ABSTRACT

Genetic polymorphisms in drug-metabolizing enzymes are now known to be the major

determinants of inter-individual variability in drug response leading to several forms of adverse

drug effects and reactions. Extensive studies has shown that identification of the genetic bases

for polymorphic expression of a gene is done through intronic or exomic Single Nucleotide

Polymorphisms (SNPs) which abolishes the need for different mechanism for explaining the

variability in drug metabolism and fully suggests that the human Cytochrome P450 (CYP)

genes are highly polymorphic. Genetic variation involved in drug metabolism may account for

variations in responses to some drugs and may affect receptor adaptation, toxicity, altered drug

effects and cross tolerance among various groups of individuals. Apart from the other relatively

known genes such as the gluthathione and N-acetyltransferase genes which are largely involved

and responsible for variances in drug metabolism and response. The Cytochrome P450 (CYP)

genes has been proved to be the most common. We have discussed the clinical importance of

the CYP2C9 gene alterations. This review may help researchers to understand the genetic

mechanism and adverse effect of the CYP2C9 gene polymorphism.

Keywords: Single Nucleotide Polymorphism, CYP 2C9, Adverse Drug Reactions, Drug

Metabolism, Warfarin

Name of Author(s) : Minari, J.B., Ogar G.O., Bello, B.

Affliation: Department of Cell Biology and Genetics, Faculty of Science, University of

Lagos.

Overview of the study

Genetic variation involved in drug metabolism may account for variations in responses to some

drugs and may affect receptor adaptation, toxicity, altered drug effects and cross tolerance.

There are several known genes which are largely responsible for variances in drug metabolism

and response (Sohayla et al., 2007). The most common are the Cytochrome P450 (CYP) genes,

which encode enzymes that influence the metabolism of more than 80 percent of current

prescription drugs (Gomes et al., 2009). Cytochrome P450 (CYP) enzymes are key players in

the Phase 1-dependent metabolism of drugs and other xenobiotics, mostly catalysing oxidations

of the substrates, but occasionally also reduction reactions. At present more than 57 active

human P450 genes and 58 pseudogenes are known. According to Berka et al. (2011) Human

CYPs are primarily membrane-associated proteins. The different alleles are summarized at the

Human CYP allele nomenclature committee home page (Figure 1) (www.cypalleles.ki.se)

(Ingelman-Sundberg et al.,2007). In the CYP gene family, the most penetrant genetic

alterations are gene deletions, missense mutations and mutations creating splicing defects and

premature stop codons (Ingelman-Sundberg and Rodriguez-Antona, 2006)). Various

physiological(e.g age, nutrition) and pathological(liver, kidney or heart diseases) factors can

also affect drug metabolism.(Rostami-Hodjegan and Tucker, 2007). Danielson (2002) has

earlier reported the contributory effects of Inhibitors (Sertraline,Sulphaphenazole) and Inducers

(Refampicin,Secobarbital) in the cyp2c9 genome.

The mutations in the CYP genes may cause absence of enzyme, diminished enzyme

expression, enzyme with altered substrate specificity or increased enzyme expression. Based

on the composition of alleles, the affected individual might be divided into four major

phenotypes: Poor Metabolizers (PMs), having two non-functional genes; Intermediate

Metabolizers (IMs), being deficient on one allele; Extensive Metabolizers (EMs), having two

copies of normal genes and Ultra rapid Metabolizers (UMs), having three or more functional

active gene copies (Ingelman-Sundberg and Rodriguez-Antona, 2005).

Figure 1: Importance of the different polymorphic CYP genes as assessed by the number of

hits at the human CYP allele nomenclature home page (www.cypalleles.ki.se)

Source: Ingelman-Sundberg et al.,2007

Figure 2: Selected CYP2C9 substrates. The site(s) of oxidation of each substrate is indicated

by an arrow(s).

Source: www.ncbi.nlm.nih.gov, 2014

Figure 3: Cytochrome P450 Catalytic Pathway

Source: Danielson, 2002

PHASE I

1. Substrate binding, 2. Electron transfer, 3. Oxidation, 4. Electron transfer, 5. Reduction

PHASE II

6. Elimination and hydroxylation (Conjugation), S. Alternative route for mono-oxygenation,

C. Reduction by CO

Figure 4: Effects of Mutation on CYP gene

Source: Ingelman‐Sundberg, 2001.

CYP2C9 is a major cytochrome P450 enzyme that is involved in the metabolic clearance of a

wide variety of therapeutic agents, including oral anticoagulants, oral hypoglycaemias, and

nonsteroidal anti-inflammatories. Disruption of CYP2C9 activity by metabolic inhibition or

pharmacogenetic variability underlies many of the adverse drug reactions that are associated

with the enzyme (Allan and Jeffery, 2005). The phenotypes CYP2C9*2 and CYP2C9*3 are the

two most common variations and are associated with reduced enzymatic activity. Impaired

metabolism of drugs metabolized by the CYP2C9 isoenzyme, such as phenytoin, S-warfarin

(Aquilante et al., 2006), tolbutamide, losartan, and nonsteroidal antiinflammatory drugs

(NSAIDs) (e.g., ibuprofen, diclofenac, piroxicam, tenoxicam, mefenamic acid) has been noted.

Noteworthy is that:

CYP2C9 gene is highly polymorphic. (www.imm.ki.se/cypalleles)

CYP2C9 gene is 55 kb long, including 9 exons.

Located on the long arm of chromosome 10.

The CYP2C9 gene encodes a protein of 492 amino acids.

(Wang et al., 2007)

CYP2C9 is a major liver P450 enzyme.

Metabolizes ~15% of the drugs that undergo phase I metabolism

(Rendic, 2002)

CYP2C9 is involved in the metabolic clearance of a wide variety of therapeutic agents.

(Allan and Jeffery, 2005)

Table 1: Common substrates for CYP2C9 by therapeutic class∗Source: Allan and Jeffery, 2005.

CLASS SUBSTRATES

Oral Anticoagulant Warfarin, Acenocoumarol(Phenprocoumon)

Oral

anticonvulsant

Phenytoin(Phenobarbital)

Non-Steroidal

Anti-inflammatory

Drugs (NSAID)

Flurbiprofen,diclofenac,Piroxicam,Suprofen,Ibuprofen,Mefenamic

acid,Celecoxib

Oral

hypoglycaemic

Tolbutamide,Glimepiride,Glyburide,Glipizide

Drug Substrate Metabolism by CYP2C9

Concerning gene variations, it is a well-known fact that CYP2C9 polymorphisms have

functional consequences for in vitro and in vivo pharmacokinetics and for clinical drug

response and side effects. There are more than 30 different SNPs described in the regulatory

and coding region of CYP2C9, but the polymorphic behaviour of CYP2C9 seems to be

determined mainly by 2 common coding variants: CYP2C9*2 (R144C) and CYP2C9*3

(I359L), both yielding enzymes with decreased activity. CYP2C9*2 and CYP2C9*3 are mainly

present in Caucasians (11% and 7% frequency, respectively) while the frequencies are lower in

Africans. In Asians, CYP2C9*2 has indeed not been detected (Kirchheiner & Brockmoller,

2005). CYP2C9 is the principal enzyme responsible for the metabolism of S-warfarin. Persons

who are CYP2C9 poor metabolizers have reduced S-warfarin clearance. Clinical studies have

shown that these persons require lower dosages of warfarin and are at an increased risk of

excessive anticoagulation (Aquilante et al., 2006). Kirchheiner and Brockmoller (2005)

observed that the most relevant CYP2C9 genetic variations which result in decreased activity

are, CYP2C9*2, mainly present in Caucasians with a 10-15% frequency, but almost present in

Africans and Asians, and CYP2C9*3, which has an allele frequency of 4-10% in Caucasians, 4-

7% in Asians and 1-2% in Africans. CYP2C9*2 and *3 were associated with a decrease in the

intrinsic clearance of S-warfarin, respectively, compared with the wild-type allele (Haining et

al., 1996). According to Aithal et al., (1999) Individuals who require a low dose of warfarin to

maintain optimum anticoagulation have a slightly higher frequency of variant alleles than those

who require a higher dose. Increased risk of bleeding was observed in patients with mutant

alleles (Poor Metabolizers), and subsequent dosage adjustments were required (Higashi et al.,

2002). CYP2C9 genotyping may help identify high-risk patients who are candidates for lower

warfarin doses, more frequent monitoring, or alternative drug treatments (Tang et al., 2001).

In vitro results have shown that the substrate affinity of CYP2C9.2 varies as compared with the

wild-type enzyme. Consequently, for some substrates, it is unaffected, but with some other

drugs such as acenocoumarol, the activity is severely decreased. The CYP2C9.3 protein,

containing the conservative amino acid substitution I359L, results in a significant reduction of

the catalytic activity with only around 10% of the intrinsic clearance of the wild-type enzyme

for most CYP2C9 substrates (Thijssen & Ritzen, 2003). CYP2C9 2 and/or 3 have been⁎ ⁎

shown to affect the oral clearance of at least 17 different CYP2C9 drugs: S-acenocoumarol, S-

phenprocoumon, S-warfarin, glimepiride, glyburide, tolbutamide, nateglinide, candesartan,

losartan, celecoxib, diclofenac, flubiprofen, S-ibuprofen, tenoxicam, fluvastatin, phenytoin and

torsemide (Kirchheiner & Brockmoller, 2005),

Figure 5: Potential Benefit of Adjusting Dose to Genotype

Source: Kirchheiner et al., 2005

GENETIC POLYMORPHISM OF CYP2C9 GENE

Genetic polymorphism exists for CYP2C9 expression because the CYP2C9 gene is

highly polymorphic (Sim, 2011).

Common Variants: CYP2C9*1 (wild-type)

CYP2C9*2

CYP2C9*3

CYP2C9*5

CYP2C9*6

CYP2C9*11

More than 50 single nucleotide polymorphisms (SNPs) have been described in the

coding regions of the CYP2C9 gene (Sim, 2011)

Genetic variants in CYP2C9 could cause great changes in the encoded protein’s

metabolic activity and cause severe adverse drug reactions(Van-Booven et al., 2010)

ADVERSE DRUG REACTIONS(ADRs):

Caused 5% of hospitalization

Experienced by 10% of the hospitalized patients

Over 2million serious ADRs yearly

4th leading cause of death

59% of drugs causing ADRs are metabolized by polymorphic enzymes.

(source: Food and Drug Administration, 2014)

Multiple in vivo studies also show that several mutant CYP2C9 genotypes are associated with

significant reduction of in metabolism and daily dose requirements of selected CYP2C9

substrate. In fact, adverse drug reactions (ADRs) often result from unanticipated changes in

CYP2C9 enzyme activity secondary to genetic polymorphisms. Especially for CYP2C9

substrates such as warfarin and phenytoin, diminished metabolic capacity because of genetic

polymorphisms or drug-drug interactions can lead to toxicity at normal therapeutic doses (Sim,

2011). The gene coding for the CYP2C9 enzyme is highly poly- morphic, including functional

variants of major pharma- cogenetic importance. Changes in metabolic activity caused by

genetic variants in CYP2C9 play a major role in pathogenesis caused by adverse drug

reactions. Patients with low enzyme activity are at risk of adverse drug reaction, especially for

CYP2C9 substrates with a narrow therapeutic window, such as S-warfarin, pheny- toin,

glipizide, and tolbutamide (Pirmohamed and Park, 2003)

The CYP2C9*3 allele has stronger pharmacokinetic effects than CYP2C9*2. For most

substrates, CYP2C9*3 heterozygous individuals had approximately 50% of the wild type total

oral clearance and CYP2C9*3 homozygous individuals had a 5- to 10-fold reduction. With

respect to CYP2C9*2, a significant effect was found for Swarfarin, acenocoumarol,

tolbutamide and celecoxib clearance but not for other drugs (Kirchheiner & Brockmoller,

2005).

Table 2: Genetic Polymorphism of Cytochrome P450 (CYP) 2C9

Source: www.pharmacologyweekly.com

Allele Location 2C9 Enzyme

Activity

CYP2C9*1 10q24.1 Normal

CYP2C9*2 Exon 3 Reduced

CYP2C9*3 Exon 7 Reduced

CYP2C9*4 Exon 7 Reduced

CYP2C9*5 Exon 7 Reduced

CYP2C9*6 Exon 5 None

CYP2C9*11 Exon 7 Reduced

Figure 8: PCR sequencing primers for CYP2C9 alleles

Source: Limdi et al., 2008

CLINICAL IMPORTANCE (CASE STUDY)

WARFARIN

• Widely prescribed anticoagulant drug used to decrease the tendency for thrombosis

and also prevent blood clots(Thrombosis). (Holbrook et al., 2006)

• Clinical consequence of CYP2C9 Poor Metabolizer is evident in decreased clearance of

warfarin. (Buzková et al., 2006)

• The only common adverse effect of warfarin is haemorrhage (bleeding) (Holbrook et

al., 2012)

• Therapeutic range: International Normalized Ratio 2-3 (2.5-3.5 for prosthetic heart

valves)

• INR <2: risk of thromboembolic event, INR >3: risk of bleeding complications

(Mushiroda et al., 2006)

• In December of 2011, the FDA warned that it was investigating Pradaxa due to a greater

amount of injury or death reports than expected. USA Today reported that in 2011, the

FDA received reports of 3,781 Pradaxa adverse effects and 542 deaths among users,

more than all other medications.

• In September of 2012, The Journal for The American Medical Association warned that

the FDA may have rushed approval of Pradaxa, overlooking key side effects like

internal bleeding.

• In its March, 2012 report, the Journal of Neurosurgery detailed a specific case and

concerns by doctors that once Pradaxa causes internal bleeding, there is very little they

can do to stop it. Source: www.drugrisk.com,2014

Figure 9: Haemorrhage

Source: www.drugrisk.com, 2014

Figure 10: CYP2C9 Polymorphisms and Warfarin Dose

Source: Gage et al., 2004

*1/*1=EMs(wild type)

*1/*2=IMs

*1/*3=PMs

*1/*5=IMs

*2/*2=IMs

*2/*3=PMs

*3/*3=PMs

Table 3: Adverse Drug Reactions of Important Substrates

Source: Ingelman‐Sundberg , 2001

DRUGS SUBSTRATES ADVERSE EFFECTS IN POOR METABOLIZERS

Oral Anticoagulants Warfarin Increased bleeding,Internal or external hemorrhage, hematuria

Oral Antiglycaemics Tolbutamide Hypoglycaemia

Oral Anticonvulsant Phenytoin Ataxia

NSAID Celecoxib Platelet dysfunction, renal and gastrointestinal toxicity

Figure 11: Examples of dose adjustments based on Phenotypes

Source: Kirchheiner et al., 2005

Table 4: Allele Frequencies of CYP2C9 Polymorphisms(Known variants)

Source: Kirchheiner and Brockmoller, 2005.

Allele and Genotype African

subjects(%)

Asian subjects(%) White subjects(%)

CYP2C9*2 4 0 11

CYP2C9*3 2 3 7

CYP2C9*4 0 ? 0

CYP2C9*5 1.8 ? 0

CYP2C9*6 0.6 ? 0

CYP2C9*11 2.7 ? 0.4

In conclusion,

The CYP2C9 polymorphism is clinically highly significant and also substrate dependent.

Genotyping of CYP2C9 polymorphism has to be taken into account for appropriate dosing in

many different therapies.

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