克拉霉素亚微乳注射液的研究

【作者】 陆岩；

【导师】 唐星；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2008， 硕士

【摘要】 亚微乳剂凭借其生物相容性好、低毒低刺激性等优点被作为静脉给药的优良载体而广泛应用。克拉霉素抗菌谱广,抗菌活性强,但其静脉注射剂有严重的血管刺激性,因此开发静脉给药新剂型十分必要。本研究的目的是运用高压均质技术,将油相及油水界面膜作为目标载药部位,制备一种静脉刺激性低且能够经受住高温灭菌的克拉霉素亚微乳注射液（ClaE）,并对其大鼠体内药代动力学行为进行全面的评价。本论文建立了克拉霉素HPLC体外分析方法,该方法重现性好,准确度高。研究显示克拉霉素的溶解度、稳定性和表观油水分配系数与介质pH值变化紧密相关。低pH值时药物稳定性差,易降解;随着pH升高,其溶解度下降而表观油水分配系数变大。克拉霉素在长链油、中链油中溶解度均低于4mg/mL,而在新型油溶剂V_E中约为52.25mg/mL。因此,可以选择V_E作为制备克拉霉素亚微乳注射液的油相。本文通过高压均质法制备了以V_E为油相的克拉霉素亚微乳注射液,并利用HPLC、动态光散射技术、电泳激光散射技术和超滤法等以亚微乳剂的理化性质（外观、粒径、ζ-电位、pH值、含量和包封率）和制剂灭菌前后稳定性作为评价指标,对主要工艺及处方因素进行了研究。最终确定处方和工艺为:按质量百分比,油相组成为克拉霉素0.25%、MCT10%、V_E 5%、豆磷脂1%,水相组成为甘油2.5%、Tween-80 0.2%、F-68 0.2%、EL-40 2%、L-半胱氨酸0.05%、注射用油酸钠0.1%,加注射用水至全量;制备初乳的油相和水相温度均为55℃,高压均质参数为600bar压力循环8次,均质后调节乳剂pH值至8.0,灭菌条件为100℃旋转水浴灭菌30min。以不同pH值、灭菌方式、灭菌时间和药物浓度为考察项,系统考察了“以V_E为油相的ClaE”的灭菌稳定性。结果表明:调节ClaE pH值为8.0,100℃旋转水浴灭菌30min,能保证制剂的理化稳定性。该制剂高温加速及室温放置稳定性研究结果显示:高温40℃、60℃加速10天及室温25℃放置3个月后,ClaE含量大幅下降,均低于标示含量的90%。另外,考虑到V_E剂量的安全使用范围和EL-40静脉给药的副作用,本文通过进一步深入的研究确定以制备磷脂复合物的方法载药来制备克拉霉素亚微乳注射液。本研究以反应溶剂、磷脂比例与种类为考察项,确定克拉霉素磷脂复合物的制备方法为克拉霉素:豆磷脂1∶10（质量比）,在65℃无水乙醇中反应3小时,蒸发除去溶剂,即得。利用DSC技术确证分析了克拉霉素磷脂复合物的形成以及药物和磷脂两者间可能存在的相互作用。研究结果表明克拉霉素磷脂复合物可以大幅度提高克拉霉素在长链油和中链油中的溶解性。运用与“以V_E为油相的克拉霉素亚微乳注射液”相同的制备方法和检测技术,确定以磷脂复合物载药的ClaE最终处方和工艺为:按质量百分比,油相组成为磷脂复合物以克拉霉素计0.25%、MCT 16%、LCT 4%、豆磷脂1%,水相组成为甘油2.5%、Tween-80 0.2%、F-68 0.2%、L-半胱氨酸0.02%、注射用油酸钠0.1%,加注射用水至全量;制备初乳的油相和水相温度分别为55℃和80℃,高压均质参数为700bar压力循环7次,均质后调节乳剂pH值至8.0,灭菌条件为100℃旋转水浴灭菌30min。以磷脂复合物载药的ClaE理化性质测定显示ClaE平均粒径140.3±47.2nm,ζ-电位-20.99mV,含量101.1%,包封率91.4%。振摇稳定性实验表明剧烈振摇该亚微乳剂,其粒径无显著变化,无絮凝、合并现象产生,可认为该克拉霉素亚微乳注射液振摇稳定性良好,适于工业化生产和运输。高温60℃加速10天含量仅降低5.7%。长期稳定性考察结果显示在25±2℃及10±2℃条件下放置6个月,本品的外观、pH值、药物含量、平均粒径以及药物包封率等都没有发生显著的变化,即在试验条件下本制剂稳定。因此,该制剂具备工业化生产和临床应用的前景。本文建立了UPLC/MS/MS法测定大鼠血浆中克拉霉素的方法,该法测定时间短,准确性,重现性良好。以克拉霉素溶液剂为参比制剂,以磷脂复合物载药的克拉霉素亚微乳注射液和溶液剂的药时过程均符合三隔室模型,两者的药动学特征曲线相似。按统计矩原理来计算两者的非隔室模型参数,结果显示除参数Vss外,两种制剂间主要药动学参数没有显著性差异（n=6,P＞0.05）,两者的AUC_0-t分别为（44.96±7.222）和（38.52±28.455）mg/L^*h,MRT分别为（4.162±0.653）和（4.896+2.809）h。更多还原

【Abstract】 Parenteral submicro-emulsion,with high biocompatibility,low toxicity and irritation,has been widely used as an excellent drug delivery carrier.Clarithromycin has wide antibacterial spectrum and strong antibacterial activity.However,clarithromycin i.v.can cause serious venous irritation,and thus developing a new preparation i.v.is quite necessary.The aim of this thesis was to prepare thermal sterile stable and lower i.v.irritation clarithromycin submicro-emulsion（ClaE）using high-pressure homogenization technology.An HPLC method was established for the analysis of ClaE in vitro.The solubility,stability and oil phase-water phase partition of clarithromycin were closely related with the pH of mediums.At low pH,the chemical stability was bad.By increasing the pH of mediums,the solubility decreased and oil phase-water phase partition turned higher.The solubility of clarithromycin in LCT,MCT and safflower oil were all below 4mg/mL while it was 52.25mg/mL in V_E.So V_E can be selected as an oil solvent for preparing ClaE.High pressure homogenization was used to prepare ClaE.HPLC,dynamic light scattering and electrophoretic light scattering technology,and ultrafiltration were also employed.Taking physical appearance,pH,particle size distribution（PSD）,ζ-potential,content,entrapment efficiency and thermal sterile stability as index,the final formulation and preparation process for ClaE using V_E as oil phase were as follows:as quality percentage,oil phase was composed of 0.25%clarityromycin,10%MCT,5%V_E,1%soybean lecithin;the water phase was composed of 2.5%glycerol,0.2%F-68,0.2%Tween-80,2%EL-40,0.1%sodium oleate and 0.05% L-Cysteine;and the oil phase and water phase were both heated to 55℃;the homogenization pressure and cycles were 600bar and 8 times;the pH was adjusted to 8.0 and then ClaE was sterilized in a rotating water bath at 100℃for 30min.The thermal sterile stability of ClaE using V_E as oil phase were carefully investigated including the effects of different sterile time,sterile methods,drug concentrations and pH values. The results showed that sterilization in a rotating water bath at 100℃for 30min can maintain the stability of ClaE while the pH was adjusted to 8.0 before sterilization.The results of high temperature accelerating test and room temperature stability investigation indicated that after storage at 40℃and 60℃for 10 days or at 25℃for 3 months,the drug content of ClaE were all below 90%of the effective content.In addition,as the safe application for large dose of V_E is unknown and EL-40 has serious adverse effects,therefore,based on more deeply research, preparing clarithromycin-phospholipid complex was employed to prepare ClaE.The reaction solvents,the ratios and types of phospholipids were investigated,and the clarithromycin-phospholipid complex was finally prepared with clarithromycin and soybean lecithin at a ratio of 1:10 in dehydrated alcohol at 65℃for 3h,and then the reaction solvent was evaporated to obtain dry residue.DSC was also employed to identify the formation of the complex and analyze the possible interaction between clarithormyin and phospholipids.The final formulation and preparation process for ClaE using clarithromycin- phospholipid complex were as follows:as quality percentage,oil phase was composed of 0.25% clarityromycin in the form of the complex,16%MCT,4%LCT,1%soybean lecithin;the water phase was composed of 2.5%glycerol,0.2%F-68,0.2%Tween-80,0.1%sodium oleate and 0.02%L-Cysteine;and the oil phase and water phase were heated to 55℃and 80℃, respectively;the homogenization pressure and cycles were 700bar and 7 times;the pH was adjusted to 8.0.and then ClaE was sterilized in a rotating water bath at 100℃for 30min.The characteristics including PSD,ζ-potential,drug content and entrapment efficiency of ClaE using clarithromycin-phospholipid complex were 140.3±47.2nm,-20.99mV,101.1%and 91.4%,respectively.Shaking stability test showed that the particle size of ClaE had no significant variation and there were also no flocculation and coalescence.After accelerating test at 60℃for 10 days,the drug content only reduced 5.7%.In addition,the results of long term stability test at 25±2℃and 10±2℃for 6 months showed that the characteristics of ClaE were nearly unchanged,and therefore,ClaE using clarithromycin-phospholipid complex was stable enough and had the potential for industrial production and clinical application.UPLC/MS/MS method was established to determine clarithromycin concentration in rat plasma.Taking clarithromycin solution（ClaS）as reference,both ClaE using clarithromycinphospholipid complex and ClaS fitted three-compartment model and their pharmacokinetic curves were similar.The main pharmacokinetic parameters of ClaE and ClaS analyzed by statistical moment method showed no statistically differences（n=6,P＞0.05）except Vss.AUC_0-t and MRT of ClaE and ClaS were（44.96±7.222）and（38.52±28.455）mg/L*h,and（4.162±0.653） and（4.896±2.809）h,respectively.更多还原