IMM018及氢吗啡酮临床前药动学研究
摘要
在新药开发过程中，探究该药物与其它药物的代谢相互作用，是评价药物安全性和有效性的一个重要组成部分。本论文建立了测定六种主要CYP酶亚型探针性底物代谢产物的LC／MS／MS法，用于评估药物对CYP酶的抑制及诱导能力。本论文阐明了一种新的抗炎候选药物血小板活化因子(PDF)拮抗剂IMM018对肝CYP酶的影响，及其在大鼠和犬体内的动力学过程，研究了IMM018在大鼠体内的排泄和组织分布，为IMM018的药理、毒理和临床研究提供参考依据。
氢吗啡酮(hydromorphone，HYD)为半合成的阿片受体拮抗剂。本文建立了液相色谱-串联质谱法测定比格犬给予HYD缓释制剂后血浆中HYD的浓度，用于评价其缓释特征。
一、LC／MS／MS法测定六种CYP酶探针底物的代谢产物
体外药物相互作用研究数据可以用于指导临床药物相互作用研究，或者指导新化合物的合成设计，为此必须确保所获得的体外数据真实可信。本论文使用重组CYP酶，与探针性底物非那西丁(CYP1A2)，甲苯磺丁脲(CYP2C9)，美芬妥英(CYP2C19)，右美沙芬(CYP2D6和CYP3A4-D)，氯唑沙宗(CYP2E1)，咪达唑仑(CYP3A4-M)孵化，建立快速测定孵化液中各探针性底物代谢物的液相色谱-串联质谱分析方法(LC／MS／MS)，用于新候选药物对六种主要CYP酶的抑制及诱导筛选研究，并对分析方法进行了确证。通过对质谱条件、色谱条件以及样品预处理方法的考察优化了分析方法。该法的线性范围为1.0～2000 ng·mL~(-1)，定量下限1.0 ng·mL~(-1)，日内和日间精密度(RSD)在3.4％～13.9％之间，准确度在-5.3％～7.3％之间。应用本文建立的分析方法测定了六种CYP酶的阳性抑制剂的IC_(50)值，获得结果与文献相符，进一步证实了方法的准确性。
二、IMM018对肝CYP酶的影响
本文对IMM018的药物代谢性相互作用进行了研究：利用人源重组CYP酶，考察了IMM018对CYP1A2，CYP2C9，CYP2D6和CYP3A4酶的抑制作用；同时通过测定连续3天灌胃给予低、高剂量IMM018(50和100 mg·kg~(-1)·d~(-1)后，大鼠肝CYP酶的活性，并与空白对照组进行比较，考察IMM018对大鼠肝CYP酶的诱导影响。采用LC／MS／MS法测定孵化样品中CYP1A(非那西丁O-去乙基)，CYP2C(甲苯磺丁脲4-羟基化)，CYP2D(右美沙芬O-去甲基)以及CYP3A(右美沙芬N-去甲基，咪达唑仑1′-羟基化)酶的活性。抑制试验结果表明IMM018对CYP1A2的IC_(50)为47.1μmol·L~(-1)，K_i为13.1μmol·L~(-1)，属于混合型抑制；对CYP2C9的IC_(50)为29.7μmol·L~(-1)，K_i为20.2μmo1·L~(-1)，属于非竞争性抑制；对CYP2D6的IC_(50)为5.4μmol·L~(-1)，K_i为10.0μmol·L~(-1)，属于非竞争性抑制。诱导实验结果显示给药组与空白对照组CYP1A，CYP2C和CYP2D酶活性无显著性差异(p＞0.05)，给药组与空白对照组CYP3A酶活性有显著性差异(p＜0.05)，给药低、高剂量组之间也存在着一定的差异(p＜0.05)。IMM018对测试的4种CYP同工酶均具有中等程度的抑制作用，且对CYP3A4抑制具有时间依赖性及底物依赖性；对大鼠肝CYP3A具有诱导作用，且存在轻微的剂量依赖性。
三、IMM018在大鼠和犬体内的药物动力学研究
以苯海拉明(diphenhydramine)为内标，建立同时测定生物样品中IMM018及其五种Ⅰ相代谢物的液相色谱-串联质谱法(LC／MS／MS)，并进行了完整的方法确证。通过对质谱条件、色谱条件以及样品预处理方法的考察优化了分析方法。色谱柱为Gemini-C_(18)柱(150×4.6 mm I．D．，5μm)，以甲醇：水：甲酸(50：50：0.25．v／v)为流动相，流速为0.5 mL·min~(-1)。采用电喷雾离子源(ESI源)，以正离子方式检测，扫描方式为选择反应监测(SRM)。该法测定IMM018及其代谢物M3，M4和M5的线性范围为3.0～4000 ng·mL~(-1)，定量下限3.0 ng·mL~(-1)；M2的线性范围为4.0～40000 ng·mL~(-1)，定量下限4.0 ng·mL~(-1)；M1的线性范围为0.4～4000 ng·mL~(-1)，定量下限0.4 ng·mL~(-1)。IMM018及其代谢物M1～M5的日内和日间精密度在2.0％～14.7％之间，准确度在-4.1％～4.3％之间。本文建立的方法在灵敏度、动态范围和测试速度等方面均达到生物样品分析方法的要求。
采用建立的分析方法研究了IMM018及其代谢物在大鼠和犬体内的药物动力学。大鼠灌胃给予IMM018(25 mg·kg~(-1))后，在血浆中仅在个别时间点检测到原形药物(低于100 ng·mL~(-1))，生成的主要代谢产物为M1，M2及M3。代谢物M1，M2和M3消除半衰期分别为4.49，2.67和2.79 h。大鼠静脉注射给予IMM018(25 mg·kg~(-1))后，代谢物M1，M2和M3在血浆中t_(1／2)分别为5.69，4.38和3.43 h。犬灌胃给予IMM018(7.5 mg·kg~(-1))后，代谢物M1，M2和M3消除半衰期分别为0.31，3.29和5.14 h。犬静脉注射给予IMM018(7.5 mg·kg~(-1))后，代谢物M1，M2和M3在血浆中t_(1／2)分别为0.512，4.76和2.36 h。
四、IMM018在大鼠体内的排泄和组织分布研究
为了进一步了解IMM018在大鼠体内的生物转化过程，建立了测定尿，粪和胆汁中IMM018及其主要代谢物的LC／MS／MS法，并且研究了IMM018自大鼠体内的排泄情况，根据尿和胆汁的排泄体积以及粪的排泄质量计算IMM018及其代谢物的排泄量、累积排泄量、累积排泄率。
大鼠灌胃给予50 mg·kg~(-1) IMM018 60 h后，在尿中未检测到原形药物，可检测到代谢物M2、M3、M1(按代谢物在尿中排泄量从大到小顺序)及微量M4和M5。尿中代谢物累积排泄总量约占剂量的79.7％，其中代谢物M2累积排泄量约占剂量的75.9％；代谢物M3累积排泄量约占剂量的3.24％；代谢物M1累积排泄量约占剂量的0.57％；代谢物M4累积排泄量约占剂量的0.0097％；代谢物M5累积排泄量约占剂量的0.012％。
大鼠灌胃给予50 mg·kg~(-1) IMM018 60 h后，在粪中可检测到少量原形、代谢物M2、M3、M1、M4及微量M5。粪中原形及代谢物累积排泄总量约占剂量的2.43％，其中原形药物累积排泄量约占剂量的0.050％；代谢物M2累积排泄量约占剂量的2.17％；代谢物M3累积排泄量约占剂量的0.17％；代谢物M1累积排泄量约占剂量的0.00404％；代谢物M4累积排泄量约占剂量的0.035％；代谢物M5累积排泄量约占剂量的0.00091％。
大鼠灌胃给予50 mg·kg~(-1) IMM018 60 h后，在胆汁中可检测到少量原形及代谢物M2、M3、M1、M4。胆汁原形及代谢物累积排泄总量约占剂量的6.00％，其中原形药物累积排泄量约占剂量的0.17％；代谢物M2累积排泄量约占剂量的1.97％；代谢物M3累积排泄量约占剂量的0.15％；代谢物M1累积排泄量约占剂量的3.69％；代谢物M4累积排泄量约占剂量的0.01％。
排泄试验结果表明，大鼠灌胃给予IMM018后主要以代谢物M2形式由尿中排泄。IMM018自大鼠体内排泄较快，不易在体内蓄积。
为了研究IMM018在大鼠体内的分布情况，建立了测定大鼠组织中IMM018及其代谢物浓度的LC／MS／MS法，对大鼠灌胃给药后药物在体内的分布情况进行了研究。
大鼠灌胃给予IMM018后，1.0，4.0，12 h三个时间点IMM018在各组织中的浓度很低。各代谢物在各组织中广泛分布，在胃内容物及肠内容物中除发现大量原形外，还发现代谢物M1＞M2＞M5＞M4＞M3(按代谢物浓度从大到小顺序)，1h时胃内容物中代谢物M1的量约为原形的7％。1 h时除胃壁、肠内容物、胃内容物外，M2在各组织中的浓度均高于原形，M3浓度次之；在采集的三个时间点样品中，IMM018各代谢物M1、M2、M4及M5，除子宫、卵巢外，其余组织均在1 h达到最大组织浓度；代谢物M3，除大肠及生殖器外，其余组织均在1 h达到最大组织浓度。相对于血浆浓度，IMM018各代谢物在大多数组织中浓度较高，特别是在胃肠道、肺、子宫、心、膀胱、胰腺和肝组织中有较高的浓度。代谢物M2、M1和M5在胃肠道中吸收较快，在4 h时的胃内容物药物浓度约为1h药物浓度的25％，小肠内容物约为1 h药物浓度的14％。各代谢物在组织中消除迅速，12 h时代谢物M1在心、肝、肾、脂肪、小肠、大肠、胃壁、卵巢、胰腺的浓度降为1 h时的百分之一。原形及代谢物在脑组织中含量低，说明药物不容易通过血脑屏障。
五、氢吗啡酮缓释制剂在犬体内的药动学研究
氢吗啡酮(hvdromorphone，HYD)是一种半合成的阿片受体拮抗剂。本实验建立快速、灵敏的液相色谱-串联质谱法测定比格犬血浆中氢吗啡酮。比格犬血浆0.1 mL经β-葡糖苷酸酶孵化16 h后，采用液-液萃取法处理，以甲醇：水：甲酸(65：35：1)为流动相，Zorbax SB C_8柱分离，采用大气压化学电离源，选择反应监测(SRM)。测定氢吗啡酮的线性范围为0.80～200.0 ng·mL~(-1)，定量下限为0.80 ng·mL~(-1)。日内、日间精密度(RSD)在2.6％～5.3％之间，准确度(RE)在-0.2％～1.0％之间。应用此法研究了6只比格犬单剂量口服盐酸氢吗啡酮缓释片4mg后的药物动力学特点。该法选择性强，灵敏度高，适用于氢吗啡酮缓释制剂的药代动力学研究。
Potent drug-drug interactions can result in serious side effects, as a result, preclinical (in vitro) and some clinical (in vivo) interaction studies are now important components of the drug candidate selection process. A rapid liquid chromatographytandem mass spectrometry (LC/MS/MS) method has been validated and employed routinely evaluate the potential for a new drug to modify cytochrome P450 (P450) activities by determining the effect of the drug on in vitro probe reactions that represent activity of specific P450 enzymes.
IMM018, 1-[5-(4-Chloro-phenyl)-3-oxo-pent-4-enyl]-pipefidine-4-carboxylic acid ethyl ester, is a drug candidate which has been shown to have analgesic and antiinflammatory actions in rats. In present paper, pharmacokinetics of IMM018 in rats and dogs were investigated. An analytical method based on liquid chromatography coupled to tandem mass spectrometry detection was developed and validated for simultaneous quantification of IMM018 and its five metabolites in rat plasma, using diphenhydramine as the internal standard. The metabolism, excretion and tissue distribution of IMM018 in rats were studied to support the pharmacology and toxicology study of IMM018.
Hydromorphone is a semi-synthetic opioid agonist. A sensitive, rapid and specific liquid chromatographic-tandem mass spectrometric (LC/MS/MS) method for the determination of hydromorphone in beagle dog plasma was developed. The method is proved to be suitable for hydromorphone control-release preparation pharmacokinetics and bioavailability in beagle dog.
1. Determination of six CYPs probe metabolites
In vitro drug interaction data can be used in guiding clinical interaction studies, or, the design of new candidates. To make such a claim, it must be assured that the in vitro data obtained is confident. To meet this need, a rapid liquid chromatography-tandem mass spectrometry (LC/MS/MS) method has been validated and employed for routine screening of new chemical entities for inhibition of six major human cytoohrome P450 (CYP) isoforms using cDNA-expressed CYPs. Probe substrates were used near the Michaelis-Menten constant (K_m) concentration values for CYP1A2 (phenacetin), CYP2C9 (tolbutamide), CYP2C19 (S-mephenytoin), CYP2D6 (dextrornethorphan), CYP2E1 (chlorzoxazone), and CYP3A4 (midazolam and dextromethorphan). The major metabolites of CYP-specific probe substrates were quantified. The LC/MS/MS method was found to be accurate and precise within the linear range of 1.0-2000 ng·mL~(-1) for each analyte in enzyme incubation mixture. The lower limit of quantification (LLOQ) was 1.0 ng·mL~(-1). The limit of detection (LOD) for the tested analytes was 0.48 ng·mL~(-1) or better based on signal-to-noise ratio＞3. The inhibition potential of the six CYP isoforms has been evaluated using their known selective inhibitors. The 50% inhibitory concentrations (IC_(50) values) measured by this method demonstrated high precision and are consistent with the literature values.
2. Studies on effect of IMM018 on hepatic CYPs
IMM018, a new anti-inflammatory agent, was being developed for the treatment of patients with rheumatoid arthritis. Drug-drug interaction studies using recombinant human cytochrome P450s were conducted to assess CYP1A2, CYP2C9, CYP2D6 and CYP3A4 inhibition potential; Induction potential of CYP1A, CYP2C, CYP2D and CYP3A by IMM018 was examined with liver microsomes from control rats or rats treated with IMM018 at 50 and 100 mg·kg~(-1)·d~(-1) for 3 consecutive days. The assays that were validated are: phenacetin O-deethylase (CYP1A2), tolbutamide methylhydroxylase (CYP2C9), dextromethorphan O-demethylase (CYP2D6), midazolam l'-hydroxylase (CYP3A4) and dextromethorphan N-demethylase (CYP3A4). High-pressure liquid chromatography-tandem mass spectrometry was applied as the analytical method. IMM018 was a mixed-type inhibitor of CYP1A2 (K_i=13.1μmol·L~(-1)) and it also appeared to be noncompetitive inhibitor of CYP2C9 (K_i=20.2μmol·L~(-1)) and CYP2D6 (K_i=10.0μmol·L~(-1)). Furthermore, IMM018 showed time-dependent inactivation for CYP3A4. In the induction study, there were no significant difference in the enzymatic activities between the control and IMM018 treated rats except CYP3A (p＞0.05). IMM018 is likely to inhibit the metabolism of comedications moderately, if their primary routes of elimination are via cytochrome. The induction of CYP3A activity by IMM018 was dose-dependent in Wistar rats.
3. Studies on pharmacokinetics of IMM018 in rats and dogs
A sensitive LC/MS/MS method was developed for the determination of IMM018 and its five major metabolites in the plasma using diphenhydramine as internal standard. The analytes and internal standard were extracted with redistilled ethyl acetate and chromatographed isocratically on a Gemini C_(18) analytical column with a mobile phase composed of methanol: water: formic acid in the ratio of 50: 50: 0.25 (v/v/v). All analytes were detected in the selected reaction monitoring mode using an electrospray ionization source. IMM018 and its five metabolites could be simultaneously determined within 6 min. Linear calibration curves were obtained in the concentration ranges of 3.0～4000 ng·mL~(-1) for IMM018, M3, M4 and M5, 0.4～4000 ng·mL~(-1) for M1, and 4.0～40000 ng·mL~(-1) for M2. The intra- and inter-day precision (RSD), calculated from quality control (QC) samples, was between 2.0%～14.7% for each analyte. The accuracy was between -4.1%～4.3%. The method was utilized to support preclinical pharmacokinetic and toxicokinetic studies of IMM018 in rats and dogs.
The pharmacokinetics of IMM018 was investigated in rats and dogs by the LC/MS/MS method. After oral administration of IMM018 to rats (25 mg·kg~(-1)), the t_(1/2) values for metabolites M1, M2 and M3 were 4.49 h, 2.67 h and 2.79 h, respectively. After intravenous injections of IMM018 to rats (25 mg·kg~(-1)), the t_(1/2) values for metabolites M1, M2 and M3 were 5.69, 4.38 and 3.43 h, respectively.
4. Studies on excretion and tissue distribution of IMM018 in rats
An LC/MS/MS method for the simultaneous determination of IMM018 and its metabolites in rat urine, feces and bile was developed and validated. The excretion of IMM018 and its metabolites was studied by this method. According to the volume of urine and bile and the weight of feces, the total accumulated excretion of IMM018 and its metabolites were calculated.
After oral administration of IMM018 at dosage of 50 mg·kg~(-1) to rats, IMM018 was not found in urine sample, accumulated excretion of M2 in urine sample was 75.90%; M3 was 3.24%; M1 was 0.57%; M4 was 0.0097%; M5 was 0.012%. The accumulation excretion of IMM018 in feces was 0.050%; M2 was 2.17%; M3 was 0.17%; M1 was 0.00404%; M4 was 0.035%; M5 was 0.00091%. The accumulation excretion of IMM018 in bile was 0.17%; M2 was 1.97%; M3 was 0.15%; M1 was 3.69%; M4 was 0.01%. These results show that IMM018 was mainly excreted in urine in the form of metabolite M2.
The rats were divided into 3 groups. After the oral administration of 50 mg·kg~(-1) IMM018, the rats were sacrificed at 1.0, 4.0 and 12 h separately. The tissue samples were collected and used to prepare homogenates. A sensitive, specific and accurate method for simultaneous quantifying IMM018 and its metabolites in rat tissues and to study the distribution of IMM018 in rat tissues was developed. After oral administration of IMM018, the drug was distributed extensively in rats in vivo. IMM018 and its major metabolites (M1～M5) were found in gastric contents and intestinal content. The concentrations of M1～M5 in lung, uterus, heart, bladder, pancreas, and liver were higher than that of the other tissues. The concentration of IMM018 and its metabolites were low in brain, which show that the blood-brain barrier was hard to be passed through for IMM018 and its metabolites.
5. Studies on pharmacokinetics of hydromorphone control-release preparation
An LC/MS/MS method for determination of hydromorphone (HYD) in Beagle dog plasma was established. After incubation withβ-glucuronidase for 16 h, an aliquot of 0.1 mL plasma was treated by liquid-liquid extraction. The analytes of interest were separated on a Zorbax SB C_8 column with the mobile phase consisting of methanol: water: formic acid (65: 35: 1). Atmospheric pressure chemical ionization source of MS was applied and operated in positive ion mode. The linear calibration curve was obtained in the concentration range of 0.80～200.0 ng·mL~(-1). The lower limit of quantification was 0.80 ng·mL~(-1). The inter-day and intra-day precision (RSD) was below 6.0%, and the accuracy (RE) was between-0.2%～1.0% calculated from QC samples. The method was used to determine the pharmacokinetic parameters of HYD after a single oral administration of 4 mg HYD sustained release tablets to Beagle dogs. The method was proved to be special, sensitive, and suitable for the pharmacokinetic study of HYD sustained release formulation.
氢吗啡酮(hydromorphone，HYD)为半合成的阿片受体拮抗剂。本文建立了液相色谱-串联质谱法测定比格犬给予HYD缓释制剂后血浆中HYD的浓度，用于评价其缓释特征。
一、LC／MS／MS法测定六种CYP酶探针底物的代谢产物
体外药物相互作用研究数据可以用于指导临床药物相互作用研究，或者指导新化合物的合成设计，为此必须确保所获得的体外数据真实可信。本论文使用重组CYP酶，与探针性底物非那西丁(CYP1A2)，甲苯磺丁脲(CYP2C9)，美芬妥英(CYP2C19)，右美沙芬(CYP2D6和CYP3A4-D)，氯唑沙宗(CYP2E1)，咪达唑仑(CYP3A4-M)孵化，建立快速测定孵化液中各探针性底物代谢物的液相色谱-串联质谱分析方法(LC／MS／MS)，用于新候选药物对六种主要CYP酶的抑制及诱导筛选研究，并对分析方法进行了确证。通过对质谱条件、色谱条件以及样品预处理方法的考察优化了分析方法。该法的线性范围为1.0～2000 ng·mL~(-1)，定量下限1.0 ng·mL~(-1)，日内和日间精密度(RSD)在3.4％～13.9％之间，准确度在-5.3％～7.3％之间。应用本文建立的分析方法测定了六种CYP酶的阳性抑制剂的IC_(50)值，获得结果与文献相符，进一步证实了方法的准确性。
二、IMM018对肝CYP酶的影响
本文对IMM018的药物代谢性相互作用进行了研究：利用人源重组CYP酶，考察了IMM018对CYP1A2，CYP2C9，CYP2D6和CYP3A4酶的抑制作用；同时通过测定连续3天灌胃给予低、高剂量IMM018(50和100 mg·kg~(-1)·d~(-1)后，大鼠肝CYP酶的活性，并与空白对照组进行比较，考察IMM018对大鼠肝CYP酶的诱导影响。采用LC／MS／MS法测定孵化样品中CYP1A(非那西丁O-去乙基)，CYP2C(甲苯磺丁脲4-羟基化)，CYP2D(右美沙芬O-去甲基)以及CYP3A(右美沙芬N-去甲基，咪达唑仑1′-羟基化)酶的活性。抑制试验结果表明IMM018对CYP1A2的IC_(50)为47.1μmol·L~(-1)，K_i为13.1μmol·L~(-1)，属于混合型抑制；对CYP2C9的IC_(50)为29.7μmol·L~(-1)，K_i为20.2μmo1·L~(-1)，属于非竞争性抑制；对CYP2D6的IC_(50)为5.4μmol·L~(-1)，K_i为10.0μmol·L~(-1)，属于非竞争性抑制。诱导实验结果显示给药组与空白对照组CYP1A，CYP2C和CYP2D酶活性无显著性差异(p＞0.05)，给药组与空白对照组CYP3A酶活性有显著性差异(p＜0.05)，给药低、高剂量组之间也存在着一定的差异(p＜0.05)。IMM018对测试的4种CYP同工酶均具有中等程度的抑制作用，且对CYP3A4抑制具有时间依赖性及底物依赖性；对大鼠肝CYP3A具有诱导作用，且存在轻微的剂量依赖性。
三、IMM018在大鼠和犬体内的药物动力学研究
以苯海拉明(diphenhydramine)为内标，建立同时测定生物样品中IMM018及其五种Ⅰ相代谢物的液相色谱-串联质谱法(LC／MS／MS)，并进行了完整的方法确证。通过对质谱条件、色谱条件以及样品预处理方法的考察优化了分析方法。色谱柱为Gemini-C_(18)柱(150×4.6 mm I．D．，5μm)，以甲醇：水：甲酸(50：50：0.25．v／v)为流动相，流速为0.5 mL·min~(-1)。采用电喷雾离子源(ESI源)，以正离子方式检测，扫描方式为选择反应监测(SRM)。该法测定IMM018及其代谢物M3，M4和M5的线性范围为3.0～4000 ng·mL~(-1)，定量下限3.0 ng·mL~(-1)；M2的线性范围为4.0～40000 ng·mL~(-1)，定量下限4.0 ng·mL~(-1)；M1的线性范围为0.4～4000 ng·mL~(-1)，定量下限0.4 ng·mL~(-1)。IMM018及其代谢物M1～M5的日内和日间精密度在2.0％～14.7％之间，准确度在-4.1％～4.3％之间。本文建立的方法在灵敏度、动态范围和测试速度等方面均达到生物样品分析方法的要求。
采用建立的分析方法研究了IMM018及其代谢物在大鼠和犬体内的药物动力学。大鼠灌胃给予IMM018(25 mg·kg~(-1))后，在血浆中仅在个别时间点检测到原形药物(低于100 ng·mL~(-1))，生成的主要代谢产物为M1，M2及M3。代谢物M1，M2和M3消除半衰期分别为4.49，2.67和2.79 h。大鼠静脉注射给予IMM018(25 mg·kg~(-1))后，代谢物M1，M2和M3在血浆中t_(1／2)分别为5.69，4.38和3.43 h。犬灌胃给予IMM018(7.5 mg·kg~(-1))后，代谢物M1，M2和M3消除半衰期分别为0.31，3.29和5.14 h。犬静脉注射给予IMM018(7.5 mg·kg~(-1))后，代谢物M1，M2和M3在血浆中t_(1／2)分别为0.512，4.76和2.36 h。
四、IMM018在大鼠体内的排泄和组织分布研究
为了进一步了解IMM018在大鼠体内的生物转化过程，建立了测定尿，粪和胆汁中IMM018及其主要代谢物的LC／MS／MS法，并且研究了IMM018自大鼠体内的排泄情况，根据尿和胆汁的排泄体积以及粪的排泄质量计算IMM018及其代谢物的排泄量、累积排泄量、累积排泄率。
大鼠灌胃给予50 mg·kg~(-1) IMM018 60 h后，在尿中未检测到原形药物，可检测到代谢物M2、M3、M1(按代谢物在尿中排泄量从大到小顺序)及微量M4和M5。尿中代谢物累积排泄总量约占剂量的79.7％，其中代谢物M2累积排泄量约占剂量的75.9％；代谢物M3累积排泄量约占剂量的3.24％；代谢物M1累积排泄量约占剂量的0.57％；代谢物M4累积排泄量约占剂量的0.0097％；代谢物M5累积排泄量约占剂量的0.012％。
大鼠灌胃给予50 mg·kg~(-1) IMM018 60 h后，在粪中可检测到少量原形、代谢物M2、M3、M1、M4及微量M5。粪中原形及代谢物累积排泄总量约占剂量的2.43％，其中原形药物累积排泄量约占剂量的0.050％；代谢物M2累积排泄量约占剂量的2.17％；代谢物M3累积排泄量约占剂量的0.17％；代谢物M1累积排泄量约占剂量的0.00404％；代谢物M4累积排泄量约占剂量的0.035％；代谢物M5累积排泄量约占剂量的0.00091％。
大鼠灌胃给予50 mg·kg~(-1) IMM018 60 h后，在胆汁中可检测到少量原形及代谢物M2、M3、M1、M4。胆汁原形及代谢物累积排泄总量约占剂量的6.00％，其中原形药物累积排泄量约占剂量的0.17％；代谢物M2累积排泄量约占剂量的1.97％；代谢物M3累积排泄量约占剂量的0.15％；代谢物M1累积排泄量约占剂量的3.69％；代谢物M4累积排泄量约占剂量的0.01％。
排泄试验结果表明，大鼠灌胃给予IMM018后主要以代谢物M2形式由尿中排泄。IMM018自大鼠体内排泄较快，不易在体内蓄积。
为了研究IMM018在大鼠体内的分布情况，建立了测定大鼠组织中IMM018及其代谢物浓度的LC／MS／MS法，对大鼠灌胃给药后药物在体内的分布情况进行了研究。
大鼠灌胃给予IMM018后，1.0，4.0，12 h三个时间点IMM018在各组织中的浓度很低。各代谢物在各组织中广泛分布，在胃内容物及肠内容物中除发现大量原形外，还发现代谢物M1＞M2＞M5＞M4＞M3(按代谢物浓度从大到小顺序)，1h时胃内容物中代谢物M1的量约为原形的7％。1 h时除胃壁、肠内容物、胃内容物外，M2在各组织中的浓度均高于原形，M3浓度次之；在采集的三个时间点样品中，IMM018各代谢物M1、M2、M4及M5，除子宫、卵巢外，其余组织均在1 h达到最大组织浓度；代谢物M3，除大肠及生殖器外，其余组织均在1 h达到最大组织浓度。相对于血浆浓度，IMM018各代谢物在大多数组织中浓度较高，特别是在胃肠道、肺、子宫、心、膀胱、胰腺和肝组织中有较高的浓度。代谢物M2、M1和M5在胃肠道中吸收较快，在4 h时的胃内容物药物浓度约为1h药物浓度的25％，小肠内容物约为1 h药物浓度的14％。各代谢物在组织中消除迅速，12 h时代谢物M1在心、肝、肾、脂肪、小肠、大肠、胃壁、卵巢、胰腺的浓度降为1 h时的百分之一。原形及代谢物在脑组织中含量低，说明药物不容易通过血脑屏障。
五、氢吗啡酮缓释制剂在犬体内的药动学研究
氢吗啡酮(hvdromorphone，HYD)是一种半合成的阿片受体拮抗剂。本实验建立快速、灵敏的液相色谱-串联质谱法测定比格犬血浆中氢吗啡酮。比格犬血浆0.1 mL经β-葡糖苷酸酶孵化16 h后，采用液-液萃取法处理，以甲醇：水：甲酸(65：35：1)为流动相，Zorbax SB C_8柱分离，采用大气压化学电离源，选择反应监测(SRM)。测定氢吗啡酮的线性范围为0.80～200.0 ng·mL~(-1)，定量下限为0.80 ng·mL~(-1)。日内、日间精密度(RSD)在2.6％～5.3％之间，准确度(RE)在-0.2％～1.0％之间。应用此法研究了6只比格犬单剂量口服盐酸氢吗啡酮缓释片4mg后的药物动力学特点。该法选择性强，灵敏度高，适用于氢吗啡酮缓释制剂的药代动力学研究。
Potent drug-drug interactions can result in serious side effects, as a result, preclinical (in vitro) and some clinical (in vivo) interaction studies are now important components of the drug candidate selection process. A rapid liquid chromatographytandem mass spectrometry (LC/MS/MS) method has been validated and employed routinely evaluate the potential for a new drug to modify cytochrome P450 (P450) activities by determining the effect of the drug on in vitro probe reactions that represent activity of specific P450 enzymes.
IMM018, 1-[5-(4-Chloro-phenyl)-3-oxo-pent-4-enyl]-pipefidine-4-carboxylic acid ethyl ester, is a drug candidate which has been shown to have analgesic and antiinflammatory actions in rats. In present paper, pharmacokinetics of IMM018 in rats and dogs were investigated. An analytical method based on liquid chromatography coupled to tandem mass spectrometry detection was developed and validated for simultaneous quantification of IMM018 and its five metabolites in rat plasma, using diphenhydramine as the internal standard. The metabolism, excretion and tissue distribution of IMM018 in rats were studied to support the pharmacology and toxicology study of IMM018.
Hydromorphone is a semi-synthetic opioid agonist. A sensitive, rapid and specific liquid chromatographic-tandem mass spectrometric (LC/MS/MS) method for the determination of hydromorphone in beagle dog plasma was developed. The method is proved to be suitable for hydromorphone control-release preparation pharmacokinetics and bioavailability in beagle dog.
1. Determination of six CYPs probe metabolites
In vitro drug interaction data can be used in guiding clinical interaction studies, or, the design of new candidates. To make such a claim, it must be assured that the in vitro data obtained is confident. To meet this need, a rapid liquid chromatography-tandem mass spectrometry (LC/MS/MS) method has been validated and employed for routine screening of new chemical entities for inhibition of six major human cytoohrome P450 (CYP) isoforms using cDNA-expressed CYPs. Probe substrates were used near the Michaelis-Menten constant (K_m) concentration values for CYP1A2 (phenacetin), CYP2C9 (tolbutamide), CYP2C19 (S-mephenytoin), CYP2D6 (dextrornethorphan), CYP2E1 (chlorzoxazone), and CYP3A4 (midazolam and dextromethorphan). The major metabolites of CYP-specific probe substrates were quantified. The LC/MS/MS method was found to be accurate and precise within the linear range of 1.0-2000 ng·mL~(-1) for each analyte in enzyme incubation mixture. The lower limit of quantification (LLOQ) was 1.0 ng·mL~(-1). The limit of detection (LOD) for the tested analytes was 0.48 ng·mL~(-1) or better based on signal-to-noise ratio＞3. The inhibition potential of the six CYP isoforms has been evaluated using their known selective inhibitors. The 50% inhibitory concentrations (IC_(50) values) measured by this method demonstrated high precision and are consistent with the literature values.
2. Studies on effect of IMM018 on hepatic CYPs
IMM018, a new anti-inflammatory agent, was being developed for the treatment of patients with rheumatoid arthritis. Drug-drug interaction studies using recombinant human cytochrome P450s were conducted to assess CYP1A2, CYP2C9, CYP2D6 and CYP3A4 inhibition potential; Induction potential of CYP1A, CYP2C, CYP2D and CYP3A by IMM018 was examined with liver microsomes from control rats or rats treated with IMM018 at 50 and 100 mg·kg~(-1)·d~(-1) for 3 consecutive days. The assays that were validated are: phenacetin O-deethylase (CYP1A2), tolbutamide methylhydroxylase (CYP2C9), dextromethorphan O-demethylase (CYP2D6), midazolam l'-hydroxylase (CYP3A4) and dextromethorphan N-demethylase (CYP3A4). High-pressure liquid chromatography-tandem mass spectrometry was applied as the analytical method. IMM018 was a mixed-type inhibitor of CYP1A2 (K_i=13.1μmol·L~(-1)) and it also appeared to be noncompetitive inhibitor of CYP2C9 (K_i=20.2μmol·L~(-1)) and CYP2D6 (K_i=10.0μmol·L~(-1)). Furthermore, IMM018 showed time-dependent inactivation for CYP3A4. In the induction study, there were no significant difference in the enzymatic activities between the control and IMM018 treated rats except CYP3A (p＞0.05). IMM018 is likely to inhibit the metabolism of comedications moderately, if their primary routes of elimination are via cytochrome. The induction of CYP3A activity by IMM018 was dose-dependent in Wistar rats.
3. Studies on pharmacokinetics of IMM018 in rats and dogs
A sensitive LC/MS/MS method was developed for the determination of IMM018 and its five major metabolites in the plasma using diphenhydramine as internal standard. The analytes and internal standard were extracted with redistilled ethyl acetate and chromatographed isocratically on a Gemini C_(18) analytical column with a mobile phase composed of methanol: water: formic acid in the ratio of 50: 50: 0.25 (v/v/v). All analytes were detected in the selected reaction monitoring mode using an electrospray ionization source. IMM018 and its five metabolites could be simultaneously determined within 6 min. Linear calibration curves were obtained in the concentration ranges of 3.0～4000 ng·mL~(-1) for IMM018, M3, M4 and M5, 0.4～4000 ng·mL~(-1) for M1, and 4.0～40000 ng·mL~(-1) for M2. The intra- and inter-day precision (RSD), calculated from quality control (QC) samples, was between 2.0%～14.7% for each analyte. The accuracy was between -4.1%～4.3%. The method was utilized to support preclinical pharmacokinetic and toxicokinetic studies of IMM018 in rats and dogs.
The pharmacokinetics of IMM018 was investigated in rats and dogs by the LC/MS/MS method. After oral administration of IMM018 to rats (25 mg·kg~(-1)), the t_(1/2) values for metabolites M1, M2 and M3 were 4.49 h, 2.67 h and 2.79 h, respectively. After intravenous injections of IMM018 to rats (25 mg·kg~(-1)), the t_(1/2) values for metabolites M1, M2 and M3 were 5.69, 4.38 and 3.43 h, respectively.
4. Studies on excretion and tissue distribution of IMM018 in rats
An LC/MS/MS method for the simultaneous determination of IMM018 and its metabolites in rat urine, feces and bile was developed and validated. The excretion of IMM018 and its metabolites was studied by this method. According to the volume of urine and bile and the weight of feces, the total accumulated excretion of IMM018 and its metabolites were calculated.
After oral administration of IMM018 at dosage of 50 mg·kg~(-1) to rats, IMM018 was not found in urine sample, accumulated excretion of M2 in urine sample was 75.90%; M3 was 3.24%; M1 was 0.57%; M4 was 0.0097%; M5 was 0.012%. The accumulation excretion of IMM018 in feces was 0.050%; M2 was 2.17%; M3 was 0.17%; M1 was 0.00404%; M4 was 0.035%; M5 was 0.00091%. The accumulation excretion of IMM018 in bile was 0.17%; M2 was 1.97%; M3 was 0.15%; M1 was 3.69%; M4 was 0.01%. These results show that IMM018 was mainly excreted in urine in the form of metabolite M2.
The rats were divided into 3 groups. After the oral administration of 50 mg·kg~(-1) IMM018, the rats were sacrificed at 1.0, 4.0 and 12 h separately. The tissue samples were collected and used to prepare homogenates. A sensitive, specific and accurate method for simultaneous quantifying IMM018 and its metabolites in rat tissues and to study the distribution of IMM018 in rat tissues was developed. After oral administration of IMM018, the drug was distributed extensively in rats in vivo. IMM018 and its major metabolites (M1～M5) were found in gastric contents and intestinal content. The concentrations of M1～M5 in lung, uterus, heart, bladder, pancreas, and liver were higher than that of the other tissues. The concentration of IMM018 and its metabolites were low in brain, which show that the blood-brain barrier was hard to be passed through for IMM018 and its metabolites.
5. Studies on pharmacokinetics of hydromorphone control-release preparation
An LC/MS/MS method for determination of hydromorphone (HYD) in Beagle dog plasma was established. After incubation withβ-glucuronidase for 16 h, an aliquot of 0.1 mL plasma was treated by liquid-liquid extraction. The analytes of interest were separated on a Zorbax SB C_8 column with the mobile phase consisting of methanol: water: formic acid (65: 35: 1). Atmospheric pressure chemical ionization source of MS was applied and operated in positive ion mode. The linear calibration curve was obtained in the concentration range of 0.80～200.0 ng·mL~(-1). The lower limit of quantification was 0.80 ng·mL~(-1). The inter-day and intra-day precision (RSD) was below 6.0%, and the accuracy (RE) was between-0.2%～1.0% calculated from QC samples. The method was used to determine the pharmacokinetic parameters of HYD after a single oral administration of 4 mg HYD sustained release tablets to Beagle dogs. The method was proved to be special, sensitive, and suitable for the pharmacokinetic study of HYD sustained release formulation.
引文
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24. Ye RD, Prossnitz ER, Zou AH, Cochrane CG. Characterization of a human cDNA that encodes a functional receptor for platelet activating factor. Biochem. Biophys. Res. Commun., 1991, 180: 105-111
25. Sugimoto T, Tsuchimochi H, McGregor CG, Mutoh H, Shimizu T, Kurachi Y. Molecular cloning and characterization of the platelet-activating factor receptor gene expressed in the human heart. Biochem. Biophys. Res. Commun., 1992, 189: 617-624
26. Iznmi T, Kishimoto S, Takano T, Nakamura M, Miyabe Y, Nakata M, Sakanaka C, Shimizu T. Expression of human platelet-activating factor receptor gene in EoL-1 cells following butyrateinduced differentiation. Biochem J., 1995, 305: 829-835
27. Nakamura M, Honda Z, Izumi T, Sakanaka C, Mutoh H, Minami M, Bite H, Seyama Y, Matsumoto T, Noma M. Molecular cloning and expression of platelet-activating factor receptor from human lenkocytes. J. Biol Chem., 1991, 266: 20400-20405
28. Godfroid JJ, Heymans F, Broquet C. Synthesis of ether phospholipids. Pharmacol Res. Commun., 1986, 18: 1-10
29. Nguer CM, Pellegrini O, Galanaud P, Benveniste J, Thomas Y, Richard Y. Regulation of pafacether receptor expression in human B cells. J. Immunol., 1992, 149: 2742-2748
30. Carmody RJ, Cotter TG. Signaling apoptosis: a radical approach. Redox. Report., 2001, 6: 77-90
31. Hourton D, Stengel D, Chapman MJ, Ninio E. Oxidized low density lipoproteins downregulate LPS-induced platelet-activating factor receptor expression in human monocyte-derived macrophages: implications for LPS-induced nuclear factor-kappaB binding activity. Ear. J. Biochem., 2001, 268: 4489-4496
32. Lambrecht G, Parnham MJ. Kadsurenone distinguishes between different platelet activating factor receptor subtypes on macrophages and polymorphonuclear lencocytes. Br. J. Phannacol., 1986, 87: 287-289
33. Hang W, Diehi JR, Grapes L, Rothschild MF, Roudebush WE. The pig platelet-activating factor receptor gene is expressed at the mRNA level in different tissues and is mapped to chromosome 6. Anita. Reprod. Sci., 2002, 70: 277-282
34. Domingo MT, Spinnewyn B, Chabrier PE, Braquet P. Presence of specific binding sites for platelet-activating factor (PAF) in brain. Biochem. Biophys. Res. Commun., 1988, 151: 730-736
35. Homma H, Tokumura A, Hanahan DJ. Binding and internalization of platelet -activating factor 1O-alkyl-2-acetyl-su-glycero-3-phosphocholine in washed rabbit platelets. J. Biol. Chem., 1987, 262: 10582-10587
36. Shaw JO, Henson PM. The binding of rabbit basophil-derived platelet-activating factor to rabbit platelets. Am. J. Pathol., 1980, 98: 791-810
37. Ukena D, Dent G, Birke FW, Robaut C, Sybrecht GW, Barnes PJ. Radiofigand binding of antagonists of platelet-activating factor to intact human platelets. FEBS Lett., 1988, 2Z8: 285-289
38. Burgos RA, Hidalgo MA, Matthei SM, Hermosilla R, Folch H, Hancke JL. Determination of specific receptor sites for platelet activating factor in bovine nentrophils. Am. J. Vet. Res., 2004, 65: 628-636
39. Sjogren P, Jensen NH, Jensen TS. Disappearance of morphine-induced hyperalgesia after discontinuing or substituting morphine with other opioid agouists, Pain, 1994, 59: 313-316
40. MacDonald N, Der L, Allan S, Champion P. Opioid hyperexcitability: the application of alternate opioid therapy. Pain. 1993, 53: 353-355
41. Manfredi PL, Borsook D, Chandler SW, Payne R. Intravenous methadone for cancer pain unrelieved by morphine and hydromorphone: clinical observations. Pain, 1997, 70: 99-101
42. Ashby MA, Martin P, Jackson KA. Opioid substitution to reduce adverse effects in cancer pain management. Med. J. Aust., 1999, 170: 68-71
43. Wright AW, Mather LE, Smith MT. Hydromorphone-3-glucuronide : A more potent neuro-excitant than its structural analogue, morphine-3-glucuronide. Life Sci., 2001, 69: 409-420
44. Hagen N, Michael P, Thirlwell, et al. Steady-state pharmacokinetics of hydromorphone and hydromorphone-3-glucuronide in cancer patients after immediate and controlled-release hydromorphone, J. Clin. Pharmacol., 1995, 35: 37-44
45. MurrayA, Hagen NA. Hydromorphone. J. Pain Symptom Manage., 2005, 29: 57-66
46. Bouquillon A, Freeman D, Moulin D. Simultaneous solid-phase extraction and chromatographic analysis of morphine and hydromorphone in plasma by high-performance liquid chromatography with electrochemical detection. J. Chromatogr., 1992, 577: 354-357
47. Wetzelsberger N, Lücker PW, Erkins W. High-pressure liquid chromatographic method for the determination of hydromorphone in human plasma with electrochemical detection. Drag Res., 1986, 36: 1707-1710
48. Zheng M, Mcerlane KM, Ong MC. LC-MS-MS analysis of hydromorphone and hydromorphone metabolites with application to a pharmacokinetic study in the male Sprague-Dawley rat. Xenobiotica, 2002, 32: 141-151
49. Naidong W, Jiang X, Newland K, et al. Development and validation of a senstive method for hydromorphone in human plasma by normal phase liquid chromatography-tandem mass spectrometry. J. Phama. Biomed. Anal, 2000, 23: 697-704
50. Peng SX, Barbone AG and Ritchie DM. High-throughput cytochrome P450 inhibition assays by ultrafast gradient liquid chromatography with tandem mass spectrometry using monolithic columns. Rapid Commun. Mass Spectrom., 2003, 17: 509-518
51. Bu HZ, Magis L, Knuth K and Teitelbaum P. High-throughpnt cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. Ⅵ. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2At, 3A4, 2C9, 2D6 and 2E1. Rapid Commun. Mass Spectrom., 2001, 15: 741-748
52. Ayrton J, Plumb R, Leavens WJ, Mailer D, Dickins M and Dear GJ. Application of a generic fast liquid chromatography tandem mass spectrometry method for the analysis of the analysis of cytochrome P450 probe substrates. Rapid Commun. Mass Spectrom., 1998, 12: 217-224
53. Dierks EA, Stams KR, Lim HK, Cornelius G, Zhang H and Ball SE. 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 spectrometry. Drug Metab. Dispos., 2001, 29: 23-29
54. Samule A, Testino SA, Jr and Patonay G. High-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry. J. Pharm. Biomed. Anal., 2003, 30: 1459-1467
55. Bu HZ, Magis L, Knuth K and Teitelbaum P. High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. Ⅰ. Development of direct injection/online guard cartridge extraction/tandem mass spectrometry for the simultaneous detection of CYP probe substrates and their metabolites. Rapid Commun. Mass Spectrom., 2000, 14: 1619-1624
56. Zhang TY, Zhu YX and Gunarani C. Rapid and quantitative determination of metabolites from multiple cytochrome P450 probe substrates by gradient liquid chromatography-electrospray ionization-ion trap mass spectrometry. J. Chromatogr. B., 2002, 780: 371-379
57. Turpeinen M, Jouko U, Jorma J and Olavi P. Multiple P450 substrates in a single day: rapid and comprehensive in vitro interaction assay. Eur. J. Pharm. Sci., 2005, 24: 123-132
58. Kim MJ, Kim H, Cha IJ, Park JS, Shon JH, Liu KH and Shin JG. High-throughput screening of inhibitory potential of nine cytochrome P 450 enzymes in vitro using liquid chromatography /tandem mass spectrometry. Rapid Commun. Mass Spectrom., 2005, 19: 2651-2658
59. Margolis JM and Obarch RS. Impact of nouspecific binding to microsomes and phospholipids on the inhibition of cytochrome P4502D6: Impficatious for relating in vitro inhibition data to in vivo drug interactions. Drug Metab. Dispos., 2003, 31: 601-611
60. Umeda S, Harakawa N, Yamamoto M and Ueno K. Effect of nonspecific binding to microsomes and metabolic elimination of buprenorphine on the inhibition of cytochrome P4502D6. Biol. Pharm. Bull., 2005, 28: 212-216
61. Tucker GT, Houston JB and Huang SM. Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential-towards a consensus. Br. J. Clin. Pharmacol., 2001, 52: 107-117
62. Zhang TY, Zhu YX and Gunarani C. Rapid and quantitative determination of metabolites from multiple cytochrome P450 probe substrates by gradient liquid chromatography-electrospray ionization-ion trap mass spectrometry. J. Chromatogr. B., 2002, 780: 371-379
63. Wang RW, Newton DJ, Liu N, Atkins W and Lu AY. Human cytochrome P-450 3A4: in vitro drug-drug interaction patterns are substrate-dependent. Drug Metab. Dispos., 2000, 28: 360-366
64. Yu A and Haining RL. Comparative contrbution to dextromethorphan metabolism by cytochrome P450 isoforms in vitro: Can dextromethorphan be used as a dual probe for both CYP2D6 and CYP3A4 activities? Drug Metab. Dispos., 2001, 29: 1514-1520
65. Yuan R, Madani S, Wei XX, Reynolds K and Huang SM. Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab. Dispos., 2002, 30: 1311-1319
66. Kenworthy KE, Bloomer JC, Clarke SE and Houston JB. CYP3A4 drug interactions: correlation of 10 in vitro probes substrates. Br. J. Clin. Pharmacol., 1999, 48: 716-727
67. Hickman D, Wang JP, Wang Y, Unadkat JD. Evaluation of the selectivity of In vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities. Drug Metab. Dispos., 1998, 26: 207-215
68. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 1951, 193: 265-275
69. Omura T, Sate R. The carbon monoxide-binding pigment of liver microsomes. Ⅰ. Evidence for its bemoprotein nature. J. Biol. Chem., 1964, 239: 2370-2378
70. Nomeir AA, Ruegg C, Shoemarker M, Faweau LV, Palamanda JR, Silber P and Lin CC. Inhibition of CYP3A4 in a rapid microtiter plate assay using recombinant enzyme and in human liver microsomes using conventional substrates. Drug Metab. Dispos., 2001, 29: 748-753
71. Turpeinen M, Korhonen LE, Tolonen A, Uusitalo J, Juvonen R, Raunio H and Pelkonen O. Cytochrome P450 (CYP) inhibition screening: Comparison of three tests. Eur. J. Pharm. Sci., 2006, 29: 130-138
72. Stresser DM, Blanchard AP, Turner SD, Erve JCL, Dandenean AA, Miller VP and Crespi CL. Substrate-depandent modulations of CY13A4 catalytic activity: analysis of 27 test compounds with four fluorometric substrates. Drug Metab. Dispos., 2 000, 28: 1440-1448
73. Tucker GT, Houston JB and Huang SM. Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential-towards a consensus. Br. J. Clin. Pharmarcol., 2001, 52: 107-117.
74. Schmidt TJ, Lyβ G, pahl HL, Merfort I. Helenanofide Type Sesquiterpene Lactones. Part 5: The Role of Glutathione Addition Under Physiological Conditions. Bioorg. Med. Chem., 1999, 7: 2849-2855
75. Chen Q, Ngui JS, Doss GA., Wang RW, Cai X, DiNinno FP., Blizzard TA, Hammond NIL, Stearns RA., Evans DC, Baillie TA, Tang W. Cytochrome P450 3A4-mediated bioactivation of raloxifane: Irreversible enzyme inhibition and thiol adduct formation. Chem. Res. Toxicol., 2002, 15: 907-914
76. Witt UG, Schultz JE, Dolker M, Eger K. Inhibition of protein phosphatases by Michael adducts of ascorbic acid analogues with α, β-uusaturated carbonyl compounds. Bioorg. Med. Chem., 2000, 8: 807-813
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8. Kenworthy KE, Bloomer JC, Clarke SE and Houston JB. CYP3A4 drug interactions: correlation of 10 in vitro probes substrates. Br. J. Clin. Pharmacol., 1999, 48: 716-727
9. Wang RW, Newton DJ, Liu N, Atldus W and Lu AY. Human cytochrome P-450 3A4: in vitro drug-drug interaction patterns are substrate-dependent. Drug Metab, Dispos., 2000, 28: 360-366
10. Nomeir AA, Ruegg C, Shoemarker M, Favreau LV, Palamanda JR, Silber P and Lin CC. Inhibition of CYP3A4 in a rapid microtiter plate assay using recombinant enzyme and in human fiver microsomes using conventional substrates. Drug Metab. Dispos., 2001, 29: 748-753
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12. Stresser DM, Blanchard AP, Turner SD, Erve JCL, Dandeneau AA, Miller VP and Crespi CL. Substrate-dependent modulatious of CYP3A4 catalytic activity: analysis of 27 test compounds with fonr fluorometrio substrates. Drug Metab. Dispos., 2000, 28: 1440-1448
13. Tucker GT, Houston JB and Huang SM. Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential-towards a consensus. Br. J. Clin. Pharmacol., 2001, 52: 107-117
14. Margolis JM and Obarch RS. Impact of nonspecific binding to microsomes and phospholipids on the inhibition of oytochrome P4502D6: Implications for relating in vitro inhibition data to in vivo drug interactions. Drug Metab. Dispos., 2003, 31: 601-611
15. Umeda S, Harakawa N, Yamamoto M and Ueno K. Effect of nenspecific binding to microsomes and metabolic elimination of buprenorphine on the inhibition of cytochrome P4502D6. Biol. Pharm. Bull., 2005, 28: 212-216
16. Walsky RL and Obach RS. Validated assays for human cytochrome P450 activities. Drug Metab. Dispos., 2004, 32: 647-660
17. Kremers P. In vitro tests for predicting drug-drug interactions: the need for validated procedures. Pharmacol. Toxicol., 2002, 91: 209-217
18. Tucker GT, Houston JB and Huang SM. Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential-towards a consensus. Br. J. Clin. Pharmacol., 2001, 52: 107-117
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21. Korth R, Zimmermann K, Richter WO. Lipoprotein-associated pal (LA-paf) was found in washed human platelets and monocyte/macrophage-like U937 cells. Chem. Phys. Lipids., 1994, 70: 109-119
22. Nakamura M, Honda Z, Izumi T, Sakanaka C, Mutoh H, Minami M, Bite H, Seyama Y, Matsumoto T, Noma M. Molecular cloning and expression of platelet-activating factor receptor from human leukocytes. J. Biol. Chem., 1991, 266: 20400-20405
23. Kunz D, Gerard NP, Gerard C. The human leukocyte platelet-activating factor receptor, cDNA cloning, cell surface expression, and construction of a novel cpitopc-bearing analog. J. Biol. Chem., 1992, 267: 9101-9106
24. Ye RD, Prossnitz ER, Zou AH, Cochrane CG. Characterization of a human cDNA that encodes a functional receptor for platelet activating factor. Biochem. Biophys. Res. Commun., 1991, 180: 105-111
25. Sugimoto T, Tsuchimochi H, McGregor CG, Mutoh H, Shimizu T, Kurachi Y. Molecular cloning and characterization of the platelet-activating factor receptor gene expressed in the human heart. Biochem. Biophys. Res. Commun., 1992, 189: 617-624
26. Iznmi T, Kishimoto S, Takano T, Nakamura M, Miyabe Y, Nakata M, Sakanaka C, Shimizu T. Expression of human platelet-activating factor receptor gene in EoL-1 cells following butyrateinduced differentiation. Biochem J., 1995, 305: 829-835
27. Nakamura M, Honda Z, Izumi T, Sakanaka C, Mutoh H, Minami M, Bite H, Seyama Y, Matsumoto T, Noma M. Molecular cloning and expression of platelet-activating factor receptor from human lenkocytes. J. Biol Chem., 1991, 266: 20400-20405
28. Godfroid JJ, Heymans F, Broquet C. Synthesis of ether phospholipids. Pharmacol Res. Commun., 1986, 18: 1-10
29. Nguer CM, Pellegrini O, Galanaud P, Benveniste J, Thomas Y, Richard Y. Regulation of pafacether receptor expression in human B cells. J. Immunol., 1992, 149: 2742-2748
30. Carmody RJ, Cotter TG. Signaling apoptosis: a radical approach. Redox. Report., 2001, 6: 77-90
31. Hourton D, Stengel D, Chapman MJ, Ninio E. Oxidized low density lipoproteins downregulate LPS-induced platelet-activating factor receptor expression in human monocyte-derived macrophages: implications for LPS-induced nuclear factor-kappaB binding activity. Ear. J. Biochem., 2001, 268: 4489-4496
32. Lambrecht G, Parnham MJ. Kadsurenone distinguishes between different platelet activating factor receptor subtypes on macrophages and polymorphonuclear lencocytes. Br. J. Phannacol., 1986, 87: 287-289
33. Hang W, Diehi JR, Grapes L, Rothschild MF, Roudebush WE. The pig platelet-activating factor receptor gene is expressed at the mRNA level in different tissues and is mapped to chromosome 6. Anita. Reprod. Sci., 2002, 70: 277-282
34. Domingo MT, Spinnewyn B, Chabrier PE, Braquet P. Presence of specific binding sites for platelet-activating factor (PAF) in brain. Biochem. Biophys. Res. Commun., 1988, 151: 730-736
35. Homma H, Tokumura A, Hanahan DJ. Binding and internalization of platelet -activating factor 1O-alkyl-2-acetyl-su-glycero-3-phosphocholine in washed rabbit platelets. J. Biol. Chem., 1987, 262: 10582-10587
36. Shaw JO, Henson PM. The binding of rabbit basophil-derived platelet-activating factor to rabbit platelets. Am. J. Pathol., 1980, 98: 791-810
37. Ukena D, Dent G, Birke FW, Robaut C, Sybrecht GW, Barnes PJ. Radiofigand binding of antagonists of platelet-activating factor to intact human platelets. FEBS Lett., 1988, 2Z8: 285-289
38. Burgos RA, Hidalgo MA, Matthei SM, Hermosilla R, Folch H, Hancke JL. Determination of specific receptor sites for platelet activating factor in bovine nentrophils. Am. J. Vet. Res., 2004, 65: 628-636
39. Sjogren P, Jensen NH, Jensen TS. Disappearance of morphine-induced hyperalgesia after discontinuing or substituting morphine with other opioid agouists, Pain, 1994, 59: 313-316
40. MacDonald N, Der L, Allan S, Champion P. Opioid hyperexcitability: the application of alternate opioid therapy. Pain. 1993, 53: 353-355
41. Manfredi PL, Borsook D, Chandler SW, Payne R. Intravenous methadone for cancer pain unrelieved by morphine and hydromorphone: clinical observations. Pain, 1997, 70: 99-101
42. Ashby MA, Martin P, Jackson KA. Opioid substitution to reduce adverse effects in cancer pain management. Med. J. Aust., 1999, 170: 68-71
43. Wright AW, Mather LE, Smith MT. Hydromorphone-3-glucuronide : A more potent neuro-excitant than its structural analogue, morphine-3-glucuronide. Life Sci., 2001, 69: 409-420
44. Hagen N, Michael P, Thirlwell, et al. Steady-state pharmacokinetics of hydromorphone and hydromorphone-3-glucuronide in cancer patients after immediate and controlled-release hydromorphone, J. Clin. Pharmacol., 1995, 35: 37-44
45. MurrayA, Hagen NA. Hydromorphone. J. Pain Symptom Manage., 2005, 29: 57-66
46. Bouquillon A, Freeman D, Moulin D. Simultaneous solid-phase extraction and chromatographic analysis of morphine and hydromorphone in plasma by high-performance liquid chromatography with electrochemical detection. J. Chromatogr., 1992, 577: 354-357
47. Wetzelsberger N, Lücker PW, Erkins W. High-pressure liquid chromatographic method for the determination of hydromorphone in human plasma with electrochemical detection. Drag Res., 1986, 36: 1707-1710
48. Zheng M, Mcerlane KM, Ong MC. LC-MS-MS analysis of hydromorphone and hydromorphone metabolites with application to a pharmacokinetic study in the male Sprague-Dawley rat. Xenobiotica, 2002, 32: 141-151
49. Naidong W, Jiang X, Newland K, et al. Development and validation of a senstive method for hydromorphone in human plasma by normal phase liquid chromatography-tandem mass spectrometry. J. Phama. Biomed. Anal, 2000, 23: 697-704
50. Peng SX, Barbone AG and Ritchie DM. High-throughput cytochrome P450 inhibition assays by ultrafast gradient liquid chromatography with tandem mass spectrometry using monolithic columns. Rapid Commun. Mass Spectrom., 2003, 17: 509-518
51. Bu HZ, Magis L, Knuth K and Teitelbaum P. High-throughpnt cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. Ⅵ. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2At, 3A4, 2C9, 2D6 and 2E1. Rapid Commun. Mass Spectrom., 2001, 15: 741-748
52. Ayrton J, Plumb R, Leavens WJ, Mailer D, Dickins M and Dear GJ. Application of a generic fast liquid chromatography tandem mass spectrometry method for the analysis of the analysis of cytochrome P450 probe substrates. Rapid Commun. Mass Spectrom., 1998, 12: 217-224
53. Dierks EA, Stams KR, Lim HK, Cornelius G, Zhang H and Ball SE. 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 spectrometry. Drug Metab. Dispos., 2001, 29: 23-29
54. Samule A, Testino SA, Jr and Patonay G. High-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry. J. Pharm. Biomed. Anal., 2003, 30: 1459-1467
55. Bu HZ, Magis L, Knuth K and Teitelbaum P. High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. Ⅰ. Development of direct injection/online guard cartridge extraction/tandem mass spectrometry for the simultaneous detection of CYP probe substrates and their metabolites. Rapid Commun. Mass Spectrom., 2000, 14: 1619-1624
56. Zhang TY, Zhu YX and Gunarani C. Rapid and quantitative determination of metabolites from multiple cytochrome P450 probe substrates by gradient liquid chromatography-electrospray ionization-ion trap mass spectrometry. J. Chromatogr. B., 2002, 780: 371-379
57. Turpeinen M, Jouko U, Jorma J and Olavi P. Multiple P450 substrates in a single day: rapid and comprehensive in vitro interaction assay. Eur. J. Pharm. Sci., 2005, 24: 123-132
58. Kim MJ, Kim H, Cha IJ, Park JS, Shon JH, Liu KH and Shin JG. High-throughput screening of inhibitory potential of nine cytochrome P 450 enzymes in vitro using liquid chromatography /tandem mass spectrometry. Rapid Commun. Mass Spectrom., 2005, 19: 2651-2658
59. Margolis JM and Obarch RS. Impact of nouspecific binding to microsomes and phospholipids on the inhibition of cytochrome P4502D6: Impficatious for relating in vitro inhibition data to in vivo drug interactions. Drug Metab. Dispos., 2003, 31: 601-611
60. Umeda S, Harakawa N, Yamamoto M and Ueno K. Effect of nonspecific binding to microsomes and metabolic elimination of buprenorphine on the inhibition of cytochrome P4502D6. Biol. Pharm. Bull., 2005, 28: 212-216
61. Tucker GT, Houston JB and Huang SM. Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential-towards a consensus. Br. J. Clin. Pharmacol., 2001, 52: 107-117
62. Zhang TY, Zhu YX and Gunarani C. Rapid and quantitative determination of metabolites from multiple cytochrome P450 probe substrates by gradient liquid chromatography-electrospray ionization-ion trap mass spectrometry. J. Chromatogr. B., 2002, 780: 371-379
63. Wang RW, Newton DJ, Liu N, Atkins W and Lu AY. Human cytochrome P-450 3A4: in vitro drug-drug interaction patterns are substrate-dependent. Drug Metab. Dispos., 2000, 28: 360-366
64. Yu A and Haining RL. Comparative contrbution to dextromethorphan metabolism by cytochrome P450 isoforms in vitro: Can dextromethorphan be used as a dual probe for both CYP2D6 and CYP3A4 activities? Drug Metab. Dispos., 2001, 29: 1514-1520
65. Yuan R, Madani S, Wei XX, Reynolds K and Huang SM. Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab. Dispos., 2002, 30: 1311-1319
66. Kenworthy KE, Bloomer JC, Clarke SE and Houston JB. CYP3A4 drug interactions: correlation of 10 in vitro probes substrates. Br. J. Clin. Pharmacol., 1999, 48: 716-727
67. Hickman D, Wang JP, Wang Y, Unadkat JD. Evaluation of the selectivity of In vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities. Drug Metab. Dispos., 1998, 26: 207-215
68. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 1951, 193: 265-275
69. Omura T, Sate R. The carbon monoxide-binding pigment of liver microsomes. Ⅰ. Evidence for its bemoprotein nature. J. Biol. Chem., 1964, 239: 2370-2378
70. Nomeir AA, Ruegg C, Shoemarker M, Faweau LV, Palamanda JR, Silber P and Lin CC. Inhibition of CYP3A4 in a rapid microtiter plate assay using recombinant enzyme and in human liver microsomes using conventional substrates. Drug Metab. Dispos., 2001, 29: 748-753
71. Turpeinen M, Korhonen LE, Tolonen A, Uusitalo J, Juvonen R, Raunio H and Pelkonen O. Cytochrome P450 (CYP) inhibition screening: Comparison of three tests. Eur. J. Pharm. Sci., 2006, 29: 130-138
72. Stresser DM, Blanchard AP, Turner SD, Erve JCL, Dandenean AA, Miller VP and Crespi CL. Substrate-depandent modulations of CY13A4 catalytic activity: analysis of 27 test compounds with four fluorometric substrates. Drug Metab. Dispos., 2 000, 28: 1440-1448
73. Tucker GT, Houston JB and Huang SM. Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential-towards a consensus. Br. J. Clin. Pharmarcol., 2001, 52: 107-117.
74. Schmidt TJ, Lyβ G, pahl HL, Merfort I. Helenanofide Type Sesquiterpene Lactones. Part 5: The Role of Glutathione Addition Under Physiological Conditions. Bioorg. Med. Chem., 1999, 7: 2849-2855
75. Chen Q, Ngui JS, Doss GA., Wang RW, Cai X, DiNinno FP., Blizzard TA, Hammond NIL, Stearns RA., Evans DC, Baillie TA, Tang W. Cytochrome P450 3A4-mediated bioactivation of raloxifane: Irreversible enzyme inhibition and thiol adduct formation. Chem. Res. Toxicol., 2002, 15: 907-914
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