O'Donnell John P, Dalvie Deepak K, Kalgutkar Amit S, Obach R Scott
Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Groton, CT 06340, USA.
Drug Metab Dispos. 2003 Nov;31(11):1369-77. doi: 10.1124/dmd.31.11.1369.
The nonsteroidal anti-inflammatory agent (+ or -)-suprofen [alpha-methyl-4-(2-thienylcarbonyl)benzeneacetic acid] was evaluated as a P450 2C9 inactivator. (+ or -)-Suprofen inactivated the diclofenac-4-hydroxylase activity of baculovirus-expressed P450 2C9 in a time- and concentration-dependent manner, which was consistent with mechanism-based inactivation. The loss of activity followed pseudo-first-order kinetics and was suprofen- and NADPH-dependent. The kinetic parameters for inactivation kinact and KI were 0.091 min-1 and 3.7 microM, respectively, and the partition ratio was 101. Although P450 2C9 substrate S-warfarin partially protected against inactivation, reactive oxygen scavengers such as superoxide dismutase and catalase did not prevent inactivation. Extensive dialysis did not regenerate enzyme activity, suggesting that inactivation proceeded via covalent modification. Inactivated P450 2C9 lost <10% of its ability to form a CO-reduced complex, suggesting that inactivation may have resulted from covalent modification of apoprotein. Addition of exogenous nucleophiles such as glutathione and semicarbazide partially protected against inactivation. Apart from the metabolism of suprofen to 5-hydroxysuprofen, the formation of a suprofen-glutathione conjugate was also discernible in microsomal mixtures containing glutathione. Time of flight mass spectrometry revealed a protonated monoisotopic mass of 566.1304 for this conjugate, consistent with an elemental composition of C24H28N3O9S2. The mass spectrum indicated that conjugation had occurred on the intact thiophene ring, presumably via a thioether linkage. Further evidence for the formation of an electrophilic intermediate in suprofen-P450 2C9 incubations was obtained via the characterization of a novel pyridazine adduct upon addition of semicarbazide to the microsomal mixtures. The pyridazine derivative had a protonated monoisotopic mass of 257.0895 that was consistent with an elemental composition of C14H13O3N2. The formation of the stable pyridazine adduct suggested the generation of an electrophilic gamma-thioketo-alpha, beta-unsaturated aldehyde, analogous to that observed during the cytochrome P450-mediated bioactivation of furan. This electrophilic alpha, beta-unsaturated aldehyde represents a possible reactive intermediate that bioalkylates P450 2C9.
非甾体抗炎药（±）舒洛芬[α-甲基-4-（2-噻吩基羰基）苯乙酸]被评估为一种细胞色素P450 2C9失活剂。（±）舒洛芬以时间和浓度依赖性方式使杆状病毒表达的细胞色素P450 2C9的双氯芬酸-4-羟化酶活性失活，这与基于机制的失活一致。活性丧失遵循假一级动力学，且依赖于舒洛芬和NADPH。失活的动力学参数kinact和KI分别为0.091 min-1和3.7 μM，分配比为101。尽管细胞色素P450 2C9底物S-华法林可部分保护其免受失活，但超氧化物歧化酶和过氧化氢酶等活性氧清除剂并不能阻止失活。广泛透析不能使酶活性恢复，这表明失活是通过共价修饰进行的。失活的细胞色素P450 2C9形成一氧化碳还原复合物的能力丧失<10%，这表明失活可能是由于脱辅基蛋白的共价修饰所致。添加谷胱甘肽和氨基脲等外源性亲核试剂可部分保护其免受失活。除了舒洛芬代谢为5-羟基舒洛芬外，在含有谷胱甘肽的微粒体混合物中也可检测到舒洛芬-谷胱甘肽共轭物的形成。飞行时间质谱显示该共轭物的质子化单同位素质量为566.1304，与元素组成C24H28N3O9S2一致。质谱表明共轭发生在完整的噻吩环上，推测是通过硫醚键。通过在微粒体混合物中添加氨基脲后对一种新型哒嗪加合物的表征，获得了舒洛芬-P450 2C9孵育中亲电中间体形成的进一步证据。哒嗪衍生物的质子化单同位素质量为257.0895，与元素组成C14H13O3N2一致。稳定哒嗪加合物的形成表明生成了亲电γ-硫代酮-α,β-不饱和醛，类似于在细胞色素P450介导的呋喃生物活化过程中观察到的情况。这种亲电α,β-不饱和醛代表了一种可能的反应中间体，可对细胞色素P450 2C9进行生物烷基化。
