A Novel Transdermal Local Anesthetic Drug-development And Pharmacodynamic Studies Of Tetracaine Ethosomes

BACKGROUNDThe outstanding characteristics of skin examination and surgery are mini-invasive and superficial,therefore,only local anesthesia other than general anesthesia is required for these operations,such as minor surgery(depilation,chemical peel,biopsy sampling,split thickness skin grafting,curettage,electric coagulation,etc.),cosmetic laser(laser hair removal,hemangioma laser therapy,facial laser rejuvenation)and percutaneous invasive examination(lumbar puncture,abdominocentesis,bone marrow puncture,thoracentesis,venous cannulation,venipuncture,etc.).Currently,there are two methods for local anesthesia:dermal infiltration and application of percutaneous local anesthetic preparation.Although the former has a rapid effect,poor compliance of patients due to pain and metal phobia ensues,especially for children.By means of dermal infiltration,it is not easy to control the precise dose,the ensuing overdose might cause neurotoxicity.Therefore,application of percutaneous local anesthetic preparation born at the right moment because it could administer without special equipment,directly apply to skin surface and painless.Traditional percutaneous local anesthetic preparation,such as gel,tincture,2ointment could acquire reliable anesthetic effect in clinical practice.At the present stage,low temperature eutectic cream of lidocaine and prilocaine(EMLA,AstraZeneca;Compound Lidocaine Cream,Unisplendour Pharmaceutical Co.Ltd.)is most widely used in clinic practice.However,there are two disadvantages for eutectic mixture of lidocaine and prilocaine.On the one hand,it always need a great amount of eutectic mixture and must be occluded long time.On the other hand,prilocaine might cause such side effects as methemoglobinemia,contact urticaria,and neurotoxicity.With the above reasons,EM LA in AstraZeneca was propelled to withdraw from the Chinese market.Some scholars employed penetration enhancer in order to shorten application time and improve the permeability of transdermal local anesthetic drug.However,the scope of application is confined because it might impair barrier function of skin and induce allergy and irritability.In consideration of limitations of dermal infiltration and application of traditional percutaneous local anesthetic drug,the development of novel transdermal carrier has become an important path and research hotspot to tackle the above problems and meet the clinic need.Due to excellent percutaneous permeability,nanometer narticles,including nanoemulsion,liposome and nanoparticles,have become the research focus of novel transdermal carrier.Although nanoemulsion have favorable percutaneous permeability,but it must contain high-dose surfactant.It is well-known that the risk for skin irritability caused by non-ionic surfactants,even for those with lowest possibility,is very high.Thus,there is a long way for nanoemulsion to become a carrier for local anesthetics.Liposomes and nanopartilcles just have a passive targeting to follicles and superficial stratum corneum.It is not easy for intact liposome or nanopartilcle to penetrate deeper epidermis and dermis.Hence,they are not opportune candidate for transdermal local anesthetic carrier.Inspiringly,ethosomes,containing high concentration of short-chain alcohol,not only possess well deformability,high encapsulation efficiency,affluent loading efficiency and excellent stability but also excellent percutaneous permeability without surfactant.Compared to ointment and conventional liposomes,ethosomes have similar spatial structure to biologic membrane and better permeability,high bioavailability.Furthermore,animal skin irritability test indicated that ethosomes is not only non-poisonous itself but non-skin irritability.Therefore,ethsomes have the real basis to become an ideal carrier for transdermal local anesthetic drug.Tetracaine,as a traditional long acting ester local anesthetic,was widely used in clinic practice such as department of stomatology and department of dermatology and was known to the world for high liposolubility,fast onset,long lasting and strong potency.Research has shown that tetracaine has superior tissue permeability and diffusion than other local anesthetic,such as shorter onset time and longer lasting time than lidocaine and bore good safety.Several studies indicated that 5%tetracaine liposomes produce better superficial local anaesthesia than EMLA when applied in equal volume and ethosomes bear better permeability than liposomes.Based on this,there is a great possibility that tetracaine ethosomes is an effective topic anesthetic preparation.The present project make ethosomes as a transdermal carrier for tetracaine to formulate tetracaine ethosomes.We believe that the outcome of the present project will provide a quick onset,long-lasting and safe novel transdermal local anesthetic drug.OBJECTIVEThe objective of the present project is to develop a novel transdermal local anesthetic drug-tetracaine ethosomes,improve the permeability of tetraciane,establish quality-control method of tetracaine ethosomes,measure the morphology and physicochemical property of tetracaine ethosomes,and investigate the in vitro release kinetics and drug efficacy of 5%tetracaine ethosomes.METHODS1.Pre-formulation study of tetracaine ethosomesThe wavelength of tetracaine was determined by full wavelength scanning,the chromatographic condition of tetracaine was ascertained and determination method of tetracaine was established.2.The preparation technology of tetracaine base ethosomesIn the present project,a Box_Behnken design based on response surface methodology(RSM)was employed to design and formulate an appropriate tetracaine ethosomes formulation and preparation technology.Thereby,the parameters such as volume fraction of absolute ethanol,mass volume ratio of egg yolk lecithin,ultrasound power were chosen as key variables and prescribed into three levels.The encapsulation efficiency and polydispersity index were further selected as the response for the combination of independent variables.Experimental design and analysis of experimental outcomes were performed by Design-Expert 8.0 to comprehensive estimate the response of independent variables.Three levels of a three factor,Box_Behnken Design were used to evaluate the critical formulation variables.Response surfaces were drawn to estimate the individual and interactive effects of independent variables.A model fitting equation was established to determine the optimal parameters for tetracaine ethosomes.3.Physico-chemical property and quality control of tetracaine ethosomesBased on optimal formulation,injection-sonification-filter method was used to prepare tetracaine ethosomes.Particle size and particle size distribution of tetracaine ethosomes was determined by Zetasizer Nano S90.The microscopic morphology of tetracaine ethosomes was studied by Transmission Electron Microscope.A method.determining the content of tetracaine in tetracaine ethosomes.was established by HPLC.4.In vitro release studies of tetracaine ethosomesIn vitro release experiments were carried out using Franz diffusion cells and the permeation membrane was rat skin.The respective formulation(tetracaine ethosomes 1mL and tetracaine tincture 1mL)was gently place in the donaor chamber to investigate the difference of permeation between tetracaine ethosomes and tetracaine tincture.The normal saline containing 5%ethanol was used as receptor fluid.The content of tetracaine in receptor fluid was analyzed by HPLC.The cumulative curve was plotted of the total amount of the tetracaine that permeated at each time interval vs.time,curve of permeation from respective formulation was fitted and average permeation rate was acquired.5.The pharmacodynamic study of tetracaine ethosomesThe rear legs of ten rats were randomly divided into five groups:5%tetracaine ethosomes group,eutectic cream of lidocaine and prilocaine group.5%tetracaine tincture group,blank ethosomes group,no-treatment control group.The comparison of local anesthetic effect between left and right rear leg on the same rat were adopted,namely,1.0 mL5%tetracaine ethosomes,1.0 mL5%tetracaine tincture,1.0 mL blank ethosomes,1.0 g eutectic cream of lidocaine and prilocaine were applied on the rear leg of the former four groups,while no treatment was applied on no-treatment control group.After 1.0 hour,Electronic Von Frey Anesthesiometer was used to determine the pain tolerance threshold for both left and right rear leg of rats,and compare the local anesthetic for 5%tetracaine ethosomes group,eutectic cream of lidocaine and prilocaine group,5%tetracaine tincture group.RESULTS1.Pre-formulation study of tetracaine ethosomesThrough full-wavelength scanning,the wavelength of tetracaine was identified as 310nm.Established chromatographic condition for tetracaine:HPLC column:America Thermo Scientific Hypersil ODS-2 C18,250mmx4.6mm,5μm;mobile phase:acetonitrile:H2O+0.25%H3PO4(80:20),flow rate:1mL·min-1,wavelength:310nm;column temperature:room temperature;injection volume:10μL.A good linear relationship was found between peak areas for various concentration,from 4μg·mL-1 to 1000μg·mL-1 and the standard curve equation in acetonitrile was Y=56.1741+49.391X.(R2=0.9997).The developed method had good precision in intraday and inter-day variation and had good accuracy,which met the experimental requirement2.The preparation technology of tetracaine base ethosomesBased on the experimental results,an optimization study was further performed to evaluate the optimal conditions for the preparation of the tetracaine ethosomes with highest encapsulation efficiency and lowest PDI with Deisgn-Expert 8.0.The optimal conditions were as follows:mass volume ratio of egg yolk lecithin:10%,volume fraction of absolute ethanol:50%,ultrasound power:104W.The total desirability of the model was 0.972,indicating the observed responses were close to the ones predicted by the design.Therefore,it is valid and predictive to determine optimal conditions through mathematic model generated from 17 runs.3.Physico-chemical property and quality control of tetracaine ethosomesThe tetracaine ethosomes was prepared through injection-ultrasound-filter method.The chromatographic condition used to determine the tetracaine content in ethosomes:HPLC column:America Thermo Scientific Hypersil ODS-2 C18,250mm×4.6mm,5μm;mobile phase:acetonitrile:H2O+0.25%H3P04(80:20),flow rate:1 mL·min-1,wavelength:310nm;column temperature:room temperature;injection volume:10μL.The method was proved to be specific.The tetracaine ethosomes prepared with optimal conditions is a faint yellow,translucent liquid.There is no discoloration and stratification for three months under normal condition.Under the optimal conditions,the loading efficiency was 97.22±1.66%,the encapsulation efficiency was 85.326±0.04%,PDI was 0.147±0.027.The average diameter determined by Zetasizer Nano S90 was 17.60±1.22nm.The microscopic morphology of tetracaine ethosomes was studied by TEM and TEM micrographs indicated that ethosomes were uniform elliptical or spherical in shape.4.In vitro release studies of tetracaine ethosomesAccording to the percutaneous permeation curve of tetracaine ethosomes and tetracaine tincture,the permeation rate for the former was 24.72±0.38μg·h-1·cm-2,while the permeation rate for the later was 13.72±0.36μg·h-1·cm-2.Therefore,the permeation rate of tetracaine ethosomes was much higher than that of tetracaine tincture.5.The pharmacodynamic study of tetracaine ethosomesThe local anesthetic effect for both 5%tetracaine ethosomes,eutectic cream of lidocaine and prilocaine are superior to 5%tetracaine tincture group.There is no significant difference of local anesthetic effect between eutectic cream of lidocaine and prilocaine group(13.639±3.880)and 5%tetracaine ethosomes group(13.159 ±3.144)(P=1.000).CONCLUSION1.Pre-formulation studies showed that the developed HPLC method had good linear relationship and strong stability and good precision.2.The preparation technology studies indicated that response surface methodology based Box_Behnken design could be successfully used to analyze and optimize the effect of formulation variables and design tetracaine ethosomes.The acquired three-dimensional response surface plots and two-dimensional contour plots were very useful to display the interactive effects of the three factors-volume fraction of absolute ethanol,mass volume ratio of egg yolk lecithin,ultrasound power-on encapsulation efficiency and PDI.3.Physico-chemical property and quality control of tetracaine ethosomes illustrated that tetracaine ethosomes were faint yellow,translucent liquid and remain stable under normal condition.4.In vitro release studies demonstrated that preparation of tetracaine ethosomes from tetracaine could significantly improve the permeability of tetracaine.5.The pharmacodynamic study of tetracaine ethosomes indicated that there is no significant difference of local anesthetic effect between 5%tetracaine ethosomes and eutectic cream of lidocaine and prilocaine,and the local anesthetic effect for both them are significantly superior to 5%tetracaine tincture.