Paradoxical urinary phenytoin metabolite (S)/(R) ratios in CYP2C19*1/*2 patients
Introduction
Although a number of antiepileptic drugs (AEDs) have been introduced in the last 15 years, PHT (5,5-diphenylhydantoin) remains among the most frequently prescribed drugs for the treatment of epilepsy. Over the past 65 years, PHT has been intensively studied; nonetheless treatment failure or adverse effects are relatively common because of its saturable metabolism and narrow therapeutic range. In addition, genetically determined variations of its major enzymes, CYP2C9 and CYP2C19, may lead to altered clearance. PHT is primarily metabolized via an arene-oxide intermediate to 5-(4′-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), an inactive metabolite that accounts for approximately 67–88% of administered PHT. p-HPPH is then excreted into the urine as a glucuronide. A dihydrodiol metabolite of PHT (via an arene oxide) accounts for an additional 7–11%. Less than 5% of unmetabolized PHT is renally eliminated (Dill et al., 1956, Browne and Leduc, 2002).
Recent advances in pharmacogenomics have provided a greater understanding of some of the factors affecting PHT metabolism. p-HPPH is formed as a mixture of stereoisomers (R)-p-HPPH and (S)-p-HPPH. The prochiral para-hydroxylation of PHT is catalyzed by CYP2C9 and CYP2C19 (Fig. 1). CYP2C9 is approximately 40 times more stereoselective towards formation of the (S) isomer, whereas hydroxylation by CYP2C19 results in an (S)/(R) ratio close to one (Bajpai et al., 1996). Both CYP2C9 and CYP2C19 are polymorphic and these polymorphisms can impact the clinical effects of PHT. A high percentage of Japanese and Chinese (18–23%) lack CYP2C19 activity, compared to 2–5% of Caucasians. Although CYP2C9*3 is the primary determinant of PHT poor metabolism, other CYP2C9 and CYP2C19 alleles have been shown to play a role in decreased metabolic clearance (Desta et al., 2002).
The most commonly occurring mutant alleles of CYP2C9 are CYP2C9*2 (p.Arg144Cys) and CYP2C9*3 (p.Ile359Leu). The corresponding single nucleotide polymorphisms (SNP) are nucleotide c.430C > T and c.1075A > T, respectively (Bhasker et al., 1997, Stubbins et al., 1996, Sullivan-Klose et al., 1996). When mutant proteins were cloned and expressed in COS-1 cells, the catalytic activity of CYP2C9.2 enzyme towards PHT was moderately affected whereas activity of CYP2C9.3 enzyme was dramatically reduced [decreased maximum velocity of metabolite formation (Vmax), increased Michealis–Menten constant (Km)] (Veronese et al., 1991). Patients with CYP2C9*2/*2 or who are heterozygous for CYP2C9*3 are slow metabolizers of PHT, showing an elevated serum PHT concentration and a marked decrease in PHT clearance. Kidd et al. (1999) determined that the clearance of PHT in a single individual with CYP2C9*1/*3 was 21% of wild type. The mean 12 h concentration of PHT in serum after a 300 mg dose to three healthy Turkish volunteers (CYP2C9*2/*2) was 41% higher than the wild type subjects (Aynacioglu et al., 1999). Allele frequencies for CYP2C9*2 are approximately 0.08 in Caucasians, 0.01 in African Americans, 0.0 in Chinese–Taiwanese, whereas those for CYP2C9*3 are 0.06, 0.005, and 0.026, respectively (Sullivan-Klose et al., 1996). Xie et al. (2002) reported the frequencies of CYP2C9*2 and CYP2C9*3 in Caucasians to range from 8 to 19% and 3.3 to 16.2%, respectively.
Most variants of CYP2C19 identified to date result in non-functional or absent enzyme. CYP2C19*2 was first reported in 1994 and it differs from the CYP2C19*1 in that it has a point mutation in exon 5, c.681G > A. This results in a splicing defect in exon 5, leading to premature termination of protein synthesis (de Morais et al., 1994a). CYP2C19*3 is a truncated protein resulting from a premature stop codon formed by a single base change c.636G > A (De Morais et al., 1994b). In a Caucasian population, CYP2C19*2 and CYP2C19*3 allelic frequencies are reported to be 15% and 0.04%, respectively (Desta et al., 2002).
The CYP2C19*2 and CYP2C9*3 polymorphisms have clinical significance and are most commonly associated with slow PHT metabolism accounting for almost all Asian poor metabolizers and 75% of Caucasian poor metabolizers of (S)-mephenytoin, a common CYP2C9 substrate (Lee et al., 2002, Smith et al., 1998). To date, the effect of genotype on (S) and (R) ratios in the urine has not been studied under steady-state conditions. We carried out the current investigation to confirm and further characterize the effect of CYP2C9 and CYP2C19 polymorphsims on PHT para-hydroxylation in epilepsy patients who were maintained on PHT without interacting co-medications.
Section snippets
Patient population
The subjects included in this report were participants in a clinical investigation of the effects of age, sex, and genotype on the disposition of AEDs. Subjects were patients recruited from epilepsy clinics and nursing homes in the Minneapolis (MN) metropolitan area. Informed consent was obtained from all subjects. The study was approved by the University of Minnesota Human Subjects Committee. All patients were on maintenance PHT therapy, were not taking any interacting drugs, and were studied
Genotyping
Forty-five patients (25 men, 20 women) participated in this study. Forty-one of the forty-five subjects provided 0–12 and 12–24 h urine collections, whereas two subjects provided a sample only during the first 12 h and two during the next sampling interval. Genotype frequency for the wild type, heterozygous, or homozygous variant alleles are presented in Table 3. The allelic frequency for CYP2C9*2 and CYP2C9*3 was 0.10 and 0.04, respectively. Genotyping of CYP2C19 revealed that the CYP2C19*3
Discussion
Individuals heterozygous for the CYP2C19*2 would be expected to produce very little (R) isomer because the mutant CYP2C19.2 enzyme would be inactive and the major elimination pathway would be CYP2C9.1 enzyme, producing predominantly the (S) isomer. The expected (S)/(R) ratio would be close to 40, but was actually surprisingly lower at 12.9 ± 1.7 in the 12 individuals heterozygous for the CYP2C19*2 polymorphism in our study. This surprising decrease could be explained by a linkage disequilibrium
Conclusions
We found an unexpected relationship between (S)/(R)-p-HPPH ratios measured under steady-state conditions and CYP2C9*1/*1, CYP2C19*1/*2 genotype. As expected, the (S)/(R)-p-HPPH ratio is reduced in patients with a CYP2C9*1/*2 mutation and is markedly decreased in those with the CYP2C9*1/*3 genotype. In patients with the CYP2C9*1/*1, CYP2C19*1/*2 haplotype, however, we found that the (S)/(R)-p-HPPH ratio paradoxically decreased, suggesting that there is an unknown SNP in the CYP2C9 gene, in turn
Acknowledgements
This study was funded by a grant from National Institute of Neurological Disorders and Stroke (NIH Grant NINDS P50 NS16308) and General Clinical Research Center Program (GCRC: M01-RR00400). We thank Dr. Erin Schuetz for valuable discussions about the genetic aspects of this project and we thank Dr. Richard Brundage and Gauri Khandekar for their assistance with statistical analysis.
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