Preformulation studies of EFdA, a novel nucleoside reverse transcriptase inhibitor for HIV prevention

Abstract

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a novel nucleoside analog of great interest because of its superior activity against wild-type and multidrug-resistant HIV-1 strains, and favorable safety profiles in vitro and in vivo. The aim of this work was to provide preformulation information of EFdA important for delivery system development. A simple, accurate and specific reverse-phase high performance liquid chromatographic (RP-HPLC) method with UV detection was developed for quantification of EFdA. In addition, physicochemical characterizations including pH solubility profile, octanol/water partition coefficient (Log P_o/w), DSC analysis, field emission scanning electron microscopy, and stability studies under various conditions were conducted. EFdA existed in planar or flake shape, with a melting point of ∼130 °C, and had a pH dependent solubility. The log P_o/w value of EFdA was −1.19. The compound was stable upon exposure to pH levels from 3 to 9 and showed good stability at elevated temperature (65 °C). In vitro cytotoxicity assessments were performed in two different epithelial cell lines. In cell-based studies, the EFdA selectivity index (50% cytotoxic concentration [CC₅₀] values/50% effective concentration [EC₅₀]) was found to be greater than 1 × 10³. Permeability studies using cell- and tissue-based models showed that EFdA had an apparent permeability coefficient (P_app) < 1×10⁻⁶cm/s and that the paracelluar pathway was the dominant transport route for EFdA. Overall, EFdA possesses favorable characteristics for further formulation development.

• Caco-2 cells
• EFdA
• permeability
• preformulation
• solubility
• transport

Acknowledgements

We gratefully acknowledge the staff associated with the Materials Micro-Characterization Laboratory, Department of Mechanical Engineering and Materials Science, University of Pittsburgh, for assistance with the scanning electron microscopy conducted in this study. We would like to thank Marilyn R. Cost for her technical support in the human tissue processing.