Database screening as a strategy to identify endogenous candidate metabolites to probe and assess mitochondrial drug toxicity.

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.

Characterization of Clofazimine Metabolism in Human Liver Microsomal Incubation In Vitro.

Clofazimine [N,5-bis(4-chlorophenyl)-3-[(propane-2-yl)rimino]-3,5-dihydrophenazin-2-amine] is an antimycobacterial agent used as a second-line antituberculosis (anti-TB) drug. Nonetheless, little information is known about the metabolic routes of clofazimine, and the enzymes involved in metabolism. This study aimed to characterize the metabolic pathways and enzymes responsible for the metabolism of clofazimine in human liver microsomes. Eight metabolites, including four oxidative metabolites, three glucuronide conjugates, and one sulfate conjugate were identified, and their structures were deduced based on tandem mass spectrometry (MS/MS) spectra. Hydroxylated clofazimine and hydrated clofazimine was generated even in the absence of the NADPH generating system presumably via a nonenzymatic pathway. Hydrolytic-dehalogenated clofazimine was catalyzed mainly by CYP1A2 whereas hydrolytic-deaminated clofazimine was formed by CYP3A4/A5. In case of glucuronide conjugates, UGT1A1, UGT1A3, and UGT1A9 showed catalytic activity toward hydroxylated and hydrated clofazimine glucuronide whereas hydrolytic-deaminated clofazimine glucuronide was catalyzed by UGT1A4, UGT1A9, UGT1A3, and UGT2B4. Our results suggested that CYP1A2 and CYP3A are involved in the formation of oxidative metabolites while UGT1A1, 1A3, 1A4, 1A9, and 2B4 are involved in the formation of glucuronide conjugates of oxidative metabolites of clofazimine.

Clofazimine enhances the efficacy of BCG revaccination via stem cell-like memory T cells.

Tuberculosis (TB) is one of the deadliest diseases, claiming ~2 million deaths annually worldwide. The majority of people in TB endemic regions are vaccinated with Bacillus Calmette Guerin (BCG), which is the only usable vaccine available. BCG is efficacious against meningeal and disseminated TB in children, but protective responses are relatively short-lived and fail to protect against adult pulmonary TB. The longevity of vaccine efficacy critically depends on the magnitude of long-lasting central memory T (TCM) cells, a major source of which is stem cell-like memory T (TSM) cells. These TSM cells exhibit enhanced self-renewal capacity as well as to rapidly respond to antigen and generate protective poly-functional T cells producing IFN-γ, TNF-α, IL-2 and IL-17. It is now evident that T helper Th 1 and Th17 cells are essential for host protection against TB. Recent reports have indicated that Th17 cells preserve the molecular signature for TSM cells, which eventually differentiate into IFN-γ-producing effector cells. BCG is ineffective in inducing Th17 cell responses, which might explain its inadequate vaccine efficacy. Here, we show that revaccination with BCG along with clofazimine treatment promotes TSM differentiation, which continuously restores TCM and T effector memory (TEM) cells and drastically increases vaccine efficacy in BCG-primed animals. Analyses of these TSM cells revealed that they are predominantly precursors to host protective Th1 and Th17 cells. Taken together, these findings revealed that clofazimine treatment at the time of BCG revaccination provides superior host protection against TB by increasing long-lasting TSM cells.

Identification of Potential Biomarkers for Thyroid Cancer Using Bioinformatics Strategy: A Study Based on GEO Datasets.

BACKGROUND: The molecular mechanisms and genetic markers of thyroid cancer are unclear. In this study, we used bioinformatics to screen for key genes and pathways associated with thyroid cancer development and to reveal its potential molecular mechanisms. METHODS: The GSE3467, GSE3678, GSE33630, and GSE53157 expression profiles downloaded from the Gene Expression Omnibus database (GEO) contained a total of 164 tissue samples (64 normal thyroid tissue samples and 100 thyroid cancer samples). The four datasets were integrated and analyzed by the RobustRankAggreg (RRA) method to obtain differentially expressed genes (DEGs). Using these DEGs, we performed gene ontology (GO) functional annotation, pathway analysis, protein-protein interaction (PPI) analysis and survival analysis. Then, CMap was used to identify the candidate small molecules that might reverse thyroid cancer gene expression. RESULTS: By integrating the four datasets, 330 DEGs, including 154 upregulated and 176 downregulated genes, were identified. GO analysis showed that the upregulated genes were mainly involved in extracellular region, extracellular exosome, and heparin binding. The downregulated genes were mainly concentrated in thyroid hormone generation and proteinaceous extracellular matrix. Pathway analysis showed that the upregulated DEGs were mainly attached to ECM-receptor interaction, p53 signaling pathway, and TGF-beta signaling pathway. Downregulation of DEGs was mainly involved in tyrosine metabolism, mineral absorption, and thyroxine biosynthesis. Among the top 30 hub genes obtained in PPI network, the expression levels of FN1, NMU, CHRDL1, GNAI1, ITGA2, GNA14 and AVPR1A were associated with the prognosis of thyroid cancer. Finally, four small molecules that could reverse the gene expression induced by thyroid cancer, namely ikarugamycin, adrenosterone, hexamethonium bromide and clofazimine, were obtained in the CMap database. CONCLUSION: The identification of the key genes and pathways enhances the understanding of the molecular mechanisms for thyroid cancer. In addition, these key genes may be potential therapeutic targets and biomarkers for the treatment of thyroid cancer.

Topical Delivery of Artemisone, Clofazimine and Decoquinate Encapsulated in Vesicles and Their In vitro Efficacy Against Mycobacterium tuberculosis.

Vesicles are widely investigated as carrier systems for active pharmaceutical ingredients (APIs). For topical delivery, they are especially effective since they create a "depot-effect" thereby concentrating the APIs in the skin. Artemisone, clofazimine and decoquinate were selected as a combination therapy for the topical treatment of cutaneous tuberculosis. Delivering APIs into the skin presents various challenges. However, utilising niosomes, liposomes and transferosomes as carrier systems may circumvent these challenges. Vesicles containing 1% of each of the three selected APIs were prepared using the thin-film hydration method. Isothermal calorimetry, differential scanning calorimetry and hot-stage microscopy indicated no to minimal incompatibility between the APIs and the vesicle components. Encapsulation efficiency was higher than 85% for all vesicle dispersions. Vesicle stability decreased and size increased with an increase in API concentration; and ultimately, niosomes were found the least stable of the different vesicle types. Skin diffusion studies were subsequently conducted for 12 h on black human female skin utilising vertical Franz diffusion cells. Transferosomes and niosomes delivered the highest average concentrations of clofazimine and decoquinate into the skin, whereas artemisone was not detected and no APIs were present in the receptor phase. Finally, efficacy against tuberculosis was tested against the Mycobacterium tuberculosis H37Rv laboratory strain. All the dispersions depicted some activity, surprisingly even the blank vesicles portrayed activity. However, the highest percentage inhibition (52%) against TB was obtained with niosomes containing 1% clofazimine.

Biophysical insights into the interaction of clofazimine with human alpha 1-acid glycoprotein: a multitechnique approach.

Alpha1-acid glycoprotein (AAG) is a major acute phase protein of human plasma. Binding of clofazimine to AAG is investigated using optical spectroscopy and molecular docking tools. We found significant quenching of intrinsic fluorescence of AAG upon the binding of clofazimine, binding mode is static with binding constant of 3.52 × 104at 298 K. The Gibbs free energy change is found to be negative for the interaction of clofazimine with AAG indicating spontaneity of the binding process. Binding of clofazimine induced ordered structure in protein and lead to molecular compaction. Molecular docking results indicate the binding site is located in the central beta barrel, hydrogen bonding and hydrophobic interactions are main bonding forces between AAG-clofazimine.

An Expandable Mechanopharmaceutical Device (2): Drug Induced Granulomas Maximize the Cargo Sequestering Capacity of Macrophages in the Liver.

PURPOSE: Drug-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. METHODS: Paralleling 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. RESULTS: Granuloma-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. CONCLUSIONS: Granuloma-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.

Delivery of a hydrophobic drug into the lower gastrointestinal system via an endogenous enzyme-mediated carrier mechanism: An in vitro study.

Clofazimine (CFZ) is a hydrophobic antibiotic agent which exhibits poor solubility. This poor solubility was overcome herein by the formulation of CFZ with the digestive enzyme pepsin. It is shown that pepsin can actively bind 11 CFZ molecules in the protein's native gastric environment, forming a CFZ-pepsin complex. A dynamic dissolution system, representing both the gastric and intestinal system, was used to analyze this CFZ-pepsin complex, revealing that only CFZ which binds to pepsin in the gastric environment remains in solution in the intestinal environment. The CFZ-pepsin complex displays adequate solution stability for the delivery of CFZ into the lower intestinal system. In vitro bioactivity assays against Clostridium difficile demonstrated the effectiveness of this CFZ-pepsin complex for the treatment of infectious diseases in the lower intestinal system.

Anti-leprosy drug Clofazimine binds to human Raf1 kinase inhibitory protein and enhances ERK phosphorylation.

Human Raf1 kinase inhibitory protein (hRKIP) is an important modulator of the Ras/Raf1/MEK/ERK signaling pathway. Here, we demonstrated that anti-leprosy drug Clofazimine can bind to hRKIP with a significantly stronger affinity than the endogenous substrate phosphatidylethanolamine (PE) by using Biolayer interference technology. Moreover, we identified that residues P74, S75, K80, P111, P112, V177, and P178 play crucial roles in the binding of hRKIP to Clofazimine by using a combination of Nuclear Magnetic Resonance spectroscopy and molecular docking approach. These residues are located at the conserved ligand-binding pocket of hRKIP. Furthermore, we found that 3.2 µM Clofazimine could significantly increase the ERK phosphorylation level by about 37%. Our results indicate that Clofazimine can enhance Ras/Raf1/MEK/ERK signaling transduction pathway via binding to hRKIP. This work provides valuable hints for exploiting Clofazimine as a potential lead compound to efficiently treat the diseases related to RKIP or the Ras/Raf/MEK/ERK pathway.

Reverse Engineering the Intracellular Self-Assembly of a Functional Mechanopharmaceutical Device.

Weakly basic, poorly soluble chemical agents could be exploited as building blocks for constructing sophisticated molecular devices inside the cells of living organisms. Here, using experimental and computational approaches, we probed the relationship between the biological mechanisms mediating lysosomal ion homeostasis and the self-assembly of a weakly basic small molecule building block (clofazimine) into a functional, mechanopharmaceutical device (intracellular Crystal-Like Drug Inclusions - "CLDIs") in macrophage lysosomes. Physicochemical considerations indicate that the intralysosomal stabilization of the self-assembled mechanopharmaceutical device depends on the pHmax of the weakly basic building block and its affinity for chloride, both of which are consistent with the pH and chloride content of a physiological lysosomal microenvironment. Most importantly, in vitro and in silico studies revealed that high expression levels of the vacuolar ATPase (V-ATPase), irrespective of the expression levels of chloride channels, are necessary and sufficient to explain the cell-type dependent formation, stabilization, and biocompatibility of the self-assembled mechanopharmaceutical device within macrophages.

Chemotherapeutic efficacies of a clofazimine and diminazene aceturate combination against piroplasm parasites and their AT-rich DNA-binding activity on Babesia bovis.

Recently, we reported that clofazimine (CF) has an anti-piroplasm activity, but it could not completely eliminate parasites in the host. The currently available anti-piroplasm drug, diminazene aceturate (DA), has sometimes been reported to have toxic side effects. In the present study, we evaluated the combination treatment with CF and DA against piroplasms both in vitro and in vivo. Additionally, mRNA level and DNA amounts were analyzed in CF‒ and DA‒treated Babesia bovis by a qPCR. The CF-DA combination had additive effects on Babesia bovis, B. bigemina, and B. caballi and synergistic effects on Theileria equi. The CF-DA combination chemotherapies against B. microti in mice were more potent than their monotherapies. In the CF‒ and DA‒treated B. bovis, CF dose-dependently down-regulated mRNA level and DNA amounts of extranuclear genes (AT-rich featured), whereas DA down-regulated only DNA amounts of extranuclear genes, but those of nuclear genes were slightly down- or up-regulated by CF and DA. In conclusion, the CF-DA combination has a higher efficiency against piroplasms than CF or DA monotherapies. CF and DA might have an AT-rich DNA-binding activity. All results suggest that the CF-DA combination chemotherapy will be a better choice to treat piroplasmosis instead of DA monotherapy.

Subcellular Partitioning and Intramacrophage Selectivity of Antimicrobial Compounds against Mycobacterium tuberculosis.

The efficacy of antimicrobial drugs against Mycobacterium tuberculosis, an intracellular bacterial pathogen, is generally first established by testing compounds against bacteria in axenic culture. However, inside infected macrophages, bacteria encounter an environment which differs substantially from broth culture and are subject to important host-dependent pharmacokinetic phenomena which modulate drug activity. Here, we describe how pH-dependent partitioning drives asymmetric antimicrobial drug distribution in M. tuberculosis-infected macrophages. Specifically, weak bases with moderate activity against M. tuberculosis (fluoxetine, sertraline, and dibucaine) were shown to accumulate intracellularly due to differential permeability and relative abundance of their ionized and nonionized forms. Nonprotonatable analogs of the test compounds did not show this effect. Neutralization of acidic organelles directly with ammonium chloride or indirectly with bafilomycin A1 partially abrogated the growth restriction of these drugs. Using high-performance liquid chromatography, we quantified the degree of accumulation and reversibility upon acidic compartment neutralization in macrophages and observed that accumulation was greater in infected than in uninfected macrophages. We further demonstrate that the efficacy of a clinically used compound, clofazimine, is augmented by pH-based partitioning in a macrophage infection model. Because the parameters which govern this effect are well understood and are amenable to chemical modification, this knowledge may enable the rational development of more effective antibiotics against tuberculosis.

Elasticity in Macrophage-Synthesized Biocrystals.

Supramolecular crystalline assembly constitutes a rational approach to bioengineer intracellular structures. Here, biocrystals of clofazimine (CFZ) that form in vivo within macrophages were measured to have marked curvature. Isolated crystals, however, showed reduced curvature suggesting that intracellular forces bend these drug crystals. Consistent with the ability of biocrystals to elastically deform, the inherent crystal structure of the principal molecular component of the biocrystals-the hydrochloride salt of CFZ (CFZ-HCl)-has a corrugated packing along the (001) face and weak dispersive bonding in multiple directions. These characteristics were previously found to be linked to the elasticity of other organic crystals. Internal stress in bent CFZ-HCl led to photoelastic effects on the azimuthal orientation of polarized light transmittance. We propose that elastic, intracellular crystals can serve as templates to construct functional microdevices with different applications.

Macrophage-Mediated Clofazimine Sequestration Is Accompanied by a Shift in Host Energy Metabolism.

Prolonged (8 weeks) oral administration of clofazimine results in a profound pharmacodynamic response-bioaccumulation in macrophages (including Kupffer cells) as intracellular crystal-like drug inclusions (CLDIs) with an associated increase in interleukin-1 receptor antagonist production. Notably, CLDI formation in Kupffer cells concomitantly occurs with the formation of macrophage-centric granulomas. Accordingly, we sought to understand the impact of these events on host metabolism using 1H-nuclear magnetic resonance metabolomics. Mice received a clofazimine or vehicle-enriched (sham) diet for at least 8 weeks. At 2 weeks, the antimicrobial activity of clofazimine was evident by changes in urine metabolites. From 2 to 8 weeks, there was a striking change in metabolite levels indicative of a reorientation of host energy metabolism paralleling the onset of CLDI and granuloma formation. This was evidenced by a progressive reduction in urine levels of metabolites involved in one-carbon metabolism with corresponding increases in whole blood, and changes in metabolites associated with lipid, nucleotide and amino acid metabolism, and glycolysis. Although clofazimine-fed mice ate more, they gained less weight than control mice. Together, these results indicate that macrophage sequestration of clofazimine as CLDIs and granuloma formation is accompanied by a profound metabolic disruption in energy homeostasis and one-carbon metabolism.

Efflux pump inhibitors: targeting mycobacterial efflux systems to enhance TB therapy.

The emergence of drug resistance continues to plague TB control, with a global increase in the prevalence of MDR-TB. This acts as a gateway to XDR-TB and thus emphasizes the urgency for drug development and optimal treatment options. Bedaquiline is the first new anti-TB drug approved by the FDA in 40 years and has been shown to be an effective treatment option for MDR Mycobacterium tuberculosis infection. Bedaquiline has also recently been included in clinical trials for new regimens with the aim of improving and shortening treatment periods. Alarmingly, efflux-mediated bedaquiline resistance, as well as efflux-mediated cross-resistance to clofazimine, has been identified in treatment failures. This mechanism of resistance results in efflux of a variety of anti-TB drugs from the bacterial cell, thereby decreasing the intracellular drug concentration. In doing so, the bacillus is able to render the antibiotic treatment ineffective. Recent studies have explored strategies to reverse the resistance phenotype conferred by efflux pump activation. It was observed that the addition of efflux pump inhibitors partially restored drug susceptibility in vitro and in vivo. This has significant clinical implications, especially in MDR-TB management where treatment options are extremely limited. This review aims to highlight the current efflux pump inhibitors effective against M. tuberculosis, the effect of efflux pump inhibitors on mycobacterial growth and the clinical promise of treatment with efflux pump inhibitors and standard anti-TB therapy.

Chemical Analysis of Drug Biocrystals: A Role for Counterion Transport Pathways in Intracellular Drug Disposition.

In mammals, highly lipophilic small molecule chemical agents can accumulate as inclusions within resident tissue macrophages. In this context, we characterized the biodistribution, chemical composition, and structure of crystal-like drug inclusions (CLDIs) formed by clofazimine (CFZ), a weakly basic lipophilic drug. With prolonged oral dosing, CFZ exhibited a significant partitioning with respect to serum and fat due to massive bioaccumulation and crystallization in the liver and spleen. The NMR, Raman, and powder X-ray diffraction (p-XRD) spectra of CLDIs isolated from the spleens of CFZ-treated mice matched the spectra of pure, CFZ hydrochloride crystals (CFZ-HCl). Elemental analysis revealed a 237-fold increase in chlorine content in CLDIs compared to untreated tissue samples and a 5-fold increase in chlorine content compared to CFZ-HCl, suggesting that the formation of CLDIs occurs through a chloride mediated crystallization mechanism. Single crystal analysis revealed that CFZ-HCl crystals had a densely packed orthorhombic lattice configuration. In vitro, CFZ-HCl formed at a pH of 4-5 only if chloride ions were present at sufficiently high concentrations (>50:1 Cl(-)/CFZ), indicating that intracellular chloride transport mechanisms play a key role in the formation of CLDIs. While microscopy and pharmacokinetic analyses clearly revealed crystallization and intracellular accumulation of the drug in vivo, the chemical and structural characterization of CLDIs implicates a concentrative, chloride transport mechanism, paralleling and thermodynamically stabilizing the massive bioaccumulation of a weakly basic drug.

Computer-guided drug repurposing: identification of trypanocidal activity of clofazimine, benidipine and saquinavir.

In spite of remarkable advances in the knowledge on Trypanosoma cruzi biology, no medications to treat Chagas disease have been approved in the last 40 years and almost 8 million people remain infected. Since the public sector and non-profit organizations play a significant role in the research efforts on Chagas disease, it is important to implement research strategies that promote translation of basic research into the clinical practice. Recent international public-private initiatives address the potential of drug repositioning (i.e. finding second or further medical uses for known-medications) which can substantially improve the success at clinical trials and the innovation in the pharmaceutical field. In this work, we present the computer-aided identification of approved drugs clofazimine, benidipine and saquinavir as potential trypanocidal compounds and test their effects at biochemical as much as cellular level on different parasite stages. According to the obtained results, we discuss biopharmaceutical, toxicological and physiopathological criteria applied to decide to move clofazimine and benidipine into preclinical phase, in an acute model of infection. The article illustrates the potential of computer-guided drug repositioning to integrate and optimize drug discovery and preclinical development; it also proposes rational rules to select which among repositioned candidates should advance to investigational drug status and offers a new insight on clofazimine and benidipine as candidate treatments for Chagas disease. One Sentence Summary: We present the computer-guided drug repositioning of three approved drugs as potential new treatments for Chagas disease, integrating computer-aided drug screening and biochemical, cellular and preclinical tests.

Macrophages sequester clofazimine in an intracellular liquid crystal-like supramolecular organization.

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.

Molecular imaging of intracellular drug-membrane aggregate formation.

Clofazimine is a lipophilic antibiotic with an extremely long pharmacokinetic half-life associated with the appearance of crystal-like drug inclusions, in vivo. Here, we studied how clofazimine accumulates inside cells in the presence of supersaturating, extracellular concentrations of the drug (in the range of physiological drug concentrations). Based on a combination of molecular imaging, biochemical analysis and electron microscopy techniques, clofazimine mass increased inside cells in vitro, over a period of several days, with discrete clofazimine inclusions forming in the cytoplasm. These inclusions grew in size, number and density, as long as the drug-containing medium was replenished. With Raman confocal microscopy, clofazimine's spectral signature in these inclusions resembled that of amorphous clofazimine precipitates and was unlike that of clofazimine crystals. Additional experiments revealed that clofazimine first accumulated in mitochondria, with ensuing changes in mitochondrial structure and function. In turn, the degenerating organelles coalesced, fused with each other and condensed to form prominent drug-membrane aggregates (dubbed autophagosome-like drug inclusions or "aldis"). Like clofazimine, it is possible that intracellular drug-membrane aggregate formation is a common phenomenon underlying the reported phenotypic effects of many other small molecule drugs.

Clofazimine inhibits human Kv1.3 potassium channel by perturbing calcium oscillation in T lymphocytes.

The Kv1.3 potassium channel plays an essential role in effector memory T cells and has been implicated in several important autoimmune diseases including multiple sclerosis, psoriasis and type 1 diabetes. A number of potent small molecule inhibitors of Kv1.3 channel have been reported, some of which were found to be effective in various animal models of autoimmune diseases. We report herein the identification of clofazimine, a known anti-mycobacterial drug, as a novel inhibitor of human Kv1.3. Clofazimine was initially identified as an inhibitor of intracellular T cell receptor-mediated signaling leading to the transcriptional activation of human interleukin-2 gene in T cells from a screen of the Johns Hopkins Drug Library. A systematic mechanistic deconvolution revealed that clofazimine selectively blocked the Kv1.3 channel activity, perturbing the oscillation frequency of the calcium-release activated calcium channel, which in turn led to the inhibition of the calcineurin-NFAT signaling pathway. These effects of clofazimine provide the first line of experimental evidence in support of a causal relationship between Kv1.3 and calcium oscillation in human T cells. Furthermore, clofazimine was found to be effective in blocking human T cell-mediated skin graft rejection in an animal model in vivo. Together, these results suggest that clofazimine is a promising immunomodulatory drug candidate for treating a variety of autoimmune disorders.