Development And Evaluation Of “Jifang E” Sustained-release Injection

Locoweed is a poisonous plant which contains swainsonine and can cause typical neurological symptoms and pathological changes.Locoweed is mainly distributed in natural grasslands in western provinces such as Tibet,Mongolia,Qinghai,Gansu,Xinjiang,Ningxia,Shaanxi and Sichuan and is also widely distributed in the grasslands of western United States,Brazil and Argentina of South America.Therapeutics have previousy been developed for the prevention and treatment of locoweed poisoning.These include"Jifang E",Fangcaoling detoxication sustained-release pills,Dangqi Shenhu Ding,SW-BSA vaccine,SW-HSA vaccine and lithium chloride conditional avoidance to prevent locoweed poisoning.And sukang detoxication oral liquid,nutrition lick block which is used to treat locoweed poisoning.At present,“Jifang E”mainly includes two formulations:powder and sustained-release pill.The former has some disadvantages such as large individual differences and difficult to control,and has been shown to have some side effects,which affect production and reduce the economic value of livestock;The latter has disadvantages such as time-consuming in application,great effort and easy to reverse,which affects the preventive effect of drugs.Therefore,there is a need to develop a novel formulation that can facilitate drug administration and achieve sustained release.In this study,a thermosensitive gel based on“Jifang E”was developed.Poloxamer 407 and poloxamer 188 were used as the sustained-release matrix,and the optimal ratio of each drug component was determined by the gelation temperature.The sustained-release injection was prepared above room temperature and below physiological temperature.By studying the micellar fluid dynamics,the influence of each formula in the sustained-release injection on the stability of the micelles was analyzed.The morphological characteristics of the gel were studied by freezing scanning electron microscopy,and the effects of various components in the sustained-release injection on the morphology of the gel were observed.By evaluating the viscosity and viscoelastic changes of the sustained-release injection under different temperatures,as well as the viscosity and viscoelastic changes under different shear forces and oscillation frequencies,the stability and properties of the sustained-release injection under different environmental factors were estimated.To preliminarily evaluate and estimate the sustained release performance of sustained-release injection through in vitro dissolution test and in vitro drug release test with and without membrane.Through the acute toxicity test,muscle irritation test and biocompatibility test,the toxic effect of“Jifang E”sustained release injection was revealed.Pharmacokinetics test was used to evaluate the sustained release effect in vivo.The following results were obtained:1.Preparation of“Jifang E”sustained release injection:The thermosensitive gel was selected as the carrier of the drug“Jifang E”.The thermosensitive gel was configured using the cold solution method.The gelling temperature was used as the screening criterion.Poloxamer 407 was used as sustained-release matrix,poloxamer 188 as temperature regulator,Vitamin C as antioxidant and PEG4000 as adhesive.A sustained release injection of“Jifang E”was prepared,which gelatinized above room temperature and below physiological temperature.The results showed that poloxamer 407 could reduce the gelling temperature,and poloxamer 188,additives and“Jifang E”could increase the gelling temperature.After screening different formulations,the optimal ratios of each formulation were finally developed as:P407（24%）,P188（6%）,Vitamin C（1%）,PEG4000（0.5%）and“Jifang E”（10%）,the remaining component is deionized water.2.In vitro drug release and dissolution evaluation of“Jifang E”sustained release injection:The in vitro drug release test of the sustained-release injection was carried out by the non-membrane method and the membrane method.The in vitro dissolution test of sustained-release injection was performed by the membrane-free method.The results showed that,when the oscillation frequency was 0 rpm and 50 rpm,the complete dissolution time of the sustained-release injection was 4 h and 12 h,the results were the same as those obtained without membrane.At 0 rpm and 50 rpm,the correlation between dissolution rate and drug release rate was R²=0.9924,R²=0.9954.The results showed that there was a strong correlation between the dissolution rate and the drug release rate of the drug-containing sustained-release gel.The drug release results showed that the“Jifang E”sustained-release injection could be released for 3 days,and the release rate of“Jifang E”solution was more than 80%in only 1hour,indicating that the“Jifang E”sustained-release injection had great sustained release performance.The drug release model was fitted by the cumulative dissolution rate of the sustained-release injection and the cumulative drug release rate and time.The results showed that the in vitro dissolution kinetic equation was in accordance with Higuchi equation,and the R²of the oscillation frequency of 0 rpm and 50 rpm was 0.98204 and 0.9991,respectively;In the in vitro drug release without membrane,the equation of in vitro drug release rate and time of“Jifang E”sustained release injection was fitted at the oscillation frequency of 0 rpm,and the R²value of the first-order equation was the largest,which was 0.98997.At the oscillation frequency of 50 rpm,Higuchi’s R²value was the largest,which was 0.98411.However,it is worth noting that the R²value of the first-order equation is close to the Higuchi equation,which is 0.98339.Therefore,the release kinetics of“Jifang E”sustained release injection conformed to the first order equation in the in vitro release without membrane;In the in vitro drug release with membrane,the R²value of the first equation was 0.9544,and the R²values of the other two equations were 0.42197 and 0.63165,respectively,which had a certain gap.These results indicated that the in vitro release kinetics of“Jifang E”sustained-release injection conformed to the first order equation.3.Characterization of“Jifang E”sustained release injection:The results of micellar fluid dynamics showed that the composition used had almost three peaks at both 25°C and37°C,but the particle size distribution was different.The micelle size of all formulations decreased with increasing temperature,indicating that there is a relationship between this parameter and the dehydration of PPO units in poloxamer as well as the viscosity of the formulation,which favors the formation of colloidal systems with uniform spherical micelles.With the addition of P188,additives and drugs,the whole micelle system was more stable and the particles were uniformly dispersed.The colloidal morphology study showed that all the formulations formed a uniform pore structure after gelling,and all were pore structures after gelling.With the addition of P188 as the additive,the stability of the gel decreased and the biological adhesion force decreased,but the gel structure became more compact with the addition of the drug.The effects of rheological properties of different formulations on drug delivery,drug release and histocompatibility were evaluated by viscosity and viscoelasticity,respectively.The results showed that the“Jifang E”sustained release injection was a pseudoplastic fluid,which was mainly viscous and increased with the increase of temperature,but the viscosity was low,which was conducive to injection.The gelling properties of“Jifang E”in vitro and in vivo showed that the“Jifang E”could form a stagnant gel at physiological temperature,and the gel was reversible when the ambient temperature was reduced.4.Safety evaluation of“Jifang E”sustained release injection:According to the toxicological test method,the acute toxicity of“Jifang E”sustained-release injection was detected by subcutaneous injection on the neck and back of mice.The results showed that the mice in the high-dose group had the first clinical symptoms,and the symptoms began to appear from 2 to 4 hours after injection.Generally,the mice died soon after the appearance of restlessness symptoms,and the peak period of death was generally 7-9 hours after injection,the mice in the low-dose group were immobile for a long time and had no food or water intake after injection of“Jifang E”sustained release injection.LD₅₀=828.323 mg/kg,SD=0.0448,LD₅₀95%confidence limit range 828.323 mg/kg（676.706-1013.911 mg/kg）for“Jingfang E”sustained release injection calculated by modified Kou’s method.According to the LD₅₀value of drug toxicity grade,“Jifang E”sustained release injection is a common drug with high safety.However,when the solvent was injected at the highest dose in the acute toxicity test,the mice showed no obvious symptoms and no death,indicating that the toxicity of the solvent was low.The muscle stimulation and biocompatibility of“Jifang E”sustained-release injection were tested by subcutaneous injection on the back and bilateral quadriceps femoris in New Zealand white rabbits.The results showed that there was only slight hyperemia in subcutaneous and muscle at the injection site,and the bleeding spots were small.Histopathological observation showed slight inflammatory cell infiltration at the injection site.5.Pharmacokinetics evaluation of“Jifang E”sustained release injection:The content of“Jifang E”was detected by microplate reader and substituted into the formula in the kit instruction to calculate the concentration of“Jifang E”in plasma.DAS2.0（Chinese Pharmacological Association,Anhui Province,China）software was used to obtain the best compartmental model of the drug,and then the pharmacokinetic parameters were obtained.The results showed that“Jifang E”sustained release injection was suitable for the non-compartmental model,which could be released for about 5 days.The absorption half-life T_1/2was 32.821±16.798 h,and the blood concentration reached the peak at 12 h,and the peak concentration C_maxwas 5.609±0.43μg/m L.The area under the curve(AUC_0-∞)was131.002±14.849μg/m L·h,and the mean retention time in vivo MRT was 24 hours.The plasma concentration of“Jifang E”sustained release injection showed a bimodal trend with time.In conclusion,this study successfully developed“Jifang E”sustained release injection for the prevention of Locoweed poisoning in livestock,which laid an important foundation for the subsequent field promotion experiment and the study of detoxification mechanism.