INTRODUCTION:Preclinical and clinical study data show that combining bedaquiline (B or BDQ), moxifloxacin (M), and pyrazinamide (Z), known as BMZ, has potent antimicrobial activity that might shorten treatment duration for drug-susceptible pulmonary tuberculosis. METHODS/DESIGN:We describe the design of Tuberculosis Trials Consortium (TBTC) Study 38/CRUSH-TB (NCT05766267), an open-label multicenter international randomized controlled phase 2C trial that compares two four-month regimens, BMZ plus rifabutin (Rb) (2BMZRb/2BMRb) or BMZ plus delamanid (D or DLM) (2BMZD/2BMD), with standard 6-months isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE). All drugs are administered seven days per week, under direct observation, at least five days per week. A total of 288 participants, aged ≥12 years, newly diagnosed with sputum smear-positive or Xpert MTB/RIF (Ultra)-positive drug-susceptible pulmonary tuberculosis, will be randomized 1:1:1 to receive BMZRb, BMZD, or HRZE. Participants are followed until 78 weeks post-randomization, or until the last enrolled participant completes 52 weeks post-randomization, whichever comes first. The primary endpoint is time to sputum culture negative in liquid media. Secondary endpoints include sustained cure, safety, and additional mycobacteriology and pharmacokinetic and pharmacodynamic outcomes. This trial has an adaptive design, wherein new arms can be added. DISCUSSION:This trial tests the hypothesis whether four-month BMZ-based regimens with Rb or D can shorten time to culture negativity while being safe and tolerable for participants. The study design is adaptive, allowing for additional study arms as new drugs become available. Findings from this trial might have important implications for clinically managing drug-susceptible pulmonary tuberculosis at individual and programmatic levels. Trial registration IND Number: 158058. IND Sponsor: U.S. Centers for Disease Control and Prevention. CLINICALTRIALS:govIdentifier:NCT05766267. Registered 13 March 2023, https://classic. CLINICALTRIALS:gov/ct2/show/NCT05766267.

Multiple in vitro potency assays are used to evaluate compounds against Mycobacterium tuberculosis, but a consensus on clinically relevant assays is lacking. We aimed to identify an in vitro assay signature that predicts preclinical efficacy and early clinical outcome. Thirty-one unique in vitro assays were compiled for 10 TB drugs. In vitro EC50 values were compared to pharmacokinetic-pharmacodynamic (PK-PD)-model-derived EC50 values from mice evaluated via multinomial regression. External validation of best-performing in vitro assay combinations was performed using five new TB drugs. Best-performing assay signatures for acute and subacute infections were described by assays that reproduce conditions found in macrophages and foamy macrophages and chronic infection by the ex vivo caseum assay. Subsequent simulated mouse bacterial burden over time using predicted in vivo EC50 was within 2-fold of observations. This study helps us identify clinically relevant assays and prioritize successful drug candidates, saving resources and accelerating clinical success.

The development of granulomas with central necrosis harboring Mycobacterium tuberculosis (Mtb) is the hallmark of human tuberculosis (TB). New anti-TB therapies need to effectively penetrate the cellular and necrotic compartments of these lesions and reach sufficient concentrations to eliminate Mtb. BTZ-043 is a novel antibiotic showing good bactericidal activity in humans in a phase IIa trial. Here, we report on lesional BTZ-043 concentrations severalfold above the minimal-inhibitory-concentration and the substantial local efficacy of BTZ-043 in interleukin-13-overexpressing mice, which mimic human TB pathology of granuloma necrosis. High-resolution MALDI imaging further reveals that BTZ-043 diffuses and accumulates in the cellular compartment, and fully penetrates the necrotic center. This is the first study that visualizes an efficient penetration and accumulation of a clinical-stage TB drug in human-like centrally necrotizing granulomas and that also determines its lesional activity. Our results most likely predict a substantial bactericidal effect of BTZ-043 at these hard-to-reach sites in TB patients. Therapy of tuberculosis is challenging, mainly due to complex structures of necrotic granulomas that often impair drug delivery. In this work, the authors show that the drug BTZ-043 fully penetrates necrotic granulomas and has potent lesional antibacterial activity.

Background BTZ-043 is a promising novel drug candidate for antituberculosis treatment. This study aimed to apply a previously developed mouse-to-human translational modeling platform for antituberculosis drugs to predict phase 2A outcomes for BTZ-043 in humans and evaluate the impact of observed drug-drug interactions on the contribution of BTZ-043 to combotherapy in a mouse model.Methods The study utilized data from mouse experiments for BTZ-043 monotherapy and combotherapy with bedaquiline, pretomanid, and linezolid, and clinical information for BTZ-043 monotherapy. The translational models were applied to predict the colony-forming units as a measure of efficacy in humans treated with BTZ-043 monotherapy and evaluate the effect of BTZ-043 on the pharmacokinetics-pharmacodynamics of combotherapy bedaquiline, pretomanid, and linezolid.Results The mouse-pharmacokinetic and mouse-pharmacodynamic data for BTZ-043 monotherapy were best described by 2-compartmental and direct Emax models, respectively. The model-based prediction of efficacy in humans was comparable to the observed phase 2A efficacy. Single-compartmental models, developed separately, best described the mouse-pharmacokinetic data for bedaquiline, pretomanid, and linezolid in combotherapy. Coadministration with BTZ-043 was associated with at least a 2-fold reduction in bedaquiline, pretomanid, and linezolid exposures in mice, and model-based simulations suggested that the observed decreases in exposure to these drugs would have resulted in even lower efficacy than what was observed when bedaquiline, pretomanid, and linezolid are coadministered with BTZ-043.Conclusions The translational modeling platform adequately predicted the efficacy of BTZ-043 monotherapy. In the absence of drug-drug interactions, coadministration of BTZ-043 with bedaquiline, pretomanid, and linezolid in combotherapy is predicted to improve treatment efficacy. Clinical Trials Registration. NCT0404400.Conclusions The translational modeling platform adequately predicted the efficacy of BTZ-043 monotherapy. In the absence of drug-drug interactions, coadministration of BTZ-043 with bedaquiline, pretomanid, and linezolid in combotherapy is predicted to improve treatment efficacy. Clinical Trials Registration. NCT0404400. The previously developed translational modeling platform for antituberculosis drugs adequately predicted phase 2A efficacy outcomes for BTZ-043. In the absence of drug-drug interactions, BTZ-043 increases efficacy outcomes when coadministered with a combination regimen of bedaquiline, pretomanid, and linezolid.

Abstract Introduction BTZ-043 is a promising novel drug candidate for anti-tuberculosis treatment. This study aimed to apply a previously developed mouse-to-human translational modeling platform for anti-tuberculosis drugs to predict phase IIA outcomes for BTZ-043 in humans and evaluate the impact of observed drug-drug interactions on the contribution of BTZ-043 to combotherapy in a mouse model. Methods The study utilized data from mouse experiments for BTZ-043 monotherapy and combotherapy with bedaquiline, pretomanid, and linezolid, and clinical information for BTZ-043 monotherapy. The translational models were applied to predict the colony-forming units as a measure of efficacy in humans treated with BTZ-043 monotherapy and evaluate the effect of BTZ-043 on the pharmacokinetics-pharmacodynamics of combotherapy bedaquiline, pretomanid, and linezolid. Results The mouse-pharmacokinetic and mouse-pharmacodynamic data for BTZ-043 monotherapy were best described by two-compartmental and direct Emax models, respectively. The model-based prediction of efficacy in humans was comparable to the observed phase IIA efficacy. Single-compartmental models, developed separately, best described the mouse-pharmacokinetic data for bedaquiline, pretomanid, and linezolid in combotherapy. Co-administration with BTZ-043 was associated with at least a 2-fold reduction in bedaquiline, pretomanid, and linezolid exposures in mice, and model-based simulations suggested that the observed decreases in exposure to these drugs would have resulted in even lower efficacy than what was observed when BPaL is co-administered with BTZ-043. Conclusion The translational modeling platform adequately predicted the efficacy of BTZ-043 monotherapy. In the absence of drug-drug interactions, co-administration of BTZ-043 with bedaquiline, pretomanid, and linezolid in combotherapy is predicted to improve treatment efficacy.

Accelerated tuberculosis drug discovery has increased the number of plausible multidrug regimens. Testing every drug combination in vivo is impractical, and varied experimental conditions make it challenging to compare results between experiments. Using published treatment efficacy data from a mouse tuberculosis model treated with candidate combination regimens, we trained and externally validated integrative mathematical models to predict relapse in mice and to rank both previously experimentally studied and unstudied regimens by their sterilization potential. We generated 18 datasets of 18 candidate regimens (comprising 11 drugs of six classes, including fluoroquinolone, nitroimidazole, diarylquinolines, and oxazolidinones), with 2965 relapse and 1544 colony-forming unit (CFU) observations for analysis. Statistical and machine learning techniques were applied to predict the probability of relapse in mice. The locked down mathematical model had an area under the receiver operating characteristic curve (AUROC) of 0.910 and showed that bacterial kill measured by longitudinal CFU cannot account for relapse alone and that sterilization is drug dependent. The diarylquinolines had the highest predicted sterilizing activity in the mouse model, and the addition of pyrazinamide to drug regimens provided the shortest estimated tuberculosis treatment duration to cure in mice. The mathematical model predicted the effect of treatment combinations, and these predictions were validated by conducting 11 experiments on previously unstudied regimens, achieving an AUROC of 0.829. We surmise that the next generation of tuberculosis drugs are highly effective at treatment shortening and suggest that there are several promising three- and four-drug regimens that should be advanced to clinical trials.

JSF-3285 is a promising preclinical candidate for tuberculosis that potently targets the Mycobacterium tuberculosis β-ketoacyl-ACP synthase KasA. In mouse models of acute, sub-acute, chronic, and relapse infection, JSF-3285 offers substantial activity in combination with bedaquiline and pretomanid, which could be applicable for both drug-sensitive and drug-resistant infections.

Recent efforts to improve tuberculosis (TB) treatment options have focused on developing molecules with novel mechanisms of action and identifying optimal treatment regimens. Inhibition of Mycobacterium tuberculosis cytochrome bc1 oxidase has emerged as a promising therapeutic target that could potentially contribute to improved TB combination regimens. Using a relapsing mouse model, we demonstrate that cytochrome bc1 inhibitors could serve as effective partner drugs, enhancing regimen sterilisation. We propose several novel regimen strategies for both multidrug-resistant TB (MDR-TB) and drug-sensitive TB (DS-TB), where cytochrome bc1 inhibitors contribute to sterilisation and treatment shortening. Additionally, we show that clinical isolates exhibit heightened susceptibility to cytochrome bc1 inhibitors compared to laboratory-adapted strains, further supporting their translational potential. These findings suggest that cytochrome bc1 inhibitors have significant potential to improve TB treatment outcomes and highlight the need for further studies to evaluate their clinical contribution to novel treatment regimens.

Isoniazid is an important first-line medicine to treat tuberculosis (TB). Isoniazid resistance increases the risk of poor treatment outcomes and development of multidrug resistance, and is driven primarily by mutations involving katG, encoding the prodrug-activating enzyme, rather than its validated target, InhA. The chemical tractability of InhA has fostered efforts to discover direct inhibitors of InhA (DIIs). In this study, we bridge the gap in understanding the potential contribution of DIIs to novel combination regimens and demonstrate a clear distinction of DIIs, like GSK693 and the newly described GSK138, from isoniazid, based on activity against clinical isolates and contribution to novel drug regimens. The results suggest that DIIs, specifically GSK138 and GSK693, could be promising partners in novel drug regimens, including those used against isoniazid-resistant TB, potentially enhancing their efficacy and/or preventing the selection of resistant mutants and supporting the continued exploration of InhA as a promising target for TB drug development.

Mycobacterium abscessus lung infection is notoriously difficult to treat due, in part, to the intrinsic resistance of this pathogen to most marketed antibiotics. β-Lactams, namely imipenem and cefoxitin, are first-line drugs in combination regimens used to treat this infection; and there is growing interest in dual-β-lactam-based regimens. Better understanding of the molecular basis of β-lactam activity through study of the genetic determinants of β-lactam susceptibility and tolerance would enable more rational drug combinations and guide discovery of novel drug targets to complement β-lactams. We recently used an inducible CRISPR interference (CRISPRi) system to silence cwlM and pbpB and confirm their essentiality for in vitro growth and resistance to sub-MIC concentrations of imipenem. Here, we extend those findings to show that silencing either gene alone is bactericidal and augments the bactericidal activity of imipenem in vitro . Furthermore, using CRISPRi in a mouse model of M. abscessus lung infection for the first time, we confirm the essentiality of each gene for in vivo survival. These results validate cwlM and pbpB as essential genes and promising drug targets in this pathogen, including for potentiation of carbapenem activity. The results further establish CRISPRi as a powerful method for validating drug targets and studying gene-gene and gene-drug interactions in vitro and in vivo .

Isoniazid is an important first-line medicine to treat tuberculosis (TB). Isoniazid resistance increases the risk of poor treatment outcomes and development of multidrug resistance, and is driven primarily by mutations involving katG , encoding the pro-drug activating enzyme, rather than its validated target, InhA. The chemical tractability of InhA has fostered efforts to discover direct inhibitors of InhA (DIIs). During the past five years, successful target engagement and in vivo efficacy have been demonstrated by diverse DIIs. In this study, we bridge the gap in understanding the potential contribution of DIIs to novel combination regimens and demonstrate a clear distinction of DIIs, like GSK693 and the newly described GSK138, from isoniazid, based on activity against clinical isolates and contribution to novel drug regimens. The results presented increase the understanding of DII mechanism of action and provide further impetus to continue exploiting InhA as a promising target for TB drug development.

Mycobacterium tuberculosis ( Mtb) is one of the leading infectious causes of death worldwide. There is no available licensed therapeutic vaccine that shortens active tuberculosis (TB) disease drug treatment and prevents relapse, despite the World Health Organization's calls. Here, we show that an intranasal DNA vaccine containing a fusion of the stringent response rel Mtb gene with the gene encoding the immature dendritic cell-targeting chemokine, MIP-3α/CCL20, shortens the duration of curative TB treatment in immunocompetent mice. Compared to the first-line regimen for drug-susceptible TB alone, our novel adjunctive vaccine induced greater Rel Mtb -specific T-cell responses associated with optimal TB control in spleen, blood, lungs, mediastinal lymph nodes, and bronchoalveolar lavage (BAL) fluid. These responses were sustained, if not augmented, over time. It also triggered more effective dendritic cell recruitment, activation, and colocalization with T cells, implying enhanced crosstalk between innate and adaptive immunity. Moreover, it potentiated a 6-month TB drug-resistant regimen, rendering it effective across treatment regimens, and also showed promising results in CD4+ knockout mice, perhaps due to enhanced Rel-specific CD8+ T-cell responses. Notably, our novel fusion vaccine was also immunogenic in nonhuman primates, the gold standard animal model for TB vaccine studies, eliciting antigen-specific T-cell responses in blood and BAL fluid analogous to those observed in protected mice. Our findings have critical implications for therapeutic TB vaccine clinical development in immunocompetent and immunocompromised populations and may serve as a model for defining immunological correlates of therapeutic vaccine-induced protection. One sentence summary:A TB vaccine shortens curative drug treatment in mice by eliciting strong TB-protective immune responses and induces similar responses in macaques.

A significant challenge in the development of long-acting injectable drug formulations, especially for anti-infective agents, is delivering an efficacious dose within a tolerable injection volume. Co-administration of the extracellular matrix-degrading enzyme hyaluronidase can increase maximum tolerable injection volumes but is untested for this benefit with long-acting injectable formulations. One concern is that hyaluronidase could potentially alter the tissue response surrounding an injection depot, a response known to be important for drug release kinetics of long-acting injectable formulations. The objective of this pilot study was to evaluate the impact of co-administration of hyaluronidase on the drug release kinetics, pharmacokinetic profiles, and injection site histopathology of the long-acting injectable paliperidone palmitate for up to four weeks following intramuscular injection in mouse and rat models. In both species, co-administration of hyaluronidase increased paliperidone plasma exposures the first week after injection but did not negate the overall long-acting release nature of the formulation. Hyaluronidase-associated modification of the injection site depot was observed in mice but not in rats. These findings suggest that further investigation of hyaluronidase with long-acting injectable agents is warranted.

Despite known treatments, tuberculosis (TB) remains the world's top infectious killer, highlighting the pressing need for new drug regimens. To prioritize the most efficacious drugs for clinical testing, we previously developed a PK-PD translational platform with bacterial dynamics that reliably predicted short-term monotherapy outcomes in Phase IIa trials from preclinical mouse studies. In this study, we extended our platform to include PK-PD models that account for drug-drug interactions in combination regimens and bacterial regrowth in our bacterial dynamics model to predict cure at the end of treatment and relapse 6 months post-treatment. The Phase III STAND trial testing a new regimen comprised of pretomanid (Pa), moxifloxacin (M), and pyrazinamide (Z) (PaMZ) was suspended after a separate ongoing trial (NC-005) suggested that adding bedaquiline (B) to the PaMZ regimen would improve efficacy. To forecast if the addition of B would, indeed, benefit the PaMZ regimen, we applied an extended translational platform to both regimens. We predicted currently available short- and long-term clinical data well for drug combinations related to BPaMZ. We predicted the addition of B to PaMZ to shorten treatment duration by 2 months and to have similar bacteriological success to standard HRZE treatment (considering only treatment success but not withdrawal from side effects and other adverse events), both at the end of treatment for treatment efficacy and 6 months after treatment has ended in relapse prevention. Using BPaMZ as a case study, we have demonstrated our translational platform can predict Phase II and III outcomes prior to actual trials, allowing us to better prioritize the regimens most likely to succeed.

Rationale: Observational studies suggest that high-dose isoniazid may be efficacious in treating multidrug-resistant tuberculosis. However, its activity against Mycobacterium tuberculosis (M.tb) with katG mutations (which typically confer high-level resistance) is not established. Objectives: To characterize the early bactericidal activity (EBA) of high-dose isoniazid in patients with tuberculosis caused by katG-mutated M.tb. Methods: A5312 was a phase IIA randomized, open-label trial. Participants with tuberculosis caused by katG-mutated M.tb were randomized to receive 15 or 20 mg/kg isoniazid daily for 7 days. Daily sputum samples were collected for quantitative culture. Intensive pharmacokinetic sampling was performed on Day 6. Data were pooled across all A5312 participants for analysis (drug-sensitive, inhA-mutated, and katG-mutated M.tb). EBA was determined using nonlinear mixed-effects modeling. Measurements and Main Results: Of 80 treated participants, 21 had katG-mutated M.tb. Isoniazid pharmacokinetics were best described by a two-compartment model with an effect of NAT2 acetylator phenotype on clearance. Model-derived maximum concentration and area under the concentration-time curve in the 15 and 20 mg/kg groups were 15.0 and 22.1 mg/L and 57.6 and 76.8 mg ⋅ h/L, respectively. Isoniazid bacterial kill was described using an effect compartment and a sigmoidal maximum efficacy relationship. Isoniazid potency against katG-mutated M.tb was approximately 10-fold lower than in inhA-mutated M.tb. The highest dose of 20 mg/kg did not demonstrate measurable EBA, except against a subset of slow NAT2 acetylators (who experienced the highest concentrations). There were no grade 3 or higher drug-related adverse events. Conclusions: This study found negligible bactericidal activity of high-dose isoniazid (15-20 mg/kg) in the majority of participants with tuberculosis caused by katG-mutated M.tb. Clinical trial registered with www.clinicaltrials.gov (NCT01936831).

Maintaining safe and potent pharmaceutical drug levels is often challenging. Multidomain peptides (MDPs) assemble into supramolecular hydrogels with a well-defined, highly porous nanostructure that makes them attractive for drug delivery, yet their ability to extend release is typically limited by rapid drug diffusion. To overcome this challenge, we developed self-assembling boronate ester release (SABER) MDPs capable of engaging in dynamic covalent bonding with payloads containing boronic acids (BAs). As examples, we demonstrate that SABER hydrogels can prolong the release of five BA-containing small-molecule drugs as well as BA-modified insulin and antibodies. Pharmacokinetic studies revealed that SABER hydrogels extended the therapeutic effect of ganfeborole from days to weeks, preventing Mycobacterium tuberculosis growth better than repeated oral administration in an infection model. Similarly, SABER hydrogels extended insulin activity, maintaining normoglycemia for six days in diabetic mice after a single injection. These results suggest that SABER hydrogels present broad potential for clinical translation.

The combination of bedaquiline, pretomanid, and linezolid (BPaL) has become a preferred regimen for treating multidrug- and extensively drug-resistant tuberculosis (TB). However, treatment-limiting toxicities of linezolid and reports of emerging bedaquiline and pretomanid resistance necessitate efforts to develop new short-course oral regimens. We recently found that the addition of GSK2556286 increases the bactericidal and sterilizing activity of BPa-containing regimens in a well-established BALB/c mouse model of tuberculosis. Here, we used this model to evaluate the potential of new regimens combining bedaquiline or the more potent diarylquinoline TBAJ-587 with GSK2556286 and the DprE1 inhibitor TBA-7371, all of which are currently in early-phase clinical trials. We found the combination of bedaquiline, GSK2556286, and TBA-7371 to be more active than the first-line regimen and nearly as effective as BPaL in terms of bactericidal and sterilizing activity. In addition, we found that GSK2556286 and TBA-7371 were as effective as pretomanid and the novel oxazolidinone TBI-223 when either drug pair was combined with TBAJ-587 and that the addition of GSK2556286 increased the bactericidal activity of the TBAJ-587, pretomanid, and TBI-223 combination. We conclude that GSK2556286 and TBA-7371 have the potential to replace pretomanid, an oxazolidinone, or both components, in combination with bedaquiline or TBAJ-587.

Abstract TBI-223, a novel oxazolidinone for tuberculosis, is designed to provide improved efficacy and safety compared to linezolid in combination with bedaquiline and pretomanid (BPaL). We aim to optimize the dosing of TBI-223 within the BPaL regimen for enhanced therapeutic outcomes. TBI-223 is investigated in preclinical monotherapy, multidrug therapy, and lesion penetration experiments to describe its efficacy and safety versus linezolid. A translational platform incorporating linezolid and BPaL data from preclinical experiments and 4 clinical trials (NCT00396084, NCT02333799, NCT03086486, NCT00816426) is developed, enabling validation of the framework. TBI-223 preclinical and Phase 1 data (NCT03758612) are applied to the translational framework to predict clinical outcomes and optimize TBI-223 dosing in combination with bedaquiline and pretomanid. Results indicate that daily doses of 1200–2400 mg TBI-223 may achieve efficacy comparable to the BPaL regimen, with >90% of patients predicted to reach culture conversion by two months.

The clinical efficacy of combination drug regimens containing the first-generation diarylquinoline (DARQ) bedaquiline in the treatment of multidrug-resistant tuberculosis has validated ATP synthesis as a vulnerable pathway in Mycobacterium tuberculosis. New DARQs in clinical development may be even more effective than bedaquiline, including against emerging bedaquiline-resistant strains. Telacebec (T) is a novel cytochrome bc(1):aa(3) oxidase inhibitor that also inhibits ATP synthesis. Based on its demonstrated efficacy as a monotherapy in mice and in a phase 2a clinical trial, we tested the contribution of T to novel combination therapies against two strains of M. tuberculosis (H37Rv and HN878) in an established BALB/c mouse model of tuberculosis in an effort to find more effective regimens. Overall, T was more effective in regimens against the HN878 strain than against the H37Rv strain, a finding supported by the greater vulnerability of the former strain to T and to genetic depletion of QcrB. Against both strains, combinations of a DARQ, clofazimine, and T were highly bactericidal. However, only against HN878 did T contribute synergistically, whereas an antagonistic effect was observed against H37Rv. These results demonstrate the therapeutic potential of T and highlight how differences in the susceptibility of M. tuberculosis strains could lead to different conclusions about a drug's potential contribution to novel drug regimens.