【作者】 李培德；

【导师】 侯加法；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2011， 博士

【摘要】 静脉全身麻醉是指将一种或几种药物经静脉注入,通过血液循环作用于中枢神经系统而产生全身麻醉的方法,由于目前尚无任何一种静脉全麻药能够单一满足手术的需要,因此临床上的静脉全麻往往是将多种药理性质各不相同的药物,先后或同时使用,以实现迅速完成诱导,麻醉过程平稳,无污染,苏醒快等目的,为外科手术创造良好的条件,确保动物的安全和术后顺利康复。氯胺酮、赛拉嗪和咪达唑仑是兽医临床麻醉常用的三种药物,可用于小动物的麻醉前用药、化学保定、诱导和维持麻醉,但各药单用时,往往副作用较大,难以使动物进入理想的麻醉状态。本研究旨在通过合理配伍三种药物,组成复方麻醉剂,经正交试验优化其配比,获得最佳处方,并研究其急性毒性、局部刺激性、稳定性、药物代谢动力学过程、临床麻醉效果和药物对免疫功能的影响,为临床上合理应用本药提供数据和参考。试验共分为以下六个部分：试验Ⅰ舒眠宁注射液处方的研究氯胺酮、赛拉嗪和咪达唑仑是三种广泛应用于小动物临床的麻醉药,本试验将三者配伍使用,试图获得更好的麻醉效果,最低的副作用。在预实验基础上,以小白鼠为试验动物,以氯胺酮、赛拉嗪和咪达唑仑的含量为因素,每个因素设置三个水平,以注射复方药物后小鼠翻正反射消失数为考察指标,选用L9(34)正交表安排试验,优化复方麻醉剂的处方,并制定生产工艺。结果：复方中三种主要成份的含量为氯胺酮(8 g·100mL-1)、赛拉嗪(0.9 g·100mL-1)和咪达唑仑(0.2g·100mL-1),定名为舒眠宁注射液,处方工艺简单,适合工厂化大批量生产。试验Ⅱ舒眠宁注射液的安全性和稳定性试验为评价舒眠宁注射液的安全性,本试验以小鼠为试验动物,进行了急性毒性试验,经序贯法测定出其ED50和LD50分别为0.706 mL·kg-1体重和2.366 mL·kg-1体重,治疗指数为3.35；在家兔进行了局部刺激性试验,结果表明本药无眼刺激性,对肌肉具有非常轻度刺激性,对静脉具有轻度刺激性,可安全、方便地应用于临床；为确定本药的有效期,以市售包装的三批舒眠宁注射液,进行长期试验,结果表明在常温、避光条件下放置两年后,本品药液澄清、无杂质产生,主药的标示量变化不显著,有效期可初步确定为2年。试验Ⅲ舒眠宁在犬体内的药代动力学研究为研究舒眠宁主要成份(氯胺酮、赛拉嗪和咪达唑仑)在犬体内的药代动力学过程。本试验建立了一种可同步测定犬血浆内氯胺酮、赛拉嗪和咪达唑仑的RP-HPLC法。采用一步液-液萃取法处理血浆样品。使用C18色谱柱,流动相为乙腈：甲醇-10 mmol·L-1庚烷磺酸钠(44：10：46,v/v),加冰醋酸调至pH 3,流速0.7 mL·min-1,检测波长210 nm,柱温30℃。6只犬静注舒眠宁后,采血,测定血药浓度。氯胺酮、赛拉嗪和咪达唑仑三种成份的药时曲线均符合开放二室模型,主要药代动力学参数分别为：氯胺酮Vc= 1.231±0.147 L·kg-1 T1/2α=4.271±0.521min, T1/2β=65.877±15.701min, AUC=147.331±20.5μg·min·mL-1, CL(s)=0.0393±0.0055L·min-1·kg-1;赛拉嗪Vc=0.918±0.299L·kg-1,T1/2α=4.627±2.056 min, T1/2β=55.773±8.62 min, AUC=20.866±1.781μg·min·mL-1, CL(s)= 0.0336±0.00289 L·min-1·kg-1;咪达唑仑Vc=0.339±0.055 L·kg-1, T1/2α=5.675±1.82 min,T,/2p=39.152±6.546min,AUC=13.554±1.059μg·min·mL-1,CL(s)=0.0131±0.00097 5L·min-1·kg-1.试验Ⅳ舒眠宁注射液对灵(?)的麻醉效果研究9只灵(?)不使用麻醉前用药,经静脉注射舒眠宁0.06 mL·kg-1体重,观察其麻醉诱导时间、维持麻醉时间、苏醒时间,记录心率、血氧饱和度、呼吸数、收缩压、舒张压和直肠温度,评价镇痛、镇静和肌松效果。结果显示：其诱导、维持麻醉、苏醒时间分别为33.11±3.76sec、23±5.83 min、15±5min;麻醉后心率和血压先升高后缓慢降低,但均在正常生理范围内；体温缓慢下降,但变化幅度较小呼吸数先降低后缓慢升高；麻醉过程平稳,未见呕吐、眼球震颤、大量流涎等副作用。试验Ⅴ赛拉嗪对小鼠脾淋巴细胞增殖和周期分布的影响本研究使用α2-AR激动剂赛拉嗪、α2-AR颉颃剂育亨宾、α2A-AR颉颃剂BRL44408、α2B-AR颉颃剂Imiloxan hydrochloride和α2c-AR颉颃剂JP1302,研究α2-AR激动剂与其颉颃剂对ConA介导的小鼠脾淋巴细胞增殖和细胞周期分布的影响。结果表明：终浓度为0.01-0.4μg·mL-1的赛拉嗪对ConA介导的脾淋巴细胞增殖无明显影响；当赛拉嗪浓度在20-80μg·mL-1范围内时,对ConA介导的脾淋巴细胞增殖产生明显抑制,与单纯ConA组比较差异极显著(P<0.01),且呈剂量依赖性。育亨宾(20μg·mL-1,40μg·mL-1)与40μg·mL-1赛拉嗪联用时,可增强赛拉嗪对脾淋巴细胞的抑制作用；α2A-AR颉颃剂和α2B-AR颉颃剂均不影响赛拉嗪的细胞增殖调节作用；而α2C-AR颉颃剂JP-1302在较低浓度(30,60ng·mL-1),就能够明显增强40μg·mL-1的赛拉嗪对淋巴细胞的抑制作用,呈剂量依赖性。高浓度赛拉嗪(40μg·mL-1)可抑制细胞进入S期,促进晚期凋亡;JP1302可显著增强高浓度赛拉嗪的促凋亡作用和对淋巴细胞周期的阻滞作用。上述试验结果提示,赛拉嗪对脾淋巴细胞活性的抑制作用可能是由α2C-AR介导的。试验Ⅵ舒眠宁对小鼠脾淋巴细胞增殖和周期分布的影响本研究使用舒眠宁注射液的三种主要成份(氯胺酮、赛拉嗪和咪达唑仑)单用或联用,研究舒眠宁对ConA介导的小鼠脾淋巴细胞增殖和细胞周期分布的影响。结果表明：体外试验中,终浓度为0.0625～12.5μg·mL-1范围内的氯胺酮对ConA介导的脾淋巴细胞增殖无明显影响；当氯胺酮浓度升高至25～100μg·mL-1时,可产生明显抑制作用。终浓度为0.01-0.4μg·mL-1的赛拉嗪对ConA介导的脾淋巴细胞增殖无明显影响；当赛拉嗪浓度达到20～80μg·mL-1后,则产生明显抑制作用,呈剂量依赖性。试验浓度范围(0.01～100μg·mL-1)内的咪达唑仑与脾淋巴细胞作用48h后,均可抑制ConA诱导的细胞增殖。低浓度舒眠宁(0.01～0.4μg·mL-1)不影响ConA诱导的脾淋巴细胞增殖和周期分布,达到较高浓度时(2～80μg·mL-1)可对其产生剂量相关性抑制,与单纯ConA组比较差异极显著(P<0.01)。体内试验结果显示,低、中、高三个剂量的舒眠宁,对ConA介导的小鼠脾淋巴细胞增殖和周期分布均无显著影响。本研究结果显示,使用舒眠宁注射液麻醉小鼠,不会显著影响其脾淋巴细胞活性。更多还原

【Abstract】 Intravenous anaesthesia is a way in which the patients are anaesthetized by mainline, lose their consciousness, rigor off their muscle, or lose ordinary respiring functions.The advantages of intravenous anaesthesia are that there is no repulsion and it can easily be used without specific anaethesia appliances. Intravenous anaesthesia is used in anaesthetic induction, anaesthetic maintenance, analgesias and pain easing after the operation. Ketamine, xylazine and midazolam are widely used for premedication, chemical restraint, and the induction and maintenance of anesthesia in small animals. The use of these three drugs as a sole anesthetic has been limited by muscle hypertonicity,myoclonus, violent recovery, convulsions, hypotension, bradycardia and respiratory depression. In the present study, we combine the three anesthetics into a compound prescription, the most appropriate prescription was determined by orthogonal experiment and orthogonal design decisive verification experiment in mice, carry out the acute toxicity, local irritation, stability test, pharmacokinetics, anesthetic effect and its effect on the immune function of the injection, providing experiment data for correct application of Shumianning injection. This study contained the follwing 6 parts.Experiment I Research on Prescription of Shumianning InjectionKetamine, xylazine and midazolam have been used extensively as small animal anesthetics for surgical procedures. Based on the results of pre-experimet, according to the facors that affect the anesthetic effect, orthogonal method was used, the concentrations of ketamine, xylazine and midazolam were selected as variable factors and table L9(34) was used to do exiperiment on the basis of loss of righting reflex in mouse. Results:The optimum prescription was:ketamine (8 g·100mL-1), xylazine (0.9 g·100mL-1) and midazolam (0.2 g·100mL-1), named as Shumianning injection. The manufacturing process is simple, which is suitable for mass production.ExperimentⅡSafety Evaluation and Long-Term Stability of Shumianning InjectionTo evaluate the safety of Shumianning injection, acute toxicity experiment was carried out in mice by up-and-down method, the results showed that ED50=0.706 mL·kg-1 body weight, LD50=2.366 mL·kg-1 body weight, TI=3.35; Injecting Shumianning into rabbits to carry out local irritation experiment, the results indicated that Shumianning had mild vein irritation and slight muscle irritation, did not cause eye irritation; To establish expiration time and suitable package for Shumianning injection, long term test was performed with 3 batches merchant injection, the results showed that the main compositions of Shumianning injection were stable, the injection looked clear, no impurity, the period of validity was 2 years at room temperature, away from light.ExperimentⅢPharmacokinetics of Shumianning in CanineTo study the pharmacokinetics of Shumianning, a reverse-phase HPLC method with UV detection is developed and validated for simultaneous determination of ketamine, xylazine and midazolam in canine plasma. Analytes are extracted from alkalinized samples into diethyl ether-methylene chloride (7:3, v/v) using single-step liquid-liquid extraction. Chromatographic separation is performed on a C18 column using a mobile phase containing acetonitrile-methanol-10mmol·L-1 sodium heptanesulfonate buffer adjusted to pH 3 with glacial acetic acid (44:10:46, v/v) at a detection wavelength of 210 nm. Shumianning was applied intravenously to 6 dogs, the concentrations of ketamine, xylazine and midazolam were measured. The concentration-time curves of ketamine, xylazine and midazolam were best described of two-compartment open model and the typical pharmacokinetics parameters of the three drugs were as follows:Ketamine Vc=1.231±0.147 L·kg-1, T1/2α=4.271±0.521 min,T1/2β=65.877±15.701min,AUC=147.331±20.54μg·min·mL-1,L(s)=0.0393±0.0055 L·min-1·kg-1;XylazineVc=0.918±0.299L·kg-1,T1/2α=4.627±2.056min,T1/2β=55.773±8.62 min, AUC=20.866±1.781μg·min·mL-1, CL(s)=0.0336±0.00289 L·min-1·kg-1; Midazolam Vc=0.339±0.055 L·kg-1, T1/2α=5.675±1.82 min, T1/2β=39.152±6.546 min, AUC=13.554±1.059μg·min·mL-1, CL(s)=0.0131±0.000975 L·min-1·kg-1. Experiment IV Anesthetic Effect of Shumianning Injection in GreyhoundIn this study the anesthetic effect of Shumianning was investigated in 9 greyhounds. The animals were injected with Shumianning (0.06 mL·kg-1Ⅳ) without premedication, to observe the time to induction, maintenance, recovery, heart rate, SpO2, respiration rate, systolic pressure, diastolic pressure, rectal temperature, effects of analgesia, sedative and muscle relaxation. The time to induction, maintenance, recovery were 33.11±3.76 sec,23±5.83 min,15±5 min, respectively; heart rate, SAP and DAP increased slowly with a peak at 5 min, then decreased steadily but remained within the normal range; rectal temperatures decreased slowly but did not vary significantly during induction and maintence of anesthesia; respiratory rate decreased rapidly at first and then increased slowly; provided good surgical anesthesia; no vomiting, nystagmus, excessive salivation.Experiment V Effect of xylazine on cell proliferation and cell cycle in mouse spleen cellsXylazine is anα2-adrenoceptor agonist extensively used in veterinary medicine. The present study is focused on the effects of xylazine with or withoutα2 antagonists (yohimbine, BRL44408, imiloxan hydrochloride and JP1302) on the proliferation and cell cycles of mouse spleen cell in culture. The results are as follows:Xylazine at doses of 0.01,0.1,0.2 and 0.4μg·mL-1 did not significantly influence the proliferation of splenocytes in cultures triggered by ConA; at concentrations ranging between 20 and 80μg·mL-1 significantly inhibited spleen cell proliferation to Con A in a dose-dependent manner. Yohimbine enhanced the inhibitory effect of xylazine(40μg·mL-1) on spleen cell proliferation, BRL 44408, imiloxan were ineffective in modulating the inhibitory effect of xylazine(40μg·mL-1); JP1302 (30,60 ng·mL-enhanced the inhibitory effect of xylazine(40μg·mL-1) on spleen cell proliferation, dose dependently. xylazine(40μg·mL-1) blocked the progression of mouse lymphocytes into S phase, enhanced the apoptosis; JP1302 enhanced the activity of cell cycle arrest of xylazine. The present results provide evidence that the inhibitory effect of xylazine on spleen cell viability probably results from activation ofα2C-adrenoceptors.Experiment VI Effect of shumianning on cell proliferation and cell cycle in mouse spleen cellsThe aim of the this study was to investigate the effects of shumianning, ketamine, xylazine and midazolam on the proliferation and cell cycles of mouse lymphocytes in culture. The results are as follows:Low concentrations of ketamine and xylazine did not significantly influence the proliferation of splenocytes in cultures triggered by ConA; at high concentrations significantly inhibited spleen cell proliferation to Con A in a dose-dependent manner. Midazolam at concentrations ranging between 0.01 and 100μg·mL-1 significantly inhibited spleen cell proliferation to ConA in a dose dependent manner. Low concentrations of shumianning (0.01-0.4μg·mL-1) did not significantly change the proliferation of splenocytes in cultures stimulated by ConA; at high concentrations (2-80μg·mL-1) significantly inhibited spleen cell proliferation to ConA, dose dependently. The in vivo administration of shumianning at doses of 0.56,2.8 and 11.2 mg·kg-1 body weight, which produced weak sedation, slight and deep anesthesia, respectively, did not significantly change the proliferation and cell cycles of splenocytes in cultures triggered by ConA. Cumulatively, these results suggest that clinically relevant concentrations of shumianning did not affect the viability of splenocytes.更多还原