1. Drug/xenobiotic metabolism 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: 1.Active drug can be inactivated 2.Active drug can be converted to an active metabolite or toxic metabolite 3.Prodrug can be converted to an active drug 4.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/aldehyde dehydrogenases 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 Microsomal drug oxidations require: 1.P450 2.P450 reductase 3.NADPH 4.Molecular oxygen Steps of P450 mediated oxidation: 1.Oxidized P450 binds with drug to form a complex 2.P450 reductase reduces the P450/drug complex 3.P450 reductase reduces molecular oxygen to form an “activated oxygen”-P450/drug complex 4.Activated oxygen is transferred to drug to form oxidized product 5.One molecule of water is produced Drug + O 2 + NADPH + H + Drug-OH + H 2 O + NADP + Electron donatedFe 3+ reduced to Fe 2+

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-CoA glycinetransferase Sulfation – Addition of a sulfate by sulfotransferase (SULT) Methylation – Addition of a methyl group by transmethylases Water conjugation – Addition of water by epoxide hydrolase

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. 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 Factors affecting drug metabolism (cont.)

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/sparteine hydroxylation – mephenytoin oxidation – 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. 12 th 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)