Polymorphisms in the drug-metabolizing enzymes represent the first recognized and, so far, the most documented examples of genetic variants with consequences in drug response and toxicity. The major phase I enzymes are the CYP superfamily of isoenzymes. N-acetyltransferase, uridine diphosphate glucuronosyltransferase (UGT), and glutathione S-transferase are examples of phase II metabolizing enzymes that exhibit genetic polymorphisms. Thiopurine S-methyltransferase (TPMT) and dihydropyrimidine dehydrogenase (DPD) are examples of nucleotide base-metabolizing enzymes. Table 9–1 lists examples of polymorphic metabolizing enzymes and corresponding drug substrates whose plasma concentrations and pharmacologic effects may be altered as a consequence of genetic variation. Examples of such effects are discussed in the following sections.
Currently, 57 different CYP isoenzymes have been documented to be present in humans, with 42 involved in the metabolism of exogenous xenobiotics and endogenous substances such as steroids and prostaglandins.11 Fifteen of these isoenzymes are known to be involved in the metabolism of drugs, but significant interindividual variabilities in enzyme activity exist as a result of induction, inhibition, and genetic inheritance. Functional genetic polymorphism has been discovered for CYP2A6, CYP2C9, CYP2C19, CYP2D6, and more recently, CYP3A4/5. A polymorphism in the regulatory region of the gene encoding for CYP1A212 has been identified, which was shown to affect inducibility of the gene and the magnitude of increased metabolism of caffeine in smokers, but its functional importance remains to be determined.
Polymorphisms in the CYP2D6 gene are the best characterized of the CYP variants. Over the years, at least 75 gene variants and 120 alleles have been identified in the CYP2D6 gene (www.cypalleles.ki.se). Despite the extensive number of alleles, Sistonen et al.13 showed that genotyping 12 SNPs that represent 20 different haplotypes could provide 90% to 95% accuracy in predicting the real phenotype (outward expression of genotypes). More specifically, different studies showed that the CYP2D6 extensive-metabolizer (EM) and poor-metabolizer (PM) phenotypes could be predicted with up to 99% confidence with six genotypic variants. CYP2D6*1 is considered the wild-type variant and exhibits normal enzyme activity. CYP2D6*2 has the same activity as CYP2D6*1 but is capable of duplication or amplification. Both these variants are present in EMs. The two null variants, CYP2D6*4 (defective splicing) and CYP2D6*5 (gene deletion), are predominantly found in Caucasian PMs (5%–10% of population) and result in an inactive enzyme and absence of enzyme, respectively. The predominant variants in people of Asian and African heritage are CYP2D6*10 (Pro34Ser) and CYP2D6*17 (Arg296Cys), respectively, both resulting in single-amino-acid substitution and consequent reduction in enzyme activity. In addition to *2, gene duplication had been documented for *1, *4, *6, *10, *17, *29, *35, *41, *43, and *45 variants,14 and intermediate metabolizer (IM) phenotypes have been associated with *9, *10, *17, *29, *36, and *41 variants.11
Poor CYP2D6 metabolizers carry two defective alleles, such as CYP2D6*3, CYP2D6*4 (more common), CYP2D6*5, and CYP2D6*6, resulting in a total absence of active enzyme and an impaired ability to metabolize CYP2D6-dependent substrates. Depending on the importance of the affected CYP2D6 pathway to overall drug metabolism and the drug's therapeutic index, clinically significant side effects may occur in PMs as a result of elevated parent drug concentrations. For example, compared with EMs, PMs have been shown to develop neuropathy after treatment with the antianginal agent perhexiline15 and have experienced more adverse effects with propafenone16 and neuroleptic agents such as perphenazine.17,18
The therapeutic implication of CYP2D6 polymorphism is different if the substrate in question is a prodrug. In this case, PMs would not be able to convert the drug into the therapeutically active metabolite. Two examples of prodrugs dependent on CYP2D6-mediated conversion to active forms are codeine and tramadol. Codeine and tramadol are converted by CYP2D6 to morphine and O-desmethyltramadol, respectively, and thus poor CYP2D6 metabolizers would experience little analgesic relief after taking these drugs.19,20 Another example is CYP2D6-catalyzed conversion of tamoxifen to the more potent antiestrogen metabolite, endoxifen, in which case PMs have been shown to have shortened time to recurrence of breast cancer and worse relapse-free survival.21
Furthermore, the potential and magnitude of drug interactions involving competitive inhibition of CYP2D6 are much greater in EMs versus PMs, who have either deficient or absent enzyme activity.22,23 For example, Hamelin and colleagues24 showed that in EMs, but not PMs, hemodynamic responses to metoprolol (a CYP2D6 substrate) were pronounced and prolonged during concomitant diphenhydramine administration. Thus potent CYP2D6 inhibitors may reduce the metabolic capacity of EMs significantly so that EMs appear phenotypically as PMs.
Patients who are EMs have a wide range of CYP2D6 activity, with ultrarapid metabolizers (UMs) on one end of the spectrum and intermediate metabolizers (IMs) with diminished activity on the other end. Both have clinical implications in terms of dosage adjustment for CYP2D6 substrates. UMs carry a duplicated or amplified mutant allele, resulting in two or multiple copies of the functional allele, and therefore show very high CYP2D6 activity. For the CYP2D6 substrate nortriptyline, a patient with three copies of CYP2D6*2 was shown to require doses three- to five-fold higher than normally recommended to achieve therapeutic plasma concentrations [50–150 ng/mL (50–150 mcg/L; 190–570 nmol/L)].25,26 In the same report, another patient with duplicated CYP2D6*2 required twice the usual recommended daily dose (300 mg vs 25–150 mg) to achieve adequate therapeutic response.26 There are similar reports of lower drug efficacy in UMs with antiemetics such as ondansetron.27 On the other hand, UMs administered the usual therapeutic dose of codeine might exhibit symptoms of narcotic overdose associated with high morphine concentration. This toxicity potential had been reported in several case reports.28,29 The UM phenotype also has been reported to affect the potential for drug interaction with paroxetine, a CYP2D6 substrate as well as a potent CYP2D6 inhibitor, whence a UM with three functional CYP2D6 gene copies had undetectable paroxetine concentration with standard dosing and showed no inhibitory effect at CYP2D6.30
The high prevalence of CYP2D6*10 (associated with lower enzyme activity) in the Asian population provides a biologic and molecular explanation for the higher drug concentrations and/or lower dosage requirements of neuroleptic medications and mianserin in people of Asian heritage.31,32 The widespread presence of the CYP2D6*17 variant among people of African heritage suggests that native African populations would metabolize CYP2D6 substrates at a slower rate than do other racial groups.33,34 However, there are no current genotype- and phenotype-based data to document the need for prescribing lower doses of psychotropics and other CYP2D6 substrates in native African populations.
In addition to the therapeutic implications of genetic polymorphisms, one study showed that the CYP2D6 polymorphism also has an economic impact.35 The annual cost of treating UMs and PMs (carriers of two nonfunctional CYP2D6 alleles) was $4,000 to $6,000 higher than the cost of treating EMs or IMs (carriers of one nonfunctional allele and one allele associated with diminished activity). The cost of genotyping can be considerably less than that incurred in a patient with a serious adverse drug reaction. Brockmoller and colleagues36 have suggested how CYP2D6 genotyping can be used to achieve higher therapeutic success with the CYP2D6 substrate haloperidol. Along these lines, the FDA approved the AmpliChip® CYP450 Test (Roche Diagnostics) in 2005 for analyzing 27 CYP2D6 alleles in addition to the CYP2C19*1, *2, and *3 alleles (discussed below) to assist clinicians in individualizing therapy with drugs metabolized through the CYP2D6 and 2C19 pathways.
The principal defective alleles for the CYP2C19 genetic polymorphism are CYP2C19*2 (aberrant splice site) and CYP2C19*3 (premature stop codon), resulting in inactive CYP2C19 enzyme and the PM phenotype. The clinical implication of the CYP2C19 polymorphism has not been examined as extensively as that of the CYP2D6 polymorphism. However, PMs for the CYP2C19 polymorphism showed up to a 10-fold increase in the area under the curve (AUC) of the CYP2C19 substrate omeprazole compared with EMs.37
The presence of a defective CYP2C19 allele has been associated with improved Helicobacter pylori cure rates after dual (omeprazole and amoxicillin)38 or triple (omeprazole, amoxicillin, and clarithromycin) therapy with omeprazole,39 as well as with lansoprazole.40 This difference likely reflects the higher achievable intragastric pH in the PM group.41 The cure rate achieved with dual therapy was 100% in PMs compared with 60% and 29% in heterozygous and homozygous EMs, respectively.38 In two studies, EMs had H. pylori eradication rates of 41% with dual therapy and 74% to 83% with triple therapy.39,40 In contrast, both dual- and triple-therapy regimens produced 100% cure rates in all 15 PMs included in the same studies. Interestingly, EMs who failed initial triple therapy (lansoprazole, clarithromycin, and amoxicillin) and were retreated with high-dose lansoprazole (30 mg 4 times daily) and amoxicillin achieved a 97% H. pylori eradication.42 A gene–dose effect in attainment of desirable intragastric pH ranges and H. pylori eradication rate, as well as the cost effectiveness of pharmacogenomic-guided dosing was shown for lansoprazole.43
Similar to the CYP2D6 polymorphism, people of Asian heritage also metabolize most CYP2C19 substrates at a slower rate than do Caucasians.44 This is a reflection of a higher prevalence of both PMs (13%–22.5% vs 2%–6% in Caucasians) and heterozygotes for the defective CYP2C19 allele in Asians.37 This genotypic difference may explain the practice of prescribing lower diazepam dosages for patients of Chinese heritage.45 Similar to CYP2D6 alleles with high enzyme activity, a recently identified CYP2C19*17 allele is associated with a very rapid metabolism phenotype, and carriers of this allelic variant would likely require higher doses of proton pump inhibitors 46,47 and other CYP2C19 substrates such as voriconazole.48 On the other hand, IMs and PMs of CYP2C19 may have reduced response to the antiplatelet agent clopidogrel and potentially require higher doses or alternative antiplatelet therapy (e.g., prasugrel) for better clinical outcomes. This is because clopidogrel is a prodrug that requires conversion via CYP2C19 to its active form. In IMs and PMs, usual doses of clopidogrel are less effective at inhibiting platelet aggregation and preventing cardiovascular events then in EMs.49
Warfarin, phenytoin, and tolbutamide are examples of narrow therapeutic index drugs that are metabolized by CYP2C9. Warfarin is a racemic mixture, and the S-isomer, which possesses about three times the anticoagulant effects of the R-isomer, is metabolized by CYP2C9. CYP2C9*2 and CYP2C9*3 are the two most common CYP2C9 variants in Caucasians, and both exhibit single-amino-acid substitutions at positions critical for enzyme activity.50 This could have clinically important consequences in warfarin-treated patients. For example, a 90% reduction in S-warfarin clearance was reported in CYP2C9*3 homozygotes compared with subjects homozygous for the wild-type (*1) variant.51 In another study, an overrepresentation of CYP2C9 variant alleles was observed in 81% of patients requiring low-dose warfarin therapy (≤1.5 mg/day).2 The low-dose group was reported to have more difficulty with warfarin induction, requiring longer hospital stays to stabilize the warfarin regimen and experiencing a higher incidence of bleeding complications. In addition, a profound therapeutic response to usual doses of warfarin was observed in a patient homozygous for the CYP2C9*3 allele, necessitating dose reduction to 0.5 mg/day.52 The clinical relevance of CYP2C9 polymorphism in warfarin dosing was recently reviewed in a meta-analysis of 39 studies.53 More recently, the CYP2C9*8 allele was shown to affect warfarin dose response.54 The CYP2C9*8 allele occurs in approximately 10% of African Americans and may have important implications for metabolism of CYP2C9 substrates in this population.
In addition to the wild type CYP2A6*1, several variants for the CYP2A6 polymorphism have been identified (www.cypalleles.ki.se): CYP2A6*2 (single amino acid substitution), CYP2A6*4 (gene deletion), CYP2A6*5 (gene conversion), and CYP2A6*20 (frameshift) and are associated with abolished enzyme activity. Deletion of the CYP2A6 gene is very common in Asian patients,55 which likely accounts for the dramatic difference in the frequency of PMs in Asian (20%) versus Caucasian populations (≤1%). Nicotine is metabolized by CYP2A6, and the clinical relevance of the CYP2A6 polymorphism lies in management of tobacco abuse. Investigators reported that nonsmokers were more likely to carry the defective CYP2A6 allele than were smokers. Smokers who had the defective CYP2A6 allele smoked fewer cigarettes and were more likely to quit. The inability to metabolize nicotine, secondary to the presence of a defective CYP2A6 allele, likely leads to enhanced nicotine tolerance and increased adverse effects from nicotine. Based on these observations, CYP2A6 inhibition may have a role in the management of tobacco dependency.55
Although the role of CYP2B6 in the metabolism of anticancer drugs such as cyclophosphamide and ifosfamide has been studied, it is with the antiretroviral agents that its clinical relevance was revisited and highlighted. The nonnucleoside reverse transcriptase inhibitor efavirenz is metabolized by CYP2B6. Many patients receiving efavirenz experience central nervous system adverse effects that are related to variable systemic exposure of the drug, which could be related to the lower metabolizing efficiency of the CYP2B6*6, *16, or *18 allelels.56 Additional studies validating the current literature results could lead to clinical application with more effective efavirenz treatment regimen.
Within the CYP3A subfamily, at least three isoenzymes, namely, CYP3A4, CYP3A5, and CYP3A7, have been characterized. Despite as much as 40-fold interindividual variability in its expression, functional CYP3A4 is expressed in most adults, with intestinal expression playing a significant role in the first-pass metabolism of numerous drugs. Although several CYP3A4 variants (e.g., *6, *17, *20) have been associated with reduced activity, their low frequency suggest limited clinical relevance.
CYP3A5 is reported to be polymorphic in 60% of African-Americans and 33% of Caucasians. In contrast to individuals with the CYP3A5*1 allele, subjects with variant alleles such as CYP3A5*3 (aberrant splice site) in intron 3 have no functional CYP3A5 enzyme.57 With overlapping substrate specificities, it remains unknown whether there are clinically used drugs that are substrates for CYP3A5 but not CYP3A4 and vice versa. Although variability exists between dose-adjusted concentration and CYP3A5 genotypes, studies have shown a correlation between pharmacokinetics of tacrolimus and CYP3A5 genetic constitution.58,59
Phase II and Nucleotide-Base Metabolizing Enzymes
The clinical relevance of genetic polymorphisms in TPMT, DPD, and UGT enzymes has been demonstrated in the treatment of cancer.4,60,61 The TPMT gene has four mutant alleles: TPMT*3A (the most common), TPMT*2, TPMT*3B, and TPMT*3C. Mercaptopurine is inactivated by TPMT, and patients who are homozygous or heterozygous for the TPMT mutant alleles are at higher risk for developing serious anemias during mercaptopurine treatment.4 DPD mediates the metabolism of 5-fluorouracil, and patients with a defective allele of the DPYD gene encoding for DPD cannot metabolize 5-fluorouracil and thus may experience enhanced drug-related neurotoxicity.60 The camptothecin derivative irinotecan (CPT-11) is activated by carboxylesterase to SN-38, which is a potent topoisomerase I inhibitor. SN-38 is inactivated by glucuronidation via the polymorphic UGT1A1 enzyme, which may play a role in CPT-11–related toxicity. A polymorphism in the promoter region of the UGT1A1 gene results in the (TA)7TAA allele (also known as UGT1A1*28), which possesses lower enzyme activity than the wild-type (TA)6TAA allele. Impaired SN-38 glucuronidation secondary to the (TA)7TAA allele may result in abnormally high SN-38 concentrations. A prospective clinical trial demonstrated more severe diarrhea and neutropenia in irinotecan-treated patients who are homozygous or heterozygous carriers of the (TA)7TAA allele.62 A more recent meta-analysis showed dose-related increases in the risk for toxic effects with irinotecan with the UGT1A1 (TA)7TAA allele.63 The FDA has approved the Invader® UGT1A1 Molecular Assay (Third Wave Technologies) to genotype for UGT1A1 alleles, and the labeling for irinotecan was revised to recommend dose adjustment for individuals who are homozygous for the (TA)7TAA allele.