盐酸双苯氟嗪在大鼠肝微粒体内的代谢特征研究
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摘要
药物代谢反应研究是药物研发中的一个重要课题,而新药研发早期,找到一个拥有良好药物代谢(DM)和药代动力学(PK)特性的化合物是其重要目标。药物在体内的代谢主要经历Ⅰ相和Ⅱ相反应,其中Ⅰ相反应对于药物小分子在体内的活性与毒性起着关键作用,而催化Ⅰ相反应的关键代谢酶系细胞色素P450混合功能氧化酶系(CYP450s)。阐明参与药物体内转化的CYP450酶,评价药物与代谢酶间的相互作用,对于理解药物代谢机制、预测药物相互作用和药物代谢多态性等方面具有重要意义。目前欧美各国也已经把CYP450s及其同工酶测定用于新药筛选及代谢研究,并把它列为新药申报必须进行的一项内容。
盐酸双苯氟嗪是河北医科大学开发的新型哌嗪类钙通道拮抗剂,研究表明该药经由多种机制保护局灶性和全脑缺血再灌注损伤,其药理作用强于同类药氟桂利嗪和桂利嗪。
本实验采用液质联用技术(LC-MS-MS)确定了盐酸双苯氟嗪在大鼠肝微粒体中代谢生成的4个特征性代谢产物的结构,明确了其代谢途径；进行了肝微粒体温孵条件优化和酶动力学研究；利用化学抑制剂实验、相关性分析和重组酶实验研究了盐酸双苯氟嗪在不同性别大鼠肝微粒体中的代谢行为,鉴定了参与代谢产物生成的CYP450酶亚型,初步探讨了其酶催化机制；通过体内和体外实验考察了盐酸双苯氟嗪对不同性别大鼠CYP450酶活性的影响。上述系列研究为探讨盐酸双苯氟嗪的代谢途径和酶催化机制、研究盐酸双苯氟嗪与其他药物相互作用及作用机制奠定基础,从而为制定安全、合理的临床用药方案、减少药物不良反应提供了科学依据。
第一部分盐酸双苯氟嗪在大鼠肝微粒体中的代谢行为及其代谢物LC-MS-MS测定条件的确立
目的：确定盐酸双苯氟嗪在不同性别大鼠肝微粒体中的代谢行为；建立盐酸双苯氟嗪及其代谢物的LC-MS-MS测定方法。
方法：雌、雄SD大鼠各10只,体重230±20g,断头处死,取肝脏,超速离心法制备肝微粒体,Lowry法测定蛋白浓度,Omura和Sato方法测定CYP450酶含量；盐酸双苯氟嗪(100μM)在500μl肝微粒体温孵反应系统中(pH 7.4)于37℃水浴中振荡温孵30min,反应结束后于冰浴中终止反应。取100μl反应液,经乙酸乙酯萃取处理后,吸取上清液20μ1进行LC-MS-MS分析。肝微粒体样品分析测定采用Dikma RP-C18分析色谱柱(250×4.6mm,5μm),流动相有机相A为甲醇/0.5%o甲酸(1000：0.5),水相B为0.5‰甲酸溶液,采用梯度洗脱方式,洗脱程序为：0～1 min, 40～95% A; 1～6 min,95% A; 6～7 min,95～60% A;柱温40℃,流速为0.8ml/ml。盐酸双苯氟嗪及其代谢物采用ESI正离子模式检测。
结果：雌、雄大鼠肝微粒体蛋白浓度分别为11.26±2.10 mg/ml和10.47±1.87 mg/ml, CYP450含量为0.627±0.10 nmol/mg protein和0.573±0.077 nmol/mg protein。盐酸双苯氟嗪在雄性大鼠肝微粒体中被迅速代谢成4个代谢物,它们分别是盐酸双苯氟嗪哌嗪环脱烷基代谢物(1-4-氟苯基)-4-哌嗪基丁酮(M1)、二苯亚甲基烷基氧化苯环还原代谢物4-羟基二苯甲酮(M2)、二苯亚甲基还原代谢物二苯甲醇(M4)和二苯亚甲基氧化代谢物二苯甲酮(M5)；在雌性大鼠肝微粒体中被迅速代谢成3个代谢物,分别是M1、M2和M5；以上结果提示,盐酸双苯氟嗪在大鼠肝微粒体中的代谢行为具有性别差异。盐酸双苯氟嗪在大鼠肝微粒体中的最初两步代谢反应是哌嗪环脱烷基生成M1和二苯亚甲基氧化生成M5,M5可以被进一步代谢为M2和M4；在两种不同性别大鼠肝微粒体中,M1的浓度均比其他代谢物为高,是盐酸双苯氟嗪在大鼠肝微粒体中生成的最主要的代谢物。
所建立的LC-MS-MS测定方法的回收率为78-86%,日内和日间RSD为0.15-14.42%,符合药代动力学分析方法的要求；盐酸双苯氟嗪在2.59-1295 ng/ml、M1在25.92-12961 ng/ml、M2在0.065-32.38 ng/ml、M4在2.63-1315 ng/ml、M5在2.46-1228 ng/ml范围内呈良好线性关系；盐酸双苯氟嗪及其代谢物短期稳定性、长期稳定性和3次冷冻-解冻循环稳定性符合相关要求。
结论：本研究建立的LC-MS-MS测定方法符合药代动力学分析方法的要求,适合盐酸双苯氟嗪及其4个代谢物的含量测定,且盐酸双苯氟嗪在大鼠肝微粒体中的代谢行为具有性别差异,M1为其最主要的代谢物。
第二部分盐酸双苯氟嗪在大鼠肝微粒体中温孵反应条件的优化和酶动力学研究
目的：对盐酸双苯氟嗪在大鼠肝微粒体中温孵反应的条件进行优化,并测定盐酸双苯氟嗪代谢物的酶动力学参数,为后续大鼠肝微粒体实验提供依据。
方法：以温孵时间、大鼠肝微粒体蛋白浓度和底物浓度为考察对象,固定其中两个温孵条件,观察第三个条件对盐酸双苯氟嗪代谢物生成量或生成速率的影响,进而优化温孵条件。用Lineweaver-Burk作图法计算盐酸双苯氟嗪在大鼠肝微粒体和CYP450重组酶中的酶动力学参数(最大速率Vmax、米氏常数Km和清除率Clint)。
结果：盐酸双苯氟嗪在雌、雄大鼠肝微粒体中进行温孵反应的最佳时间分别为60min和30min,肝微粒体蛋白浓度均为1mg/ml,底物浓度分别为10gM和25gM。在雄性大鼠肝微粒体中,M1、M2、M4和M5的Km值分别是3.97μM、0.0064μM、4.14μM和3.69μM, Vmax分别是650.26、0.98、108.66和100.92 pmol/min/nmol P450;在雌性大鼠肝微粒体中,M1、M2和M5的Km值分别是3.89μM、0.0055μM和5.63μM,Vmax分别是330.17、0.58和6.54 pmol/min/nmol P450。在重组酶CYP3A1温孵液中,M1、M2、M4和M5的Km值分别是3.52μM、0.0067μM、5.67μM和3.90μM, Vmax分别是683.15、1.14、136.40和115.41 pmol/min/nmol P450;在重组酶CYP3A2温孵液中,M1、M2、M4和M5的Km值分别是3.49μM、0.0081μM、5.03μM和3.95μM, Vmax分别是719.44、1.57、144.91和132.80 pmol/min/nmol P45。
结论：盐酸双苯氟嗪在雌性或雄性大鼠肝微粒体的最佳温孵时间分别是60min和30min,最佳肝微粒体蛋白浓度均为1mg/mL;最佳盐酸双苯氟嗪浓度为10μM或25μM。在此优化的温孵条件下,代谢物生成速率均能达到饱和状态,所测定的大鼠肝微粒体和CYP450重组酶的各项酶动力学参数(Vmax、Km和Clint)准确、可靠,可满足盐酸双苯氟嗪体外代谢研究要求,为后续大鼠肝微粒体和CYP450重组酶实验提供依据。
第三部分参与盐酸双苯氟嗪代谢的大鼠肝微粒体CYP450酶的鉴别
目的：研究不同性别大鼠肝微粒体中,参与盐酸双苯氟嗪代谢的CYP450酶亚型。
方法：运用CYP450酶选择性抑制剂、相关性分析和重组酶实验探讨参与盐酸双苯氟嗪代谢的CYP450酶亚型。
化学抑制剂实验,选用P450酶特异性抑制剂[]呋拉茶碱(CYP1A2, 1-100μM)、毛果芸香碱(CYP2A1,0.5-100μM)、邻甲苯海拉明(CYP2B1, 1-100μM)、磺胺苯毗唑(CYP2C6,1-200μM)、西咪替丁(CYP2C11和CYP2C12,0.5-100μM)、奎尼丁(CYP2D1, 1-100μM)、二乙基二硫代氨基甲酸酯钠(CYP2E1,2.5-200μM)和酮康唑(CYP3A,0.5-200μM)分别加入不同性别大鼠肝微粒体中与盐酸双苯氟嗪共同温孵,测定反应系统中代谢产物生成量；用含抑制剂的样品溶液中代谢物的生成量与不含抑制剂的阴性对照溶液中代谢物生成量的百分比定量,评价不同抑制剂对各代谢物生成的影响。
相关性分析实验,将盐酸双苯氟嗪分别与15个来自不同大鼠的肝微粒体样本共同温孵,测定盐酸双苯氟嗪代谢反应与P450酶活性的相关性。以下列酶亚型特异性探针药物的代谢反应生成速率来测定P450酶的活性：非那西丁O-脱乙基反应(CYP1A2)、睾酮7a-羟基化反应(CYP2A1)、睾酮16p-羟基化反应(CYP2B1)、双氯芬酸4-羟基化反应(CYP2C6)、睾酮16α-羟基化反应(CYP2C11) 5α-androstane-3α,17β-diol-3,17-disulphate 15β-羟基化反应(CYP2C12)、右美沙芬O-脱甲基反应(CYP2D1)、氯唑沙宗6-羟基化反应(CYP2E1)和咪达唑仑1’-羟基化反应(CYP3A)。将盐酸双苯氟嗪代谢物的生成速率与探针药物代谢物的生成速率进行线性回归,计算相关系数。
CYP450重组酶实验,盐酸双苯氟嗪与重组酶(CYP1A2, CYP2A1, CYP2B1, CYP2C6, CYP2C11/12, CYP2D1, CYP2E1和CYP3A1/2)进行温孵,测定盐酸双苯氟嗪代谢物的生成速率,确证参与盐酸双苯氟嗪代谢的CYP450酶亚型。
结果：抑制剂研究、相关性分析和重组酶实验三种分析方法测定结果均显示,雄性大鼠肝微粒体中,代谢盐酸双苯氟嗪生成M1的最主要的酶亚型是CYP2A1,其他依次为CYP3A、CYP1A2和CYP2E1,而CYP2C11的作用最小。代谢盐酸双苯氟嗪生成M2的CYP450酶按作用大小依次为CYP3A、CYP2A1、CYP1A2、CYP2C11和CYP2E1。转化盐酸双苯氟嗪生成M4的酶亚型活性高低依次为CYP3A、CYP2A1、CYP2E1和CYP2C11,而参与M5生成的酶亚型分别为CYP3A、CYP2A1和CYP2C6。其中CYP3A和CYP2A1均参与了雄性大鼠肝微粒体中4种代谢物的生成反应。在雌性大鼠肝微粒体中,CYP1A2和CYP3A参与了M1的生成,且CYP3A的作用比CYP1A2稍强。参与M2生成的5种P450酶按作用大小依次为CYP3A、CYP1A2、CYP2C6、CYP2A1和CYP2E1。而CYP3A、CYP2A1、CYP2D1和CYP1A2则是参与M5生成的4种酶亚型。其中,CYP3A和CYP1A2均参与了雌性大鼠肝微粒体中3种代谢物的生成反应。由此可见,CYP3A是盐酸双苯氟嗪在不同性别大鼠肝微粒体中代谢的最主要的酶亚型,重组酶实验结果表明,CYP3A2显示出比CYP3A1更强的活性。
结论：三种方法分析结果共同确证了CYP3A是两种不同性别大鼠肝微粒体中参与盐酸双苯氟嗪代谢的最主要CYP450酶亚型,其中CYP3A2的活性比CYP3A1更强；均能参与盐酸双苯氟嗪代谢物生成反应的CYP450酶亚型,在雌性大鼠为CYP1A2,在雄性大鼠为CYP2A1,具有明显的性别差异。第四部分盐酸双苯氟嗪对大鼠CYP450酶活性的影响
目的：通过大鼠体内、外实验,观察盐酸双苯氟嗪对不同性别大鼠CYP450酶的影响。
方法：在正常雌、雄大鼠肝微粒体温孵反应系统中加入不同浓度盐酸双苯氟嗪(0-200μM)和探针药物共同温孵,反应结束后,样品经处理,进行LC-MS-MS分析,测定探针药物代谢产物生成量；用加入盐酸双苯氟嗪的样品溶液中探针药物代谢物的生成量与不加盐酸双苯氟嗪的阴性对照溶液中代谢物生成量的百分比定量,观察盐酸双苯氟嗪对CYP450的影响。
SD大鼠,雌、雄各半,随机分为空白组、盐酸双苯氟嗪低、中、高剂量组和苯巴比妥组,每组6只。实验组分别灌胃给予盐酸双苯氟嗪30、60、90 mg/kg,苯巴比妥组灌胃给予苯巴比妥120 mg/kg,空白对照组灌胃给予等容积0.5%羧甲基纤维素钠,连续给药14天。第15天分别灌胃给予Cocktail探针药物非那西丁、甲苯磺丁脲、奥美拉唑、右美沙芬、氯唑沙宗和氨苯砜,于灌胃后5、15、40、60、90、120、180、240、360、480、720min时,眼内眦取血,置肝素锂抗凝管中,离心,分离出血浆,经乙酸乙酯萃取后,进样20μL进行LC-MS-MS分析,绘制药-时曲线,计算药代动力学参数；测定给药360min时血浆样品中探针药物及其代谢物的浓度。取血结束后,断头处死大鼠,取肝脏,称重,制备肝微粒体。在肝微粒体温孵反应系统中加入探针药物,反应结束后,样品经处理,进行LC-MS-MS分析,测定探针药物及其代谢物的浓度,计算给药360min时血浆样品和肝微粒体温孵系统中探针药物的代谢率。通过比较实验组和空白对照组的相对肝脏重量、蛋白浓度、CYP450酶含量、药-时曲线、药代动力学参数和探针药物的代谢率,确定盐酸双苯氟嗪对CYP450酶活性的影响。
结果：在正常雄性大鼠肝微粒体温孵液中,盐酸双苯氟嗪对CYP2C6、CYP2D1和CYP2C11有抑制作用,其IC50值分别是8.85、20.93和69.45μg/mL;在正常雌性大鼠肝微粒体温孵液中,盐酸双苯氟嗪对CYP2C12、CYP2C6、CYP2D1和CYP3A显示抑制作用,其IC5o值分别是19.3、35.01、36.69和138.53μg/ML。
雌、雄大鼠被不同剂量盐酸双苯氟嗪诱导14天后,低、中、高剂量盐酸双苯氟嗪对雌、雄大鼠相对肝脏重量、蛋白浓度和CYP450酶含量没有影响；苯巴比妥使雌、雄大鼠相对肝脏重量、蛋白浓度和CYP450酶含量均显著增加。药动学参数和探针药物实验结果均显示,在雌性大鼠,盐酸双苯氟嗪低剂量仅对CYP2D1有抑制作用,中高剂量则不仅抑制CYP2D1,尚能抑制CYP2C12和CYP3A,与雌性大鼠肝微粒体温孵结果基本一致,所不同的是在体外试验盐酸双苯氟嗪抑制CYP2C6,而在整体试验其三个剂量均对CYP2C6有诱导作用。在雄性大鼠,盐酸双苯氟嗪低剂量仅对CYP2C11有诱导作用,并不抑制任何CYP450酶；中剂量则可抑制CYP2D1和诱导CYP2C11及CYP3A;高剂量既可抑制CYP2C6和CYP2D1,又能诱导CYP2C11和CYP3A,而在雄性大鼠肝微粒体温孵试验盐酸双苯氟嗪则对这4个CYP450酶均呈抑制作用。苯巴比妥对雌雄大鼠的CYP1A2、CYP2C11(雄性大鼠特异性)、CYP2C12(雌性大鼠特异性)、CYP2D1、CYP3A均有诱导作用。
结论：大鼠肝微粒体体外温孵研究结果表明,盐酸双苯氟嗪对CYP450酶活性均呈抑制作用,但存在明显的性别差异。而在整体动物试验研究,盐酸双苯氟嗪对某些CYP450酶活性的抑制作用与体外研究结果基本一致,但对另外一些CYP450酶活性有诱导作用,也存在明显的性别差异。临床用药应避免将盐酸双苯氟嗪与经这些酶代谢的药物共同应用,以减少药物相互作用和不良反应的发生。
Metabolism is a important topic in drug research, and its main aim is to find lead compound which is characterized good DM and PK in the initial stage of drug development. Drug is metabolized in the liver through two general sets of reactions, called PhaseⅠand PhraseⅡ. The Phrase I reactions may increase or decrease the activity or toxicity of micromolecular compounds in body, and CYP450s are the critical enzymes to catalyze the reactions. So, it is meaningful to identify CYP450 isoenzymes which is invovled in the drug metabolism and evaluate the interaction between the drug and P450 isoenzymes. These studies can elucidate metabolism-mechanism, anticipate drug-drug interaction and metabolism-polymorphism. Therefore in Europe and America, identification of CYP450s and their isoenzymes has been used to screen new drugs or study metabolism, and it is one of content in applying for new drug.
Dipfluzine Hydrochloride 1-diphenylmethyl-4-(3-(4-fluorobenzoyl))-piperazine hydrochloride, Dip), a novel diphenylpiperazine calcium channel blocker, was first synthetized by Hebei Medical University. Studies have demonstrated that Dip exerts protective effects against focal or whole cerebral ischemic injury via multiple mechanisms and its pharmacological effects are more potent than its analogs, cinnarizine (CZ) or flunarizine (FZ).
In the present study, the LC-MS-MS method was established to determine four metabolites of Dip in rat liver micrsomes, and illuminate its metabolism-pathway. The conditions of rat liver microsomes incubation were optimized and enzymes kinetics was investigated at the initial stage. And then, inhibitory effects of known CYP450 isoform-selective inhibitors, correlation analysis and recombinant rat CYP450 isoforms were used to identify the isoform of CYP450s invovled in the formation of metabolites and explore the enzyme catalyzing mechanism of Dip. Isoform-specific reaction markers were used to evaluate the effects of Dip on CYP450s activities in vitro and in vivo in rats. These studies are useful to illuminate metabolism-pathway or enzyme catalyzing mechanism of Dip and to understand the interactions of drugs or interacting mechanism, thus drug-drug interaction and adverse effect can be decreased based on these results.
Part one The metabolism of Dip in different sex rat liver microsomes and the establishment of LC-MS-MS methoad for the determination of Dip and its metabolites.
Aim:To determine the metabolism of Dip in different sex rat liver microsomes and establish the LC-MS-MS method for Dip or its metabolites.
Methods:20 Sprague-Dawley rats (10 Male and 10 female), weighing 230±20g were acrificed by decapitation and the livers were removed rapidly. Then the rat liver microsomes were prepared and the concentration of the protein or CYP450 was determined by the method of Lowry or Omura and Sato, respectively. Dip (100μM) was incubated in 500μl pooled rat liver microsomes deluted in potassium phosphate buffer (100 mM, pH 7.4) for 30min at 37℃. Reaction was terminated by placing tubes on ice and the reaction solution was extracted by ethyl acetate, and then 20μl of the organic layer was transferred into LC-MS-MS. Chromatographic separation was performed on a Diamonsil C18 column (250×4.6 mm,5μm, Dikma Technologies), at a flow rate of 0.8 ml/min using a linear gradient elution of A (0.5‰formic acid in methanol) and B (0.5‰formic acid in water). An 8-min linear gradient from 40% A to 95% A was applied, followed by a 5-min elution at 95% A and a 1-min wash to return to the starting condition. The column was maintained at 40℃. Mass spectra was recorded by electrospray ionization with a positive mode.
Results:The contents of total protein and CYP450s in male or female rat liver microsomes were 11.26±2.10 versus 10.47±1.87 mg/ml and 0.627±0.10 versus 0.573±0.077 nmol/mg protein(means±S.D., n=10), respectively. Dip was rapidly metabolized and four metabolites were identified in male rat liver microsomes, which were 1-(4-fluoro-benzene)-4- piperazine-butanone (M1), 4- OH-benzophenone (M2), benzhydrol (M4) and benzophenone (M5), respectively. But three metabolites of Dip were generated in female rat liver microsomes, which were M1, M2 and M5. So metabolite profiles in male rat liver microsomes were different from those in females, indicating there is the gender difference in the metabolism of Dip in rat liver microsomes. The initial two metabolic reaction of Dip were the dealkylation of piperazine ring and the oxidation of diphenyl methylene, which formed M1 and M5, respectively. And then M5 could be further metabolized to form metabolites M2 and M4. Comparing with other metabolites, the content of Ml was the highest in male or female rat liver microsomes, suggesting that M1 is the main metabolite of Dip.
The recovery of Dip and its metabolites was 78-86%, and the precisions of intra- and inter-day were 0.15-14.42% for the developed LC-MS-MS method. The calibration curve was linear in the range from 2.59 to 1295 ng/mL for Dip, from 25.92 to 12961 ng/ml for M1, from 0.065 to 32.38 ng/mL for M2, from 2.63 to 1315 ng/ml for M4, and from 2.46 to 1228 ng/ml for M5. Dip and its metabolites were found to be stable for short-term and long- term or after three cycles of freeze and thaw. These results showed this methoad is qualified to the pharmacokinetic study.
Conclusion:Dip and its metabolites were determined and the metabolic pathway was identified. The developed LC-MS-MS method can satisfy the requirement of pharmacokinetic study and was suitable for simultaneous analysis of Dip and its four metabolites in vitro. There is the gender difference in the metabolism of Dip in rat liver microsomes and M1 is the main metabolite of Dip.
Part two The optimization of incubation condition and the study of enzyme kinetic of Dip in rat liver microsomes
Aim:To optimize the conditions for microsomal incubation and measure kinetic parameters of the metabolites for Dip to provide the reference for the further research of rat liver microsomes.
Methods:Time for microsomal incubation, protein concentration of rat liver microsomes and substrate concentration were used as investigating objects. When one of them was changed after the immobilization of the other, the conditions for microsomal incubation could be optimized by observing the concentrations of metabolites. The kinetic parameters (Km, Vmax and Clint) in rat liver microsomes or recombined CYP450s were calculated by Lineweaver-Burk plot
Results:Incubation time of 60 min or 30min, protein concentration of 1 mg/ml and substrate concentration of 10μM or 25μM were found to be optimal for the metabolism of Dip in female or male rat liver microsomes, respectively. In male rat liver microsomes, Km and Vmax for M1, M2, M4 and M5 were 3.97,0.0064,4.14μM,3.69μM, and 650.26,0.98,108.66, 100.92 pmol/min/nmol P450, respectively. In female rat liver microsomes, Km and Vmax for M1, M2 and M5 were 3.89,0.0055,5.63μM and 330.17, 0.58,6.54 pmol/min/nmol P450, respectively. In the recombinant CYP450s incubation,3.52,0.0067,5.67,3.90μM and 683.15,1.14,136.40,115.41 pmol/min/nmol P450 were Km and Vmax for M1, M2, M4, M5 in CYP3Al,and 3.49,0.0081,5.03μM,3.95μM and 719.44,1.57,144.91,132.80 pmol/min/nmol P450 were Km and Vmax for M1, M2, M4, M5 in CYP3A2.
Conclusion:The optimal incubation time in male or female rat liver microsomes is 30min or 60min, respectively, the optimal concentration of protein is all 1mg/mL, the optimal concentration of Dip in male or female rat microsomal incubation is 25μM or 10μM, respectively. Under these optimal conditions, the formation velocity for all metabolites is saturable, and measured enzyme kinetic parameters of rat liver microsomes and recombined CYP450s, such as Vmax, Km and Clint, were accdurate and reliable, which could satisfy the requests for Dip metabolism research. Thus these results can provide the reference for further research on rat liver microsomes or recombinant CYP450s.
Part three Identification of rat liver microsomal CYP450 enzymes involved in the metabolism of Dip
Aim:To study the CYP450 enzymes involved in the metabolism of Dip in different gender rat liver microsomes.
Methods:The CYP450 isoforms involved in the metabolism of Dip were investigated by the experiments of selective CYP450 inhibitors, correlation analysis and recombinant CYP450 enzymes.
The following P450 isoform-selective inhibitors were used:furafylline (CYP1A2 inhibitor used at 1～100μM), pilocarpine (CYP2A1 inhibitor used at 0.5～100μM), orphenadrine (CYP2B1 inhibitor used at 1～100μM), sulfaphenazole (CYP2C6 inhibitor used at 1～200μM), cimetidine (CYP2C11 or CYP2C12 inhibitor used at 0.5～100μM), quinidine (CYP2D1 inhibitor used at 1～100μM), diethyldithiocarbamate (CYP2E1 inhibitor used at 2.5～200μM) and ketoconazole (CYP3A inhibitor used at 0.5～200μM). Dip was added and incubated with inhibitors in male or female rat liver microsomes or recombinant CYP450 isoforms, and then the concentration of metabolites was determined. The ratio of metabolites in samples with and without inhibitor was calculatedand the effect of chemical inhibitors on the metabolism of Dip was evaluated.
Dip was incubated with microsomes from 15 different rat livers to test the correlation of Dip metabolism and the activity of individual CYP450s. The following isoform-specific reaction markers were used to determine the activity of each rat CYP450 isoform: phenacetin O-deethylation (CYP1A2), testosterone 7a-hydroxylation (CYP2A1), testosterone 16β-hydroxylation (CYP2B1), diclofenac 4-hydroxylation (CYP2C6), testosterone 16a-hydroxylation (CYP2C11),5α-androstane-3α,17β-diol-3,17-disulphate 15β- hydroxylation (CYP2C12), dextromethorphan O-demethylation (CYP2D1), chlorzoxazone 6-hydroxylation (CYP2E1) and midazolam 1'-hydroxylation (CYP3A). The correlation coefficients between the formation rates of Dip metabolites and the activity of each CYP450 isoform in the different rat liver microsomes were calculated.
Dip was incubated with a panel of recombinant rat CYP450 isoforms (Rat CYP1A2, CYP2A1, CYP2B1, CYP2C6, CYP2C11, CYP2D1, CYP2E1 and CYP3A1/2) to assess the involvement of each individual recombinant rat CYP450 enzyme in metabolite formation
Results:The results from experiments of selective CYP450 inhibitors, correlation analysis and recombinant rat CYP450 isoforms all confirmed that in male rat liver microsomes in order of contribution, CYP2A1, CYP3A, CYP1A2, CYP2E1 and CYP2C11 contributesto M1 generation. CYP3A, CYP2A1, CYP1A2, CYP2C11 and CYP2E1 metabolize Dip to M2, CYP3A, CYP2A1, CYP2E1 and CYP2C11 contributed to M4 formation, and CYP3A, CYP2Aland CYP2C6 involved in the M5 formation. So, CYP3A and CYP2A1 were the major CYP isoenzymes responsible for catalyzing Dip to four metabolites in male rat liver microsomes. For female rat liver microsomes, CYP1A2 and CYP3A metabolize Dip to M1, CYP3A, CYP1A2, CYP2C6, CYP2A1and CYP2E1 involved in M2 formation, CYP3A, CYP2A1, CYP2D1 and CYP1A2 have major roles to M5 formation. Thus, CYP3A and CYP1A2 were the major isoenzymes for all four metabolites in female rat liver microsomes. These results above suggest that CYP3A is the most important isoenzyme for the metabolism of Dip in rat liver microsomes for different sex, and CYP3A2 exhibited more activity than CYP3A1.
Conclusion:CYP3A is the most important isoenzyme for the metabolism of Dip in two gender rat liver microsomes, and CYP3A2 exhibited more potent activity than CYP3A1. Both CYP1A2 in female rats and CYP2A1 in the male rats involve in the formation reactions of four metabolites of Dip, which reveals predominant noticeable gender difference.
Part four Effects of Dip on CYP450s activities in rats
Aim:To evaluate the effects of Dip on CYP450s activities in vitro and in vivo in rats.
Methods:Markers were incubated in normal rat liver microsomes with Dip (0-200μM). After reactions were terminated and extracted by ethyl acetate, then the organic layer was transferred into LC-MS-MS and determined the concentration of metabolites for markers. Calculate ratio of content of metabolites in samples (added Dip) and in the control (no Dip), and evaluate the effect of Dip on the CYP450s.
Male and female SD rats were divided into five groupes. Dip was administered by orally to rats at doses of 30,60 and 90 mg·kg-1 body weight for fourteen days. At the fifteen day, rats were orally administered Cocktail probe markers:phenacetin, tolbutamide, omeprazole, dextromethorphan, chlorzoxazone and aminophenylsulfone. Blood samples were collected via medial angle of eye at 5,15,40,60,90,120,180,240,360,480 and 720 min after administration of Dip, put into heparinized centrifuge tube, respectively, and centrifuged. The separated plasma was extracted by ethyl acetate and 20μl of the supernatants were transferred to LC-MS-MS for determination. The concentrations of probe markers and their metabolites were determined in plasma samples at 360 min after administration of Dip andthe drug-time curve was plotted, by which pharmacokinetics parameteres were calculated.
Rats were sacrificed by decapitation, the livers were weighed, removed rapidly and rat liver microsomes were prepared. Probe markers were added into incubation samples, and after reaction was completed, samples were extracted by ethyl acetate,20μL of the supernatants were transferred to LC-MS-MS for determining the concentration of probe markers and its metabolites. The metabolism ratio of probe in plasma and in liver microsome at 360 min after administration of Dip were calculated and the effects of Dip on CYP450s activities were evaluated by comparing the relative liver weight, concentration of protein and CYP450 content, the drug concentration-time cueves, pharmacokinetics parameteres and the metabolism ratio of probes between the experimental groups and control group..
Results:In the normal male rat liver microsomes, Dip had inhibitive effects on CYP2D1, CYP2C6 and CYP2C11, IC50 were 8.85,20.93 and 69.45μg/mL, respectively. However, for the female, Dip showed inhibitor on CYP2C12, CYP2C6, CYP2D1and CYP3A, IC50 were 19.3,35.01,36.69 and 138.53μg/mL, respectively.
Dip had no effect on the relative weight of livers, protein concentration and CYP450 content for male and female rats after they were feeded on Dip for fourteen days, but these indexes were raised remarkably when phenobarbital was administered by orally to rats. Results from pharmacokinetics parameteres and markers displayed that low-dose Dip inhibited the activities of CYP2D1 in female rats, or induced the activities of CYP2C11 in male rats and CYP2C6 in female rats. Moderate-dose Dip showed the ability to inhibit CYP2D1 in male rats and CYP2C12, CYP2D1and CYP3A in female rats or induce CYP2C11, CYP3A in male rats and CYP2C6 in female rats. High-dose Dip had certain inhibitive effect to CYP2C6, CYP2D1 in male rats and CYP2C12, CYP2D1, CYP3A in female rats and inductive effect to CYP2C11, CYP3A in male rats and CYP2C6 in female rats. CYP1A2, CYP2C11, CYP2C12, CYP2D1 and CYP3A in male or female rats were all induced after administration of phenobarbital.
Conclusion:The results of normal rat liver microsomes incubation show that Dip inhibits the activity of CYP450s in either male or female rats, in whom there is a significant gender difference. The results of all Dip-induced rat liver microsomes incubation show that Dip had inhibitive effects on the activity of certain CYP450s, which is essentially consistent with those from rat liver microsomes incubation, but Dip can also induce the activity of some CYP450s. These results also exist the gender difference. Thus, co-administration of Dip and other drugs that were metabolized by the P450s must be avoided to decrease drug-drug interaction and adverse effect.
盐酸双苯氟嗪是河北医科大学开发的新型哌嗪类钙通道拮抗剂,研究表明该药经由多种机制保护局灶性和全脑缺血再灌注损伤,其药理作用强于同类药氟桂利嗪和桂利嗪。
本实验采用液质联用技术(LC-MS-MS)确定了盐酸双苯氟嗪在大鼠肝微粒体中代谢生成的4个特征性代谢产物的结构,明确了其代谢途径；进行了肝微粒体温孵条件优化和酶动力学研究；利用化学抑制剂实验、相关性分析和重组酶实验研究了盐酸双苯氟嗪在不同性别大鼠肝微粒体中的代谢行为,鉴定了参与代谢产物生成的CYP450酶亚型,初步探讨了其酶催化机制；通过体内和体外实验考察了盐酸双苯氟嗪对不同性别大鼠CYP450酶活性的影响。上述系列研究为探讨盐酸双苯氟嗪的代谢途径和酶催化机制、研究盐酸双苯氟嗪与其他药物相互作用及作用机制奠定基础,从而为制定安全、合理的临床用药方案、减少药物不良反应提供了科学依据。
第一部分盐酸双苯氟嗪在大鼠肝微粒体中的代谢行为及其代谢物LC-MS-MS测定条件的确立
目的：确定盐酸双苯氟嗪在不同性别大鼠肝微粒体中的代谢行为；建立盐酸双苯氟嗪及其代谢物的LC-MS-MS测定方法。
方法：雌、雄SD大鼠各10只,体重230±20g,断头处死,取肝脏,超速离心法制备肝微粒体,Lowry法测定蛋白浓度,Omura和Sato方法测定CYP450酶含量；盐酸双苯氟嗪(100μM)在500μl肝微粒体温孵反应系统中(pH 7.4)于37℃水浴中振荡温孵30min,反应结束后于冰浴中终止反应。取100μl反应液,经乙酸乙酯萃取处理后,吸取上清液20μ1进行LC-MS-MS分析。肝微粒体样品分析测定采用Dikma RP-C18分析色谱柱(250×4.6mm,5μm),流动相有机相A为甲醇/0.5%o甲酸(1000：0.5),水相B为0.5‰甲酸溶液,采用梯度洗脱方式,洗脱程序为：0～1 min, 40～95% A; 1～6 min,95% A; 6～7 min,95～60% A;柱温40℃,流速为0.8ml/ml。盐酸双苯氟嗪及其代谢物采用ESI正离子模式检测。
结果：雌、雄大鼠肝微粒体蛋白浓度分别为11.26±2.10 mg/ml和10.47±1.87 mg/ml, CYP450含量为0.627±0.10 nmol/mg protein和0.573±0.077 nmol/mg protein。盐酸双苯氟嗪在雄性大鼠肝微粒体中被迅速代谢成4个代谢物,它们分别是盐酸双苯氟嗪哌嗪环脱烷基代谢物(1-4-氟苯基)-4-哌嗪基丁酮(M1)、二苯亚甲基烷基氧化苯环还原代谢物4-羟基二苯甲酮(M2)、二苯亚甲基还原代谢物二苯甲醇(M4)和二苯亚甲基氧化代谢物二苯甲酮(M5)；在雌性大鼠肝微粒体中被迅速代谢成3个代谢物,分别是M1、M2和M5；以上结果提示,盐酸双苯氟嗪在大鼠肝微粒体中的代谢行为具有性别差异。盐酸双苯氟嗪在大鼠肝微粒体中的最初两步代谢反应是哌嗪环脱烷基生成M1和二苯亚甲基氧化生成M5,M5可以被进一步代谢为M2和M4；在两种不同性别大鼠肝微粒体中,M1的浓度均比其他代谢物为高,是盐酸双苯氟嗪在大鼠肝微粒体中生成的最主要的代谢物。
所建立的LC-MS-MS测定方法的回收率为78-86%,日内和日间RSD为0.15-14.42%,符合药代动力学分析方法的要求；盐酸双苯氟嗪在2.59-1295 ng/ml、M1在25.92-12961 ng/ml、M2在0.065-32.38 ng/ml、M4在2.63-1315 ng/ml、M5在2.46-1228 ng/ml范围内呈良好线性关系；盐酸双苯氟嗪及其代谢物短期稳定性、长期稳定性和3次冷冻-解冻循环稳定性符合相关要求。
结论：本研究建立的LC-MS-MS测定方法符合药代动力学分析方法的要求,适合盐酸双苯氟嗪及其4个代谢物的含量测定,且盐酸双苯氟嗪在大鼠肝微粒体中的代谢行为具有性别差异,M1为其最主要的代谢物。
第二部分盐酸双苯氟嗪在大鼠肝微粒体中温孵反应条件的优化和酶动力学研究
目的：对盐酸双苯氟嗪在大鼠肝微粒体中温孵反应的条件进行优化,并测定盐酸双苯氟嗪代谢物的酶动力学参数,为后续大鼠肝微粒体实验提供依据。
方法：以温孵时间、大鼠肝微粒体蛋白浓度和底物浓度为考察对象,固定其中两个温孵条件,观察第三个条件对盐酸双苯氟嗪代谢物生成量或生成速率的影响,进而优化温孵条件。用Lineweaver-Burk作图法计算盐酸双苯氟嗪在大鼠肝微粒体和CYP450重组酶中的酶动力学参数(最大速率Vmax、米氏常数Km和清除率Clint)。
结果：盐酸双苯氟嗪在雌、雄大鼠肝微粒体中进行温孵反应的最佳时间分别为60min和30min,肝微粒体蛋白浓度均为1mg/ml,底物浓度分别为10gM和25gM。在雄性大鼠肝微粒体中,M1、M2、M4和M5的Km值分别是3.97μM、0.0064μM、4.14μM和3.69μM, Vmax分别是650.26、0.98、108.66和100.92 pmol/min/nmol P450;在雌性大鼠肝微粒体中,M1、M2和M5的Km值分别是3.89μM、0.0055μM和5.63μM,Vmax分别是330.17、0.58和6.54 pmol/min/nmol P450。在重组酶CYP3A1温孵液中,M1、M2、M4和M5的Km值分别是3.52μM、0.0067μM、5.67μM和3.90μM, Vmax分别是683.15、1.14、136.40和115.41 pmol/min/nmol P450;在重组酶CYP3A2温孵液中,M1、M2、M4和M5的Km值分别是3.49μM、0.0081μM、5.03μM和3.95μM, Vmax分别是719.44、1.57、144.91和132.80 pmol/min/nmol P45。
结论：盐酸双苯氟嗪在雌性或雄性大鼠肝微粒体的最佳温孵时间分别是60min和30min,最佳肝微粒体蛋白浓度均为1mg/mL;最佳盐酸双苯氟嗪浓度为10μM或25μM。在此优化的温孵条件下,代谢物生成速率均能达到饱和状态,所测定的大鼠肝微粒体和CYP450重组酶的各项酶动力学参数(Vmax、Km和Clint)准确、可靠,可满足盐酸双苯氟嗪体外代谢研究要求,为后续大鼠肝微粒体和CYP450重组酶实验提供依据。
第三部分参与盐酸双苯氟嗪代谢的大鼠肝微粒体CYP450酶的鉴别
目的：研究不同性别大鼠肝微粒体中,参与盐酸双苯氟嗪代谢的CYP450酶亚型。
方法：运用CYP450酶选择性抑制剂、相关性分析和重组酶实验探讨参与盐酸双苯氟嗪代谢的CYP450酶亚型。
化学抑制剂实验,选用P450酶特异性抑制剂[]呋拉茶碱(CYP1A2, 1-100μM)、毛果芸香碱(CYP2A1,0.5-100μM)、邻甲苯海拉明(CYP2B1, 1-100μM)、磺胺苯毗唑(CYP2C6,1-200μM)、西咪替丁(CYP2C11和CYP2C12,0.5-100μM)、奎尼丁(CYP2D1, 1-100μM)、二乙基二硫代氨基甲酸酯钠(CYP2E1,2.5-200μM)和酮康唑(CYP3A,0.5-200μM)分别加入不同性别大鼠肝微粒体中与盐酸双苯氟嗪共同温孵,测定反应系统中代谢产物生成量；用含抑制剂的样品溶液中代谢物的生成量与不含抑制剂的阴性对照溶液中代谢物生成量的百分比定量,评价不同抑制剂对各代谢物生成的影响。
相关性分析实验,将盐酸双苯氟嗪分别与15个来自不同大鼠的肝微粒体样本共同温孵,测定盐酸双苯氟嗪代谢反应与P450酶活性的相关性。以下列酶亚型特异性探针药物的代谢反应生成速率来测定P450酶的活性：非那西丁O-脱乙基反应(CYP1A2)、睾酮7a-羟基化反应(CYP2A1)、睾酮16p-羟基化反应(CYP2B1)、双氯芬酸4-羟基化反应(CYP2C6)、睾酮16α-羟基化反应(CYP2C11) 5α-androstane-3α,17β-diol-3,17-disulphate 15β-羟基化反应(CYP2C12)、右美沙芬O-脱甲基反应(CYP2D1)、氯唑沙宗6-羟基化反应(CYP2E1)和咪达唑仑1’-羟基化反应(CYP3A)。将盐酸双苯氟嗪代谢物的生成速率与探针药物代谢物的生成速率进行线性回归,计算相关系数。
CYP450重组酶实验,盐酸双苯氟嗪与重组酶(CYP1A2, CYP2A1, CYP2B1, CYP2C6, CYP2C11/12, CYP2D1, CYP2E1和CYP3A1/2)进行温孵,测定盐酸双苯氟嗪代谢物的生成速率,确证参与盐酸双苯氟嗪代谢的CYP450酶亚型。
结果：抑制剂研究、相关性分析和重组酶实验三种分析方法测定结果均显示,雄性大鼠肝微粒体中,代谢盐酸双苯氟嗪生成M1的最主要的酶亚型是CYP2A1,其他依次为CYP3A、CYP1A2和CYP2E1,而CYP2C11的作用最小。代谢盐酸双苯氟嗪生成M2的CYP450酶按作用大小依次为CYP3A、CYP2A1、CYP1A2、CYP2C11和CYP2E1。转化盐酸双苯氟嗪生成M4的酶亚型活性高低依次为CYP3A、CYP2A1、CYP2E1和CYP2C11,而参与M5生成的酶亚型分别为CYP3A、CYP2A1和CYP2C6。其中CYP3A和CYP2A1均参与了雄性大鼠肝微粒体中4种代谢物的生成反应。在雌性大鼠肝微粒体中,CYP1A2和CYP3A参与了M1的生成,且CYP3A的作用比CYP1A2稍强。参与M2生成的5种P450酶按作用大小依次为CYP3A、CYP1A2、CYP2C6、CYP2A1和CYP2E1。而CYP3A、CYP2A1、CYP2D1和CYP1A2则是参与M5生成的4种酶亚型。其中,CYP3A和CYP1A2均参与了雌性大鼠肝微粒体中3种代谢物的生成反应。由此可见,CYP3A是盐酸双苯氟嗪在不同性别大鼠肝微粒体中代谢的最主要的酶亚型,重组酶实验结果表明,CYP3A2显示出比CYP3A1更强的活性。
结论：三种方法分析结果共同确证了CYP3A是两种不同性别大鼠肝微粒体中参与盐酸双苯氟嗪代谢的最主要CYP450酶亚型,其中CYP3A2的活性比CYP3A1更强；均能参与盐酸双苯氟嗪代谢物生成反应的CYP450酶亚型,在雌性大鼠为CYP1A2,在雄性大鼠为CYP2A1,具有明显的性别差异。第四部分盐酸双苯氟嗪对大鼠CYP450酶活性的影响
目的：通过大鼠体内、外实验,观察盐酸双苯氟嗪对不同性别大鼠CYP450酶的影响。
方法：在正常雌、雄大鼠肝微粒体温孵反应系统中加入不同浓度盐酸双苯氟嗪(0-200μM)和探针药物共同温孵,反应结束后,样品经处理,进行LC-MS-MS分析,测定探针药物代谢产物生成量；用加入盐酸双苯氟嗪的样品溶液中探针药物代谢物的生成量与不加盐酸双苯氟嗪的阴性对照溶液中代谢物生成量的百分比定量,观察盐酸双苯氟嗪对CYP450的影响。
SD大鼠,雌、雄各半,随机分为空白组、盐酸双苯氟嗪低、中、高剂量组和苯巴比妥组,每组6只。实验组分别灌胃给予盐酸双苯氟嗪30、60、90 mg/kg,苯巴比妥组灌胃给予苯巴比妥120 mg/kg,空白对照组灌胃给予等容积0.5%羧甲基纤维素钠,连续给药14天。第15天分别灌胃给予Cocktail探针药物非那西丁、甲苯磺丁脲、奥美拉唑、右美沙芬、氯唑沙宗和氨苯砜,于灌胃后5、15、40、60、90、120、180、240、360、480、720min时,眼内眦取血,置肝素锂抗凝管中,离心,分离出血浆,经乙酸乙酯萃取后,进样20μL进行LC-MS-MS分析,绘制药-时曲线,计算药代动力学参数；测定给药360min时血浆样品中探针药物及其代谢物的浓度。取血结束后,断头处死大鼠,取肝脏,称重,制备肝微粒体。在肝微粒体温孵反应系统中加入探针药物,反应结束后,样品经处理,进行LC-MS-MS分析,测定探针药物及其代谢物的浓度,计算给药360min时血浆样品和肝微粒体温孵系统中探针药物的代谢率。通过比较实验组和空白对照组的相对肝脏重量、蛋白浓度、CYP450酶含量、药-时曲线、药代动力学参数和探针药物的代谢率,确定盐酸双苯氟嗪对CYP450酶活性的影响。
结果：在正常雄性大鼠肝微粒体温孵液中,盐酸双苯氟嗪对CYP2C6、CYP2D1和CYP2C11有抑制作用,其IC50值分别是8.85、20.93和69.45μg/mL;在正常雌性大鼠肝微粒体温孵液中,盐酸双苯氟嗪对CYP2C12、CYP2C6、CYP2D1和CYP3A显示抑制作用,其IC5o值分别是19.3、35.01、36.69和138.53μg/ML。
雌、雄大鼠被不同剂量盐酸双苯氟嗪诱导14天后,低、中、高剂量盐酸双苯氟嗪对雌、雄大鼠相对肝脏重量、蛋白浓度和CYP450酶含量没有影响；苯巴比妥使雌、雄大鼠相对肝脏重量、蛋白浓度和CYP450酶含量均显著增加。药动学参数和探针药物实验结果均显示,在雌性大鼠,盐酸双苯氟嗪低剂量仅对CYP2D1有抑制作用,中高剂量则不仅抑制CYP2D1,尚能抑制CYP2C12和CYP3A,与雌性大鼠肝微粒体温孵结果基本一致,所不同的是在体外试验盐酸双苯氟嗪抑制CYP2C6,而在整体试验其三个剂量均对CYP2C6有诱导作用。在雄性大鼠,盐酸双苯氟嗪低剂量仅对CYP2C11有诱导作用,并不抑制任何CYP450酶；中剂量则可抑制CYP2D1和诱导CYP2C11及CYP3A;高剂量既可抑制CYP2C6和CYP2D1,又能诱导CYP2C11和CYP3A,而在雄性大鼠肝微粒体温孵试验盐酸双苯氟嗪则对这4个CYP450酶均呈抑制作用。苯巴比妥对雌雄大鼠的CYP1A2、CYP2C11(雄性大鼠特异性)、CYP2C12(雌性大鼠特异性)、CYP2D1、CYP3A均有诱导作用。
结论：大鼠肝微粒体体外温孵研究结果表明,盐酸双苯氟嗪对CYP450酶活性均呈抑制作用,但存在明显的性别差异。而在整体动物试验研究,盐酸双苯氟嗪对某些CYP450酶活性的抑制作用与体外研究结果基本一致,但对另外一些CYP450酶活性有诱导作用,也存在明显的性别差异。临床用药应避免将盐酸双苯氟嗪与经这些酶代谢的药物共同应用,以减少药物相互作用和不良反应的发生。
Metabolism is a important topic in drug research, and its main aim is to find lead compound which is characterized good DM and PK in the initial stage of drug development. Drug is metabolized in the liver through two general sets of reactions, called PhaseⅠand PhraseⅡ. The Phrase I reactions may increase or decrease the activity or toxicity of micromolecular compounds in body, and CYP450s are the critical enzymes to catalyze the reactions. So, it is meaningful to identify CYP450 isoenzymes which is invovled in the drug metabolism and evaluate the interaction between the drug and P450 isoenzymes. These studies can elucidate metabolism-mechanism, anticipate drug-drug interaction and metabolism-polymorphism. Therefore in Europe and America, identification of CYP450s and their isoenzymes has been used to screen new drugs or study metabolism, and it is one of content in applying for new drug.
Dipfluzine Hydrochloride 1-diphenylmethyl-4-(3-(4-fluorobenzoyl))-piperazine hydrochloride, Dip), a novel diphenylpiperazine calcium channel blocker, was first synthetized by Hebei Medical University. Studies have demonstrated that Dip exerts protective effects against focal or whole cerebral ischemic injury via multiple mechanisms and its pharmacological effects are more potent than its analogs, cinnarizine (CZ) or flunarizine (FZ).
In the present study, the LC-MS-MS method was established to determine four metabolites of Dip in rat liver micrsomes, and illuminate its metabolism-pathway. The conditions of rat liver microsomes incubation were optimized and enzymes kinetics was investigated at the initial stage. And then, inhibitory effects of known CYP450 isoform-selective inhibitors, correlation analysis and recombinant rat CYP450 isoforms were used to identify the isoform of CYP450s invovled in the formation of metabolites and explore the enzyme catalyzing mechanism of Dip. Isoform-specific reaction markers were used to evaluate the effects of Dip on CYP450s activities in vitro and in vivo in rats. These studies are useful to illuminate metabolism-pathway or enzyme catalyzing mechanism of Dip and to understand the interactions of drugs or interacting mechanism, thus drug-drug interaction and adverse effect can be decreased based on these results.
Part one The metabolism of Dip in different sex rat liver microsomes and the establishment of LC-MS-MS methoad for the determination of Dip and its metabolites.
Aim:To determine the metabolism of Dip in different sex rat liver microsomes and establish the LC-MS-MS method for Dip or its metabolites.
Methods:20 Sprague-Dawley rats (10 Male and 10 female), weighing 230±20g were acrificed by decapitation and the livers were removed rapidly. Then the rat liver microsomes were prepared and the concentration of the protein or CYP450 was determined by the method of Lowry or Omura and Sato, respectively. Dip (100μM) was incubated in 500μl pooled rat liver microsomes deluted in potassium phosphate buffer (100 mM, pH 7.4) for 30min at 37℃. Reaction was terminated by placing tubes on ice and the reaction solution was extracted by ethyl acetate, and then 20μl of the organic layer was transferred into LC-MS-MS. Chromatographic separation was performed on a Diamonsil C18 column (250×4.6 mm,5μm, Dikma Technologies), at a flow rate of 0.8 ml/min using a linear gradient elution of A (0.5‰formic acid in methanol) and B (0.5‰formic acid in water). An 8-min linear gradient from 40% A to 95% A was applied, followed by a 5-min elution at 95% A and a 1-min wash to return to the starting condition. The column was maintained at 40℃. Mass spectra was recorded by electrospray ionization with a positive mode.
Results:The contents of total protein and CYP450s in male or female rat liver microsomes were 11.26±2.10 versus 10.47±1.87 mg/ml and 0.627±0.10 versus 0.573±0.077 nmol/mg protein(means±S.D., n=10), respectively. Dip was rapidly metabolized and four metabolites were identified in male rat liver microsomes, which were 1-(4-fluoro-benzene)-4- piperazine-butanone (M1), 4- OH-benzophenone (M2), benzhydrol (M4) and benzophenone (M5), respectively. But three metabolites of Dip were generated in female rat liver microsomes, which were M1, M2 and M5. So metabolite profiles in male rat liver microsomes were different from those in females, indicating there is the gender difference in the metabolism of Dip in rat liver microsomes. The initial two metabolic reaction of Dip were the dealkylation of piperazine ring and the oxidation of diphenyl methylene, which formed M1 and M5, respectively. And then M5 could be further metabolized to form metabolites M2 and M4. Comparing with other metabolites, the content of Ml was the highest in male or female rat liver microsomes, suggesting that M1 is the main metabolite of Dip.
The recovery of Dip and its metabolites was 78-86%, and the precisions of intra- and inter-day were 0.15-14.42% for the developed LC-MS-MS method. The calibration curve was linear in the range from 2.59 to 1295 ng/mL for Dip, from 25.92 to 12961 ng/ml for M1, from 0.065 to 32.38 ng/mL for M2, from 2.63 to 1315 ng/ml for M4, and from 2.46 to 1228 ng/ml for M5. Dip and its metabolites were found to be stable for short-term and long- term or after three cycles of freeze and thaw. These results showed this methoad is qualified to the pharmacokinetic study.
Conclusion:Dip and its metabolites were determined and the metabolic pathway was identified. The developed LC-MS-MS method can satisfy the requirement of pharmacokinetic study and was suitable for simultaneous analysis of Dip and its four metabolites in vitro. There is the gender difference in the metabolism of Dip in rat liver microsomes and M1 is the main metabolite of Dip.
Part two The optimization of incubation condition and the study of enzyme kinetic of Dip in rat liver microsomes
Aim:To optimize the conditions for microsomal incubation and measure kinetic parameters of the metabolites for Dip to provide the reference for the further research of rat liver microsomes.
Methods:Time for microsomal incubation, protein concentration of rat liver microsomes and substrate concentration were used as investigating objects. When one of them was changed after the immobilization of the other, the conditions for microsomal incubation could be optimized by observing the concentrations of metabolites. The kinetic parameters (Km, Vmax and Clint) in rat liver microsomes or recombined CYP450s were calculated by Lineweaver-Burk plot
Results:Incubation time of 60 min or 30min, protein concentration of 1 mg/ml and substrate concentration of 10μM or 25μM were found to be optimal for the metabolism of Dip in female or male rat liver microsomes, respectively. In male rat liver microsomes, Km and Vmax for M1, M2, M4 and M5 were 3.97,0.0064,4.14μM,3.69μM, and 650.26,0.98,108.66, 100.92 pmol/min/nmol P450, respectively. In female rat liver microsomes, Km and Vmax for M1, M2 and M5 were 3.89,0.0055,5.63μM and 330.17, 0.58,6.54 pmol/min/nmol P450, respectively. In the recombinant CYP450s incubation,3.52,0.0067,5.67,3.90μM and 683.15,1.14,136.40,115.41 pmol/min/nmol P450 were Km and Vmax for M1, M2, M4, M5 in CYP3Al,and 3.49,0.0081,5.03μM,3.95μM and 719.44,1.57,144.91,132.80 pmol/min/nmol P450 were Km and Vmax for M1, M2, M4, M5 in CYP3A2.
Conclusion:The optimal incubation time in male or female rat liver microsomes is 30min or 60min, respectively, the optimal concentration of protein is all 1mg/mL, the optimal concentration of Dip in male or female rat microsomal incubation is 25μM or 10μM, respectively. Under these optimal conditions, the formation velocity for all metabolites is saturable, and measured enzyme kinetic parameters of rat liver microsomes and recombined CYP450s, such as Vmax, Km and Clint, were accdurate and reliable, which could satisfy the requests for Dip metabolism research. Thus these results can provide the reference for further research on rat liver microsomes or recombinant CYP450s.
Part three Identification of rat liver microsomal CYP450 enzymes involved in the metabolism of Dip
Aim:To study the CYP450 enzymes involved in the metabolism of Dip in different gender rat liver microsomes.
Methods:The CYP450 isoforms involved in the metabolism of Dip were investigated by the experiments of selective CYP450 inhibitors, correlation analysis and recombinant CYP450 enzymes.
The following P450 isoform-selective inhibitors were used:furafylline (CYP1A2 inhibitor used at 1～100μM), pilocarpine (CYP2A1 inhibitor used at 0.5～100μM), orphenadrine (CYP2B1 inhibitor used at 1～100μM), sulfaphenazole (CYP2C6 inhibitor used at 1～200μM), cimetidine (CYP2C11 or CYP2C12 inhibitor used at 0.5～100μM), quinidine (CYP2D1 inhibitor used at 1～100μM), diethyldithiocarbamate (CYP2E1 inhibitor used at 2.5～200μM) and ketoconazole (CYP3A inhibitor used at 0.5～200μM). Dip was added and incubated with inhibitors in male or female rat liver microsomes or recombinant CYP450 isoforms, and then the concentration of metabolites was determined. The ratio of metabolites in samples with and without inhibitor was calculatedand the effect of chemical inhibitors on the metabolism of Dip was evaluated.
Dip was incubated with microsomes from 15 different rat livers to test the correlation of Dip metabolism and the activity of individual CYP450s. The following isoform-specific reaction markers were used to determine the activity of each rat CYP450 isoform: phenacetin O-deethylation (CYP1A2), testosterone 7a-hydroxylation (CYP2A1), testosterone 16β-hydroxylation (CYP2B1), diclofenac 4-hydroxylation (CYP2C6), testosterone 16a-hydroxylation (CYP2C11),5α-androstane-3α,17β-diol-3,17-disulphate 15β- hydroxylation (CYP2C12), dextromethorphan O-demethylation (CYP2D1), chlorzoxazone 6-hydroxylation (CYP2E1) and midazolam 1'-hydroxylation (CYP3A). The correlation coefficients between the formation rates of Dip metabolites and the activity of each CYP450 isoform in the different rat liver microsomes were calculated.
Dip was incubated with a panel of recombinant rat CYP450 isoforms (Rat CYP1A2, CYP2A1, CYP2B1, CYP2C6, CYP2C11, CYP2D1, CYP2E1 and CYP3A1/2) to assess the involvement of each individual recombinant rat CYP450 enzyme in metabolite formation
Results:The results from experiments of selective CYP450 inhibitors, correlation analysis and recombinant rat CYP450 isoforms all confirmed that in male rat liver microsomes in order of contribution, CYP2A1, CYP3A, CYP1A2, CYP2E1 and CYP2C11 contributesto M1 generation. CYP3A, CYP2A1, CYP1A2, CYP2C11 and CYP2E1 metabolize Dip to M2, CYP3A, CYP2A1, CYP2E1 and CYP2C11 contributed to M4 formation, and CYP3A, CYP2Aland CYP2C6 involved in the M5 formation. So, CYP3A and CYP2A1 were the major CYP isoenzymes responsible for catalyzing Dip to four metabolites in male rat liver microsomes. For female rat liver microsomes, CYP1A2 and CYP3A metabolize Dip to M1, CYP3A, CYP1A2, CYP2C6, CYP2A1and CYP2E1 involved in M2 formation, CYP3A, CYP2A1, CYP2D1 and CYP1A2 have major roles to M5 formation. Thus, CYP3A and CYP1A2 were the major isoenzymes for all four metabolites in female rat liver microsomes. These results above suggest that CYP3A is the most important isoenzyme for the metabolism of Dip in rat liver microsomes for different sex, and CYP3A2 exhibited more activity than CYP3A1.
Conclusion:CYP3A is the most important isoenzyme for the metabolism of Dip in two gender rat liver microsomes, and CYP3A2 exhibited more potent activity than CYP3A1. Both CYP1A2 in female rats and CYP2A1 in the male rats involve in the formation reactions of four metabolites of Dip, which reveals predominant noticeable gender difference.
Part four Effects of Dip on CYP450s activities in rats
Aim:To evaluate the effects of Dip on CYP450s activities in vitro and in vivo in rats.
Methods:Markers were incubated in normal rat liver microsomes with Dip (0-200μM). After reactions were terminated and extracted by ethyl acetate, then the organic layer was transferred into LC-MS-MS and determined the concentration of metabolites for markers. Calculate ratio of content of metabolites in samples (added Dip) and in the control (no Dip), and evaluate the effect of Dip on the CYP450s.
Male and female SD rats were divided into five groupes. Dip was administered by orally to rats at doses of 30,60 and 90 mg·kg-1 body weight for fourteen days. At the fifteen day, rats were orally administered Cocktail probe markers:phenacetin, tolbutamide, omeprazole, dextromethorphan, chlorzoxazone and aminophenylsulfone. Blood samples were collected via medial angle of eye at 5,15,40,60,90,120,180,240,360,480 and 720 min after administration of Dip, put into heparinized centrifuge tube, respectively, and centrifuged. The separated plasma was extracted by ethyl acetate and 20μl of the supernatants were transferred to LC-MS-MS for determination. The concentrations of probe markers and their metabolites were determined in plasma samples at 360 min after administration of Dip andthe drug-time curve was plotted, by which pharmacokinetics parameteres were calculated.
Rats were sacrificed by decapitation, the livers were weighed, removed rapidly and rat liver microsomes were prepared. Probe markers were added into incubation samples, and after reaction was completed, samples were extracted by ethyl acetate,20μL of the supernatants were transferred to LC-MS-MS for determining the concentration of probe markers and its metabolites. The metabolism ratio of probe in plasma and in liver microsome at 360 min after administration of Dip were calculated and the effects of Dip on CYP450s activities were evaluated by comparing the relative liver weight, concentration of protein and CYP450 content, the drug concentration-time cueves, pharmacokinetics parameteres and the metabolism ratio of probes between the experimental groups and control group..
Results:In the normal male rat liver microsomes, Dip had inhibitive effects on CYP2D1, CYP2C6 and CYP2C11, IC50 were 8.85,20.93 and 69.45μg/mL, respectively. However, for the female, Dip showed inhibitor on CYP2C12, CYP2C6, CYP2D1and CYP3A, IC50 were 19.3,35.01,36.69 and 138.53μg/mL, respectively.
Dip had no effect on the relative weight of livers, protein concentration and CYP450 content for male and female rats after they were feeded on Dip for fourteen days, but these indexes were raised remarkably when phenobarbital was administered by orally to rats. Results from pharmacokinetics parameteres and markers displayed that low-dose Dip inhibited the activities of CYP2D1 in female rats, or induced the activities of CYP2C11 in male rats and CYP2C6 in female rats. Moderate-dose Dip showed the ability to inhibit CYP2D1 in male rats and CYP2C12, CYP2D1and CYP3A in female rats or induce CYP2C11, CYP3A in male rats and CYP2C6 in female rats. High-dose Dip had certain inhibitive effect to CYP2C6, CYP2D1 in male rats and CYP2C12, CYP2D1, CYP3A in female rats and inductive effect to CYP2C11, CYP3A in male rats and CYP2C6 in female rats. CYP1A2, CYP2C11, CYP2C12, CYP2D1 and CYP3A in male or female rats were all induced after administration of phenobarbital.
Conclusion:The results of normal rat liver microsomes incubation show that Dip inhibits the activity of CYP450s in either male or female rats, in whom there is a significant gender difference. The results of all Dip-induced rat liver microsomes incubation show that Dip had inhibitive effects on the activity of certain CYP450s, which is essentially consistent with those from rat liver microsomes incubation, but Dip can also induce the activity of some CYP450s. These results also exist the gender difference. Thus, co-administration of Dip and other drugs that were metabolized by the P450s must be avoided to decrease drug-drug interaction and adverse effect.
引文
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3 HOUSTONJ B, CARLILEDJ. Prediction of hepatic clearance frommicrosomes, hepatocytes and liver slices[J]. Drug Metab Rev,1997, 29(4):891-922
4 D A Smith, HvandeWaterbeemd, D KWalker, et al. Pharmacokinetics and metabolism in drug design [M]. Newyork:John Wiley and Sons,2001: 137
5 Waxman D J. Rat hepatic cytochrome P-450 isoenzyme 2c[J]. J Biol Chem,1984,259:1548-1549
6 Waxman D J, Dannan G A, Guengerich F. P1 Regulation of rat hepatic cytochrome P2450:Age2dependent Expression, Hormonal Imprinting and Xenobiotic Inducibility of Sex-specific isoenzymes[J]. Biochemstry,1985, 24:4409-4417
1 FAN H R, FAN H, LIU C X. Cocktail the drug probe method used to evaluate cytochrome P450 isozymes of the impact of research[J]. Chin Pharm J(中国药学杂志),2006,41(14)：1045-1048
2 BJORNSSON TD, CALLAGHAN JT, EINOLF HJ, et al. The conduct of in vitro and in vivo drug-drug interaction studies:a pharmaceutical research and manufacturers of america (PhRMA) perspective[J]. Drug Metab Dispos,2003,31(7):518-532
3 DONATO MT, CASTELL JV. Strategies and molecular probes to investigate the role of cytochrome P450 in drug metabolism [J]. Clin Pharmacokinet.2003,42(2):153-178
4 DIERKS EA, STAMS KR, LIM HK, et al. A method for the simultaneous evaluation of the activities of seven major human drug metabolizing cytochrome p450s using an in vitro cocktail of probe substrates and fast gradient liquid chromatography tandem mass spectrometryl[J]. Drug Metab Dispos,2001,29(1):23-29
5 Eagling VA, Tjia JF, and Back DJ. Differential selectivity of cytochrome P450 inhibitors against probe substrates in human and rat liver microsomes. Br J Clin Pharmacol 1998,45:107-114
6刘会臣,杜玉民,王永利.双苯氟嗪在大鼠体内的代谢途径[J].药学学报.2005,40(2)：168-172
7 Meuldermans W, Hendrickx J, Hurkmans R, et al. Excretion and metabolism of flunarizine in rats and dogs[J]. Arzneimittelforschung 1983, 33:1142-1151
8 Kariya S, Isozaki S, Narimatsu S, and Suzuki T. Oxidative metabolism of flunarizine in rat liver microsomes[J]. Res Commun Chem Pathol Pharmacol 1992,78:85-95
9 Lavrijsen K, van Houdt J, van Dyck D, et al. Comparative metabolism of flunarizine in rats, dogs and man:an in vitro study with subcellular liver fractions and isolated hepatocytes. Xenobiotica 1992,22:815-836
10 Narimatsu S, Kariya S, Isozaki S, et al. Involvement of CYP2D6 in oxidative metabolism of cinnarizine and flunarizine in human liver microsomes. Biochem Biophys Res Commun[J] 1993,193:1265-1268
11 Kariya S, Isozaki S, Uchino K, et al. Oxidative metabolism of flunarizine and cinnarizine by microsomes from B-lymphoblastoid cell lines expressing human cytochrome P450 enzymes[J]. Biol Pharm Bull 1996,19: 1511-1514
12 W axman D J. Rat hepatic cytoch rome P-450 isoenzyme 2c[J]. J Biol Chem,1984,259:15481-15490
13 W axman D J, Dannan G A, Guengerich F P. Regulation of rat hepatic cytochrome P-450:A ge-dependent Expression, Hormonal Imprinting, and Xenobiotic Inducibility of Sex specific isoenzymes[J]. B iochem istry,1985,24:4409-4417
14 Kariya S, Isozaki S, Uchino K, et al. Oxidative metabolism of flunarizine and cinnarizine by microsomes from B-lymphoblastoid cell lines expressing human cytochrome P450 enzymes[J]. Biol Pharm Bull 1996,19: 1511-1514
15 Kobayashi K, Urashima K, Shimada N, et al. Selectivities of human cytochrome P450 inhibitors toward rat P450 isoforms:study with cDNA-expressed systems of the rat[J]. Drug Metab Dispos 2003, 31:833-836
16 Eagling VA, Tjia JF and Back DJ. Differential selectivity of cytochrome P450 inhibitors against probe substrates in human and rat liver microsomes[J]. Br J Clin Pharmacol 1998,45:107-114
17赵阳,柳晓泉,钱之玉,等.药物代谢的性别差异[J].药学进展,2001,25(5)：289-293
1 LAHOZ A, DONATO MT, PICAZO L, et al. Determination of major human cytochrome P450s activities in 96-well plates using liquid chromatography tandem mass spectrometry[J]. Toxicol In Vitro,2007, 21(7):1247-1252
2 LAINE JE, AURIOLA S, PASANEN M, et al. Acetaminophen bioactivation by human cytochrome P450 enzymes and animal microsomes[J]. Xenobiotica,2009,39(1):11-21
3 ISOHERRANEN N, HACHAD H, YEUNG CK, et al. Qualitative analysis of the role of metabolites in inhibitory drug-drug interactions:literature evaluation based on the metabolism and transport drug interaction database[J]. Chem Res Toxicol,2009,22(2):294-298
4 KROSSER S, NEUGEBAUER R, DOLGOS H, et al. Investigation of sarizotan's impact on the pharmacokinetics of probe drugs for major cytochrome P450 isoenzymes:a combined cocktail trial[J]. Eur J Clin Pharmacol,2006,62(4):277-284
5 Reginald FF. Probing the world of cytochrome P450 enzymes [J]. Mol Interv,2004,4(3):157-162
6 Siwaporn C, Anne NN, Steven L, et al. Combined phenotypic assessment of cytochrome P450 1A2,2C9,2C19,2D6 and 3A, N-acetyltransferase-2 and xanthineoxidase activities with the "Cooperstown 5+ 1 cocktail" [J].Clin Pharmacol Ther,2003,74(5):437-447
7 Magnus C, Katarina A, Per D, et al. The Karolinska cocktail for phenotyping of five human cytochrome P450 enzymes[J]. Clin Pharmacol Ther,2003,73(6):517-528
8 BROWN HS, GALETIN A, HALLIFAX D, et al. Prediction of in vivo drug-drug interactions from in vitro data:factors affecting prototypic drug-drug interactions involving CYP2C9, CYP2D6 and CYP3A4[J]. Clin Pharmacokinet,2006,45(10):1035-1050
9 TOMALIK-SCHARTE D, JETTER A, KINZIG-SCHIPPERS M, et al. Effect of propiverine on cytochrome P450 enzymes:a cocktail interaction study in healthy volunteers[J]. Drug Metab Dispos,2005,33(12): 1859-1866
10 Zhou HH, Zeen T, James FM. "Cocktail" approaches and strategies in drug development:valuable tool or flawed science[J]. J Clin Pharmacol,2004, 44:120-134
11 TANG H, MIN G, GE B, et al. Evaluation of protective effects of Chi-Zhi-Huang decoction on Phase I drug metabolism of liver injured rats by cocktail probe drugs[J]. J Ethnopharmacol,2008,117(3):420-426
12 KUMAR V, WAHLSTROM JL, ROCK DA, et al. CYP2C9 inhibition: impact of probe selection and pharmacogenetics on in vitro inhibition profiles[J]. Drug Metab Dispos,2006,34(12):1966-1975
13于卫江,黄丽军,朱大岭.应用大鼠肝微粒体筛选对细胞色素P4502D6有抑制作用的中药[J].中草药,2007,38(3)：397-401
14于卫江,黄丽军,刘艳,等.奥扎格雷钠对大鼠CYP2D6亚型酶的影响.[J]中国药理学通报,2007,23(1)：67-72
15 Zhao B C. Enzyme[M]//Zhou A R. Biochemistry.5 th ed. Beijing:People Medical Publishing House,2001:49-70
16 Kaoru K, Kikuko U, Noriaki S, et al. Substrate specificity for tar cytochrome P450 isoforms:screening wiyh cDNA-expressed systems of rat [J]. Biochemical Pharmacology,2002,63:889-896
17 LA Vignati, A Bogni, P Grossi, et al. A human and mouse pregnane X receptor reporter gene assay in combination with cytotoxicity measurement s as a tool to evaluate species specific CYP3A induction[J]. Toxicology, 2004,199:23-33
1 Prentis RA, Lis Y, Walker SR. Pharmaceutical innovation by seven U K-owned pharmaceutical companies (1964-1985) [J]. Br J Clin Pharmacol, 1998,25:387-396
2刘昌孝.我国药代动力学研究发展的回顾[J].中国药学杂志,2010,45(2)：81-89
3 成碟,徐为人,刘昌孝.细胞色素 P450(CYP450)遗传多态性研究进展[J].中国药理学通报,2006,22(12)：1409-1414
4 Guengerich FP, Wu Z, Bartleson CJ. Function of human cytochrome P450s: characterisation of the orphans. Biochem Biophys Res Commun 2005, (338):465-469
5 Seliskar M, Rozman D. Mammalian cytochromes P450-importance of tissue specificity. Biochim Biophys Acta,2007, (1770):458-466
6 Hrycay EG, Bandiera SM. Cytochrome P450 Enzymes. In:Gad SC (ed) Preclinical development handbook. ADME and biopharmaceutical properties.2008, Wiley, New York
7 Shimada T, Yamazaki H, Mimura M, et al. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals:studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther,1994,270:414-423
8 Smith DA, Abel SM, Hyland R, et al. Human cytochrome P450s: selectivity and measurement in vivo. Xenobiotica,1998(28):1095-1128
9 Wienkers LC, Heath TG. Predicting in vivo drug interactions from in vitro drug discovery data. Nat Rev Drug Discov 2005 (4):825-833
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22 Reginald FF. Probing the world of cytochrome P450 enzymes [J]. Mol Interv,2004,4(3):157-162
23 Siwaporn C, Anne NN, Steven L, et al. Combined phenotypic assessment of cytochrome P450 1A2,2C9,2C 19,2D6 and 3A, N-acetyltransferase-2 and xanthineoxidase activities with the Cooperstown 5+1 cocktail"[J]. Clin Pharmacol Ther,2003,74(5):437-447
24 Magnus C, Katarina A, Per D, et al. The Karolinska cocktail for phenotyping of five human cytochrome P450 enzymes[J]. Clin Pharmacol Ther,2003,73(6):517-528
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43 Hyland R, Jones BC, Smith DA. Identification of the cytochrome P450 enzymes involved in the N-oxidation of voriconazole. Drug Metab Dispos, 2003,31:540-547
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7 Yune-Jung Yoon, Kwon-Bok Kim, Hyunmi Kim, et al. Characterization of Benidipine and Its Enantiomers'Metabolism by Human Liver Cytochrome P450 Enzymes[J]. Drug metabolism and disposition,2007,35 (9): 1518-1524
8 Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem,2003,75:3019-3030
9刘会臣,杜玉民,王永利.双苯氟嗪在大鼠体内的代谢途径[J].药学学报,2005,40(2)：168-172
1 OBACH R S. The prediction of human clearance from hepatic microsomal metabolism data[J]. Curr Opin Drug Discov Devel,2001,4(1):36-44
2 OBACH R S.Nonspecific binding to microsomes:Impact on scale-up of in vitro intrinsic clearance to hepatic clearance as assessed through examination of warfarin,imipramine,and propranolol[J]. DrugMetab Dispos,1997,25(12):1359-1369
3 HOUSTONJ B, CARLILEDJ. Prediction of hepatic clearance frommicrosomes, hepatocytes and liver slices[J]. Drug Metab Rev,1997, 29(4):891-922
4 D A Smith, HvandeWaterbeemd, D KWalker, et al. Pharmacokinetics and metabolism in drug design [M]. Newyork:John Wiley and Sons,2001: 137
5 Waxman D J. Rat hepatic cytochrome P-450 isoenzyme 2c[J]. J Biol Chem,1984,259:1548-1549
6 Waxman D J, Dannan G A, Guengerich F. P1 Regulation of rat hepatic cytochrome P2450:Age2dependent Expression, Hormonal Imprinting and Xenobiotic Inducibility of Sex-specific isoenzymes[J]. Biochemstry,1985, 24:4409-4417
1 FAN H R, FAN H, LIU C X. Cocktail the drug probe method used to evaluate cytochrome P450 isozymes of the impact of research[J]. Chin Pharm J(中国药学杂志),2006,41(14)：1045-1048
2 BJORNSSON TD, CALLAGHAN JT, EINOLF HJ, et al. The conduct of in vitro and in vivo drug-drug interaction studies:a pharmaceutical research and manufacturers of america (PhRMA) perspective[J]. Drug Metab Dispos,2003,31(7):518-532
3 DONATO MT, CASTELL JV. Strategies and molecular probes to investigate the role of cytochrome P450 in drug metabolism [J]. Clin Pharmacokinet.2003,42(2):153-178
4 DIERKS EA, STAMS KR, LIM HK, et al. A method for the simultaneous evaluation of the activities of seven major human drug metabolizing cytochrome p450s using an in vitro cocktail of probe substrates and fast gradient liquid chromatography tandem mass spectrometryl[J]. Drug Metab Dispos,2001,29(1):23-29
5 Eagling VA, Tjia JF, and Back DJ. Differential selectivity of cytochrome P450 inhibitors against probe substrates in human and rat liver microsomes. Br J Clin Pharmacol 1998,45:107-114
6刘会臣,杜玉民,王永利.双苯氟嗪在大鼠体内的代谢途径[J].药学学报.2005,40(2)：168-172
7 Meuldermans W, Hendrickx J, Hurkmans R, et al. Excretion and metabolism of flunarizine in rats and dogs[J]. Arzneimittelforschung 1983, 33:1142-1151
8 Kariya S, Isozaki S, Narimatsu S, and Suzuki T. Oxidative metabolism of flunarizine in rat liver microsomes[J]. Res Commun Chem Pathol Pharmacol 1992,78:85-95
9 Lavrijsen K, van Houdt J, van Dyck D, et al. Comparative metabolism of flunarizine in rats, dogs and man:an in vitro study with subcellular liver fractions and isolated hepatocytes. Xenobiotica 1992,22:815-836
10 Narimatsu S, Kariya S, Isozaki S, et al. Involvement of CYP2D6 in oxidative metabolism of cinnarizine and flunarizine in human liver microsomes. Biochem Biophys Res Commun[J] 1993,193:1265-1268
11 Kariya S, Isozaki S, Uchino K, et al. Oxidative metabolism of flunarizine and cinnarizine by microsomes from B-lymphoblastoid cell lines expressing human cytochrome P450 enzymes[J]. Biol Pharm Bull 1996,19: 1511-1514
12 W axman D J. Rat hepatic cytoch rome P-450 isoenzyme 2c[J]. J Biol Chem,1984,259:15481-15490
13 W axman D J, Dannan G A, Guengerich F P. Regulation of rat hepatic cytochrome P-450:A ge-dependent Expression, Hormonal Imprinting, and Xenobiotic Inducibility of Sex specific isoenzymes[J]. B iochem istry,1985,24:4409-4417
14 Kariya S, Isozaki S, Uchino K, et al. Oxidative metabolism of flunarizine and cinnarizine by microsomes from B-lymphoblastoid cell lines expressing human cytochrome P450 enzymes[J]. Biol Pharm Bull 1996,19: 1511-1514
15 Kobayashi K, Urashima K, Shimada N, et al. Selectivities of human cytochrome P450 inhibitors toward rat P450 isoforms:study with cDNA-expressed systems of the rat[J]. Drug Metab Dispos 2003, 31:833-836
16 Eagling VA, Tjia JF and Back DJ. Differential selectivity of cytochrome P450 inhibitors against probe substrates in human and rat liver microsomes[J]. Br J Clin Pharmacol 1998,45:107-114
17赵阳,柳晓泉,钱之玉,等.药物代谢的性别差异[J].药学进展,2001,25(5)：289-293
1 LAHOZ A, DONATO MT, PICAZO L, et al. Determination of major human cytochrome P450s activities in 96-well plates using liquid chromatography tandem mass spectrometry[J]. Toxicol In Vitro,2007, 21(7):1247-1252
2 LAINE JE, AURIOLA S, PASANEN M, et al. Acetaminophen bioactivation by human cytochrome P450 enzymes and animal microsomes[J]. Xenobiotica,2009,39(1):11-21
3 ISOHERRANEN N, HACHAD H, YEUNG CK, et al. Qualitative analysis of the role of metabolites in inhibitory drug-drug interactions:literature evaluation based on the metabolism and transport drug interaction database[J]. Chem Res Toxicol,2009,22(2):294-298
4 KROSSER S, NEUGEBAUER R, DOLGOS H, et al. Investigation of sarizotan's impact on the pharmacokinetics of probe drugs for major cytochrome P450 isoenzymes:a combined cocktail trial[J]. Eur J Clin Pharmacol,2006,62(4):277-284
5 Reginald FF. Probing the world of cytochrome P450 enzymes [J]. Mol Interv,2004,4(3):157-162
6 Siwaporn C, Anne NN, Steven L, et al. Combined phenotypic assessment of cytochrome P450 1A2,2C9,2C19,2D6 and 3A, N-acetyltransferase-2 and xanthineoxidase activities with the "Cooperstown 5+ 1 cocktail" [J].Clin Pharmacol Ther,2003,74(5):437-447
7 Magnus C, Katarina A, Per D, et al. The Karolinska cocktail for phenotyping of five human cytochrome P450 enzymes[J]. Clin Pharmacol Ther,2003,73(6):517-528
8 BROWN HS, GALETIN A, HALLIFAX D, et al. Prediction of in vivo drug-drug interactions from in vitro data:factors affecting prototypic drug-drug interactions involving CYP2C9, CYP2D6 and CYP3A4[J]. Clin Pharmacokinet,2006,45(10):1035-1050
9 TOMALIK-SCHARTE D, JETTER A, KINZIG-SCHIPPERS M, et al. Effect of propiverine on cytochrome P450 enzymes:a cocktail interaction study in healthy volunteers[J]. Drug Metab Dispos,2005,33(12): 1859-1866
10 Zhou HH, Zeen T, James FM. "Cocktail" approaches and strategies in drug development:valuable tool or flawed science[J]. J Clin Pharmacol,2004, 44:120-134
11 TANG H, MIN G, GE B, et al. Evaluation of protective effects of Chi-Zhi-Huang decoction on Phase I drug metabolism of liver injured rats by cocktail probe drugs[J]. J Ethnopharmacol,2008,117(3):420-426
12 KUMAR V, WAHLSTROM JL, ROCK DA, et al. CYP2C9 inhibition: impact of probe selection and pharmacogenetics on in vitro inhibition profiles[J]. Drug Metab Dispos,2006,34(12):1966-1975
13于卫江,黄丽军,朱大岭.应用大鼠肝微粒体筛选对细胞色素P4502D6有抑制作用的中药[J].中草药,2007,38(3)：397-401
14于卫江,黄丽军,刘艳,等.奥扎格雷钠对大鼠CYP2D6亚型酶的影响.[J]中国药理学通报,2007,23(1)：67-72
15 Zhao B C. Enzyme[M]//Zhou A R. Biochemistry.5 th ed. Beijing:People Medical Publishing House,2001:49-70
16 Kaoru K, Kikuko U, Noriaki S, et al. Substrate specificity for tar cytochrome P450 isoforms:screening wiyh cDNA-expressed systems of rat [J]. Biochemical Pharmacology,2002,63:889-896
17 LA Vignati, A Bogni, P Grossi, et al. A human and mouse pregnane X receptor reporter gene assay in combination with cytotoxicity measurement s as a tool to evaluate species specific CYP3A induction[J]. Toxicology, 2004,199:23-33
1 Prentis RA, Lis Y, Walker SR. Pharmaceutical innovation by seven U K-owned pharmaceutical companies (1964-1985) [J]. Br J Clin Pharmacol, 1998,25:387-396
2刘昌孝.我国药代动力学研究发展的回顾[J].中国药学杂志,2010,45(2)：81-89
3 成碟,徐为人,刘昌孝.细胞色素 P450(CYP450)遗传多态性研究进展[J].中国药理学通报,2006,22(12)：1409-1414
4 Guengerich FP, Wu Z, Bartleson CJ. Function of human cytochrome P450s: characterisation of the orphans. Biochem Biophys Res Commun 2005, (338):465-469
5 Seliskar M, Rozman D. Mammalian cytochromes P450-importance of tissue specificity. Biochim Biophys Acta,2007, (1770):458-466
6 Hrycay EG, Bandiera SM. Cytochrome P450 Enzymes. In:Gad SC (ed) Preclinical development handbook. ADME and biopharmaceutical properties.2008, Wiley, New York
7 Shimada T, Yamazaki H, Mimura M, et al. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals:studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther,1994,270:414-423
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