【作者】 蒋华芳；

【导师】 范国荣；

【作者基本信息】 第二军医大学 ， 药物分析， 2013， 博士

【摘要】 五肽PEP801（Met-Gln-Cys-Asn-Ser）是一种合成的小分子五肽，它是首次在痢疾阿米巴中提取产生，并被发现具有抗炎活性的的小分子五肽。在体内能够抑制人体单核吞噬细胞的活动，具有抗炎活性。近来有研究证明，五肽PEP801（Met-Gln-Cys-Asn-Ser）能够保护脑缺血损伤，对脑缺血中风具有治疗意义。目前尚处于临床前研究阶段，国内、外未见有关五肽PEP801（Met-Gln-Cys-Asn-Ser）药代动力学研究的报道。本文的工作旨在对其临床前药代动力学和毒代动力学作一整体评价，以支持人体药代动力学研究并为临床用药及剂型开发提供参考依据。一、五肽PEP801代谢和稳定性研究五肽PEP801（Met-Gln-Cys-Asn-Ser）含有甲硫氨酸和半胱氨酸，这两个氨基酸残基容易发生氧化，尤其是半胱氨酸含有巯基，容易发生金属催化的氧化反应。同时生物基质中广泛存在的各种蛋白水解酶容易使多肽类化合物快速降解，导致极短的半衰期。因此本研究在实验之初，用液相-质谱联用技术详细进行了五肽PEP801（Met-Gln-Cys-Asn-Ser）的代谢稳定性研究。在溶液稳定性考察中发现，酸性体系和有机相体系可以稳定PEP801。但是在全血稳定性中发现，抗氧剂、低温和蛋白酶抑制剂均不能有效抑制PEP801的降解。而直接快速甲醇沉淀蛋白可以阻止PEP801的降解。同时通过PEP801在大鼠全血、肝匀浆和肾匀浆体外温孵实验中发现，全血和肝脏可能是PEP801代谢主要场所。而PEP801在不同种属SD大鼠、比格犬和人全血和肝微粒中代谢稳定性研究发现，PEP801的降解速度：SD大鼠>比格犬>人。因此通过AB公司Q-Trap四极杆-线性离子阱串联质谱仪和Agilent6200高效液相色谱‐高分辨飞行时间质谱联用系统两种仪器对五肽PEP801在SD大鼠、比格犬以及人全血和肝微粒体体外温孵样本的主要代谢产物进行了鉴别。实验结果表明：PEP801（Met-Gln-Cys-Asn-Ser）在生物基质中主要发生了肽链的断裂，以及可能是肽链断裂后进一步脱氢反应。在生物基质中总共发现四个主要代谢产物和两个微量代谢产物。四个主要代谢产物为M1(Asn-Ser)、 M2(Gln-Cys)、M3(Cys-Asn)和M4(Met-Gln-Cys-Asn)；两个微量代谢产物为M5（Gln-Cys-Asn），而M6可能为M3（Cys-Asn）进一步脱氢的代谢产物。大鼠、比格犬与人全血、肝微粒体体外温孵代谢物分布基本类似，因此大鼠和比格犬可以作为毒性研究的种属。通过固相合成四个主要主要代谢产物M1(Asn-Ser)、 M2(Gln-Cys)、M3(Cys-Asn)和M4(Met-Gln-Cys-Asn)，用液质联用方法对大鼠体内全血样本进行定量、定性分析。发现二肽M1(Asn-Ser)为相对稳定和降解较慢的主要代谢产物。考虑到五肽PEP801极短的半衰期，同时测定二肽M1(Asn-Ser)在全血中的浓度，可以表征五肽PEP801（Met-Gln-Cys-Asn-Ser）在体内的药代和毒代动力学过程。二、SD大鼠、比格犬全血中PEP801和代谢产物二肽（ASN-SER）分析方法的建立用液相-质谱联用技术进行五肽PEP801（Met-Gln-Cys-Asn-Ser）和二肽（Asn-Ser）在SD大鼠、比格犬全血中浓度的方法学研究。PEP801在全血中极不稳定，直接用含有0.2%甲酸的甲醇立刻进行蛋白沉淀，可阻止蛋白酶对PEP801的降解，并进一步进行蛋白沉淀前处理。同时根据快速定量取血要求，在大鼠取血时用100μl的定量毛细管在眼眶定量取血方式；而比格犬采用0.5ml的定量胰岛素注射器准确取血100μl方式进行快速定量取血。取血完毕后立刻置于先前加有0.2%甲酸的甲醇中进行样本前处理。在液质联用方法建立过程中，考虑到五肽PEP801（Met-Gln-Cys-Asn-Ser）和其主要代谢产物二肽M1(Asn-Ser)不同的化学属性，用不同的液相分离条件分别测定PEP801和二肽（Asn-Ser）在大鼠、比格犬全血中的浓度，并同时对其进行了系统的方法学验证。经验证，方法的准确度、精密度、灵敏度、专属性及定量线性等均符合生物样品的分析要求，可用于SD大鼠、比格犬药代动力学和毒代动力学研究。三、PEP801在动物体内的药代动力学研究利用建立的液质联用方法进行了大鼠静脉给药0.5mg/kg、1mg/kg、2mg/kg三个剂量以及比格犬静脉推注0.2mg/kg、0.4mg/kg、0.6mg/kg三个剂量的药代动力学研究。SD大鼠静脉注射PEP8010.5mg/kg、1mg/kg、2mg/kg三个剂量后体内半衰期极短，降解快速。PEP801和代谢物二肽（Asn-Ser）的药时曲线的末端相消除半衰期（t1/2）分别为0.24±0.02min、0.24±0.01min、0.28±0.03min以及1.94±0.04min、2.93±0.23min、2.81±0.06min； PEP801和二肽（Asn-Ser）的AUC0-τ分别为65.46±3.17ng min/mL、154.51±6.90ng min/ml、355.19±15.43ng min/ml和1133.28±32.70ng min/ml、2385.88±83.26ng min/ml、4263.99±74.85ng min/ml。 PEP801（Met-Gln-Cys-Asn-Ser）和二肽（Asn-Ser）的AUC0-τ与给药剂量基本呈正相关，相关系数分别为r2=0.9936和r2=0.9921。可认为在该剂量水平下，SD大鼠体内药代动力学行为是线性的。鉴于其较短的半衰期，血药浓度维持时间极短。临床中建议采用静脉滴注给药。而比格犬模拟临床用药采用静脉推注给药方式，静脉推注PEP801（Met-Gln-Cys-Asn-Ser）0.2mg/kg、0.4mg/kg、0.6mg/kg三个剂量给药后体内半衰期极短，降解快速。PEP801和二肽（Asn-Ser）的药时曲线的末端相消除半衰期（t1/2）分别为0.47±0.05min、0.40±0.05min、0.56±0.06min以及8.55±0.47min、12.50±1.42min、13.06±1.20min； PEP801和代谢物二肽（Asn-Ser）的AUC0-τ分别为1546.09±65.50ng min/ml、2675.17±54.4ng min/ml、6666.53±151.43ng min/ml和1258.04±102.95ng min/ml、2771.37±103.71ng min/ml、5117.49±352.76ng min/ml。 PEP801和代谢物二肽（Asn-Ser）的AUC0-τ与给药剂量基本呈正相关，相关系数分别为r2=0.9763和r2=0.9833。可认为在该三个剂量水平下，比格犬内药代动力学行为是线性的。四、PEP801在动物体内的毒代动力学研究利用建立的液质联用方法进行SD大鼠和比格犬28天多剂量静脉慢推给药15mg/kg、30mg/kg、60mg/kg三个剂量组的毒代动力学研究。具体分析与综合比较毒代动力学研究给药首日和给药末期相应的SD大鼠体内PEP801（Met-Gln-Cys-Asn-Ser）及其代谢产物二肽（Asn-Ser）实验数据。SD大鼠28天长期毒性条件下，考虑动物个体差异，低剂量、中剂量、高剂量组对应的SD大鼠体内PEP801（Met-Gln-Cys-Asn-Ser）及其代谢产物二肽（Asn-Ser）的最大血药浓度(C0)、各自单位剂量的药时曲线下面积AUC0～τ/Dose以及血药全身清除率(Cl)随多剂量给药次数与时间的推移无明显变化（p>0.05）。因此，从体内PEP801及其代谢产物二肽（Asn-Ser）主要毒代动力学参数角度，提示在低剂量、中剂量及高剂量三个剂量组28天长期、多剂量静脉慢推给药过程中，药物对机体没有明显损伤，并未造成药代、毒代属性的明显变化。比格犬28天长期毒性条件下，考虑动物个体差异，低剂量、中剂量、高剂量组对应的比格犬体内PEP801及其代谢产物（Asn-Ser）的静脉推注给药完毕后即刻血药浓度(C0)、各自单位剂量的药时曲线下面积AUC0～τ/Dose以及血药全身清除率(Cl)随多剂量给药次数与时间的推移无明显变化（p>0.05）。因此，从体内PEP801（Met-Gln-Cys-Asn-Ser）及其代谢产物（Asn-Ser）主要毒代动力学参数角度，提示在低剂量、中剂量及高剂量三个剂量组28天长期、多剂量静脉慢推给药过程中，药物对机体没有明显损伤，并未造成药代、毒代属性的明显变化。更多还原

【Abstract】 PEP801(Met-Gln-Cys-Asn-Ser), a synthetic pentapeptide produced by Entamoebahistolytic in axenic culture, was found to be an anti-inflammatory functional peptide.In vivostudies have suggested that it can inhibit the locomotion of human monocytes. Latestresearches demonstrated PEP801can protect the brain from ischemic injury. Up to now,PEP801is in the stage of preclinical-study. The pre-clinical pharmacokinetic research data ofPEP801has not been reported in recent years. This study was designed to evaluate the pre-clinical pharmacokinetics of PEP801in animals to support the pharmacokinetics study inhuman, clinical dosage and dosage form development.1. Preliminary investigation on the stability and metabolism of PEP801in animalsPEP801(Met-Gln-Cys-Asn-Ser) has Cys and Met which easily undergo oxidation,especially for Cys which contains thiol. In addition, in biological matrix, peptides oftenundergo rapid degradation in presence of different metabolic enzymes to obtain extreme shorthalf-life time. Therefore, a preliminary investigation on stabiliity and metabolism of PEP801(Met-Gln-Cys-Asn-Ser) using LC-MS/MS was performed.Through basic acid/base/organicsolvent stability test, PEP801was found to be more stable in acid enviroment and organicsolvent. However, remarkable degradation was found in blood. Low temperature, antioxidantand enzyme inhibitors could not prevent its rapid degradation in blood. However, directmethonal addition could stabilize PEP801in blood, which was selected as the samplepretreatment. The main degradation site might be blood and liver vial rat blood and tissuehomogenate incubaton stability tests. In addition, the metabolic stability of PEP801in bloodand liver microsome of different species such as SD rat, beagle dog and human demonstratedthe labile of PEP801: SD rat> beagle dog> human.Thus, in vitro metabolites in rat blood and liver microsome have been indentified usingQ-Trap and Q-TOF. The main metabolism pathway was the cleavage of amide acid bone andpotential dehydrogenation after amide acid bone cleavage. Four main metabolits, tetrapeptide(Gln-Cys-Asn-Ser), dipeptide (Met-Gln, Gln-Cys, Asn-Ser) have been indentified andsynthesized. Meanwhile from preliminary determination of in vivo metabolites study,dipeptide (Asn-Ser) was relatively dominant and stable, which was selected as acharacterization of main metabolites and monitored with PEP801(Met-Gln-Cys-Asn-Ser), inorder to acknowledge the comprehensive pharmacokinetic and toxicokinetics of PEP801(Met- 2. Methods establishment of PEP801and metabolite, dipeptide (Asn-Ser) in biologicalmatrixMethods of determination of PEP801(Met-Gln-Cys-Asn-Ser) and metabolite, dipeptide(Asn-Ser) in rat and beagle dog blood by LC-MS/MS were developed. PEP801(Met-Gln-Cys-Asn-Ser) was very unstable in blood. Therefore, direct deactivate enzyme by methanolcontaining0.2%FA was used, which was followed by simple protein precipitation.Considering different chemical behavior of PEP801and metabolite dipeptide (Asn-Ser),different LC conditions were used to determine PEP801and metabolite dipeptide (Asn-Ser) inrat and dog blood. After methods validation, the intra-and inter-day precision and accuracy,sensitivity, specificity and linear range met the acceptance criteria, which can be applied forthe pre-clinical pharmacokinetic and toxicokinetics studies of PEP801in rat and dog.3. Pharmacokinetic study of PEP801in animalsThe validated methods were successfully applied in measuring levels of PEP801(Met-Gln-Cys-Asn-Ser) and metabolite, dipeptide (Asn-Ser) in blood following intravenousinfusion of low, middle and high doses of pentapeptide in rats and dogs, respectively.Pharmacokinetic parameters were calculated using non-compartment alanalysis method.The blood concentration-time profile demonstrated that the concentrations of PEP801(Met-Gln-Cys-Asn-Ser) and dipeptide (Asn-Ser) in rat blood were reduced quickly followingintravenous injection administration of PEP801at0.5,1,2mg/kg. The estimated eliminationhalf-life (T1/2) of PEP801and dipeptide (Asn-Ser) was0.24±0.02min,0.24±0.01min,0.28±0.03min and1.94±0.04min,2.93±0.23min,2.81±0.06min, respectively.Thearea under curve (AUC0-τ) of PEP801and dipeptide (Asn-Ser) was65.46±3.17ng min/ml,154.51±6.90ng min/ml,355.19±15.43ng min/ml and1133.28±32.70ng min/ml,2385.88±83.26ng min/ml,4263.99±74.85ng min/ml, erspectively. AUC0-τofPEP801and metabolite dipeptide (Asn-Ser) were positively correlated with the doses and thecorrelation coefficients which were r2=0.9936and r2=0.9921, respectively. Considering theshort half-life time, intravenous drip was recommended for clinical usage.It was also shown by the blood concentration-time data that the concentrations ofPEP801and dipeptide (Asn-Ser) in dog blood were reduced quickly following intravenousinjection administration of PEP801at0.2,0.4,0.8mg/kg by syringe pumps. The estimatedelimination half-life (T1/2) of PEP801and dipeptide (Asn-Ser) was0.47±0.05min,0.40±0.05 min,0.56±0.06min;8.55±0.47min,12.50±1.42min,13.06±1.20min, respectively.The areaunder curve (AUC0-τ) of PEP801and dipeptide (Asn-Ser) was1546.09±65.50ng min/ml,2675.17±54.4ng min/ml,6666.53±151.43ng min/ml and1258.04±102.95ng min/ml,2771.37±103.71ng min/ml,5117.49±352.76ng min/ml, erspectively. AUC0-τofPEP801and metabolite, dipeptide (Asn-Ser) were positively correlated with the doses and thecorrelation coefficients which were r2=0.9763and r2=0.9833, respectively. Considering theshort half-life time, intravenous drip was recommended for clinical usage.4. Toxicokinetics study of PEP801in animalsPEP801(Met-Gln-Cys-Asn-Ser) and metabolite, dipeptide (Asn-Ser) levels weredetermined in rat and dog blood following28days of repeat-dose intravenous infusion of15mg/kg,30mg/kg, and60mg/kg doses of PEP801. Toxicokinetics parameters were calculatedusing non-compartment alanalysis method.Comparing the blood concentration-time data in rat and blood of the first day and the28thday, no distinctish differences of the maximum concentration (Cmax), clearance (Cl) andAUC0～τwere found as admister time was prolonged, which indicated no significantmetabolism damage was observed under28days of repeating intravenous infusion amongthree levels for rat and dog species.更多还原