Biotransformation of Xenobiotics

BIOTRANSFORMATION OF XENOBIOTICS

Overview Phase I and Phase II enzymes Reaction mechanisms, substrates Enzyme inhibitors and inducers Genetic polymorphism Detoxification Metabolic activation

Introduction

Purpose Converts lipophilic to hydrophilic

compounds Facilitates excretion

Consequences Changes in PK characteristics Detoxification Metabolic activation

Comparing Phase I & Phase II

Enzyme Phase I Phase II

Types of reactions Hydrolysis Oxidation Reduction

Conjugations

Increase in hydrophilicity

Small Large

General mechanism

Exposes functional group

Polar compound added to functional group

Consquences May result in metabolic activation

Facilitates excretion

First Pass Effect

Biotransformation by liver or gut enzymes before compound reaches systemic circulation

Results in lower systemic bioavailbility of parent compound

Examples: Propafenone, Isoniazid, Propanolol

Phase I reactions

Hydrolysis in plasma by esterases (suxamethonium by cholinesterase)

Alcohol and aldehyde dehydrogenase in liver cytosol (ethanol)

Monoamine oxidase in mitochondria (tyramine, noradrenaline, dopamine, amines)

Xanthine oxidase (6-mercaptopurine, uric acid production)

Enzymes for particular substrates (tyrosine hydroxylase, dopa-decarboxylase etc.)

Phase I: Hydrolysis

Carboxyesterases & peptidasesHydrolysis of esters

eg: valacyclovir, midodrine Hydrolysis of peptide bonds

e.g.: insulin (peptide)

Epoxide hydrolaseH2O added to epoxides

eg: carbamazepine

Phase I: Reductions

Azo ReductionN=N to 2 -NH2 groups

eg: prontosil to sulfanilamide

Nitro ReductionN=O to one -NH2 group

eg: 2,6-dinitrotoluene activationN-glucuronide conjugate hydrolyzed by gut

microfloraHepatotoxic compound reabsorbed

Reductions

Carbonyl reductionChloral hydrate is reduced to trichlorothanol

Disulfide reductionFirst step in disulfiram metabolism

Reductions

Quinone reductionCytosolic flavoprotein NAD(P)H quinone

oxidoreductasetwo-electron reduction, no oxidative stresshigh in tumor cells; activates diaziquone to

more potent form

Flavoprotein P450-reductaseone-electron reduction, produces

superoxide ionsmetabolic activation of paraquat,

doxorubicin

Reductions

DehalogenationReductive (H replaces X)

Enhances CCl4 toxicity by forming free radicals

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

acylhalide intermediatesDehydrodechlorination (2 X’s removed, form

C=C)DDT to DDE

Phase I: Oxidation-ReductionAlcohol dehydrogenase

Alcohols to aldehydesGenetic polymorphism; Asians metabolize

alcohol rapidlyInhibited by ranitidine, cimetidine, aspirin

Aldehyde dehydrogenaseAldehydes to carboxylic acidsInhibited by disulfiram

Phase I: Monooxygenases

Monoamine OxidasePrimaquine, haloperidol, tryptophan are

substratesActivates 1-methyl-4-phenyl-1,2,5,6-

tetrahydropyridine (MPTP) to neurotoxic toxic metabolite in nerve tissue, resulting in Parkinsonian-like symptoms

MonoOxygenases

Peroxidases couple oxidation to reduction of H2O2 & lipid hydroperoxidaseProstaglandin 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

Flavin-containing Mono-oxygenasesGenerally results in detoxificationMicrosomal enzymesSubstrates: Nicotine, Cimetidine,

Chlopromazine, Imipramine

Phase I: Cytochrome P450

Microsomal enzyme ranking first among Phase I enzymes

Heme-containing proteinsComplex formed between Fe2+ and CO

absorbs light maximally at 450 (447-452) nm

Cytochrome P450 reactions

Hydroxylation

Testosterone to 6-hydroxytestosterone (CYP3A4)


EPOXIDATION OF DOUBLE BONDSCarbamazepine to 10,11-epoxide

HETEROATOM OXYGENATIONOmeprazole to sulfone (CYP3A4)


HETEROATOM DEALKYLATIONO-dealkylation (e.g., dextromethorphan to

dextrophan by CYP2D6)N-demethylation of caffeine to:

theobromine (CYP2E1)paraxanthine (CYP1A2)theophylline (CYP2E1)


Oxidative Group TransferN, S, X replaced with OParathion to paroxon (S by O)Activation of halothane to

trifluoroacetylchloride (immune hepatitis)


Cleavage of EstersCleavage of functional group, with O

incorporated into leaving groupLoratadine to Desacetylated loratadine

(CYP3A4, 2D6)


DehydrogenationAbstraction of 2 H’s with formation of C=CActivation of Acetaminophen to

hepatotoxic metabolite N-acetylbenzoquinoneimine

Cytochrome P450 expression

Gene family, subfamily names based on amino acid sequences

At least 15 P450 enzymes identified in human Liver Microsomes

Cytochrome P450 expressionVARIATION IN LEVELS activity due to

Genetic PolymorphismEnvironmental Factors: inducers,

inhibitors, diseaseMultiple P450’s can catalyze same

reaction

A single P450 can catalyze multiple pathways

Major P450 Enzymes in Humans

CYP1A1/ 2

Expressedin:

Substrates Inducers Inhibitors

LiverLungSkinGIPlacenta

CaffeineTheophylline

Cigarrettesmoke;Cruciferousveggies;Charcoal-broiled meat

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


CYP2B6

Expressedin:


Liver DiazepamPhenanthrene

??? Orphenadrine(mechanism-based)


CYP2C19

Genetic polymorphism Substrates Inducers Inhibitors

Poor metabolizers have defective CYP2C9

Phenytoin Piroxicam Tolbutamide Warfarin

Rifampin

Sulfafenazole


CYP2C19


Rapid and slowmetabolizers of S-mephenytoin

N-demethylationpathway of S-mephenytoinmetabolismpredominates in slowmetabolizers

S-mephenytoin(4’-hydroxylationis catalyzed byCYP2C19)

Rifampin Tranylcypromine


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


CYP2E1

Expressed in: Substrates Inducers Inhibitors

LiverLungKidneyLympocytes

EthanolAcetaminophenDapsoneCaffeineTheophyllineBenzene

EthanolIsoniazid

Disulfiram


CYP3A4

Expressedin:


Liver;Kidney;Intestine;MostabundantP450enzyme inliver

AcetaminophenCarbamazepineCyclosporineDapsoneDigitoxinDiltiazemDiazepamErythromycinEtoposideLidocaineLoratadineMidazolamLovasatinNifedipineRapamycinTaxolVerapamil

RifampinCarbamazepinePhenobarbitalPhenytoin

Ketoconazole;Ritonavir;Grapefruit juice;Troleandomycin


CYP4A9/ 11

Expressedin:


Liver Fatty acids andderivaties;Catalzyes - and 1-hyroxylation

??? ???

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)

Inhibition of P450

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

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)

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

Glucuronidation & genetic polymorphism

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 & -glucuronidase

Conjugates excreted in bile or urine (MW)

-glucuronidase from gut microflora cleaves glucuronic acid

Aglycone can be reabsorbed & undergo enterohepatic recycling

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

PHASE 2 Reactions

CONJUGATIONS -OH, -SH, -COOH, -CONH with glucuronic acid to give

glucuronides -OH with sulphate to give sulphates -NH2, -CONH2, amino acids, sulpha drugs with acetyl-

to give acetylated derivatives -halo, -nitrate, epoxide, sulphate with glutathione to

give glutathione conjugates

all tend to be less lipid soluble and therefore better excreted (less well reabsorbed)

Phase II: Sulfation

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)

Sulfation

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

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)

Phase II: Methylation

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)

Methylation & genetic polymorphism

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

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

Acetylation & genetic polymorphism

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

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

Amino Acid Conjugation

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

Phase II:Glutathione Conjugation

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

Glutathione Conjugation

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


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


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

Distinct microsomal and cytosolic glutathione-S-transferases

Genetic polymorphism

Glutathione-S-transferase

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


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

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

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

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

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


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


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


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: 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

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

Biotransformation of Xenobiotics

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