【作者】 王胜浩；

【导师】 曾苏；

【作者基本信息】 浙江大学 ， 药物分析学， 2006， 博士

【摘要】 为延长药物在体内的作用时间,采用新颖的药物释放系统（drug deliverysystems,DDS）不失为一种理想方法,而可生物降解的可注射植入剂（injectableimplants）是其中一种具有良好发展前景的释放系统。注射植入剂以可生物降解的辅料如乳酸—乙醇酸共聚物（DL-PLGA）等为制剂骨架,药物溶解或混悬于PLGA溶液中得到的一种可供注射的溶液或混悬液。这种可注射溶液的粘度相对较低,能通过常用注射针头,经皮下注射给药后,制剂与体内水性环境接触,即可形成凝胶状骨架,药物通过骨架向外缓慢释放。骨架PLGA在体内最终可被降解成乳酸和乙醇酸单体。注射植入剂可通过控制辅料PLGA的用量等,到达控制药物持续释放时间的目的。酮洛芬（ketoprofen,KET）的半衰期比较短,普通口服制剂需一天给药三次,市售的口服缓释制剂也至少需一天给药一次,其它如透皮制剂的给药一般为每3天一次。由于关节炎等慢性病的治疗要求病人长期用药,制备一次给药可维持药效4周以上的长效制剂显得十分重要。在缓控释制剂的研究中,药物释放行为往往作为制剂学特性的主要评价指标,但针对手性药物制剂,大多以非立体选择性的观点,即只评价外消旋体的递释行为,而不考察单个对映体的释放行为。因此,以非立体选择性的观点研制的长效制剂不但可能无法真实反应药效,有时甚至产生误导作用。如果从制剂学的角度仍将外消旋体药物当作单一化合物来处理,那么类似沙利度胺（thalidomide）的悲剧可能会重演。本项研究以外消旋体酮洛芬（rac-KET）为模型药物,首先采用常规的非立体选择性检测方法,进行rac-KET可注射植入剂制剂研究,然后以立体选择性拆分方法考察给药系统体外立体选择性释放特点,进一步,采用生物样品中的对映体的拆分方法评价植入剂在生物体内的对映体释放特性。通过上述研究,即首先从非立体选择性释放评价,到对映体专属性的体外释放研究最后到生物体内立体选择性考察,从三个层次评价KET释放系统的释药特性,为最终的创新制剂研究提供依据。本项目的研究成功可为科学设计生物降解型长效手性药物制剂的处方,加强有效对映体的控制释放,增加有效对映体生物利用度和降低毒性,提供理论与实验依据。一、可生物降解的注射植入型rac-KET控释给药系统的制剂学研究在已有生物降解PLGA微球研究的基础上,通过对PLGA中LA与LG不同比例的筛选,不同PLGA浓度对药物释放速率的评价,以及PEG400对药物突释的影响等的考察,认为PLGA可以作为新颖控释给药系统——rac-KET注射植入型控释给药系统的骨架。该生物降解型植入剂为溶液制剂,具有在水性介质中形成骨架型凝胶的特点。通过系统研究,成功制备了该长效控释制剂;建立了专属性强的rac-KET控释给药系统的含量测定方法。含量测定的方法学考察结果显示:空白辅料不干扰制剂的测定;在2.0μg/ml～12.0μg/ml的浓度范围内,符合线性要求;高中低三个浓度的回收率试验结果为99.0～100.8%。表明所建立的经回流提取步骤的测定方法适用于植入剂的含量测定。同时,由于植入剂的药物释放要求维持比较长的时间,针对制剂在水性介质中形成凝胶的特点,建立了体外释放度试验模型和释放度的测定方法。rac-KET控释给药系统的体外释放研究表明,不同药物浓度的植入剂和添加不同量的PEG400能使药物到达缓慢释放的目的,即T₈₀%均大于30天,其中KET浓度为10%的植入剂,其T₈₀%大于60天。以首日释放量（以考察突释效应）和维持药物释放时间对植入剂进行评价,5%药物浓度制剂中适量添加PEG400,可以减小植入剂的突释效应,但对延长药物释放时间基本无作用。5%和10%PEG400添加的植入剂,其T₈₀分别为50天和44.2天,而对照（不添加PEG400,药物浓度为5%）则为46.5天。总体而言,5%KET（添加5%PEG400）植入剂基本可到达降低首日突释效应的目的。从不同药物浓度对植入剂的首日释放量和维持药物释放时间的考察结果观察,10%药物浓度要优于5%和15%药物浓度的制剂;同时,与5%KET（添加5%PEG400）植入剂比较,10%KET不但突释效应比较小,而且维持的时间更持久。因此,我们认为10%KET植入剂具有比较显著的延缓药物释放的作用。对目标制剂的数学模型研究结果显示,rac-KET从植入剂中释放更符合Cubic方程（Mt/M_∞=18.5259+2.0079t-0.0238t²+0.001t³,方程的相关系数为R²=0.994;F=473.23,p＜0.001）。二、注射植入型rac-KET控释给药系统体外对映体选择性释放研究采用手性流动相添加剂万古霉素,以常规的C₁₈ODS为色谱柱,建立了可用于分离测定植入剂体外释放液中两个KET对映体的RP-HPLC方法。同时对所建立的方法进行了灵敏度、准确度、精密度、线性和专属性等的评价,认为该方法完全适用于体外释放液中KET对映体的分离测定。通过考察不同KET浓度植入剂以及不同释放介质pH值对植入剂的立体选择性释放影响,认为（1）不同浓度的rac-KET PLGA植入剂（4%、7%和10%）在pH 7.4的释放介质中均存在显著的立体选择性释放现象,其中7%制剂更显著。（2）不同pH值的释放介质对制剂的立体选择性也有比较大的影响,当释放介质pH为7.4时,7%rac-KET植入剂释放的立体选择性大于其在pH 10.0的介质中释放。对映体的这种立体选择性释放特性可能与辅料PLGA的可生物降解性有关。从数学模型研究的角度考察,7%rac-KET植入剂的S-对映体体外释放更符合Higuchi方程(M_t/M_∞=0.134+0.07736t^1/2),而R-对映体的释放则符合一级方程（ln（1-Mt/M∞）=-0.0145t-0.23）。由于植入剂中对映体释放速率的差异最终导致两者释放数学模型的差异。三、可生物降解的注射植入型rac-KET控释给药系统在大鼠体内的立体选择性释放研究建立了柱前衍生化的RP-HPLC方法分离测定大鼠血浆中的R-KET和S-KET。结果表明:在0.03-10.0μg/ml对映体浓度范围内呈良好的线性关系;绝对回收率大于77%;日内日间精密度小于13%;对映体的LOQ（定量限）均为0.03μg/ml。Rac-KET注射植入剂经单次大鼠皮下给药后,可维持有效浓度约8周。与普通注射剂比较,KET植入剂的有效对映体（S-（+）-KET）峰浓度显著降低[C_maxl25.20μg/ml;111.13μg/ml（普通注射剂;用纯溶剂配制,不含辅料）]];MRT显著延长[10.60天:0.49天（普通注射剂）];AUC为77.79day.μg/ml,与普通注射剂相当（73.54 day.μg/ml）。Rac-KET implants经大鼠皮下给药后,具有S-KET始终显著高于R-KET的特点（给药后至释放结束）,首个C_maxl为25.20±7.45μg/ml（S-KET）和8.96±2.80μg/ml（R-KET）。R-KET血浓度不但比S-对映体低,而且在较多时间点无法被检出。表明该rac-KET注射植入剂在大鼠体内的对映体浓度存在显著的差异。Rac-KET implants组的S/R对映体比例明显小于普通注射剂组（纯溶剂配制）,尤其在5,12和24小时（第24小时后solution组无法检测）,显示在大鼠体内的对映体浓度差异,除了KET的手性转化因素外,植入剂中的KET对映体与生物降解过程中的PLGA存在某种作用可能是另外一个原因。更多还原

【Abstract】 In connection with aim of prolonging the duration of action of conventional drugs in vivo,new drug delivery systems（DDS）continue to attract much attention.The controlled or sustained release of drugs represents one such approach.And in this regard report,DDS using biodegradable polymers which includes copolymer of lactic and glycolic acid（DL-PLGA）was studied.The biodegradable injectable implant using PLGA as matrix is one kind of DDS.The injectable implants is a solution with low viscosity and has a liquid consistency which facilitates injection through a needle.It can turn to a gel matrix immediately once contact with aqueous fluids and the release of the drug takes place slowly through this formed matrix.The matrix will be biodegraded by hydrolyzing into lactic and glycolic acid ultimately.In implant preparation, PLGA is used in diffusion controlled,swelling controlled and chemically controlled delivery system.This kind of DDS is able to provide pre-programmed durations of action and offer several advantages over the conventional dosage forms.A biodegradable injectable implant of Ketoprofen（KET）we developed was one kind of such DDS described above.It is expected to release slowly and maintain drug concentration within the therapeutic range for a long time.For KET is one of the most common pain relievers for Rheumatoid arthritis and t_1/2of KET is very short,to develop controlled and sustained DDS of KET is necessary.Meanwhile,although there are differences between two KET enantiomers’ actions in the clinical effect,a limited number of literatures about enantioselective release of KET oral DDS have been reported.Moreover, evaluations of the enantiomer release are mainly limited to studies in vitro and little attention has been paid to the in vivo release difference between the two KET enantiomers.Onishi et al.have prepared biodegradable KET implant tablets which could keep the effective release in vivo for 4 days（administrated by inserting subcutaneously by surgical procedures in rats）using PLGA as matrix,however no attention was paid to the possible enantioselectity of the enantiomers in vivo.In order to investigate the enantioselective release,the biodegradable injectable implants containing racemaic KET（rac-KET）were elaborated with PLGA,and the enantioselective release in vitro and in vivo were observed by the special reversed-phase HPLC method developed1.Incorporation of ketprofen injectalbe implantsIn this study Poly（DL-lactide-co-glycolide）（PLGA）were used in a formulation（injectable implants）which forms a gel matrix immediatedly on contact with aqueous fluids.The biodegradable injectable implants containing a non-sterodial anti-inflammatory durg,racemic ketoprofen（rac-KET）were prepared by dissolving KET in DL-PLGA solution.To evaluate the initial fast release（initial burst）and the release profile of the formulation,the implants were prepared using the PLGA with the different lactide/glycolide ratio,PEG 400 used as additive in implants and the different drug loadings of KET,respectively.The results show that PLGA（7:3） was an ideal excipent for implants to sustain release of KETThe UV spectrophotometer method was applied to determine the drug loading of rac-KET injectable implants at 255nm,drug was extracted with the solvent,alcohol,by refluxing at 70℃. The analytical method afforded recovery ranged from 99.0～100.8%,the assay was linear from 2.0μg/ml～12.0μg/ml.The similar UV spectrophotometer method with the detected wavelength of 260nm was employed to determine the KET released from the injectable implants in vitro release,theassay was also special,accurate and simple.The different drug loadings of implants and the different amount of PEG 400 added to the implants,the formulation could provide sustained release of the drug lasting about 2 months in vitro release.The 10%drug loading of rac-KET implants,it’s T80%was longer than 60 days. The initial fast release（burst effect）declined with the addition of 5%PEG400 to 5%KET implants.Compared with other KET preparations,10%rac-KET implants was a promising drug deliver system with weaker initial burst effect and the longer release period.In order to understand the drug release mode from 10%drug loading of rac-KET biodegradable injectable implants,the in vitro release data were fitted to5 power law equations,the cubic mathematical equation appeared to describe rac-KET release from the implants much better than other equations,The cubic equation（Mt/M_∞=18.5259+2.0079t-0.0238t²+0.001t³,R²=0.994; F=473.23,p＜0.O01）,can only simulate the release pattern but does not describe the underlying physical drug releasing mechanism.2.The in vitro stereoselective release from rac-KET injectable implantsA stereoselective reversed-phase HPLC assay was developed that could simultaneously quantify S-（+）and R-（-）enantiomers of ketoprofen in release samples.Racemic ketoprofen（rac-KET） and its S-（+）enantiomer（S-（+）-KET）were dissolved in an injectable viscous polymer solution consisting of the biodegradable poly（D,L-lactide-co-glycolide,70:30）（D,L-PLG）and a solvent, N-methyl-2-pyrrolidone（NMP）.Once injected into an aqueous environment,the polymeric mixture solidified into a solid implant due to the leaching of NMP.In vitro release studies show that such implants with ketoprofen can provide sustained release of the drug lasting about three months in a pH 7.4 release medium.Moreover,a preferential faster S-（+）-KET release over R-（-）-KET was observed for the implants containing 4%,7%,and 10%of racemic ketoprofen in the neutral pH 7.4 release medium.Stereoselective release was minimal in the first 42 days in vitro but became very pronounced at later time points.When S-（+）-KET was incorporated into the polymeric mixture,its release was also faster than that of the racemic ketoprofen,confirming the stereoselective release of ketoprofen from the D,L-PLG implants.The observed stereoselective release of KET at pH 7.4 was most likely produced by chiral interactions between KET enantiomers and transiently produced D-lactic acid or L-lactic acid rich domains within the implants during D,L-PLG degradation.However,such stereoselective release was not observed in pH 10.0 release medium,probably due to a much faster and homogeneous polymer degradation.The study suggests possible stereoselective release of racemic drugs from D,L-PLG microspheres and implants in vivo.In addition,to understand the enantiomer release mode from 7%rac-KET injectable implants, the in vitro release data were fitted to some power law equations.Higuchi equation appeared to describe S-KET release from the implants(M_t/M_∞=0.134+0.07736t^1/2)much better than other equations;and first order equation could only simulate the release pattern of R-KET（ln（1-Mt/M∞）=-0.0145t-0.23）.The difference of enantiomer release between R-KET and S-KET probably causes the different release pattern. 3.The Stereoselective release from KET injectable implants in ratsA stereoselective reversed-phase HPLC method has been developed and validated to separate and quantify the S-（+）and R-（-）enantiomers of ketoprofen（KET）as their diastereoisomeric amides with（S）-（-）-alpha-（1-Naphthyl）ethylamine（S-NEA）from a novel biodegradable injectable implant for the in vivo release in rats.The method involved liquid-liquid extraction of S-（+）and R-（-）enantiomers from rat plasma,using R-（-）-flurbiprofen（FBF）as the internal standard,and employed S-NEA as a pre-column chiral derivatization reagent.The derivatized products were separated on a 5μm reversed-phase C₁₈column with a mixture of methanol and 0.01 mol·L^-1KH₂PO₄（pH4.5）with a volume ratio of 71:29 as mobile phase.The detection of ketoprofen derivatives was made atλ=244 nm with UV detector.The assay was linear from 0.03 to 10.0μg/ml for each enantiomer.The absolute recoveries for each enantiomer were greater than 77%.The intra-day and inter-day variations’ were less than 13%.For each enantiomer,the limit of quantification（LOQ）was 0.03μg/ml with RSD of 15.0%（n=5）for R-（-）-KET and 13.5%（n=5）for S-（+）-KET.The reproducibility of the assay was satisfactory.The enantioselective release of the biodegradable injectable implant containing racemic KET（rac-KET）elaborated with PLGA in SD rats was investigated using pre-column chiral derivatization RP-HPLC.The rac-KET injectable implant,once injected subcutaneously in rats, produced long lasting the plasma S-enantiomer level and the plasma S-（+）-KET level was always higher than that of R-（-）-KET in rats.The difference of enantiomer concentration was related with the chiral inversion of R-（-）-KET to S-（+）-KET in rats and the biodegardable properties of the achiral excipient of Poly(D,L-lactide-co-glycolide（DL-PLGA）degraded in biological system.The rac-KET injectable implant also provided the sustained release of S-KET, the duration above the effective plasma level was about 8 weeks after a single injection.更多还原