Purpose: Clofazimine (CFZ) is a weakly basic, pigmented (red colored) antibiotic, included in the WHO list of essential medications for the treatment of leprosy and is currently used against multi-drug-resistant tuberculosis. CFZ undergoes extensive intracellular bioaccumulation in tissue macrophages, forming Crystal-Like Drug Inclusions (CLDIs). In CLDIs, CFZ is present as an insoluble, hydrochloride salt form (CFZ-HCl) that displays great stability and biocompatibility within tissue macrophages. CLDIs were shown to be less cytotoxic and have anti-inflammatory properties in vitro compared to soluble CFZ free base. Therefore, even though CFZ-HCl and CFZ free base have the same active pharmaceutical ingredient, we hypothesize that CFZ-HCl is a much more biocompatible, therapeutically efficacious form of CFZ that alters cellular response and drug’s pharmacodynamics that could be potentially used in repurposing this drug for other indications. Skin pigmentation is the most common side effect that is observed in more than 94% of the patients treated with orally administered CFZ. The most viable alternative approach to reduce CFZ-induced skin hyperpigmentation would be to minimize the concentrations of drug in the circulation via local administration. Therefore, we are currently working towards reformulating CFZ, both as a locally injectable suspension of micronized, biomimetic crystals of CFZ hydrochloride, as well as an inhalable dry micronized powder.
Methods: Formulation process consists of five major steps: bulk synthesis of CFZ-HCl crystals; micronization/jet milling of the synthesized crystals to a homogeneous size that is less than 5 microns; sterilization via dry heat; characterization via proton NMR, single crystal Raman micro-spectroscopy, and powder XRD to make sure the chemical and crystal structure is preserved throughout the process; and stability studies in vitro and in vivo of CFZ-HCl microcrystals.
Results: First, CFZ-HCl crystals were synthesized in bulk and jet milled into the homogeneous particle size distribution of less than 5 microns, with the actual size ranging from 0.62 ± 0.03 to 1.11 ± 0.16 um, depending on the suspension media / diluent used. Proton NMR, single crystal Raman micro-spectroscopy, and powder XRD analysis confirmed that our CFZ-HCl microparticles preserved all of chemical and crystal structure properties of CLDIs throughout the formulation process. Furthermore, our in vitro studies in RAW 264.7 macrophages showed that CFZ-HCl microparticles have the same macrophage targeting properties and intracellular stability as CLDIs while keeping the high cell viability (>90%) (no statistical significant difference, p<0.05).
Conclusion: In conclusion, we were able to successfully formulate the biomimetic formulation of clofazimine hydrochloride (CFZ-HCl) microcrystals that can be potentially used for other indications by using either injectable suspension of these particles or inhalable dry micronized powder for local administration. By advancing solid, micro/nanoparticles of CFZ, it may be possible to achieve prolonged, therapeutic drug concentrations at the site of action while avoiding CFZ-induced skin pigmentation. This would result in more targeted formulation that could be useful to repurpose this drug for other indications. In fact, several of our in vivo studies are currently investigating these claims.