山楂叶总黄酮缓释片的研究
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摘要
基于山楂叶总黄酮(HLF)大鼠肠道吸收动力学考察结果,以体外释放度为指标,优化处方制得日服2次的HLF缓释片;以总牡荆素为指标成分考察Beagle犬单剂量口服缓释片后的药代动力学特征。
1.HLF大鼠肠道吸收特性研究
离体翻转肠囊法和在体单向灌流法考察HLF大鼠肠吸收特性。HLF在大鼠各肠段吸收良好;离体试验表明HLF在十二指肠、空肠、回肠和结肠中的表观渗透系数(P_(app))分别为(1.32±0.41)×10~(-5)、(1.62±0.27)×10~(-5)、(1.51±0.34)×10~(-5)和(0.92±0.11)×10~(-5)cm/sec;在体试验表明HLF在十二指肠、空肠、回肠和结肠中的有效渗透系数(P_(eff))分别为(4.25±0.47)×10~(-5)、(2.33±0.27)×10~(-5)、(2.80±0.72)×10~(-5)和(2.91±0.97)×10~(-5)cm/sec;HLF吸收具有浓度依赖性,可能受某种Na~+依赖性分泌蛋白和多药耐药蛋白2(Multidrug resistance-associated protein 2,MRP2)外排作用的影响。
2.HLF缓释片的制备及体外释药机理研究
紫外分光光度法测定缓释片HLF含量和体外释放度;建立水解测定HLF中总牡荆素含量的HPLC方法;以HLF体外释放度为指标,经单因素考察和预试验筛选得较优处方及制备工艺,按选定处方制得三批样品,测定含量和释放度;考察HLF缓释片中总牡荆素的释放行为,作为体内外相关性考察依据。结果:所得缓释片均一性和重现性较好,体外释放符合缓释制剂设计要求;各时间点总牡荆素累积释放量均大于HLF,表明牡荆素及其糖苷属于HLF中水溶性较好的一类成分。
建立HLF缓释片的质量标准。本品为棕黄色至棕色片;本品2、6、10 h的释放量应分别为标示量的20～40%、50～85%、75%以上:本品含HLF应为标示量的90～110%。
初步研究缓释片体外释药机理。各释药模型拟合结果表明Weibull方程拟合度最高(r=0.9731),表明其能较好地描述缓释片释药行为;Ritger-Peppas方程拟合度也较大,释放特征参数n=0.7601,介于0.45～0.89之间,表明药物释放为药物扩散和骨架溶蚀协同作用:Harland方程分析结果表明药物释放过程中90%以上的药物通过扩散机制释放。
3.HLF缓释片Beagle犬体内药代动力学研究及体内外相关性评价
建立水解测定Beagle犬血浆中总牡荆素含量的HPLC法。方法学研究表明该方法在0.2～10μg·ml~(-1)浓度范围内线性良好,低、中、高三种浓度提取回收率均大于80%,日内、日间RSD均小于5%,符合生物样品检测要求。
以HLF普通片为参比制剂,研究HLF缓释片在Beagle犬体内药代动力学。单剂量分别给予Beagle犬受试制剂和参比制剂后总牡荆素药时曲线下面积AUC_(0-t)分别为253.05±73.69 ng·h·ml~(-1)和213.21±75.19 ng·h·ml~(-1);AUC_(0-∞)分别为423.90±1156.35 ng·h·ml~(-1)与442.09±1156.76 ng·h·ml~(-1);达峰时间T_(max)分别为2.71±0.71 h和0.58±0.49 h;峰浓度C_(max)分别为43.09±9.92 ng·ml~(-1)和66.89±35.35ng·ml~(-1);t_(1/2)分别为4.52±2.99 h和5.12±2.29 h;MRT分别为2.49±0.93 h和1.29±0.46h。经配对t检验,受试制剂的MRT和T_(max)较参比制剂显著延长(P＜0.05),表明两制剂在吸收速度方面生物不等效:HLF缓释片相对生物利用度为97.94±18.85%,在80～125%的生物等效范围内,表明两制剂在吸收程度方面生物等效。体内外相关性研究表明HLF缓释片体外累积释放百分率与Beagle犬体内药物吸收分数之间相关性较好,线性相关系数r=0.981(P＜0.02),表明体外释放度可为HLF缓释片内在质量控制提供有效保证。
In the present study,twice a day HLF sustained-release(SR) tablets were developed according to the results of hawthorn leaves flavonoids(HLF) intestinal absorption study in rats.Based on in vitro drug release profile,optimal formulation was achieved through the single-factor tests.Pharmacokinetics of SR tablets were investigated in Beagle dogs with total vitexins(TV) as marker compound.
1.Intestinal absorption kinetics of HLF in rats
The regional intestinal absorption of HLF in rats was investigated using in vitro everted gut sacs and in situ single pass intestinal perfusion(SPIP).Results showed that HLF was well absorbed at all intestinal segments.In vitro studies showed that the apparent permeabilities(P_(app)) of HLF at duodenum,jejunum,ileum and colon were (1.32±0.41)×10~(-5),(1.62±0.27)×10~(-5),(1.51±0.34)×10~(-5) and(0.92±0.11)×10~(-5) cm/sec, respectively.In situ studies showed the effective permeabilities(P_(eff)) of HLF at duodenum was(4.25±0.47)×10~(-5),(2.33±0.27)×10~(-5),(2.80±0.72)×10~(-5) and (2.91±0.97)×10~(-5) cm/sec,respectively.The absorption of HLF was concentration dependent and some kind of Na~+ dependent transporter as well as the multidrug resistance-associated protein 2(MRP2) might be involved in the efflux of HLF.
2.Preparation of HLF SR tablets and preliminary study on drug release mechanism
Ultraviolet spectrophotometry(UV) method was developed and validated for the determination of drug content and release behavior.TV was detected by hydrolyzing vitexin glycosides to vitexin and the hydrolysis condition was optimized.An accurate, reliable and reproducible high performance liquid chromatography(HPLC) method was applied for the detection of TV.Optimal formulation and preparation technique were screened base on the single-factor tests.The release profiles of three batches of tablets indicated that HLF SR tablets had well reproducibility and homogeneity,and accorded with the demand of SR formulation.The release profile of TV from the optimal SR tablets was investigated,providing data for the in vitro/in vivo correlation study.Results showed TV release was generally faster than that of HLF,suggesting that vitexin and its glucosides were among the highly water-soluble components in HLF.
The quality standard for the HLF SR tablet was established.HLF SR tablets were expected to be yellow to brown.The release ratio of HLF from the SR tablet at 2,6 and 10 h should be 20～40%,50～85%,and above 75%,respectively.The content of HLF should be in the range of 80～120%.
Several drug release models were used to study the in vitro drug release mechanism of the HLF SR tablet.Weibull model was proved to be the best model for the description of drug release profile(r=0.9731).The release parameter n obtained from Ritger-Peppas equation was 0.7601,between 0.45 and 0.89,indicating that drug release mechanism was of non-Fickian diffusion where drug diffusion and matrix erosion were synergetic.Harland equation analysis further indicated that the release amount of drug through diffusion was more than 90%throughout the release process.
3.Pharmacokinetics of HLF SR tablets in Beagle dogs and in vitro/in vivo correlation
Hydrolysis-HPLC method was developed and validated for the determination of plasma concentration of TV.The calibration curve was linear over the range of 0.2～10μg·ml~(-1)(r=0.9997).The mean absolute recovery of vitexin over three concentrations was above 80%.The intra- and inter-day precisions were within 5%.
The pharmacokinetics of HLF SR tablets were investigated in Beagle dogs using HLF immediate-release tablets as reference.After single-dose administration of the test preparations and the reference preparations to dogs,AUC_(0-t) were 253.05±73.69 ng·h·ml~(-1) and 213.21±75.19 ng·h·ml~(-1),AUC_(0-∞) was 423.90±156.35 ng·h·ml~(-1) and 442.09±156.76 ng·h·ml~(-1),T_(max) were 2.71±0.71 h and 0.58±0.49 h,C_(max) were 43.09±9.92 ng·ml~(-1) and 66.89±35.35 ng·ml~(-1),t_(1/2) were 4.52±2.99 h and 5.12±2.29 h, MRT were 2.49±0.93 and1.29±0.46 h,respectively.Statistical analysis showed that T_(max) and MRT of the SR tablet was significantly prolonged(P＜0.05).Relative bioavailability of single-dose administration was 97.94±18.85%,between 80～120%, suggesting the HLF SR tablet was bioequivalent to the reference tablet.
The good linear correlation between the fraction of absorption in vivo and the release rate in vitro was obtained,suggesting that in vitro drug release could be used to control the internal quality of HLF SR tablet.
1.HLF大鼠肠道吸收特性研究
离体翻转肠囊法和在体单向灌流法考察HLF大鼠肠吸收特性。HLF在大鼠各肠段吸收良好;离体试验表明HLF在十二指肠、空肠、回肠和结肠中的表观渗透系数(P_(app))分别为(1.32±0.41)×10~(-5)、(1.62±0.27)×10~(-5)、(1.51±0.34)×10~(-5)和(0.92±0.11)×10~(-5)cm/sec;在体试验表明HLF在十二指肠、空肠、回肠和结肠中的有效渗透系数(P_(eff))分别为(4.25±0.47)×10~(-5)、(2.33±0.27)×10~(-5)、(2.80±0.72)×10~(-5)和(2.91±0.97)×10~(-5)cm/sec;HLF吸收具有浓度依赖性,可能受某种Na~+依赖性分泌蛋白和多药耐药蛋白2(Multidrug resistance-associated protein 2,MRP2)外排作用的影响。
2.HLF缓释片的制备及体外释药机理研究
紫外分光光度法测定缓释片HLF含量和体外释放度;建立水解测定HLF中总牡荆素含量的HPLC方法;以HLF体外释放度为指标,经单因素考察和预试验筛选得较优处方及制备工艺,按选定处方制得三批样品,测定含量和释放度;考察HLF缓释片中总牡荆素的释放行为,作为体内外相关性考察依据。结果:所得缓释片均一性和重现性较好,体外释放符合缓释制剂设计要求;各时间点总牡荆素累积释放量均大于HLF,表明牡荆素及其糖苷属于HLF中水溶性较好的一类成分。
建立HLF缓释片的质量标准。本品为棕黄色至棕色片;本品2、6、10 h的释放量应分别为标示量的20～40%、50～85%、75%以上:本品含HLF应为标示量的90～110%。
初步研究缓释片体外释药机理。各释药模型拟合结果表明Weibull方程拟合度最高(r=0.9731),表明其能较好地描述缓释片释药行为;Ritger-Peppas方程拟合度也较大,释放特征参数n=0.7601,介于0.45～0.89之间,表明药物释放为药物扩散和骨架溶蚀协同作用:Harland方程分析结果表明药物释放过程中90%以上的药物通过扩散机制释放。
3.HLF缓释片Beagle犬体内药代动力学研究及体内外相关性评价
建立水解测定Beagle犬血浆中总牡荆素含量的HPLC法。方法学研究表明该方法在0.2～10μg·ml~(-1)浓度范围内线性良好,低、中、高三种浓度提取回收率均大于80%,日内、日间RSD均小于5%,符合生物样品检测要求。
以HLF普通片为参比制剂,研究HLF缓释片在Beagle犬体内药代动力学。单剂量分别给予Beagle犬受试制剂和参比制剂后总牡荆素药时曲线下面积AUC_(0-t)分别为253.05±73.69 ng·h·ml~(-1)和213.21±75.19 ng·h·ml~(-1);AUC_(0-∞)分别为423.90±1156.35 ng·h·ml~(-1)与442.09±1156.76 ng·h·ml~(-1);达峰时间T_(max)分别为2.71±0.71 h和0.58±0.49 h;峰浓度C_(max)分别为43.09±9.92 ng·ml~(-1)和66.89±35.35ng·ml~(-1);t_(1/2)分别为4.52±2.99 h和5.12±2.29 h;MRT分别为2.49±0.93 h和1.29±0.46h。经配对t检验,受试制剂的MRT和T_(max)较参比制剂显著延长(P＜0.05),表明两制剂在吸收速度方面生物不等效:HLF缓释片相对生物利用度为97.94±18.85%,在80～125%的生物等效范围内,表明两制剂在吸收程度方面生物等效。体内外相关性研究表明HLF缓释片体外累积释放百分率与Beagle犬体内药物吸收分数之间相关性较好,线性相关系数r=0.981(P＜0.02),表明体外释放度可为HLF缓释片内在质量控制提供有效保证。
In the present study,twice a day HLF sustained-release(SR) tablets were developed according to the results of hawthorn leaves flavonoids(HLF) intestinal absorption study in rats.Based on in vitro drug release profile,optimal formulation was achieved through the single-factor tests.Pharmacokinetics of SR tablets were investigated in Beagle dogs with total vitexins(TV) as marker compound.
1.Intestinal absorption kinetics of HLF in rats
The regional intestinal absorption of HLF in rats was investigated using in vitro everted gut sacs and in situ single pass intestinal perfusion(SPIP).Results showed that HLF was well absorbed at all intestinal segments.In vitro studies showed that the apparent permeabilities(P_(app)) of HLF at duodenum,jejunum,ileum and colon were (1.32±0.41)×10~(-5),(1.62±0.27)×10~(-5),(1.51±0.34)×10~(-5) and(0.92±0.11)×10~(-5) cm/sec, respectively.In situ studies showed the effective permeabilities(P_(eff)) of HLF at duodenum was(4.25±0.47)×10~(-5),(2.33±0.27)×10~(-5),(2.80±0.72)×10~(-5) and (2.91±0.97)×10~(-5) cm/sec,respectively.The absorption of HLF was concentration dependent and some kind of Na~+ dependent transporter as well as the multidrug resistance-associated protein 2(MRP2) might be involved in the efflux of HLF.
2.Preparation of HLF SR tablets and preliminary study on drug release mechanism
Ultraviolet spectrophotometry(UV) method was developed and validated for the determination of drug content and release behavior.TV was detected by hydrolyzing vitexin glycosides to vitexin and the hydrolysis condition was optimized.An accurate, reliable and reproducible high performance liquid chromatography(HPLC) method was applied for the detection of TV.Optimal formulation and preparation technique were screened base on the single-factor tests.The release profiles of three batches of tablets indicated that HLF SR tablets had well reproducibility and homogeneity,and accorded with the demand of SR formulation.The release profile of TV from the optimal SR tablets was investigated,providing data for the in vitro/in vivo correlation study.Results showed TV release was generally faster than that of HLF,suggesting that vitexin and its glucosides were among the highly water-soluble components in HLF.
The quality standard for the HLF SR tablet was established.HLF SR tablets were expected to be yellow to brown.The release ratio of HLF from the SR tablet at 2,6 and 10 h should be 20～40%,50～85%,and above 75%,respectively.The content of HLF should be in the range of 80～120%.
Several drug release models were used to study the in vitro drug release mechanism of the HLF SR tablet.Weibull model was proved to be the best model for the description of drug release profile(r=0.9731).The release parameter n obtained from Ritger-Peppas equation was 0.7601,between 0.45 and 0.89,indicating that drug release mechanism was of non-Fickian diffusion where drug diffusion and matrix erosion were synergetic.Harland equation analysis further indicated that the release amount of drug through diffusion was more than 90%throughout the release process.
3.Pharmacokinetics of HLF SR tablets in Beagle dogs and in vitro/in vivo correlation
Hydrolysis-HPLC method was developed and validated for the determination of plasma concentration of TV.The calibration curve was linear over the range of 0.2～10μg·ml~(-1)(r=0.9997).The mean absolute recovery of vitexin over three concentrations was above 80%.The intra- and inter-day precisions were within 5%.
The pharmacokinetics of HLF SR tablets were investigated in Beagle dogs using HLF immediate-release tablets as reference.After single-dose administration of the test preparations and the reference preparations to dogs,AUC_(0-t) were 253.05±73.69 ng·h·ml~(-1) and 213.21±75.19 ng·h·ml~(-1),AUC_(0-∞) was 423.90±156.35 ng·h·ml~(-1) and 442.09±156.76 ng·h·ml~(-1),T_(max) were 2.71±0.71 h and 0.58±0.49 h,C_(max) were 43.09±9.92 ng·ml~(-1) and 66.89±35.35 ng·ml~(-1),t_(1/2) were 4.52±2.99 h and 5.12±2.29 h, MRT were 2.49±0.93 and1.29±0.46 h,respectively.Statistical analysis showed that T_(max) and MRT of the SR tablet was significantly prolonged(P＜0.05).Relative bioavailability of single-dose administration was 97.94±18.85%,between 80～120%, suggesting the HLF SR tablet was bioequivalent to the reference tablet.
The good linear correlation between the fraction of absorption in vivo and the release rate in vitro was obtained,suggesting that in vitro drug release could be used to control the internal quality of HLF SR tablet.
引文
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