Studies On The Ophthalmic Submicron Emulsion Of Cyclosporin A

Cyclosporin A (CsA) was a cyclic polypeptide, including eleven amino acid. It was now routinely used as a selective immunosuppressor for organ transplantation. The immunosuppression of CsA was with high performance and non-toxicity on bone marrow. Over the past years, CsA had been evaluated for numerous potential applications in ophthalmology. It was effective in preventing recurrence of graft rejection after keratoplasty, dry eye, uveitis and so on. At present, CsA ophthalmic formulations were prepared and applied only in a few large-scale hospital in domestic and there were no CsA ophthalmic preparations in the market. Because of the poor solubility in water, CsA was usually utilized in clinic as the oily eye drop. However, the oily eye drop had severe irritation to the eye and sometimes the patients had the allergic response. As one of the excellent drug delivery systems, submicron emulsion had received extensive interests in the fields of ophthalmic preparations now. It could increase the amount of drugs with poor solubility loaded in the carrier efficiently. Furthermore, it could improve the ophthalmic bioavailability of drugs, because submicron emulsion had large dispersity and drugs were absorbed quickly. In this study, CsA ophthalmic submicron emulsion was prepared to decrease the eye’s irritation and elevate the ocular bioavailability of CsA. This study would provide a safe and effective ophthalmic immunosuppressant for clinic. A series of contents in this study were as follow:An HPLC method was established for determination of CsA. The method is simple, rapid, sensitive, reproducible and suitable for the quality control of CsA emulsion. The partition coefficient of CsA in oil/water showed that CsA had poor solubility in water but good liposolubility.High-speed stirring method was used to prepare CsA original emulsion. Then the original emulsion was homogenized with ultrasound method to obtain CsA ophthalmic submicron emulsion. The parameters such as Ke, particle size distribution,ζ-potential, the pH value change before or after sterilizing and the appearance, were selected as the index to screen excipients and preparation conditions. The composition of oil phase, the amount and proportion of surfactant was optimized. On the basis of the best formula, the preparation temperature, the pH value, the added method of surfactant, the power and times of ultrasound, the condition of sterilization were investigated to improve the stability of the submicron emulsion system.The appearance, pH value, drug contents, related substances were the main evaluation parameters to study the stability of CsA ophthalmic submicron emulsion. The result of affecting factors testing showed that the preparation was sensitive to light and temperature. It should be kept at proper temperature with protection from light. The parameters didn’t have conspicuous changes after the accelerated and long-term stabilitytesting.The positive and reverse dialysis bag techniques were performed to determine the release profiles of CsA in vitro, and the effect of different rotating speed on drug release was studied. The results of release tests showed that CsA was released more rapidly by reverse dialysis technique than positive one. The release rate increased obviously with the increase of rotating speed. The permeability characteristics in vitro of CsA through the isolated cornea of rabbits were studied. Compared with the CsA oily eye drop, the lag time of CsA ophthalmic submicron emulsion shortened and the apparent permeation coefficient of the emulsion increased 1.46 times. The results showed that emulsion can improve the permeability characteristics of CsA through the cornea.The results of rabbit ocular irritation tests of single dose and multiple dose suggested that CsA emulsion was safe to ocular tissues, but CsA oil solution was slightly irritating. The acute toxicity studies were carried out in mice by using maximum tolerable dose (MTD) method. The result showed that the MTD of mice was 0.3ml/10g. This dose in mice were approximately 250 times greater than the daily human dose, assuming that the entire dose of the CsA ophthalmic emulsion was absorbed.To evaluate the immunosuppressive effect of CsA ophthalmic emulsion the allogeneic corneal transplantation models in rabbit were established. CsA ophthalmic emulsion and dexamethasone (DM) eye drop were applied in the corneal transplantation models. The results indicated that CsA, DM, and the combined group could relieve the turbidity, edema and vascularization of corneal implant. These three groups could markly prolong the survival time of corneal grafts in corneal transplantation. The CsA group was superior to DM group and the combined therapy group was superior to CsA or DM group alone.