| Ivermectin (IVM) is a semi-synthetic macrolide multicomponent antiparasitic, with advantages of broad-spectrum, high efficiency, small dosage and safety. IVM is of highly efficient against parasites and arthropods. IVM is widely used to control livestock-related parasitic infections with several administration ways such as oral administration subcutaneous and poured. In this paper, biodegradable polymer RB as matrix of precipitated in situ forming gel and high polymer sustained-release material PLA were used to prepare IVM injectable in situ forming gel, achieving goals of sustained release, prolonged drug duration of action, saving manpower, material and financial resources and better playing its role in prevention and treatment of parasitic infections. The details are as follows:1. Preformulation studies The equilibrium solubility and partition coefficient of IVM were determined and high performance liquid chromatography (HPLC) method for detection of IVM in vitro was established. IVM was soluble in ethyl acetate, NMP and other organic reagents; The LogP was 2.02, showing its highly lopophilic. Chromatographic conditions:mobile phase was methanol:water (95:5), UV detection wavelength was set at 245 nm, column temperature was set at 35℃, flow rate was set at 0.8 mL/min. IVM concentration was of good linear relationship at 0.2～50μg/mL for the liner regression equation was Ar=23393C-1218.6(r2=0.9997). Specific, precision and recoveries were all in line with the methodology requirements.2. Screening of prescription component content Prescription component content were screened based on 24 h burst release 30 d cumulative release. The results showed that: â‘ when conten of solvents decreased from 25% to 15% of IVM in situ forming gel that were based on ethyl acetate, NMP and benzyl benzoate, the burst release were respectively decreased from 52.3% to 33.5%, from 46.4% to 30.1%, from 54.1% to 35.6%, and the cumulative release decreased from 82.4% to 62.1%, from 73.3% to 53.9%, from 92% to 56.3%; the minimum burst release and cumulative release were found in formulations with NMP and maximum burst release and cumulative release were found in formulations with benzyl benzoate of the same solvent concentration. â‘¡When concentration of IVM raised to 2% and 4% from 1%, cumulative release from the gel of the same sampling time point was 2.4-2.9 and 3.1-3.7 times that of 1%. â‘¢with the addition of 0%,1%,3%,5% of PLA in the NMP gel, burst release and cumulative release were respectively 38.5%,27.0%, 23.1%,13.9% and 67.7%,66.8%,53.9%,43.8%. â‘£The optimal prescription was:Each 100mL contains 4 g IVM,15 mL NMP,5 g PLA, the rest of the matrix RB。3. Prescription process reproducibility, physicochemical properties and preliminary stability study Three sample batches were prepared based on the optimal formulation and process of IVM injectable in situ forming gel. Results showed that:three were no significant difference in drug content and release among the three batches of IVM injectable in situ forming gel, indicating good reproducibility of prescription and process; cumulative release was about 80% in 80 d, and the drug release mechanism might be synergistic effect of drug diffusion and matrix erosion. The gels prepared were of good formability, low viscosity, and were easy to inject. Gels were sensitive to strong light and stable to heat. IVM injectable in situ forming gel behaved good stability when packed with brown vials and placed at 40℃, RH 75% for 6 months.4. Establishment of HPLC-fluorescence detection method to quantify IVM concentration in plasma Chromatographic conditions:mobile phase was methanol:water (97:3), excitation wavelength was (Ex) 365 nm and emission wavelength was (Em) 465 nm of the fluorescence detector, column temperature was set at 40℃, flow rate was set at 1.0 mL/min. IVM concentration was of good linear relationship at 1～250 ng/mL for the liner regression equation was Ar=54174X+124396(r2=0.9991); the method was specific; limit of detection was 0.5 ng/mL and quantitation limit was 1 ng/mL; precision and recoveries were all in line with the methodology requirements. Plasma samples were extracted with ethyl acetate and acetonitrile, and then derivatized with N-methyl-imidazole solution and trifluoroacetic anhydride solution; the etraction and derivation method were of high recovery rate, high degree of separation and no impurity peaks.5. Pharmacokinetics study of 4% IVM injectable in situ forming gel Twelve dogs were randomLy divided into two groups, which were subcutaneously injected with 4 mg/kg of IVM in situ forming gel and 0.5 mg/kg IVM conventional injection respectably. Plasma concentrations were analyzed by method of HPLC-fluorescence; then the plasma concentration-time data were performed using pharmacokinetic software Winnonlin with non-compartment model. The main pharmacokinetic parameters were as followed:For IVM injectable in situ forming gel, AUC0â†'∞ were (5383.12±241.97) d-ng/mL; AUC0â†'∞ were (5379.68±241.97) d-ng/mL; t1/2λz were (24.37±1.71)d; tmax were (4.83±2.48) d; Cmax were (182.93±26.47) ng/mL; MRT were (32.86±0.91)d. For IVM conventional injection, AUC0â†'∞,were (772.89±44.48) d-ng/mL; AUC0â†'t were (767.53±48.13) d·ng/mL; t1/2λz were (4.80±1.0) d; tmax were (2.00±0.00) d; Cmax were (149.54±15.33) ng/mL; MRT were (7.24±0.69) d. Compared with IVM conventional injection, IVM injectable in situ forming gel displayed a much longer terminal elimination half life and mean residence time in vivo and effective blood concentration could be maintained around 110～120 d, suggesting the significant effect of sustained release. |
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