No abstract

fThe antileprosy drug clofazimine has shown potential for shortening tuberculosis treatment; however, the current dosing of the drug is not evidence based, and the optimal dosing is unknown. Our objective was to conduct a preclinical evaluation of the pharmacokinetics and pharmacodynamics of clofazimine in the mouse model of tuberculosis, with the goal of providing useful information on dosing for future studies. Pharmacokinetic parameters were evaluated in infected and uninfected BALB/c mice. Pharmacodynamic parameters were evaluated in Mycobacterium tuberculosis-infected mice that were treated for 12 weeks with one of six different clofazimine dosing regimens, i.e., doses of 6.25, 12.5, and 25 mg/kg of body weight/day and 3 regimens with loading doses. Clofazimine progressively accumulated in the lungs, livers, and spleens of the mice, reaching levels of greater than 50 g/g in all tissues by 4 weeks of administration, while serum drug levels remained low at 1 to 2 g/ml. Elimination of clofazimine was extremely slow, and the half-life was dependent on the duration of drug administration. Clofazimine exhibited dosedependent tissue and serum concentrations. At any dose, clofazimine did not have bactericidal activity during the first 2 weeks of administration but subsequently demonstrated potent, dose-independent bactericidal activity. The antituberculosis activity of clofazimine was dependent on neither the dose administered nor the drug concentrations in the tissues, suggesting that much lower doses could be effectively used for tuberculosis treatment. C lofazimine (CFZ) is a phenazine dye developed in the 1950s for the treatment of tuberculosis (TB) (1, 2). Despite promising activity against Mycobacterium tuberculosis both in vitro and in vivo, clofazimine was not advanced as a TB drug but instead became incorporated decades later into the multidrug treatment of leprosy, where it remains a key drug (3, 4). Interest in clofazimine for TB treatment has been revitalized following the 2010 report by Van Deun and colleagues that a clofazimine-containing regimen not only was highly effective for the treatment of multidrug-resistant (MDR) TB, but was also associated with a significantly decreased duration of therapy (5); 9 months of a clofazimine-containing regimen resulted in 87.9% relapse-free cure, which is in sharp contrast to the limited efficacy (less than 50% relapse-free cure) of the World Health Organization (WHO)-recommended, at least 20-month-long MDR-TB treatment regimen (6, 7). This report was complemented by experimental chemotherapy studies in which the inclusion of clofazimine in first-and secondline regimens significantly shortened the duration of treatment needed for relapse-free cure in mouse models of drug-susceptible and MDR TB, respectively (8, 9). These clinical and preclinical data suggest that clofazimine has great potential for TB treatment.Because clofazimine was abandoned as an option for TB treatment shortly after its discovery, and also because the use of clofazimine for leprosy is not ba...

SummaryThe immunometabolic mechanisms underlying suboptimal T cell immunity in tuberculosis remain undefined. Here, we examine how chronic Mycobacterium tuberculosis (Mtb) and M. bovis BCG infections rewire metabolic circuits and alter effector functions in lung CD8+ T cells. As Mtb infection progresses, mitochondrial metabolism deteriorates in CD8+ T cells, resulting in an increased dependency on glycolysis that potentiates inflammatory cytokine production. Over time, these cells develop bioenergetic deficiencies that reflect metabolic “quiescence.” This bioenergetic signature coincides with increased mitochondrial dysfunction and inhibitory receptor expression and was not observed in BCG infection. Remarkably, the Mtb-triggered decline in T cell bioenergetics can be reinvigorated by metformin, giving rise to an Mtb-specific CD8+ T cell population with improved metabolism. These findings provide insights into Mtb pathogenesis whereby glycolytic reprogramming and compromised mitochondrial function contribute to the breakdown of CD8+ T cell immunity during chronic disease, highlighting opportunities to reinvigorate immunity with metabolically targeted pharmacologic agents.

Our data suggest that clofazimine's delayed antimicrobial activity may be due more to its mechanism of action rather than to host-related factors.

The antileprosy drug clofazimine was recently repurposed as part of a newly endorsed short-course regimen for multidrug-resistant tuberculosis. It also enables significant treatment shortening when added to the first-line regimen for drug-susceptible tuberculosis in a mouse model. However, clofazimine causes dose- and duration-dependent skin discoloration in patients, and the optimal clofazimine dosing strategy in the context of the first-line regimen is unknown. We utilized a well-established mouse model to systematically address the impacts of duration, dose, and companion drugs on the treatment-shortening activity of clofazimine in the first-line regimen. In all studies, the primary outcome was relapse-free cure (culture-negative lungs) 6 months after stopping treatment, and the secondary outcome was bactericidal activity, i.e., the decline in the lung bacterial burden during treatment. Our findings indicate that clofazimine activity is most potent when coadministered with first-line drugs continuously throughout treatment and that equivalent treatment-shortening results are obtained with half the dose commonly used in mice. However, our studies also suggest that clofazimine at low exposures may have negative impacts on treatment outcomes, an effect that was evident only after the first 3 months of treatment. These data provide a sound evidence base to inform clofazimine dosing strategies to optimize the antituberculosis effect while minimizing skin discoloration. The results also underscore the importance of conducting long-term studies to allow the full evaluation of drugs administered in combination over long durations.

Emergence of mutant SARS-CoV-2 strains associated with an increased risk of COVID-19-related death necessitates better understanding of the early viral dynamics, host responses and immunopathology. Single cell RNAseq (scRNAseq) allows for the study of individual cells, uncovering heterogeneous and variable responses to environment, infection and inflammation. While studies have reported immune profiling using scRNAseq in terminal human COVID-19 patients, performing longitudinal immune cell dynamics in humans is challenging. Macaques are a suitable model of SARS-CoV-2 infection. Our longitudinal scRNAseq of bronchoalveolar lavage (BAL) cell suspensions from young rhesus macaques infected with SARS-CoV-2 (n = 6) demonstrates dynamic changes in transcriptional landscape 3 days post- SARS-CoV-2-infection (3dpi; peak viremia), relative to 14-17dpi (recovery phase) and pre-infection (baseline) showing accumulation of distinct populations of both macrophages and T-lymphocytes expressing strong interferon-driven inflammatory gene signature at 3dpi. Type I interferon response is induced in the plasmacytoid dendritic cells with appearance of a distinct HLADR+CD68+CD163+SIGLEC1+ macrophage population exhibiting higher angiotensin-converting enzyme 2 (ACE2) expression. These macrophages are significantly enriched in the lungs of macaques at 3dpi and harbor SARS-CoV-2 while expressing a strong interferon-driven innate anti-viral gene signature. The accumulation of these responses correlated with decline in viremia and recovery.

dExperimental and clinical studies have indicated that the antileprosy drug clofazimine may contribute treatment-shortening activity when included in tuberculosis treatment regimens. Clofazimine accumulates to high levels in tissues, has a long half-life, and remains in the body for months after administration is stopped. We hypothesized that in tuberculosis treatment, accumulated clofazimine may contribute sustained antimicrobial activity after treatment cessation, and we used the BALB/c mouse model of chronic tuberculosis chemotherapy to address this hypothesis. Mycobacterium tuberculosis-infected mice were treated for 4 weeks or 8 weeks with either isoniazid alone, clofazimine alone, the first-line regimen rifampin-isoniazid-pyrazinamideethambutol, or a first-line regimen where clofazimine was administered in place of ethambutol. To evaluate posttreatment antimicrobial activity, bacterial regrowth in the lungs and spleens was assessed at the day of treatment cessation and 2, 4, 6, and 8 weeks after treatment was stopped. Bacterial regrowth was delayed in all mice receiving clofazimine, either alone or in combination, compared to the mice that did not receive clofazimine. This effect was especially evident in mice receiving multidrug therapy. In mice not receiving clofazimine, bacterial regrowth began almost immediately after treatment was stopped, while in mice receiving clofazimine, bacterial regrowth was delayed for up to 6 weeks, with the duration of sustained antimicrobial activity being positively associated with the time that serum clofazimine levels remained at or above the 0.25-g/ml MIC for M. tuberculosis. Thus, sustained activity of clofazimine may be important in the treatment-shortening effect associated with this drug.

This is the first report of clofazimine (CFZ) penetration and distribution in normal mouse brain. Mice were administered 25 mg/kg CFZ or 100 mg/kg CFZ orally, daily for 2 weeks. Animals were sacrificed and blood and brain tissues were harvested. Liquid chromatography tandem mass spectrometry showed high concentrations of CFZ in homogenized brain, with 100 mg/kg dose having significantly higher concentration than 25 mg/kg. Matrix-assisted laser desorption/ionization spectrometric imaging of brain sections showed widespread tissue distribution of CFZ. Our results show dose dependent localization in brain.