Pharmacogenetic Relevance of Diazepam Metabolism in Human Liver Samples

Diazepam 대사의 약물유전학적 규명

Background : It has been well documented that the disposition of diazepam may differ markedly between different individuals. There is evidence to suggest a possible association between the variability of N-demethylation of diazepam and the S-mephenytoin hydroxylase polymorphism. However, the pharmacogenetic relevance of other metabolic pathways of diazepam is obscure at present. Method : Fresh human liver samples (n=14) were obtained as an excess material removed during surgery on the liver. After preparing the microsomes, the kinetic parameters of S-mephenytoin 4-hydroxylase in human liver microsomes were assessed. Enzyme kinetic parameters were obtained for the formation of oxazepam, temazepam and demethyldiazepam. The ability of S-mephenytoin to inhibit the formation of the metabolites was determined by measuring the velocity of production of these metabolites from diazepam in human liver microsomes in the presence of mephenytoin. Results : After incubating with $200{\mu}M$ diazepam, the average velocities of formation of demethyldiazepam, temazepam and oxazepam in 14 different liver preparations were $11.3{\pm}5.9,\;23.7{\pm}16.9\;and\;1.5{\pm}1.1$pmole/mg protein per min, respectively. Among the individual Michaelis-Men-ten parameters of diazepam metabolism, only the $V_{max}$ of N-demethylation gave a close correlation $(r_s=0.821,\;p<0.05)$ with the $V_{max}$ of S-mephenytoin hydroxylation. Three liver samples showed a 9-fold smaller mean $V_{max}$ of diazepam demethylation than that of the remaining 11 samples. Conclusion : This study provided further evidence that S-mephenytoin 4-hydroxylase is primarily responsible for the formation of demethylation but not for the $C_3$-hydroxylation of diazepam in Korean liver samples.

배경: 개체 간에 다양한 반응을 보이는 diazepam의 대사는 N-demethylation, $C_3$-hydroxylation등의 경로에 의해 이루어지며, 대사정도에 의해 각 개체의 약물효과가 결정된다. 이들의 대사경로 중 N-demethylation과정은 in vivo 연구에서 약물유전학적으로 결정된 S-mephenytoin hydroxylase에 의해 이루어진다고 하나 그 외의 과정은 아직 규명되어 있지 않다. 방법 : 한국인의 간 조직을 이용하여 이들의 S-mephenytoin hydroxylation 능력을 결정 한 후 S-mephenytoin hydroxylation의 대사능력 에 따르는 diazepam의 대사산물인 demethyldiazepam, temazepam및 oxazepam의 형성되는 정도를 HPLC로 측정하였다. 결과 : 14례의 한국인 간 조직에서 $200{\mu}M$의 diazepam을 incubation시켰을 때 demethyldiazepam, temazepam 및 oxazepam의 평균 형성속도는 각각 $11.3{\pm}5.9,\;23.7{\pm}16.9\;and\;1.5{\pm}1.1$pmole/mg protein/min이었다. 각종 Michaelis-Menten 지수 중 N-demethylation의 $V_{max}$와 S-mephenytoin hydroxylation의 $V_{max}$만이 통계적으로 유의한 상관관계 $(r_{s}=0.821,\;p<0.05)$가 있었다. 14례의 간 조직 중 3례는 상대적으로 낮은 $V_{max}$ 및 높은 $K_{m}$을 보였고 이 조직들은 나머지 11례의 간 조직 보다 diazepam의 N-demethylation의 $V_{max}$가 1/9에 불과하였다. 결론 : Diazepam의 대사물질인 demethyldiazepam의 형성에 S-mephenytoin hydroxylase가 밀접한 관계가 있음이 분명하였으나 한국인 간 조직에서 diazepam의 $C_{3}$-hydroxylation에는 다른 효소가 관여할 것으로 사료된다.