Bio-Trans of Xenobiotics.

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
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 Types of reactions Phase I Hydrolysis Oxidation Reduction Small Exposes functional group May result in metabolic activation Phase II Conjugations
Increase in hydrophilicity General mechanism Consquences
Large Polar compound added to functional group 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
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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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Reductions
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
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Reductions
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
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Reductions
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 Xs removed, form C=C)

DDT to DDE
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Phase I: Oxidation-Reduction
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
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Phase I: Monooxygenases
Monoamine oxidase
Primaquine, haloperidol, tryptophan are substrates Activates 1-methyl-4-phenyl-1,2,5,6tetrahydropyridine (MPTP) to neurotoxic toxic metabolite in nerve tissue, resulting in Parkinsonian-like symptoms
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Monooxygenases
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
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Monooxygenases
Flavin-containing mono-oxygenases
Generally results in detoxification Microsomal enzymes Substrates: nicotine, cimetidine, chlopromazine, imipramine Repressed rather than induced by phenobarbital, 3-methylcholanthrene
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Phase I: Cytochrome P450

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+

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Cytochrome P450
catalytic cycle
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Cytochrome P450 reactions

Hydroxylation of aliphatic or aromatic carbon
(S)-mephenytoin to 4-hydroxy-(S)mephenytoin (CYP2C19) Testosterone to 6-hydroxytestosterone (CYP3A4)
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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 Hs with formation of C=C Activation of Acetaminophen to hepatotoxic metabolite N-acetylbenzoquinoneimine
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Cytochrome P450 expression

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

Variation in levels, activity due to:
Genetic polymorphism Environmental factors: inducers, inhibitors, disease Multiple P450s 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
Expressed in: Liver Lung Skin GI Placenta Substrates Caffeine Theophylline Inducers Cigarrette smoke; Cruciferous veggies; Charcoalbroiled meat Inhibitors Furafylline (mechanismbased); -naphthoflavone (reversible)
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CYP2B6 Expressed in: Liver Substrates Inducers Inhibitors Orphenadrine (mechanismbased)
Diazepam ??? Phenanthrene
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CYP2C19
Genetic polymorphism Substrates Inducers Rifampin Inhibitors Sulfafenazole
Poor metabolizers have defective Phenytoin CYP2C9 Piroxicam Tolbutamide Warfarin
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CYP2C19 Genetic polymorphism Rapid and slow metabolizers of Smephenytoin N-demethylation pathway of Smephenytoin metabolism predominates in slow metabolizers Substrates Inducers Inhibitors Tranylcypromine
S-mephenytoin Rifampin (4-hydroxylation is catalyzed by CYP2C19)
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CYP2D6
Genetic polymorphism
Substrates
Inducers
Inhibitors
Poor metabolizers lack CYP2D6 Debrisoquine causes marked, prolonged hypotension in slow metabolizers No effect on response to propanolol in poor metabolizers; alternate pathway (CYP2C19) will predominate 5-10% of Caucasians are poor metabolizers < 2% of Asians, African Americans are poor metabolizers
Propafenone None known Desipramine Propanolol Codeine Dextromethorphan Fluoxetine Clozapine Captopril Poor metabolizers identified by urinary exrection of Dextrorphan
Fluoxetine Quinidine
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CYP2E1 Expressed in: Liver Lung Kidney Lympocytes Substrates Ethanol Acetaminophen Dapsone Caffeine Theophylline Benzene Inducers Ethanol Isoniazid Inhibitors Disulfiram
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CYP3A4 Expressed in: Liver; Kidney; Intestine; Most abundant P450 enzyme in liver Substrates Inducers Inhibitors
Acetaminophen Carbamazepine Cyclosporine Dapsone Digitoxin Diltiazem Diazepam Erythromycin Etoposide Lidocaine Loratadine Midazolam Lovasatin Nifedipine Rapamycin Taxol Verapamil
Rifampin Carbamazepine Phenobarbital Phenytoin
Ketoconazole; Ritonavir; Grapefruit juice; Troleandomycin
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CYP4A9/11
Expressed Substrates in: Liver Inducers Inhibitors ???
Fatty acids and ??? derivaties; 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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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
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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, 3methylcholanthrene, cigarette smoking Substrates include dextrophan, methadone, morphine, p-nitrophenol, valproic acid, NSAIDS, bilirubin, steroid hormones
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Glucuronidation & genetic polymorphism
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
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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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Phase II: Sulfation

Sulfotransferases are widely-distributed enzymes Cofactor is 3-phosphoadenosine-5phosphosulfate (PAPS) Produce highly water-soluble sulfate esters, eliminated in urine, bile Xenobiotics & endogenous compounds are sulfated (phenols, catechols, amines, hydroxylamines)
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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
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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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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)
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Methylation & genetic polymorphism
Several types of methyltransferases in human tissues

Phenol O-methyltransferase, Catechol Omethyltransferase, N-methyltransferase, Smethyltransferase
Genetic polymorphism in thiopurine metabolism

high activity allele, increased toxicity low activity allele, decreased efficacy
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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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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
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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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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 48 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
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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
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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-Stransferase ( , , , ) Distinct microsomal and cytosolic glutathioneS-transferases Genetic polymorphism
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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
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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 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
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Guidance focuses on P450 enzymes Other hepatic enzymes not as wellcharacterized 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 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):899-910
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Bio-Trans of Xenobiotics.

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Bio-Trans of Xenobiotics.

Hochgeladen von

Copyright:

Verfügbare Formate

Biotransformation of Xenobiotics

FDA guidances related to biotransformation

Comparing Phase I & Phase II

Increase in hydrophilicity General mechanism Consquences

Large Polar compound added to functional group Facilitates excretion

First Pass Effect

Oxidative (X and H replaced with =O)

Dehydrodechlorination (2 Xs removed, form C=C)

Lactoperoxidase (mammary gland) Myleoperoxidase (bone marrow)

Phase I: Cytochrome P450

Overall reaction proceeds by catalytic cycle: RH+O2+H++NADPH ROH+H2O+NADP+

Cytochrome P450 reactions

Cytochrome P450 reactions

Heteroatom oxygenation, N-hydroxylation

Cytochrome P450 reactions

Cytochrome P450 reactions

Cytochrome P450 reactions

Cytochrome P450 reactions

Cytochrome P450 expression

Cytochrome P450 expression

Major P450 Enzymes in Humans

Major P450 Enzymes in Humans

Diazepam ??? Phenanthrene

Major P450 Enzymes in Humans

Poor metabolizers have defective Phenytoin CYP2C9 Piroxicam Tolbutamide Warfarin

Major P450 Enzymes in Humans

S-mephenytoin Rifampin (4-hydroxylation is catalyzed by CYP2C19)

Major P450 Enzymes in Humans

Major P450 Enzymes in Humans

Major P450 Enzymes in Humans

Rifampin Carbamazepine Phenobarbital Phenytoin

Ketoconazole; Ritonavir; Grapefruit juice; Troleandomycin

Major P450 Enzymes in Humans

Fatty acids and ??? derivaties; Catalzyes - and 1-hyroxylation

Metabolic activation by P450

Formation of pharmacologically active species

Induction and P450

Phase II: Glucuronidation

Glucuronidation & genetic polymorphism

Glucuronidation & glucuronidase

Glucuronidation and glucuronidase

Metabolic activation of 2.6-dinitrotoluene) by -glucuronidase

Phase II: Sulfation

Capacity limited by low PAPS levels

Phase II: Methylation

Methylation & genetic polymorphism

Several types of methyltransferases in human tissues

Genetic polymorphism in thiopurine metabolism

Phase II: Acetylation

Humans express two forms Substrates include sulfanilamide, isoniazid, dapsone

Acetylation & genetic polymorphism

Rapid and slow acetylators

Drug toxicities in slow acetylators

Phase II:Amino Acid Conjugation

Amino Acid Conjugation

Phase II:Glutathione Conjugation

Activation of xenobiotics following GSH conjugation

FDA-CDER Guidances for Industry

In vitro guidance: assumptions

In vitro guidance: techniques/approaches

In vitro guidance: techniques/approaches

In vitro guidance: techniques/approaches

In vitro guidance: techniques/approaches

In vitro guidance: timing of studies