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

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

www.dvmdocs.webs.com

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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


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Introduction

• Purpose– Converts lipophilic to hydrophilic compounds– Facilitates excretion

• Consequences– Changes in PK characteristics– Detoxification– Metabolic activation


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Comparing Phase I & Phase II

Enzyme Phase I Phase II

Types of reactions HydrolysisOxidationReduction

Conjugations

Increase inhydrophilicity

Small Large

General mechanism Exposes functionalgroup

Polar compound addedto functional group

Consquences May result inmetabolic activation

Facilitates excretion


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• 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


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Phase I: Hydrolysis

• Carboxyesterases & peptidases– hydrolysis of esters– eg: valacyclovir, midodrine – hydrolysis of peptide bonds– e.g.: insulin (peptide)

• Epoxide hydrolase– H2O added to expoxides

– eg: carbamazepine


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Phase I: Reductions

• Azo reduction– N=N to 2 -NH2 groups

– eg: prontosil to sulfanilamide

• Nitro reduction– N=O to one -NH2 group

– eg: 2,6-dinitrotoluene activation• N-glucuronide conjugate hydrolyzed by gut microflora

• Hepatotoxic compound reabsorbed


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• 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


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• 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


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• Dehalogenation– Reductive (H replaces X)

• Enhances CCl4 toxicity by forming free radicals

– Oxidative (X and H replaced with =O)• Causes halothane hepatitis via reactive acylhalide

intermediates

– Dehydrodechlorination (2 X’s removed, form C=C)• DDT to DDE

Reductions


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• 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


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• 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


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• Peroxidases couple oxidation to reduction of H2O2 & 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


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• Flavin-containing mono-oxygenases– Generally results in detoxification– Microsomal enzymes– Substrates: nicotine, cimetidine,

chlopromazine, imipramine– Repressed rather than induced by

phenobarbital, 3-methylcholanthrene

Monooxygenases


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• Microsomal enzyme ranking first among Phase I enzymes with respect to catalytic versatility

• Heme-containing proteins– Complex formed between Fe2+ and CO absorbs light

maximally at 450 (447-452) nm

• Overall reaction proceeds by catalytic cycle:

RH+O2+H++NADPH ROH+H2O+NADP+

Phase I: Cytochrome P450


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Cytochrome P450

catalytic cycle


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• Hydroxylation of aliphatic or aromatic carbon– (S)-mephenytoin to 4’-hydroxy-(S)-

mephenytoin (CYP2C19)– Testosterone to 6-hydroxytestosterone

(CYP3A4)

Cytochrome P450 reactions


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• Expoxidation of double bonds– Carbamazepine to 10,11-epoxide

• Heteroatom oxygenation, N-hydroxylation– Amines to hydroxylamines– Omeprazole to sulfone (CYP3A4)


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• Heteroatom dealkylation– O-dealkylation (e.g., dextromethorphan to

dextrophan by CYP2D6)– N-demethylation of caffeine to:

theobromine (CYP2E1)

paraxanthine (CYP1A2)

theophylline (CYP2E1)



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• Oxidative group transfer– N, S, X replaced with O– Parathion to paroxon (S by O)– Activation of halothane to

trifluoroacetylchloride (immune hepatitis)


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• Cleavage of esters– Cleavage of functional group, with O incorporated

into leaving group– Loratadine to Desacetylated loratadine (CYP3A4,

2D6)


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• Dehydrogenation– Abstraction of 2 H’s with formation of C=C– Activation of Acetaminophen to hepatotoxic

metabolite N-acetylbenzoquinoneimine


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• Gene family, subfamily names based on amino acid sequences

• At least 15 P450 enzymes identified in human liver microsomes

Cytochrome P450 expression


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Cytochrome P450 expression

• Variation in levels, activity due to:– Genetic polymorphism– Environmental factors: inducers, inhibitors,

disease– Multiple P450’s can catalyze same reaction

(lowest Km is predominant)

– A single P450 can catalyze multiple pathways


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Major P450 Enzymes in Humans

CYP1A1/ 2

Expressedin:

Substrates Inducers Inhibitors

LiverLungSkinGIPlacenta

CaffeineTheophylline

Cigarrettesmoke;Cruciferousveggies;Charcoal-broiled meat

Furafylline(mechanism-based); -naphtho-flavone(reversible)


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CYP2B6

Expressedin:


Liver DiazepamPhenanthrene

??? Orphenadrine(mechanism-based)


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CYP2C19

Genetic polymorphism Substrates Inducers Inhibitors

Poor metabolizers have defectiveCYP2C9

PhenytoinPiroxicamTolbutamideWarfarin

Rifampin Sulfafenazole



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CYP2C19


Rapid and slowmetabolizers of S-mephenytoin

N-demethylationpathway of S-mephenytoinmetabolismpredominates in slowmetabolizers

S-mephenytoin(4’-hydroxylationis catalyzed byCYP2C19)

Rifampin Tranylcypromine



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CYP2D6


Poor metabolizers lackCYP2D6

Debrisoquine causes marked,prolonged hypotension inslow metabolizers

No effect on response topropanolol in poormetabolizers; alternatepathway (CYP2C19) willpredominate

5-10% of Caucasians arepoor metabolizers

< 2% of Asians, AfricanAmericans are poormetabolizers

PropafenoneDesipraminePropanololCodeineDextromethorphanFluoxetineClozapineCaptopril

Poor metabolizersidentified byurinary exrection ofDextrorphan

None known FluoxetineQuinidine



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CYP2E1

Expressed in: Substrates Inducers Inhibitors

LiverLungKidneyLympocytes

EthanolAcetaminophenDapsoneCaffeineTheophyllineBenzene

EthanolIsoniazid

Disulfiram



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CYP3A4

Expressedin:


Liver;Kidney;Intestine;MostabundantP450enzyme inliver

AcetaminophenCarbamazepineCyclosporineDapsoneDigitoxinDiltiazemDiazepamErythromycinEtoposideLidocaineLoratadineMidazolamLovasatinNifedipineRapamycinTaxolVerapamil

RifampinCarbamazepinePhenobarbitalPhenytoin

Ketoconazole;Ritonavir;Grapefruit juice;Troleandomycin



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CYP4A9/ 11

Expressedin:


Liver Fatty acids andderivaties;Catalzyes - and 1-hyroxylation

??? ???



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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)


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• 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


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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)


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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


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• Crigler-Nijar syndrome (severe): inactive enzyme; severe hyperbilirubinemia; inducers have no effect

• Gilbert’s 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


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Glucuronidation & -glucuronidase

• Conjugates excreted in bile or urine (MW) -glucuronidase from gut microflora cleaves

glucuronic acid

• Aglycone can be reabsorbed & undergo enterohepatic recycling


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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


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• 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


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• 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


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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)


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• Common, minor pathway which generally decreases water solubility

• Methyltransferases– Cofactor: S-adenosylmethionine (SAM)

– -CH3 transfer to O, N, S, C

• Substrates include phenols, catechols, amines, heavy metals (Hg, As, Se)

Phase II: Methylation


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• 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


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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


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• 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


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Phase II:Amino Acid Conjugation

• Alternative to glucuronidation

• Two principle pathways– -COOH group of substrate conjugated with -NH2

of glycine, serine, glutamine, requiring CoA activation

• e.g: conjugation of benzoic acid with glycine to form hippuric acid

– Aromatic -NH2 or NHOH conjugated with -COOH of serine, proline, requiring ATP activation


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• 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


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• 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


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• 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


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• 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



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• Four classes of soluble glutathione-S-transferase ( , , , )

• Distinct microsomal and cytosolic glutathione-S-transferases

• Genetic polymorphism



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• 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 B1 not carcinogenic in mice which

can conjugate with glutathione very rapidly

Glutathione-S-transferase


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• 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



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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


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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:– www.fda.gov/cder/guidance/index.htm


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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


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In vitro guidance: techniques/approaches

• Identify a drug’s 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


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• 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



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• 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



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• 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***



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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


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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


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References

• Casarett and Doull’s 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):899-910

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

Documents

cytochrome p450 slide

biotransformation slide

carbamazepine slide

2d6 slide

oxidationreduction slide

sulfone cyp3a4 slide

reduction of h

pass effect slide