Wei Xiaohui, Dai Guowei, Liu Zhongfa, Cheng Hao, Xie Zhiliang, Klisovic Rebecca, Marcucci Guido, Chan Kenneth K
Division of Pharmaceutics, College of Pharmacy, Ohio State University, Columbus, OH 43210, USA.
Drug Metab Dispos. 2008 Nov;36(11):2227-33. doi: 10.1124/dmd.108.021295. Epub 2008 Jul 24.
Enzyme kinetics of GTI-2040 (5'-GGC TAA ATC GCT CCA CCA AG-3'), a phosphorothioate ribonucleotide reductase antisense, were investigated for the first time in 3' exonuclease solution and human liver microsomes (HLMs), using the ion-pair high-performance liquid chromatogram method for quantification of the parent drug and two major 3'N-1 and 3'N-2 metabolites. Enzyme kinetics of GTI-2040 in 3'-exonuclease solution were found to be well characterized by the Michaelis-Menten model, using the sum of formation rates of 3'N-1 and 3'N-2 (approximately total metabolism) because of sequential metabolism. In HLMs, a biphasic binding was observed for GTI-2040 with high- and low-affinity constants (K(d)s) of 0.03 and 3.8 microM, respectively. Enzyme kinetics of GTI-2040 in HLMs were found to deviate from Michaelis-Menten kinetics when the total GTI-2040 substrate was used. However, after correction for the unbound fractions, the formation rate of total metabolites could be described by Michaelis-Menten kinetics. Using the free substrate fraction, the K(m) and V(max) of GTI-2040 were determined to be 6.33 +/- 3.2 microM and 16.5 +/- 8.4 nmol/mg/h, respectively. Using these values, in vitro hepatic intrinsic clearance (CL(int)) in HLM was estimated to be 2.61 +/- 0.56 ml/h. The CL(int) was then used to predict GTI-2040's in vivo intrinsic clearance in humans by a microsomal protein scaling factor, which gave a mean value of 182.7 l/h, representing 24.1% of the observed in vivo mean scaled hepatic intrinsic clearance of 758.7 l/h in patients with acute myeloid leukemia. We concluded that the saturable nonspecific binding of GTI-2040 in HLMs complicated the interpretation of its enzyme kinetics, and scaled intrinsic clearance from HLMs only partially predicted the in vivo intrinsic clearance.
首次在3'外切核酸酶溶液和人肝微粒体（HLMs）中，使用离子对高效液相色谱法对硫代磷酸核糖核苷酸还原酶反义寡核苷酸GTI-2040（5'-GGC TAA ATC GCT CCA CCA AG-3'）的酶动力学进行了研究，以定量母体药物和两种主要的3'N-1和3'N-2代谢物。由于顺序代谢，使用3'N-1和3'N-2的形成速率之和（近似总代谢），发现GTI-2040在3'-外切核酸酶溶液中的酶动力学可用米氏模型很好地表征。在HLMs中，观察到GTI-2040具有双相结合，高亲和力常数（K(d)s）和低亲和力常数分别为0.03和3.8 microM。当使用总GTI-2040底物时，发现GTI-2040在HLMs中的酶动力学偏离米氏动力学。然而，在校正未结合部分后，总代谢物的形成速率可用米氏动力学描述。使用游离底物部分，GTI-2040的K(m)和V(max)分别确定为6.33±3.2 microM和16.5±8.4 nmol/mg/h。使用这些值，估计HLM中的体外肝内在清除率（CL(int)）为2.61±0.56 ml/h。然后，通过微粒体蛋白比例因子，使用CL(int)预测GTI-2040在人体内的体内内在清除率，其平均值为182.7 l/h，占急性髓性白血病患者体内观察到的平均比例化肝内在清除率758.7 l/h的24.1%。我们得出结论，GTI-2040在HLMs中的可饱和非特异性结合使其酶动力学的解释复杂化，并且从HLMs按比例缩放的内在清除率仅部分预测了体内内在清除率。
