1. Genetic Polymorphism in Drug Metabolism – CYP450 Isoenzymes
Presented by: Lahari Paladugu (PharmD 09-10)
Presented on: February 7, 2014

2. 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.

3. 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.

4. 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).

5. 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.

6. 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.

7. 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.

8. 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

9. 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

10. 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

11. 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.

12. 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.

13. 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

14. 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.

15. 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.

16. 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.

17. **NOTE** The effect of inhibitor is great in EMs than in PMs.
The effect of inducer is greater in PMs than in EMs.

18. 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

19. Genetic Polymorphisms in Genes that Can Influence Drug Metabolism – CYP450 Isoforms

20. Phase I Enzymes Enzyme 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

21. CYP2E1
Acetaminophen, ethanol
Possible effects on alc consumption
Possible 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

22. 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.

23. 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%

24. 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

25. 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.

26. 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.

27. 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

28. 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

29. 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.

30. 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.

31. 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.

32. 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.

33. 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.

34. 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.

35. Phase 2 Enzymes Enzyme Substrate Clinical Consequence
N-acetyltransferase (NAT1)
Aminosalicylic acids, aminobenzoic acid, sulfamethoxazole
Possible increased cancer risk
Hypersensitivity 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

36. 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

37. 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.

38. 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, Print. Pages
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