Genetic Basis of Drug Metabolism

Margaret K. Ma, Michael H. Woo, Howard L. Mcleod

Disclosures

Am J Health Syst Pharm. 2002;59(21) 

In This Article

Genetic Polymorphisms in the CYP Isoenzymes

The CYP isoenzyme superfamily comprises over 50 heme-containing proteins that catalyze the oxidative metabolism of many structurally diverse drugs and chemicals. It is one of the most widely studied drug-metabolizing-enzyme systems. Its name is derived from the characteristic maximum spectral absorbance at 450 nm, when it is in its reduced state. CYP exists as multiple forms or isoenzymes, with each having variable distribution in different tissues. The basis for nomenclature is dependent on the similarity among the genetic sequences that encode the isoenzymes.[30] When the DNA sequence of the gene for one CYP isoenzyme is less than 40% similar to that of another, the two isoenzymes belong to different CYP families. Each enzyme family is represented by an Arabic number (e.g., CYP1) and further divided into subfamilies in which the isoenzymes' corresponding DNA sequences are approximately 70% identical. For example, CYP1A2 belongs to family 1 and sub-family A; the last Arabic number indicates a particular gene encoding for the isoenzyme within the A subfamily. More detailed descriptions of the CYP nomenclature are found in the literature.[31,32]

CYP2D6

CYP2D6 isoenzyme metabolizes 25-30% of all clinically used medications, including dextromethorphan, -blockers (e.g., metoprolol), antiarrhythmics, anti-depressants (e.g., fluvoxamine, fluoxetine, imipramine, nortriptyline), antipsychotics (e.g., haloperidol, risperidone), morphine derivatives, and many other drugs. Variability in the interindividual responses to these agents is often caused by genetic polymorphisms in CYP2D6, also termed the debrisoquin/sparteine genetic polymorphism in reference to the drugs that are its substrates that led to its discovery.[33] Unlike the CYP3A family, CYP2D6 is a noninducible enzyme; thus, its genotype offers a high predictability of CYP2D6-mediated metabolism.

CYP2D6, the gene encoding CYP2D6 isoenzyme, has the most variations of all genes for CYP isoenzymes, with more than 75 allelic variants identified to date, resulting from point mutations, single base-pair deletions or additions, gene rearrangements, and deletion of the entire gene. These mutations result in either a reduction or complete loss of activity.[34] Administering a CYP2D6 substrate as a probe drug (e.g., bufuralol, dextromethorphan, debrisoquin, sparteine) and measuring the metabolite-to-parent drug ratio in the urine (metabolic ratio) differentiate extensive metabolizers from poor metabolizers. Genotype-phenotype studies have revealed that poor metabolizers possess two nonfunctional alleles and that the phenotype is an autosomal recessive trait.[35,36] An ultrarapid metabolizer phenotype has also been identified and found to result from gene duplication (up to 13 copies of CYP2D6).[37] Poor metabolizers are more likely to have adverse effects from drugs that are substrates of the isoenzyme and decreased efficacy from drugs requiring CYP2D6-mediated activation (e.g., codeine is converted into morphine by CYP2D6), while extensive and ultrarapid metabolizers may have therapeutic failure with drugs activated by CYP2D6 (e.g., standard antidepressant doses).[38] Because CYP2D6 isoenzyme metabolizes such a large number of drugs used in the clinical setting, pharmacists have an important role in drug monitoring, including identifying CYP2D6 substrates, monitoring for drug efficacy and toxicity, and understanding the phenotypic and genotypic tools available.

The frequency of the phenotype of poor metabolizers differs among ethnic groups. Less than 1% of Asians, 2-5% of African-Americans, and 6- 10% of Caucasians are poor metabolizers of CYP2D6.[4] The most common variant alleles in Caucasians are CYP2D6*3, *4, *5, and *6, which account for about 98% of poor metabolizers.[39] The CYP2D6*3A allele is a frameshift mutation caused by a single adenine deletion in exon 5 that results in a premature stop codon. CYP2D6*4A contains a G-to-A transition in the last nucleotide of intron 3, producing a splicing defect and subsequent frameshift in the open-reading frame and premature stop codon.[40] The CYP2D6*5 variant is caused by a deletion of the entire CYP2D6 gene.[41] Although poor metabolizers may be homozygous for one particular defective allele (e.g., CYP2D6*4/*4), compound heterozygosity (e.g., CYP2D6*4/*6) is common. Despite a lower frequency of poor metabolizers, Asian and African-American populations tend to have reduced CYP2D6 activity compared with Caucasians because of a lower occurrence of nonfunctional alleles (e.g., *3, *4, *5,*6), but a higher frequency of alleles associated with reduced activity (e.g., *10, *17).[42]

Genotyping CYP2D6 has been shown to successfully predict the clearance of fluoxetine, fluvoxamine, desipramine, and mexiletine.[43,44,45,46] In some instances, the genotype for CYP2D6 has been useful in predicting adverse effects associated with antidepressants and neuroleptics. Arrhythmias, nausea, and vomiting occurred selectively in poor metabolizers during treatment with mexiletine, propafenone, and dexfenfluramine largely because of elevated plasma drug concentrations.[46,47,48] Currently, preliminary dosage recommendations based on CYP2D6 genotypes are available for antidepressants.[49] This gives us a glimpse of how pharmacogenetics can suggest dose regimens for a small population of patients. Prospective studies are warranted to address whether genotype-based dose recommendations have a positive outcome on therapy.

CYP2C9

Impaired metabolism of drugs metabolized by the CYP2C9 isoenzyme, such as phenytoin, S-warfarin,[50] tolbutamide, losartan, and nonsteroidal antiinflammatory drugs (NSAIDs) (e.g., ibuprofen, diclofenac, piroxicam, tenoxicam, mefenamic acid) has been noted.[51,52]

The CYP2C9 genotype was first observed to be correlative with the pharmacokinetics of tolbutamide. Three allelic variants of the CYP2C9 gene have been identified that are associated with decreased enzyme activity.[52,53] The variant alleles CYP2C9*2 (Arg144Cys) and *3 (Ile359Leu) contain single nucleotide polymorphisms that result in single amino acid substitutions. CYP2C9*2 and *3 were associated with a 5.5- and 27.0-fold decrease in the intrinsic clearance of S-warfarin, respectively, compared with the wild-type allele.[54,55] As such, clinical consequences of the CYP2C9*3 allele are likely to be more dramatic than those of CYP2C9*2. Homozygous CYP2C9*3 alleles were found in poor metabolizers of phenytoin, glipizide, tolbutamide, and losartan. Increased risks of bleeding were observed in patients with mutant alleles (poor metabolizers), and subsequent dosage adjustments were required.[50,56,57]

Phenytoin is a substrate of both CYP2C9 and CYP2C19 isoenzymes, but CYP2C9 is responsible for its metabolism to a greater extent; thus, mutant alleles encoding the CYP2CP gene have a greater effect on the clinical toxicity of phenytoin. The CYP2C9*3 mutant allele occurs in approximately 6-9% of Caucasians and Asians.[52]CYP2C9*2 occurs in approximately 8-20% of Caucasians and less frequently in African-Americans and is virtually absent in Asians. Individuals who require a low dose of warfarin to maintain optimum anticoagulation have a slightly higher frequency of variant CYP2C9 alleles than those who require a higher dose.[58] One study found that life-threatening bleeding was four times more likely in a group of patients requiring a lower dose of warfarin. These patients also had more difficulty establishing therapeutic anti-coagulation, which led to multiple visits to the hospital and prolonged hospital stays resulting from serious or life-threatening bleeding events, and additional required laboratory testing.[56] CYP2C9 genotyping may help identify high-risk patients who are candidates for lower warfarin doses, more frequent monitoring, or alternative drug treatments.

In addition to the metabolism of warfarin and phenytoin, polymorphisms in CYP2C9 have the potential to affect the toxicity of several NSAIDS. For example, homozygous CYP2C9*3 alleles may result in slower metabolism of these drugs. However, there have been no reports correlating CYP2C9 genotype to the pharmacokinetics of NSAIDs. Since NSAIDs have relatively high therapeutic indices, these polymorphisms may have less of an impact on clinical consequences. It is worth mentioning that a recent study has elucidated the role of CYP2C9 genotype on the metabolism of celecoxib, a cyclooxygenase-2 inhibitor.[59] The study found that patients either heterozygous or homozygous who have at least one copy of the CYP2C9*2 (i.e., CYP2C9*1/*2 or CYP2C9*2/*2) or CYP2C9*3 (i.e., CYP2C9*1/*3 or CYP2C9*3/*3) allele would have an increased celecoxib plasma area-under-the-concentration-time curve as a result of the reduced drug metabolism. The clinical significance of this observation remains unclear.

CYP2C9 genotyping may affect the clinical use of warfarin because of the relatively high prevalence of poor metabolizers, severe outcomes as a consequence of drug overdose, and frequency with which it is prescribed. CYP2C9 genotyping assays are available only for clinical research, but commercial assays are being developed.[60] Genotype testing for CYP2C9 will allow pharmacists to develop dose recommendations to reduce the risk of adverse drug reactions in patients receiving warfarin and screen for high-risk patients who are candidates for lower initial warfarin doses.[56]

CYP2C19

CYP2C19 isoenzyme metabolizes several pharmacologically important therapeutic agents. Extensive and poor metabolizers exist for S-mephenytoin, omeprazole and other proton-pump inhibitors, diazepam, propranolol, imipramine, and amitriptyline. The phenotype was initially determined using mephenytoin as a probe drug because of the significant correlation between formation of the 4-hydroxymephenytoin metabolite and the amount of CYP2C19 in human liver microsomes.[61] Phenotyping of CYP2C19 with mephenytoin is limited because of concerns about the use of the probe drug, low urinary metabolite concentration, and stability of the metabolite in urine. More recently, omeprazole and other substrates have been used in phenotyping studies. The poor metabolizer phenotype is a result of two nonfunctional alleles and is inherited as an autosomal recessive trait. In contrast, the extensive metabolizer phenotype consists of both heterozygous and homozygous dominant genotypes, but they usually cannot be distinguished by phenotyping methods.

At least five mutant alleles have been identified.[62] The most common variant alleles in poor metabolizers, CYP2C19*2 and *3, arise from single base-pair substitutions in exons 4 (CYP2C19*3) and 5 (CYP2C19*2) that introduce premature stop codons and truncated polypeptide chains with no functional activity.[63]CYP2C19*2 is identified with a 40 base-pair deletion at the beginning of the exon, which shifts the reading frame to create an aberrant splice site and produces a premature stop codon about 20 amino acids downstream.[64]CYP2C19*3 is mainly found in the Japanese population. There are also ethnic differences in the frequency of the poor metabolizer phenotype. About 3-5% of Caucasians and 12- 23% of most Asian populations are poor metabolizers.[65,66]

Although relatively few drugs are metabolized by CYP2C19, pronounced pharmacodynamic effects tend to be seen in Asians treated with omeprazole because of the higher frequency of poor metabolizers.[67] Poor metabolizers of mephenytoin also have higher serum omeprazole- metabolite ratios than extensive metabolizers because of impaired omeprazole metabolism.[68] Results of a study by Furuta et al.[69] suggested that the CYP2C19 genotype might influence the cure rates for Helicobacter pylori infection in patients with peptic ulcers. The cure rate was 100% in poor metabolizers, 60% in patients with heterozygous genotypes, and 29% in patients with homozygous wild-types. This may be explained by the higher accumulation of plasma omeprazole concentrations in poor metabolizers, resulting in a greater degree of gastric acid suppression.[67] Similar to omeprazole, the extent of lansoprazole and pantoprazole metabolism is highly dependent on CYP2C19 genotype. Thus, interindividual differences in plasma concentrations of these proton-pump inhibitors may be prospectively predicted by genetically determined CYP2C19 status. However, the contributions of CYP2C19 isoenzyme to the metabolism of these proton-pump inhibitors are not evenly distributed; omeprazole is more extensively metabolized by CYP2C19 than pantoprazole, lansoprazole, and rabeprazole.[70]

Diazepam is another example of a CYP2C19 substrate affected by this polymorphism. The half-life of diazepam in plasma is significantly prolonged in individuals who are homozygous for the mutant CYP2C19*2 allele (80 hours) compared with heterozygotes (64 hours) and people with homozygous wild-type CYP2C19 (20 hours).[71,72] Asian populations have been reported to exhibit slower diazepam metabolism than Caucasians, possibly due to a higher frequency of CYP2C19*2 and CYP2C19*3 genotypes. Poor metabolizers of CYP2C19 may be at a higher risk for diazepam toxicity, and caution must be exercised in dosing diazepam.

CYP3A Subfamily


CYP3A isoenzymes are the predominant subfamily of CYP enzymes, making it one of the most important drug-metabolizing enzymes. The genes for CYP3A isoenzymes are expressed primarily in the liver and small intestines.[73,74] Hepatic CYP3A4 isoenzyme has been estimated to metabolize almost 50% of currently used drugs as well as endogenous and exogenous corticosteroids. Intestinal CYP3A4 isoenzyme contributes significantly to the first-pass metabolism of orally administered drugs.[75] There is large interindividual variability in genetic expression for, CYP3A exceeding 30-fold in some populations,[76] but evidence for polymorphic activity has been elusive until recently. Consequently, these variations play a significant role in the variability of oral bioavailability and metabolism of CYP3A substrates, including HIV protease inhibitors, benzodiazepines, calcium channel blockers, hydroxymethylglutaryl coenzyme A- reductase inhibitors, antineoplastic drugs, nonsedating antihistamines, and immunosuppressants. These variations can result in differences in drug efficacy and toxicity among individuals.

CYP3A activities are the sum of the activities of at least three CYP3A isoenzymes: CYP3A4, CYP3A5, and CYP3A7.[77,78] Interindividual variability due to CYP3A4 activity alone may vary by up to 50-fold. Functional consequences of a polymorphism in the CYP3A4 promoter region (A290G) (i.e., CYP3A4*1B) have been studied as a possible cause of this variability,[79,80,81,82] but the effects of this SNP have not been clearly defined. Ethnic variation has been suggested as a possible reason, which may explain the higher frequency of the homozygous mutation in Ghanaian (51%) than in Scottish Caucasian (0%) and Saudi populations (1%).[83]

It is estimated that CYP3A5 isoenzyme is only present in 10-30% of liver samples tested.[84,85] Recently, CYP3A5 isoenzyme was found to account for at least 50% of the total CYP3A content in people who carry the wild-type CYP3A5*1 allele (96- 98% of the combined population), suggesting that CYP3A5 may play a significant role in the metabolism of CYP3A substrates.[86] Only people with at least one wild-type CYP3A5*1 allele express large amounts of CYP3A5 isoenzyme, resulting in a 2.5-fold increase in clearance of the probe drug (midazolam). The most common cause of loss of the expression of the CYP3A5 gene in the liver is an SNP at nucleotide 22,893 in intron 3 (CYP3A5*3), which causes alternative splicing (exon 3B) and protein truncation. The CYP3A5*6 allele contains a G30597A mutation in exon 7, causing the deletion of exon 7 and reducing CYP3A5 activity. CYP3A5*1 is more frequently expressed in non-Caucasian populations (30% of Caucasians, Japanese, and Mexicans; 40% of Chinese; and 60% of African-Americans, Southeast Asians, Pacific Islanders, and Southwestern American Indians); thus, these populations may metabolize CYP3A substrates more rapidly.[86] CYP3A5 appears to be an important genetic contributor to interindividual and interracial differences in CYP3A-dependent drug metabolism. Patients expressing both wild-type CYP3A4 and CYP3A5 genotypes extensively metabolize CYP3A substrates, which may lead to a lack of therapeutic effect. Prediction of drug interactions involving the inhibition and induction of CYP3A will continue to be a challenge and clinically important because of the diverse role CYP3A plays in the metabolism of currently available and future drugs. However, the variability observed in CYP3A activity may not be solely due to polymorphisms in the genes for these isoenzymes. The recent discovery of the PXR gene, a regulator of CYP gene expression, provides another possible factor for the variability in drug metabolism and a molecular basis for drug interactions. Identifying the regulatory mechanisms of enzyme induction and polymorphisms within these regulatory elements and high-throughput screening for future drugs may allow accurate prediction of CYP3A enzyme induction and drug- drug interactions.[87,88,89,90,91] Specific guidelines for the use of CYP3A pharmacogenetics to modulate drug therapy are under development.

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