莪术醇静脉注射亚微乳剂的研究
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摘要
莪术醇是从莪术油中分离得到的单体化合物,是莪术油的主要活性成分。文献报道莪术油具有抗菌、抗病毒、抗肿瘤、抗早孕、抗突变、保肝、活血化瘀等药理作用,但由于其成分复杂,性质不稳定,质量可控性差,且处方中含有高浓度的吐温-80作增溶剂,临床应用过程中易引起不同程度的不良反应。作为莪术油的主要活性成分和质量控制指标,近年来,莪术醇药理活性及作用机理的研究受到广泛关注,尤其是对抗菌、抗病毒和抗肿瘤活性的探讨。但莪术醇几乎不溶于水,且在酸、碱及高温条件下易发生降解,传统的助溶剂既存在安全性隐患,又不能满足注射剂对药物浓度的要求。理化性质的缺陷严重限制了莪术醇的进一步研究与发展。为此,本课题对“莪术醇静脉注射亚微乳”进行立项研究。
课题建立了气相色谱法用于莪术醇原料及其亚微乳剂的含量及有关物质测定,并进行了系统方法学验证。通过对莪术醇原料药在不同溶剂系统中的溶解度和稳定性等理化性质的考察,为新型释药系统的处方设计提供依据。利用莪术醇亲脂性较强的特点,将其溶于精制植物油中,以大豆卵磷脂(S75)为乳化剂,泊洛沙姆(F68)为助乳化剂,高压均质法制备水包油亚微乳剂。采用单因素和正交试验方法,系统考察了油相组成及用量、乳化剂种类及用量、辅助乳化剂和制备工艺参数对乳剂灭菌前后的外观、粒径、离心分层和含量变化的影响,成功制备了粒径分布均匀、平均粒径小于150nm、zeta电位小于-25mv、耐热压灭菌(115℃、30min)的亚微乳。载药量为1mg/mL～6mg/mL的亚微乳剂经室温和4℃放置9个月,以及30℃和40℃加速实验6个月,外观、粒径、pH值、含量和有关物质均未发生明显变化,质量稳定。
以布格呋喃为内标,建立了气相色谱法用于莪术醇注射剂体内血药浓度和组织浓度的测定。在选定的色谱条件下,内标物与原形药莪术醇均达基线分离,空白血浆,心、肝、脾、肺、肾等空白组织样品中内源性物质及代谢产物对测定无干扰,血浆和各组织的方法学均符合生物样品测定的要求。以聚氧乙烯蓖麻油增溶的莪术醇注射液为参比制剂,考察了莪术醇亚微乳在大鼠体内的药时曲线及小鼠体内的组织分布。药时曲线结果表明,两种制剂的AUC无明显差异,但亚微乳剂表观分布容积显著高于参比注射液,且体内消除速率略快,提示亚微乳载体注射后可快速向周边组织转运,具备微粒制剂的一般特性。此外,在4h内亚微乳平均滞留时间MRT较参比注射液明显缩短,而长时间内MRT则明显延长。组织分布结果显示,与参比注射液相比,莪术醇亚微乳剂在心和肾中的分布减少,肝和脾中的分布无明显变化,而肺组织中的药物浓度明显升高。
特殊安全性考察结果表明,莪术醇亚微乳剂在临床使用浓度下无溶血现象发生;兔耳缘静脉注射亚微乳剂后,注射部位均未见红肿、充血、硬结等血管刺激现象;豚鼠按20mg/kg剂量隔日腹腔注射莪术醇亚微乳剂致敏,连续3次,末次给药后第10日静脉注射40mg/kg激发,动物出现轻微的躁动、步态不稳现象,但5min内即恢复,表现为弱阳性过敏反应,与之相比,参比注射液给药后豚鼠立即出现步态不稳、跳跃、抽搐、萎靡等症状,表现为强阳性过敏反应。
体外抗菌试验表明,莪术醇对26种菌株的体外最低抑菌浓度(MIC)均高于6.72mg/mL,无明显抗菌活性。体外抗病毒试验表明,莪术醇在体外细胞培养中对流感甲型病毒具有一定的抑制作用,IC_(50)为243.71μg/mL,SI为11.84;对柯萨奇病毒B3有明显的抑制作用,IC_(50)为78.79μg/mL,SI为12.2。小鼠体内抗病毒试验结果显示,莪术醇亚微乳剂在10mg/kg/d～40mg/kg/d剂量范围内,能够显著改善流感病毒FM_1致肺炎小鼠的肺脂数,其中高剂量组还可显著改善脾脂数,提示本品可提高小鼠机体免疫能力,对小鼠病毒性肺炎具有较强的治疗作用。体内抗肿瘤试验表明,小鼠尾静脉注射莪术醇亚微乳剂,在30mg/kg/d～120mg/kg/d剂量范围内未观察到抑瘤(U14)作用。
本课题制备的莪术醇亚微乳剂,有效提高了制剂中的药物载药量,处方中不含有机溶剂、吐温-80或聚氧乙烯蓖麻油等增溶剂,降低了注射给药的过敏反应及毒副作用,改善了药物组织分布,使心、肾药物浓度下降,而肺组织药物水平提高。莪术醇亚微乳剂质量稳定,可满足注射给药的要求,有望发展成为病毒性肺炎或病毒性肠炎的治疗药物。
Curcumol is one of the monomer compounds separated from Zedoary turmeric oil, which has pharmacologic actions of anti-bacteria, anti-virus, anti-tumor, anti-mutation, protecting liver and so on. On account of its complex component, poor stability and quality control, and the high concentration of tween 80 in the formulation, Zedoary turmeric oil was found to have some adverse reactions in clinical application. As the main active ingredient and quality control index of Zedoary turmeric oil, curcumol has been paid wide attention to the research of its pharmacological activity and mechanism, especially on anti-bacteria, anti-virus and anti-tumor. Poor water-solubility and instability of curcumol restricts further research and development of curcumol seriously. The research project "Curcumol Submicroemulsion for Intravenous Injection" was established to develop the drug delivery system without conventional cosolvent.
A GC method was established by systematic method verification to determine the content and related substances of Curcumol and its submicroemulsion. The solubility and stability of curcumol bulk material in different solvent system were inspected and evaluated to provide references and basis for the formulation design of new drug delivery system. Curcumol with higher lipophilicity was dissolved in refined vegetable oils, then the lecithin (S75) and poloxamer (F68) were taken as emulsifiers, and then the mixture was prepared into a O/W submicroemulsion by high pressure homogenizer. Taking appearance, mean particle size and size distribution, demixing or not after centrifugalization and content changes of emulsion before and after the sterilization as index, the single factor test and orthogonal test were designed to evaluate and investigate the influence of the oil, emulsifier, preparation process parameters etc.on the stabilization of emulsion. Curcumol submicroemulsion was prepared successfully and its mean particle size is less than 150nm with a normal size distribution, its zeta potential less than -25mV, and it can be sterilized by autoclave(115℃,30min). The results of determination of the appearance, the particle size, pH, the content and related substances indicated that the emulsion with drug loading of 1mg/mL～6mg/mL were stable at 4℃and 25℃for 9 months and at the acceleration condition of 30℃and 40℃for 6 months.
Taking buagafuran as internal standard, a GC method was developed for the determination of curcumol in rat plasma and mice tissue. Under the assay condition, the internal standard and curcumol are for good separation; the endogenous substance and metabolites from blank plasma and tissues including heart, liver, spleen, lung and kidney is non-interference to the determination. Using curcumol injection prepared with Cremophor EL as reference preparation, the concentration-time curve in rat and tissue distribution in mice were investigated after intravenous administration of curcumol submicroemulsion. The pharmacokinetics results demonstrated that the AUC was no significant differences on the two Curcumol preparations. But the apparent distribution volume of emulsion was evidently higher and its elimination rate was slightly faster than those of the injection. It clued to that submicroemulsion after injecting administrated can be transferred quickly to peripheral tissues which meet general characteristics of particulate preparation. Moreover, the mean residence time (MRT) of emulsion was obviously shortened in 4h and prolonged in long time than injection. The result of tissue distribution study in mice showed that compared with the curcumol injection,the drug distribution of curcumol submicroemulsion was reduced in heart and kidney, and no obvious changes in liver and spleen, while in lung the drug concentration was increased significantly.
The results of special safety tests indicated that there was no hemolyzation and agglomeration on rabbit erythrocyte in vitro with the clinical using concentration; no vascular stimulation such as turgescence, congestion, sclerosis etc. Curcumol submicroemulsion were injected to guinea pigs at the dose of 20mg/kg by i.p. q.o.d×3, and only slight allergic reaction on guinea pig were observed after i.v. administration stimulation of curcumol submicroemulsion at the dose of 40mg/kg in the 10th day at the end of i.p. administration, and all of the symptoms disappeared in 5min. Compared with curcumol submicroemulsion, severe hypersensitive reaction with the symptoms such as unsteady gait, jumping, twitch and cachexia occurred immediately after administrated curcumol injection in the same way.
Curcumol anti-bacterial experiments in vitro showed that the value of MIC to 26 strains were more than 6.72mg/mL, that is, there is no obvious anti-bacterial effect. The external anti-virus effect of curcumol by using cell infection model were judged by means of the observation to the cell changes. It was showed that curcumol was able to correspondingly restrain the influenza virus A, with IC_(50) 243.71μg/mL, SI 11.84; and was markedly against the CVB3, with IC_(50) 78.79μg/mL, SI 12.2. The anti-virus effect of curcumol in mice indicated that pulmonary-index was decreased significantly after intravenous administration of curcumol submicroemulsion at the dose of 10mg/kg/d (P<0.05)～40mg/kg/d (P<0.01), and splenic-index was increased obviously at the dose of 40mg/kg/d (P<0.01). It comes to the conclusion that curcumol submicroemulsion can improve the immunity and has better therapeutic effect to viral pneumonia in mice caused by influenza virus. The effect of anti-tumor(uterine cervical carcinoma,U14) in mice vivo was not observed after intravenous administration of curcumol submicroemulsion at the dose of 30mg/kg/d～120mg/kg/d.
There are no organic solvents, tween 80 or polyoxyethylene castor oil etc.as cosolvent in the formulation of Curcumol submicroemulsion. It can efficiently improve the drug loading, decrease allergic reaction and adverse reaction, meliorate drug tissue distribution to make the drug concentration decreased in heart and kidney, while increased in lung. Curcumol submicroemulsion has good quality stability, so it is fitted to be used for injecting administration. Curcumol submicroemulsion is expected to develop into a therapeutic drug on viral pneumonia or viral enteritis in the near future.
课题建立了气相色谱法用于莪术醇原料及其亚微乳剂的含量及有关物质测定,并进行了系统方法学验证。通过对莪术醇原料药在不同溶剂系统中的溶解度和稳定性等理化性质的考察,为新型释药系统的处方设计提供依据。利用莪术醇亲脂性较强的特点,将其溶于精制植物油中,以大豆卵磷脂(S75)为乳化剂,泊洛沙姆(F68)为助乳化剂,高压均质法制备水包油亚微乳剂。采用单因素和正交试验方法,系统考察了油相组成及用量、乳化剂种类及用量、辅助乳化剂和制备工艺参数对乳剂灭菌前后的外观、粒径、离心分层和含量变化的影响,成功制备了粒径分布均匀、平均粒径小于150nm、zeta电位小于-25mv、耐热压灭菌(115℃、30min)的亚微乳。载药量为1mg/mL～6mg/mL的亚微乳剂经室温和4℃放置9个月,以及30℃和40℃加速实验6个月,外观、粒径、pH值、含量和有关物质均未发生明显变化,质量稳定。
以布格呋喃为内标,建立了气相色谱法用于莪术醇注射剂体内血药浓度和组织浓度的测定。在选定的色谱条件下,内标物与原形药莪术醇均达基线分离,空白血浆,心、肝、脾、肺、肾等空白组织样品中内源性物质及代谢产物对测定无干扰,血浆和各组织的方法学均符合生物样品测定的要求。以聚氧乙烯蓖麻油增溶的莪术醇注射液为参比制剂,考察了莪术醇亚微乳在大鼠体内的药时曲线及小鼠体内的组织分布。药时曲线结果表明,两种制剂的AUC无明显差异,但亚微乳剂表观分布容积显著高于参比注射液,且体内消除速率略快,提示亚微乳载体注射后可快速向周边组织转运,具备微粒制剂的一般特性。此外,在4h内亚微乳平均滞留时间MRT较参比注射液明显缩短,而长时间内MRT则明显延长。组织分布结果显示,与参比注射液相比,莪术醇亚微乳剂在心和肾中的分布减少,肝和脾中的分布无明显变化,而肺组织中的药物浓度明显升高。
特殊安全性考察结果表明,莪术醇亚微乳剂在临床使用浓度下无溶血现象发生;兔耳缘静脉注射亚微乳剂后,注射部位均未见红肿、充血、硬结等血管刺激现象;豚鼠按20mg/kg剂量隔日腹腔注射莪术醇亚微乳剂致敏,连续3次,末次给药后第10日静脉注射40mg/kg激发,动物出现轻微的躁动、步态不稳现象,但5min内即恢复,表现为弱阳性过敏反应,与之相比,参比注射液给药后豚鼠立即出现步态不稳、跳跃、抽搐、萎靡等症状,表现为强阳性过敏反应。
体外抗菌试验表明,莪术醇对26种菌株的体外最低抑菌浓度(MIC)均高于6.72mg/mL,无明显抗菌活性。体外抗病毒试验表明,莪术醇在体外细胞培养中对流感甲型病毒具有一定的抑制作用,IC_(50)为243.71μg/mL,SI为11.84;对柯萨奇病毒B3有明显的抑制作用,IC_(50)为78.79μg/mL,SI为12.2。小鼠体内抗病毒试验结果显示,莪术醇亚微乳剂在10mg/kg/d～40mg/kg/d剂量范围内,能够显著改善流感病毒FM_1致肺炎小鼠的肺脂数,其中高剂量组还可显著改善脾脂数,提示本品可提高小鼠机体免疫能力,对小鼠病毒性肺炎具有较强的治疗作用。体内抗肿瘤试验表明,小鼠尾静脉注射莪术醇亚微乳剂,在30mg/kg/d～120mg/kg/d剂量范围内未观察到抑瘤(U14)作用。
本课题制备的莪术醇亚微乳剂,有效提高了制剂中的药物载药量,处方中不含有机溶剂、吐温-80或聚氧乙烯蓖麻油等增溶剂,降低了注射给药的过敏反应及毒副作用,改善了药物组织分布,使心、肾药物浓度下降,而肺组织药物水平提高。莪术醇亚微乳剂质量稳定,可满足注射给药的要求,有望发展成为病毒性肺炎或病毒性肠炎的治疗药物。
Curcumol is one of the monomer compounds separated from Zedoary turmeric oil, which has pharmacologic actions of anti-bacteria, anti-virus, anti-tumor, anti-mutation, protecting liver and so on. On account of its complex component, poor stability and quality control, and the high concentration of tween 80 in the formulation, Zedoary turmeric oil was found to have some adverse reactions in clinical application. As the main active ingredient and quality control index of Zedoary turmeric oil, curcumol has been paid wide attention to the research of its pharmacological activity and mechanism, especially on anti-bacteria, anti-virus and anti-tumor. Poor water-solubility and instability of curcumol restricts further research and development of curcumol seriously. The research project "Curcumol Submicroemulsion for Intravenous Injection" was established to develop the drug delivery system without conventional cosolvent.
A GC method was established by systematic method verification to determine the content and related substances of Curcumol and its submicroemulsion. The solubility and stability of curcumol bulk material in different solvent system were inspected and evaluated to provide references and basis for the formulation design of new drug delivery system. Curcumol with higher lipophilicity was dissolved in refined vegetable oils, then the lecithin (S75) and poloxamer (F68) were taken as emulsifiers, and then the mixture was prepared into a O/W submicroemulsion by high pressure homogenizer. Taking appearance, mean particle size and size distribution, demixing or not after centrifugalization and content changes of emulsion before and after the sterilization as index, the single factor test and orthogonal test were designed to evaluate and investigate the influence of the oil, emulsifier, preparation process parameters etc.on the stabilization of emulsion. Curcumol submicroemulsion was prepared successfully and its mean particle size is less than 150nm with a normal size distribution, its zeta potential less than -25mV, and it can be sterilized by autoclave(115℃,30min). The results of determination of the appearance, the particle size, pH, the content and related substances indicated that the emulsion with drug loading of 1mg/mL～6mg/mL were stable at 4℃and 25℃for 9 months and at the acceleration condition of 30℃and 40℃for 6 months.
Taking buagafuran as internal standard, a GC method was developed for the determination of curcumol in rat plasma and mice tissue. Under the assay condition, the internal standard and curcumol are for good separation; the endogenous substance and metabolites from blank plasma and tissues including heart, liver, spleen, lung and kidney is non-interference to the determination. Using curcumol injection prepared with Cremophor EL as reference preparation, the concentration-time curve in rat and tissue distribution in mice were investigated after intravenous administration of curcumol submicroemulsion. The pharmacokinetics results demonstrated that the AUC was no significant differences on the two Curcumol preparations. But the apparent distribution volume of emulsion was evidently higher and its elimination rate was slightly faster than those of the injection. It clued to that submicroemulsion after injecting administrated can be transferred quickly to peripheral tissues which meet general characteristics of particulate preparation. Moreover, the mean residence time (MRT) of emulsion was obviously shortened in 4h and prolonged in long time than injection. The result of tissue distribution study in mice showed that compared with the curcumol injection,the drug distribution of curcumol submicroemulsion was reduced in heart and kidney, and no obvious changes in liver and spleen, while in lung the drug concentration was increased significantly.
The results of special safety tests indicated that there was no hemolyzation and agglomeration on rabbit erythrocyte in vitro with the clinical using concentration; no vascular stimulation such as turgescence, congestion, sclerosis etc. Curcumol submicroemulsion were injected to guinea pigs at the dose of 20mg/kg by i.p. q.o.d×3, and only slight allergic reaction on guinea pig were observed after i.v. administration stimulation of curcumol submicroemulsion at the dose of 40mg/kg in the 10th day at the end of i.p. administration, and all of the symptoms disappeared in 5min. Compared with curcumol submicroemulsion, severe hypersensitive reaction with the symptoms such as unsteady gait, jumping, twitch and cachexia occurred immediately after administrated curcumol injection in the same way.
Curcumol anti-bacterial experiments in vitro showed that the value of MIC to 26 strains were more than 6.72mg/mL, that is, there is no obvious anti-bacterial effect. The external anti-virus effect of curcumol by using cell infection model were judged by means of the observation to the cell changes. It was showed that curcumol was able to correspondingly restrain the influenza virus A, with IC_(50) 243.71μg/mL, SI 11.84; and was markedly against the CVB3, with IC_(50) 78.79μg/mL, SI 12.2. The anti-virus effect of curcumol in mice indicated that pulmonary-index was decreased significantly after intravenous administration of curcumol submicroemulsion at the dose of 10mg/kg/d (P<0.05)～40mg/kg/d (P<0.01), and splenic-index was increased obviously at the dose of 40mg/kg/d (P<0.01). It comes to the conclusion that curcumol submicroemulsion can improve the immunity and has better therapeutic effect to viral pneumonia in mice caused by influenza virus. The effect of anti-tumor(uterine cervical carcinoma,U14) in mice vivo was not observed after intravenous administration of curcumol submicroemulsion at the dose of 30mg/kg/d～120mg/kg/d.
There are no organic solvents, tween 80 or polyoxyethylene castor oil etc.as cosolvent in the formulation of Curcumol submicroemulsion. It can efficiently improve the drug loading, decrease allergic reaction and adverse reaction, meliorate drug tissue distribution to make the drug concentration decreased in heart and kidney, while increased in lung. Curcumol submicroemulsion has good quality stability, so it is fitted to be used for injecting administration. Curcumol submicroemulsion is expected to develop into a therapeutic drug on viral pneumonia or viral enteritis in the near future.
引文
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