T-OA And T-DA Solid Dispersions And Microemulsions Are Studied In Vivo And In Vitro, And The Concept Of Early Preparation Intervention Is Proposed

Objectives:The goal of this study was to put forward Early-stage Preparation Discovery Concept(EPDC),introduce its development principles,and carry out further studies to validate it.We chose a water-insoluble lead compound with clear anti-tumor function(T-OA)as the model drug.Applied solid dispersion as a method of early-stage preparations,to enhance the solubility and bioavailability of T-OA.We also evaluated the effects of solid dispersions on improving the solubility and bioavailability by applying in vitro dissolution study and in vivo pharmacokinetics study.After designed several early-stage preparation methods,we compared these methods,validated their effect on improving the water solubility and in vivo absorption.At last we chose one of the methods to perform preliminary studies on a lead compound T-DA with similar structure as T-OA,in order to test the feasibility of EPDC.Meanwhile we built up and improved EPDC technology system by comparing different EPDC methods.Methods:This study included three major parts.The first part was about studying solid dispersions formulation and pharmacokinetics of T-OA.High performance liquid chromatography(HPLC)was used to determine the content.In the formulation studies,we applied melt method and then solvent method on some comlon drug delivery carriers,PEG 6000,PEG 4000 and Poloxamer F68.The formulation was optimized,based on the studies of drug carrier ratio,surfactant and dissolution medium.We also used X-ray diffraction,Infrared spectroscopy,DSC and Scanning Electron Microscopy to further study solid dispersion.We used SD rats as model axnimals,built a method for plasma sample treatment and measuring drug concentration in plasma.The pharmacokinetics of pure drug and its solid dispersions were compared.We measured the degree of enhancement that solid dispersions had on T-OA absorption by analyzing the data of pharmacokinetics with Kinetica 4.4 software's noncompartmental model.The second part was about studying microemulsion formulation and pharmacokinetics of T-OA.High performance liquid chromatography(HPLC)was used to determine the content.In the formulation studies,we investigated effects of several oils,surfactants and cosurfactants on T-OA's solubility.Through building Pseudo-ternary phase diagram,we found the best ratio of surfactant and cosurfactants(Km)and the optimized oil-in-water microemulsion formulation.We measured the viscosity,pH,particle size and Zeta potential in vitro and did Transmission Electron Microscopy analysis as well.We compared the pharmacokinetics of oleic acid solution and microemulsion of T-OA on SD rats.The data of pharmacokinetics were analyzed with Kinetica 4.4 software's noncompartmental model.Meanwhile,we compared microemulsion to solid dispersions on how various early-stage methods differently affect the degree of enhancement of T-OA absorption.At last,we did in vitro dilution experiment to further analyze the in vivo results.The third part was about studying microemulsion formulation and pharmacokinetics of T-DA.High performance liquid chromatography(HPLC)was used to determine the content.T-DA I was prepared according to T-OA microemulsion formulation based on EPDC.In the formulation studies,we investigated effects of several oils,surfactants and eosurfactants on T-DA's solubility.Through building Pseudo-temarn phase diagram,we found the best ratio of surfactant and cosurfactants(Km)and got the optimized oil-in-water microemulsion formulation T-DA ?.We measured the viscosity,pH,particle size,Zeta potential and electron microscopy analysis on T-DA ? and ?.Also the pharmacokinetics of those two microemulsion formulations of T-DA on SD rats was studied.The data of pharmacokinetics were analyzed with Kinetica 4.4 software's noncompartmental model.So the feasibility of the application of EPDC on the formulations of other similar compounds was preliminarily evaluated.Results:HPLC was applied to detect T-OA in each preparation and the methodology validation was proved.Methodology results indicated that all the measurements meet the requirements.The melt method of T-OA showed that PEG 4000,PEG 6000 or Poloxamer F 68 could not form good solid dispersions,which means the melt method should be given up in accordance with EPDC development principles.The best ratio of drug and delivery carrier was 1:5 for solvent method solid dispersions.Adding SDS could improve the dissolution uniformity among different batches of solid dispersions,and the best drug/carrier/SDS ratio of 1:5:0.06.More than 90%of T-OA dissolved in pH 4.5,pH 6.8 or water in 10 minutes,while only 50%of T-OA dissolved in pH 1.0,and the dissolution decreased very fast,which indicated that the formulation was suitable to be made enteric solid dispersions in the future.The DSC test showed that the crystal type changed in the solid dispersions and further X-ray diffraction result showed the drug was in amorphous form.Infrared spectroscopy results indicated the drug's hydroxyl bond and carbonyl bond could have physical and chemical reactions with the carrier in solid dispersions.Scanning Electron Microscopy results showed that the drug's crystal form disappeared in solid dispersions and the added SDS could help prevent the drug from forming crystals in the solid dispersion process.In vivo tests of solid dispersions showed four times the bioavailability of pure drug.In the solubility study of T-OA,oleic acid as oleic phase,Tween 80 as the surfactant and anhydrous ethanol as the cosurfacant were selected.Pseudo-ternary phase diagram analysis found the best surfactant/cosurfactant ratio(Km)to be 3:7.Under consideration of low surfactant amount and high drug loaded,we prepared T?OA oil-in-water microemulsions.In the microemulsion preparation,several results showed the drug concentration 20mg/ml,droplet size 70 nm,viscosity 15.57 mpa·s,electrical conductivity 44.1?S·cm-1 and Zeta electric potential-0.174mV.The result of Transmission Electron Microscopy confirmed the formation of oil-in-water microemulsion and the particle size was consistent with Melvin measurement.The pharmacokinetics of T-OA microemulsion showed that its bioavailability was 57 times greater than that of pure drug,14 times greater than that of solid dispersions.Similarly,bioavailability of T-OA oleic acid solution was also increased significantly compared to solid dispersions as an early-stage preparation.Therefore,we added extra dilution experiments for both solid dispersions and microemulsions to find the mechanism underlying the difference.The results indicated that the ability of the drug in solid dispersions to dissolve declined very quickly,the dissolution ratio decreased from 11.6%to 1.0%in 4 h,and remained insoluble until 48h,which means the drug could form crystals in the process and therefore affect the bioavailability;on the other hand,the drug concentration in microemulsion kept increasing,the dissolution ratio increased from 4.3%to 9.3%in 6 h,and remained 12.8%until 48 h.So it was deduced that in microemulsions and oleic acid solution,as two EPDC methods,drug was easier to maintain its molecular state to benefit drug absorption in vivo.EPDC was validated in the T-DA's microemulsion study.T-DA microemulsions I was prepared in the formulation optimized for T-OA microemulsions.In the solubility study of T-DA,oleic acid as oleic phase,Tween 80 as the surfactant and isopropanol as the cosurfacant were selected.Pseudo-temary phase diagram analysis found the best surfactant/cosurfactant ratio(Km)to be 4:6.Under consideration of low surfactant amount and high water loaded,we prepared T-DA oil-in-water microemulsions ?.Both T-DA1 and ? were prepared in 50 mg/ml oil-in-water microemulsions and were compared side to side.T-DA I was composed with oleic acid/Tween 80/anhydrous ethanol/water at the weight ratio of 0.20:0.16:0.38:0.26,T-DA? was composed with oleic acid/Tween 80/isopropanol/water at the weight ratio of 0.12:0.20:0.30:0.38.More measurements were 50 nm(particle size),2.80 mpa·s(viscosity),43.8 ?S·cm-1(electrical conductivity),4.91(pH)and-0.147 mV(Zeta potential)for T-DA I,and 76 nm(particle size),2.80 mpa·s(viscosity),75.2?S·cm-1(electrical conductivity),5.21(pH)and-0.144 mV(Zeta potential)for T-DA ?.Therefore both T-DA ? and T-DA ? had similar in vitro properties.The analysis of pharmacokinetics in rats showed that there is no significant difference between T-DA ? and T-DA ? in Tmax,Cmax,t1/2,MRT and AUC.Conclusions:The three early-stage preparations of T-OA,solid dispersions,microemulsion,and oleic acid solution,all can improve drug's in vitro dissolving performance and in vivo bioavailability.The solid dispersions had 4 times bioavailability of pure drug;microemulsion had 57 times bioavailability of pure drug and 14 times bioavailability of solid dispersions.These three technologies all meet the standards required by EPDC,and so they all can be components of EPDC technology system.T-OA formulation could be directly applied to T-DA,also the solubility of T-DA was enhanced,which proved EPDC could be successfully used.In the study of T-DA microemulsion,formulation ? optimized for T-DA microemulsion and formulation I optimized for T-OA,there was no significant difference between them,which means that T-OA formulation based on EPDC is applicable to similar compound,i.e.T-DA.This study shows preliminary results validating the feasibility of EPDC and also indicated that EPDC based formulation has the versatility to be applied to similar compounds.