Iwai Megumi, Suzuki Hiroshi, Ieiri Ichiro, Otsubo Kenji, Sugiyama Yuichi
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.
Pharmacogenetics. 2004 Nov;14(11):749-57. doi: 10.1097/00008571-200411000-00006.
Two kinds of single nucleotide polymorphism (SNP; Asn130Asp and Val174Ala) are frequently observed in the liver specific transporter, organic anion transporting polypeptide 1B1 (OATP1B1/OATP-C) gene. Although these two SNPs occur independently in European-Americans, Val174Ala is mostly associated with Asn130Asp in Japanese. Our previous in-vivo studies in Japanese subjects indicated that the non-renal clearance of pravastatin was decreased to 13% of that in wild-type subjects (Nishizato et al. Clin Pharmacol Ther 2003;73(6):554-564). The purpose of the present study is to characterize the function of SNPs variants of OATP1B1 in cDNA transfected cells.
The localization and transport activity were analyzed in HEK293 cells stably expressing wild-type OATP1B1 (OATP1B11a), OATP1B11b (Asn130Asp), OATP1B15 (Val174Ala) and OATP1B115 (Asn130Asp and Val174Ala). To characterize the intrinsic Vmax, observed Vmax in uptake study were normalized by the expression level estimated from Western blotting.
All SNP variants are predominantly located on the cell surface. No significant alteration was observed in Km values for the transport of 17beta-estradiol 17beta-d-glucuronide (E217betaG), a typical substrate of OATP1B1, among these SNP variants. However, the normalized Vmax value for OATP1B115 was drastically decreased to less than 30% compared with OATP1B11a. In contrast, the transport activity of OATP1B11b (Asn130Asp) and OATP1B15 (Val 174Ala) was similar to that of OATP1B1*1a.
These results are consistent with the results of our previous clinical studies. It is thus suggested that in-vivo disposition may be predicted from in-vitro results using recombinant transporters.
在肝脏特异性转运体有机阴离子转运多肽1B1（OATP1B1/OATP-C）基因中经常观察到两种单核苷酸多态性（SNP；Asn130Asp和Val174Ala）。虽然这两种SNP在欧裔美国人中独立出现，但在日本人中Val174Ala大多与Asn130Asp相关。我们之前在日本受试者中的体内研究表明，普伐他汀的非肾清除率降至野生型受试者的13%（Nishizato等人，《临床药理学与治疗学》2003年；73(6):554 - 564）。本研究的目的是在cDNA转染细胞中表征OATP1B1的SNP变体的功能。
在稳定表达野生型OATP1B1（OATP1B11a）、OATP1B11b（Asn130Asp）、OATP1B15（Val174Ala）和OATP1B115（Asn130Asp和Val174Ala）的HEK293细胞中分析定位和转运活性。为了表征内在Vmax，摄取研究中观察到的Vmax通过蛋白质印迹法估计的表达水平进行归一化。
所有SNP变体主要位于细胞表面。在这些SNP变体中，对于OATP1B1的典型底物17β-雌二醇17β-D-葡萄糖醛酸（E217βG）的转运，Km值未观察到显著变化。然而，与OATP1B11a相比，OATP1B115的归一化Vmax值急剧下降至不到30%。相比之下，OATP1B11b（Asn130Asp）和OATP1B15（Val174Ala）的转运活性与OATP1B1*1a相似。
这些结果与我们之前的临床研究结果一致。因此表明，使用重组转运体的体外结果可以预测体内处置情况。
