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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.

Biomarker discovery and implementation are at the forefront of the precision medicine movement. Modern advances in the field of metabolomics afford the opportunity to readily identify new metabolite biomarkers across a wide array of disciplines. Many of the metabolites are derived from or directly reflective of mitochondrial metabolism. L-carnitine and acylcarnitines are established mitochondrial biomarkers used to screen neonates for a series of genetic disorders affecting fatty acid oxidation, known as the inborn errors of metabolism. However, L-carnitine and acylcarnitines are not routinely measured beyond this screening, despite the growing evidence that shows their clinical utility outside of these disorders. Measurements of the carnitine pool have been used to identify the disease and prognosticate mortality among disorders such as diabetes, sepsis, cancer, and heart failure, as well as identify subjects experiencing adverse drug reactions from various medications like valproic acid, clofazimine, zidovudine, cisplatin, propofol, and cyclosporine. The aim of this review is to collect and interpret the literature evidence supporting the clinical biomarker application of L-carnitine and acylcarnitines. Further study of these metabolites could ultimately provide mechanistic insights that guide therapeutic decisions and elucidate new pharmacologic targets.

Many weakly basic, lipophilic drugs accumulate in lysosomes and exert complex, pleiotropic effects on organelle structure and function. Thus, modeling how perturbations of lysosomal physiology affect the maintenance of lysosomal ion homeostasis is necessary to elucidate the key factors which determine the toxicological effects of lysosomotropic agents, in a cell-type dependent manner. Accordingly, a physiologically-based mathematical modeling and simulation approach was used to explore the dynamic, multi-parameter phenomenon of lysosomal stress. With this approach, parameters that are either directly involved in lysosomal ion transportation or lysosomal morphology were transiently altered to investigate their downstream effects on lysosomal physiology reflected by the changes they induce in lysosomal pH, chloride, and membrane potential. In addition, combinations of parameters were simultaneously altered to assess which parameter was most critical for recovery of normal lysosomal physiology. Lastly, to explore the relationship between organelle morphology and induced stress, we investigated the effects of parameters controlling organelle geometry on the restoration of normal lysosomal physiology following a transient perturbation. Collectively, our results indicate a key, interdependent role of V-ATPase number and membrane proton permeability in lysosomal stress tolerance. This suggests that the cell-type dependent regulation of V-ATPase subunit expression and turnover, together with the proton permeability properties of the lysosomal membrane, is critical to understand the differential sensitivity or resistance of different cell types to the toxic effects of lysosomotropic drugs.

Prostate cancer was the most common form and had the second highest death rate of male cancer in the United States in 2015. Current diagnosis techniques, such as prostate-specific antigen tests, transrectal ultrasound scans, and biopsies, are often inconclusive, and in the latter case, invasive. Here, we explore the use of clofazimine hydrochloride nanoparticles (CFZ-HCl NPs), a repurposed formulation from an FDA-approved antimycobacterial agent, as a photoacoustic contrast agent for the evaluation of prostate cancer due to its macrophage-targeting capabilities and high optical absorbance at 495 nm. Using a transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model, our results indicate a preferential accumulation of intravenously injected CFZ-HCl NPs in cancerous prostates over normal prostates. Differences in accumulation of CFZ-HCl NPs between cancerous and normal prostates were determined using a two-wavelength unmixing technique via ex vivo photoacoustic imaging. Thus, intravenous injection of CFZ-HCl NPs leads to differences in the interactions of the particles with cancerous vs normal prostates, while allowing for photoacoustic detection and analysis of prostate cancer. These findings could lead to the development of a new noninvasive technique for the detection and monitoring of prostate cancer progression in an animal model that can potentially be translated to human patients.

Adverse drug reactions (ADRs) are considered an inherent risk of medication use, and some ADRs have been associated with off-target drug interactions with mitochondria. Metabolites that reflect mitochondrial function may help identify patients at risk of mitochondrial toxicity. We employed a database strategy to identify candidate mitochondrial metabolites that could be clinically useful to identify individuals at increased risk of mitochondrial-related ADRs. This led to l -carnitine being identified as the candidate mitochondrial metabolite. l -carnitine, its acetylated metabolite, acetylcarnitine and other acylcarnitines are mitochondrial biomarkers used to detect inborn errors of metabolism. We hypothesized that changes in l -carnitine disposition, induced by a “challenge test” of intravenous l -carnitine, could identify mitochondrial-related ADRs by provoking variation in l -carnitine and/or acetylcarnitine blood levels. To test this hypothesis, we induced mitochondrial drug toxicity with clofazimine (CFZ) in a mouse model. Following CFZ treatment, mice received an l -carnitine “challenge test”. CFZ-induced changes in weight were consistent with previous work and reflect CFZ-induced catabolism. l -carnitine induced differences in whole blood acetylcarnitine concentrations in a manner that was dependent on CFZ treatment. This supports the usefulness of a database strategy for the discovery of candidate metabolite biomarkers of drug toxicity and substantiates the potential of the l -carnitine “challenge test” as a “probe” to identify drug-related toxicological manifestations.

Mitochondrial health declines with age, and older patients can demonstrate dysfunction in mitochondrial-rich tissues, such as cardiac and skeletal muscle. Aged mitochondria may make older adults more susceptible to adverse drug reactions (ADRs). We assessed mitochondrial metabolic function by measuring two metabolites, l-carnitine and acetylcarnitine, to determine their effectiveness as candidate clinical biomarkers for age-related, drug-induced alterations in mitochondrial metabolism. To study age- and medication-related changes in mitochondrial metabolism, we administered the FDA-approved mitochondriotropic drug, clofazimine (CFZ), or vehicle for 8 weeks to young (4-week-old) and old (61-week-old) male C57BL/6J mice. At the end of treatment, whole blood and cardiac and skeletal muscle were analyzed for l-carnitine, acetylcarnitine, and CFZ levels; muscle function was measured via a treadmill test. No differences were found in blood or cardiac carnitine levels of CFZ-treated mice, but CFZ-treated mice displayed lost body mass and alterations in endurance and levels of skeletal muscle mitochondrial metabolites. These findings demonstrate the age-related susceptibility of the skeletal muscle to mitochondria drug toxicity. Since drug-induced alterations in mitochondrial metabolism in skeletal muscle were not reflected in the blood by l-carnitine or acetylcarnitine levels, drug-induced catabolism and changes in muscle function appear more relevant to stratifying individuals at increased risk for ADRs.

Photoacoustic Tomography (PAT) is a deep-tissue imaging modality, with potential clinical applications in the diagnosis of arthritis, cancer and other disease conditions. Here, we identified Clofazimine (CFZ), a red-pigmented dye and anti-inflammatory FDA-approved drug, as a macrophage-targeting photoacoustic (PA) imaging agent. Spectroscopic experiments revealed that CFZ and its various protonated forms yielded optimal PAT signals at wavelengths −450 to 540 nm. CFZ’s macrophage-targeting chemical and structural forms were detected with PA microscopy at a high c ontrast-to- n oise r atio (CNR > 22 dB) as well as with macroscopic imaging using synthetic gelatin phantoms. In vivo , natural and synthetic CFZ formulations also demonstrated significant anti-inflammatory activity. Finally, the injection of CFZ was monitored via a real-time ultrasound-photoacoustic (US-PA) dual imaging system in a live animal and clinically relevant human hand model. These results demonstrate an anti-inflammatory drug repurposing strategy, while identifying a new PA contrast agent with potential applications in the diagnosis and treatment of arthritis.

The antimycobacterial drug clofazimine (CFZ) is used as a single agent at high doses, to suppress the exaggerated inflammation associated with leprosy. Paradoxically, increasing doses of CFZ leads to bioaccumulation of CFZ in the spleen and other organs under physiologically relevant dosing regimens, without accompanying dose-dependent elevation in the concentrations of the circulating drug in the blood. In long-term oral dosing regimens, CFZ induces immunological and metabolic changes resulting in splenomegaly, while the mass of other organs decreases or remains unchanged. As an organ that extensively sequesters CFZ as insoluble drug precipitates, the spleen likely influences drug-induced inflammatory signaling. To probe the role of systemic drug concentrations vs. drug bioaccumulation in the spleen, healthy mice were treated with six different dosing regimens. A subgroup of these mice underwent surgical splenectomies prior to drug treatment to assess the bioaccumulation-dependent changes in immune system signaling and immune-system-mediated drug distribution. Under increasing drug loading, the spleen was observed to grow up to six times in size, sequestering over 10% of the total drug load. Interestingly, when the spleen was removed prior to CFZ administration, drug distribution in the rest of the organism was unaffected. However, there were profound cytokine elevations in the serum of asplenic CFZ-treated mice, indicating that the spleen is primarily involved in suppressing the inflammatory signaling mechanisms that are upregulated during CFZ bioaccumulation. Thus, beyond its role in drug sequestration, the spleen actively modulates the systemic effect of CFZ on the immune system, without impacting its blood concentrations or distribution to the rest of the organism.

PurposeDrug-induced liver injuries (DILI) comprise a significant proportion of adverse drug reactions leading to hospitalizations and death. One frequent DILI is granulomatous inflammation from exposure to harmful metabolites that activate inflammatory pathways of immune cells of the liver, which may act as a barrier to isolate the irritating stimulus and limit tissue damage.MethodsParalleling the accumulation of CFZ precipitates in the liver, granulomatous inflammation was studied to gain insight into its effect on liver structure and function. A structural analog that does not precipitate within macrophages was also studied using micro-analytical approaches. Depleting macrophages was used to inhibit granuloma formation and assess its effect on drug bioaccumulation and toxicity.ResultsGranuloma-associated macrophages showed a distinct phenotype, differentiating them from non-granuloma macrophages. Granulomas were induced by insoluble CFZ cargo, but not by the more soluble analog, pointing to precipitation being a factor driving granulomatous inflammation. Granuloma-associated macrophages showed increased activation of lysosomal master-regulator transcription factor EB (TFEB). Inhibiting granuloma formation increased hepatic necrosis and systemic toxicity in CFZ-treated animals.ConclusionsGranuloma-associated macrophages are a specialized cell population equipped to actively sequester and stabilize cytotoxic chemotherapeutic agents. Thus, drug-induced granulomas may function as drug sequestering “organoids” –an induced, specialized sub-compartment– to limit tissue damage.

Lysosomes are acidic organelles and are involved in various diseases, the most prominent is malaria. Accumulation of molecules in the cell by diffusion from the external solution into cytosol, lysosome and mitochondrium was calculated with the Fick–Nernst–Planck equation. The cell model considers the diffusion of neutral and ionic molecules across biomembranes, protonation to mono- or bivalent ions, adsorption to lipids, and electrical attraction or repulsion. Based on simulation results, high and selective accumulation in lysosomes was found for weak mono- and bivalent bases with intermediate to high log  K ow . These findings were validated with experimental results and by a comparison to the properties of antimalarial drugs in clinical use. For ten active compounds, nine were predicted to accumulate to a greater extent in lysosomes than in other organelles, six of these were in the optimum range predicted by the model and three were close. Five of the antimalarial drugs were lipophilic weak dibasic compounds. The predicted optimum properties for a selective accumulation of weak bivalent bases in lysosomes are consistent with experimental values and are more accurate than any prior calculation. This demonstrates that the cell model can be a useful tool for the design of effective lysosome-targeting drugs with minimal off-target interactions.