Genetic Polymorphism in Drug Metabolism – CYP450 Isoenzymes

Presented by: Lahari Paladugu (PharmD 09-10)Presented on: February 7, 2014

What is Genetic Polymorphism?(1) The existence together of many forms of DNA sequences at a locus within the population.

(2) A discontinuous genetic variation that results in different forms or types of individuals among the members of a single species.

Genetic Polymorphism & Drug Metabolism• inter-individual variation of drug effects • Genetic polymorphisms of drug-metabolizing enzymes give

rise to distinct subgroups in the population that differ in their ability to perform certain drug biotransformation reactions. • Polymorphisms are generated by mutations in the genes for

these enzymes, which cause decreased, increased, or absent enzyme expression or activity by multiple molecular mechanisms.

Drug Metabolism• The metabolism of drugs and other xenobiotics into

more hydrophilic metabolites is essential for their elimination from the body, as well as for termination of their biological and pharmacological activity. •Drug metabolism or biotransformation reactions are

classified as either phase I functionalization reactions or phase II biosynthetic (conjugation reactions).

Drug Metabolism (cont.)• The enzyme systems involved in the biotransformation

of drugs are localized primarily in the liver. •Other organs with significant metabolic capacity

include the GI tract, kidneys, and lungs. • These biotransformation reactions are carried out by

CYPs (cytochrome CYP450 isoforms) and by a variety of transferases.

Drug Metabolism (cont.)•Pathways of drug metabolism are classified as either:•Phase I reactions: oxidation, reduction, hydrolysis•Phase II reactions: acetylation, glucuronidation,

sulfation, methylation•Both types of reactions convert relatively lipid soluble

drugs into relatively inactive and more water soluble metabolites, allowing for more efficient systemic elimination.

Polymorphisms•Genetic differences in drug metabolism are the result of

genetically based variation in alleles for genes that code for enzymes responsible for the metabolism of drugs.• In polymorphisms, the genes contain abnormal pairs or

multiples or abnormal alleles leading to altered enzyme function. •Differences in enzyme activity occur at different rates

according to racial group.

Single Nucleotide Polymorphisms (SNPs)• Single changes in one allele of a gene responsible for a variety of

metabolic processes including enzymatic metabolism.• The combination of alleles encoding the gene determines the activity

and effectiveness of the enzyme. • The overall function of the enzyme is the phenotype of enzyme

function. • Phenotype: the observable physical or biochemical characteristics

determined by both genetic makeup and environmental influences

• Poor metabolizers• two defective alleles (ex: CYP2D6*4/*5 and CYP2D6*4/*4)• Combination of alleles including one resulting in no enzyme

(ex: CYP2D6*5 and CYP2D6*4 deletion)• Intermediate metabolizers• Heterozygous – having only one wild type allele and one

defective allele• Normal metabolizers• Carry wild type alleles (ex: CYP2D6*1/*3).•Wild type alleles encode genes for normal enzyme function

• Extensive metabolizers• Carry one wild type and one amplified gene • ex: CYP2D6*1/*2, CYP2D6*A/*1a, and CYP2D6*1A/*5

• Ultra-rapid metabolizers• Carry two or more copies of amplified gene • ex: CYP2D6*2/*3

•Genetic changes may inactivate or reduce enzyme activity leading to increase in the substrate drug.

•Genetic duplication may increase enzyme activity resulting in lower levels of substrate drug.

Inhibitors & Inducers• Polymorphisms affect drug interactions by altering the effect of

inhibitors and inducers on the enzyme. • results in an exaggerated effect or minimal effect on the substrate

• Inhibitor: An enzyme inhibitor is a molecule, which binds to enzymes and decreases their activity.

• Inducer: An enzyme inducer is a type of drug that increases the metabolic activity of an enzyme either by binding to the enzyme and activating it, or by increasing the expression of the gene coding for the enzyme.

Extensive Metabolizers - Inhibitors• Extensive metabolizer ----- level of substrate drug is normally

low due to rapid metabolism by the enzyme. • An inhibitor to the enzyme will inhibit the extensive

metabolism and cause significant elevations in the substrate drug. • Effect of inhibitors is much greater in an EM inc. level of

substrate levels

Poor Metabolizers - Inhibitors• In a poor metabolizer, the level of substrate drug remains

high because the metabolism of the substrate is much less than normal. •When an inhibitor is added, the additional inhibition of

metabolism is not much greater than is already occurring in the PM. • The effect of inhibitor is less in a PM than in normal

metabolizers.• The drug interaction might not occur.

Extensive Metabolizers - Inducers• Level of substrate drug is lower than in a normal

metabolizer due to rapid metabolism.• The addition of an inducer does not cause a greater

difference in the level of substrate because the metabolism is already increased greatly.• The drug interaction might not occur.

Poor Metabolizers - Inducers• Level of substrate drug is higher than expected in normal

metabolizer because of the lower metabolism of substrate. • The addition of inducer will cause a signification increase in

the metabolism of the substrate much lower level of substrate than expected in a normal metabolizer. • Drug interaction may occur to a greater extent.• Drug interaction may result in substrate levels similar to

those of normal metabolizers.

**NOTE**• The effect of inhibitor is great in EMs than in PMs.

• The effect of inducer is greater in PMs than in EMs.

Complex Drug Interactions• Can be seen when a substrate is metabolized through more than one

enzyme systems where one or more enzymes are affected by polymorphism.

Substrate is metabolized through a polymorphic enzyme

Substrate becomes active metabolite

This active metabolite acts as an inhibitor or inducer in second system

Genetic Polymorphisms in Genes that Can Influence

Drug Metabolism – CYP450 Isoforms

Phase I EnzymesEnzyme Substrate Clinical Consequence

CYP1A1 Benzopyrine, phenacetin Inc. or dec. cancer risk

CYP1A2 Acetaminophen, amonafide, caffeine, paraxanthine, ethoxyresorufin, propranolol, fluvoxamine

Decreased theophylline metabolism

CYP1B1 Estrogen metabolites Possible inc. cancer risk

CYP2A6 Coumarin, nicotine, halothane Dec. nicotine metabolism and cigarette addiction

CYP2B6 Cyclophosphamide, aflatozin, mephenytoin Significance unknown

CYP2C8 Retinoic acid, paclitaxel Significance uknown

CYP2C9 Tolbutamide, warfarin, phenytoin, NSAIDS Anticoagulant effect on warfarin

CYP2C19 Mephenytoin, omeprazole, hexobarbital, mephobartibal, propranolol, proquanil, phenytoin

Peptic ulcer response to omeprazole

CYP2D6 Betablockers, antidepressants, antipsychotics, codeine, debrisoquin, dextromethorphan, encainide, flecanide, fluoxetine, guanoxan, methxyamphetamine, phenacetin, propafenone, sparteine

Tardive dyskinesia from antipsychotics; narcotic side effects, efficacy and dependency, imipramine dose requirement; beta blocker effects

CYP2E1 Acetaminophen, ethanol Possible effects on alc consumptionPossible inc cancer risk

CYP3A4/3A7/3A7 Macrolides, cyclosporine, tacrolimus, calcium channel blockers, midazolam, tefrenadie, lidocaine, dapsone, quinidine, triazolam, etoposide, teniposide, loastatian, alfentanil, tamoxifen, steroids, benzo(a)pyrene

Tacrolimus dose requirement in pediatric cancer patients

Aldehyde dehydrogenase

Cyclophosphamide, vinyl chloride SCE frequency in lymphocytes

Alcohol dehydrogenase

Ethanol Inc. alc consumption and dependence

Dihydrodyrimidine dehydrogenase (DPD)

5-fluorouracil Inc. 5-flurorouracil toxicity

NQO1 Ubiquinone, menadione, mitomycin C Menadione-associated orlithiasis, dec tumor sensitivity to mitomycin-C; possible inc. cancer risk

P450 Enzymes in Drug Metabolism• The polymorphic P450 (CYP) enzyme superfamily is the most

important system involved in the biotransformation of many endogenous and exogenous substances including drugs, toxins, and carcinogens.• Genotyping for CYP polymorphisms provides important genetic

information that help to understand the effects of xenobiotics on human body. • For drug metabolism, the most important polymorphisms are those of

the genes coding for CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5, which can result in therapeutic failure or severe adverse reactions.

CYTOCHROME P4502D6• Most extensively studied polymorphic drug metabolizing enzyme• Debrisoquin --- marked hypotension• Impaired ability to hydroxylate, and therefore, inactivate debrisoquin• 5-10% of white subjects have relative deficiency in ability to oxidize

debrisoquin• Also have impaired ability to metabolize the antiarrhythmic and oxytocic drug

sparteine• PM lower urinary concentration, higher plasma concentrations• Subjects inherited two copies of a gene or genes that encoded an enzyme

with either decreased CYP2D6 activity or no activity at all• Prominent in East African population – frequency as high as 29%

CYTOCHROME P4502C SUBFAMILY• Accounts for approximately 18% of the CYP content in the liver • Catalyzes roughly 20% of the CYP-mediated metabolism of drugs

CYP2C19• Study using mephenytoin as probe drug determined that individuals

can be segregated into EMs and PMs.• PM trait is autosomal recessive – present in 3-5% of Caucasians & 12-23% of

Asian populations

CYP2C19 (cont.)

•Also catalyzes the metabolism of several proton pump inhibitors (i.e. omeprazole), diazepam, thalidomide, and some barbiturates. •Responsible for inactivation or propranolol and metabolic activation of antimalarial drug proquanil.

CYP2C19 & Diazepam• Diazepam is demethylated by CYP2C19• Plasma diazepam half-life is longer in individuals who are homozygous

for the defective CYP2C19*2 allele compared to those who are homozygous for the wild type allele. • Half-life of the desmethyldiazepam metabolite is also longer in

CYP2C19 poor metabolizers.• High frequency in Asian population. • Diazepam induced toxicity may occur as a result of slower metabolism

– careful dosing in Asian population.

CYP2C9• Major CYP2C subfamily member in the liver• Primarily responsible for the oxidative metabolism of important

compounds – warfarin, phenytoin, tolbutamide, glipizide, losartan, etc.• 6 different polymorphisms – CYP2C9*1, *2, *3, *4, *5, *6• CYP2C9*1 – wild type allele, CYP2C9*2-*6 – variants• Variants *2 and *3 alleles are common in Caucasians (≈35%)• CYP2C9*2 and *3 alleles associated with significant reduction in the

metabolism and clearance of selected CYP2C9 substrates

CYP2C9 & Warfarin• Polymorphisms linked to both toxicity and dosage

requirements for optimal anticoagulation with warfarin. • *2 and *3 variants – higher risk of acute bleeding

complications than patients with *1 wild type genotype. • Require 15-30% lower maintenance dose of warfarin to

achieve target INR• Patients with variant CYP2C9 genotype take a median of

95 days longer to achieve stable dosing compared to wild-type group

Dihydropyrimide Dehydrogenase• Metabolism of antineoplastic agent fluorouracil.• In the 1980s, fatal CNS toxicity developed in several patients after

treatment with standard doses fluorouracil.• Patients had inherited deficiency of dihyropyrimidine dehydrogenase.

• DPD metabolizes fluorouracil and endogenous pyrimidines. • Severe fluorouracil toxicity occurs when DPD activity < 100 pmol/min/mg

protein.• 3% of population carries heterozygous mutations that inactivate DPD and 1%

are homozygous for the inactivating mutations.• Heterozygous individuals do not exhibit no phenotype until challenged with

fluorouracil.

CYTOCHROME P4503A SUBFAMILY• CYP3A subfamily plays a critical role in the metabolism of more drugs

than any other phase I enzyme. • Expressed in liver and small intestine

• Contribute to oral absorption, first-pass, and systemic metabolism

• Expression is highly inducible – enzyme activity influence by factors such as variable homeostatic control mechanisms, up- or down- regulation by environment factors, and polymorphisms.

CYP3A4• More than 30 SNPs have been identified for CYP3A4 gene• Unlike other P450s, there is no evidence for deleted or null allele for

CYP3A4.• The most common variant in CYP3A4, CYP3A4*1B is an A392G

transition in the promoter region referred to as the nifedipine response element. • One study shows that this variant may be associated with a slower clearance

of cyclosporine. • This is a rather controversial finding.

CYP3A5• Polymorphically expressed in adults in about 10-20% in Caucasians,

33% in Japanese, and 55% in African Americans.• The variable CYP3A5*3 is a result of improper mRNA splicing and

reduced translation of functional protein. • CYP3A5 is the primary extra-hepatic CYP3A isoform, its polymorphic

expression has been implicated in disease risk and the metabolism of endogenous steroids or drug in tissues other than liver. • CYP3A5 has been linked to tacrolimus dose requirements to maintain

adequate immunosuppression in solid organ transplant patients.

CYP3A7• Expressed in fetal liver during development• Hepatic expression is generally down-regulated after birth, but the

CYP3A7 protein has been detected in some adults• Increased CYP3A7 expression has been associated with the

replacement of 60 nucleotide fragment of the CYP3A7 promoter with the corresponding region form of the CYP3A4 promoter (CYP3A7*1C allele.)• This promoter swap results in increased gene expression of the

pregnane X receptor response element.

• PXR signaling serves as a central regulator of inducible CYP3A4 expression as well as several other genes involved in drug detoxification. • Polymorphisms in PXR suggest observed variability in CYP3A4

enzymatic activity may be due to, in part, inherited differences in the upstream signaling proteins that control induction of gene expression.

Phase 2 EnzymesEnzyme Substrate Clinical Consequence

N-acetyltransferase (NAT1) Aminosalicylic acids, aminobenzoic acid, sulfamethoxazole

Possible increased cancer riskHypersensitivity to sulfonamides; amonafide toxicity; hydralazine-induced lupus, isoniazid neurotoxicity and hepatitis

N-acetyltransferase (NAT2) Isoniazid, hydralazine, sulfonamides, amonifidide, procainamine, dapsone, caffeine

Glutathione transferase (GSTM1, M3, T1)

Busulfan, aminochrome, dopachrome, adrenochrome, noradrenochrome

Possible inc cancer risk; cisplatin induced ototoxicity

Glutathione transferase (GSTP1) 13-cis retinoic acid, busulfan, ethacrynic acid, epirubicin

Possible inc cancer risk

Sulfotransferases Steroids, acetaminophen, tamoxifen, estrogens, dopamine

Possible inc or dec cancer risk; clinical outcomes in women receiving tamoxifen for breast cancer

Catechol-O-methyltransferases Estrogens, levodopa, ascorbic acid Decreased response to amphetamine, substance abuse, levodopa response

Thiopurine methyltransferase Mercatopurine, thioguanine, azathioprine

Thiopurine toxicity and efficacy, risk of second cancers

UDP-glucuronosyl-transferase (UGT1A1)

Irinotecan, troglitazone, bilirubin Irinotecan glucuronidation and toxicity, hyperbilirubinemia (Crigler-Najjar syndrome, Gilbert’s syndrome)

UDP-glucuronosyl-transferase (UGT2B) Opioids, morphine, naproxen, ibuprofen, epirubicin

Significance unknown

N-ACETYLTRANSFERASE• N-acetylation of isoniazid to acetylisoniazid• Individuals are slow or rapid acetylators • Ethnic variation is seen

• Slow acetylation: Japanese (10%), Chinese (20%), Caucasians (60%)

• NAT2 protein is the specific protein isoform that acetylates isoniazid.• 27 unique NAT2 alleles identified• NAT2*4 is the wild type allele• NAT2 alleles containing the G191A, T341C, A434C, G590A, and/or G857A

missense associated substitutions are associated with slow acetylator phenotype.

References• Shargel, Leon. Chapter 12 – Pharmacogenetics. Applied Biopharmaceutics and Pharmacokinetics, 5th edition. E-book.• Shargel, Leon. Comprehensive Pharmacy Review, 7th Edition. Philadelphia: Lipincott- William & Wilkins, 2010. Print.

Pages 430-433.• David B. Troy, Paul Beringer. Remington: The Science and Practice of Pharmacy, 21st Edition. Pages 1230 – 1239. • Brunton, Laurence. Chabner, Bruce. Knollman, Bjorn. Goodman & Gilman’s The Pharmacological Basis of

Therapeutics, 12th edition. Pages 124-130.

• http://www.biology-online.org/dictionary/Genetic_polymorphism• http://en.wikipedia.org/wiki/Drug_metabolism• http://www.medscape.com/viewarticle/444804_5• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1934960/• http://dmd.aspetjournals.org/content/29/4/570.full