Search Results (61)

Background and Objectives: Infections caused by non-tuberculous mycobacteria (NTM), including Mycobacterium abscessus complex (MABc), are increasing globally and are notoriously difficult to treat due to the intrinsic resistance of these bacteria to many common antibiotics. The aims of this study were to demonstrate the in vitro activity of imipenem/relebactam against MABc clinical isolates and to determine any in vitro synergism between imipenem/relebactam and other antimicrobials. Methods: A nationwide collection of 175 MABc clinical respiratory isolates obtained from 24 hospitals in Spain (August 2022–April 2023) was studied. Fifteen different antimicrobial agents were comprised, including imipenem/relebactam. MICs were determined according to CLSI criteria, and the synergism studies were performed with the selected clinical isolates. Results: Of the 175 isolates obtained, 110 were identified as M. abscessus subsp. abscessus (62.9%), 51 as M. abscessus subsp. massiliense (29.1%), and 14 as M. abscessus subsp. bolleti (8%). The antibiotics yielding the highest susceptibility rates were tigecycline, eravacycline, and omadacycline (100%); followed by imipenem/relebactam and clofazimine (97.6%); and finally amikacin (94.6%). Only four isolates were resistant to imipenem/relebactam, three of which were further characterized by WGS, revealing MABc mutations in Bla_Mab as well as D,D- and L,D-transpeptidades and mspA porin, which may play an important role in reduced susceptibility to imipenem/relebactam, even though none were previously described or associated with resistance to β-lactams. Conclusions: Our data demonstrate that relebactam improved the anti-MABc activity of imipenem, representing a β-lactam for the treatment of MABc infections. Furthermore, imipenem/relebactam demonstrated in vitro synergism with other anti-MABc treatments, thus supporting its use as part of dual regimens. Full article

Rabies, a viral encephalitis caused by rabies virus (RABV), is 100% fatal upon the onset of symptoms. Effective post-exposure prophylaxis (PEP) measures are available, but they are often difficult to access in low-income countries. WHO estimates about 59,000 deaths due to rabies globally, and the majority are contributed by developing countries. Hence, developing drugs for the treatment of post-symptomatic rabies is an urgent and unmet demand. It is worth noting that previous efforts regarding antiviral strategies, such as small-interfering RNA, antibodies and small-molecule inhibitors, against the rabies virus have failed to show efficacy in pre-clinical studies, especially when the virus has reached the central nervous system (CNS). Therefore, drug repurposing seems to be an alternative tool for the development of new anti-rabies drugs. We validated and used a high-throughput, FITC-conjugated antibody-based flow cytometry assay to expedite the identification of repurposable new drug candidates against the RABV. The assay was validated using ribavirin and salinomycin as reference compounds, which showed EC50 values of 10.08 µM and 0.07 µM, respectively. We screened a SelleckChem library comprising 3035 FDA-approved compounds against RABV (CVS-11) at 10 µM concentration. Five compounds (clofazimine, tiamulin, difloxacin, harringtonine and homoharringtonine) were active against RABV, with greater than 90% inhibition. Homoharringtonine (HHT) identified in the present study is active against laboratory-adapted RABV (CVS-11) and clinical isolates of RABV, with an average EC50 of 0.3 µM in both BHK-21 and Neuro-2a cell lines and exhibits post-entry inhibition. Full article

Background/Objectives: Clofazimine (CFZ) is a versatile antimicrobial active against several bacterial species, although its reduced aqueous solubility and the occurrence of side effects limit its use. Nanostructured lipid carriers (NLCs) constitute an interesting approach to increase drug bioavailability and safety. However, the development of nanoparticle-based formulations is challenging. In the present work, a combination of smart pharmaceutical technology approaches was proposed to develop CFZ-loaded NLCs, taking advantage of previous knowledge on NLCs screening. Methods: A design space previously established using Artificial Intelligence (AI) tools was applied to develop CFZ-loaded NLC formulations. After formulation characterization, Neurofuzzy Logic (NFL) and in silico docking simulations were employed to enhance the understanding of lipid nanocarriers. Then, the performance of formulations designed following NFL guidelines was characterized in terms of biocompatibility, using murine fibroblasts, and antimicrobial activity against several strains of Staphylococcus aureus. Results: The followed approach enabled CFZ-loaded NLC formulations with optimal properties, including small size and high antimicrobial payload. NFL was useful to investigate the existing interactions between NLC components and homogenization conditions, that influence CFZ-loaded NLCs’ final properties. Also, in silico docking simulations were successfully applied to examine interactions and affinity between the drug and the lipid matrix components. Finally, the designed CFZ-loaded formulations demonstrated suitable biocompatibility, together with antimicrobial activity. Conclusions: The implementation of smart strategies during nanoparticle-based therapeutics development, such as those described in this manuscript, would enable the more efficient design of new systems for suitable antimicrobial delivery. Full article

Mycobacterium abscessus complex (MABc) poses a major therapeutic challenge due to its intrinsic multidrug resistance and ability to form biofilms. This study evaluated the in vitro activity of three antimycobacterial agents—bedaquiline, delamanid, and clofazimine—on 20 clinical MABc isolates, including M. abscessus subsp. abscessus, massiliense, and bolletii, with a focus on biofilm-forming phenotypes. Biofilm analysis showed that the rough colony morphotypes were mostly weak biofilm formers, while the smooth and mixed morphotypes were predominantly moderate or strong biofilm formers. A statistically significant association was observed between the mixed colony morphology and strong biofilm formation (p = 0.032). Importantly, bedaquiline exhibited potent and consistent activity across all isolates, regardless of the biofilm-forming ability, with MIC values ranging from 0.125 to 1 µg/mL. In contrast, delamanid and clofazimine showed limited efficacy, with MIC values exceeding 16 µg/mL and 8 µg/mL, respectively. These findings strongly support the role of bedaquiline as a promising core agent for future combination therapies targeting drug-resistant MABc infections, including biofilm-associated infections. Our results, among the first from Poland, highlight the critical need for incorporating novel agents such as bedaquiline into therapeutic strategies against this difficult-to-treat pathogen. Full article

Background: Mycobacterium abscessus (MABS) is an opportunistic pathogen that causes chronic, difficult-to-treat pulmonary infections, particularly in people with cystic fibrosis (PwCF), leading to rapid lung function decline and increased morbidity and mortality. Treatment is particularly challenging due to the pathogen’s resistance mechanisms and the need for prolonged multidrug therapy, which is characterized by poor clinical outcomes and highlights the urgent need for novel therapeutic strategies. Imipenem/relebactam, a novel β-lactam-β-lactamase inhibitor combination, demonstrates in vitro activity against resistant MABS strains and effective pulmonary penetration. Prior research indicates synergistic activity of imipenem with various antibiotics against M. abscessus. Objectives: This study aims to evaluate the in vitro activity of imipenem/relebactam, alone and in combination with various antibiotics, against MABS clinical isolates from PwCF (n = 28). Methods: Susceptibility and synergy were assessed using broth microdilution and checkerboard assays. Extracellular time-kill assays were performed to evaluate the bactericidal activity of synergistic three-drug combinations containing imipenem/relebactam. Results: Imipenem/relebactam demonstrated potent in vitro activity against clinical MABS isolates, exhibiting substantial synergy with cefuroxime, cefdinir, amoxicillin, and cefoxitin. Rifabutin, azithromycin, moxifloxacin, clofazimine, and minocycline also demonstrated additive effects with imipenem/relebactam. Extracellular time-kill assays identified imipenem/relebactam + cefoxitin + rifabutin and imipenem/relebactam + cefoxitin + moxifloxacin as the most effective combinations. Conclusions: These findings suggest that imipenem/relebactam may offer a significant advancement in the management of MABS infections in PwCF. The promising efficacy of multidrug regimens combining imipenem/relebactam with agents like cefoxitin, azithromycin, moxifloxacin, clofazimine, and rifabutin highlights potential therapeutic strategies. Full article

Background/Objectives: Four years after the COVID-19 pandemic, a very limited number of drugs has been marketed; thus, the search for new medications still represents a compelling need. In our previous work on antiviral, antiparasitic, and antiproliferative agents, we described several compounds (1–13 and 16–20) structurally related to clofazimine, chloroquine, and benzimidazole derivatives. Thus, we deemed it worthwhile to test them against the replication of SARS-CoV-2, together with a few other compounds (14, 15 and 21–25), which showed some analogy to miscellaneous anti-coronavirus agents. Methods: Twenty-five structurally assorted compounds were evaluated in vitro for cytotoxicity against Vero E6 and for their ability to inhibit SARS-CoV-2 replication. Results: Several compounds (2, 3, 10, 11, 13–15, 18–20) demonstrated antiviral activity (IC₅₀ range 1.5–28 µM) and six of them exhibited an interesting selectivity index in the range 4.5–20. The chloroquine analogs 10 and 11 were more potent than the reference chloroquine itself and doubled its SI value (20 versus 11). Also, the benzimidazole ring emerged as a valuable scaffold, originating several compounds (13–15 and 18–20) endowed with anti-SARS-CoV-2 activity. Despite the modest activity, the cytisine and the arylamino enone derivatives 23 and 25, respectively, also deserve further consideration as model compounds. Conclusions: The investigated chemotypes may represent valuable hit compounds, deserving further in-depth biological studies to define their mechanisms of action. The derived information will guide the subsequent chemical optimization towards the development of more efficient anti-SARS-CoV-2 agents. Full article

Multidrug-resistant tuberculosis (MDR-TB) patients are treated with a standardised, short World Health Organization (WHO) regimen which includes clofazimine (CFZ) and bedaquiline (BDQ) antibiotics. These two antibiotics lead to the development of QT prolongation in patients, inhibiting potassium (K⁺) uptake by targeting the voltage-gated K⁺ (Kv)11.1 (hERG) channel of the cardiomyocytes (CMs). However, the involvement of these antibiotics to regulate other K⁺ transporters of the CMs, as potential mechanisms of QT prolongation, has not been explored. This study determined the effects of CFZ and BDQ on sodium, potassium–adenosine triphosphatase (Na⁺,K⁺-ATPase) activity of CMs using rat cardiomyocytes (RCMs). These cells were treated with varying concentrations of CFZ and BDQ individually and in combination (1.25–5 mg/L). Thereafter, Na⁺,K⁺-ATPase activity was determined, followed by intracellular adenosine triphosphate (ATP) quantification and cellular viability determination. Furthermore, molecular docking of antibiotics with Na⁺,K⁺-ATPase was determined. Both antibiotics demonstrated dose–response inhibition of Na⁺,K⁺-ATPase activity of the RCMs. The greatest inhibition was demonstrated by combinations of CFZ and BDQ, followed by BDQ alone and, lastly, CFZ. Neither antibiotic, either individually or in combination, demonstrated cytotoxicity. Molecular docking revealed an interaction of both antibiotics with Na⁺,K⁺-ATPase, with BDQ showing higher protein-binding affinity than CFZ. The inhibitory effects of CFZ and BDQ, individually and in combination, on the activity of Na⁺,K⁺-ATPase pump of the RCMs highlight the existence of additional mechanisms of QT prolongation by these antibiotics. Full article

Background/Objectives: Clofazimine (CFZ) is a Biopharmaceutics Classification System (BCS) II drug introduced in the US market in 1986 for the treatment of leprosy. However, CFZ was later withdrawn from the market due to its extremely low aqueous solubility and low absorption. In the literature, the intrinsic solubility of CFZ has been estimated to be <0.01 μg/mL, and solubilities of its different salt forms in simulated gastric and intestinal fluids are <10 µg/mL. These are extremely low solubilities for the dissolution of a drug administered orally at 100–200 mg doses. Methods: In the present investigation, seven weak organic acids (adipic, citric, glutaric, maleic, malic, succinic, and tartaric) were tested by determining the aqueous solubility of CFZ as the function of acid concentration to investigate whether any of the acids would lead to the supersolubilization of CFZ. Results: There were only minimal increases in solubilities when concentrations of acids in water were increased up to 2.4 M. The solubilities, however, increased to 0.32, 1.23, and 10.68 mg/mL, respectively, in 5 M solutions of tartaric, malic, and glutaric acids after equilibration for 24 h at 25 °C. Crystalline solids were formed after the equilibration of CFZ with all acids. Apparently, salts or cocrystals were formed with all acids, except for glutaric acid, as their melting endotherms in DSC scans were in the range of 207.6 to 248.5 °C, which were close to that of CFZ itself (224.8 °C). In contrast, the adduct formed with glutaric acid melted at the low temperature of 77 °C, and no other peak was observed at a higher temperature, indicating that the material converted to an amorphous state. Conclusions: The increase in CFZ solubility to >10 mg/mL in the presence of 5 M glutaric acid could be called supersolubilization when compared to the intrinsic solubility of the basic drug. Such an increase in CFZ solubility and the conversion of the glutarate adduct to an amorphous state are being exploited to develop rapidly dissolving dosage forms. Full article

Tuberculosis (TB) presents significant medical challenges, largely due to the genetic diversity of Mycobacterium tuberculosis, which enhances the resilience and resistance of the pathogen to first-line treatments. In response to the global rise of drug-resistant TB, second-line antitubercular drugs like bedaquiline (BDQ), linezolid (LZD), and clofazimine (CFZ) have become critical treatment options. Understanding the molecular changes these drugs induce is essential for optimizing TB therapy. To contribute to this effort, we investigated their impact on genome maintenance and stability using Mycobacterium smegmatis as a model organism. Using mutation accumulation assays and whole-genome sequencing, we found that the second-line antibiotics did not significantly increase mutation rates, unlike the positive control UV treatment. However, upon BDQ treatment, we detected mutations in transporter proteins and transcription factors without any increase in the minimal inhibitory concentration. Additionally, BDQ and CFZ were found to alter DNA repair pathways and reduce cellular dNTP levels, particularly CFZ, which depleted dGTP, impacting DNA synthesis. CFZ also upregulated DNA repair enzymes, enhancing error-free repairs. Despite minimal mutagenic effects, both drugs displayed distinct impacts on cellular mechanisms, suggesting additional modes of action. Full article

The emergence of drug-resistant mycobacteria has rendered many clinical drugs and regimens ineffective, imposing significant economic and healthcare burden on individuals and society. Repurposing drugs intended for treating other diseases is a time-saving, cost-effective, and efficient approach for identifying excellent antimycobacterial candidates or lead compounds. This study is the first to demonstrate that rupatadine (RTD), a drug used to treat allergic rhinitis, possesses excellent activity against mycobacteria without detectable resistance, particularly Mycobacterium tuberculosis and Mycobacterium marinum, with a minimal inhibitory concentration as low as 3.13 µg/mL. Furthermore, RTD exhibited moderate activity against nonreplicating M. tuberculosis with minimal inhibitory concentrations lower than drugs targeting the cell wall, suggesting that RTD has great potential to be modified and used for the treatment of nonreplicating M. tuberculosis. Additionally, RTD exhibits partial synergistic effects when combined with clofazimine, pretomanid, and TB47 against M. tuberculosis, providing the theoretical foundation for the development of treatment regimens. Transcriptomic profiling leads us to speculate that eight essential genes may be the targets of RTD or may be closely associated with mycobacterial resistance to RTD. In summary, RTD may be a promising hit for further antimycobacterial drug or regimen optimization, especially in the case of nonreplicating mycobacteria. Full article

This study, conducted between 4 October 2013, and 30 November 2018, tested the hypothesis that triple antimicrobial therapy, targeting Mycobacterium avium subspecies paratuberculosis (MAP), long considered a putative cause, would favorably affect Crohn’s disease. A double-blind multicenter study of adults with active Crohn’s disease, (i.e., Crohn’s Disease Activity Index [CDAI] 220–450 plus C-reactive protein ≥ 1.0 mg/dL, fecal calprotectin (FCP) >162.9 µg/g stool, or recent endoscopic or radiographic confirmation of active disease) receiving concomitant standard-of-care Crohn’s disease treatment (Clinicaltrials.gov: NCT01951326) were stratified by anti-tumor necrosis factor use and randomized (1:1) to anti-MAP RHB-104 (clarithromycin 95 mg, rifabutin 45 mg, and clofazimine 10 mg per capsule) (n = 166), resulting in clarithromycin 950 mg/day, rifabutin 450 mg/day, and clofazimine 100 mg/day, or placebo (n = 165) for up to 52 weeks. A greater proportion of RHB-104 versus placebo-treated patients met the primary endpoint—remission (i.e., CDAI < 150)—at week 26 (36.7% [61/166] vs. 22.4% [37/165], respectively; 95% CI for difference: 4.6, 24.0, p = 0.0048; chi-square test). Clinical response (reduction of CDAI by ≥100 points from baseline) at week 26 (first secondary endpoint) was also higher among the patients treated with RHB-104 (73/166 [44.0%]) compared with placebo (50/165 [30.3%]; 95% CI for difference: 3.4, 24.0, p = 0.0116), and it remained higher at week 52 among the patients treated with RHB-104 (59/166 [35.5%] vs. (35/165 [21.2%] for placebo; 95% CI for difference: 4.7, 23.9, p = 0.0042). A statistically significantly greater decline in FCP (another prospective efficacy endpoint) was also observed in RHB-104-treated patients, compared with placebo, at weeks 12, 26, and 52. The rates of serious adverse events were similar between groups (RHB-104: 18.7%; placebo: 18.8%). No patient died during the study. Antimicrobial therapy directed against MAP resulted in significantly greater improvement in clinical and laboratory (FCP) measures of active Crohn’s disease. Full article

Tuberculosis (TB) is an airborne bacterial infection caused by Mycobacterium tuberculosis (M. tb), resulting in approximately 1.3 million deaths in 2022 worldwide. Oral therapy with anti-TB drugs often fails to achieve therapeutic concentrations at the primary infection site (lungs). In this study, we developed a dry powder inhalable formulation (DPI) of clofazimine (CFZ) to provide localized drug delivery and minimize systemic adverse effects. Poly (lactic acid-co-glycolic acid) (PLGA) microparticles (MPs) containing CFZ were developed through a single emulsion solvent evaporation technique. Clofazimine microparticles (CFZ MPs) displayed entrapment efficiency and drug loading of 66.40 ± 2.22 %w/w and 33.06 ± 1.45 µg/mg, respectively. To facilitate pulmonary administration, MPs suspension was spray-dried to yield a dry powder formulation (CFZ SD MPs). Spray drying had no influence on particle size (~1 µm), zeta potential (−31.42 mV), and entrapment efficiency. Solid state analysis (PXRD and DSC) of CFZ SD MPs studies demonstrated encapsulation of the drug in the polymer. The drug release studies showed a sustained drug release. The optimized formulation exhibited excellent aerosolization properties, suggesting effective deposition in the deeper lung region. The in vitro antibacterial studies against H37Ra revealed improved (eight-fold) efficacy of spray-dried formulation in comparison to free drug. Hence, clofazimine dry powder formulation presents immense potential for the treatment of tuberculosis with localized pulmonary delivery and improved patient compliance. Full article

Objectives: Slow-growing nontuberculous mycobacteria (NTMs) are highly prevalent and routinely cause opportunistic intracellular infectious disease in immunocompromised hosts. Methods: The activity of the triple combination of antibiotics, clarithromycin (CLR), rifabutin (RFB), and clofazimine (CFZ), was evaluated and compared with the activity of single antibiotics as well as with double combinations in an in vitro biofilm assay and an in vivo murine model of Mycobacterium avium subsp. hominissuis (M. avium) lung infection. Results: Treatment of 1-week-old biofilms with the triple combination exerted the strongest effect of all (0.12 ± 0.5 × 10⁷ CFU/mL) in reducing bacterial growth as compared to the untreated (5.20 ± 0.5 × 10⁷/mL) or any other combination (≥0.75 ± 0.6 × 10⁷/mL) by 7 days. The treatment of mice intranasally infected with M. avium with either CLR and CFZ or the triple combination provided the greatest reduction in CLR-sensitive M. avium bacterial counts in both the lung and spleen compared to any single antibiotic or remaining double combination by 4 weeks posttreatment. After 4 weeks of treatment with the triple combination, there were no resistant colonies detected in mice infected with a CLR-resistant strain. No clear relationships between treatment and spleen or lung organ weights were apparent after triple combination treatment. Conclusions: The biofilm assay data and mouse disease model efficacy results support the further investigation of the triple-antibiotic combination. Full article

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

The aim of this study was to determine the drug loading capacity of phosphatidylcholine-based formulations for four poorly water-soluble drug substances (clofazimine, fenofibrate, artemether, cannabidiol). Two self-dispersing lipid formulations were investigated, which consisted of soybean phospholipids, medium-chain triglycerides and ethanol with a different phospholipid–oil ratio. The direct loading of the bulk formulation was conducted with dual centrifugation, which proved to be a suitable method for screening experiments with the highly viscous formulations. To estimate possible precipitation after dispersion in the gastrointestinal fluids, the solubility of the drugs was investigated in the dispersed formulations. For this purpose, nanodispersions were prepared from the bulk formulations via high pressure homogenization and subsequently subjected to passive loading. A newly developed HPLC method with Charged Aerosol Detection allowed a simultaneous evaluation of the content of soybean lecithin and medium-chain triglycerides in the nanodispersions. When comparing the two phosphatidylcholine-based formulations, a high content of oil was advantageous with regard to a high loading capacity. Drug substances with melting points below 150 °C exhibited a high solubility in the phospholipid-based formulations. A surprisingly high solubility was observed for artemether and cannabidiol with up to 13.0% and 33.3% drug loaded to the formulations, respectively. In the dispersions, a similar solubility as in the bulk formulations was obtained for fenofibrate and cannabidiol. Clofazimine yielded a higher loading result in the nanodispersions than in the bulk formulation. Full article