Biopharmaceutical characterization of the telomerase inhibitor BRACO19

To characterize the telomerase inhibitor and G-quadruplex stabilizing substance 9-[4-(N,N-dimethylamino)phenylamino]-3,6-bis (3-pyrrolodino-propionamido) acridine x 3HCl (BRACO19) in terms of biopharmaceutical properties such as solubility, protein binding, interaction with membrane lipids, cytotoxicity, and permeability across pulmonary epithelial cells. Protein binding and interaction with membrane lipids were investigated by two high-performance liquid chromatography methods with immobilized human serum albumin and immobilized phosphatidylcholine, respectively. Cytotoxicity (methyl-thiazolyl-tetrazolium assay) and transport studies were performed with the bronchial cell lines 16HBE14o(-) and Calu-3, primary human alveolar epithelial cells, and the intestinal cell line Caco-2. Transport experiments were also done in the presence of cyclosporin A (10 mu M) and tetraethylammonium chloride (5 mM) and at low temperature (4 degrees C). BRACO19 has good solubility of at least 2 mg/mL in water and in physiological buffers of pH 7.4 and below. Protein binding to human serum albumin was 38%. No interaction with membrane lipids could be found. Cytotoxicity in 16HBE14o(-), Calu-3, and human alveolar epithelial cells was in the range of IC50 = 3.5 to 13.5 mu M. Caco-2 cells were not affected at concentrations up to 50 mu M. No transport of BRACO19 was detected across either cell monolayer in absorptive direction. In secretory direction, permeability was very low, with P-app values in the range of 0.25x10(-7) to 0.98x10(-7) cm/s for all epithelial cell cultures tested. The transport was not influenced by cyclosporin A or tetraethylammonium chloride or at 4 degrees C, indicating that no efflux/influx systems or active transport are involved. From these results, we conclude that the very poor permeability of BRACO19 is its main biopharmaceutical limitation. Further applications will require a suitable formulation to warrant adequate delivery across cellular barriers.