人肝CYP3A参与了山冈橐吾碱的代谢及其肝毒性代谢物的形成(英文)

目的　在体外研究山冈橐吾碱在人肝微粒体内的代谢及参与其代谢的主要的CYP4 5 0酶 ,探讨其代谢致毒机理。方法　采用人肝微粒体研究山冈橐吾碱的主要代谢方式和代谢物。在体外运用CYP4 5 0酶的选择性抑制剂和cDNA表达的人肝CYP4 5 0酶 ,探讨其对山冈橐吾碱的代谢及肝毒性的吡咯代谢物形成的影响及参与山冈橐吾碱代谢的主要的CYP4 5 0酶。结果　山冈橐吾碱在人肝微粒体内的主要代谢物为肝毒性的吡咯代谢物 :去氢倒千里光裂碱 ,7 谷胱甘肽基 去氢倒千里光裂碱 ,7,9 二谷胱甘肽基去氢倒千里光裂碱和山冈囊吾酸。CYP4 5 0特异性抑制剂α 萘黄酮 (抑制CYP1A2 )、黄胺苯吡唑 (抑制CYP2C)、奎尼丁 (抑制CYP2D6 )和二乙基二硫代氨基甲酸钠 (抑制CYP2E1)对山冈橐吾碱的代谢无明显的影响。但CYP3A的特异性抑制剂酮康唑和三乙酰竹桃霉素可以显著地抑制山冈橐吾碱的代谢及其吡咯代谢物和结合型吡咯物的形成。此外 ,在cDNA表达的人肝CYP3A4的温孵液中 ,山冈橐吾碱被代谢成相应的吡咯代谢物 ,而山冈橐吾碱在cDNA表达的人肝CYP1A2、CYP2C9、CYP2D6和CYP2E1温孵液中无代谢。结论　山冈橐吾碱在人肝微粒体内的主要代谢方式是形成肝毒性吡咯代谢物 ,CYP3A作为主要的CYP4 5 0酶参与了山冈橐吾碱的代谢及其肝毒性吡咯代谢?     

尼莫地平在人肝微粒体内的代谢

:采用人肝微粒体在体外研究尼莫地平 (Nim)在人体内的代谢物及代谢途径 . Nim在人肝微粒体内被迅速代谢成 3个代谢物 ,分别是 Nim二氢吡啶环脱氢代谢物 M1,二氢吡啶环侧链脱甲基代谢物M2 ,二氢吡啶环脱氢及其侧链脱甲基代谢物 M3.Nim在人肝微粒体中的最初的两步代谢反应是其二氢吡啶环脱氢氧化及其侧链脱甲基反应 ,两者的代谢产物可以被进一步代谢为代谢物 M3.CYP3A的特异性抑制剂醋竹桃霉素和酮康唑可以抑制Nim的二氢吡啶环脱氢氧化及其侧链脱甲基反应 ,使 Nim的代谢速率明显下降 ,结果提示 CYP3A参与了 Nim在人肝微粒体内的代谢     

西尼地平在人肝微粒体内代谢及代谢抑制

目的：在体外研究西尼地平在人肝微粒体内的代谢及选择性细胞色素P-450（CYP450）酶抑制剂对其代谢的影响。方法：在体外用人肝微粒体研究西尼地平的代谢,并用CYP450酶的选择性抑制剂探讨其对西尼地平代谢的影响及人肝微粒体中参与西尼地平二氢吡啶环脱氢代谢的CYP450酶。结果：西尼地平在人肝微粒体内被迅速代谢物M1,二氢吡啶环侧链脱甲基代谢物M2,二氢吡嘧环脱氢及其侧链脱甲基代谢物M3,酮康唑竞争性地抑制西尼地平二氢吡啶环的脱氢代谢,同时降低西尼地平的代谢速率,而其它抑制剂,奎尼丁,α-Naphthoflavone,diethyldithiocarbamate,sulfaphenazole和tra-nylcypromine对西尼地平二氢吡啶环的脱氢代谢没有明显的影响。结论：西尼地平在人肝微粒体内被迅速代谢,其二氢吡啶环的脱氢代谢是其代谢的关键性的步骤,CYP3A作为主要的CYP酶参与了西尼地平二氢吡啶环的脱氢代谢,CYP3A的抑制剂可能会与西尼地平发生代谢相互作用。     

大豆苷元在人肝微粒体中的单羟化代谢机制大豆苷元在人肝微粒体中的单羟化代谢机制

目的探讨大豆苷元在人肝微粒中羟基化代谢所涉及的肝细胞色素P450(CYP)同工酶，为研究其在人体内的代谢提供基础。方法通过分析大豆苷元在肝微粒体中和重组CYP酶中形成的单羟化代谢物的酶促动力学,分析其酶学模型，然后用不同CYP同工酶选择性抑制剂或底物进行抑制实验，初步筛选出介导大豆苷元单羟化代谢所涉及的CYP同工酶。结果代谢物的形成动力学符合米氏方程单酶模型。CYP1A2选择性抑制剂呋喃茶碱和CYP1A2单克隆抗体均能明显抑制3种单羟化代谢物的形成。而其他CYP选择性的抑制剂对3种代谢物的形成没有或较小产生抑制作用。用重组酶实验得出相同结果。结论体外肝微粒体研究表明，大豆苷元的单羟基代谢主要由CYP1A2所介导。     

雷公藤甲素在大鼠肝微粒体中代谢及酶促反应动力学研究

目的:研究雷公藤甲素在大鼠肝微粒体中代谢及酶促反应动力学。方法:将雷公藤甲素与5种不同诱导剂(地塞米松(DEX)、苯巴比妥(PB)、β-萘黄酮(β-NF)、吡啶(PD))诱导的大鼠肝微粒体进行体外共孵育;并与8种选择性CYP酶抑制剂(酮康唑、醋竹桃霉素、磺胺苯吡唑、二乙基二硫代氨基甲酸酯(盐)、奎尼丁、呋拉茶碱、毛果芸香碱、奥芬那君)在空白肝微粒体中共孵育。采用液相色谱-质谱联用技术测定孵育后剩余雷公藤甲素的含量。结果:酮康唑和醋竹桃霉素能明显抑制雷公藤甲素的代谢;磺胺苯吡唑和奥芬那君对其代谢也有一定抑制作用,但不及酮康唑和醋竹桃霉素。结论:雷公藤甲素在大鼠肝微粒体中代谢主要由CYP3A介导,其次由CYP2C和CYP2B介导。     

建立用大鼠肝微粒体快速考察药物体外CYP3A1活性抑制的方法

目的研究酮康唑对大鼠肝微粒体细胞色素P450同工酶3A1（CYP3A1）活性的作用,建立一种用大鼠肝微粒体快速考察药物体外CYP3A1活性抑制的方法。方法采集大鼠肝微粒体,随机分为对照组和药物处理组。酮康唑药物处理组分别加入不同浓度的酮康唑,对照组只加入培养液,混匀后放入37℃培养箱中孵育15 min,然后加入CYP 3A1底物睾酮,继续孵育10 min,最后加入内标物氢化可的松。用HPLC法测定睾酮经大鼠肝微粒体温孵后生成6-β羟基睾酮的量,代表CYP3A1的活性。结果各个处理组与对照组的比较差异均有显著性差异（P〈0.05）;提示在一定浓度范围内,大鼠同工酶CYP3A1的相对活性百分比随着酮康唑浓度的增加而逐渐减低,各处理组与对照组比较差异均有显著性意义（P〈0.5）。半数抑制浓度（IC50）值为3.25μg/ml。结论酮康唑对大鼠肝微粒体细胞色素P450同工酶3A1的活性有强抑制作用,建立的通过大鼠肝微粒体快速考察药物体外CYP3A1探针抑制的方法重复性和稳定性均良好,为评价新药对大鼠肝微粒体CYP3A1的活性的体外作用提供可靠的检测手段。     

甲基莲心碱在大鼠肝脏中的代谢产物及其途径

大鼠灌胃给予甲基莲心碱 20 mg·kg^-1，采用液相色谱-串联质谱联用法对大鼠肝脏中的代谢产物进行分析；并建立肝微粒体温浴及NADPH再生体系，采用高效液相色谱-紫外检测法研究CYP450亚型的特异性抑制剂对甲基莲心碱体外代谢的影响。在正离子检测方式下，除甲基莲心碱外共检测到4种代谢产物M₁、M₂(主要代谢产物)、M₃和M₄。其中，M₂和M₄通过与对照品的色谱和质谱比对，确认为莲心碱和异莲心碱，而M₁ 和M₃可能为去甲基莲心碱和去甲基异莲心碱。CYP3A1的特异性抑制剂酮康唑和CYP2D1的特异性抑制剂奎尼丁均可抑制甲基莲心碱在肝微粒体温孵液中的代谢，其主要代谢产物莲心碱的生成抑制率分别为25.7%和80.5%。因此提示，甲基莲心碱在肝脏中的主要代谢途径是苄基和喹啉环上的甲氧基脱甲基化，其主要代谢物为莲心碱，CYP2D1和CYP3A1均参与了其生物转化。     

白屈菜碱在大鼠肝微粒体中代谢的酶动力学及CYP450酶特异性抑制剂对其代谢的影响

目的研究白屈菜碱在大鼠肝微粒体中代谢的酶动力学及CYP450酶特异性抑制剂对其代谢的影响。方法将系列浓度的白屈菜碱与大鼠肝微粒体进行体外共孵育,采用HPLC法测定孵育液中剩余白屈菜碱的浓度,利用Graph Pad Prism 6.0软件进行数据拟合并计算酶动力学参数;分别将5种CYP450酶的特异性抑制剂与白屈菜碱进行共孵育,考察抑制剂对白屈菜碱代谢的影响,探讨参与其代谢的酶亚型。结果在大鼠肝微粒体中,白屈菜碱的Vmax为(3.52±0.18)mmol·min-1·kg-1;Km为(12.02±2.92)μmol·L-1;CLint为292.4 L·min-1·kg-1;CYP450酶特异性抑制剂酮康唑、α-萘黄酮、氟康唑和奎尼丁可以显著地抑制白屈菜碱的代谢,而噻氯匹定对白屈菜碱的代谢没有明显影响。结论白屈菜碱在大鼠肝微粒体中广泛代谢,CYP3A4、CYP1A2、CYP2D6、CYP2C9是参与其代谢的主要代谢酶。     

诃子酸在不同种属肝微粒体中的代谢稳定性和代谢酶表型研究

目的 建立测定肝微粒体孵育体系中诃子酸浓度的方法,并比较其在人、犬、猴、小鼠、大鼠肝微粒体中的Ⅰ相、Ⅱ相代谢稳定性及种属差异,确定其在人肝微粒中的代谢表型。方法 将诃子酸与不同种属肝微粒体共同孵育,应用UPLCMS/MS检测孵育液中诃子酸的含量,考察其代谢稳定性及体外动力学参数。采用化学抑制剂法确定其在人肝微粒中的代谢表型。将诃子酸与各CYP450同工酶CYP1A2、CYP2A6、CYP2C9、CYP2C19、CYP2D6、CYP2E1和CYP3A4的特异性抑制剂（α-萘黄酮、香豆素、磺胺苯吡唑、噻氯匹定、奎尼丁、二乙基二硫代氨基甲酸钠、酮康唑）共同孵育,确定其代谢酶表型。结果 诃子酸在Ⅰ相、Ⅱ相孵育体系中均可代谢,在Ⅰ相代谢中,犬肝微粒体孵育与人最为相似,半衰期（t_1/2）分别为115.50 min和121.58 min;Ⅱ相代谢中5个种属代谢稳定性均中等,其中猴与人肝微粒体代谢趋势最为相近。诃子酸在人肝微粒中的代谢是由多种CYP酶共同介导的,其中CYP2C9、CYP2E1和CYP3A4是主要的同工酶。结论 建立的UPLCMS/MS方法简便、快速、专属性强、灵敏性高,可用于肝微粒体孵育体系中诃子酸浓度的测定及体外代谢的研究。诃子酸在人、犬、猴、小鼠、大鼠肝微粒体中代谢存在一定种属差异,且其代谢过程与多种CYP酶相关。     

布格呋喃与大鼠肝脏药物代谢酶的相互作用

目的：研究参与布格呋喃代谢的CYP450同工酶类型和体外代谢的酶促动力学特征,并观察口服布格呋喃对大鼠肝脏药物代谢酶的影响。方法：采用比色／动态荧法光及UV—HPLC法检测大鼠口服布格呋喃（8mg·kg^-1·d^-1,连续3日）后肝脏CYP450同工酶（CYP1A2、2C6、2C11、2D2、2E1和3A2）和Ⅱ相酶-谷胱甘肽巯基转移酶（GST）、尿苷二磷酸葡萄糖醛酸转移酶（UDPGT）和谷胱甘肽还原酶（GR）活性。应用肝微粒体温孵法测定布格呋喃体外代谢速率（Vmax和米氏常数Km）。比较布格呋喃在正常及高诱导大鼠肝微粒体中代谢速率的差异以及CYP1A2、2C6、2C11、2D2、2E1和3A2的选择l生抑制剂（呋拉茶碱、磺基苯吡唑、奥美拉唑、奎尼丁、戒酒硫和酮康唑）对布格呋喃代谢的抑制程度,鉴定参与布格呋喃代谢的CYP450同工酶类型。结果：布格呋喃在乙醇、地塞米松和3-甲基胆葸诱导大鼠肝微粒体中的Km、Vmax分别为正常组的11倍、6倍和1.3倍;应用CYP3A2、2E1和IA2的选择性抑制剂酮康唑、戒酒硫和呋拉茶碱可不同程度地抑制布格呋喃的代谢,使代谢速率下降为对照组的34％、47％和78％。     

Gender differences in microsomal metabolic activation of hepatotoxic clivorine in rat

The gender differences in the in vitro microsomal metabolic activation of hepatotoxic clivorine, a representative naturally occurring hepatotoxic otonecine type pyrrolizidine alkaloid, in Sprague-Dawley rats and their relation to the gender differences in susceptibility to clivorine intoxication were reported in the present study. Clivorine-induced liver damage in the male rat via metabolic activation to form the reactive pyrrolic ester followed by covalent binding to liver tissue constituents has been reported previously by our research group. The present study demonstrated, for the first time, that cytochromes p450 3A1 and 3A2, which are constitutively expressed in the male rat, might play a significant role in the metabolic activation of clivorine in the rat. Thus, in the male rat, the metabolic activation by liver microsomes to form the reactive pyrrolic ester was found as the only direct metabolic pathway of clivorine followed by subsequent formation of the toxic tissue-bound pyrroles leading to hepatotoxicity. In the case of the female rat, a less significant metabolic activation was observed, whereas the formations of two novel nonpyrrolic metabolites were determined as the predominant biotransformations. None of the four cDNA-expressed rat enzymes (cytochrome p450 2C12, 2E1, 3A1, 3A2) tested could catalyze the formation of these two new metabolites. Furthermore, the female rat (LD(50) = 114 +/- 9 mg/kg, i.p.) was found to be significantly less susceptible to clivorine intoxication than the male rat (LD(50) = 91 +/- 3 mg/kg, i.p.). Therefore, the results suggested that a significantly lower metabolic activation due to the lack of cytochrome p450 3A1 and p450 3A2 activities mainly accounted for the smaller susceptibility of the female rat to clivorine intoxication.     

Species differences in the in vitro metabolic activation of the hepatotoxic pyrrolizidine alkaloid clivorine

Clivorine is a representative naturally occurring hepatotoxic otonecine-type pyrrolizidine alkaloid. Our previous study has demonstrated that clivorine induces liver damage via metabolic activation to form the reactive pyrrolic ester followed by covalent binding to liver tissue constituents. The present study investigated species differences in the in vitro metabolic activation of clivorine in the male rat and guinea pig of both sexes. In the male rat, the activation of clivorine to form the reactive pyrrolic ester was found as the only metabolic pathway. Moreover, the toxic tissue-bound pyrroles and four isolatable metabolites identified, namely DHR, 7-GSH-DHR, 7,9-diGSH-DHR, and clivoric acid, were all generated from further metabolism of this reactive intermediate. In the case of both sexes of guinea pig, the same activation was observed as the minor biotransformation, while an additional metabolic pathway, a direct hydrolysis of clivorine to form novel clivopic acid was identified as the predominant detoxification pathway. Furthermore, the formation rates for the toxic tissue-bound pyrroles and less toxic DHR were significantly slower and higher, respectively, compared with those in the male rat. In addition, the formation of the reactive pyrrolic ester was mediated by the CYP3A subfamily in both animals, while carboxylesterases might be responsible for the detoxification hydrolysis in guinea pig. The results suggest that the higher metabolic rates for detoxification hydrolyses and the lower formation rate for the toxic tissue-bound pyrroles play the key roles in guinea pig resistance to clivorine intoxication. Therefore, the male rat and guinea pig should be the suitable animal models for further studies of bioactivation and deactivation of otonecine-type PA, respectively.     

Characterization of rat liver microsomal metabolites of clivorine, an hepatotoxic otonecine-type pyrrolizidine alkaloid.

The metabolism of the hepatotoxic otonecine-type pyrrolizidine alkaloid (PA), clivorine, was investigated using rat liver microsomes. The metabolites dehydroretronecine (DHR), 7-glutathionyldehydroretronecine (7-GSH-DHR), 7, 9-diglutathionyldehydroretronecine (7,9-diGSH-DHR), and clivoric acid were identified using chromatographic and mass spectrometric analyses. NMR characterizations were also performed on the isolated clivoric acid and the synthetic 7-GSH-DHR and 7,9-diGSH-DHR. The results indicated that the two glutathione (GSH) conjugates were formed by reaction of the unstable toxic pyrrolic ester with GSH added in the microsomal incubation system, whereas DHR was generated from hydrolysis of the unstable pyrrolic ester, and that clivoric acid was produced from all these further conversions of the unstable pyrrolic ester. Furthermore, tissue-bound pyrroles were also determined to be present after microsomal incubation of clivorine. Clivoric acid has not been previously identified, and DHR and 7, 9-diGSH-DHR were found, for the first time, as metabolites of an otonecine-type PA, while 7-GSH-DHR was previously reported by us to be a microsomal metabolite of clivorine. The in vitro metabolic pathway of clivorine was delineated to be the initial formation of the unstable pyrrolic ester, which then may undergo hydrolysis, GSH conjugations, or covalent binding with hepatic tissues that may lead to hepatotoxicity. The present definitive identification of four pyrrolic ester-related metabolites of clivorine and indirect determination of bound pyrroles provide the strongest evidence to date to support the hypothesis that the formation of an unstable pyrrolic ester plays a key role in otonecine-type PA-induced hepatotoxicity.     

Cytochrome P450 and flavin-containing monooxygenase enzymes are responsible for differential oxidation of the anti-thyroid-cancer drug vandetanib by human and rat hepatic microsomal systems

We studied the in vitro metabolism of the anti-thyroid-cancer drug vandetanib in a rat animal model and demonstrated that N-desmethylvandetanib and vandetanib N-oxide are formed by NADPH- or NADH-mediated reactions catalyzed by rat hepatic microsomes and pure biotransformation enzymes. In addition to the structural characterization of vandetanib metabolites, individual rat enzymes [cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO)] capable of oxidizing vandetanib were identified. Generation of N-desmethylvandetanib, but not that of vandetanib N-oxide, was attenuated by CYP3A and 2C inhibitors while inhibition of FMO decreased formation of vandetanib N-oxide. These results indicate that liver microsomal CYP2C/3A and FMO1 are major enzymes participating in the formation of N-desmethylvandetanib and vandetanib N-oxide, respectively. Rat recombinant CYP2C11 > >3A1 > 3A2 > 1A1 > 1A2 > 2D1 > 2D2 were effective in catalyzing the formation of N-desmethylvandetanib. Results of the present study explain differences between the CYP- and FMO-catalyzed vandetanib oxidation in rat and human liver reported previously and the enzymatic mechanisms underlying this phenomenon.     

Identification of human cytochrome P450 enzymes involved in the metabolism of IN-1130, a novel activin receptor-like kinase-5 (ALK5) inhibitor

1.?The in vitro metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1130), a selective activin receptor-like kinase-5 (ALK5) inhibitor and a candidate drug for fibrotic disease, was studied.

2.?The cytochrome P450s (CYPs) responsible for metabolism of IN-1130 in liver microsomes of rat, mouse, dog, monkey and human, and in human CYP supersomes?, were identified using specific CYP inhibitors. The order of disappearance of IN-1130 in various liver microsomal systems studied was as follows: monkey, mouse, rat, human, and dog.

3.?Five distinct metabolites (M1–M5) were identified in all the above microsomes and their production was substantially inhibited by CYP inhibitors such as SKF-525A and ketoconazole. Among nine human CYP supersomes? examined, CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19 were involved in the metabolism of IN-1130, and the production of metabolites were significantly inhibited by specific CYP inhibitors. IN-1130 disappeared fastest in CYP2C8 supersomes. CYP3A4 produced four metabolites of IN-1130 (M1–M4), whereas supersomes expressing human FMO cDNAs, such as FMO1, FMO3, and FMO5, produced no metabolites.

4.?Hence, it is concluded that metabolism of IN-1130 is mediated by CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19.     

Imidacloprid insecticide metabolism: human cytochrome P450 isozymes differ in selectivity for imidazolidine oxidation versus nitroimine reduction

Many metabolites of imidacloprid (IMI) have been identified, but the enzymatic basis for their formation has not been reported. This study with individual recombinant cytochrome P450 (CYP450) isozymes from human liver shows that the principal organoextractable NADPH-dependent metabolites are the 5-hydroxy (major) and olefin (minor) derivatives from hydroxylation and desaturation of the imidazolidine moiety and the nitrosoimine (major), guanidine (minor) and urea (trace) derivatives from reduction and cleavage of the nitroimine substituent. Isozymes selective for imidazolidine oxidation in order of decreasing overall activity are CYP3A4>CYP2C19 or CYP2A6>CYP2C9, while those selective for nitroimine reduction are CYP1A2, CYP2B6, CYP2D6 and CYP2E1. Three flavin monooxygenase isozymes (FMO1, FMO3, and FMO5) with NADPH are not active as assayed. These observations establish site specificity in IMI metabolism by CYP450 isozymes and that a single enzyme (CYP3A4) both oxidizes and reduces IMI at the imidazolidine and nitroimine moieties, respectively.     

Identification of cytochrome P450 enzymes involved in the metabolism of zotepine,an antipsychotic drug,in human liver microsomes

1. Studies using human liver microsomes and recombinant human cytochrome P450 (P450) enzymes and flavin-containing monooxygenase (FMO) were performed to identify the enzymes responsible for the formation of zotepine metabolites in man. 2. Human liver microsomes produced four metabolites and a tentative order of importance was: norzotepine, 3-hydroxyzotepine, zotepine S-oxide and 2-hydroxyzotepine. Zotepine N-oxide was also detected, but it could not be quantified. 3. The rates of formation of the major metabolite, norzotepine, and zotepine S-oxide (at a substrate concentration of 20 μM) were significantly correlated with the testosterone 6β-hydroxylase activities and CYP3A4 contents of the 12 different human liver microsomal samples. Inhibition studies with P450 enzyme selective inhibitors and anti-rat CYP3A2 antibodies also indicated a predominant role of CYP3A4 in the formation of norzotepine and zotepine S-oxide. Furafylline and sulphaphenazole inhibited the N-demethylation of zotepine by up to ～ 30%. 4. Correlation and inhibition data for the 2- and 3-hydroxylation of zotepine were consistent with the predominant role of CYP1A2 and 2D6 in the formation of these metabolites, respectively. 5. Recombinant CYP1A1, 1A2, 2B6, 2C19, 3A4 and 3A5 efficiently catalysed N-demethylation of zotepine. CYP1A1, 1A2, 2B6 and 3A4 were also active for S-oxidation. CYP1A2 and 2D6*1-Val374 efficiently produced 2-hydroxyzotepine and 3-hyroxyzotepine, respectively. Recombinant human FMO3 did not catalyse zotepine S-oxidation. 6. These results suggest that both the N-demethylation and S-oxidation of zotepine are mediated mainly by CYP3A4, and that CYP1A2 and 2D6 play an important role in the 2- and 3-hydroxylation of zotepine, respectively.     

西格列他钠的体外代谢

目的研究拟治疗2型糖尿病的创新化合物西格列他钠(chiglitazar)的体外代谢速率、代谢酶和代谢转化,为临床应用提供参考。方法采用高效液相-紫外检测(HPLC-UV)的方法测定肝微粒体孵育液中西格列他钠的浓度,用特异性抑制剂的方法分析化合物的代谢酶,用大鼠肝微粒体体外研究西格列他钠可能的代谢产物和代谢途径。结果建立了可靠的测定大鼠肝微粒体中西格列他钠的HPLC分析方法;体外半衰期方法求得西格列他钠的t1/2为27.2min,固有清除率(Clint)为50.9mL.min-1.g-1蛋白;代谢酶研究表明,西格列他钠主要被P450酶中的CYP3A亚型代谢;西格列他钠在大鼠肝微粒体中的代谢主要为羟基化和O-脱烷基化,采用LC/MSn分析共发现了8个代谢物。结论西格列他钠是代谢活跃的化合物,有必要研究代谢产物的活性及临床注意药物相互作用。     

Identification of human cytochrome P450 enzymes involved in the metabolism of IN-1130, a novel activin receptor-like kinase-5 (ALK5) inhibitor

1. The in vitro metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1,130), a selective activin receptor-like kinase-5 (ALK5) inhibitor and a candidate drug for fibrotic disease, was studied. 2. The cytochrome P450s (CYPs) responsible for metabolism of IN-1,130 in liver microsomes of rat, mouse, dog, monkey and human, and in human CYP supersomestrade mark, were identified using specific CYP inhibitors. The order of disappearance of IN-1,130 in various liver microsomal systems studied was as follows: monkey, mouse, rat, human, and dog. 3. Five distinct metabolites (M1-M5) were identified in all the above microsomes and their production was substantially inhibited by CYP inhibitors such as SKF-525A and ketoconazole. Among nine human CYP supersomestrade mark examined, CYP3A4, CYP2C8, CYP2D6 1, and CYP2C19 were involved in the metabolism of IN-1,130, and the production of metabolites were significantly inhibited by specific CYP inhibitors. IN-1,130 disappeared fastest in CYP2C8 supersomes. CYP3A4 produced four metabolites of IN-1,130 (M1-M4), whereas supersomes expressing human FMO cDNAs, such as FMO1, FMO3, and FMO5, produced no metabolites. 4. Hence, it is concluded that metabolism of IN-1,130 is mediated by CYP3A4, CYP2C8, CYP2D6 1, and CYP2C19.