Abstract
Clofazimine is a poorly-soluble but orally-bioavailable small molecule drug that massively accumulates in macrophages when administered over prolonged periods of time. To determine whether crystal-like drug inclusions (CLDIs) that form in subcellular spaces correspond to pure clofazimine crystals, macrophages of clofazimine-fed mice were elicited with an intraperitoneal thioglycollate injection. Inside these cells, CLDIs appeared uniform in size and shape, but were sensitive to illumination. Once removed from cells, CLDIs were unstable. Unlike pure clofazimine crystals, isolated CLDIs placed in distilled water burst into small birefringent globules, which aggregated into larger clusters. Also unlike pure clofazimine crystals, CLDIs fragmented when heated, and disintegrated in alkaline media. In contrast to all other organelles, CLDIs were relatively resistant to sonication and trypsin digestion, which facilitated their biochemical isolation. The powder x-ray diffraction pattern obtained from isolated CLDIs was consistent with the diffraction pattern of liquid crystals and inconsistent with the expected molecular diffraction pattern of solid, three dimensional crystals. Observed with the transmission electron microscope (TEM), CLDIs were bounded by an atypical double-layered membrane, approximately 20 nanometers thick. CLDIs were polymorphic, but generally exhibited an internal multilayered organization, comprised of stacks of membranes 5 to 15 nanometers thick. Deep-etch, freeze-fracture electron microscopy of unfixed snap-frozen tissue samples confirmed this supramolecular organization. These results suggest that clofazimine accumulates in macrophages by forming a membrane-bound, multilayered, liquid crystal-like, semi-synthetic cytoplasmic structure.
Highlights
Clofazimine is an antibiotic and anti-inflammatory drug that is very poorly soluble yet orally bioavailable [1,2,3,4]
Unlike crystal-like drug inclusions (CLDIs) from intact tissues (Fig. 6), the internal layers of isolated CLDIs appeared to be peeling off (Fig. 7C), suggesting a partial disassembly. This is the first study to directly probe the physical and chemical properties of CLDIs, and to directly reveal the morphology of CLDIs formed in clofazimine-treated animals
And physically, the intracellular CLDIs appeared very different from chemically-pure clofazimine crystals: they were uniform in size and shape, and were highly responsive to changes in medium tonicity, pH, temperature and illumination
Summary
Clofazimine is an antibiotic and anti-inflammatory drug that is very poorly soluble yet orally bioavailable [1,2,3,4]. Clofazimine possesses three ionizable amine groups that become protonated and charged at acidic pH. It is a highly hydrophobic molecule, with a logP .7. In the gastrointestinal tract, clofazimine could form supersaturated solutions as it passes from the acidic pH of the stomach to the more alkaline pH of the intestine. Clofazimine could bind to proteins and form complexes with intracellular membranes [6]. It could precipitate out as particulate aggregates or crystals that may be actively phagocytosed by cells of the mononuclear phagocyte system
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