Research On New Sustained-release Preparations For Controlling The Source Of Echinococcosis And Schistosomiasis Infection

ObjectiveThe in situ sustained-release injection is a research focus in the sustained-release formulation field recently,based on the advantages as follows:the simple preparation process,easily controlled dosage,convenient injection via syringes,sustained-release for several months without surgery to remove residues due to biodegradation of polymers.It's a composition of drug and polymer dissolved in a suitable solvent.When it is administered to the body in a liquid state,the composition coagulates a polymer matrix with the water-soluble solvents diffusing away from the composition quickly.At last,drug is sustained-re lease in the biodegradable polymer matrix.Therefore,it is an economic and effective idea to prevent and control echionococcosis and schistosomiasis transmission by the application of in situ sustained-release injection technology with antiparasite drugs,such as praziquantel,niclosamide.Methods1.A new sustained-release formulation to block echionococcosis transmission1.1 Screening the formulation of praziquantel in situ sustained elease injectionEstablishing praziquantel(PZQ)analysis method in vitro by high performance liquid chromatography(HPLC)methods,determining solubilities of praziquantel in a variety of disperse medium.According to the release of praziquantel in vitro,polycaprolactone(PCL)as polymer and N-methyl pyrrolidone(NMP)as solvent were chosen for praziquantel in situ sustained-release injection by single factor screening.We optimized the formulation by Box-Behnken design of three factors and three levels of three centers in Design-Expert.8.0.At last,the final formulation was determined by the praziquantel release in mice.1.2 The stereoselective release in vitro and pharmacokinetics in vivo of praziquantel enantiomer suspensions and rac-praziquantel in situ sustained-release injectionEstablishing praziquantel enantiomers analysis method in vitro and in vivo by high performance liquid chromatographic-high resolution mass spectrum(HPLC-HRMS)method.To study stereoselective pharmacokinetics of praziquantel enantiomers and their pharmacokinetic interactions by HPLC-HRMS after oral administration of racemic-praziquantel(rac-PZQ)suspension(100 mg/kg),R-(-)-praziquantel(R-PZQ)suspension(50 mg/kg)and S-(+)-praziquantel(S-PZQ)suspension(50 mg/kg),respectively.To study stereoselective release in vitro and pharmacokinetics in vivo of rac-PZQ(1 g/kg),R-PZQ(1 g/kg)and S-PZQ(1 g/kg)in situ sustained-release injections in mice by HPLC-HRMS.To study stereoselective pharmacokinetics in vivo of rac-PZQ in situ sustained-release injection(100 mg/kg)in beagle dogs by HPLC-HRMS.1.3 The therapeutic efficacy of praziquantel in situ sustained-re lease injection against echionococcus infection in the field trialWe chose Ganzi Tibetan Autonomous Region to start the field trial,where the positive rate of dogs infected echinococcus was higher.In the field,the dogs were divided into two groups randomly:drug group and control group.The praziquantel in situ sustained-re lease injection(100 mg/kg)was subcutaneously administered into the dogs in drug group.The other group was untreated as control group.Fecal samples were taken every month from each dog for 6 months to confirm the echinococcus infection by coproantigen determination.The efficacy of the treatment was determined by comparing positive rates of dogs infected echinococcus between drug group and control group.2.A new sustained-release formulation to block schistosmasis transmission2.1 Screening the formulation of niclosamide and praziquantel in situ sustained-release injectionsEstablishing niclosamide analysis method in vitro by ultraviolet visible spectrophotometry(UV)and high performance liquid chromatography(HPLC)methods,and determining solubilities of niclosamide in a variety of disperse medium.Establishing praziquantel analysis method in vitro by ultraviolet visible spectrophotometry(UV).According to the release of drug in vitro,the polymers,several kinds of solvent and pore-forming agents were screened for niclosamide and praziquantel in situ sustained-release injection,respectively.2.2 The protective effect of niclosamide and praziquantel in situ sustained-re lease against Schistosoma japonicum infection in miceSixty-five female mice were randomly divided into 13 groups,each with 5 mice.Among them,25 mice were subcutaneously injected the niclosamide in situ sustained-release injection at a single dose of 450 mg/kg,15 mice were subcutaneously injected the praziquantel in situ sustained-release injection at a single dose of 450 mg/kg.The other 25 mice were untreated as control group.5 mice were infected with 80±14 Schistosoma japonicum cercarials on 1,15,43,57 and 71 days after treatment in each group.5 weeks later,mean worm burdens and worm reductions were calculated.2.3 The protective effect of niclosamide in situ sustained-release against Schistosoma japonicum infection in bovinesThe niclosamide in situ sustained-release injection was prepared.In the field trial,10 bovines uninfected Schistosoma japonicum were divided into 2 groups.Among them,4 bovines were injected the injection at a single dose of 30 mg/kg.The other 6 bovines were untreated as control group.1 and 3 months later,the number of bovines infected Schistosoma japonicum was calculated.Results1.A new sustained-release formulation to block echionococcosis transmission 1.1 Screening the formulation of praziquantel in situ sustained-release injectionAccording to screening result,the optimum formulation was the PLGA(Poly(D,L-lactide-co-glycolide))formulation "PLGA:NMP:PZQ=13:2(w/v/w)".The percentage of praziquantel residues was 95.8%at 2 h,and reduced to 0.2%at 130 day in mice after subcutaneous administration of praziquantel in situ sustained-re lease injection.We speculated the praziquantel in situ sustained-release injection should be sustained-re lease for about 4 months in mice.We chose PLGA formulation of in situ sustained-release injection for further study considering the need of the field application.1.2 The stereoselective release in vitro and pharmacokinetics in vivo of praziquantel enantiomer suspensions and rac-praziquantel in situ sustained-release iejectionData showed that there were enantioselective metabolic differences and stereoselective pharmacokinetics of R-PZQ and S-PZQ in vivo after oral administration of praziquantel enantiomer suspensions.Enantioselectivity in the kinetic disposition between R-PZQ and S-PZQ was observed in the study.The average MRT(Mean retention time)value of R-PZQ in plasma were higher than that of S-PZQ by 45.1%(p<0.01).And the average Cma,(Maximum concentration)value and AUC(Area under the curve)value of S-PZQ in plasma were higher than that of R-PZQ by 408.9%(p<0.01)and 212.9%(p<0.05),respectively.The praziquantel in situ sustained-release subcutaneous injection has sustained-release for 4 months in mice and over 6 months in beagle dogs.The average AUC and Cmax value of S-(+)-praziquantel in plasma were higher than that of R-(-)-praziquantel by 79.2%(P<0.01)and 114.0%(P<0.05).However,the average MRT value of R-(-)-praziquantel in plasma was higher than that of S-(+)-praziquantel by 96.3%(P<0.05).The average T1/2(Half time)of R-(-)-praziquantel was higher than that of S-(+)-praziquantel by 101.9%without significant difference.1.3 The therapeutic efficacy of praziquantel in situ sustained-release injection against echionococcus infection in the field trialIn the first time,the positive rate of dogs infected echionococcus in control group was 2.0%in forth month after treatment,and the positive rates of dogs infected infected echionococcus in drug group were always 0.0%for 6 months.In the second time,the positive rates of dogs infected echionococcus in both groups were 0.0%.There was no significant difference between drug group and control group.2.A new sustained-release formulation to block schistosmasis transmission2.1 Screening the formulation of niclosamide and praziquantel in situ sustained-re lease injectionsAccording to screening result,the optimum formulations were the formulation"PLGA:NMP:NCA:T80(Polysorbate 80)=1:6:0.3:1(w/v/w/w)" for niclosamide and the formulation "PLGA:NMP:PZQ=1:6:0.3(w/v/w)" for praziquantel releasing2 months in vitro.2.2 The protective effect of niclosamide and praziquantel in situ sustained-release against Schistosoma japonicum infection in miceIn mice,the mean burdens in niclosamide group were significantly lower than that of control group(P<0.05)with worm reductions over 50.0%on 1,15,43,57 and 71 days after treatment in each group.The mean burdens in praziquantel group were significantly lower than that of control group(P<0.05)on 1 and 43 days after treatment in each group.But the mean burden was 15.1%on 15 days after treatment without significant difference,so we chose the niclosamide in situ sustained-release injection in field trial.2.3 The protective effect of niclosamide in situ sustained-release against Schistosoma japonicum infection in bovinesIn Bovines,the positive rates in control group were 33.3%and 50.0%respectively as 0.0%in drug group on 1 and 3 months later after treatment in each group.The niclosamide in situ sustained-release injection could protect bovines over 3 months in the field,which was a potential formulation to prevent and control schistosomiasis.Conclusion1.We chose praziquantel in situ sustained-re lease injection“PLGA:NMP:PZQ=1:3:2(w/v/w)" for further study considering the need of the field application to block echionococcosis transmission for 6 months and chose niclosamide in situ sustained-re lease injection "PLGA:NMP:NCA:T80=1:6:0.3:1(w/v/w/w)" for further study considering the need of the field application to block schistosomiasis transmission for 3 months.2.Enantioselectivity in the metabolism of R-PZQ and S-PZQ was observed in the study obviously after rac-PZQ in situ sustained-release injection(100 mg/kg)injected subcutaneously in beagle dogs.We speculated that the release time of R-PZQ was longer than S-PZQ in the new formulation so that the therapy time was longer as the R-PZQ was the effective enantiomer.The reason lies on the enantioselective metabolic differences and possible stereoselective release of racemic drugs from polymer,such as PLGA.So it's necessary to study the praziquantel enantiomer concentrations in plasma after subcutaneous administration of the formulation,not only for determining the minimize effective concentration of R-PZQ in the field trial,but also for optimizing the treatment protocol to block echionococcosis transmission based SDGs(Sustainable Development Goals).3.There was no significant difference between drug group and control group in the field trial to investigate the therapeutic efficacy of praziquantel in situ sustained-release injection against echionococcosis.The number of positive dogs may decresed rapidly after the government of China conduct a method as every dog is treated with praziquantel once every month,which is a significant measurement to control and prevent dog echinococcus.So the positive rate was 0.0%-2.0%in control group.In order to ensure the comparability of the results,we may choose areas with a higher positive rate of dogs in the next step,such as Tibet.At the same time,we should establish the laboratory model of dogs infected echinococcus to evaluate efficacy.4.The niclosamide in situ sustained-release injection could protect mice against Schistosoma japonicum infection over 2 months.The praziquantel in situ sustained-release injection could protect mice against Schistosoma japonicum infection over 1 month.But the mean burden was 15.1%on 15 days after treatment without significant difference,so we chose the niclosamide in situ sustained-release injection in field trial.The niclosamide in situ sustained-release injection could protect bo vines over 3 months in the field,which was a potential formulation to block schistosomiasis transmission.