Drug/xenobiotic metabolism and pharmacogenetics

George Howell III, Ph.D

Sites of drug metabolism

• Liver – responsible for the majority of drug metabolism– First pass metabolism

• Kidney• GI• Lung• Skin• Brain

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 inactivated2. Active drug can be converted to an active

metabolite or toxic metabolite3. Prodrug can be converted to an active drug4. Unexcretable drug can be converted to an

excretable metabolite

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

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%

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%

P450 cycle

Microsomal drug oxidations require:1. P4502. P450 reductase3. NADPH4. Molecular oxygen

Steps of P450 mediated oxidation:5. Oxidized P450 binds with drug to

form a complex6. P450 reductase reduces the

P450/drug complex7. P450 reductase reduces

molecular oxygen to form an “activated oxygen”-P450/drug complex

8. Activated oxygen is transferred to drug to form oxidized product

9. One molecule of water is produced

Drug + O2 + NADPH + H+ Drug-OH + H2O + NADP+

Electron donated Fe3+ reduced to Fe2+

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

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

Conjugation reactions

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

• 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.)

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

Pharmacogenetics

How genetic variability affects drug metabolism

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

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)

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)

GENETIC POLYMORPHISMS

• S-mephenytoin hydroxylation – CYP2C19Caucasians (3%) Orientals (15-20%)

• Results in poor metabolizer phenotype• Substrates – Acids, bases or neutral compounds• Diazepam, imipramine, propranolol• Proguanil (antimalarial) activated by CYP2C19

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)

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)

N-ACETYLATION

• N-acetylation– Slow acetylators• Isoniazid- induced peripheral polyneuropathy• Drug - induced lupus erythematosus – 35 drugs with

primary amino group

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

Butyrylcholinesterase deficiency

• Autosomal recessive• Succinylcholine is metabolized by BchE• Increased accumulation of succinylcholine

(depolarizing neuromuscular blocker)• Increased muscle paralysis including respiratory

paralysis (succinylcholine apnea)