本文選題：丙泊酚 + 阿芬太尼��； 參考：《第四軍醫(yī)大學(xué)》2014年博士論文

【摘要】：研究背景 麻醉劑是中國古代外科成就之一，早在2000年以前，名醫(yī)華佗發(fā)明的“麻沸散”成為具有最早記錄的麻醉劑，成為中國古代醫(yī)學(xué)的世界之最。1846年Cliver WendellHolmes首次提出“麻醉”名詞，使用笑氣、氯仿、乙醚等化學(xué)麻醉劑進行外科手術(shù)。麻醉藥物的發(fā)明和使用開創(chuàng)了外科手術(shù)的新時代，對外科學(xué)發(fā)展起到了極大的推動作用。 在人類醫(yī)學(xué)史上，不斷研發(fā)能夠滿足各種手術(shù)，不同人群的要求，達到滿意的鎮(zhèn)靜、鎮(zhèn)痛、肌松效果，一直是麻醉藥物研發(fā)的熱點和難點。但遺憾的是迄今尚無任何一種麻醉藥物單獨使用能夠滿足手術(shù)麻醉的需要，目前臨床上常采用復(fù)合麻醉，即將幾種麻醉藥物或麻醉方法聯(lián)合使用來達到理想的麻醉效果，這種復(fù)合麻醉已成為當今臨床麻醉實踐的主流趨勢，是達到“平衡麻醉”最常用的策略。未來的麻醉應(yīng)向更簡捷、更易控、更安全的方向發(fā)展，探索和開發(fā)符合這一要求的麻醉藥物將是研究人員不懈努力的方向。 本研究基于平衡麻醉理論，設(shè)想如果能夠?qū)煞N藥物聯(lián)合使用并制備成一種復(fù)方制劑，使其能夠兼具鎮(zhèn)痛和鎮(zhèn)靜雙效作用，使麻醉全過程更為便捷。要實現(xiàn)這一目標，面臨諸多新藥研究中的關(guān)鍵問題，如復(fù)方制劑中各組分間是否存在相互作用？如果存在相互作用是何種類型（相加、協(xié)同、拮抗）及相互作用可能的機制是什么？藥物各組分間混合后理化性質(zhì)是否穩(wěn)定？是否具有穩(wěn)定、成熟的制劑工藝？復(fù)方麻醉藥物能否固定比例并篩選最佳比例，滿足臨床麻醉的需要？圍繞復(fù)方藥物研發(fā)中的關(guān)鍵問題本課題完成以下研究： 1.丙泊酚和阿芬太尼相互作用研究 在《固定復(fù)方制劑的臨床開發(fā)指導(dǎo)原則》中明確指出：固定復(fù)方制劑中各組分藥效的相加或協(xié)同作用常是開發(fā)復(fù)方制劑的依據(jù)和基礎(chǔ)。因此，我們采用熱板法、甩尾法、醋酸扭體法三種常用的疼痛模型，測量丙泊酚、阿芬太尼及二者聯(lián)合使用時鎮(zhèn)痛作用的ED50，采用等輻射分析方法研究丙泊酚與阿芬太尼的鎮(zhèn)痛相互作用。結(jié)果發(fā)現(xiàn)，丙泊酚和阿芬太尼起效迅速，可顯著緩解熱板法和熱甩尾法引起的急性疼痛及醋酸扭體法引起的炎性疼痛。在熱板實驗中，測得丙泊酚的ED50為8.82mg/kg，阿芬太尼ED50為41.44μg/kg，聯(lián)合用藥組ED50mix丙泊酚為2.09mg/kg，阿芬太尼為9.91μg/kg；在甩尾實驗中，測得丙泊酚的ED50為7.50mg/kg，阿芬太尼ED50為40.16μg/kg，聯(lián)合用藥組ED50mix丙泊酚為1.88mg/kg，阿芬太尼為10.16μg/kg；在醋酸扭體實驗中測得丙泊酚的ED50為15.00mg/kg，阿芬太尼ED50為12.50μg/kg，聯(lián)合用藥組ED50為丙泊酚3.75mg/kg，阿芬太尼3.13μg/kg。用等輻射分析法分析后，三個模型中的聯(lián)合用藥組ED50mix均顯著小于理論相加ED50add，p 0.05；聯(lián)合用藥組ED50mix落在相加線的左側(cè)，并且相互作用指數(shù)(interaction index) γ均小于1，提示二者在三種疼痛模型上均產(chǎn)生明顯協(xié)同疼痛效應(yīng)。該部分研究文章已發(fā)表在SCI收錄雜志Pharmacol Biochem Behav，2014，116:25-9。 2.復(fù)方丙泊酚阿芬太尼乳劑工藝研究、理化性質(zhì)及穩(wěn)定性考察 在處方前研究的基礎(chǔ)上，采用高壓均質(zhì)法制備靜脈注射用復(fù)方丙泊酚阿芬太尼乳劑。考察了磷脂加入方法、油相溶解溫度、水相油相加入順序、高壓均質(zhì)壓力及次數(shù)對乳劑穩(wěn)定性的影響，優(yōu)化了乳劑中油相種類、油酸鈉的用量、pH，并考察了滅菌溫度及時間對乳劑的影響。從粒徑、電位、pH、滲透壓、乳液粘度、酸值和過氧化值幾個方面考察了乳劑的理化性質(zhì)。建立了丙泊酚和阿芬太尼體外高效液相色譜分析方法，通過加速試驗、長期試驗、光照試驗和凍融試驗考察了乳劑的穩(wěn)定性。結(jié)果表明：本研究成功研制了復(fù)方丙泊酚阿芬太尼乳劑。確定最終處方及制備工藝為：以5%大豆油和5%的中鏈脂肪酸甘油三酯為油相，1.2%的蛋黃卵磷脂和0.03%的油酸鈉為乳化劑，均質(zhì)前調(diào)節(jié)pH至8.0，60MPa壓力均質(zhì)6次，充氮灌封，滅菌。以上方法制得的三批乳劑的平均粒徑為221.6nm，-電位為-22.83mV，丙泊酚和阿芬太尼藥物含量均為99.0%。加速試驗、長期試驗、光照試驗和凍融試驗結(jié)果顯示乳劑在40±2℃、濕度75%±5%條件下放置6個月，25±2℃，濕度60±10%的條件下放置12個月，（4500±500）Lx光照條件下放置10天以及-20℃和-50℃至4℃凍融，乳劑的外觀、pH、粒徑、電位和藥物含量等均無顯著性變化。該部分研究已申請國家發(fā)明專利，專利申請?zhí)枺?01210069537.4。 3.成犬持續(xù)輸注復(fù)方丙泊酚-阿芬太尼乳劑麻醉作用評價 將研制不同劑量比的復(fù)方丙泊酚阿芬太尼乳劑應(yīng)用于成犬部分脾切除手術(shù)的麻醉誘導(dǎo)和麻醉維持過程，通過觀察比較MAP, SpO2, EtCO2, T, HR, BIS, RR及麻醉時間、蘇醒時間等指標來評價不同比例的復(fù)方丙泊酚阿芬太尼乳劑的麻醉鎮(zhèn)痛效果。結(jié)果顯示各劑量組MAP, SpO2, EtCO2, T均無統(tǒng)計學(xué)差異，但復(fù)方丙泊酚阿芬太尼乳劑劑量比為75:1組相對于陽性對照組會引起成犬麻醉深度過深，并且會導(dǎo)致嚴重的心動過緩，術(shù)后動物蘇醒時間與對照組相比明顯延長。劑量比為300:1組心率加快，提示可能鎮(zhèn)痛藥物劑量存在不足，劑量比為150:1組相對于對照組沒有顯著性差異。本實驗結(jié)果提示復(fù)方丙泊酚阿芬太尼乳劑劑量比為150:1時為最佳比例，能夠較好滿足手術(shù)中麻醉要求。該部分研究結(jié)果已投向SCI收錄雜志Vet J，目前正在審稿中。 4.丙泊酚與阿芬太尼協(xié)同作用機制的探索 采用全細胞膜片鉗技術(shù)記錄丙泊酚和阿芬太尼作用于小鼠ACC腦薄片的mIPSC和eIPSC電流頻率和幅度的變化。免疫細胞化學(xué)和Western-blot檢測細胞膜GABA受體的分布及其磷酸化的改變。激光共聚焦及流式細胞儀檢測丙泊酚和阿芬太尼對神經(jīng)元細胞內(nèi)Ca2+離子濃度的影響；cAMP試劑盒檢測丙泊酚和阿芬太尼對神經(jīng)元胞內(nèi)cAMP表達的影響；PLC試劑盒檢測丙泊酚和阿芬太尼對神經(jīng)元內(nèi)PLC活性的影響。結(jié)果表明：丙泊酚和阿芬太尼可協(xié)同激活GABA受體。兩者聯(lián)合使用與單用丙泊酚處理組相比，顯著增加了mIPSC和eIPSC的幅度(P0.05)，提示聯(lián)合用藥可增強丙泊酚對突觸后GABA受體的作用。進一步分析發(fā)現(xiàn)，聯(lián)合使用可促進GABA受體Aβ3在胞膜的表達及其磷酸化。丙泊酚和阿芬太尼聯(lián)用能夠降低細胞內(nèi)cAMP的濃度，PLC的活性，進而導(dǎo)致Ca2+內(nèi)流減少，從而對μ受體下游信號產(chǎn)生協(xié)同效應(yīng)。該部分研究結(jié)果已投向SCI收錄雜志Pain Med，目前正在審稿中。 結(jié)論：綜上所述，本課題研究結(jié)果證實了丙泊酚和阿芬太尼之間具有良好的協(xié)同作用并初步探討了其協(xié)同作用機制。利用藥劑學(xué)技術(shù)，首次成功研制了復(fù)方丙泊酚阿芬太尼乳劑，目前國內(nèi)外尚無此類復(fù)方制劑上市；并通過隨機，對照方法，對實施部分脾切除術(shù)的成犬進行藥效學(xué)評價，，探討了兩者固定比例的可行性并篩選出最佳比例，結(jié)果證明固定比例且兩者劑量比為150:1時能夠滿足手術(shù)中的麻醉要求。這些研究解決了復(fù)方制劑開發(fā)過程中的關(guān)鍵性問題，為進一步研發(fā)該復(fù)方制劑奠定了堅實的基礎(chǔ)。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R943

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