(W1230-07-40) Preparation of Highly Soluble Anti-tubercular Salt: Clofazimine mesylate

Purpose: Regardless of the fact that tuberculosis (TB) infections have significantly decreased in developed nations, 10 million people worldwide develop symptoms each year, making it the greatest infectious killer. Moreover, Mycobacterium tuberculosis, a causative organism for TB, is developing resistance against first-line medications like isoniazid and rifampicin. Therefore, there is a need to develop/ modify other drugs effective against Mycobacterium tuberculosis. Clofazimine, a second-line anti-tubercular drug, has been proven effective against tuberculosis (Minimum inhibitory concentration of 0.12 – 0.24 ug/mL). This BCS Class II drug has high permeability but very low aqueous solubility, resulting in poor oral absorption. Therefore, it is necessary to enhance the solubility profile of clofazimine. Salt formation is one of the most basic and old methods to enhance drug solubility. This study aimed to increase the solubility of the highly effective anti-tubercular medication clofazimine through the salt formation.

Methods: Clofazimine salts were prepared by solvent evaporation technique using a rotary evaporator for the first time. The counterions were selected based on their pKa value (the difference in the pKa of drug and counterion > 3). Clofazimine and the counterions (Methane sulfonic acid, benzene sulfonic acid, citric acid, lactic acid, and phosphoric acid) were mixed in a molar ratio of 1:1.2 and then added to acetone. The rotary evaporator was operated for 30 minutes at 40°C with a speed of 100 rpm. The pressure was decreased from 500 mbar to 300 mbar in intervals of 10 minutes. After the solvent's evaporation, the salts were air dried, stored in a desiccator, and characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), infrared spectroscopy (IR), and X-ray powder diffraction (XRD) and solubility determination using an incubator shaker. Excess clofazimine and its salts were added to the scintillation vials containing 3 ml of 60% ethanol-water mixture to examine the equilibrium solubility. The vials were sealed and placed in an incubator shaker for 24 hours ¹. The samples were centrifuged, diluted with mobile phase, and then analyzed at 492 nm using a validated UPLC method ².

Results: Solvent evaporation via rotary evaporator, a commercial instrument, proved to be a suitable technique for clofazimine salt formation. Stable and crystalline salts were obtained using this method. All clofazimine salts prepared in this study were dark brown, green, or black, in contrast to dark orange colored clofazimine. The color variation is attributable to distinct structures arising from the interaction of clofazimine and the counterion. All the salts were crystalline as per the sharp endothermic peaks in the DSC thermogram and diffraction patterns in the XRD data. Negligible weight loss of samples (exhibited by TGA) before degradation/ melting peak implies the absence of hydrates or solvates. In the IR spectra, a slight change in the imine region at 1690- 1600 cm^-1 (for all salts), specific carbonyl peaks at 1700- 1725 cm^-1(clofazimine citrate and clofazimine lactate), peaks at 1375- 1300 cm^-1(clofazimine mesylate and clofazimine besylate) indicating sulfone group, the peaks at 1080 cm^-1 (clofazimine phosphate) indicating phosphate group and short - broad peaks observed at 2800 – 3400 cm^-1(hydroxyl group) also confirm salt formation ³. All the clofazimine salts had greater solubility than the clofazimine-free base. Clofazimine mesylate was 85 times more soluble than the clofazimine base, while clofazimine lactate, clofazimine phosphate­, clofazimine citrate, and clofazimine besylate had a solubility of 24, 22, 18 and 12 times than that of clofazimine base, respectively.

Conclusion: The solubility of clofazimine was enhanced by salt formation via a rotary evaporator. The characterization of salts using DSC and XRD confirmed the crystalline nature of clofazimine and its salts. The results from the IR spectra further validated the salt formation. The primary aim of the study, i.e., to increase the solubility of clofazimine, was achieved. Clofazimine mesylate had the highest solubility of 84.96 + 0.189 mg/mL (85 times the solubility of clofazimine base). This high solubility of clofazimine mesylate salt could be attributed to specific properties of methane sulfonic acid like high aqueous solubility, low pKa (-1.2), and low molecular weight (96 gm/mol). All other salts were found to be more soluble than the clofazimine-free base.

References: (1) Bolla, G.; Nangia, A. Clofazimine Mesylate: A High Solubility Stable Salt. Cryst. Growth Des. 2012, 12 (12), 6250–6259. https://doi.org/10.1021/cg301463z.
(2) Sabnis, A. A. Inhaled Clofazimine Delivery for the Treatment of Pulmonary Tuberculosis. Thesis, Creighton University, 2015.
(3) Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Vyvyan, J. A. Introduction to Spectroscopy; Cengage Learning, 2008.

Acknowledgments: Grateful to the School of Pharmacy and Health Professions for funding this project.

X-ray powder diffraction of clofazimine and its salts.



Equilibrium solubility of clofazimine and its salts



Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) thermograms of Clofazimine and its salts.