【作者】 单婷婷；

【导师】 刘泽源；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药理学， 2010， 硕士

【摘要】 药代动力学研究是评价药品质量的重要手段,它通过对药物的药代动力学参数如血药浓度-时间曲线下面积(AUC)、峰浓度(Cmax)和达峰时间(tmax)等进行分析,来考察药物活性成分的质量及其在体内的量变规律。因此,药代动力学的研究、评估是药品申报与评审的重要内容,己成为药品研制、开发管理方面的重要工作。阿仑膦酸钠(Alendronate sodium)是一类治疗钙代谢性疾病的二膦酸盐类药物,例如治疗骨质疏松、高血钙症和Paget’s病等骨疾病。阿仑膦酸钠为水溶性药物,分子量>150,带有负电荷,在血浆中的滞留时间短,大部分有选择性的蓄积在骨骼中,口服平均生物利用度大约为0.7%[1]。因此,血浆中阿仑膦酸钠的浓度极低。同时,该化合物分子极性大,无生色基团,不能直接用常规的紫外检测器或荧光检测器检测[2],给血浆中阿仑膦酸钠的测定带来极大难度。目的:建立血浆中阿仑膦酸钠浓度的检测方法,用该方法考察中国健康男性志愿者口服70 mg阿仑膦酸钠片后的药动学参数,并对两种制剂的药代动力学参数及相对生物利用度进行评价;采用体外Caco-2细胞模型、崩解度和溶出度试验对两种制剂的吸收、崩解、溶出进行对比,寻找两制剂药代动力学参数有显著性差异的原因。方法:采用高效液相色谱法(HPLC)测定阿仑膦酸钠在不同介质中的浓度;利用蛋白沉淀法与固相萃取法进行血浆样品前处理;MTT法检测细胞对药物的敏感程度;建立Caco-2细胞模型,考察药物的吸收及转运情况;利用崩解度和溶出度试验,测定体外药物崩解、溶出情况。内容:1.建立阿仑膦酸钠在人血浆中的高效液相-荧光检测法(HPLC-FLD)及在HBSS溶液、去离子水中的高效液相-紫外检测法(HPLC-UV)。以帕米膦酸钠为内标(I.S.);色谱柱:Kromasil-C18柱(4.6×150 mm,5μm);流动相:甲醇-乙腈-柠檬酸与焦磷酸钠混合液;用梯度洗脱的方法进行洗脱;荧光检测波长:激发波长(λex)=260 nm,发射波长(λem)=310 nm;紫外检测波长:265 nm。同时进行各种介质中阿仑膦酸钠样品定量分析方法的确证研究。2.阿仑膦酸钠的临床试验。试验设计为双周期随机自身交叉试验,6名受试者随机编号分为两组;试验前受试者签署知情同意书并配合完成规定的各项检查;试验中单剂量口服给药,于不同时间点采集上肢静脉血,离心后分离取得血浆;试验后,经过前处理的血浆样品采用建立的HPLC-FLD法测定阿仑膦酸钠的浓度,计算药代动力学参数,并纪录、分析。将A制剂与B制剂单次给药的Cmax、AUC0-t和AUC0-∞经对数转换后进行方差分析及在α=0.05水平上进行双单侧t检验,对tmax进行非参数检验,评价两种制剂的药动学参数。3.利用Caco-2细胞模型考察阿仑膦酸钠的吸收及转运情况。建立Caco-2细胞单层模型,以细胞单层跨膜电阻、标志物的表观通透系数和形态学三个指标评价该模型的完整性。进行MTT试验,确定阿仑膦酸钠的敏感浓度。将已知浓度的阿仑膦酸钠溶液分别加在Caco-2细胞单层膜的基底侧(Basolateral,BL)或绒毛面侧(Apical,AP),从两个方向考察药物转运;在设定时间点,分别从接受池侧取一定量的生物样品;该样品经过前处理后,用建立的HPLC-UV法测定其中阿仑膦酸钠的浓度,计算表观通透系数值(Papp);并进行纪录、统计及分析。4.阿仑膦酸钠的崩解度及溶出度试验。试验方法均依照《中国药典》2005版中的要求进行。取两种制剂的阿仑膦酸钠片各6片,分别置崩解仪的吊篮玻璃管中,启动崩解仪进行试验,依次记录崩解时间。同样各取两种制剂的阿仑膦酸钠6片,以700 mL去离子水为溶出介质,介质温度37℃,桨速50 r·min-1,分别在设定的时间点取一定量的样品;样品经过前处理后,用建立的HPLC-UV法测定其中阿仑膦酸钠的浓度,纪录、统计后计算出各自的溶出度。结果:1.血浆中阿仑膦酸钠的HPLC-FLD检测分析方法具有较高的灵敏度和专属性,方法学考察结果符合生物样本分析的要求,血浆内源性杂质对样品的测定没有干扰。阿仑膦酸钠和帕米膦酸钠(内标)的保留时间分别为8.9 min和7.6 min。阿仑膦酸钠在1～100 ng·mL-1范围内线性良好(r=0.9999),最低定量浓度为1 ng·mL-1。阿仑膦酸钠的批内、批间精密度和准确度良好,样品长期冻融(30天)和多次冻融(n=3)稳定性良好。该方法灵敏、准确,可用于临床药动学及生物等效性研究。建立的HBSS溶液及去离子水中的HPLC-UV检测方法简单、快速、灵敏、专一。两种介质中,阿仑膦酸钠线性关系均较良好,均可用于对应介质中阿仑膦酸钠浓度的检测。2.6名健康男性志愿者单剂量口服阿仑膦酸钠A制剂和B制剂70 mg后主要药动学参数分别为:AUC0-t(μg·L-1·h): 102.06±42.19和164.42±50.47;AUC0-∞(μg·L-1·h): 129.96±67.84和184.34±55.04 ; Cmax(μg·L-1): 35.48±12.33和55.59±17.02;tmax(h): 1.33±0.41和1.25±0.59; t1/2(h): 2.04±0.66和2.03±0.79;Frel(%): 61.60±16.64。统计结果显示两种制剂的药代动力学参数差异较大,A制剂相对生物利用度较低。3.两种阿仑膦酸钠制剂通过Caco-2细胞膜的转运量均极少,Papp均﹤10-7cm·s-1,属于吸收较差类的药物(即吸收﹤1%)。两者转运量之间的差异没有统计学意义。4.去离子水中阿仑膦酸钠的HPLC-UV检测方法符合《中国药典》2005版规定,可以用于阿仑膦酸钠片的溶出度试验测定。记录试验数据,并使用SAS 9.1软件分析试验数据得出:①两种制剂崩解时间的差异具有统计学意义,B制剂崩解时间短于A制剂;②两种制剂均可以在15 min内溶出完全,A、B制剂的平均溶出速率差异没有统计学意义。但A制剂6片的片间溶出度差异较大,具有统计学意义(P<0.05),B制剂6片的片间溶出度差异较小,不具有统计学意义(P>0.05),说明两种制剂本身的溶出仍存在一定的区别。结论:本研究主要建立了阿仑膦酸钠在血浆、HBSS液及去离子水三种介质中各自样品的检测方法。三种样品的定量分析方法灵敏度高,重现性好,均能准确定量相应介质中阿仑膦酸钠的浓度。本研究对6名中国健康男性志愿者口服两种阿仑膦酸钠片后的药动学参数进行了分析,结果发现两种制剂的主要药代动力学参数有显著性差异,A制剂相对B制剂的相对生物利用度较低,平均61.60±16.64%。本研究探索了两种制剂药动学参数差异性较大的可能原因。①通过建立体外Caco-2细胞模型来考察两种制剂的吸收及转运情况。结果发现两种制剂的阿仑膦酸钠通过Caco-2细胞膜的转运量均极少,吸收较差,二者差异没有统计学意义。②本研究亦进行了两种制剂阿仑膦酸钠片的崩解度和溶出度试验,来考察各自的崩解时间及溶出度方面的差异。统计结果表明,两种制剂的崩解度及溶出度均有一定的差异。综合上述试验结果,分析两种制剂药代动力学参数有显著性差异的原因可能是由于处方工艺方面的不同,导致崩解度和溶出度的差异,进而影响药物的体内吸收过程,造成药代动力学参数的差异。同时,可能还存在其它因素的影响,这有待于进一步研究。更多还原

【Abstract】 Pharmacokinetics study is an important means of drug quality assessment, pharmacokinetic parameters of the drugs such as the plasma concentration - time area under the curve (AUC), peak concentration (Cmax) and peak time (tmax) are used to evaluate the quantitative change disciplinarian in vivo. Therefore, pharmacokinetics is very important in the drug report and review, it is also very important in the drug research, development and management.Alendronate sodium is a bisphosphonate drug used to treat Calcium metabolism disorders disease, such as osteoporosis, hypercalcemia, Paget’s disease and so on. Alendronate sodium is water-soluble with negative charge; molecular weight is more than 150. The residence time of alendronate sodium is short in plasma, it is selectively accumulated in the skeleton and its oral absorption rate is approximately 0.7%[1]. Therefore, the plasma concentration of Alendronate sodium is very low. Moreover, because the molecule polarity is great, the structure is without chromophore or fluorophore groups, Alendronate sodium is not directly determined with routine UV or fluorescence detector [2],it is difficult to determination of alendronate sodium .Objective: To establish the determination method of alendronate sodium concentration in plasma, study the pharmacokinetic of Chinese healthy male volunteers after 70mg alendronate sodium was taken orally. Pharmacokinetic parameters of two preparations were carried out. In vitro Caco-2 cell model, disintegration and dissolution tests were used to compare the absorption, disintegration and dissolution of two preparations to study why pharmacokinetic parameters of two preparations were significant difference.Methods: alendronate sodium concentration in different media was determined by high performance liquid chromatography (HPLC). The protein precipitation and solid phase extraction were used in pretreatment of the plasma sample. Cell sensitivity to the drug was determined by MTT assay. Caco-2 cell model was established to study drug absorption and transport. In vitro drug disintegration was determined by disintegration and dissolution tests. Contents:1. Alendronate sodium concentration in human plasma was determined by HPLC- fluorescence detection method (HPLC-FLD), and Alendronate sodium concentration in the HBSS solution and deionized water was determined by HPLC-UV detection method. Pamidronate sodium was internal standard(I.S.), column: Kromasil-C18 column (4.6×150 mm, 5μm);mobile phase: methanol-acetonitrile-the mixture of citric acid and pyrophosphate sodium, gradient elution was used, fluorescence detection wavelength: excitation wavelength (λex) was 260 nm, emission wavelength (λem) was 310 nm. UV detection wavelength was 265nm. The quantitative analysis method was verified in various media.2. Clinical trials of Alendronate sodium were two-period randomized crossover trials. Six subjects were divided into two groups according to random numbers. All subjects signed an informed consent and carried out the various inspections according to the provision prior to the trials. Single dose oral administration was taken in the trials. Venous blood at upper limbs were collected at different time points and centrifuged to obtain plasma. The concentration of the alendronate sodium in the plasma after pretreatment was determined by HPLC-FLD method. Pharmacokinetic parameters were calculated and recorded and analyzed. single dose oral administration Cmax、AUC0-t and AUC0-∞data of the test preparation and reference preparation were logarithmically transformed, analysis of variance and two-sided t test (α= 0.05 ) and non-parametric test of Tmax were taken. Pharmacokinetic parameters of two preparations were evaluated.3. The transport and absorption of alendronate sodium were studied on the Caco-2 cell model. Caco-2 cell monolayer model was established, cell single transmembrane resistance and the apparent permeability coefficient of markers and morphology were used to evaluate the integrity of the model. Sensitivity of the alendronate sodium concentration was determined in the MTT test. The known concentration of alendronate sodium Lin was added to Caco-2 cell monolayer based low side (Basolateral, BL) or villous surface side (Apical, AP). Drug transportion from both directions was studied. A certain amount of the biological samples were taken from receive pool respectively at set time points. After pretreatment of sample, alendronate sodium concentration is determined by established HPLC-UV method. Apparent permeability coefficient (Papp) was calculated; data were recorded and statistically analyzed. 4. The disintegration and dissolution test of alendronate sodium. Test methods were in accordance with "Chinese Pharmacopoeia 2005 Edition". 6 tablets from every two preparations were taken and placed into the basket glass tube of the disintegration apparatus respectively, the disintegration apparatus were started, and the disintegration time was recorded successively. In the same way, other 6 tablets from every two preparations were taken and placed into the basket glass tube of the disintegration apparatus respectively. The dissolution medium was 700 mL deionized water, medium temperature was 37℃. Paddle speed was 50 r·min-1. A certain amount of the samples were taken respectively at set time points; after pretreatment of sample, alendronate sodium concentration was determined by established HPLC-UV method., data were recorded and statistically analyzed to calculate their dissolution respectively.Results:1. HPLC-FLD determination method for plasma samples has a high Sensitivity and specificity. The results of method validation meet the requirements of the analysis of biological samples. Endogenous impurity of the plasma samples did not interfered with sample determination. Alendronate sodium and pamidronate sodium (internal standard) of the retention time were 8.9 min and 7.6 min respectively. Linear range of the alendronate sodium was between 1 ng·mL-1 and 100 ng·mL-1(r = 0.9999). The quantification limit was 1 ng·mL-1. The long-term freeze-thaw and repeated freeze-thaws stability of alendronate sodium were good. The within-day precision and day to day precision and accuracy of determination method for alendronate sodium are good. This method was sensitive and accurate, so it could be used in clinical pharmacokinetics and bioequivalence studies.Because the HPLC-UV determination method in HBSS solution and deionized water was simple, rapid, sensitive and specific and linear relations of determination method were good, both media apply to the determination method for the alendronate sodium.2. After single dose oral administration of alendronate sodium test tablets and reference tablets by 6 healthy male volunteers, statistical results of pharmacokinetic parameters: AUC0-t(μg·L-1·h) are 102.06±42.19 and 164.42±50.47 respectively; AUC0-∞(μg·L-1·h) are 129.96±67.84和184.34±55.04 respectively; Cmax(μg·L-1) are 35.48±12.33和55.59±17.02 respectively; tmax(h) is 1.33±0.41 and 1.25±0.59 respectively; t1/2(h)are 2.04±0.66和2.03±0.79 respectively. The statistical results showed that pharmacokinetic parameters of two preparations were significant difference and the relative bioavailability of A preparation was low.3. Transfer volume of two preparations in the Caco-2 cell model was less. The Papp of alendronate sodium were less than 10-7cm·s-1, so absorption of alendronate sodium was poor (absorption rate is less than 1%). Transfer volume between two preparations was not significant difference statistically.4. The HPLC-UV determination method of alendronate sodium in the deionized water meet the requirement of“Chinese Pharmacopoeia" 2005 version provisions”, and this method was applied to the dissolution test of alendronate sodium tablets. Test data obtained through the SAS 9.1 software analysis: the disintegration time difference of two formulations was significant .The disintegration time of A preparation was less than that of B preparation; two preparations were dissolved in complete within 15 min; The average dissolution rate of two preparations were not significant difference statistically. But the dissolution difference of six tablets of A preparation were large and significant statistically. The dissolution difference of six tablets of B preparation were small, they were not significant statistically .The results indicate that the dissolution of two preparations, there was still difference.Conclusion:The determination methods of alendronate sodium in plasma and HBSS and deionized water were established in this paper. The sensitivity and good reproducibility of quantitative analytical methods in three media were good, so the method was applied to determine the alendronate sodium concentration accurately and quantitatively.After oral administration of two alendronate sodium preparations by six healthy male Chinese volunteers, pharmacokinetic parameters are analyzed, bioequivalence was evaluated, the results show pharmacokinetic parameters of two preparations were significant difference and the relative bioavailability of A preparation was low.In vitro Caco-2 cell model was established to study the absorption and transport of two preparations to discuss pharmacokinetic parameters of two preparations were significant difference. The results showed that transfer volume of two preparations in the Caco-2 cell model was less, the absorption of two preparations was poor. Transfer volumes between the two were not significant difference statistically. The disintegration test and dissolution test of two alendronate sodium preparations were conducted to study the difference of the disintegration time and dissolution rate between two preparations.The above results show prescription process of two preparations are different that causes the pharmacokinetic parameters of two formulations is significant difference, resulting in disintegration and dissolution of the differences, thereby affecting the drug absorption process in vivo, resulting in differences in pharmacokinetic parameters . At the same time, there may be other factors, which need further study.更多还原