Narayanan Rangaraj, LeDuc Barbara, Williams David A
Nonclinical Pharmacokinetics, Bristol-Myers-Squibb, Princeton, NJ 08543, USA.
Life Sci. 2004 Apr 2;74(20):2527-39. doi: 10.1016/j.lfs.2003.10.009.
The purposes of this study were to develop a HPLC method to assay for haloperidol glucuronide (HALG); to apply this assay method to the in vitro determination of haloperidol (HAL) UDP-glucuronosyltransferase (UGT) enzyme kinetics in rat liver microsomes (RLM); and to identify the UGT isoforms catalyzing glucuronidation of HAL in rats. Incubation of Brij-activated RLM with HAL and UDP-glucuronic acid (UDPGA) in TRIS pH 7.4 buffer resulted in the formation of a single peak in the HPLC chromatogram at 270 nm. The identity of this peak was confirmed to be that of HALG by 1) beta-glucuronidase hydrolysis; 2) incubation without UDPGA; 3) UV spectral analysis; and 4) LC/MS/MS to yield the expected mass of 552.1. Enzyme kinetic studies using single enzyme Michaelis-Menton model showed an apparent Vmax = 271.9 +/- 10.1 pmoles min(-1) mg protein(-1) and Km = 61 +/- 7.2 microM. Glucuronidation activity in homozygous Gunn (j/j) rats was approximately 80% as compared to Sprague-Dawley RLM. HALG formation was approximately doubled in PB-induced RLM. There was no increase in glucuronidation activities in 3MC-induced RLM. The Gunn rat and the PB-induced RLM data suggest predominant but not exclusive involvement of the UGT2B family in the formation of HALG. Because the UGTs exhibit overlapping substrate specificities and most substrates are glucuronidated by more than one isoform, inhibition studies with UGT2B1 substrate probe testosterone and the UGT2B12 substrate probe borneol were conducted. UGT2B1 and UGT2B12 exhibited 40% and 90% inhibition of HAL glucuronidation, respectively. Thus, UGT2B12 and UGT 2B1 isoforms are responsible for catalyzing HAL glucuronidation in rats. Our HPLC assay provides a specific and sensitive technique for the measurement of in vitro HAL-UGT activity.
本研究的目的是开发一种用于测定氟哌啶醇葡萄糖醛酸苷（HALG）的高效液相色谱（HPLC）方法；将该测定方法应用于体外测定大鼠肝微粒体（RLM）中氟哌啶醇（HAL）UDP - 葡萄糖醛酸基转移酶（UGT）的酶动力学；并鉴定催化大鼠体内HAL葡萄糖醛酸化的UGT同工型。在TRIS pH 7.4缓冲液中，将经Brij活化的RLM与HAL和UDP - 葡萄糖醛酸（UDPGA）一起孵育，在HPLC色谱图中于270 nm处形成一个单峰。通过以下方法确认该峰为HALG：1）β - 葡萄糖醛酸酶水解；2）不添加UDPGA进行孵育；3）紫外光谱分析；4）液相色谱/串联质谱（LC/MS/MS），得到预期质量数为552.1。使用单酶米氏模型进行的酶动力学研究表明，表观Vmax = 271.9±10.1 pmol min⁻¹ mg蛋白⁻¹，Km = 61±7.2 μM。与Sprague - Dawley RLM相比，纯合Gunn（j/j）大鼠的葡萄糖醛酸化活性约为80%。在苯巴比妥（PB）诱导的RLM中，HALG的形成增加了约一倍。在3 - 甲基胆蒽（3MC）诱导的RLM中，葡萄糖醛酸化活性没有增加。Gunn大鼠和PB诱导的RLM数据表明，UGT2B家族在HALG的形成中起主要但非唯一的作用。由于UGT表现出重叠的底物特异性，且大多数底物由一种以上的同工型进行葡萄糖醛酸化，因此使用UGT2B1底物探针睾酮和UGT2B12底物探针冰片进行了抑制研究。UGT2B1和UGT2B12分别对HAL葡萄糖醛酸化表现出40%和90%的抑制作用。因此，UGT2B12和UGT 2B1同工型负责催化大鼠体内HAL的葡萄糖醛酸化。我们的HPLC测定法为体外HAL - UGT活性的测量提供了一种特异且灵敏的技术。
