Polymorphism affecting drug metabolism

1. 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.

2. 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.

3. 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).

4. 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.

5. 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.

6. 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.

7. 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

8. • 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

9. • 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

10. •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.

11. 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.

12. 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

13. 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.

14. 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.

15. 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.

16. **NOTE**
• The effect of inhibitoris great in EMs than in PMs.
• The effect of inducer is greater in PMs than in EMs.

17. 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

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

19. 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

20. 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

21. 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.

22. 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%

23. 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

24. 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.

25. 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.

26. 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

27. 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

28. 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.

29. 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.

30. 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.

31. 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.

32. 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.

33. •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.

34. 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

35. 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

36. 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.

37. 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