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
Download ReportTranscript 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
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 Increase in Small hydrophilicity General Exposes functional mechanism group Conjugations Consquences Facilitates excretion May result in metabolic activation Large Polar compound added to functional group 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 & peptidases Hydrolysis of esters eg: valacyclovir, midodrine Hydrolysis of peptide bonds e.g.: insulin (peptide) Epoxide hydrolase H2O added to epoxides eg: carbamazepine 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 Reductions Carbonyl reduction Chloral hydrate is reduced to trichlorothanol Disulfide reduction First step in disulfiram metabolism 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 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 X’s removed, form C=C) DDT to DDE 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 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 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 Monooxygenases Flavin-containing Mono-oxygenases Generally results in detoxification Microsomal enzymes Substrates: Nicotine, Cimetidine, Chlopromazine, Imipramine Phase I: Cytochrome P450 Microsomal enzyme ranking first among Phase I enzymes Heme-containing proteins Complex formed between Fe2+ and CO absorbs light maximally at 450 (447-452) nm Cytochrome P450 reactions Hydroxylation Testosterone to 6-hydroxytestosterone (CYP3A4) Cytochrome P450 reactions EPOXIDATION OF DOUBLE BONDS Carbamazepine to 10,11-epoxide HETEROATOM OXYGENATION Omeprazole to sulfone (CYP3A4) Cytochrome P450 reactions 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 Oxidative Group Transfer N, S, X replaced with O Parathion to paroxon (S by O) Activation of halothane to trifluoroacetylchloride (immune hepatitis) Cytochrome P450 reactions Cleavage of Esters Cleavage of functional group, with O incorporated into leaving group Loratadine to Desacetylated loratadine (CYP3A4, 2D6) Cytochrome P450 reactions Dehydrogenation Abstraction of 2 H’s with formation of C=C Activation 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 expression VARIATION IN LEVELS activity due to Genetic Polymorphism Environmental Factors: inducers, inhibitors, disease Multiple P450’s can catalyze same reaction A single P450 can catalyze multiple pathways Major P450 Enzymes in Humans CYP1A1/2 Expressed in: Substrates Inducers Inhibitors Liver Lung Skin GI Placenta Caffeine Theophylline Cigarrette smoke; Cruciferous veggies; Charcoalbroiled meat Furafylline (mechanismbased); -naphthoflavone (reversible) Major P450 Enzymes in Humans CYP2B6 Expressed in: Substrates Inducers Inhibitors Liver Diazepam ??? Phenanthrene Orphenadrine (mechanismbased) Major P450 Enzymes in Humans CYP2C19 Genetic polymorphism Substrates Inducers Inhibitors Poor metabolizers have Phenytoin Rifampin Sulfafenaz defective CYP2C9 Piroxicam ole Tolbutami de Warfarin Major P450 Enzymes in Humans CYP2C19 Genetic polymorphism Substrates Inducers Rapid and slow metabolizers of Smephenytoin N-demethylation pathway of Smephenytoin metabolism predominates in slow metabolizers S-mephenytoin Rifampin (4’-hydroxylation is catalyzed by CYP2C19) Inhibitors Tranylcypromine Major P450 Enzymes in Humans CYP2D6 Genetic polymorphism Substrates 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 Inducers Inhibitors Fluoxetine Quinidine Major P450 Enzymes in Humans CYP2E1 Expressed in: Substrates Inducers Inhibitors Liver Lung Kidney Lympocytes Ethanol Acetaminophen Dapsone Caffeine Theophylline Benzene Ethanol Isoniazid Disulfiram Major P450 Enzymes in Humans CYP3A4 Expressed in: Substrates Inducers Inhibitors Liver; Kidney; Intestine; Most abundant P450 enzyme in liver 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 Major P450 Enzymes in Humans CYP4A9/11 Expressed Substrates in: Inducers Inhibitors Liver ??? Fatty acids and ??? Metabolic activation by P450 Formation of toxic species De-chlorination of chloroform to phosgene De-hydrogenation 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 sub-therapeutic plasma concentrations eg, co-administration of Rifampin and oral contraceptives is contraindicated Some drugs induce, inhibit same enzyme (Isoniazid, Ethanol (2E1), Ritonavir (3A4) 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 acetylto 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: Glucuronidation Major Phase II pathway in mammals UDP-glucuronyltransferase forms O-, N-, S-, Cglucuronides; 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 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 II: Sulfation Sulfo-transferases 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 Omethyltransferase, N-methyltransferase, Smethyltransferase 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 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 (amine) 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 Metabolic activation Serine or proline N-esters of hydroxyl-amines are unstable & degrade to reactive electrophiles. Phase II Glutathione Conjugation Glutathione-S-transferase catalyzes conjugation with glutathione Glutathione is tripeptide of glycine, cysteine, glutamic acid Formed by -glutamyl-cysteine synthetase, glutathione synthetase Buthione-S-sulfoxine is inhibitor Glutathione Conjugation Two types of reactions with glutathione 1.Displacement of halogen, sulfate, sulfonate, phospho, nitro group 2.Glutathione added to activated double bond Glutathione substrates Hydrophobic, Containing electrophilic atom Can react with glutathione non-enzymatically 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 Glutathione Conjugation Four classes of soluble glutathione-S-transferase microsomal and cytosolic glutathione-Stransferases Genetic polymorphism Glutathione-S-transferase Inducers (include phenobarbital, corticosteroids, antioxidants) Over expression 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 Conjugation Excretion of Glutathione Conjugates Excreted in bile Converted to Mercapturic Acids in kidney, excreted in urine Enzymes involved are -glutamyl-transpeptidase, aminopeptidase M