceptives is contraindicated Some drugs induce, inhibit same enzyme (isoniazid, ethanol (2E1), ritonavir (3A4)

36 36 Phase II: Glucuronidation Major Phase II pathway in mammals UDP-glucuronyltransferase forms O-, N-, S-, C- glucuronides; six forms in human liver –Cofactor is UDP-glucuronic acid –Inducers: phenobarbital, indoles, 3- methylcholanthrene, cigarette smoking –Substrates include dextrophan, methadone, morphine, p-nitrophenol, valproic acid, NSAIDS, bilirubin, steroid hormones

37 37 Crigler-Nijar syndrome (severe): inactive enzyme; severe hyperbilirubinemia; inducers have no effect Gilberts syndrome (mild): reduced enzyme activity; mild hyperbilirubinemia; phenobarbital increases rate of bilirubin glucuronidation to normal Patients can glucuronidate p-nitrophenol, morphine, chloroamphenicol Glucuronidation & genetic polymorphism

38 38 Glucuronidation & - glucuronidase Conjugates excreted in bile or urine (MW) -glucuronidase from gut microflora cleaves glucuronic acid Aglycone can be reabsorbed & undergo enterohepatic recycling

39 39 Glucuronidation and - glucuronidase Metabolic activation of 2.6-dinitrotoluene) by -glucuronidase – -glucuronidase removes glucuronic acid from N-glucuronide –nitro group reduced by microbial N-reductase –resulting hepatocarcinogen is reabsorbed

40 40 Sulfotransferases are widely-distributed enzymes Cofactor is 3-phosphoadenosine-5- phosphosulfate (PAPS) Produce highly water-soluble sulfate esters, eliminated in urine, bile Xenobiotics & endogenous compounds are sulfated (phenols, catechols, amines, hydroxylamines) Phase II: Sulfation

41 41 Sulfation is a high affinity, low capacity pathway –Glucuronidation is low affinity, high capacity Capacity limited by low PAPS levels –Acetaminophen undergoes both sulfation and glucuronidation –At low doses sulfation predominates –At high doses, glucuronidation predominates Sulfation

42 42 Sulfation Four sulfotransferases in human liver cytosol Aryl sulfatases in gut microflora remove sulfate groups; enterohepatic recycling Usually decreases pharmacologic, toxic activity Activation to carcinogen if conjugate is chemically unstable –Sulfates of hydroxylamines are unstable (2-AAF)

43 43 Common, minor pathway which generally decreases water solubility Methyltransferases –Cofactor: S-adenosylmethionine (SAM) –-CH 3 transfer to O, N, S, C Substrates include phenols, catechols, amines, heavy metals (Hg, As, Se) Phase II: Methylation

44 44 Several types of methyltransferases in human tissues –Phenol O-methyltransferase, Catechol O- methyltransferase, N-methyltransferase, S- methyltransferase Genetic polymorphism in thiopurine metabolism –high activity allele, increased toxicity –low activity allele, decreased efficacy Methylation & genetic polymorphism

45 45 Phase II: Acetylation Major route of biotransformation for aromatic amines, hydrazines Generally decreases water solubility N-acetyltransferase (NAT) –Cofactor is AcetylCoenzyme A Humans express two forms Substrates include sulfanilamide, isoniazid, dapsone

46 46 Rapid and slow acetylators –Various mutations result in decreased enzyme activity or stability –Incidence of slow acetylators 70% in Middle Eastern populations; 50% in Caucasians; 25% in Asians –Drug toxicities in slow acetylators nerve damage from dapsone; bladder cancer in cigarette smokers due to increased levels of hydroxylamines Acetylation & genetic polymorphism

47 47 Phase II:Amino Acid Conjugation Alternative to glucuronidation Two principle pathways –-COOH group of substrate conjugated with - NH 2 of glycine, serine, glutamine, requiring CoA activation e.g: conjugation of benzoic acid with glycine to form hippuric acid –Aromatic -NH 2 or NHOH conjugated with - COOH of serine, proline, requiring ATP activation

48 48 Substrates: bile acids, NSAIDs Species specificity in amino acid acceptors –mammals: glycine (benzoic acid) –birds: ornithine (benzoic acid) –dogs, cats, taurine (bile acids) –nonhuman primates: glutamine Metabolic activation –Serine or proline N-esters of hydroxylamines are unstable & degrade to reactive electrophiles Amino Acid Conjugation

49 49 Enormous array of substrates Glutathione-S-transferase catalyzes conjugation with glutathione Glutathione is tripeptide of glycine, cysteine, glutamic acid –Formed by -glutamylcysteine synthetase, glutathione synthetase –Buthione-S-sulfoxine is inhibitor Phase II:Glutathione Conjugation

50 50 Two types of reactions with glutathione –Displacement of halogen, sulfate, sulfonate, phospho, nitro group –Glutathione added to activated double bond or strained ring system Glutathione substrates –Hydrophobic, containing electrophilic atom –Can react with glutathione nonenzymatically Glutathione Conjugation

51 51 Conjugation of N-acetylbenzoquinoneimine (activated metabolite of acetaminophen) O-demethylation of organophosphates Activation of trinitroglycerin –Products are oxidized glutathione (GSSG), dinitroglycerin, NO (vasodilator) Reduction of hydroperoxides –Prostaglandin metabolism Glutathione Conjugation

52 52 Four classes of soluble glutathione-S- transferase (,,, ) Distinct microsomal and cytosolic glutathione- S-transferases Genetic polymorphism Glutathione Conjugation

53 53 Inducers (include 3-methylcholanthrene, phenobarbital, corticosteroids, anti-oxidants) Overexpression of enzyme leads to resistance (e.g., insects to DDT, corn to atrazine, cancer cells to chemotherapy) Species specificity –Aflatoxin B 1 not carcinogenic in mice which can conjugate with glutathione very rapidly Glutathione-S-transferase

54 54 Excretion of glutathione conjugates –Excreted intact in bile –Converted to mercapturic acids in kidney, excreted in urine Enzymes involved are -glutamyltranspeptidase, aminopeptidase M Activation of xenobiotics following GSH conjugation –Four mechanisms identified Glutathione Conjugation

55 55 FDA-CDER Guidances for Industry Recommendations, not regulations Discuss aspects of drug development Used in context of planning drug development to achieve marketing approval Among guidances are those dealing with in vitro and in vivo drug interaction studies

56 56 In vitro guidance CDER Guidance for Industry: Drug Metabolism/Drug Interaction Studies in the Drug Development Process: Studies in Vitro, April 1997, CLIN 3 Availability: –

57 57 In vitro guidance: assumptions Circulating concentrations of parent drug and/or active metabolites are effectors of drug actions Clearance is principle regulator of drug concentration Large differences in blood levels can occur because of individual differences Assay development critical

58 58 In vitro guidance: techniques/approaches Identify a drugs major metabolic pathways Anticipate drug interactions Recommended methods –Human liver microsomes –rCYP450s expressed in various cell lines –Intact liver systems –Effects of specific inhibitors –Effects of antibodies on metabolism

59 59 Guidance focuses on P450 enzymes Other hepatic enzymes not as well- characterized Gastrointestinal drug metabolism is discussed Metabolism studies in animals (preclinical phase) should be conducted early in drug development In vitro guidance: techniques/approaches

60 60 Correlation between in vitro and in vivo studies Should use in vitro concentrations that approximate in vivo plasma concentrations Should be used in combination with in vivo studies; e.g., a mass balance study may show that metabolism makes small contribution to elimination pathways In vitro guidance: techniques/approaches

61 61 Can rule out a particular pathway If in vitro studies suggest a potential interaction, should consider investigation in vivo ***When a difference arises between in vivo and in vitro findings, in vivo should take precedence*** In vitro guidance: techniques/approaches

62 62 In vitro guidance: timing of studies Early understanding of metabolism can help in designing clinical regimens Best to complete in vitro studies prior to start of Phase III

63 63 In vitro guidance: labeling In vivo findings should take precedence in drug product labeling If it is necessary to include in vitro information, should explicitly state conditions of extrapolation to in vivo Assumption: if a drug is a substrate for a particular enzyme, then certain interactions may be anticipated

64 64 References Casarett and Doulls Toxicology, The Basic Sciences of Poisons, 5th Edition, Klassen, Amdur & Doull (eds), Macmillan Publishing Co. CDER Guidance for Industry: Drug Metabolism/Drug Interaction Studies in the Drug Development Process: Studies in Vitro, April 1997, CLIN 3 Davit B, Reynolds K, Yuan R et al. FDA evaluations using in vitro metabolism to predict and interpret in vivo metabolic drug-drug interactions: impact on labeling. J Clin Pharmacol 1999 Sep;39(9):

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