1. Drug/xenobioticmetabolism and pharmacogenetics George Howell III, Ph.D

2. Sites of drug metabolism • Liver – responsible for the majority of drug metabolism • First pass metabolism • Kidney • GI • Lung • Skin • Brain

3. Drug metabolism and its effects • Drug metabolism – the processes by which biochemical reactions alters drugs within the body • 4 ways a drug can be altered: • Active drug can be inactivated • Active drug can be converted to an active metabolite or toxic metabolite • Prodrug can be converted to an active drug • Unexcretable drug can be converted to an excretable metabolite

4. Major types of biotransformation reactions • Oxidation/reduction reactions (Phase I) • Typically transform drug into more hydrophilic metabolites by adding or exposing a polar functional group • Catabolic • Can be more reactive and toxic than the parent compound • Can be excreted is sufficiently polar • Conjugation/hydrolysis reactions (Phase II) • Further modifications to compounds to improve hydrophilicity • Anabolic • Conjugate drug with an endogenous substrate such as glucuronic acid, sulfuric acid, acetic acid, or an amino acid to form a highly polar compound

5. Oxidation/reduction reactions (Phase I) • More than 95% of oxidative biotransformations are performed by the cytochrome P450 monoxygenases • ~75% of all drugs currently used are oxidized by the P450s • Cytochrome P450 enzymes • Broad substrate specificity • Metabolize xenobiotics • Can play a role in formation of endogenous substances (steroid production) • Alcohol/aldehydedehydrogenases • Monoamine oxidase • Esterases • Acetylcholinesterase (AchE) • Butylcholinesterase (BchE) • Carboxylesterase (CES) Percentage of total liver P450 content CYP3A4 = 30% CYP2C9 = 20% CYP1A2 = 15% CYP2E1 = 10% CYP2D6 = 5% CYP2A6 = 4% CYP2B6 = 1%

6. Cytochrome P450s • Located within the endoplasmic reticulum • 74 CYP gene families • Three main ones involved in drug metabolism in the liver • CYP1, CYP2, CYP3 • ~50% of all rx drugs metabolized by CYP3A4 • P450 3A4 (CYP3A4) • 3 = number of the enzyme family • A = letter of the subfamily • 4 = specifies specific enzyme • Broad substrate specificity due in part to the activated oxygen of the complex (powerful oxidizing agent that can easily react) • Can be induced or inhibited by a variety of compounds • Leads to significant drug interactions ~50%

7. P450 cycle Drug + O2 + NADPH + H+ Drug-OH + H2O + NADP+ Microsomal drug oxidations require: • P450 • P450 reductase • NADPH • Molecular oxygen Steps of P450 mediated oxidation: • Oxidized P450 binds with drug to form a complex • P450 reductase reduces the P450/drug complex • P450 reductase reduces molecular oxygen to form an “activated oxygen”-P450/drug complex • Activated oxygen is transferred to drug to form oxidized product • One molecule of water is produced Electron donated Fe3+ reduced to Fe2+

8. P450 substrates, inducers, and inhibitors • P450 induction • Increase expression by increased synthesis or decreased degradation • Results in increased metabolism of substrates • Decreased substrate plasma concentrations • P450 inhibition • Decrease enzyme activity • Decrease rate of metabolism of other substrates • Increase substrate plasma concentrations

9. Conjugation/hydrolysis reactions (Phase II) • Glucuronidation (highest % of drug metabolism of phase II) • Addition of UDP glucuronic acid catalyzed by UDP glucuronosyltransferase (UGT) • Acetylation • Addition of acetate by N-acetyltransferase (NAT) • Glutathione conjugation • Addition of glutathione by glutathione-S-transferase (GST) • Glycine conjugation • Addition of glycine by Acyl-CoAglycinetransferase • Sulfation • Addition of a sulfate by sulfotransferase (SULT) • Methylation • Addition of a methyl group by transmethylases • Water conjugation • Addition of water by epoxidehydrolase

10. Conjugation reactions

11. Factors affecting drug metabolism • Genetic variability (pharmacogenomics) • Certain populations have polymorphisms or mutations in metabolic enzymes with make them rapid or poor metabolizers • Race and ethnicity • Polymorphisms in metabolic genes among races • CYP2D6 polymorphisms among races • Age • Many biotransformations are slowed in young and elderly • Neonates can carry out most but not all oxidative reactions • Enzyme systems mature over the first two weeks and through childhood • Neonates can have decreased conjugating ability • Jaundice as a result of deficient bilirubin conjugation by UGT • Gray baby syndrome – decreased conjugation of chloramphenicol metabolite • Gender • Males metabolize ethanol, propranolol, some benzodiazepines, estrogens, and salicylates more rapidly

12. Factors affecting drug metabolism (cont.) • Diet • Chargrilled foods and cruciferous vegetables induce CYP1A enzymes • Grapefruit juice inhibits CYP3A • Environment • Cigarette smoke induces P450 enzymes via Ahr dependent mechanism • Industrial workers exposed to some pesticides metabolize more rapidly • Drug interactions • See tables 4-5 and 4-6 in Lange for known inducers and inhibitors • Disease • Liver diseases (hepatitis, cirrhosis, cancer, hemochromatosis, fatty liver) can impair P450 activity • Cardiac disease can slow blood flow to liver and therefore decrease metabolism • Thyroid disease • Hyperthyroid – increase metabolism • Hypothyroid – decrease metabolism

14. Acetaminophen toxicity • Normally undergoes glucuronidation and/or sulfation • Remaining drug undergoes P450 mediated metabolism • Excess acetaminophen saturates conjugation pathways…..shunts to P450 mediated metabolism • Role of ethanol • Hepatic glutathione (GSH) is depleted faster than is regenerated and N-acetylbenzoiminoquinone (toxic metabolite that reacts with proteins) accumulates • N-acetylcysteine is administered w/I 8-16 hours to protect from hepatotoxicity

15. Pharmacogenetics How genetic variability affects drug metabolism

16. GENETIC POLYMORPHISMS • Major factor accounting for differences in pharmacokinetic and pharmacodynamic parameters of individuals • Approximately 25 polymorphisms identified • Clinically important • N-acetylation • debrisoquine/sparteinehydroxylation • mephenytoinoxidation • aldehyde oxidation • butyrylcholinesterase (BchE) deficiency

17. HYDROXYLATION POLYMORPHISMS • Debrisoquine (old antihypertensive) – CYP2D6 • 5-10% most populations are poor metabolizers(1-2% Chinese, Japanese) • Predominant enzyme for amines with hydrophobic planer unit • Approximately 15 variants of CYP2D6 • 4 - no activity, 5 - reduced activity, 3 - increased activity, 2 - no effect • Variation of 1000 fold can be found in extensive metabolizers (heterozygous, allelic variants)

18. IMPACT of DEFICIENT CYP2D6 • Debrisoquine – single dose • Primary effect on first pass metabolism • Little change in ½ life • Increased peak plasma concentration (Clinical effects) • Sparteine – single dose • Primary effect – increased ½ life • No appreciable change in peak plasma concentration • (no observable clinical effects)

19. GENETIC POLYMORPHISMS • S-mephenytoin hydroxylation – CYP2C19 Caucasians (3%) Orientals (15-20%) • Results in poor metabolizer phenotype • Substrates • Acids, bases or neutral compounds • Diazepam, imipramine, propranolol • Proguanil (antimalarial) activated by CYP2C19

20. 12th Edition of Basic and Clinical has a more extensive table…….look up metabolizer phenotype for the prevalent polymorphisms (can be inferred from clinical consequence)

21. N-ACETYLATION • Incidence – slow acetylators • 90% Moroccans, 5% Canadian Eskimos, 30-67% Caucasians and persons from African • Slow acetylators • Phenytoin-isoniazid -inhibition of CYP450 • Arylamine – induced bladder cancer – benzidine • Rapid acetylators • Drug ineffective – dose must be increased • Hepatitis (insignificant)

22. N-ACETYLATION • N-acetylation • Slow acetylators • Isoniazid- induced peripheral polyneuropathy • Drug - induced lupus erythematosus – 35 drugs with primary amino group

23. ALDEHYDE DEHYDROGENASE • About 50% of people of Oriental descent are slow metabolizers of acetaldehyde • Rare outside the Oriental population • Significant acetaldehyde build up associated with ethanol intake – flushing, increased heart rate, nausea

24. Butyrylcholinesterase deficiency • Autosomal recessive • Succinylcholine is metabolized by BchE • Increased accumulation of succinylcholine (depolarizing neuromuscular blocker) • Increased muscle paralysis including respiratory paralysis (succinylcholine apnea)