双效麻醉药复方丙泊酚-阿芬太尼乳剂的制备及药效学研究

发布时间：2018-05-25 10:12

本文选题：丙泊酚 + 阿芬太尼　；参考：《第四军医大学》2014年博士论文

【摘要】：研究背景麻醉剂是中国古代外科成就之一，早在2000年以前，名医华佗发明的“麻沸散”成为具有最早记录的麻醉剂，成为中国古代医学的世界之最。1846年Cliver WendellHolmes首次提出“麻醉”名词，使用笑气、氯仿、乙醚等化学麻醉剂进行外科手术。麻醉药物的发明和使用开创了外科手术的新时代，对外科学发展起到了极大的推动作用。在人类医学史上，不断研发能够满足各种手术，不同人群的要求，达到满意的镇静、镇痛、肌松效果，一直是麻醉药物研发的热点和难点。但遗憾的是迄今尚无任何一种麻醉药物单独使用能够满足手术麻醉的需要，目前临床上常采用复合麻醉，即将几种麻醉药物或麻醉方法联合使用来达到理想的麻醉效果，这种复合麻醉已成为当今临床麻醉实践的主流趋势，是达到“平衡麻醉”最常用的策略。未来的麻醉应向更简捷、更易控、更安全的方向发展，探索和开发符合这一要求的麻醉药物将是研究人员不懈努力的方向。本研究基于平衡麻醉理论，设想如果能够将两种药物联合使用并制备成一种复方制剂，使其能够兼具镇痛和镇静双效作用，使麻醉全过程更为便捷。要实现这一目标，面临诸多新药研究中的关键问题，如复方制剂中各组分间是否存在相互作用？如果存在相互作用是何种类型（相加、协同、拮抗）及相互作用可能的机制是什么？药物各组分间混合后理化性质是否稳定？是否具有稳定、成熟的制剂工艺？复方麻醉药物能否固定比例并筛选最佳比例，满足临床麻醉的需要？围绕复方药物研发中的关键问题本课题完成以下研究： 1.丙泊酚和阿芬太尼相互作用研究在《固定复方制剂的临床开发指导原则》中明确指出：固定复方制剂中各组分药效的相加或协同作用常是开发复方制剂的依据和基础。因此，我们采用热板法、甩尾法、醋酸扭体法三种常用的疼痛模型，测量丙泊酚、阿芬太尼及二者联合使用时镇痛作用的ED50，采用等辐射分析方法研究丙泊酚与阿芬太尼的镇痛相互作用。结果发现，丙泊酚和阿芬太尼起效迅速，可显著缓解热板法和热甩尾法引起的急性疼痛及醋酸扭体法引起的炎性疼痛。在热板实验中，测得丙泊酚的ED50为8.82mg/kg，阿芬太尼ED50为41.44μg/kg，联合用药组ED50mix丙泊酚为2.09mg/kg，阿芬太尼为9.91μg/kg；在甩尾实验中，测得丙泊酚的ED50为7.50mg/kg，阿芬太尼ED50为40.16μg/kg，联合用药组ED50mix丙泊酚为1.88mg/kg，阿芬太尼为10.16μg/kg；在醋酸扭体实验中测得丙泊酚的ED50为15.00mg/kg，阿芬太尼ED50为12.50μg/kg，联合用药组ED50为丙泊酚3.75mg/kg，阿芬太尼3.13μg/kg。用等辐射分析法分析后，三个模型中的联合用药组ED50mix均显著小于理论相加ED50add，p 0.05；联合用药组ED50mix落在相加线的左侧，并且相互作用指数(interaction index) γ均小于1，提示二者在三种疼痛模型上均产生明显协同疼痛效应。该部分研究文章已发表在SCI收录杂志Pharmacol Biochem Behav，2014，116:25-9。 2.复方丙泊酚阿芬太尼乳剂工艺研究、理化性质及稳定性考察在处方前研究的基础上，采用高压均质法制备静脉注射用复方丙泊酚阿芬太尼乳剂。考察了磷脂加入方法、油相溶解温度、水相油相加入顺序、高压均质压力及次数对乳剂稳定性的影响，优化了乳剂中油相种类、油酸钠的用量、pH，并考察了灭菌温度及时间对乳剂的影响。从粒径、电位、pH、渗透压、乳液粘度、酸值和过氧化值几个方面考察了乳剂的理化性质。建立了丙泊酚和阿芬太尼体外高效液相色谱分析方法，通过加速试验、长期试验、光照试验和冻融试验考察了乳剂的稳定性。结果表明：本研究成功研制了复方丙泊酚阿芬太尼乳剂。确定最终处方及制备工艺为：以5%大豆油和5%的中链脂肪酸甘油三酯为油相，1.2%的蛋黄卵磷脂和0.03%的油酸钠为乳化剂，均质前调节pH至8.0，60MPa压力均质6次，充氮灌封，灭菌。以上方法制得的三批乳剂的平均粒径为221.6nm，-电位为-22.83mV，丙泊酚和阿芬太尼药物含量均为99.0%。加速试验、长期试验、光照试验和冻融试验结果显示乳剂在40±2℃、湿度75%±5%条件下放置6个月，25±2℃，湿度60±10%的条件下放置12个月，（4500±500）Lx光照条件下放置10天以及-20℃和-50℃至4℃冻融，乳剂的外观、pH、粒径、电位和药物含量等均无显著性变化。该部分研究已申请国家发明专利，专利申请号：201210069537.4。 3.成犬持续输注复方丙泊酚-阿芬太尼乳剂麻醉作用评价将研制不同剂量比的复方丙泊酚阿芬太尼乳剂应用于成犬部分脾切除手术的麻醉诱导和麻醉维持过程，通过观察比较MAP, SpO2, EtCO2, T, HR, BIS, RR及麻醉时间、苏醒时间等指标来评价不同比例的复方丙泊酚阿芬太尼乳剂的麻醉镇痛效果。结果显示各剂量组MAP, SpO2, EtCO2, T均无统计学差异，但复方丙泊酚阿芬太尼乳剂剂量比为75:1组相对于阳性对照组会引起成犬麻醉深度过深，并且会导致严重的心动过缓，术后动物苏醒时间与对照组相比明显延长。剂量比为300:1组心率加快，提示可能镇痛药物剂量存在不足，剂量比为150:1组相对于对照组没有显著性差异。本实验结果提示复方丙泊酚阿芬太尼乳剂剂量比为150:1时为最佳比例，能够较好满足手术中麻醉要求。该部分研究结果已投向SCI收录杂志Vet J，目前正在审稿中。 4.丙泊酚与阿芬太尼协同作用机制的探索采用全细胞膜片钳技术记录丙泊酚和阿芬太尼作用于小鼠ACC脑薄片的mIPSC和eIPSC电流频率和幅度的变化。免疫细胞化学和Western-blot检测细胞膜GABA受体的分布及其磷酸化的改变。激光共聚焦及流式细胞仪检测丙泊酚和阿芬太尼对神经元细胞内Ca2+离子浓度的影响；cAMP试剂盒检测丙泊酚和阿芬太尼对神经元胞内cAMP表达的影响；PLC试剂盒检测丙泊酚和阿芬太尼对神经元内PLC活性的影响。结果表明：丙泊酚和阿芬太尼可协同激活GABA受体。两者联合使用与单用丙泊酚处理组相比，显著增加了mIPSC和eIPSC的幅度(P0.05)，提示联合用药可增强丙泊酚对突触后GABA受体的作用。进一步分析发现，联合使用可促进GABA受体Aβ3在胞膜的表达及其磷酸化。丙泊酚和阿芬太尼联用能够降低细胞内cAMP的浓度，PLC的活性，进而导致Ca2+内流减少，从而对μ受体下游信号产生协同效应。该部分研究结果已投向SCI收录杂志Pain Med，目前正在审稿中。结论：综上所述，本课题研究结果证实了丙泊酚和阿芬太尼之间具有良好的协同作用并初步探讨了其协同作用机制。利用药剂学技术，首次成功研制了复方丙泊酚阿芬太尼乳剂，目前国内外尚无此类复方制剂上市；并通过随机，对照方法，对实施部分脾切除术的成犬进行药效学评价，，探讨了两者固定比例的可行性并筛选出最佳比例，结果证明固定比例且两者剂量比为150:1时能够满足手术中的麻醉要求。这些研究解决了复方制剂开发过程中的关键性问题，为进一步研发该复方制剂奠定了坚实的基础。
[Abstract]:Research background
Narcotics is one of the ancient Chinese surgical achievements. Before 2000, the famous doctor Hua Tuo invented "Ma bun powder" as the earliest anesthetic. It was the first.1846 year Cliver WendellHolmes in the world of ancient Chinese medicine to put forward the term "anaesthesia", and used chemical anesthetics such as laughing gas, chloroform, ether and other chemical anesthetics to perform surgery. The invention and use of narcotic drugs have opened up a new era of surgery and played a great role in promoting the development of foreign science.
In the history of human medicine, continuous research and development to meet the requirements of various operations, different groups of people, to achieve satisfactory sedation, analgesia, and muscle relaxation has always been a hot and difficult point in the development of narcotic drugs. But it is regrettable that there is no one kind of narcotic drugs that can be used for full operation anesthesia alone so far, and complex anesthesia is often used in clinic. Drunken, combined use of several narcotic drugs or anesthesia methods to achieve an ideal anesthetic effect. This compound anesthesia has become the mainstream trend in the clinical anesthesia practice today. It is the most commonly used strategy to achieve "balanced anesthesia". Future anaesthesia should be developed in a more simple, easier and safer direction. Exploration and development conform to this requirement. Anesthetic drugs will be the direction for researchers to make unremitting efforts.
Based on the equilibrium anesthesia theory, it is conceived that if two kinds of drugs can be combined and prepared into a compound preparation, it can have both analgesic and sedative effects and make the whole process more convenient. To achieve this goal, the key problems in the research of new drugs are faced, such as whether there is a phase between the components in the compound preparation. Interaction? What are the possible mechanisms of interaction if there is interaction (adding, synergism, antagonism) and the possible mechanisms of interaction? Is the physical and chemical properties of a mixture of drugs stable? Is there a stable, mature preparation process? Can compound narcotic drugs be fixed to the best proportion to meet the needs of clinical anesthesia? The key issues in drug research and development are as follows:
Study on the interaction of 1. propofol and oft
In the guiding principle of the clinical development of the fixed compound preparation, it is clearly pointed out that the addition or synergistic effect of each component in the fixed compound preparation is often the basis and basis for the development of compound preparation. Therefore, we use three common pain models, hot plate method, tail flick method, and acetic acid torsional body method, to measure propofol, fueneni and two combination The analgesic effect of ED50 with time analgesic effect was studied by means of equal radiation analysis. The results showed that propofol and anfentanil had rapid effects and could significantly alleviate the acute pain caused by hot plate method and hot tail flick and the inflammatory pain caused by acetic acid writhing method. In the hot plate experiment, the ED50 of propofol was measured. 8.82mg/kg, enifeni ED50 is 41.44 mu g/kg, ED50mix propofol of combined drug group is 2.09mg/kg, and enifeni is 9.91 u g/kg; in tail flick experiment, the ED50 of propofol is 7.50mg/kg, and the Ageno ED50 is 40.16 mu g/kg. The combined drug group ED50mix propofol is 1.88mg/kg, and 10.16 mu. The ED50 of propofol was 15.00mg/kg, the ED50 of the ED50 was 12.50 u g/kg, the combination group ED50 was propofol 3.75mg/kg, and the 3.13 UA g/kg. was analyzed by the equal radiation analysis. The ED50mix in the three models was significantly smaller than the theory plus ED50add, P 0.05; the combination group ED50mix fell on the left side of the addition line and phase The interaction index (interaction index) gamma is less than 1, suggesting that the two people have obvious synergistic pain effects on the three pain models. This part of the study has been published in the SCI magazine Pharmacol Biochem Behav, 2014116:25-9.
Study on the physicochemical properties and stability of 2. compound propofol and its emulsion
On the basis of the study before the prescription, the compound propofol compound propofol was prepared by high pressure homogenization. The effect of phospholipid addition method, oil phase dissolving temperature, water phase oil phase, high pressure homogenization pressure and number of times on emulsion stability were investigated, and the oil phase, sodium oleate dosage, pH were optimized. The effects of sterilization temperature and time on emulsion were investigated. The physicochemical properties of emulsion were investigated from particle size, potential, pH, osmotic pressure, viscosity of emulsion, acid value and peroxide value. The method of high performance liquid chromatographic analysis of propofol and ain was established, and the stability of emulsion was investigated by accelerated test, long period test, illumination test and freezing thawing test. The results showed that the compound propofol acyfant emulsion was successfully developed in this study. The final formulation and preparation process were as follows: 5% soybean oil and 5% medium chain fatty acid triglyceride as oil phase, 1.2% egg yolk lecithin and 0.03% sodium oleate as emulsifier, the homogenization of pH to 8.0,60MPa pressure homogenization 6 times, nitrogen filling and sterilization before homogenization. The average particle size of the three batch of three batch emulsion obtained by the above method is -22.83mV, the content of propofol and ain is 99.0%. acceleration test. Long term test, light and freeze thawing test results show that the emulsion is placed at 40 + 2 C, humidity 75% + 5%, 25 + 2 degrees, humidity 60 + 10% for 12 months (4500). There were no significant changes in the appearance, appearance, pH, particle size, potential and drug content of the emulsion for 10 days and the freezing thawing at -20 and -50 and 4 C under Lx light conditions. This part of the study has applied for the national invention patent. The patent application number: 201210069537.4.
Evaluation of anesthetic effect of continuous infusion of compound propofol and anaesthetized emulsion in 3. adult dogs
The anesthetic induction and anesthesia maintenance process of compound propofol, a compound propofol with different dose ratio, were used to evaluate the anesthetic induction and anesthesia maintenance process of partial splenectomy in dogs. The anesthetic and analgesic effects of MAP, SpO2, EtCO2, T, HR, BIS, RR and the time of anesthesia, the time of anesthesia, and the awakening time were evaluated and compared. The results showed that there was no statistical difference in MAP, SpO2, EtCO2, and T in each dose group, but the dose ratio of Fufang propofol and anfutaeni, compared with the positive control group, would cause a deep depth of anaesthesia in the dog and cause a serious bradycardia. The recovery time of the animals after the operation was significantly longer than that of the control group. The dose ratio of the group of 300:1 to the control group was significantly longer than that of the control group. The results suggest that the dose ratio of the 150:1 group has no significant difference compared with the control group. The results of this experiment suggest that the dose ratio of the compound propofol of compound propofol is the best ratio when the dose ratio is 150:1, and it can meet the anesthesia requirements in the operation. The results have been put to the SCI magazine Vet J. In the manuscript.
4. the synergistic mechanism of propofol and offee
Total cell patch clamp technique was used to record the changes in the frequency and amplitude of mIPSC and eIPSC in ACC brain slices of mice. Immunocytochemistry and Western-blot were used to detect the distribution of GABA receptor in cell membrane and the changes of phosphorylation. Laser confocal and flow cytometry was used to detect propofol and the treatment of the nerve of propofol. The effect of Ca2+ ion concentration in the cells; the effect of propofol and anfup on the intracellular cAMP expression in neurons by cAMP kit; the effect of PLC kit on PLC activity in neurons by propofol and Anfen. The results showed that propofol and alfannicol were co activated by GABA receptor. Both combined use and single use Propol Compared with the phenol treatment group, the amplitude of mIPSC and eIPSC increased significantly (P0.05), suggesting that the combined use of propofol can enhance the effect of propofol on postsynaptic GABA receptor. Further analysis shows that combined use of GABA receptor A beta 3 can promote the expression and phosphorylation of A beta in the cell membrane. The combined use of propofol and amitnei can reduce the concentration of intracellular cAMP and the activity of PLC. Sex, which leads to a decrease in the Ca2+ influx, has a synergistic effect on the downstream signal of the micron receptor. This part of the study has been directed to the SCI magazine Pain Med, which is currently under review.
Conclusion: in summary, the results of this study confirm that the synergism between propofol and arfuabortion is good and the mechanism of synergism is preliminarily discussed. In the first time, the compound propofol compound of propofol is successfully developed by the use of pharmaceutical technology. At present, there is no such compound preparation at home and abroad. The method is to evaluate the pharmacodynamics of the adult dog with partial splenectomy, and discuss the feasibility of the fixed proportion and select the best proportion. The results show that the fixed proportion and the dose ratio of 150:1 can meet the anesthesia requirements during the operation. These studies have solved the key problems in the development process of compound preparation, and further research on the key problems in the development of the compound preparation. The preparation of the compound preparation laid a solid foundation.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R943

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