The impact of experimental design on assessing mechanism-based inactivation of CYP2D6 by MDMA (Ecstasy)
The impact of experimental design on assessing mechanism-based inactivation of CYP2D6 by MDMA (Ecstasy)
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DOI：
10.1177/0269881106062902
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发表时间：
2006-11-01
影响因子：
4.1
通讯作者：
Rostami-Hodjegan, Amin
中科院分区：
文献类型：
--
作者：
Van, Linh M.;Heydari, Amir;Rostami-Hodjegan, Amin
MDMA（3-4-亚甲二氧基甲基苯丙胺，通常称为Ectamphetamine）是细胞色素P450 2D 6（CYP 2D 6）的一种有效的基于机制的抑制剂（MBI），可在体外引起酶的准不可逆抑制。对这种现象的体内影响的评价取决于定义体外抑制的参数估计值的准确性，即k（完整）（最大抑制率）和K-I（灭活常数）。这些值分两步测定，酶与抑制剂预孵育（酶失活），然后稀释并进一步孵育，以测量探针底物的残留酶活性。本研究的目的是评估不同稀释度和探针底物浓度对使用重组表达的CYP 2D 6估计的k（无效）和K-I的影响。通过将右美沙芬（DEX）转化为右啡烷（DOR）来测量酶活性。稀释因子为1.25、2、5、10、25和50（DEX，30 μ M）的k（无效）（min（-1））的平均（+/- SE）值为0.20 +/- 0.06，0.21 +/- 0.05，0.31 +/- 0.06，0.37 +/- 0.11，分别为0.51 +/- 0.10和0.58 +/- 0.08，K-I（μ M）值（校正非特异性微粒体结合后）为2.22 +/- 1.90、2.80 +/- 1.34、5.78 +/- 2.07、6.36 +/- 2.93、3.99 +/- 1.57和4.86 +/- 1.37，分别因此，高（例如50倍）和低（例如1.25倍）稀释度与动力学值的统计学显著差异相关（p < 0.05）。当使用5倍稀释液时，不同的DEX浓度（10-100 μ M）与k（无效）和K-I值的显著变化无关（10 μ M DEX时K-I较低除外）。高稀释度也显示出减少MDMA的非特异性微粒体结合。两个动力学参数的变化取决于实验程序，并且显示不太可能对MDMA典型娱乐剂量（50-100 mg）后体内预期的CYP 2D 6最大抑制产生实质性影响，因为抑制效力较高。预测动力学参数的不同值对重新合成CYP 2D 6后酶活性恢复的时间有轻微影响。
MDMA (3-4-methylenedioxymethamphetamine, commonly known as Ecstasy) is a potent mechanism-based inhibitor (MBI) of cytochrome P450 2D6 (CYP2D6), causing quasi-irreversible inhibition of the enzyme in vitro. An evaluation of the in vivo implications of this phenomenon depends on the accuracy of the estimates of the parameters that define the inhibition in vitro, namely k(intact) (the maximal inhibition rate) and K-I (the inactivation constant). These values are determined in two steps, pre-incubation of the enzyme with the inhibitor (enzyme inactivation), followed by dilution and further incubation to measure residual enzyme activity with a probe substrate. The aim of this study was to assess the impact of different dilutions and probe substrate concentrations on the estimates of k(inact) and K-I using recombinantly expressed CYP2D6. Enzyme activity was measured by the conversion of dextromethorphan (DEX) to dextrorphan (DOR). Dilution factors of 1.25, 2, 5, 10, 25 and 50 (DEX at 30 mu M) gave mean (+/- SE) values of k(inact) (min(-1)) of 0.20 +/- 0.06, 0.21 +/- 0.05, 0.31 +/- 0.06, 0.37 +/- 0.11, 0.51 +/- 0.10 and 0.58 +/- 0.08, respectively, and K-I (mu M) values (after correction for non-specific microsomal binding) of 2.22 +/- 1.90, 2.80 +/- 1.34, 5.78 +/- 2.07, 6.36 +/- 2.93, 3.99 +/- 1.57 and 4.86 +/- 1.37, respectively. Accordingly, high (e.g. 50 fold) and low (e.g. 1.25 fold) dilutions were associated with statistically significant differences in kinetic values (p < 0.05). Varying DEX concentration (10-100 mu M) was not associated with significant changes in k(inact) and K-I values when a five-fold dilution was used (with the exception of a Lower K-I at 10 mu M DEX). High dilution was also shown to reduce non-specific microsomal binding of MDMA. The changes in the two kinetic parameters were dependent on the experimental procedure and shown to be unlikely to have a material influence on the maximum inhibition of CYP2D6 expected in vivo after typical recreational doses of MDMA (50-100 mg), since the potency of inhibition was high. The different values of the kinetic parameters were predicted to have a marginal influence on the time for recovery of enzyme activity following re-synthesis of CYP2D6.