| BackgroundAnesthesia is one of the world-known surgery achievement of ancient China. The“Ma Fei San”, an anesthesia invented by famous Chinese doctor Tuo Hua2000years agowas regarded as the earliest anesthesia in the world. Since the use of “anesthesia” byCliver Wendell Holmes in1846, it has been170years for surgery with anesthesia, whichcreates an era for surgery and promotes greatly the development of surgery.To achieve satisfactory sedative, analgesia and muscle relax that meets therequirements of multiple surgery and populations, is always the hot and hard points ofanesthesia development. However, up to date, none of any one single vein anesthesia canmeet the demands of surgery. Currently, combined anesthesia in which several anaestheticwere co-administrated has been adopted to achieve ideal anesthetic effects. This kind ofanesthesia has been the trend of clinical application, and is the mostly adopt strategy toobtain “balanced anesthesia”. The future anesthesia will be easier, more controllable, andsafer. Explore and development novel anesthesia which meets these requirements will bethe direction of our continuous hard working.Based on the theory of balanced anesthesia, the present study aims to make acompound consists of two drugs, which can be both analgesia and anesthesia, and needs less administrations, thus smoothening the whole process of anesthesia. To achieve thisgoal, we needs to solve the following critical issues for the druggabiltiy of the compound.Is there any interactions between the ingredients? If is, is it additive, synergistic orantagonistic? What is the mechanism of the drug-drug interaction? How is the stability ofthe drug physicochemical property in the compound? Is the procedure steady andsuccessful? Could the optional ratio be fixed, and meet the clinical requirement? Therefore,we performed the following studies focused on the druggability of the propofol-alfentanilcompound.1. Interactions of propofol-alfentanil co-administrationThe principles of clinical development of solid compounds clearly states that theadditive or the synergistic effects of the intergradient are the ground of making acompound formula. Therefore, we measured the ED50of Propofol, Alfentanil, andPropofol-Alfentanil combination by hot-plate, tail-flip, and acetic acid induced writhing.The interaction between Propofol and Alfentanil was measured by isobolographic analysis.The results showed that propofol and alfentanil functioned fast, can release the acute paininduced by heat, and the inflammatory pain by acetic acid injection. In the hot-plate assay,the ED50of propopol is8.82mg/kg, of alfentanil is41.44μg/kg. The ED50mixin thecombined drug administration group is2.09mg/kg for propofol，and9.91μg/kg foralfentanil. In the tail-flip test, the ED50of propofol is7.50mg/kg, of alfentanil is40.16μg/kg. The ED50mixin the combined drug administration group is1.88mg/kg for propofol，and10.16μg/kg for alfentanil. In the acetic acid induced writhing test, the ED50ofpropofol is15.00mg/kg, of alfentanil is12.50μg/kg. The ED50mixin the combined drugadministration group is3.75mg/kg for propofol，and3.13μg/kg for alfentanil. Accordingto isobolographic analysis, the ED50mixin all three pain models are significantly smallerthan their ED50add. The ED50mixalways locates at the left side of additive line. In addition,the interaction index is less than1, indicating that propofol and alfentanil are synergistic inall three pain models. This part of work has been published by SCI journal of PharmcolBiochem Behav (2014,116,25-9). 2.The procedure, physicochemical property, and stability of the emulsion of propofolcompoundBased on previous pre-prescription study, we prepared the emulsion of propofol-alfentanil compound by high pressure homogeneity method and explored the effects ofphospholipid addition, the dissolve temperature in oil phase, and the adding sequence ofwater-oil phase and high pressure homogeneity on the stability of the emulsion. We furtheroptimized the types of oil, the dosages of odium oleate, pH, and explored the effects ofsterile temperature and timing on the emulsion. We investigated the physicochemicalproperties of the emulsion by the particle diameters, electrical potential, ion, pH, osmoticpressure, viscosity of the emulsion, acid value and peroxide value. We established a HPLCmethod for propofol and alfentanil, and investigated the stability of the emulsion by speedassay, long term test, light exposure and repeated freeze. The results showed that we havesuccessfully made the compound emulsion of propofol-alfentanil. The final procedure formaking compound emulsion of propofol-alfentanil is: make oil phase with5%soy beanoil and5%Medium-chain Triglycerides, make emulgator with1.2%egg yolk lecithin and0.03%sodium oleate, adjust pH to8.0before homogeneity, and finally pressurehomogeneity6times with60Mpa with final nitrogen sealing and sterile. The averagediameter of the emulsion of three batches is221.6nm, the-potential is-22.83mV. Thecontent of propofol and alfentanil is99.0%. The emulsion is stable for6month at40±2℃with humidity at75%±5%, for12month at25±2℃with humidity at60±10%, and for10days at the exposure of （4500±500）Lx light. A freezing procedure of-20℃to-50℃, andthen4℃does not significantly affect the appearance, pH, diameter, potential and drugcontents. This part of work has been patented with patent code201210069537.4.3. Evaluation of the anesthesic effects of continuous infusion of compound emulsionof propofol-alfentanil.Anesthesia induction and maintenance in adult dogs of partial spleen surgery wasachieved by different doses of compound emulsion propofol-alfentanil. The anestheticeffects of compound emulsion propofol-alfentanil were evaluated through comparing the mean arterial pressure, oxygen saturation, CO2partial pressure, body temperature, heartrate, the BIS, respiratory frequency, time and awakening time of anesthesia index. Theresults showed no difference in terms of mean arterial pressure, blood oxygen saturation,CO2partial pressure and body temperature between each dose groups.However, the compound emulsion propofol-fentanyl with dosage ratio of75:1cancause deep anesthesia and severe bradycardia in adult dogs. In such cases, slow wakeupwas observed post-operation. Dosage ratio of300:1can speed the heart rate, indicating apossible insufficient analgesia drug dose. Relative to the positive control, dosage ratio of150:1showed no significant difference. Therefore, we believe that the combination ofpropofol-fentanyl at150:1may be the best proportion, and can meet the needs ofanesthesia in the operation. The manuscript of this part of research is currentlyunder-review by SCI journal Vet J.4. Possible synergistic mechanisms propofol-fentanyl combination.mIPSC and eIPSC was recorded by using whole cell patch clamp method.Immunocytochemistry and Western blot were performed to detect cell membranedistribution and phosphorylation of GABA receptor. Confocal and flow cytometry wereperformed to detect the intraneuronal Ca2+concentrations upon propofol-fentanyltreatment. The intracellular cAMP concentration, PLC activity were evaluated bycommercial detecting kits. The results showed that propofol and alfentanil arecoordinative in activation of GABA receptors. Propofol-alfentanil combinationsignificantly increased the amplitude of mIPSC and eIPSC (P <0.05), in comparison withpropofol treatment alone. These results suggest that the combination may strengthen theeffects of propofol on postsynaptic GABA receptors. Further analysis found that thecombined application can promote the surface expression and the phosphorylation ofGABAAβ3. In addition, Propofol-alfentanil combination can reduce the concentration ofintracellular cAMP, the activity of the PLC, leading to a decrease intracellular Ca2+, andthus exhibit synergistic effect on the downstream signals of mu receptor. This part of workis currently under review by the Journal of Pain Medicine. Conclusion: To sum up, this study demonstrated that propofol and alfentanil has goodsynergy with each other, and preliminary explored the mechanism of synergy. Usingpharmacy technology, we successfully developed a compound emulsion of propofol-alfentanil, and screened the best proportion by evaluating the anesthesia effect on partialsplenectomy. The results show that the ratio of150:1can satisfy the requirements for thebalanced anesthesia in the operation. This research has solved the key problems in thedevelopment of compound propofol-alfentanil, completed pre-clinic drug evaluation,laying a solid foundation for the further development of this compound. |
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