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"Tofacitinib Is a Mechanism-Based Inactivator of Cytochrome P450 3A4": Revisiting the Significance of the Epoxide Intermediate and Glutathione Trapping.
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2019-12-18 , DOI: 10.1021/acs.chemrestox.9b00455
Vijayabhaskar Veeravalli 1, 2 , Ranjeet P Dash 1, 2, 3
Affiliation  

Tofacitinib is an orally administered medication with a mechanism of action that involves JAK1, JAK2, JAK3, and TYK2 pathway inhibition.(1) There is increasing interest in the use of this drug in the management of psoriatic arthritis, following its success and wide use as an approved treatment option for patients with rheumatoid arthritis.(2) Pharmacodynamically, the use of tofacitinib resulted in the reduction of circulating CD16/56+ natural killer cells, which was reversed in 2–6 weeks after stopping the medication.(3) Pharmacokinetic studies on tofacitinib demonstrated its rapid absorption with a peak plasma concentration at 0.5–1 h and a rapid elimination with a mean half-life of 2–3 h.(4) Clearance mechanisms for tofacitinib are approximately 70% by hepatic metabolism and 30% via renal excretion of the parent drug.(5) The metabolism of tofacitinib is primarily mediated by CYP3A4 with minor contribution from CYP2C19. In vitro metabolic studies have identified hydroxy, dihydroxy, and glucuronide of the dihydroxy tofacitinib as the major metabolites.(6) Recently, Guo et al. reported a well-designed and executed in vitro metabolism study to elucidate the mechanism-based inactivation of tofacitinib by trapping reactive metabolites generated in microsomal incubations.(7) The results identified tofacitinib as a concentration-, time-, and NADPH-dependent irreversible inhibitor of CYP3A4. Glutathione (GSH) and superoxide dismutase/catalase offered minor or little protection against the CYP3A4 inactivation.(7) Of the epoxide (6) and α-keto-aldehyde (7) intermediates (Figure 1) trapped and characterized in microsomal incubations, aldehyde intermediate was proposed to be the key for the CYP3A4 enzyme inactivation.(7) Although multiple enzymes, including CYP 1A2, 2C19, 2D6, and 3A4, participated in the metabolism of tofacitinib to the epoxide, it was concluded that CYP3A4 primarily catalyzed the formation of the aldehyde.(7) Figure 1. Chemical structures of epoxide (6) and aldehyde (7) intermediates generated following CYP-mediated metabolism of tofacitinib. While the present data(7) will promote more scientific thinking and rationalization to possibly explain the mechanistic basis of the side effects induced by tofacitinib, especially liver injury, there are certain aspects of the data that need introspection to understand and validate the proposed concept. The intent of this Letter is to highlight the concerns of undermining the significant contribution of the epoxide intermediate (6) as well as the role of glutathione trapping process in the enzyme inactivation. First, Figure 8 as represented by Guo et al. shows that enzyme activity is significantly lost in the presence of tofacitinib (23.3 ± 4.1% of remaining enzyme activity) as well as analogue 2 (43.9 ± 4.8% of remaining enzyme activity) as compared to the control group, which was attributed to the formation of the aldehyde intermediate (7) in the metabolic process.(7) Chemically, tofacitinib can form both aldehyde (7) and epoxide intermediates (6), whereas analogue 2 could only form the aldehyde intermediate during the metabolic process upon incubation with the liver microsomes.(7) Although the aldehyde intermediate was considered to be the key factor in enzyme inactivation, based on the observed higher enzyme inactivation by tofacitinib there arises a question, “does the epoxide intermediate along with the aldehyde intermediate of tofacitinib exhibit synergism and contribute to the observed higher loss in enzyme activity from tofacitinib, which is in congruence with the comparatively lesser loss of enzyme activity by analogue 2 due to its ability to generate only the aldehyde intermediate?”. Taking into confidence the differences in magnitude of CYP3A4 inactivation observed between tofacitinib and analogue 2 (Figure 8), the proposed hypothesis of only aldehyde involvement in enzyme inactivation needs to be revisited. Second, it has been inferred that the nucleophilic agent GSH exhibited limited ability to attenuate tofacitinib-induced CYP3A4 inactivation.(7) Ideally, epoxide intermediates are trapped using thiol containing trapping reagents, namely GSH and N-acetyl-l-cysteine (NAC).(8−10) Thus, epoxide intermediates that are trapped with NAC should also be trapped in the presence of GSH and cause reversal of enzyme inactivation. Therefore, the question is, “are epoxide intermediates of tofacitinib not getting trapped by GSH, and is this why GSH offered limited protection for tofacitinib induced CYP3A4 inactivation?”. The potential reason for this could be the requirement of cytosolic glutathione-s-transferases (GST) for GSH adduct formation, as cytosolic GSTs are functionally different from microsomal GSTs.(11,12) Considering the reports that epoxide intermediates could also cause enzyme inactivation, additional insights need to be drawn to understand the limited ability of GSH in mitigating CYP3A4 inactivation as observed in this study. Third, in Scheme 2, it has been depicted that the epoxide intermediate (6) conjugates with NAC to form two isomers.(7) However, the possible isomers have been named as M1/M2/M3, indicating that there is a chance of formation of three isomers.(7) In general, epoxide intermediate results in formation of a diol, further leading to a plausible genesis of two adducts.(13) Hence, Scheme 2 needs clarification for the readers who would be interested to know the probable mechanism of formation of three isomers. This Letter to the Editor was prepared to promote an exchange of thoughts on a topic of great interest in glutathione trapping in drug metabolism. The authors declare no competing financial interest. Ranjeet P. Dash is a current employee of Charles River Laboratories, Ashland. This article references 13 other publications.

中文翻译:

“ Tofacitinib是一种基于机理的细胞色素P450 3A4灭活剂”:重新探讨了环氧中间体和谷​​胱甘肽捕获的意义。

(5)托法替尼的代谢主要由CYP3A4介导,而CYP2C19的贡献较小。体外代谢研究已确定二羟基托法替尼的羟基,二羟基和葡萄糖醛酸是主要代谢产物。(6)最近,Guo等人。报道了一项精心设计和执行的体外代谢研究,旨在通过捕获微粒体温育中产生的反应性代谢产物来阐明托法替尼的机制失活。(7)结果确定托法替尼是浓度,时间和NADPH依赖性不可逆抑制剂CYP3A4。谷胱甘肽(GSH)和超氧化物歧化酶/过氧化氢酶对CYP3A4失活提供了很少或几乎没有保护。(7)(6)最近,Guo等人。报道了一项精心设计和执行的体外代谢研究,旨在通过捕获微粒体温育中产生的反应性代谢产物来阐明托法替尼的机制失活。(7)结果确定托法替尼是浓度,时间和NADPH依赖性不可逆抑制剂CYP3A4。谷胱甘肽(GSH)和超氧化物歧化酶/过氧化氢酶对CYP3A4失活提供了很少或几乎没有保护。(7)(6)最近,Guo等人。报道了一项精心设计和执行的体外代谢研究,旨在通过捕获微粒体温育中产生的反应性代谢产物来阐明托法替尼的机制失活。(7)结果确定托法替尼是浓度,时间和NADPH依赖性不可逆抑制剂CYP3A4。谷胱甘肽(GSH)和超氧化物歧化酶/过氧化氢酶对CYP3A4失活提供了很少或几乎没有保护。(7)(7)结果确定tofacitinib是浓度,时间和NADPH依赖性的CYP3A4不可逆抑制剂。谷胱甘肽(GSH)和超氧化物歧化酶/过氧化氢酶对CYP3A4失活提供了很少或几乎没有保护。(7)(7)结果确定tofacitinib是浓度,时间和NADPH依赖性的CYP3A4不可逆抑制剂。谷胱甘肽(GSH)和超氧化物歧化酶/过氧化氢酶对CYP3A4失活提供了很少或几乎没有保护。(7)6)和α-酮醛(7)中间体(图1)在微粒体温育中被捕获和表征,醛中间体被认为是CYP3A4酶失活的关键。(7)尽管包括CYP 1A2、2C19在内的多种酶, 2D6和3A4参与了tofacitinib代谢成环氧化物的作用,得出的结论是CYP3A4主要催化醛的形成。(7)图1.环氧化物(6)和醛(7)的化学结构)CYP介导的tofacitinib代谢后产生的中间体。虽然目前的数据(7)将促进更科学的思考和合理化,以可能解释托法替尼(尤其是肝损伤)引起的副作用的机理基础,但数据的某些方面需要进行自省以理解和验证所提出的概念。这封信的目的是强调担心破坏环氧化物中间体(6)的重要作用以及谷胱甘肽捕获过程在酶失活中的作用。首先,由Guo等人代表的图8。表明在tofacitinib(剩余酶活性的23.3±4.1%)和类似物2的存在下酶活性显着丧失(43.9±4.8%的剩余酶活性),与对照组相比,这归因于在代谢过程中形成了醛中间体(7)。(7)在化学上,托法替尼既可以形成醛(7)也可以形成环氧化物中间体(6),而类似物2(7)尽管醛中间体被认为是酶失活的关键因素,但根据观察到的tofacitinib更高的酶失活作用,仍产生了一个问题,“环氧化物中间体与托法替尼的醛中间体之间是否显示出协同作用,并有助于观察到的托法替尼酶活性的更高损失,这与类似物2的酶活性损失相对较小有关,这是由于类似物2仅产生醛的能力中间的?”。确信托法替尼和类似物2之间的CYP3A4失活幅度不同(图8),仅醛参与酶灭活的假说需要重新研究。其次,已推论亲核试剂GSH减弱减弱tofacitinib诱导的CYP3A4失活的能力。(7)理想地,使用含硫醇的捕获剂GSH和N-乙酰基-l捕获环氧中间体。-半胱氨酸(NAC)。(8-10)因此,被NAC捕集的环氧中间体也应在GSH存在下被捕集,并导致酶失活的逆转。因此,问题是,“托法替尼的环氧中间体没有被GSH捕获,这就是为什么GSH为托法替尼诱导的CYP3A4失活提供有限保护的原因?”。造成这种情况的潜在原因可能是由于谷胱甘肽S转移酶(GST)在功能上不同于微粒体GST,因此需要谷胱甘肽S转移酶(GST)形成谷胱甘肽加合物。(11,12)考虑到环氧化物中间体也可能导致酶失活的报道,如本研究中所观察到的,还需要更多的见解来了解GSH在缓解CYP3A4失活方面的有限能力。第三,在方案2中,已经描述了环氧化物中间体(6)与NAC共轭形成两个异构体。(7)但是,可能的异构体已被命名为M1 / M2 / M3,表明存在形成三个异构体的可能性。(7)通常,环氧中间体会导致形成二醇,进一步导致两个加合物的合理生成。(13)因此,方案2需要澄清,以便有兴趣了解三种异构体形成的可能的读者。致编辑的这封信旨在促进就谷胱甘肽在药物代谢中的诱捕非常感兴趣的话题交换思想。作者宣称没有竞争性的经济利益。Ranjeet P. Dash是Ashland Charles River Laboratories的现任雇员。本文引用了其他13个出版物。
更新日期:2019-12-19
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