Study On Injectable In Situ Forming Implants Of Niclosamide Against Cercaria

Schistosomiasis is a kind of parasitic disease with serious damage to human health,which is caused by parasitic schistosoma in human blood system.Schistosomiasis is epidemic mainly in Africa,Asia,and South America.The population suffering from schistosomiasis had reached 200 million.Schistosomiasis is the second serious parasitic disease after malaria.Schistosoma japonica is the only human blood fluke that occurs in China.Prevention and treatment drugs for schistosomiasis include praziquantel,artemisinin and its derivatives,niclosamide,etc.Praziquantel served as the first-line drug for the treatment of schistosomiasis over a long period,which is likely to develop drug resistance for a wide range and repeated use in the long-term.Reports have covered that it is possible to induce drug resistance to praziquantel for Schistosoma mansoni and Schistosoma japonica.Praziquantel has weak effects on schistosomulum.There exist different developmental stages of schistosoma in patients because of the repeated infections which are frequently occurred in endemic area.It is difficult to cure for the single use of praziquantel.Artemisinin drugs are effective for schistosomulum,but the effect is weaker for adult worms,and they are usually acted as prevention drug and treatment for acute infection.Niclosamide has a high and long efficiency for snails.Besides,it presents good killing effect against snails eggs and cercaria.But the toxicity to aquatic species is a disadvantages that can not be conqured.Studies on the drugs against schistosoma cercaria has aroused people's attention because cercaria is the only stage infected people and livestock.Schistosomiasis epidemic situation in lake region is closely related to the characteristics of geographical environment.Mashland in epidemic area is large,which is natural breeding land for snails-an intermediate host of schistosoma.The grass in lake region are flourishing,and there is a large number of farm cattle and scattered cattle.The cattle suffered from schistosomiasis are the main source of the infection.These animals spend most of the time outside during the grazing season,which increase the chance for frequent contact with contaminated water.Transdermal preparations for animals usually works for several days and is easily influenced by external factors,such as water and skin metabolism,thus causing a drug reduction in skin.So controlled drug delivery systems are needed to improve extended efficiencies and relieve handling stresses.In situ forming implants technology is that the biodegradable polymers,such as PLGA or PLA,are dissolved in water-soluble solvents,and then represent a low viscosity of injectable fluid.Upon inject into the body,the water-soluble solvents quickly diffuse away from the initial solution into the body fluid,then polymer precipitates and form a solid biodegradable implants.Drug incorporated in the polymer matrix is then released by the biodegradation mechanism of the polymers.In persent study,injectable in situ forming implants of niclosamide of different release time were prepared by different molecular weigh of PLGA and different LA/GA ratios.Our study mainly includes preparation and formulation selection of injectable in situ forming implants of niclosamide,pharmacokinetics of niclosamide in mice,research on the minimum effective concentration of niclosamide against S.japonica cercaria,pharmacokinetics of injectable in situ forming implants of niclosamide and pharmacodynamics effects against cercaria.I Preparation and formulation screening of injectable in situ forming implants of niclosamideWe preliminarily determined the types of biodegradable polymers and biocompatible solvents for the preparation of injectable in situ forming implants of niclosamide through the pre-formulation.HPLC method was adopted to establish the in vitro analysis of niclosamide and used to determine the equilibrium solubility of niclosamide in aqueous medium and organic solvents.Phosphate buffer with 0.5%tween 80(pH 7.4)was chosen as the release medium.Single factor which may influence the release time of in situ forming implants was investigated by the first day release rate and release profiles.Orthogonal test was designed for the formulation screening,and we had done variance analysis for the influencing factors.Fitting process was conducted on release profiles,and the releasing mechanism were mainly composed of dissolution and skeleton diffusion.Two kinds of PLGA were combined with different proportions according to their different degradation rate,which served as the biodegradable polymers for the preparation of injectable in situ forming implants of niclosamide.Drug release time could be controlled by changing the ratio of the two kinds of PLGA.2 Pharmacokinetics of niclosamide in mice and the minimum effective concentration against cercariaHPLC and LC-MS methods were employed to establish the in vivo analysis of niclosamide.Niclosamide was orally administered with a dose of 120 mg/kg,collecting the plasma samples in different time.HPLC was ued to detect the drug concentration,and relative pharmacokinetics parameters was calculated by DAS 2.0.We observed pharmacodynamics effects of niclosamide on S.japornicum cercaria under different doses and different time points.LC-MS was used for further analysis of niclosamide in plasma,skin and muscle.Correlation of drug concentration and pharmacodynamics effects on cercaria was acquired.We used S.japornicum cercaria to attack the mice 0.25 hour after an oral administration of niclosamide with a dose of 200 mg/kg,and the worm reduction was 79.1%.The drug concentrations in plasma,skin and muscle determined at the same points were 889.122±1015.461 ng/ml,2369.013±495.618 ng/g and 230.54±61.789 ng/g,respectively,which could refer to the minimum effective concentration of cercaricide.3 Pharmacokinetics and pharmacodynamics of injectable in situ forming implants of niclosamide in miceTwo formulations with different release durations(named form land form 2)were selected and subcutaneously injected into mice.LC-MS was used to determine drug concentration in plasma,skin and muscle at 1 d,15 d,30 d,60 d and 90 d post treatment.The results indicated that drug concentration of form 1 of niclosamide after 1 d of the treatment was significantly higher than the minimum effective concentration in plasma,skin and muscle,while form 2 surpass the minimum effective concentration at 30 d.And at the other time points,the two implants couldn't meet the requirement of minimum effective concentration.Form 1 were subcutaneously injected into mice.All the mice were infected with(40±2)japonicum cercariae at the 90th day post treatment.Worm reduction of this formulation is 86.75%compared with control group.