【作者】 王蕾；

【导师】 唐星；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2009， 博士

【摘要】 胸腺五肽（Thymopentin,TP5）是一个人工合成的寡肽,为胸腺生成素Ⅱ的免疫活性中心。TP5可诱导T细胞的分化、增殖和成熟,活化或调节不同T细胞亚群的数目和功能,实现对整个机体免疫系统的双向调节。临床上采用肌内或静脉注射给药,用于恶性肿瘤、慢性肝炎、严重感染、糖尿病及自身免疫性疾病等的治疗与辅助治疗。本文研究了用于肺部给药的TP5干粉吸入剂,可作为注射剂的替代给药方式,以解决长期频繁注射引起的患者依从性差的问题。本文首先建立了包含制剂理化性质和雾化性能两类指标的干粉吸入剂质量评价体系。制剂理化性质包括引湿性、水分含量、粒子表面形态、粒度及其分布、密度和流动性的测定;雾化性能指标为排空率和体外沉积率。以扫描电镜、激光散射、热重分析及双级液体碰撞器等作为分析手段。选择HPLC法测定TP5含量,该方法专属性好,重现性、精密度、回收率均符合规定,TP5在0.15 mg·mL^-1～2.25 mg·mL^-1浓度范围内,浓度与峰面积线性关系良好,回归方程及相关系数为A=910687·C-53447,r=0.9999。采用肺灌洗法提取大鼠肺表面的酶,灌洗液（bronchoalveolar lavage fluid,BALF）收率大于80%,酶解反应重现性良好。实验结果表明肺部酶对TP5有较强的水解能力,在稀释10倍的BALF中TP5半衰期为49.2 min;稀释300倍的BALF对TP5仍有水解能力,但反应速率降低。在选定的反应条件下,酶解反应呈零级过程。考察了常用的吸入剂添加剂,如乳糖、甘露醇、氨基酸、泊洛沙姆188及壳聚糖等对TP5酶解速率的影响,结果表明亮氨酸和苯丙氨酸可延长TP5的酶解半衰期至原来的2倍以上,分别为112.7 min和136.2 min;壳聚糖也有类似作用,可使TP5半衰期延长至85 min,且酶抑制效果与其分子量无关。上述辅料的酶抑制机制为:辅料分子与TP5分子在溶液中带相反电荷,两者通过静电引力作用形成离子复合物;复合物可改变TP5分子的伸展性,并使空间位阻增加,从而阻碍了酶催化中心与TP5分子的结合,使酶解反应速率降低。经分析可能有两种肺酶参与TP5水解:中性内肽酶从肽链的C末端水解掉两个氨基酸残基,氨肽酶从肽链N末端水解掉精氨酸。两种酶解途径中,内肽酶的催化速率要高于氨肽酶。制备了以甘露醇、乳糖和亮氨酸为载体的TP5普通干粉吸入剂,对不同处方组成的吸入剂的理化性质及吸入性能进行考察,结果表明处方组成可显著影响吸入剂的理化性质,进而影响粉末的雾化性能。流动性良好且具有较小空气动力径的轻质粉末表现出较好的吸入效果。亮氨酸能降低粉末密度,提高干粉吸入剂体外沉积率。当处方中亮氨酸含量>63%时,粉雾剂获得较佳的雾化性能。另外,处方中加入少量F68可以显著改善粉末的流动性。优化的处方组成为TP5/甘露醇/亮氨酸=10/18/72,含2%的F68,其振实密度为0.31 g·cm^-3,空气动力径为1.9μm,体外沉积率达为45%。采用喷雾干燥法制备TP5壳聚糖微球,以收率、粉末流动性对处方进行初步评价,结果表明微球处方中必须加入一定量的亮氨酸才能满足肺部吸入要求。优选的处方为TP5/壳聚糖/亮氨酸=10/45/45,含2%F68。另外发现TP5在喷雾干燥过程中与壳聚糖相互作用,形成了不可逆的复合物,导致粉末中药物含量下降至75%。制备了含壳聚糖纳米粒的TP5干粉吸入剂。首先利用离子凝胶化法制备TP5壳聚糖纳米粒,对制备工艺进行单因素考察。以纳米粒粒径和包封率为指标,分别考察了CS和TPP溶液的浓度、CS与TPP质量比、pH值和药物加入量对纳米粒质量的影响,结果表明上述因素均对纳米粒的粒径和包封率均有影响。最终确定壳聚糖纳米粒的处方为:CS:TPP为3:1,CS:TP5为2:1。纳米粒粒径为276.1±50.7 nm,包封率为17.2%,载药量为5.4%。载药纳米粒的酶解实验显示TP5部分被物理包裹入纳米粒,部分吸附于纳米粒表面。将载药的纳米粒与亮氨酸以76:24的质量比混合后喷雾干燥,制得含纳米粒的干粉吸入剂。此干粉吸入剂与壳聚糖微球相比,粉末的流动性和排空率相当,但引湿性显著下降。含量测定的结果显示药物百分含量提高至97%,表明将药物载入纳米粒后再喷雾干燥减少了TP5的损失。在处方中药物比例均为20%时,含高浓度亮氨酸的普通干粉吸入剂沉积率最高,达41.2%,壳聚糖微球的沉积率为25.4%,略高于含纳米粒的干粉吸入剂（20.8%）。采用多指标综合评价法优化TP5干粉吸入剂的制备工艺,极差分析结果表明,雾化压力对吸入剂的质量影响最大,其他工艺因素依次为干燥风速>供液速度>入口温度,最佳工艺条件为雾化压力:190 kPa;干燥风速:0.7 m³·min^-1;供液速度:7.0mL·min^-1;入口温度110℃。应用HPLC结合微渗析法测定大鼠肺粘液中TP5的局部药代动力学。实验结果表明,微渗析探针的相对回收率与传递率不等,相对回收率较低,其平均值为3.7%。考察了大鼠气管内滴注TP5溶液及气管内给予不同处方组成的TP5干粉吸入剂后,TP5在肺粘液层中的浓度-时间变化情况。结果显示,干粉吸入剂可迅速溶解于肺粘液中。经过剂量校正后,普通干粉吸入剂、壳聚糖微球、含纳米粒的干粉吸入剂在肺粘液中的AUC_ELF/dose分别为25.15±10.98,14.94±7.35和8.81±6.11,均大于溶液剂滴注的AUC_ELF/dose 1.55±1.02;干粉吸入剂在肺内的滞留时间分别为8.92±1.19 min,10.71±1.18 min和8.94±2.74 min,高于溶液剂的6.71±1.49 min。说明粉末雾化给药有效延长了TP5在肺部的滞留时间,有利于增加药物的吸收。进行了干粉吸入剂给药后大鼠体内的药效学研究。以环磷酰胺为造模药物,建立大鼠免疫抑制模型。分别以静脉注射TP5及肺部吸入TP5的方式对免疫失衡大鼠进行治疗。选择外周血中T细胞亚群CD₄⁺/CD₈⁺比值作为检测指标,采用流式细胞术对各组大鼠进行测定。实验结果显示,大鼠连续三天腹腔注射环磷酰胺后,血中CD₄⁺/CD₈⁺均值由正常的1.84下降至1.03,说明造模成功。免疫抑制大鼠经过静脉注射TP5或给予不同处方吸入剂干预后,其CD₄⁺/CD₈⁺均值分别恢复至2.29,1.94,1.52和1.81,说明肺部吸入TP5可产生与静脉注射相同的疗效。不同处方的干粉吸入剂疗效与体外沉积率有一定相关性,处方组成为TP5/甘露醇/亮氨酸的粉末体外沉积率最高,治疗效果最好。考察大鼠肺部连续给予TP5干粉吸入剂的安全性,结果表明吸入剂对肺部有一定的刺激性。组织学检查显示支气管及肺泡上皮的通透性有所改变,有炎性细胞浸润,但无严重刺激性及成纤维细胞增生现象,损伤具有可恢复性。经统计分析,含TP5/甘露醇/亮氨酸的干粉吸入剂及含壳聚糖纳米粒的干粉吸入剂刺激性较小,与空白对照组相比无显著性差异,说明这两种处方的安全性更高。结合药效学实验结果,处方组成为TP5/甘露醇/亮氨酸=20/16/64（2%F68）和TP5纳米粒/亮氨酸=74/26（含20%TP5,w/w）的TP5干粉吸入剂更适于肺部给药。更多还原

【Abstract】 Thymopentin（TP5）,a synthetic pentapeptide（Arg-Lys-Asp-Val-Tyr）,consists of the residues 32-36 of the 49-amino acid human hormone thymopoietin.This pentapeptide exhibits a similar biological activity to thymopoietin and is,therefore,considered to be the active sequence.TP5,acting as an immunomodulator,can bring the immune dysequilibrium,which may be either hyperresponsiveness or hyporesponsiveness,towards normal state.A multitude of in vivo studies have shown the efficacy of TP5 treatment for the therapy of a variety of diseases,including primary and secondary immune deficiencies, autoimmunity,infections,cancer,hepatitis and AIDS.TP5 has been used clinically in the form of injections and a course of TP5 treatment usually lasts weeks to months.However, the long-term and repeated injections are associated with problems of poor patient compliance.Therefore,the development of a TP5 dry powder inhalation（DPI） would expand the range of delivery strategies available to the physician,and potentially overcome some of the drawbacks of the other alternative delivery routes.Firstly,a quality evaluation system including two types of index,physicochemical properties（i.e.hygroscopicity,moisture content,morphology,particle size and distribution,density and flowability） and aerosol characteristics（emitted dose and respirable fraction）,was established.Analyses by scanning electron microscopy,laser diffractometry,thermogravimetry,Twin Stage Impactor were performed to characterize the manufactured powders.HPLC method was developed for the assay of TP5 in vitro. The stability,specificity and reproducibility of the method were good.The concentration of TP5 showed a good linear relationship to peak area in the range of 0.15 mg·mL^-1-2.25 mg·mL^-1.The regression equation was A=910687·C- 53447,with a correlation coefficient of 0.9999.Bronchoalveolar lavage was employed to extract the enzyme on rat lung surface. More than 80%of bronchoalveolar lavage fluid（BALF） was recovered.Lung enzyme presented a strong proteolysis to TP5.The degradation half-time of TP5 in 10%BALF was 49.2 min.BALF showed proteolytic activity with decreased reaction rate even though it was diluted 300 times.Under the selected reaction conditions,the enzymatic cleavage of TP5 followed zero order.Some commonly used additives for inhalation,such as lactose, mannitol,aminoacids,poloxamer 188 and chitosan,were selected to study the influence of additives on the stability of TP5 in BALF.The results revealed that leucine and phenylalanine could decrease proteolysis rate and prolong the degradation half-time of TP5 to 112.7 min and 136.2 min,respectively.In addition,chitosan showed a similar effect and prolonged the degradation half-time to 85 min,which was independent of its molecular weight.The fact that the three additives and TP5 were with opposite charges in solution and therefore attracted each other electronically to form an ionic complex assumably explained the mechanism of their enzyme inhibitions.The ionic complex could restrict the flexibility of TP5 molecule or induce its steric hindrance.Both explanations can possibly contribute to hinder the attack of enzyme on TP5 molecule.Two kinds of lung enzyme, neutral endopeptidase and aminopeptidase,were possibly involved in the enzymatic cleavage of TP5.The former cleaved two aminoacid residues from C-terminal and the latter hydrolyzed TP5 from N-terminal with a smaller rate.Dry powders were produced by co-spray drying TP5 with lactose or mannitol as a bulking agent,leucine as a dispersibility enhancer and poloxamer 188 as a drug stabilizer. The results revealed that formulation compositions greatly influenced the physicochemical characteristics of the powders,which in turn affected their aerodynamic behavior.A higher loading of leucine in the formulations（>63%by dry weight） improved the aerosolization properties of the powders by producing aerodynamically lighter particles.The optimum formulation,which had a tapped density of 0.31 g·cm^-3,an aerodynamic diameter of 1.9μm and an in vitro deposition of 45%,was obtained by combining TP5/mannitol/leucine（TP5-ML） in the ratio of 10/18/72.In addition,it was interesting to find that poloxamer 188 had a significant impact on improving the powder flowability rather than stabilizing TP5.TP5-loaded chitosan microspheres were prepared by spray drying.The primary evaluations were performed by choosing yield and flowability as indexes.It was found that leucine was necessary in formulation to enhance the powder dispersibility.The optimum formulation consists of TP5/chitosan/leucine 10/45/45（TP5-MP）.However, chitosan had a negtively effect on the recovery of TP5 from spray-dried powders,which may be due to formation of a partially irreversible complex between the peptide and chitosan during the spray-drying process.To obtain a DPI containing nanoparticles,TP5-loaded chitosan nanoparticles were prepared firstly by an approach of ionic gelation.Briefly,a cross-linking reaction based on electronical attraction between chitosan and TPP occurred to form drug-containing nanoparticles.Taking the particle size and entrapment efficiency as indicator,we inspected the concentration of CS and TPP,the mass ratio of CS and TPP,pH value as well as the addition of drugs,and finally determined the formula:chitosan:TPP 3:1, chitosan:TP5 2:1.The resultant nanoparticles had a particle size of 276.1±50.7 run,an entrapment efficiency of 17.2%and a loading capacity of 5.4%.The result of proteolytic experiments indicated that partial TP5 was physically encapsulated in nanoparticles and the other part was absorbed on the surface of nanoparticles.DPI containing nanoparticles （TP5-NP） was produced by co-spray drying the mixture of TP5-loaded nanoparticles and leucine at a ratio of 76:24.By comparison with chitosan microspheres,TP5-NP showed a lower hygroscopicity and a similar flowability and emitted dose.Recovery of TP5 from powder TP5-NP increased significantly,indicating encapsulation had a positive effect on the chemical stability of TP5 during the spray-drying process.With a same TP5 proportion of 20%by dry weight,TP5-ML,which contained more leucine,performed a higher in vitro deposition of 41.2%.The respirable fractions of TP5-MP were 25.4%,more than that of TP5-NP.Multi-index evaluation was performed to optimize the preparation process of TP5 DPI.The extreme difference demonstrated that the factors affecting the quality of DPIs following the order of atomizing pressure>air flow>feed flow rate>inlet temperature. According to the Z-score results,the statistical optimization of the spray drying variables was:atomizing pressure 190 kPa;air flow 0.7 m³·min^-1;feed flow rate 7.0 mL·min^-1 and inlet temperature 110℃.Microdialysis coupled with HPLC was used to study the pharmacokinetics of TP5 in rat epithelial lung fluid（ELF）.It was found that the recovery detected by gain was not equal to that by loss.The mean in vitro recovery of probe was 3.7%.The local pharmacokinetics of TP5 DPIs in ELF after tracheal administration was investigated.For comparison,the pharmacokinetic behavior of TP5 solution after intratracheal instillation was also investigated.The values of AUC_ELF/dose obtained from TP5-ML,TP5-MP and TP5-NP were 25.15±10.98,14.94±7.35 and 8.81±6.11,respectively,which was higher than that of solution instillation,1.55±1.02.Furthermore,solutions showed a shorter residence time in ELF,which could be prolonged by DPIs to 8.92±1.19 min, 10.71±1.18 min and 8.94±2.74 min,respectively.Increasing the residence time is favorable to the absorption of TP5 in lung.Pharmacodynamics study in vivo was executed by giving the rats TP5 DPIs periodically,which were injected with immunosuppressant（Cyclophosphamide,CTX） three days before the administration.CTX caused an evident decrease of CD₄⁺/CD₈⁺ in blood from the normal level of 1.84 to 1.03.After administrating TP5 for seven days,the CD₄⁺/CD₈⁺ was reversed,which showed TP5 DPIs had the same effects as what TP5 injection acted.DPIs with different formulations presented various therapy effectiveness, which were related to their in vitro depositions.The stimulation tests revealed that DPIs caused slight lung injury after sequential administration of seven days.Histopathologic findings demonstrated more infiltrating neutrophils and destructive changes of the alveolar wall.But neither serious injury nor pulmonary fibrosis was observed.The statistical analysis showed that groups of TP5-ML, TP5-NP and control were not significantly different,indicating powder TP5-ML and powder TP5-NP had a better tolerance.In conclusion,two DPI formulations,TP5-ML and TP5-NP,were preferred to the pulmonary delivery for TP5.更多还原