Late relapses in leprosy patients in Brazil: 10-year post-trial of uniform multidrug therapy (U-MDT/CT-BR).

BACKGROUND: Leprosy is a neglected dermato-neurologic, infectious disease caused by Mycobacterium leprae or M. lepromatosis. Leprosy is treatable and curable by multidrug therapy/MDT, consisting of 12 months rifampicin, dapsone and clofazimine for multibacillary/MB patients and for 6 months for paucibacillary/PB patients. The relapse rate is considered a crucial treatment outcome. A randomized Controlled Clinical Trial (U-MDT/CT-BR) conducted from 2007‒2012 compared clinical outcomes in MB patients after 12 months regular MDT/R-MDT and 6 months uniform MDT/U-MDT in two highly endemic Brazilian areas. OBJECTIVES: To estimate the 10 years relapse rate of MB patients treated with 6 months U-MDT. METHODS: The statistical analyses treated the data as a case-control study, sampled from the cohort generated for the randomized trial. Analyses estimated univariate odds ratio and applied logistic regression for multivariate analysis, controlling the confounding variables. RESULTS: The overall relapse rate was 4.08 %: 4.95 % (16 out of 323) in the U-MDT group and 3.10 % (9 out of 290) in the regular/R-MDT group. The difference in relapse proportion between U-MDT and R-MDT groups was 1.85 %, not statistically significant (Odds Ratio = 1.63, 95 % CI 0.71 to 3.74). However, misdiagnosis of relapses, may have introduced bias, underestimating the force of the association represented by the odds ratio. CONCLUSIONS: The relapse estimate of 10 years follow-up study of the first randomized, controlled study on U-MDT/CT-BR was similar to the R-MDT group, supporting strong evidence that 6 months U-MDT for MB patients is an acceptable option to be adopted by leprosy endemic countries worldwide. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00669643.

The anti-leprosy drug clofazimine reduces polyQ toxicity through activation of PPARγ.

BACKGROUND: PolyQ diseases are autosomal dominant neurodegenerative disorders caused by the expansion of CAG repeats. While of slow progression, these diseases are ultimately fatal and lack effective therapies. METHODS: A high-throughput chemical screen was conducted to identify drugs that lower the toxicity of a protein containing the first exon of Huntington's disease (HD) protein huntingtin (HTT) harbouring 94 glutamines (Htt-Q94). Candidate drugs were tested in a wide range of in vitro and in vivo models of polyQ toxicity. FINDINGS: The chemical screen identified the anti-leprosy drug clofazimine as a hit, which was subsequently validated in several in vitro models. Computational analyses of transcriptional signatures revealed that the effect of clofazimine was due to the stimulation of mitochondrial biogenesis by peroxisome proliferator-activated receptor gamma (PPARγ). In agreement with this, clofazimine rescued mitochondrial dysfunction triggered by Htt-Q94 expression. Importantly, clofazimine also limited polyQ toxicity in developing zebrafish and neuron-specific worm models of polyQ disease. INTERPRETATION: Our results support the potential of repurposing the antimicrobial drug clofazimine for the treatment of polyQ diseases. FUNDING: A full list of funding sources can be found in the acknowledgments section.

Having the cake and eating it? Clofazimine boosts immunotherapy while limiting side effects.

Combined immune checkpoint blockade (ICB) for cancer exhibits good efficacy in a subset of patients but also associates with immune-related adverse events. Xue et al. use an elegant drug screening strategy to identify the antimicrobial drug clofazimine as an agent that both potentiates ICB efficacy and decreases immune-related adverse events.

Assessing potential drug-drug interactions between clofazimine and other frequently used agents to treat drug-resistant tuberculosis.

Clofazimine is included in drug regimens to treat rifampicin/drug-resistant tuberculosis (DR-TB), but there is little information about its interaction with other drugs in DR-TB regimens. We evaluated the pharmacokinetic interaction between clofazimine and isoniazid, linezolid, levofloxacin, and cycloserine, dosed as terizidone. Newly diagnosed adults with DR-TB at Klerksdorp/Tshepong Hospital, South Africa, were started on the then-standard treatment with clofazimine temporarily excluded for the initial 2 weeks. Pharmacokinetic sampling was done immediately before and 3 weeks after starting clofazimine, and drug concentrations were determined using validated liquid chromatography-tandem mass spectrometry assays. The data were interpreted with population pharmacokinetics in NONMEM v7.5.1 to explore the impact of clofazimine co-administration and other relevant covariates on the pharmacokinetics of isoniazid, linezolid, levofloxacin, and cycloserine. Clofazimine, isoniazid, linezolid, levofloxacin, and cycloserine data were available for 16, 27, 21, 21, and 6 participants, respectively. The median age and weight for the full cohort were 39 years and 52 kg, respectively. Clofazimine exposures were in the expected range, and its addition to the regimen did not significantly affect the pharmacokinetics of the other drugs except levofloxacin, for which it caused a 15% reduction in clearance. A posteriori power size calculations predicted that our sample sizes had 97%, 90%, and 87% power at P < 0.05 to detect a 30% change in clearance of isoniazid, linezolid, and cycloserine, respectively. Although clofazimine increased the area under the curve of levofloxacin by 19%, this is unlikely to be of great clinical significance, and the lack of interaction with other drugs tested is reassuring.

The Dual-Targeted Fusion Inhibitor Clofazimine Binds to the S2 Segment of the SARS-CoV-2 Spike Protein.

Clofazimine and Arbidol have both been reported to be effective in vitro SARS-CoV-2 fusion inhibitors. Both are promising drugs that have been repurposed for the treatment of COVID-19 and have been used in several previous and ongoing clinical trials. Small-molecule bindings to expressed constructs of the trimeric S2 segment of Spike and the full-length SARS-CoV-2 Spike protein were measured using a Surface Plasmon Resonance (SPR) binding assay. We demonstrate that Clofazimine, Toremifene, Arbidol and its derivatives bind to the S2 segment of the Spike protein. Clofazimine provided the most reliable and highest-quality SPR data for binding with S2 over the conditions explored. A molecular docking approach was used to identify the most favorable binding sites on the S2 segment in the prefusion conformation, highlighting two possible small-molecule binding sites for fusion inhibitors. Results related to molecular docking and modeling of the structure-activity relationship (SAR) of a newly reported series of Clofazimine derivatives support the proposed Clofazimine binding site on the S2 segment. When the proposed Clofazimine binding site is superimposed with other experimentally determined coronavirus structures in structure-sequence alignments, the changes in sequence and structure may rationalize the broad-spectrum antiviral activity of Clofazimine in closely related coronaviruses such as SARS-CoV, MERS, hCoV-229E, and hCoV-OC43.

Sub-MIC levels of bedaquiline and clofazimine can select Mycobacterium tuberculosis mutants with increased MIC.

Multidrug-resistant tuberculosis (MDR-TB) patients not cured at the time of stopping treatment are exposed to Minimum Inhibitory Concentration (MIC) and sub-MIC levels for many months after discontinuing bedaquiline (BDQ) or clofazimine (CFZ) treatment. In vitro cultures treated with BDQ and CFZ sub-MIC concentrations clearly showed enrichment in the Rv0678 mutant population, demonstrating that pre-existing Rv0678 mutants can be selected by sub-MIC concentrations of BDQ and CFZ if not protected by an alternative MDR-TB treatment.

Clinical drug screening reveals clofazimine potentiates the efficacy while reducing the toxicity of anti-PD-1 and CTLA-4 immunotherapy.

Emerging as the most potent and durable combinational immunotherapy, dual anti-PD-1 and CTLA-4 immune checkpoint blockade (ICB) therapy notoriously increases grade 3-5 immune-related adverse events (irAEs) in patients. Accordingly, attempts to improve the antitumor potency of anti-PD-1+CTLA-4 ICB by including additional therapeutics have been largely discouraged due to concerns of further increasing fatal toxicity. Here, we screened ∼3,000 Food and Drug Administration (FDA)-approved drugs and identified clofazimine as a potential third agent to optimize anti-PD-1+CTLA-4 ICB. Remarkably, clofazimine outperforms ICB dose reduction or steroid treatment in reversing lethality of irAEs, but unlike the detrimental effect of steroids on antitumor efficacy, clofazimine potentiates curative responses in anti-PD-1+CTLA-4 ICB. Mechanistically, clofazimine promotes E2F1 activation in CD8+ T cells to overcome resistance and counteracts pathogenic Th17 cells to abolish irAEs. Collectively, clofazimine potentiates the antitumor efficacy of anti-PD-1+CTLA-4 ICB, curbs intractable irAEs, and may fill a desperate clinical need to improve patient survival.

Inhalable porous particles as dual micro-nano carriers demonstrating efficient lung drug delivery for treatment of tuberculosis.

Inhalation therapy treating severe infectious disease is among the more complex and emerging topics in controlled drug release. Micron-sized carriers are needed to deposit drugs into the lower airways, while nano-sized carriers are of preference for cell targeting. Here, we present a novel and versatile strategy using micron-sized spherical particles with an excellent aerodynamic profile that dissolve in the lung fluid to ultimately generate nanoparticles enabling to enhance both extra- and intra-cellular drug delivery (i.e., dual micro-nano inhalation strategy). The spherical particles are synthesised through the condensation of nano-sized amorphous silicon dioxide resulting in high surface area, disordered mesoporous silica particles (MSPs) with monodispersed size of 2.43 µm. Clofazimine (CLZ), a drug shown to be effective against multidrug-resistant tuberculosis, was encapsulated in the MSPs obtaining a dry powder formulation with high respirable fraction (F.P.F. <5 µm of 50%) without the need of additional excipients. DSC, XRPD, and Nitrogen adsorption-desorption indicate that the drug was fully amorphous when confined in the nano-sized pores (9-10 nm) of the MSPs (shelf-life of 20 months at 4 °C). Once deposited in the lung, the CLZ-MSPs exhibited a dual action. Firstly, the nanoconfinement within the MSPs enabled a drastic dissolution enhancement of CLZ in simulated lung fluid (i.e., 16-fold higher than the free drug), increasing mycobacterial killing than CLZ alone (p = 0.0262) and reaching concentrations above the minimum bactericidal concentration (MBC) against biofilms of M. tuberculosis (i.e., targeting extracellular bacteria). The released CLZ permeated but was highly retained in a Calu-3 respiratory epithelium model, suggesting a high local drug concentration within the lung tissue minimizing risk for systemic side effects. Secondly, the micron-sized drug carriers spontaneously dissolve in simulated lung fluid into nano-sized drug carriers (shown by Nano-FTIR), delivering high CLZ cargo inside macrophages and drastically decreasing the mycobacterial burden inside macrophages (i.e., targeting intracellular bacteria). Safety studies showed neither measurable toxicity on macrophages nor Calu-3 cells, nor impaired epithelial integrity. The dissolved MSPs also did not show haemolytic effect on human erythrocytes. In a nutshell, this study presents a low-cost, stable and non-invasive dried powder formulation based on a dual micro-nano carrier to efficiently deliver drug to the lungs overcoming technological and practical challenges for global healthcare.

Detection of a historic reservoir of bedaquiline/clofazimine resistance-associated variants in Mycobacterium tuberculosis.

BACKGROUND: Drug resistance in tuberculosis (TB) poses a major ongoing challenge to public health. The recent inclusion of bedaquiline into TB drug regimens has improved treatment outcomes, but this advance is threatened by the emergence of strains of Mycobacterium tuberculosis (Mtb) resistant to bedaquiline. Clinical bedaquiline resistance is most frequently conferred by off-target resistance-associated variants (RAVs) in the mmpR5 gene (Rv0678), the regulator of an efflux pump, which can also confer cross-resistance to clofazimine, another TB drug. METHODS: We compiled a dataset of 3682 Mtb genomes, including 180 carrying variants in mmpR5, and its immediate background (i.e. mmpR5 promoter and adjacent mmpL5 gene), that have been associated to borderline (henceforth intermediate) or confirmed resistance to bedaquiline. We characterised the occurrence of all nonsynonymous mutations in mmpR5 in this dataset and estimated, using time-resolved phylogenetic methods, the age of their emergence. RESULTS: We identified eight cases where RAVs were present in the genomes of strains collected prior to the use of bedaquiline in TB treatment regimes. Phylogenetic reconstruction points to multiple emergence events and circulation of RAVs in mmpR5, some estimated to predate the introduction of bedaquiline. However, epistatic interactions can complicate bedaquiline drug-susceptibility prediction from genetic sequence data. Indeed, in one clade, Ile67fs (a RAV when considered in isolation) was estimated to have emerged prior to the antibiotic era, together with a resistance reverting mmpL5 mutation. CONCLUSIONS: The presence of a pre-existing reservoir of Mtb strains carrying bedaquiline RAVs prior to its clinical use augments the need for rapid drug susceptibility testing and individualised regimen selection to safeguard the use of bedaquiline in TB care and control.

Why do we use 100†mg of clofazimine in TB and NTM treatment?

Current tuberculosis and non-tuberculous mycobacterial disease guidelines recommend the use of clofazimine in a 100 mg once-daily dose. The rationale behind this exact dose is not provided. I performed a literature review to determine the reasoning behind the current dosing regimen. The current 100 mg once-daily dose of clofazimine stems from a deliberate attempt to find the minimum effective daily dose in leprosy treatment, driven by efficacy, economical and toxicity considerations. While this dose is safe, economical and practical, a higher dose with a loading phase may add relevant efficacy and treatment-shortening potential to both tuberculosis and non-tuberculous mycobacterial disease treatment. We need to revisit dose-response and maximum tolerated dose studies to get the best out of this drug, while continuing efforts to generate more active r-iminophenazine molecules that accumulate less in skin and intestinal tissues and have pharmacokinetic properties that do not require loading doses.

Clofazimine-induced cutaneous hyperpigmentation as a source of stigma in the treatment of leprosy: A cross-sectional study.

OBJECTIVES: Cutaneous hyperpigmentation is one of the main adverse effects encountered in patients undergoing leprosy treatment with multidrug therapy (WHO-MDT). This adverse effect has been described as intolerable and capable of contributing to social stigma. The objectives of this study were to quantify the variation in skin colour induced by clofazimine during and after treatment and to assess the related stigma. METHODS: This observational cross-sectional study objectively measured skin colour in 51 patients by reading the individual typology angle (ITA°) with a spectrophotometer, followed by the application of the Stigma Scale of the Explanatory Model Interview Catalogue (EMIC). RESULTS: Skin hyperpigmentation was observed in 100% of the individuals. They showed more negative ITA° values in lesion areas than non-lesion areas, particularly in sun-exposed regions. Clofazimine-induced cutaneous hyperpigmentation was not homogeneous and seemed to follow the lesion locations. The mean EMIC score was 18.8 points. CONCLUSION: All patients presented skin hyperpigmentation caused by clofazimine, detectable through spectrophotometry. Hyperpigmentation strongly impacted the social domain, indicating the intersectionality of disease and skin colour stigma, contributing to the social isolation of these patients. Health authorities should consider the negative impact of clofazimine on treatment adherence.

Pre-formulation of an additive combination of two antimicrobial agents, clofazimine and nisin A, to boost antimicrobial activity.

According to the World Health Organization, antimicrobial resistance is one of the top ten issues that pose a major threat to humanity. The lack of investment by the pharmaceutical industry has meant fewer novel antimicrobial agents are in development, exacerbating the problem. Emerging drug design strategies are exploring the repurposing of existing drugs and the utilization of novel drug candidates, like antimicrobial peptides, to combat drug resistance. This proactive approach is crucial in fighting global health threats. In this study, an additive combination of a repurposed anti-leprosy drug, clofazimine, and an antimicrobial peptide, nisin A, are preformulated using liquid antisolvent precipitation to generate a stable amorphous, ionized nanoparticle system to boost antimicrobial activity. The nanotechnology aims to improve the physicochemical properties of the inherently poorly water-soluble clofazimine molecules while also harnessing the previously unreported additive effect of clofazimine and nisin A. The approach transformed clofazimine into a more water-soluble salt, yielding amorphous nanoparticles stabilized by the antimicrobial peptide; and combined the two drugs into a more soluble and more active formulation. Blending pre-formulation strategies like amorphization, salt formation, and nanosizing to improve the inherent low aqueous solubility of drugs can open many new possibilities for the design of new antimicrobial agents. This fusion of pre-formulation technologies in combination with the multi-hurdle approach of selecting drugs with different effects on microbes could be key in the design platform of new antibiotics in the fight against antimicrobial resistance.

Effect of Bedaquiline and Delamanid Pharmacokinetics on Sputum Culture Conversion and Adverse Events in Drug-Resistant Tuberculosis.

BACKGROUND: Pharmacokinetic studies of bedaquiline and delamanid in patients with pre-extensively drug-resistant tuberculosis (pre-XDR TB) will help in the optimization of these drugs for both culture conversion and adverse events. METHODS: A prospective cohort of 165 adult patients (56% male with mean [SD] age 29 [9.7] years) with pre-XDR TB was treated with bedaquiline, delamanid, clofazimine, and linezolid for 24 weeks at 5 sites in India. Bedaquiline was administered at 400 mg daily for 2 weeks followed by 200 mg thrice weekly for 22 weeks, whereas delamanid was administered at 100 mg twice daily. In 23 consenting participants at 8 and 16 weeks of treatment, blood was collected at 0, 2, 4, 5, 6, 8, 12, and 24 hours postdosing for an intense pharmacokinetic study. Pharmacokinetic parameters were correlated with sputum culture conversion and adverse events. RESULTS: The mean (SD) age and weight of patients were 30 (10) years and 54 kg, respectively. The median minimum concentration (C min ) and time-concentration curve (AUC) for bedaquiline, respectively, were 0.6 mcg/mL and 27 mcg/mL·h at week 8 and 0.8 mcg/mL and 36 mcg/mL·h at week 16, suggesting drug accumulation over time. The median C min and AUC of delamanid, respectively, were 0.17 mcg/mL and 5.1 mcg/mL·h at week 8 and 0.20 mcg/mL and 7.5 mcg/mL·h at week 16. Delay in sputum conversion was observed in patients with drug concentrations lower than the targeted concentration. At weeks 8 and 16, 13 adverse events were observed. Adverse events were resolved through symptomatic treatment. Body mass index was found to be significantly associated with drug-exposure parameters. CONCLUSIONS: Bedaquiline and delamanid when co-administered exhibit plasma drug levels within the targeted concentrations, showing an exposure-response relationship.

Clofazimine pharmacokinetics in HIV-infected adults with diarrhea: Implications of diarrheal disease on absorption of orally administered therapeutics.

Oral drug absorption kinetics are usually established in populations with a properly functioning gastrointestinal tract. However, many diseases and therapeutics can alter gastrointestinal physiology and cause diarrhea. The extent of diarrhea-associated impact on drug pharmacokinetics has not been quantitatively described. To address this knowledge gap, we used a population pharmacokinetic modeling approach with data collected in a phase IIa study of matched human immunodeficiency virus (HIV)-infected adults with/without cryptosporidiosis and diarrhea to examine diarrhea-associated impact on oral clofazimine pharmacokinetics. A population pharmacokinetic model was developed with 428 plasma samples from 23 HIV-infected adults with/without Cryptosporidium infection using nonlinear mixed-effects modeling. Covariates describing cryptosporidiosis-associated diarrhea severity (e.g., number of diarrhea episodes, diarrhea grade) or HIV infection (e.g., viral load, CD4+ T cell count) were evaluated. A two-compartment model with lag time and first-order absorption and elimination best fit the data. Maximum diarrhea grade over the study duration was found to be associated with a more than sixfold reduction in clofazimine bioavailability. Apparent clofazimine clearance, intercompartmental clearance, central volume of distribution, and peripheral volume of distribution were 3.71 L/h, 18.2 L/h (interindividual variability [IIV] 45.0%), 473 L (IIV 3.46%), and 3434 L, respectively. The absorption rate constant was 0.625 h-1 (IIV 149%) and absorption lag time was 1.83 h. In conclusion, the maximum diarrhea grade observed for the duration of oral clofazimine administration was associated with a significant reduction in clofazimine bioavailability. Our results highlight the importance of studying disease impacts on oral therapeutic pharmacokinetics to inform dose optimization and maximize the chance of treatment success.

Case Report: Borderline Lepromatous Leprosy Therapy Complicated by Type 1 Leprosy Reaction and Adverse Reactions with Dapsone and Clofazimine.

Leprosy is a global health issue, causing long-term functional morbidity and stigma. Rapid diagnosis and appropriate treatment are important; however, early diagnosis is often challenging, especially in nonendemic areas. Here, we report a case of borderline lepromatous leprosy accompanied by dapsone-induced (neutropenia, anemia, and methemoglobinemia) and clofazimine-induced (skin discoloration and ichthyosis) side effects and type 1 leprosy reactions during administration of the multidrug therapy. The patient completely recovered without developing any deformities or visual impairment. To ensure early diagnosis and a favorable outcome, clinicians should be aware of the diminished sensation of skin lesions as a key physical finding and manage the drug toxicities and leprosy reactions appropriately in patients on multidrug therapy.