Clofazimine broadly inhibits coronaviruses including SARS-CoV-2.

The COVID-19 pandemic is the third outbreak this century of a zoonotic disease caused by a coronavirus, following the emergence of severe acute respiratory syndrome (SARS) in 20031 and Middle East respiratory syndrome (MERS) in 20122. Treatment options for coronaviruses are limited. Here we show that clofazimine-an anti-leprosy drug with a favourable safety profile3-possesses inhibitory activity against several coronaviruses, and can antagonize the replication of SARS-CoV-2 and MERS-CoV in a range of in vitro systems. We found that this molecule, which has been approved by the US Food and Drug Administration, inhibits cell fusion mediated by the viral spike glycoprotein, as well as activity of the viral helicase. Prophylactic or therapeutic administration of clofazimine in a hamster model of SARS-CoV-2 pathogenesis led to reduced viral loads in the lung and viral shedding in faeces, and also alleviated the inflammation associated with viral infection. Combinations of clofazimine and remdesivir exhibited antiviral synergy in vitro and in vivo, and restricted viral shedding from the upper respiratory tract. Clofazimine, which is orally bioavailable and comparatively cheap to manufacture, is an attractive clinical candidate for the treatment of outpatients and-when combined with remdesivir-in therapy for hospitalized patients with COVID-19, particularly in contexts in which costs are an important factor or specialized medical facilities are limited. Our data provide evidence that clofazimine may have a role in the control of the current pandemic of COVID-19 and-possibly more importantly-in dealing with coronavirus diseases that may emerge in the future.

Safety and Effectiveness of 3 Novel All-Oral Shortened Regimens for Rifampicin- or Multidrug-Resistant Tuberculosis in Kazakhstan.

BACKGROUND: In 2019, the World Health Organization called for operational research on all-oral shortened regimens for multidrug- and rifampicin-resistant tuberculosis (MDR/RR-TB). We report safety and effectiveness of three 9-month all-oral regimens containing bedaquiline (Bdq), linezolid (Lzd), and levofloxacin (Lfx) and reinforced with cycloserine (Cs) and clofazimine (Cfz), delamanid (Dlm) and pyrazinamide (Z), or Dlm and Cfz. METHODS: We conducted a prospective cohort study of patients initiating treatment for pulmonary MDR/RR-TB under operational research conditions at public health facilities in Kazakhstan. Participants were screened monthly for adverse events. Participants with baseline resistance were excluded from the study and treated with a longer regimen. We analyzed clinically relevant adverse events of special interest in all participants and sputum culture conversion and end-of-treatment outcomes among individuals who were not excluded. RESULTS: Of 510 participants, 41% were women, the median age was 37 years (25th-75th percentile: 28-49), 18% had a body mass index <18.5 kg/m2, and 51% had cavitary disease. A total of 399 (78%) initiated Bdq-Lzd-Lfx-Cs-Cfz, 83 (16%) started Bdq-Lzd-Lfx-Dlm-Z, and 28 (5%) initiated Bdq-Lzd-Lfx-Dlm-Cfz. Fifty-eight individuals (11%) were excluded from the study, most commonly due to identification of baseline drug resistance (n = 52; 90%). Among the remaining 452 participants, treatment success frequencies were 92% (95% CI: 89-95%), 89% (95% CI: 80-94%), and 100% (95% CI: 86-100%) for regimens with Cs/Cfz, Dlm/Z, and Dlm/Cfz, respectively. Clinically relevant adverse events of special interest were uncommon. CONCLUSIONS: All regimens demonstrated excellent safety and effectiveness, expanding the potential treatment options for patients, providers, and programs.

Drug Exposure and Treatment Outcomes in Patients With Multidrug-Resistant Tuberculosis and Diabetes Mellitus: A Multicenter Prospective Cohort Study From China.

BACKGROUND: The management of multidrug-resistant tuberculosis (MDR-TB) remains challenging. Treatment outcome is influenced by multiple factors; the specific roles of diabetes and glycemic control remain uncertain. This study aims to assess the impact of glycemic control on drug exposure, to investigate the association between drug exposure and treatment outcomes, and to identify clinically significant thresholds predictive of treatment outcome, among patients with diabetes. METHODS: This multicenter prospective cohort study involved patients with confirmed MDR-TB and diabetes. Drug exposure level was estimated by noncompartmental analysis. The minimum inhibitory concentrations (MICs) were determined for the individual Mycobacterium tuberculosis isolates. The influence of poor glycemic control (glycated hemoglobin ≥7%) on drug exposure and the associations between drug exposure and treatment outcome were evaluated by univariate and multivariate analysis. Classification and regression tree analysis was used to identify the drug exposure/susceptibility thresholds. RESULTS: Among the 131 diabetic participants, 43 (32.8%) exhibited poor glycemic control. Poor glycemic control was independently associated with decreased exposure to moxifloxacin, linezolid, bedaquiline, and cycloserine, but not clofazimine. Additionally, a higher ratio of drug exposure to susceptibility was found to be associated with a favorable MDR-TB treatment outcome. Thresholds predictive of 6-month culture conversion and favorable outcome were bedaquiline area under the concentration-time curve (AUC)/MIC ≥245 and moxifloxacin AUC/MIC ≥67, demonstrating predictive accuracy in patients, regardless of their glycemic control status. CONCLUSIONS: Glycemic control and optimal TB drug exposure are associated with improved treatment outcomes. This dual management strategy should be further validated in randomized controlled trials of patients with MDR-TB and diabetes.

Pharmacokinetics and cardiac safety of clofazimine in children with rifampicin-resistant tuberculosis.

Clofazimine is recommended for the treatment of rifampicin-resistant tuberculosis (RR-TB), but there is currently no verified dosing guideline for its use in children. There is only limited safety and no pharmacokinetic (PK) data available for children. We aimed to characterize clofazimine PK and its relationship with QT-interval prolongation in children. An observational cohort study of South African children <18 years old routinely treated for RR-TB with a clofazimine-containing regimen was analyzed. Clofazimine 100 mg gelatin capsules were given orally once daily (≥20 kg body weight), every second day (10 to <20 kg), or thrice weekly (<10 kg). PK sampling and electrocardiograms were completed pre-dose and at 1, 4, and 10 hours post-dose, and the population PK and Fridericia-corrected QT (QTcF) interval prolongation were characterized. Fifty-four children contributed both PK and QTcF data, with a median age (2.5th-97.5th centiles) of 3.3 (0.5-15.6) years; five children were living with HIV. Weekly area under the time-concentration curve at steady state was 79.1 (15.0-271) mg.h/L compared to an adult target of 60.9 (56.0-66.6) mg.h/L. Children living with HIV had four times higher clearance compared to those without. No child had a QTcF ≥500 ms. A linear concentration-QTcF relationship was found, with a drug effect of 0.05 (0.027, 0.075) ms/µg/L. In some of the first PK data in children, we found clofazimine exposure using an off-label dosing strategy was higher in children versus adults. Clofazimine concentrations were associated with an increase in QTcF, but severe prolongation was not observed. More data are required to inform dosing strategies in children.

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.

Clofazimine as a substitute for rifampicin improves efficacy of Mycobacterium avium pulmonary disease treatment in the hollow-fiber model.

Mycobacterium avium complex pulmonary disease is treated with an azithromycin, ethambutol, and rifampicin regimen, with limited efficacy. The role of rifampicin is controversial due to inactivity, adverse effects, and drug interactions. Here, we evaluated the efficacy of clofazimine as a substitute for rifampicin in an intracellular hollow-fiber infection model. THP-1 cells, which are monocytes isolated from peripheral blood from an acute monocytic leukemia patient, were infected with M. avium ATCC 700898 and exposed to a regimen of azithromycin and ethambutol with either rifampicin or clofazimine. Intrapulmonary pharmacokinetic profiles of azithromycin, ethambutol, and rifampicin were simulated. For clofazimine, a steady-state average concentration was targeted. Drug concentrations and bacterial densities were monitored over 21 days. Exposures to azithromycin and ethambutol were 20%-40% lower than targeted but within clinically observed ranges. Clofazimine exposures were 1.7 times higher than targeted. Until day 7, both regimens were able to maintain stasis. Thereafter, regrowth was observed for the rifampicin-containing regimen, while the clofazimine-containing regimen yielded a 2 Log10 colony forming unit (CFU) per mL decrease in bacterial load. The clofazimine regimen also successfully suppressed the emergence of macrolide tolerance. In summary, substitution of rifampicin with clofazimine in the hollow-fiber model improved the antimycobacterial activity of the regimen. Clofazimine-containing regimens merit investigation in clinical trials.

How much should we still worry about QTc prolongation in rifampicin-resistant tuberculosis? ECG findings from TB-PRACTECAL clinical trial.

Regimens for the treatment of rifampicin-resistant tuberculosis currently rely on the use of QT-prolonging agents. Using data from the randomized controlled trial, TB-PRACTECAL, we investigated differences in QTcF among participants in the three interventional arms: BPaL (bedaquiline, pretomanid, and linezolid), BPaLC (BPaL with clofazimine), and BPaLM (BPaL with moxifloxacin). Additionally, we assessed whether age, body mass index, and country were causally associated with QTcF prolongation. The trial included participants from South Africa, Uzbekistan, and Belarus. A post hoc analysis of electrocardiogram data was undertaken. Random effects regression was used to model QTcF longitudinally over 24 weeks and causal frameworks guided the analysis of non-randomized independent variables. 328 participants were included in BPaL-based arms. The longitudinal analysis of investigational arms showed an initial QTcF steep increase in the first week. QTcF trajectories between weeks 2 and 24 differed slightly by regimen, with highest mean peak for BPaLC (QTcF 446.5 ms). Overall, there were 397 QTcF >450 ms (of 3,744) and only one QTcF >500 ms. The odds of QTcF >450 ms among participants in any investigational arm, was 8.33 times higher in Uzbekistan compared to Belarus (95% confidence interval: 3.25-21.33). No effect on QTcF prolongation was found for baseline age or body mass index (BMI). Clinically significant QTc prolongation was rare in this cohort of closely monitored participants. Across BPaL-based regimens, BPaLC showed a slightly longer and sustained effect on QTcF prolongation, but the differences (both in magnitude of change and trajectory over time) were clinically unimportant. The disparity in the risk of QTc prolongation across countries would be an important factor to further investigate when evaluating monitoring strategies. CLINICAL TRIALS: This study is registered with ClinicalTrials.gov as NCT02589782.

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.

Nine months of bedaquiline, linezolid, levofloxacin, clofazimine, and cycloserine chemotherapy for rifampicin/multidrug-resistant tuberculosis: a multicenter, randomized, open-label non-inferiority trial in China.

BACKGROUND: We concurrently developed a prospective study to assess clinical outcomes among patients receiving 9-month bedaquiline (BDQ)-containing regimens, aiming to provide valuable data on the use of this short-course regimen in China. METHODS: This open-label, randomized, controlled, multicenter, non-inferiority trial was conducted at sixteen hospitals, and enrolled participants aged 18 years and older with pulmonary rifampicin/multidrug tuberculosis. Participants were randomly assigned, in a 1:1 ratio. Individuals within the standard-regimen group received 6 months of BDQ, linezolid, levofloxacin, clofazimine, and cycloserine plus 12 months of levofloxacin, and any three potentially effective drugs from clofazimine, cycloserine pyrazinamide, ethambutol and protionamide, whereas individuals within shorter-regimen group received 9 months of BDQ, linezolid, levofloxacin, clofazimine and cycloserine. The primary outcome was the percentage of participants with a composite unfavorable outcome (treatment failure, death, treatment discontinuation, or loss to follow-up) by the end of the treatment course after randomization in the modified intention-to-treat population. The noninferiority margin was 10%. This trial was registered with www.chictr.org.cn , ChiCTR2000029012. RESULTS: Between Jan 1, 2020, and Dec 31, 2023, 264 were screened and randomly assigned, 132 of 264 participants were assigned to the standard-regimen group and 132 were assigned to the shorter-regimen. Thirty-three (12.55%) of 264 participants were excluded from the modified intention-to-treat analysis. As a result, 231 participants were included in the modified intention-to-treat analysis (116 in the standard-regimen group and 115 in the shorter-regimen group).In the modified intention-to-treat population, unfavorable outcomes were reported in 19 (16.5%) of 115 participants for whom the outcome was assessable in the shorter-regimen group and 26 (22.4%) of 116 participants in the standard care group (risk difference 5.9 percentage points (97.5% CI - 5.8 to 17.5)). One death was reported in the standard-regimen group. The incidence of QTcF prolongation in the shorter-regimen group (22.6%, 26/115) was similar to the standard-regimen group (24.1%, 28/116). CONCLUSIONS: The 9-month, all-oral regimen is safe and efficacious for the treatment of pulmonary rifampicin/multidrug-resistant tuberculosis. The high incidence of QTc prolongation associated with the use of BDQ highlights the urgent need of routine electrocardiogram monitoring under treatment with BDQ-containing regimens in the Chinese population.

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.

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.

Effectiveness of Bedaquiline Use beyond Six Months in Patients with Multidrug-Resistant Tuberculosis.

Rationale: Current recommendations for the treatment of rifampicin- and multidrug-resistant tuberculosis include bedaquiline (BDQ) used for 6 months or longer. Evidence is needed to inform the optimal duration of BDQ. Objectives: We emulated a target trial to estimate the effect of three BDQ duration treatment strategies (6, 7-11, and ⩾12 mo) on the probability of successful treatment among patients receiving a longer individualized regimen for multidrug-resistant tuberculosis. Methods: To estimate the probability of successful treatment, we implemented a three-step approach comprising cloning, censoring, and inverse probability weighting. Measurements and Main Results: The 1,468 eligible individuals received a median of 4 (interquartile range, 4-5) likely effective drugs. In 87.1% and 77.7% of participants, this included linezolid and clofazimine, respectively. The adjusted probability of successful treatment was 0.85 (95% confidence interval [CI], 0.81-0.88) for 6 months of BDQ, 0.77 (95% CI, 0.73-0.81) for 7-11 months, and 0.86 (95% CI, 0.83-0.88) for ⩾12 months. Compared with 6 months of BDQ, the ratio of treatment success was 0.91 (95% CI, 0.85-0.96) for 7-11 months and 1.01 (95% CI, 0.96-1.06) for ⩾12 months. Naive analyses that did not account for bias revealed a higher probability of successful treatment with ⩾12 months (ratio, 1.09 [95% CI, 1.05-1.14]). Conclusions: BDQ use beyond 6 months did not increase the probability of successful treatment among patients receiving longer regimens that commonly included new and repurposed drugs. When not properly accounted for, immortal person-time bias can influence estimates of the effects of treatment duration. Future analyses should explore the effect of treatment duration of BDQ and other drugs in subgroups with advanced disease and/or receiving less potent regimens.

Cotreatment With Clofazimine and Rapamycin Eliminates Drug-Resistant Tuberculosis by Inducing Polyfunctional Central Memory T-Cell Responses.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is acquiring drug resistance at a faster rate than the discovery of new antibiotics. Therefore, alternate therapies that can limit the drug resistance and disease recurrence are urgently needed. Emerging evidence indicates that combined treatment with antibiotics and an immunomodulator provides superior treatment efficacy. Clofazimine (CFZ) enhances the generation of T central memory (TCM) cells by blocking the Kv1.3+ potassium channels. Rapamycin (RAPA) facilitates M. tuberculosis clearance by inducing autophagy. In this study, we observed that cotreatment with CFZ and RAPA potently eliminates both multiple and extensively drug-resistant (MDR and XDR) clinical isolates of M. tuberculosis in a mouse model by inducing robust T-cell memory and polyfunctional TCM responses. Furthermore, cotreatment reduces the expression of latency-associated genes of M. tuberculosis in human macrophages. Therefore, CFZ and RAPA cotherapy holds promise for treating patients infected with MDR and XDR strains of M. tuberculosis.

Pharmacokinetic-Pharmacodynamic Determinants of Clinical Outcomes for Rifampin-Resistant Tuberculosis: A Multisite Prospective Cohort Study.

BACKGROUND: Rifampin-resistant and/or multidrug-resistant tuberculosis (RR/MDR-TB) treatment requires multiple drugs, and outcomes remain suboptimal. Some drugs are associated with improved outcome. It is unknown whether particular pharmacokinetic-pharmacodynamic relationships predict outcome. METHODS: Adults with pulmonary RR/MDR-TB in Tanzania, Bangladesh, and the Russian Federation receiving local regimens were enrolled from June 2016 to July 2018. Serum was collected after 2, 4, and 8 weeks for each drug's area under the concentration-time curve over 24 hours (AUC0-24). Quantitative susceptibility of the M. tuberculosis isolate was measured by minimum inhibitory concentrations (MICs). Individual drug AUC0-24/MIC targets were assessed by adjusted odds ratios (ORs) for favorable treatment outcome, and hazard ratios (HRs) for time to sputum culture conversion. K-means clustering algorithm separated the cohort of the most common multidrug regimen into 4 clusters by AUC0-24/MIC exposures. RESULTS: Among 290 patients, 62 (21%) experienced treatment failure, including 30 deaths. Moxifloxacin AUC0-24/MIC target of 58 was associated with favorable treatment outcome (OR, 3.75; 95% confidence interval, 1.21-11.56; P = .022); levofloxacin AUC0-24/MIC of 118.3, clofazimine AUC0-24/MIC of 50.5, and pyrazinamide AUC0-24 of 379 mg × h/L were associated with faster culture conversion (HR >1.0, P < .05). Other individual drug exposures were not predictive. Clustering by AUC0-24/MIC revealed that those with the lowest multidrug exposures had the slowest culture conversion. CONCLUSIONS: Amidst multidrug regimens for RR/MDR-TB, serum pharmacokinetics and M. tuberculosis MICs were variable, yet defined parameters to certain drugs-fluoroquinolones, pyrazinamide, clofazimine-were predictive and should be optimized to improve clinical outcome. CLINICAL TRIALS REGISTRATION: NCT03559582.

Clofazimine Inhalation Suspension Demonstrates Promising Toxicokinetics in Canines for Treating Pulmonary Nontuberculous Mycobacteria Infection.

Pulmonary nontuberculous mycobacteria (NTM) infection is recognized as a major global health concern due to its rising prevalence worldwide. As an opportunistic pathogen with increasing antibiotics resistance, prolonged systemic dosing with multiple antibiotics remains the primary treatment paradigm. These prolonged treatments, administered predominantly by oral or parenteral routes, often lead to systemic toxicity. A novel inhaled formulation of clofazimine may finally resolve issues of toxicity, thereby providing for improved NTM therapy. Clofazimine inhalation suspension was evaluated in canines to determine toxicity over 28 days of once-a-day dosing. The good laboratory practice (GLP) repeat dosing study evaluated low, mid, and high dosing (2.72 mg/kg and 2.95 mg/kg; 5.45 mg/kg and 5.91 mg/kg; and 10.87 mg/kg and 10.07 mg/kg, average male versus female dosing) of nebulized clofazimine over 30, 60, and 120 min using a jet nebulizer. Toxicokinetic analyses were performed on study days 29, 56, and 84. All three dose levels showed significant residual drug in lung tissue, demonstrating impressive lung loading and long lung residence. Drug concentrations in the lung remained well above the average NTM MIC at all time points, with measurable clofazimine levels at 28 and 56 days postdosing. In contrast, plasma levels of clofazimine were consistently measurable only through 14 days postdosing, with measurements below the limit of quantitation at 56 days postdosing. Clofazimine inhalation suspension may provide an effective therapy for the treatment of NTM infections through direct delivery of antibiotic to the lungs, overcoming the systemic toxicity seen in oral clofazimine treatment for NTM.

Bedquiline Resistance Mutations: Correlations with Drug Exposures and Impact on the Proteome in M. tuberculosis.

Bedaquiline (BDQ) is an effective drug for the treatment of drug-resistant tuberculosis. Mutations in atpE, which encodes the target of BDQ, are associated with large increases in MICs. Mutations in Rv0678 that derepress the transcription of the MmpL5-MmpS5 efflux transporter are associated with smaller increases in MICs. However, Rv0678 mutations are the most common mutations that are associated with BDQ resistance in clinical isolates, and they also confer cross-resistance to clofazimine (CFZ). To investigate the mechanism of BDQ resistance and the correlation between Rv0678 mutations and target-based atpE mutations, M. tuberculosis strains were exposed to different concentrations of BDQ or CFZ to select Rv0678 mutations and atpE mutations. Gene overexpression strains were constructed to illustrate the roles of MmpL5 and MmpS5. A quantitative proteome analysis was performed to compare the BDQ-resistant mutants to the isogenic strain H37Rv. Here, we report that the Rv0678 mutations were more readily selected than were the atpE mutations at low concentrations of BDQ or CFZ. The atpE mutations were selected by high concentrations of BDQ exposure. The overexpression of both mmpL5 and mmpS5 reduced the susceptibility of Mycobacterium tuberculosis to BDQ and CFZ. Secreted immunogenic proteins and proteins involved in the biosynthesis and transport of phthiocerol dimycocerosates were associated with Rv0678 mutations conferring BDQ resistance in the proteome analysis. In conclusion, exposure to different bedaquiline concentrations resulted in the selection of different mutations. The coexpression of MmpL5 and MmpS5 contributed to drug resistance and upregulated pathogenic proteins in M. tuberculosis, suggesting MmpL5-MmpS5 as a new potential target for antituberculosis drug development. These results warrant further surveillance for the evolution of BDQ resistance during clinical usage.

Emergence of Canonical and Noncanonical Genomic Variants following In Vitro Exposure of Clinical Mycobacterium tuberculosis Strains to Bedaquiline or Clofazimine.

In Mycobacterium tuberculosis, bedaquiline and clofazimine resistance occurs primarily through Rv0678 variants, a gene encoding a repressor protein that regulates mmpS5/mmpL5 efflux pump gene expression. Despite the shared effect of both drugs on efflux, little else is known about other pathways affected. We hypothesized that in vitro generation of bedaquiline- or clofazimine-resistant mutants could provide insight into additional mechanisms of action. We performed whole-genome sequencing and determined phenotypic MICs for both drugs on progenitor and mutant progenies. Mutants were induced through serial passage on increasing concentrations of bedaquiline or clofazimine. Rv0678 variants were identified in both clofazimine- and bedaquiline-resistant mutants, with concurrent atpE SNPs occurring in the latter. Of concern was the acquisition of variants in the F420 biosynthesis pathway in clofazimine-resistant mutants obtained from either a fully susceptible (fbiD: del555GCT) or rifampicin mono-resistant (fbiA: 283delTG and T862C) progenitor. The acquisition of these variants possibly implicates a shared pathway between clofazimine and nitroimidazoles. Pathways associated with drug tolerance and persistence, F420 biosynthesis, glycerol uptake and metabolism, efflux, and NADH homeostasis appear to be affected following exposure to these drugs. Shared genes affected by both drugs include Rv0678, glpK, nuoG, and uvrD1. Genes with variants in the bedaquiline resistant mutants included atpE, fadE28, truA, mmpL5, glnH, and pks8, while clofazimine-resistant mutants displayed ppsD, fbiA, fbiD, mutT3, fadE18, Rv0988, and Rv2082 variants. These results show the importance of epistatic mechanisms as a means of responding to drug pressure and highlight the complexity of resistance acquisition in M. tuberculosis.

QT Interval Prolongation with One or More QT-Prolonging Agents Used as Part of a Multidrug Regimen for Rifampicin-Resistant Tuberculosis Treatment: Findings from Two Pediatric Studies.

Rifampicin-resistant tuberculosis (RR-TB) involves treatment with many drugs that can prolong the QT interval; this risk may increase when multiple QT-prolonging drugs are used together. We assessed QT interval prolongation in children with RR-TB receiving one or more QT-prolonging drugs. Data were obtained from two prospective observational studies in Cape Town, South Africa. Electrocardiograms were performed before and after drug administration of clofazimine (CFZ), levofloxacin (LFX), moxifloxacin (MFX), bedaquiline (BDQ), and delamanid. The change in Fridericia-corrected QT (QTcF) was modeled. Drug and other covariate effects were quantified. A total of 88 children with a median (2.5th-to-97.5th range) age of 3.9 (0.5 to 15.7) years were included, of whom 55 (62.5%) were under 5 years of age. A QTcF interval of >450 ms was observed in 7 patient-visits: regimens were CFZ+MFX (n = 3), CFZ+BDQ+LFX (n = 2), CFZ alone (n = 1), and MFX alone (n = 1). There were no events with a QTcF interval of >500 ms. In a multivariate analysis, CFZ+MFX was associated with a 13.0-ms increase in change in QTcF (P < 0.001) and in maximum QTcF (P = 0.0166) compared to those when other MFX- or LFX-based regimens were used. In conclusion, we found a low risk of QTcF interval prolongation in children with RR-TB who received at least one QT-prolonging drug. Greater increases in maximum QTcF and ΔQTcF were observed when MFX and CFZ were used together. Future studies characterizing exposure-QTcF responses in children will be helpful to ensure safety with higher doses if required for effective treatment of RR-TB.

Investigation of genomic mutations and their association with phenotypic resistance to new and repurposed drugs in Mycobacterium tuberculosis complex clinical isolates.

BACKGROUND: WGS has the potential to detect resistance-associated mutations and guide treatment of MDR TB. However, the knowledge base to confidently interpret mutations associated with the new and repurposed drugs is sparse, and phenotypic drug susceptibility testing is required to detect resistance. METHODS: We screened 900 Mycobacterium tuberculosis complex genomes from Ireland, a low TB incidence country, for mutations in 13 candidate genes and assessed their association with phenotypic resistance to bedaquiline, clofazimine, linezolid, delamanid and pretomanid. RESULTS: We identified a large diversity of mutations in the candidate genes of 195 clinical isolates, with very few isolates associated with phenotypic resistance to bedaquiline (n = 4), delamanid (n = 4) and pretomanid (n = 2). We identified bedaquiline resistance among two drug-susceptible TB isolates that harboured mutations in Rv0678. Bedaquiline resistance was also identified in two MDR-TB isolates harbouring Met146Thr in Rv0678, which dated back to 2007, prior to the introduction of bedaquiline. High-level delamanid resistance was observed in two isolates with deletions in ddn, which were also resistant to pretomanid. Delamanid resistance was detected in two further isolates that harboured mutations in fbiA, but did not show cross-resistance to pretomanid. All isolates were susceptible to linezolid and clofazimine, and no mutations found were associated with resistance. CONCLUSIONS: More studies that correlate genotypic and phenotypic drug susceptibility data are needed to increase the knowledge base of mutations associated with resistance, in particular for pretomanid. Overall, this study contributes to the development of future mutation catalogues for M. tuberculosis complex isolates.