The Study Of Formulation Optimization, Acute Toxicity, And Pharmacokinetics Of Shuangteng Microemulsion Gel

ObejiectiveShuangteng microemulsion gel developed by our laboratory with traditional Chinese medicine of tripterygium wilfordii extract and sinomenium extract, used in the treatment of rheumatoid arthritis was a partial external preparation. Previous studies have identified the Shuangteng microemulsion gel formulation and process of the research. In order to further optimize the Shuangteng microemulsion gel of tripterygium wilfordii extract and sinomenium extract in proportion, to evaluate the optimized Shuangteng microemulsion gel in vivo and in vitro, as well as to provide experiment basis for the feasibility of Shuangteng microemulsion gel developed as a percutaneous prepation, this study of Shuangteng microemulsion gel ratio optimization, acute toxicity study, in vitro release of inspection, and skin pharmacokinetics in rats in vivo and in vivo rabbit articular cavity, plasma pharmacokinetic study were carreid out.Methods and Results1 The ratio optimization and acute toxicity study of microemulsion gel1.1 Optimization of ratio and dosage of extract of Tripterygium wilfordii and extract of Sinomenium acutum in ShuangTeng microuemulsion gel uing central composite designThe dosage of tripterygium wilfordii extract and sinomenium extract were used as independent variables and difference amount of serum GPI, IL-1, TNF-a between before and after administration of ShuangTeng microemulsion gel was used as dependent variables. Experiment was designed by Design expert 8.0.6 soft and carried out, then a second order polynomial equation was fitted to the variables and the resulting equation was used to predict response in optimal region. Results:The ratio of sinomenium extract and triptergium wilfordii extract was 5.21:1, and the best dosage of ShuangTeng microemulsion gel was 1.33g/kg.1.2 The acute toxicity study of Shuangteng microemulsion gelAccording to the classical mouse acute toxicity test method, the acute toxicity was carried out by oral administration with Shuangteng microemulsion and transdermal administration with Shuangteng microemulsion gel. Test data that was used to calculate the half lethal dose (LD50) was got by observation 14 days after administration. Results:The LD50 was 16.72mL/kg by oral means and the 95% confidence range was 13.09-21.58 mL/kg. Percutaneous drug delivery can’t make the LD50 in mice. In addition, mice did not show any toxic effects under dosage of 5 times the normal dosage.2 Investigation of release properties of sinomenine and triptolide in ShuangTeng microemulsion gelDialysis bag method was used to research the release characteristics of-ShuangTeng microemulsion gel in vitro drug release properties, with 20% ethanol/saline used as release medium. Concentration of sinomenine and Triptolide was determined by high performance liquid chromatography (HPLC). Results:The release equation of sinomenine and Triptolide were Q=363.55t-133.98 and Q=1.25t+2.67 respectively. The accumulative release rate of sinomenine and Triptolide in 12h reached 95% and 81.4% respectively and the cumulative release rate equation of time accords with zero order release rate equation.3 pharmacokinetics study of Shuangteng microemulsion gel3.1 Assessment of subcutaneous and articular microdialysis sampling for sinomenine and triptolide in vivoThe impact of the flow rate from 0.5 to 2.5 μL min-1 to the probe recovery rate was evaluated by in vitro microdialysis method and the optimal flow rate was determined by recovery rate and factual sampling volmue; the impact of drug concentration to probe recovery rate was also evaluated to ensure that the reverse dialysis method can be used in vivo microdialysis experiment; the skin and articular probe recovery rate stability of sinomenine and triptolide in vivo was studied in 10 hours and the determind probe recovery of sinomenine and triptolide could later be used to revise the actual drug concentraion in dialyaste. Results:1 μL min-1 was chose as the optimal flow rate, at which the probe showed the best recovery rate and the sampling volume can reached to 30 μL in 30 minutes, which meet to the requirement of HPLC and LC-MS/MS; the drug concentration made no difference to the probe recovery rate, whichi denoted that microdialysis method can be used in the reserch of pharmacokinetics study; the probe recovery rate of sinominine and triptolide generally stay stable in 10 hours and the mean recovery rate of sinomenine and triptolide in skin probe was (44.68+1.28)%、(51.35±1.15)%respectively in 10 hours and mean recovery rate of sinomenine and triptolide in articular probe was (44.68±1.28)%、(51.35±1.15)% respectively, which can be used to revise the drug concentraion in dialysate.3.2 Dermal pharmacokinetic study of ShuangTeng microemulsion gel by microdialysis combined with HPLCHaving ShuangTeng microemulsion gel applied to the abdomen of rats, dialysate was collected using micro dialysis sampling technology and the index compounds, triptolide and sinomenine in the dialysate, which were one of the effective components of tripterygium wilfordii and caulis sinomenii respectively, were determined by high performance liquid chromatography method. Drug concentration in the skin was calculated through the relative loss of correction and relevant parameters of statistical moment was calculated by non-compartment model using PKSolver2.0 software. The steady absorption rate was calculated by linear regression of cumulative release to time. Results:The time to peak (tmax) of triptolide was 2.3 h, which was the maximum skin concentration appeared at 2.3 h after drug administration. The mean residence time (MRT) was about 14 h and the half time (t1/2) was about 8 h. The steady absorption rate was 54.54 ng/h; the time to peak (tmax) of sinomenine was 2.3 h, which was the maximum skin concentration appeared at 2.8 h after drug administration. The mean residence time (MRT) was about 9.18 h and the half time (t1/2) was about 4.7 h. The steady absorption rate was 3.54μg/h.3.3 Joint and blood pharmacokinetic study of ShuangTeng microemulsion gel by microdialysis combined with LC-MSSinomenine was used as index ingredient in Shuangteng microemulsion gel. After local joint administration and oral administration of Shuangteng microemulsion gel, dialysate in joint and serum sample extracted from the rat’ s vein was collected every 1 hour. The sinomenine concentration was determined by LC-MS and PK-Solver soft ware was used to calculate the pharmacokinetics parameters. Results:After local joint administration of local joint administration and oral administration of Shuangteng microemulsion gel, the pharmacokinetics parameters in joint and blood were blow. The t1/2 were 2.97 h,5.86 h respectively; Tmax were 3 h,4 h respectively; Cmax were 1723.83 ng/mL; 33.45 ng/mL respectively; AUCO-t were 4712.62 ng/mL* h,184.50 ng/mL * h respectively; MRTO inf_obs were 5.29 h,9.24 h respectively. After local joint administration of oral administration and oral administration of Shuangteng microemulsion gel, the pharmacokinetics parameters in joint and blood were blow. The t1/2 were 2.22 h,4.99 h, Tmax were 1 h,1 h respectively; Cmax were 128.24 ng/mL,705.75 ng/mLrespectively; AUCO-t were 359.96 ng/mL* h,3718.50 ng/mL* h respectively; MRTO inf_obs were 3.62 h,7.98 h respectively.The Cmax (723.83 ng/mL) of sinomenine in articular cavity after topical administration was significantly higher than that (128.24 ng/mL) after oral administration The Cmax (33.45 ng/mL) of sinomenine in blood after topical administration was significantly lower than that (705.75 ng/mL) after oral administration, which indicated that topical administration promoted curative effect for RA and reduced drug toxicity compared with oral administrationConclusionThe optimized ratio of sinomenium extract and triptergium wilfordii extract was 5.21:1, and the best dosage of ShuangTeng microemulsion gel was 1.33g/kg. The optimized Shuangteng microemulsion gel showed relatively better efficacy for freund’s adjuvant model rat than primary prescription (1:1) and did not showed any acute toxicity symptoms on mouse after topical administration. The Cmax (723.83 ng/mL) of sinomenine in articular cavity after topical administration was significantly higher than that (128.24 ng/mL) after oral administration The Cmax (33.45 ng/mL) of sinomenine in blood after topical administration was significantly lower than that (705.75 ng/mL) after oral administration, which indicated that topical administration promoted curative effect for RA and reduced drug toxicity compared with oral administration. Above all, Shuangteng microemulsion gel are suitable to be made into trandermal formulation for RA.