五酯胶囊的药代动力学及与他克莫司相互作用研究
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摘要
中药在我国及其他亚洲国家历史悠久,已经被证明对慢性病具有肯定的治疗优势,具有不良反应少、毒性低等特点,在欧美国家逐步受到重视。近年来,随着全球“回归自然”呼声的提高,中药应用大幅增加。而且多是与化学合成药物同时使用,尤其是人类免疫缺陷病毒(HIV)感染和癌症患者为改善精神和生理状况服用各种中药的比例更高。以往大多数人认为中药来源于天然植物,安全无毒,也不会像化学药一样易发生药物间相互作用。事实上,中药含有多种生物活性成分,其化学组成与常用化学药类似,可通过协同或者拮抗作用共同发挥生理调节作用,也存在与多种类型的药物发生相互作用的可能。以往人们多重视化学合成药物之间的相互作用,最近有关中药和化学药相互作用而降低疗效和导致毒副反应的报道日益增多,已引起了医药界的广泛关注。
本课题以临床常用免疫抑制剂他克莫司及纯天然保肝药南五味子为主要研究对象,以HPLC、LC-MS/MS联用技术为主要监测手段,在对中药南五味子药材质量控制研究和他克莫司及五酯胶囊体内定性定量分析方法建立的基础上,深入探讨两者合并用药后体内代谢动力学特征和体外相互作用机制,具体内容包括：
1、本文以南五味子中的主要活性成分木脂素为研究对象,采用反相HPLC梯度洗脱法对南五味子药材中8种主要木脂素成分进行了多成分含量测定分析,同时运用LC-MS/MS技术对各特征成分进行了鉴别确证。并将检测方法运用到不同产地15批南五味子药材及13批制剂五酯胶囊中,方法学考察良好。同时建立15批不同产地南五味子药材及五酯胶囊的HPLC指纹图谱,该方法将多成分含量测定与指纹图谱结合可操作性强,重现性好,为更好地控制南五味子药材质量提供了科学依据,并为体内药代动力学研究提供质量稳定可靠的供试样品。
2、本文采用LC-MS/MS技术首次建立了五酯胶囊中5种主要活性成分在大鼠血浆中的定量分析方法,即五味子醇甲、五味子醇乙、五味子酯甲、五味子酚及五味子甲素。采用液-液萃取为血浆样品前处理条件,以联苯双酯为内标,色谱分析采用Agilent Zorbax SB-C18 (3.5μm,50x2.1 mm, I.D.),流动相为乙睛-甲醇-0.1%甲酸水(7：2：1,V/V/V)内含0.1%甲酸水溶液,流速0.3mL/min,柱温35℃。运用Agilent G6410A型串联质谱的多反应监测(MRM)模式正离子检测,并进行了系统的方法学评价。结果显示,在各自的生物样品浓度范围内,均呈良好线性,r>0.99；其各成分的线性范围分别为：0.50-200ng/mL,0.50-50.0ng/mL,0.50-200ng/mL, 0.50-50.Ong/mL和0.50-50.0ng/mL,最低定量限分别为：0.5 ng/mL,日内、日间精密度均小于15%,提取回收率均大于86%,该方法操作简单,选择性好,灵敏度高,成功用于五酯胶囊大鼠血浆的动力学研究。同时建立了灵敏度高、专属性强、简便、准确的LC-MS/MS方法同时测定人体血浆中的五酯胶囊中5种木脂素成分浓度,并研究五酯胶囊在中国健康人体内的药代动力学特征,为临床合理用药提供参考依据。
3、本文建立了他克莫司在大鼠血浆中的定量分析方法,方法学考察均符合要求,并成功用于他克莫司与五酯胶囊合并用药后药代动力学的相互作用研究。结果显示,他克莫司单独使用,其药代动力学参数为：Tmax为0.38±0.21h；Cmax为18.87±10.29ng/mL;AUCO→t为40.98±37.07ng h/mL;单次服用五酯胶囊后同时给药他克莫司,大鼠全血中他克莫司药代动力学参数显示：Tmax为1.54±0.15h；Cmax为59.42±30.32ng/mL;AUCO→t为239.71±28.86ng h/mL,其达峰时间及达峰浓度与单独服用他克莫司时均提高了近5倍；连续12天多次给药五酯胶囊,在第12天同时给药五酯胶囊及他克莫司,其他克莫司药代动力学参数显示：Tmax为0.32±0.09h；Cmax为43.16±10.61 ng/mL;AUCO→t为89.21±26.39 ng h/mL。其各代谢参数与空白组相比有所提高或延长,但与单次同服五酯胶囊及他克莫司相比,他克莫司的各药代参数有所下降,这一结果暗示五酯胶囊在体内不同用量与时间上对他克莫司药效上可能存在不同程度的协同或抑制,其作用机理可能更为复杂,当协同强于抑制时,表现为协同作用。但就单次给药五酯胶囊对他克莫司药代动力学产生显著的协同作用是肯定的。在此基础上,实验采取随机分组大鼠灌胃不同剂量五酯胶囊5min后,灌胃他克莫司(1.2mg/kg),通过对各成分在大鼠血样中测定所得的血药浓度求算出各成分药代动力学参数。结果显示,他克莫司达峰浓度(Cmax)呈五酯胶囊剂量依赖性变化；他克莫司曲线下面积(AUC)随五酯胶囊剂量的递增也逐渐增大,直至五酯胶囊灌胃剂量为450mg/kg时,他克莫司曲线下面积(AUC)上升为最大值825.34 ng h/mL,随后呈降低趋势,出现这一平台期可能与给药剂量增大,胃中药量过多难以及时吸收有关。
4、本文采用Caco-2细胞模型为工具,在细胞水平上考察了五酯胶囊及其活性成分对他克莫司吸收的影响,试图阐明五酯胶囊及其活性成分影响他克莫司吸收过程的作用机制。结果显示,五味子醇乙、五味子甲素、五味子酯甲及五酯胶囊溶液可明显抑制他克莫司的吸收外排作用,且五味子甲素的这种抑制作用最强。Caco-2细胞转运实验证明了五酯胶囊可通过抑制P-gp对他克莫司的外排作用而使他克莫司口服生物利用度增加。
5、本文采用人肝微粒体体外孵育实验,系统考察了五酯胶囊及其活性成分对CYP3A4、CYP1A2、CYP2C9、CYP2C19和CYP2D6这5种酶活性是否产生作用,从而影响他克莫司在肝微粒体的代谢。结果显示,五酯胶囊及五味子中8种主要活性成分对5个酶均有不同程度的抑制作用,其中主要成分对CYP3A4及CYP2C19抑制作用较强,以五酯胶囊及其主要成分酯甲和甲素作用最为明显。实验进一步证实了五酯胶囊是通过抑制CYP450酶系从而抑制他克莫司的代谢使其生物利用度升高。
Traditional Chinese medicine (TCM), which has a long history in China and other Asian countries, has already been proven to have a certain advantage of chronic disease treatment that hardly leads to adverse or toxic effects and gradually taken seriously by North America and Europe. In recent years, under the influence of the "back to nature" trend of thought in the world, the use of TCM increased dramatically. Most of them are often co-administered with chemical synthetic drugs, especially for people with HIV infection or cancer who are taking a higher proportion of TCM for improving psychical and physiological conditions. The past, most people think of TCM derived from natural plants, safe and non-toxic, unlike the chemical drugs as vulnerable to drug interactions. In fact, TCM contains a variety of bio-active ingredients, whose chemical composition is similar with commonly used chemical drugs, thus can play a role in physiological regulation together through synergistic or antagonistic effects and is also possible to interact with any type of drug. Although most people paid attention to drug-drug interaction in the past years, recently, there are an increased number of reports that interactions between TCM and chemical drugs resulted in decreased efficacy and toxicity, which has aroused widespread concern in the medical community.
Here, we investigated quality control of Nan-wuweizi (TCM) and developed quantitative analysis of tacrolimus, one of common clinical immunosuppressants, and Wuzhi-capsule (WZC) in vivo by HPLC and LC-MS/MS for understanding pharmacokinetic interactions of both drugs combination. The results are as follows:
1. A simple and sensitive high performance liquid chromatography method with photodiode array detection (HPLC-DAD) was developed for simultaneous determination of eight bioactive constituents (schisandrin, schisandrol B, schisantherin A, schisanhenol, anwulignan, deoxyshisandrin, schisandrin B and schisandrin C) in the ripe fruit of Schisandra sphenanthera and its traditional Chinese herbal preparations Wuzhi capsule by optimizing the extraction, separation and analytical conditions of HPLC-DAD. The chemical fingerprint of S. sphenanthera was established using raw materials of 15 different origins in China. The chromatographic separations were obtained by an Agilent Eclipse XDB-C18 reserved-phase column (250mm×4.6mm i.d.,5μm) using gradient elution with water-formic acid (100:0.1, v/v) and acetonitrile, at a flow rate of 1.0 mLmin-1, an operating temperature of 35℃, and a wavelength of 230 nm. The constituents were confirmed by (+) electrospray ionization LC-MS. The new method was validated and was successfully applied to simultaneous determination of components in 13 batches of Wuzhi-capsule. The results indicate that this multi-component determination method in combination with chromatographic fingerprint analysis is suitable for quantitative analysis and quality control of S. sphenanthera and its preparation. It is also concluded that the quality of Wuzhi-capsule is stable.
2. A rapid sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of multiple bioactive lignan constituents of Wuzhi capsule in rat plasma. The extraction, separation, and analytical conditions were optimized. Five constituents of the Wuzhi-capsule (schisandrin, schisandrol B, schisantherin A, schisanhenol, and deoxyshisandrin) were determined by the LC-MS/MS method. Liquid-liquid extraction with methyl tert-butyl ether was carried out using bifendate as the internal standard. The five bioactive constituents were separated on a Zorbax SB-C18 reserved-phase column (100mm×2.1mm i.d.,3.5μm) by isocratic elution using amobile phase consisting of acetonitrile, methanol, and 0.1% aqueous formic acid (72:18:10, v/v/v) at aflow rate of 0.3 mL/min. The total run time was only 3.5 min. All analytes showed good linearity over a wide concentration range (r2> 0.99) and their lower limit of quantification was 0.5 ng/mL. The average extraction recovery of the five analytes from rat plasma was more than 85%, and the intra-day and inter-day accuracy and precision of the assay were less than 15%. Our method was successfully used for pharmacokinetic study of the five components in the Wuzhi capsule. Meantime, quantitative analysis of tacrolimus was developed in rat plasma. The method meets the current FDA criteria for bioanalytical method validation and successfully applied to pharmacokinetic interactions with Wuzhi capsule. We also developed a sensitive, selective, convenient and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of five lignan constituents of Wuzhi capsule in human plasma and their pharmacokinetics in healthy Chinese volunteers, which provided reference for clinical reasonable medication.
3. Quantitative analysis method of tacrolimus was developed in rat plasma, which meets the current FDA criteria for bioanalytical method validation and successfully applied to pharmacokinetic interactions with Wuzhi capsule. The results showed pharmacokinetic parameters of tacrolimus (Cmax,59.42±30.32ng/mL; Tmax,1.54±0.15h; AUC0-t, 239.71±28.86 ng h/mL) in rat plasma after single administration of Wuzhi capsule were remarkably enhanced five times, compared to administration of tacrolimus alone(Cmax, 18.87±10.29ng/mL; Tmax,0.38±0.21h; AUC0-t, 40.98±37.07 ng h/mL). After 12d administration of tacrolimus, the pharmacokinetic parameters of tacrolimus (Cmax, 43.16±10.61 ng/mL; Tmax,0.32±0.09h; AUC0-t,89.21±26.39 ng h/mL) were obtained among the rats orally administered tacrolimus and Wuzhi capsule on the 12th day. Compared to blank group, the pharmacokinetic parameters improved or extended a little, but compared to single administration of tacrolimus and Wuzhi capsule, the pharmacokinetic parameters have somewhat decreased. These results implied that Wuzhi capsule at in vivo different dose and times has synergistic or antagonistic effects on tacrolimus efficacy. Its mechanism may be more complicated, when the collaboration is stronger than inhibition, showed synergy. However, it is affirmative that Wuzhi capsule at single dose makes significant synergies effects on the pharmacokinetics of tacrolimus. The above validated method was successfully applied to the pharmacokinetic study of tacrolimus. The SD rats were randomly divided into groups. Different dosage Wuzhi capsule was orally administrated at a dosage of 1.2mg/kg tacrolimus for each group. The pharmacokinetic parameters of tacrolimus were calculated on the plasma concentrations of tacrolimus in rats plasma samples. The results indicated that Cmax depends on Wuzhi capsule dosage; AUC with increasing doses of Wuzhi capsule gradually increased, until orally administrated dosage of 450mg/kg Wuzhi capsule, AUC increased to maximum825.34 ng h/mL, then decreased. The reason probably was that excessive stomach medicine is difficult to absorb in time along with increasing dosage.
4. The absorption and metabolism of tacrolimus and major index component of ethanol extracts of Nan-wuweizi in vitro were systematically investigated through Caco-2 cell model and liver microsomes incubation, which determined the role of cytochrome P450 enzymes (CYP450) in their metabolic pathway for the knowledge of the metabolism mechanism and prediction of herb-drug interaction. The results showed that schisandrol B, deoxyschizandrin, schisantherin A and Wuzhi capsule solution can obviously inhibit uptake and efflux function of tacrolimus, especially deoxyschizandrin most. The transport of tacrolimus using Caco-2 cell monolayers demonstrated that its bioavailability was enhanced when Wuzhi capsule inhibited the P-gp efflux pump.
5. The effects of Wuzhi capsule and its bioactive constituents on CYP3A4、CYP1A2、CYP2C9、CYP2C19 and CYP2D6 enzyme activity were investigated through liver microsomes incubation, which could further affect tacrolimus metabolism in the liver microsomes. The results showed Wuzhi capsule and eight bioactive constituents in the ripe fruit of Schisandra sphenanthera had different degrees of inhibition on five enzymes, among which main constituents showed stronger inhibition on CYP3A4 and CYP2C19. Our studies have further proved that Wuzhi capsule inhibited tacrolimus metabolism and enhanced its bioavailability through inhibition of CYP450.
本课题以临床常用免疫抑制剂他克莫司及纯天然保肝药南五味子为主要研究对象,以HPLC、LC-MS/MS联用技术为主要监测手段,在对中药南五味子药材质量控制研究和他克莫司及五酯胶囊体内定性定量分析方法建立的基础上,深入探讨两者合并用药后体内代谢动力学特征和体外相互作用机制,具体内容包括：
1、本文以南五味子中的主要活性成分木脂素为研究对象,采用反相HPLC梯度洗脱法对南五味子药材中8种主要木脂素成分进行了多成分含量测定分析,同时运用LC-MS/MS技术对各特征成分进行了鉴别确证。并将检测方法运用到不同产地15批南五味子药材及13批制剂五酯胶囊中,方法学考察良好。同时建立15批不同产地南五味子药材及五酯胶囊的HPLC指纹图谱,该方法将多成分含量测定与指纹图谱结合可操作性强,重现性好,为更好地控制南五味子药材质量提供了科学依据,并为体内药代动力学研究提供质量稳定可靠的供试样品。
2、本文采用LC-MS/MS技术首次建立了五酯胶囊中5种主要活性成分在大鼠血浆中的定量分析方法,即五味子醇甲、五味子醇乙、五味子酯甲、五味子酚及五味子甲素。采用液-液萃取为血浆样品前处理条件,以联苯双酯为内标,色谱分析采用Agilent Zorbax SB-C18 (3.5μm,50x2.1 mm, I.D.),流动相为乙睛-甲醇-0.1%甲酸水(7：2：1,V/V/V)内含0.1%甲酸水溶液,流速0.3mL/min,柱温35℃。运用Agilent G6410A型串联质谱的多反应监测(MRM)模式正离子检测,并进行了系统的方法学评价。结果显示,在各自的生物样品浓度范围内,均呈良好线性,r>0.99；其各成分的线性范围分别为：0.50-200ng/mL,0.50-50.0ng/mL,0.50-200ng/mL, 0.50-50.Ong/mL和0.50-50.0ng/mL,最低定量限分别为：0.5 ng/mL,日内、日间精密度均小于15%,提取回收率均大于86%,该方法操作简单,选择性好,灵敏度高,成功用于五酯胶囊大鼠血浆的动力学研究。同时建立了灵敏度高、专属性强、简便、准确的LC-MS/MS方法同时测定人体血浆中的五酯胶囊中5种木脂素成分浓度,并研究五酯胶囊在中国健康人体内的药代动力学特征,为临床合理用药提供参考依据。
3、本文建立了他克莫司在大鼠血浆中的定量分析方法,方法学考察均符合要求,并成功用于他克莫司与五酯胶囊合并用药后药代动力学的相互作用研究。结果显示,他克莫司单独使用,其药代动力学参数为：Tmax为0.38±0.21h；Cmax为18.87±10.29ng/mL;AUCO→t为40.98±37.07ng h/mL;单次服用五酯胶囊后同时给药他克莫司,大鼠全血中他克莫司药代动力学参数显示：Tmax为1.54±0.15h；Cmax为59.42±30.32ng/mL;AUCO→t为239.71±28.86ng h/mL,其达峰时间及达峰浓度与单独服用他克莫司时均提高了近5倍；连续12天多次给药五酯胶囊,在第12天同时给药五酯胶囊及他克莫司,其他克莫司药代动力学参数显示：Tmax为0.32±0.09h；Cmax为43.16±10.61 ng/mL;AUCO→t为89.21±26.39 ng h/mL。其各代谢参数与空白组相比有所提高或延长,但与单次同服五酯胶囊及他克莫司相比,他克莫司的各药代参数有所下降,这一结果暗示五酯胶囊在体内不同用量与时间上对他克莫司药效上可能存在不同程度的协同或抑制,其作用机理可能更为复杂,当协同强于抑制时,表现为协同作用。但就单次给药五酯胶囊对他克莫司药代动力学产生显著的协同作用是肯定的。在此基础上,实验采取随机分组大鼠灌胃不同剂量五酯胶囊5min后,灌胃他克莫司(1.2mg/kg),通过对各成分在大鼠血样中测定所得的血药浓度求算出各成分药代动力学参数。结果显示,他克莫司达峰浓度(Cmax)呈五酯胶囊剂量依赖性变化；他克莫司曲线下面积(AUC)随五酯胶囊剂量的递增也逐渐增大,直至五酯胶囊灌胃剂量为450mg/kg时,他克莫司曲线下面积(AUC)上升为最大值825.34 ng h/mL,随后呈降低趋势,出现这一平台期可能与给药剂量增大,胃中药量过多难以及时吸收有关。
4、本文采用Caco-2细胞模型为工具,在细胞水平上考察了五酯胶囊及其活性成分对他克莫司吸收的影响,试图阐明五酯胶囊及其活性成分影响他克莫司吸收过程的作用机制。结果显示,五味子醇乙、五味子甲素、五味子酯甲及五酯胶囊溶液可明显抑制他克莫司的吸收外排作用,且五味子甲素的这种抑制作用最强。Caco-2细胞转运实验证明了五酯胶囊可通过抑制P-gp对他克莫司的外排作用而使他克莫司口服生物利用度增加。
5、本文采用人肝微粒体体外孵育实验,系统考察了五酯胶囊及其活性成分对CYP3A4、CYP1A2、CYP2C9、CYP2C19和CYP2D6这5种酶活性是否产生作用,从而影响他克莫司在肝微粒体的代谢。结果显示,五酯胶囊及五味子中8种主要活性成分对5个酶均有不同程度的抑制作用,其中主要成分对CYP3A4及CYP2C19抑制作用较强,以五酯胶囊及其主要成分酯甲和甲素作用最为明显。实验进一步证实了五酯胶囊是通过抑制CYP450酶系从而抑制他克莫司的代谢使其生物利用度升高。
Traditional Chinese medicine (TCM), which has a long history in China and other Asian countries, has already been proven to have a certain advantage of chronic disease treatment that hardly leads to adverse or toxic effects and gradually taken seriously by North America and Europe. In recent years, under the influence of the "back to nature" trend of thought in the world, the use of TCM increased dramatically. Most of them are often co-administered with chemical synthetic drugs, especially for people with HIV infection or cancer who are taking a higher proportion of TCM for improving psychical and physiological conditions. The past, most people think of TCM derived from natural plants, safe and non-toxic, unlike the chemical drugs as vulnerable to drug interactions. In fact, TCM contains a variety of bio-active ingredients, whose chemical composition is similar with commonly used chemical drugs, thus can play a role in physiological regulation together through synergistic or antagonistic effects and is also possible to interact with any type of drug. Although most people paid attention to drug-drug interaction in the past years, recently, there are an increased number of reports that interactions between TCM and chemical drugs resulted in decreased efficacy and toxicity, which has aroused widespread concern in the medical community.
Here, we investigated quality control of Nan-wuweizi (TCM) and developed quantitative analysis of tacrolimus, one of common clinical immunosuppressants, and Wuzhi-capsule (WZC) in vivo by HPLC and LC-MS/MS for understanding pharmacokinetic interactions of both drugs combination. The results are as follows:
1. A simple and sensitive high performance liquid chromatography method with photodiode array detection (HPLC-DAD) was developed for simultaneous determination of eight bioactive constituents (schisandrin, schisandrol B, schisantherin A, schisanhenol, anwulignan, deoxyshisandrin, schisandrin B and schisandrin C) in the ripe fruit of Schisandra sphenanthera and its traditional Chinese herbal preparations Wuzhi capsule by optimizing the extraction, separation and analytical conditions of HPLC-DAD. The chemical fingerprint of S. sphenanthera was established using raw materials of 15 different origins in China. The chromatographic separations were obtained by an Agilent Eclipse XDB-C18 reserved-phase column (250mm×4.6mm i.d.,5μm) using gradient elution with water-formic acid (100:0.1, v/v) and acetonitrile, at a flow rate of 1.0 mLmin-1, an operating temperature of 35℃, and a wavelength of 230 nm. The constituents were confirmed by (+) electrospray ionization LC-MS. The new method was validated and was successfully applied to simultaneous determination of components in 13 batches of Wuzhi-capsule. The results indicate that this multi-component determination method in combination with chromatographic fingerprint analysis is suitable for quantitative analysis and quality control of S. sphenanthera and its preparation. It is also concluded that the quality of Wuzhi-capsule is stable.
2. A rapid sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of multiple bioactive lignan constituents of Wuzhi capsule in rat plasma. The extraction, separation, and analytical conditions were optimized. Five constituents of the Wuzhi-capsule (schisandrin, schisandrol B, schisantherin A, schisanhenol, and deoxyshisandrin) were determined by the LC-MS/MS method. Liquid-liquid extraction with methyl tert-butyl ether was carried out using bifendate as the internal standard. The five bioactive constituents were separated on a Zorbax SB-C18 reserved-phase column (100mm×2.1mm i.d.,3.5μm) by isocratic elution using amobile phase consisting of acetonitrile, methanol, and 0.1% aqueous formic acid (72:18:10, v/v/v) at aflow rate of 0.3 mL/min. The total run time was only 3.5 min. All analytes showed good linearity over a wide concentration range (r2> 0.99) and their lower limit of quantification was 0.5 ng/mL. The average extraction recovery of the five analytes from rat plasma was more than 85%, and the intra-day and inter-day accuracy and precision of the assay were less than 15%. Our method was successfully used for pharmacokinetic study of the five components in the Wuzhi capsule. Meantime, quantitative analysis of tacrolimus was developed in rat plasma. The method meets the current FDA criteria for bioanalytical method validation and successfully applied to pharmacokinetic interactions with Wuzhi capsule. We also developed a sensitive, selective, convenient and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of five lignan constituents of Wuzhi capsule in human plasma and their pharmacokinetics in healthy Chinese volunteers, which provided reference for clinical reasonable medication.
3. Quantitative analysis method of tacrolimus was developed in rat plasma, which meets the current FDA criteria for bioanalytical method validation and successfully applied to pharmacokinetic interactions with Wuzhi capsule. The results showed pharmacokinetic parameters of tacrolimus (Cmax,59.42±30.32ng/mL; Tmax,1.54±0.15h; AUC0-t, 239.71±28.86 ng h/mL) in rat plasma after single administration of Wuzhi capsule were remarkably enhanced five times, compared to administration of tacrolimus alone(Cmax, 18.87±10.29ng/mL; Tmax,0.38±0.21h; AUC0-t, 40.98±37.07 ng h/mL). After 12d administration of tacrolimus, the pharmacokinetic parameters of tacrolimus (Cmax, 43.16±10.61 ng/mL; Tmax,0.32±0.09h; AUC0-t,89.21±26.39 ng h/mL) were obtained among the rats orally administered tacrolimus and Wuzhi capsule on the 12th day. Compared to blank group, the pharmacokinetic parameters improved or extended a little, but compared to single administration of tacrolimus and Wuzhi capsule, the pharmacokinetic parameters have somewhat decreased. These results implied that Wuzhi capsule at in vivo different dose and times has synergistic or antagonistic effects on tacrolimus efficacy. Its mechanism may be more complicated, when the collaboration is stronger than inhibition, showed synergy. However, it is affirmative that Wuzhi capsule at single dose makes significant synergies effects on the pharmacokinetics of tacrolimus. The above validated method was successfully applied to the pharmacokinetic study of tacrolimus. The SD rats were randomly divided into groups. Different dosage Wuzhi capsule was orally administrated at a dosage of 1.2mg/kg tacrolimus for each group. The pharmacokinetic parameters of tacrolimus were calculated on the plasma concentrations of tacrolimus in rats plasma samples. The results indicated that Cmax depends on Wuzhi capsule dosage; AUC with increasing doses of Wuzhi capsule gradually increased, until orally administrated dosage of 450mg/kg Wuzhi capsule, AUC increased to maximum825.34 ng h/mL, then decreased. The reason probably was that excessive stomach medicine is difficult to absorb in time along with increasing dosage.
4. The absorption and metabolism of tacrolimus and major index component of ethanol extracts of Nan-wuweizi in vitro were systematically investigated through Caco-2 cell model and liver microsomes incubation, which determined the role of cytochrome P450 enzymes (CYP450) in their metabolic pathway for the knowledge of the metabolism mechanism and prediction of herb-drug interaction. The results showed that schisandrol B, deoxyschizandrin, schisantherin A and Wuzhi capsule solution can obviously inhibit uptake and efflux function of tacrolimus, especially deoxyschizandrin most. The transport of tacrolimus using Caco-2 cell monolayers demonstrated that its bioavailability was enhanced when Wuzhi capsule inhibited the P-gp efflux pump.
5. The effects of Wuzhi capsule and its bioactive constituents on CYP3A4、CYP1A2、CYP2C9、CYP2C19 and CYP2D6 enzyme activity were investigated through liver microsomes incubation, which could further affect tacrolimus metabolism in the liver microsomes. The results showed Wuzhi capsule and eight bioactive constituents in the ripe fruit of Schisandra sphenanthera had different degrees of inhibition on five enzymes, among which main constituents showed stronger inhibition on CYP3A4 and CYP2C19. Our studies have further proved that Wuzhi capsule inhibited tacrolimus metabolism and enhanced its bioavailability through inhibition of CYP450.
引文
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[2]缪海均,刘皋林,邓渝林.环丙沙星在兔体内对他克莫司药代动力学的影响[J].中国药房,2003,(01)：17-18
[3]中华人民共和国药典委员会.中华人民共和国药典(2005年版),一部[Z].北京：化学工业出版社,2000,197
[4]Yang F, Yuan J, Fu P. The Research survey of Schisand chinensis (Turcz.) Baill [J]. West China J Pharm Sciraia,2003,18(06):438-440
[5]Zhou Y, Yang J S, Wang L W, et al. The triterpenoids components in Schisandra sphenanthera Rehd. et Wils [J].Chin Pharm J.2003,38(02):81-83
[6]秦国伟,陈业高,五味子科植物化学及药理研究概况,香港科技大学生物技术研究所,中药研究与开发综述[M].北京：科学技术出版社,2000,280-318
[7]吴笑春,辛华雯,李罄.五酯胶囊对健康受试者他克莫司药动学的影响[J].中国新药杂志,2007,16(08)：647-650
[8]肖健强,邓笑媚.五酯胶囊防治抗结核药物肝损害的临床观察[J].中国防病杂志,2003,25(06)： 371-374
[9]宋金德,张兴树,王应鸿.五酯胶囊对抗结核病药物所致肝损害的作用[J].临床肺科杂志,2003,8(2)：150
[10]陈玮莹,温博贵,郑瑞明,等.五味子对CCl4中毒大鼠肝细胞核基质蛋白和结构的保护作用[J].免疫学杂志,2006,22(4)：455
[11]孟德宝,黎朝晖,刘玉爱.他克莫司的药物不良反应[J].中国误诊学杂志,2005,(04)：760-761
[12]徐芳,翟所迪,胡永芳.他克莫司发生药物相互作用的分子机制[J].中国药学杂志,2007,42(13)：965-968
[13]缪海均,刘皋林.他克莫司与其它药物的相互作用[J].中国医院用药评价与分析,2001,(03)：167-178
[14]Iwata H, Tezuka Y, Kadota S, et al. Identification and characterization of potent CYP3A4 in hibitors in Schisandra fruit extract[J]. Drug Metab Dispos,2004,(32):1351-1358
[15]Yoo HH, Lee M, Lee MW, et al. Effects of Schisandra lignans on P-glycoprotein-mediated drug efflux in human intestinal Caco-2[J]. Planta Med.2007,73 (5):444-450
[16]裴彬,蔡雄,缪晓辉.五味子甲素对大鼠肝细胞色素P4503A影响的研究[J].肝脏,2006,(04)：261-263
[17]LIU KT, CRESTEIL T, LE PROVOST E. Specific evidence that schizandrins induce a
phenobarbital-like cytochrome P-450 from separated rat liver[J]. Biochem Biophys Res. Commun, 1981,103(4):1131-1137
[18]LIU KT, LESCA P. Pharmacological properties of dibenzo [a, c] cyclooctene derivatives isolated fromFructus Schizandraechinensis. I. Interactionwith rat liver cytochromeP-450 and inhibition of xenobioticmetabolism and mutagenicity [J]. Chem Biol Interact,1982,39(3):301-314
[19]Yamauchi A, leiri I, Kataoka Y, Tanabe M, Nishizaki T, Oishi R, Higuchi S, Otsubo K, Sugimachi K. Neurotoxicity induced by tacrolimus after liver transplantation:relation to genetic polymorphisms of the ABCB1 (MDR1) gene. transplantation,2002,74(4):571-572
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