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
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Overview
Major Phase I and Phase II enzymes Reaction mechanisms, substrates Enzyme inhibitors and inducers Genetic polymorphism Detoxification Metabolic activation
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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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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
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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
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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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Cytochrome P450
catalytic cycle
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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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Acetaminophen Carbamazepine Cyclosporine Dapsone Digitoxin Diltiazem Diazepam Erythromycin Etoposide Lidocaine Loratadine Midazolam Lovasatin Nifedipine Rapamycin Taxol Verapamil
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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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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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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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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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Sulfation
Sulfation is a high affinity, low capacity pathway
Glucuronidation is low affinity, high capacity
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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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Substrates include phenols, catechols, amines, heavy metals (Hg, As, Se)
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Aromatic -NH2 or NHOH conjugated with COOH of serine, proline, requiring ATP activation
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Metabolic activation
Serine or proline N-esters of hydroxylamines are 48 unstable & degrade to reactive electrophiles
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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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Glutathione Conjugation
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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Glutathione Conjugation
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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Glutathione Conjugation
Excretion of glutathione conjugates
Excreted intact in bile Converted to mercapturic acids in kidney, excreted in urine
Enzymes involved are -glutamyltranspeptidase, aminopeptidase M
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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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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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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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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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