Physicochemical surrogates for in vitro toxicity assessment of liposomal amphotericin B.

Pharmaceutical toxicity evaluations often use in vitro systems involving primary cells, cell lines or red blood cells (RBCs). Cell-based analyses ('bioassays') can be cumbersome and typically rely on hard-to-standardize biological materials. Amphotericin B (AmB) toxicity evaluations are primarily based on potassium release from RBCs and share these limitations. This study evaluates the potential substitution of two physicochemical AmB toxicity approaches for the bioassay: Ultraviolet-visible spectroscopy (UV-vis) and in vitro drug release kinetics. UV-vis spectral analyses indicated that liposomal AmB's (L-AmB) main peak position (λmax) and peak ratio (OD346/OD322) are potential toxicity surrogates. Similarly, two first-order release parameters derived from USP-4 in vitro drug release analyses also provided linear relationships with toxicity. These were the initial, overall drug release rate and the ratio of loose to tight AmB pools. Positive slopes and high correlation coefficients (R2 > 0.9) characterized all interrelations between physicochemical parameters and toxicity. These tests converted the manufacturing variables' nonlinear (i.e., curvilinear) relationships with in vitro toxicity to linear responses. Three different toxicity attenuation approaches (2 manufacturing, 1 formulation), covering formulation composition and process aspects, support this approach's universality. These data suggest that one or more spectral and kinetic physicochemical tests can be surrogates for L-AmB in vitro toxicity testing.

Cyclosporine release and distribution in ophthalmic emulsions determined by pulsatile microdialysis.

An in vitro release test based on pulsatile microdialysis (PMD) is presented for the purpose of measuring the release of cyclosporine from ophthalmic emulsions, along with a method to determine the drug distribution within the oil-rich globule, surfactant-rich micelle and aqueous phases of the emulsion formulation. Compositionally equivalent formulations containing 0.05% cyclosporine were prepared with similar physical parameters (globule size, viscosity, surface tension zeta potential, osmolality, pH) but made with different manufacturing conditions. Emulsions were made by ultrasonication, using different ultrasonication times (22-49 min) and temperatures (50-82 °C). Formulations were stored at room temperature (20 °C) and PMD was performed under two conditions, one in which the receiving medium temperature was 20 °C, and another in which the receiving medium temperature was 35 °C to mimic the temperature change expected when a drop of formulation is administered to the eye. The PMD release data were taken at release times of 20, 40, 60, 90, 120, 180, 300 and 600 s. All experiments showed a qualitatively similar release pattern, with a rapid initial rate of drug release (Release-1) for the first few minutes, followed by a much slower release (Release-2). In addition, imposing a sudden temperature change on the formulation was observed to affect the release, with some formulations releasing faster into receiver media at 35 °C than at 20 °C, while others released faster into 20 °C than 35 °C receiver media. The drug distribution was also calculated from PMD release data into 20 °C receiver media using a novel release kinetics model. The drug distribution varied among the formulations, with 54-77% of the cyclosporine in the oil phase of the emulsions. PMD is a promising method to evaluate how manufacturing-induced differences affect the distribution and release kinetics of cyclosporine within the emulsion formulation.