Résumé
In order to meet the clinical needs of long-acting sustained-release thienorphine, injectable thienorphine loaded microspheres were developed, and the accelerated stability study was carried out to explore the suitable storage and transportation conditions of the microspheres. Using poly(lactic-co-glycolic acid) (PLGA) as carrier material, 3 batches of microspheres were prepared in pilot scale with emulsion solvent evaporation method. By investigating the in vitro release of thienorphine loaded microspheres at 37, 45, 52, and 60 ℃, and applying the Arrhenius equation, the linear relationship between the release rate constant (lgk) and the temperature (1/T) was established to obtain the equation: lgk = -8.073/T + 24.35 (R2 = 0.985 3), which showed that the release of microspheres at high temperature can be used to predict the release in vitro at 37 ℃, and 52.0 ± 0.5 ℃ was selected as the accelerated release condition in vitro. The quality research methods were established to investigate the changes of critical quality attributes such as microsphere morphology, drug loading, particle size and distribution, polymer molecular weight, and the related substances under accelerated conditions. The difference factor f1 and similarity factor f2 were used to assess the similarity of release behavior under accelerated conditions. The results showed that under the accelerated experimental conditions of 25 ± 2 ℃ and relative humidity (RH) 60% ± 5%, the critical quality attributes of injectable thienorphine loaded microspheres had no significant change in 6 months, suggesting that the long-term storage condition could be 5 ± 3 ℃.
Résumé
Objective: To establish a high performance liquid chromatography(HPLC)method for the determi- nation of the drug content in thienorphine microspheres. Methods: A COSMOSIL C18 column was used for the HPLC analysis. The mobile phase consisted of acetonitrile -0.02 mol/L phosphate buffer solution(pH=3,containing 0.2% trieth- ylamine)(45:55,V/V)with a flow rate of 1.0 ml/min,and the detection wavelength was 232 nm. The column tempera- ture was 35℃ and the injection volume was 20 μl. Results The quantification limit was 0.10 μg/ml and the detection limit was 0.05 μg/ml. The linear equation for the thienorphine was Y=34.615X,and the calibration curves were linear within the range of 2.14-40.27 μg/m(l r=0.9999). The average recovery for each concentration ranged within 98%-102% (RSD%=1.12%). The drug loading for four batches of microspheres with the same prescription and preparation process was measured by two different Methods:,which produced similar results. Conclusion: The detection method is of good specificity,simplicity,rapidity,sensitivity,reproducibility and high precision,and can be used for the determination of the drug content in thienorphine microspheres.
Résumé
The inter-species differences of thienorphine metabolism were investigated in human, Beagle dog and rat liver microsomes, by comparing enzyme kinetics of the parent drug and the formation of its major metabolites. The incubation systems of thienorphine with liver microsomes of the three species were optimized in terms of thienorphine concentration, microsomal protein content and incubation time. The concentrations of thienorphine and its metabolites in incubates were measured by a LC-MS/MS method. The biotransformation of thienorphine by human liver microsomes was the lowest among the three species. The Km, Vmax, CLint and T1/2 of thienorphine obtained from human liver microsomes were (4.00 ? 0.59) ?mol?L-1, (0.21 ? 0.06) ?mol?L-1?min-1, (117 ? 3.19) mL?min-1?kg-1 and (223 ? 6.10) min, respectively. The corresponding kinetic parameters for dog and rat liver microsomes were (3.57 ? 0.69) and (3.28 ? 0.50) ?mol?L-1, (0.18 ? 0.04) and (0.14 ? 0.04) ?mol?L-1?min-1, (213 ? 1.06) and (527 ? 7.79) mL?min-1?kg-1, (244 ? 1.21) and (70.7 ? 1.05) min, respectively. A total of six phase I metabolites were observed in liver microsomes, including one N-dealkylated metabolite, three oxidative metabolites and two N-dealkylated oxidation metabolites. All these six metabolites were detected in the liver microsomes of the three species. However, the relative amounts of the metabolites generated were different in three species. The results indicated that the major phase I metabolic pathway of thienorphine was similar in the liver microsomes from all three species. However, the inter-species differencesobserved were relative amounts of the metabolites as well as the metabolic characteristics of thienorphine in liver microsomal incubates.