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

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 H 2 O added to epoxides eg: carbamazepine

Phase I: Reductions Azo Reduction N=N to 2 -NH 2 groups eg: prontosil to sulfanilamide Nitro Reduction N=O to one -NH 2 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 CCl 4 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-1,2,5,6- tetrahydropyridine (MPTP) to neurotoxic toxic metabolite in nerve tissue, resulting in Parkinsonian-like symptoms

MonoOxygenases Peroxidases couple oxidation to reduction of H 2 O 2 & 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 Fe 2+ and CO absorbs light maximally at 450 ( ) 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

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 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: 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 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) -CH 3 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 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 -NH 2 (amine) of glycine, serine, glutamine, requiring CoA activation e.g: conjugation of Benzoic acid with Glycine to form hippuric acid Aromatic -NH 2 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-S- transferases Genetic polymorphism

Glutathione-S-transferase Inducers (include phenobarbital, corticosteroids, anti- oxidants) Over expression of enzyme leads to resistance (e.g., insects to DDT, corn to atrazine, cancer cells to chemotherapy) Species Specificity Aflatoxin B 1 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-trans- peptidase, aminopeptidase M