In Vivo Functional Effects of CYP2C9 M1L, a Novel and Common Variant in the Yup'ik Alaska Native Population

Abstract

Alaska Native people are under-represented in genetic research but have unique gene variation that may critically impact their response to pharmacotherapy. Full resequencing of CYP2C9 in a cross-section of this population identified CYP2C9 Met1Leu (M1L), a novel, relatively common single nucleotide polymorphism hypothesized to confer CYP2C9 poor metabolizer phenotype by disrupting the start codon. M1L is present at a minor allele frequency of 6.3% in Yup'ik Alaska Native people and thus can contribute to the risk of an adverse drug response from narrow-therapeutic-index CYP2C9 substrates such as (S)-warfarin. This study's objective was to characterize the catalytic efficiency of the Leu1 variant enzyme in vivo by evaluating the pharmacokinetic behavior of naproxen, a probe substrate for CYP2C9 activity, in genotyped Yup'ik participants. We first confirmed the selectivity of (S)-naproxen O-demethylation by CYP2C9 using activity-phenotyped human liver microsomes and selective cytochrome P450 inhibitors and then developed and validated a novel liquid chromatography mass spectrometry method for simultaneous quantification of (S)-naproxen, (S)-O-desmethylnaproxen, and naproxen acyl glucuronide in human urine. The average ratio of (S)-O-desmethylnaproxen to unchanged (S)-naproxen in urine was 18.0 ± 8.0 (n = 11) for the homozygous CYP2C9Met1 reference group and 10.3 ± 6.6 (n = 11) for the Leu1 variant carrier group (P = 0.011). The effect of M1L variation on CYP2C9 function and its potential to alter the pharmacokinetics of drugs metabolized by the enzyme has clinical implications and should be included in a variant screening panel when pharmacogenetic testing in the Alaska Native population is warranted. SIGNIFICANCE STATEMENT: The novel CYP2C9 Met1Leu variant in Alaska Native people was recently identified. This study validated (S)-naproxen as a CYP2C9 probe substrate to characterize the in vivo functional activity of the CYP2C9 Leu1 variant. The results of this pharmacogenetic-pharmacokinetic study suggest that the CYP2C9 Leu1 variant exhibits loss of enzyme activity. This finding may be important to consider when administering narrow-therapeutic-index medications metabolized by CYP2C9 and also compels further investigation to characterize novel genetic variation in understudied populations.

Fig. 1.

Study inclusion based on CYP2C9 M1L screening results.

Fig. 2.

Representative Michaelis-Menten plot of (S)-O-desmethylnaproxen formation in pooled HLMs. Data are means ± S.D. Individual data points represent means of technical triplicates at a given (S)-naproxen concentration from the same experimental replicate, and the solid line reflects the fit of a single-enzyme Michaelis-Menten model to the data.

Fig. 3.

P450 Supersome screen at a sub-K_m (S)-naproxen concentration (25 μM, closed bars) and a saturating concentration (1000 μM, open bars). Data are mean values across two repeated experiments, each with technical triplicates.

Fig. 4.

Michaelis-Menten plot of (S)-O-desmethylnaproxen formation by CYP2C9, CYP1A2, and CYP2C8 Supersomes. The displayed results are from a representative experiment. Individual data points represent the means of technical duplicates at a given (S)-naproxen concentration, and the solid lines reflect the fit of a single-enzyme Michaelis-Menten model to the data.

Fig. 5.

Effects of selective CYP2C9 and CYP1A2 inhibitors on (S)-O-desmethylnaproxen formation in pooled HLMs. Sulfaphenazole (SLF) was dissolved in methanol (MeOH), <0.2% final concentration, and furafylline (FF) was dissolved in DMSO, <0.1% final concentration. (S)-Naproxen substrate concentration was 20 μM. Percent inhibition was calculated relative to the control containing no inhibitor. Data are means ± S.D. across three repeated experiments, with technical triplicates.

Fig. 6.

Urinary metabolite-to-parent ratio of (S)-O-desmethylnaproxen to unchanged (S)-naproxen by M1L genotype. The regression analysis for the comparison between the CYP2C9 Met1/Met1 reference group (n = 11) and Leu1 variant carrier group (combined Met1/Leu1 heterozygotes and Leu1/Leu1 homozygotes) (n = 11) allowed for heteroscedasticity; *P < 0.05.