Clofazimine Research Articles

Effects of Bedaquiline Combined with Fluoroquinolone and/or Clofazimine on QT Interval in Patients with Multidrug-Resistant Tuberculosis: a Retrospective Study.

With the application of bedaquiline (Bdq), the success rate of multidrug-resistant tuberculosis (MDR-TB) treatment has been significantly improved; however, the cardiac safety of the patients during treatment cannot be ignored. Hence, this study compared the effects of bedaquiline alone and bedaquiline combined with fluoroquinolones (FQs) and/or clofazimine (CFZ) on the QT interval. This single-center retrospective cohort study analyzed the clinical data of MDR-TB patients treated with bedaquiline for 24 weeks from January 2020 to May 2021 in Xi'an Chest Hospital and compared the changes in QTcF between the two groups. Eighty-five patients were included in the study and grouped by types of anti-TB drugs affecting the QT interval they used. Group A included bedaquiline (n = 33), and group B included bedaquiline in combination with fluoroquinolones and/or clofazimine (n = 52). Out of patients with available corrected QT interval by Fridericia's formula (QTcF) data, 2.4% (2/85) experienced a postbaseline QTcF of ≥500 ms, and 24.7% (21/85) had at least one change of QTcF of ≥60 ms from baseline. In group A, 9.1% (3/33) had at least one ΔQTcF of >60 ms, as did 34.6% (18/52) of group B. Multivariate Cox regression analysis showed that the adjusted risk of QT prolongation was 4.82 times higher in group B (95% confidence interval [CI], 1.406 to 16.488). Bedaquiline combined with other anti-TB drugs affecting QT interval significantly increased the incidence of grade 3 or 4 QT prolongation; however, no serious ventricular arrhythmia and permanent drug withdrawal occurred. The use of bedaquiline combined with fluoroquinolone and/or clofazimine is an independent risk factor affecting QT interval. IMPORTANCE Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis. The emergence of MDR-TB is caused by an organism that is resistant to at least isoniazid and rifampin and is currently considered the major challenge for the global control of TB. Bedaquiline is the first new TB drug in 50 years with a unique mechanism of action, strong anti-M. tuberculosis activity. Yet unexplained excess deaths in the bedaquiline arms have been found in some phase II clinical trials; thus, the FDA has issued a "boxed warning." However, the cardiac safety of the patients during treatment cannot be ignored. Accordingly, further investigations are needed to establish whether bedaquiline combined with clofazimine, fluoroquinolones, or anti-TB drugs affecting the QT interval in a long-course or short-course treatment increases the risk of QT prolongation.

Co-treatment with Clofazimine and Rapamycin eliminates drug-resistant tuberculosis by inducing polyfunctional central memory T cell responses.

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), 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 evidences indicate 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.tb clearance by inducing autophagy. In this study, we observed that the co-treatment with CFZ and Rapa potently eliminates both multiple and extensively drug-resistant (MDR and XDR) clinical isolates of M.tb in a mouse model by inducing robust T cell memory and polyfunctional TCM responses. Furthermore, co-treatment reduces the expression of latency-associated genes of M.tb in human macrophages. Therefore, CFZ and Rapa co-therapy holds promise for treating patients infected with MDR and XDR strains of M.tb.

Clofazimine-Mediated, Age-Related Changes in Skeletal Muscle Mitochondrial Metabolites.

Mitochondrial health declines with age, and older patients can demonstrate dysfunction in mitochondrial-rich tissues, such as cardiac and skeletal muscle. Aged mitochondria may make older adults more susceptible to adverse drug reactions (ADRs). We assessed mitochondrial metabolic function by measuring two metabolites, l-carnitine and acetylcarnitine, to determine their effectiveness as candidate clinical biomarkers for age-related, drug-induced alterations in mitochondrial metabolism. To study age- and medication-related changes in mitochondrial metabolism, we administered the FDA-approved mitochondriotropic drug, clofazimine (CFZ), or vehicle for 8 weeks to young (4-week-old) and old (61-week-old) male C57BL/6J mice. At the end of treatment, whole blood and cardiac and skeletal muscle were analyzed for l-carnitine, acetylcarnitine, and CFZ levels; muscle function was measured via a treadmill test. No differences were found in blood or cardiac carnitine levels of CFZ-treated mice, but CFZ-treated mice displayed lost body mass and alterations in endurance and levels of skeletal muscle mitochondrial metabolites. These findings demonstrate the age-related susceptibility of the skeletal muscle to mitochondria drug toxicity. Since drug-induced alterations in mitochondrial metabolism in skeletal muscle were not reflected in the blood by l-carnitine or acetylcarnitine levels, drug-induced catabolism and changes in muscle function appear more relevant to stratifying individuals at increased risk for ADRs.

Development of low-cost, weight-adjustable clofazimine mini-tablets for treatment of tuberculosis in pediatrics

Clofazimine (CFZ) is an important component of the World Health Organization's (WHO) recommended all-oral drug regimen for treatment of multi-drug resistant tuberculosis (MDR-TB). However, the lack of a dividable oral dosage form has limited the use of the drug in pediatric populations, who may require lowering of the dose to reduce the likelihood of adverse drug events. In this study, pediatric-friendly CFZ mini-tablets were prepared from micronized powder via direct compression. Rapid disintegration and maximized dissolution in GI fluids was achieved using an iterative formulation design process. Pharmacokinetic (PK) parameters of the optimized mini-tablets were obtained in Sprague-Dawley rats and compared against an oral suspension of micronized CFZ particles to examine the effect of processing and formulation on the oral absorption of the drug. Differences in maximum concentration and area under the curve between the two formulations were non-significant at the highest dosing level tested. Variability between rats prevented bioequivalence from being determined according to guidelines outlined by the Food and Drug Administration (FDA). These studies provide an important proof-of-concept for an alternative, low-cost formulation and processing approach for the oral delivery of CFZ in manner that is suitable for children as young as 6 months of age.

Encapsulation of Clofazimine by Cyclodextran: Preparation, Characterization, and In Vitro Release Properties.

This study aimed to evaluate and compare the efficacy of cyclodextrans (CIs) and cyclodextrins (CDs) in improving the water solubility of a poorly water-soluble drug, clofazimine (CFZ). Among the evaluated CIs and CDs, CI-9 exhibited the highest percentage of drug inclusion and the highest solubility. Additionally, CI-9 showed the highest encapsulation efficiency, with a CFZ:CI-9 molar ratio of 0.2:1. SEM analysis indicated successful formation of inclusion complexes CFZ/CI and CFZ/CD, accounting for the rapid dissolution rate of the inclusion complex. Moreover, CFZ in CFZ/CI-9 demonstrated the highest drug release ratio, reaching up to 97%. CFZ/CI complexes were found to be an effective means of protecting the activity of CFZ against various environmental stresses, particularly UV irradiation, compared to free CFZ and CFZ/CD complexes. Overall, the findings provide valuable insights into designing novel drug delivery systems based on the inclusion complexes of CIs and CDs. However, further studies are needed to investigate the effects of these factors on the release properties and pharmacokinetics of encapsulated drugs in vivo, in order to ensure the safety and efficacy of these inclusion complexes. In conclusion, CI-9 is a promising candidate for drug delivery systems, and CFZ/CI complexes could be a potential formulation strategy for the development of stable and effective drug products.

In Vitro Synergistic Effects of Omadacycline with Other Antimicrobial Agents against Mycobacterium abscessus.

The clinical importance of Mycobacterium abscessus species (MABS) infections has been increasing. However, the standard treatment regimens recommended in the current guidelines often result in unfavorable outcomes. Therefore, we investigated the in vitro activity of omadacycline (OMC), a novel tetracycline, against MABS to explore its potential as a novel therapeutic option. The drug susceptibilities of 40 Mycobacterium abscessus subsp. abscessus (Mab) clinical strains obtained from the sputum of 40 patients from January 2005 to May 2014 were investigated. The MIC results for OMC, amikacin (AMK), clarithromycin (CLR), clofazimine (CLO), imipenem (IPM), rifabutin (RFB), and tedizolid (TZD) alone and their combined effects (with OMC) were examined using the checkerboard method. Additionally, we studied the differences in the effectiveness of the antibiotic combinations based on the colony morphotype of Mab. The MIC50 and MIC90 of OMC alone were 2 and 4 μg/mL, respectively. The combinations of OMC with AMK, CLR, CLO, IPM, RFB, and TZD showed synergy against 17.5%, 75.8%, 25.0%, 21.1%, 76.9%, and 34.4% of the strains, respectively. Additionally, OMC combined with CLO (47.1% versus 9.5%, P = 0.023) or TZD (60.0% versus 12.5%, P = 0.009) showed significantly higher synergy against strains with rough morphotypes than those with smooth morphotypes. In conclusion, the checkerboard analyses revealed that the synergistic effects of OMC were observed most frequently with RFB, followed by CLR, TZD, CLO, IPM, and AMK. Furthermore, OMC tended to be more effective against rough-morphotype Mab strains.

Epidemiology of Nontuberculous Mycobacteria in Nanjing and MAB_0540 Mutations Associated with Clofazimine Resistance in Mycobacterium abscessus.

Nontuberculous mycobacteria (NTM) are easily misdiagnosed as multidrug-resistant tuberculosis (MDR-TB), and treatment drugs are very limited. The main objective of our study was to evaluate the minimal inhibitory concentration (MIC) in vitro of bedaquiline (BDQ), clofazimine (CFZ), linezolid (LZD), delamanid (DLM), and pretomanid (PA-824) for treatment of M. abscessus and M. intracellulare. Furthermore, we determined whether MAB_1448, MAB_4384, MAB_2299c, MAB_1483, MAB_0540, rplD, rplC, and rrl were related to drug resistance to provide an experimental basis for the use of these five drugs in the treatment of NTM. We identified sample characteristics of epidemics in 550 patients with suspected NTM infection in Nanjing from 2019 to 2021 using the PCR-reverse spot hybrid method. Furthermore, we evaluated the MIC of BDQ, CFZ, DLM, LZD, and PA-824 against 155 clinical isolates of NTM using the microbroth dilution method. The resistant isolates were sequenced using Sanger sequencing. The top three dominant species of NTM distributed in Nanjing were M. intracellulare, M. avium, and M. abscessus. Notably, the proportion of M. abscessus infections increased. The proportion of M. abscessus increased from 12% in 2019 to 18% in 2021. Demographic analysis showed that female infection rates were substantialy greater than male for M. abscessus (P=0.017, <0.05). Our results demonstrate that NTM are highly sensitive to bedaquiline and clofazimine in vitro. However, delamanid and pretomanid had little effect on M. abscessus and M. intracellulare. In addition, we found 30-41 nucleotide deletion mutations and some novel point mutations in the MAB_0540 gene of M. abscessus that are resistant to clofazimine. Bedaquiline, clofazimine, and linezolid were more successful in vitro treatments against M. abscessus and M. intracellulare. The MAB_0540 mutation may be associated with resistance of M. abscessus to clofazimine.

Clofazimine pKa Determination by Potentiometry and Spectrophotometry: Reverse Cosolvent Dependence as an Indicator of the Presence of Dimers in Aqueous Solutions.

The weakly basic antibiotic and anti-inflammatory drug, clofazimine (CFZ), was first described in 1957. It has been used therapeutically, most notably in the treatment of leprosy. However, the compound is extremely insoluble in aqueous media, and, indeed, there is poor consensus about what its intrinsic solubility is since the reported values range from 0.04 to 11 ng/mL. To understand the speciation and solubilization of CFZ as a function of pH, it is of paramount importance to know the true aqueous pKa. However, there is also poor consensus about the value of the pKa (reported measured values range from 6.08 to 9.11). In the present study, we report the determination of the CFZ ionization constant using two independent techniques. A state-of-the-art potentiometric analysis was performed, drawing on titration data in methanol-water solutions (46-75 wt % MeOH) of CFZ, using the bias-reducing consensus of two different procedures of extrapolating the apparent psKa values to zero cosolvent to approximate the true aqueous pKa as 9.43 ± 0.12 (25 °C, I = 0.15 M reference ionic strength). In parallel, spectrophotometric UV/vis titration data were acquired (250-600 nm at different pH) in 10 mM HEPES buffer solutions containing up to 54 wt % MeOH. The alternating least squares (ALS) method was used in the analysis of the absorbance-pH spectra. Uncharacteristically, the cosolvent UV/vis data in our study showed reverse cosolvent dependence (apparent pKa values increased with increasing cosolvent) which could be explained by a dimerization of the free base. The analysis of UV/vis data obtained from 54 wt % MeOH-water solution containing 20 μM CFZ yielded the apparent pKa 9.51 ± 0.17 (I ≈ 0.005 M). To assess whether self-assembly of CFZ was energetically feasible, density functional theory (DFT) calculations were used to study the putative CFZ dimers in aqueous and methanol media. The DFT-optimized geometries and infrared spectra of CFZ dimers using water and methanol as solvents were calculated and analyzed. Based on the lack of negative frequencies in calculated infrared spectra, it was confirmed that optimized geometries correspond to the true energetic minima. Visual analysis of optimized structures indicates the presence of stacking interactions between two CFZ molecules. The protonation site (the imine nitrogen atom) was determined by 1H NMR spectroscopy.

Antimicrobial Susceptibility Testing Using the MYCO Test System and MIC Distribution of 8 Drugs against Clinical Isolates of Nontuberculous Mycobacteria from Shanghai.

Given the increased incidence and prevalence of nontuberculous mycobacterial (NTM) diseases and the natural resistance of NTM to multiple antibiotics, in vitro susceptibility testing of different NTM species against drugs from the MYCO test system and new applied drugs is required. A total of 241 NTM clinical isolates were analyzed, including 181 slowly growing mycobacteria (SGM) and 60 rapidly growing mycobacteria (RGM). The Sensititre SLOMYCO and RAPMYCO panels were used for testing susceptibility to commonly used anti-NTM antibiotics. Furthermore, MIC distributions were determined against 8 potential anti-NTM drugs, including vancomycin (VAN), bedaquiline (BDQ), delamanid (DLM), faropenem (FAR), meropenem (MEM), clofazimine (CLO), cefoperazone-avibactam (CFP-AVI), and cefoxitin (FOX), and epidemiological cutoff values (ECOFFs) were analyzed using ECOFFinder. The results showed that most of the SGM strains were susceptible to amikacin (AMK), clarithromycin (CLA), and rifabutin (RFB) from the SLOMYCO panels and BDQ and CLO from the 8 applied drugs, while RGM strains were susceptible to tigecycline (TGC) from the RAPMYCO panels and also BDQ and CLO. The ECOFFs of CLO were 0.25, 0.25, 0.5, and 1 μg/mL for the mycobacteria M. kansasii, M. avium, M. intracellulare, and M. abscessus, respectively, and the ECOFF of BDQ was 0.5 μg/mL for the same four prevalent NTM species. Due to the weak activity of the other 6 drugs, no ECOFF was determined. This study on the susceptibility of NTM includes 8 potential anti-NTM drugs and a large sample size of Shanghai clinical isolates and demonstrates that BDQ and CLO had efficient activities against different NTM species in vitro, which can be applied to the treatment of NTM diseases. IMPORTANCE We designed customized panel that contains 8 repurposed drugs, including vancomycin (VAN), bedaquiline (BDQ), delamanid (DLM), faropenem (FAR), meropenem (MEM), clofazimine (CLO), cefoperazone-avibactam (CFP-AVI), and cefoxitin (FOX) from the MYCO test system. To better understand the efficacy of these 8 drugs against different NTM species, we determined the MICs of 241 NTM isolates collected in Shanghai, China. We attempted to define the tentative epidemiological cutoff values (ECOFFs) for the most prevalent NTM species, which is an important factor in setting up the breakpoint for a drug susceptibility testing. We used the MYCO test system as an automatic quantitative drug sensitivity test of NTM and extended the method to BDQ and CLO in this study. The MYCO test system complements commercial microdilution systems that currently lack BDQ and CLO detection.

Extemporaneously compounded liquid formulations of clofazimine.

BACKGROUND: Clofazimine (CFZ) is routinely used worldwide for the treatment of leprosy and TB. However, no liquid or dispersible tablet formulations of CFZ are currently available commercially for patients with challenges ingesting soft gelatin capsules or solid formulations. The aim of this research was to develop stable extemporaneous liquid formulations of CFZ that can be stored at room temperature for several weeks to enable practical dosing in the field. METHODS: Two formulations were prepared in syrup and sugar-free vehicles with CFZ tablets using a simple method that can be used in a routine pharmacy. Suspensions were stored at room temperature and at 30°C for 30 days. Formulation aliquots were tested on Days 0, 15 and 30 for appearance, pH, potency and microbial counts. RESULTS: Appearance remained unchanged during storage. The pH of both formulations was between 4.0 and 6.0. Potency was between 90% and 110% for 30 days in the syrup formulation and for 15 days in the sugar-free formulation. Microbial counts met United States Pharmacopeia 1111 limits for oral aqueous liquids and specific organisms were absent. CONCLUSIONS: A simple field-friendly method was successfully developed for the preparation of CFZ liquid formulations using commonly available ingredients. This will permit practical dosing and titration for children and other patients with swallowing challenges.

Application of engineered myoglobins for biosynthesis of clofazimine by integration with chemical synthesis.

Significant efforts have been made in the design of artificial metalloenzymes. Myoglobin (Mb), an O2 carrier, has been engineered to exhibit different functions. Herein, we applied a series of engineered Mb mutants with peroxidase activity for biosynthesis of clofazimine (CFZ), a potential drug with a broad-spectrum antiviral activity, by integration with chemical synthesis. Two of those mutants, F43Y Mb and F43Y/T67R Mb, have been shown to efficiently catalyze the oxidative coupling of 2-N-(4-chlorophenyl) benzene-1,2-diamine (N-4-CPBDA) in the presence of H2O2, with 97% yields. The overall catalytic efficiency (kcat/Km) is 46-fold and 82-fold higher than that of WT Mb, respectively. By further combination of this reaction with chemical synthesis, the production of CFZ was accomplished with an isolated yield of 72%. These results showed that engineered Mbs containing the Tyr-heme cross-link (F43Y Mb and F43Y/T67R Mb) exhibit enhanced activity in the oxidative coupling reaction. This study also indicates that the combination of biocatalysis and chemical synthesis avoids the need for the separation of intermediate products, which offers a convenient approach for the total synthesis of the biological compound CFZ.

Polymeric nano-system for macrophage reprogramming and intracellular MRSA eradication.

Intracellular Methicillin-Resistant Staphylococcus aureus (MRSA) remains a major factor of refractory and recurrent infections, which cannot be well addressed by antibiotic therapy. Here, we design a cellular infectious microenvironment-activatable polymeric nano-system to mediate targeted intracellular drug delivery for macrophage reprogramming and intracellular MRSA eradication. The polymeric nano-system is composed of a ferrocene-decorated polymeric nanovesicle formulated from poly(ferrocenemethyl methacrylate)-block-poly(2-methacryloyloxyethyl phosphorylcholine) (PFMMA-b-PMPC) copolymer with co-encapsulation of clofazimine (CFZ) and interferon-γ (IFN-γ). The cellular-targeting PMPC motifs render specific internalization by macrophages and allow efficient intracellular accumulation. Following the internalization, the ferrocene-derived polymer backbone sequentially undergoes hydrophobic-to-hydrophilic transition, charge reversal and Fe release in response to intracellular hydrogen peroxide over-produced upon infection, eventually triggering endosomal escape and on-site cytosolic drug delivery. The released IFN-γ reverses the immunosuppressive status of infected macrophages by reprogramming anti-inflammatory M2 to pro-inflammatory M1 phenotype. Meanwhile, intracellular Fe2+-mediated Fenton reaction together with antibiotic CFZ contributes to increased intracellular hydroxyl radical (•OH) generation. Ultimately, the nano-system achieves robust potency in ablating intracellular MRSA and antibiotic-tolerant persisters by synchronous immune modulation and efficient •OH killing, providing an innovative train of thought for intracellular MRSA control.

1712. In Vitro Activity of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, in Drug Combinations Against Nontuberculous Mycobacteria Including Resistance Frequency and MIC Characterization of Mycobacterium avium ATCC 700898 Epetraborole-resistant Mutants

Abstract Background Epetraborole (EBO) is a boron-containing, oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria (NTM). The standard of care therapy for Mycobacterium avium complex (MAC) lung disease consists of a combination of a macrolide, ethambutol (EMB) and a rifamycin. A total of 5 strains of MAC, M. abscessus ATCC 19977 and M. peregrinum ATCC 700686 were tested in checkerboard assays to evaluate the effects of combining EBO with other agents. The spontaneous resistance frequency (RF) was determined for EBO singly and in combination against M. avium ATCC 700898. Methods The effects of combining EBO with clarithromycin (CLR), rifabutin (RBT), EMB, amikacin (AMK) or bedaquiline (BDQ) were evaluated in 7 strains of NTM. Synergy, additive effects, indifference or antagonism was characterized in the checkerboard assay using EUCAST criteria. The RF of M. avium ATCC 700898 at 2x, 4x and 8x the MIC (8 mg/L) of EBO was determined, as was the RF of EBO combined with CLR, RBT, AMK or EMB. MICs of selected EBO mutants were determined against AMK, BDQ, CLR, RBT, EMB, and clofazimine (CFZ) and the mutants were further characterized. Results The RF of EBO ranged from 1.58x10-7 to 8.48x10-9 when selected on 2 - 8x agar MIC. The MIC for EBO increased 32- &amp;gt; 256-fold for the resistant mutants; however, the MICs for the other drugs tested against EBO-resistant strains did not change significantly. The activity of EBO was not antagonized, and was mainly indifferent to the addition of CLR, RBT, AMK, or BDQ for all the NTM strains tested. Synergy with EMB was observed with 2 strains and additivity with 2 additional strains of MAC. The addition of EMB (24 mg/L), CLR (32 mg/L), RFB (2 mg/L) or AMK (128 mg/L) to agar plates containing 2x MIC of EBO lowered the RF to at least &amp;lt; 2.13x10-10 (Table 1). Table 1.In Vitro Resistance Frequency for the Components of SOC with and without EBO in Mycobacterium avium ATCC 700898 Conclusion In the checkerboard studies, no evidence of antagonism was observed with any strain or EBO combination; interactions were largely indifferent. EBO combined with EMB in MAC resulted in synergy or additive effects in the checkerboard assay. The addition of EMB, CLR, RFB or AMK to EBO led to a &amp;gt; 700-fold reduction in RF. Activity of other drugs was not impacted by EBO resistance suggesting that cross-resistance did not occur. Disclosures Michelle S. DeStefano, n/a, AN2 Therapeutics: Grant/Research Support Carolyn M. Shoen, PhD, AN2 Therapeutics: Grant/Research Support MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Michael H. Cynamon, MD, AN2: Grant/Research Support|AN2: Grant/Research Support.

MmpL3 Inhibition as a Promising Approach to Develop Novel Therapies against Tuberculosis: A Spotlight on SQ109, Clinical Studies, and Patents Literature.

Tuberculosis (TB) is accountable for considerable global morbidity and mortality. Effective TB therapy with multiple drugs completes in about six months. The longer duration of TB therapy challenges patient compliance and contributes to treatment collapse and drug resistance (DR) progress. Therefore, new medications with an innovative mechanism of action are desperately required to shorten the TB therapy's duration and effective TB control. The mycobacterial membrane protein Large 3 (MmpL3) is a novel, mycobacteria-conserved and recognized promiscuous drug target used in the development of better treatments for multi-drug resistance TB (MDR-TB) and extensively drug-resistant TB (XDR-TB). This article spotlights MmpL3, the clinical studies of its inhibitor (SQ109), and the patent literature. The literature on MmpL3 inhibitors was searched on PubMed and freely available patent databases (Espacenet, USPTO, and PatentScope). SQ109, an analog of ethambutol (EMB), is an established MmpL3 inhibitor and has completed Phase 2b-3 clinical trials. Infectex and Sequella are developing orally active SQ109 in partnership to treat MDR pulmonary TB. SQ109 has demonstrated activity against drug-sensitive (DS) and drug-resistant (DR) Mycobacterium tuberculosis (Mtb) and a synergistic effect with isoniazid (INH), rifampicin (RIF), clofazimine (CFZ), and bedaquiline (BNQ). The combination of SQ109, clofazimine, bedaquiline, and pyrazinamide (PZA) has been patented due to its excellent anti-TB activity against MDR-TB, XDR-TB, and latent-TB. The combinations of SQ109 with other anti-TB drugs (chloroquine, hydroxychloroquine, and sutezolid) have also been claimed in the patent literature. SQ109 is more potent than EMB and could substitute EMB in the intensive stage of TB treatment with the three- or four-drug combination. Developing MmpL3 inhibitors is a promising approach to fighting the challenges associated with DS-TB and DR-TB. The authors foresee MmpL3 inhibitors such as SQ109 as future drugs for TB treatment.

Development and Assessment of a Novel Whole-Gene-Based Targeted Next-Generation Sequencing Assay for Detecting the Susceptibility of Mycobacterium tuberculosis to 14 Drugs.

Targeted next-generation sequencing (tNGS) has emerged as an alternative method for detecting drug-resistant tuberculosis (DR-TB). To provide comprehensive drug susceptibility information and to address mutations missed by available commercial molecular diagnostics, we developed and evaluated a tNGS panel with 22 whole-gene targets using the Ion Torrent platform to predict drug resistance to 14 drugs, namely, rifampicin (RIF), isoniazid (INH), ethambutol (EMB), pyrazinamide (PZA), moxifloxacin (MFX), levofloxacin (LFX), amikacin (AMK), capreomycin (CM), kanamycin (KM), streptomycin (SM), bedaquiline (BDQ), clofazimine (CFZ), linezolid (LZD), and delamanid (DLM). We selected 50 and 35 Mycobacterium tuberculosis isolates with various DR profiles as the training set and the challenge set, respectively. Comparative variant analyses of the DR genes were performed using Sanger sequencing and whole-genome sequencing (WGS). Phenotypic drug susceptibility testing (pDST) results were used as gold standards. Regarding the limit of detection, the tNGS assay detected 2.9 to 3.8% minority variants in 4% mutant mixtures. The sensitivity and specificity of tNGS were 97.0% (95% confidence interval [CI] = 93.1 to 98.7%) and 99.1% (95% CI = 97.7 to 99.7%), respectively. The concordance of tNGS with pDST was 98.5% (95% CI = 97.2 to 99.2%), which was comparable to that of WGS (98.7%, 95% CI = 97.4 to 99.3%) and better than that of Sanger sequencing (96.9%, 95% CI = 95.3 to 98.0%). The agreement between tNGS and pDST was almost perfect for RIF, INH, EMB, MFX, LFX, AMK, CM, KM, SM, BDQ, and LZD (kappa value = 0.807 to 1.000) and substantial for PZA (kappa value = 0.791). Our customized novel whole-gene-based tNGS panel is highly consistent with pDST and WGS for comprehensive and accurate prediction of drug resistance in a strengthened and streamlined DR-TB laboratory program. IMPORTANCE We developed and validated a tNGS assay that was the first to target 22 whole genes instead of regions of drug resistance genes and comprehensively detected susceptibility to 14 anti-TB drugs, with great flexibility to include new or repurposed drugs. Notably, we demonstrated that our custom-designed Ion AmpliSeq TB research panel platform had high concordance with pDST and could significantly reduce turnaround time (by approximately 70%) to meet a clinically actionable time frame. Our tNGS assay is a promising DST solution for providing needed clinical information for precision medicine-guided therapies for DR-TB and allows the rollout of active pharmacovigilance.

Bedaquiline-Based Short Course Regime for MDR-TB -“The Indian Scenario”

India ranks highest in number of Tuberculosis (TB) and Multi Drug Resistant (MDR) TB patients globally (World Health Organization. Global tuberculosis report (2018). Maximum number of deaths due to TB also occur in India. A short course regime for treating Fluroquinolone (FQ) Sensitive MDR TB has been recommended by the World Health Organization (WHO) in 2020 with a duration of 9-11 months. India has also adopted the BDQ Short course regime for MDR-TB patients with multiple exclusion criteria. The excluded patients would receive a longer BDQ based regime which may last 18-20 months. The purpose of this mini-review is to emphasize the possibilities of high failure rates in the BDQ based short course regime, especially in the Context of India due to high baseline FQ resistance and an acute shortage of Drug Sensitivity Tests (DST) tests especially for FQ. Moreover, the need of High dose Isoniazid (Hh ) in the regime may be only a toxic addition in about 80% of patients leading to adverse effects, some of which may be fatal. Failure of BDQ-based short course regime would lead to a catastrophic amplification of the resistance of critically essential drugs such as BDQ and Clofazimine (CFZ). Chances of cure in such patients with Extensively Drug Resistant (XDR) TB would be slim, if not altogether absent.

Proteomic analysis and identification reveal the anti-inflammatory mechanism of clofazimine on lipopolysaccharide-induced acute lung injury in mice.

Background and objectiveAcute lung injury (ALI)/ acute respiratory distress syndrome (ARDS) was increasingly recognized as one of the most severe acute hyperimmune response of coronavirus disease 2019 (COVID-19). Clofazimine (CFZ) has attracted attention due to its anti-inflammatory property in immune diseases as well as infectious diseases. However, the role and potential molecular mechanism of CFZ in anti-inflammatory responses remain unclear.MethodsWe analyze the protein expression profiles of CFZ and LPS from Raw264.7 macrophages using quantitative proteomics. Next, the protective effect of CFZ on LPS-induced inflammatory model is assessed, and its underlying mechanism is validated by molecular biology analysis.ResultsLC–MS/MS-based shotgun proteomics analysis identified 4746 (LPS) and 4766 (CFZ) proteins with quantitative information. The key proteins and their critical signal transduction pathways including TLR4/NF-κB/HIF-1α signaling was highlighted, which was involved in multiple inflammatory processes. A further analysis of molecular biology revealed that CFZ could significantly inhibit the proliferation of Raw264.7 macrophages, decrease the levels of TNF-α and IL-1β, alleviate lung histological changes and pulmonary edema, improve the survival rate, and down-regulate TLR4/NF-κB/HIF-1α signaling in LPS model.ConclusionThis study can provide significant insight into the proteomics-guided pharmacological mechanism study of CFZ and suggest potential therapeutic strategies for infectious disease.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00011-022-01623-w.

Repurposing clofazimine for malignant pleural mesothelioma treatment — In-vitro assessment of efficacy and mechanism of action

AimsMalignant pleural mesothelioma (MPM) is a rare cancer of lungs' pleural cavity, with minimally effective therapies available. Thus, there exists a necessity for drug repurposing which is an attractive strategy for drug development in MPM. Repurposing of an old FDA-approved anti-leprotic drug, Clofazimine (CFZ), presents an outstanding opportunity to explore its efficacy in treating MPM. Main methodsCytotoxicity, scratch assay, and clonogenic assays were employed to determine CFZ's ability to inhibit cell viability, cell migration, and colony growth. 3D Spheroid cell culture studies were performed to identify tumor growth inhibition potential of CFZ in MSTO-211H cell line. Gene expression analysis was performed using RT-qPCR assays to determine the CFZ's effect of key genes. Western blot studies were performed to determine CFZ's ability to induce apoptosis its effect to induce autophagy marker. Key findingsCFZ showed significant cytotoxicity against both immortalized and primary patient-derived cell lines with IC50 values ranging from 3.4 μM (MSTO-211H) to 7.1 μM (HAY). CFZ significantly impaired MPM cell cloning efficiency, migration, and tumor spheroid formation. 3D Spheroid model showed that CFZ resulted in reduction in spheroid volume. RT-qPCR data showed downregulation of genes β-catenin, BCL-9, and PRDX1; and upregulation of apoptosis markers such as PARP, Cleaved caspase 3, and AXIN2. Additionally, immunoblot analysis showed that CFZ down-regulates the expression of β-catenin (apoptosis induction) and up-regulates p62, LC3B protein II (autophagy inhibition). SignificanceIt can be concluded that CFZ could be a promising molecule to repurpose for MPM treatment which needs numerous efforts from further studies.

Pharmacokinetics and Adverse Effects of Clofazimine in the Treatment of Pulmonary Non-Tuberculous Mycobacterial Infection.

Clofazimine (CFZ) is used to treat pulmonary non-tuberculous mycobacterial (NTM) infection; however, its pharmacokinetics remain unexplored in patients with pulmonary NTM, and the relationship between CFZ serum concentration and adverse effects has not been investigated. The objectives of this study were to characterize the pharmacokinetics of CFZ in pulmonary NTM disease treatment and to investigate the relationship between the steady-state CFZ serum concentration and adverse effects. A prospective observational study was conducted on 45 patients with pulmonary NTM treated with CFZ (UMIN000041053). A maximum of five serum samples per patient were taken at the CFZ trough, and serum concentration was measured using high-performance liquid chromatography-mass spectrometry (HPLC-MS). The pharmacokinetics of CFZ were analyzed using a nonlinear mixed effect model. The relationships among steady-state CFZ serum concentration and adverse effects, pigmentation, and heart rate-corrected QT (QTc) interval were investigated. Twenty-six patients had M. avium or M. intracellulare infection and nineteen had M. abscessus infection. The primary CFZ dosage was 50 mg/day. The estimated apparent CFZ clearance, apparent volume of distribution, and half-life were 2.4 L/h, 2,960 L, and 36 days, respectively. The combined use of rifampicin and CFZ significantly reduced CFZ exposure by 22%. Although there was no relationship between CFZ serum concentration and pigmentation intensity, the QTc interval was significantly correlated with CFZ serum concentration. The estimation of accurate pharmacokinetics for CFZ required approximately 5 months of monitoring. The relationship between the serum concentration and specific adverse effects of CFZ confirmed that CFZ serum concentration was not associated with pigmentation but did affect the QTc interval.

Simultaneously Determining Seven Second-Line Anti-TB Drugs by UHPLC- MS: Application for TDM in HIV-TB Patients

Background: To optimize therapy for patients with human immunodeficiency virus-tuberculosis (HIV-TB) coinfection, we developed an ultra-high-performance liquid chromatography/- tandem mass spectrometry (UHPLC-MS) method to monitor seven second-line anti-tuberculosis drugs. Methods: Blood samples (n = 70) were collected from 35 patients with HIV-TB coinfection; the plasma sample was protein-precipitated and diluted with a solution containing heptafluorobutyric acid. The plasma concentrations of rifabutin (RBT), clofazimine (CLO), moxifloxacin (MFX), prothionamide (PTH), levofloxacin (LFX), amikacin (AMK), and para-aminosalicylic acid (PAS) were detected by UHPLC-MS/MS method. Results: In these 70 samples, the mean concentrations of RBT, CLO, MFX, PTH, LFX, and AMK were 173.8 (10.0–550.0), 61.1 (54.4–67.7), 646.6 (25.0–2480.0), 120.5 (50.0–597.0), 1565.9 (100.0–3480.0), and 10753.0 (400.0–76 700.0) μg/L, respectively. Only one sample was detected to have PAS with a concentration less than the lower limit of quantification. Most of the drug concentrations detected in these patients were lower than the targeted concentrations in TB patients. Conclusion: We created a simple UHPLC-MS method for simultaneously quantifying anti-TB drugs. The plasma concentrations in HIV-TB co-infected patients were lower than the targeted concentrations. It is important to monitor anti-TB drugs in the future. This method will facilitate the monitoring of anti-TB drugs in the future.