Toxicity Evaluation of a Novel Rapamycin Liposomal Formulation After Subconjunctival and Intravitreal Injection.

Purpose: Safety and toxicity evaluation of a novel, liposome-encapsulated rapamycin formulation, intended for autoimmune ocular disorders. Methods: The formulation was assessed by micronucleus polychromatic erythrocyte production, irritability by Hen's Egg Test-Chorioallantoic Membrane (HET CAM), sterility, and pyrogenicity testing. Subconjunctival (SCJ) and intravitreal (IVT) administration of the formulation were performed to evaluate subacute and acute toxicity, respectively. Differences between groups in biochemical and hematological parameters were evaluated by analysis of variance and t-tests. Numeric score was assigned to histopathological classification. Electroretinography (ERG) testing was also performed. Data were analyzed by a 1 way no parametric Kruskal-Wallis and the Mann-Whitney tests. Significance was considered when P < 0.05. Results: No significant toxicity directly related to the preparation was detected. Micronucleus count, mucous irritation score, and pyrogenicity results were negative. Pathology demonstrated no damage related to the formulation after SCJ injection. After IVT injection, only lens injury associated with technique was observed. Retinal function was also conserved in ERG. Conclusions: The preparation evaluated offers a good toxicity and safety profile when injected in a SCJ or IVT manner in an animal model. A clinical trial conducted in humans is highly warranted, as it could reveal an alternative immunosuppressive treatment for ophthalmological immune-mediated pathologies.

Preformulation Studies of a Liposomal Formulation Containing Sirolimus for the Treatment of Dry Eye Disease.

PURPOSE: The aim of this study was to develop and characterize a liposomal product containing sirolimus to be administered subconjunctivally for the treatment of nonresponsive keratoconjunctivitis sicca (KCS) or dry eye. METHODS: Formulations were prepared using an ethanol injection method and an adaptation of the heating method in pursuance of the most suitable methodology for future industrial production. Liposomes were loaded with either a high dose of 1 mg/mL of sirolimus or a less toxic dose of 0.4 mg/mL. The effects of critical process and formulation parameters were investigated. Liposomes were characterized in terms of size, zeta potential, polydispersity, differential scanning calorimetry, morphology, entrapment efficiency, phospholipid content, thermal stability, and sterility. The formulation was evaluated clinically in dogs with spontaneous KCS. RESULTS: Sterile liposomal dispersions with sizes ranging from 140 to 211 nm, were successfully obtained. High entrapment efficiency of 93%-98% was achieved. The heating method allowed an easier production of liposomes with high entrapment efficiency, to significantly shorten production time and the elimination of the use of alcohol. The poor stability of the obtained liposomes in aqueous dispersion made the inclusion of a lyophilization step necessary to the manufacturing process. In vivo testing of the liposomal sirolimus formulations in the spontaneous KCS dog model have produced promising results, particularly with a sirolimus dose of 1 mg/mL, indicating the need for further development and study of proposed formulations in the treatment of canine KCS. Clinical improvement in tear production in dogs with spontaneous KCS treated with the 1 mg/mL dose product was observed. CONCLUSIONS: The heating method allowed easier production of high entrapment efficiency liposomes to significantly shorten production time and the elimination of the use of alcohol. Tear production was increased in dogs administered with the formulation.