Studies On 1,25-dihvdroxyvitamin D2 Nanoemulsion Injections

1,25-dihydroxy vitamin D2, a derivant of vitamin D2, is used to treat the secondary hyperparathyroidiem(SHPT). It shows a low incidence of hypercalcemia,and hyperphospheremia compared to some other drugs.The commercial available formulations of 1,25-dihydroxy vitamin D2 are soft capsules and injections at present.Most studies focused on solubilizing it into water by adding surfactants and cosurfactants due to its insolubility. In this paper, we prepared the1,25-dihydroxy vitamin D2 nanoemulsion injection which can decrease the content of surfactant and cosurfactant. The results showed that the formulation has a good stability and the in vivo testing suggested a low level of toxicity and an improvement of clinical applicability of the nanoemulsion injection. Since the process of preparation is simple and cost-effective, the production of the 1,25-dihydroxy vitamin D2 nanoemulsion injection could be enlarged easily, and this study might offer some novel ideas for further studies.A HPLC method was established for 1,25-dihydroxy vitamin D2 determination,drug loading and the impurities of the formulation. The UV wavelength scaning showed that the maximum absorption wavelengthes of 1,25-dihydroxy vitamin D2 and its related substance were 265 nm and 274 nm respectively. The chromatographic column was octadecylsilane chemically bonded silica and mobile phase was acetonitrile:water 85:15(V/V). It had good linear at the rangeof concentration of 0.06μg/mL to 4.00μg/mL, which could be used for determination of the 1,25-dihydroxy vitamin D2 in the nanoemulsion injection. The method was proved to be reproducible,accurate, sensitive and specific through the methodology validation.The solubility of 1,25-dihydroxy vitamin D2 varies from different adjuvants, so we screened different kinds of surfacntants, cosurfactants, and oils as well as their proportion based on ternary phase diagram. The optimal formulationconsist oftween80 as the surfactant, ethanol as the cosurface and MCT as the oil phase was ultimately determined through the stability and solubility testing. However, the 1,25-dihydroxy vitamin D2 is prone to being oxidized, so we also added BHT into the formulation as the antioxidant.The 1,25-dihydroxy vitamin D2 nanoemulsion was characterized in terms of content, related substances, pH, particle size distribution and osmotic pressure. The nanoemulsion particle size was between 20 nm and 50 nm; the pH of the injectionwas between 6 and 8 and the osmotic pressure was between 285 m Osmol/Kg and340mOsmol/Kg. The results of stress testing showed that 1,25-dihydroxy vitamin D2 was sensitive to high temperature and light. Therefore the preparation should be stored at low temperature and in dark place. The results of the accelerated test(25℃)and long-term stability test(2~6 ℃) showed its stability with the same packages as market ones. Compared with the market injections, our preparation is more stable and has the lower side effect. The content of 1,25-dihydroxy vitamin D2 in the injection decreased less and the related substances increased less than the market injection in40℃ temperature test and room temperature.Comparaed with the market injection, no hemolysis and swelling were observed at 48 h and 96 h in the safety evaluation and the blood vessels stimulatory test after injecting the nanoemulsion at a high concentration through rabbit’s auricular vein,while the market injection can cause a significant swelling on skin in tests above.Moreover, observations of pathological sections showed that no inflammatory was caused by injecting nanoemulsion of high drug concentration and the blood vessel wall was integrated, demonstrating the high level of safety of our formulation.In conclusion, the nanoemulsion injection of 1,25-dihydroxy vitamin D2 was prepared with an improvement of drug loading and a low usage of surfactant and cosurfactant. It also promoted the stability of 1,25-dihydroxy vitamin D2 and lowered the side effects in vivo. The preparation had the feasible preparing technology,controllable quality and stable process that was promising to be enlarged in factory.