Novel FtsZ inhibitor with potent activity against Staphylococcus aureus.

OBJECTIVES: FtsZ is an essential bacterial protein and an unexplored target for the development of antibacterial drugs. The development of a novel inhibitor targeting FtsZ offers a potential opportunity to combat drug resistance. DS01750413, a new derivative of PC190723, is a novel FtsZ inhibitor with improved in vitro and in vivo activity. The objective of this study was to investigate the efficacy of DS01750413 against Staphylococcus spp., including MRSA, in in vitro and in vivo models. METHODS: In vitro activities of DS01750413 and standard-of-care antibiotics were evaluated against clinical isolates of Gram-positive pathogens. The in vivo efficacy was evaluated in a murine systemic infection model caused by MRSA. RESULTS: DS01750413 showed potent in vitro activity against MRSA clinical isolates with MIC ranges of 0.5-1 mg/L and also demonstrated concentration-dependent bactericidal killing. In the murine bacteraemia infection model of MRSA, treatment with DS01750413 resulted in prolonged survival of animals compared with placebo-treated animals and exhibited a significant reduction in the bacterial load in liver, spleen, lungs and kidneys. CONCLUSIONS: DS01750413 showed encouraging in vitro and in vivo activity against MRSA. As a novel chemical class, DS01750413 has the potential to become clinically viable antibiotics to address the drug resistance problem by its unique novel targeting mechanism of action.

DS86760016, a Leucyl-tRNA Synthetase Inhibitor with Activity against Pseudomonas aeruginosa.

DS86760016 is a new leucyl-tRNA-synthetase inhibitor at the preclinical development stage. DS86760016 showed potent activity against extended-spectrum multidrug-resistant Pseudomonas aeruginosa isolated from clinical samples and in vitro biofilms. In a murine catheter-associated urinary tract infection model, DS86760016 treatment resulted in significant eradication of P. aeruginosa from the kidney, bladder, and catheter without developing drug resistance. Our data suggest that DS86760016 has the potential to act as a new drug for the treatment of Pseudomonas infections.

Advancing the Therapeutic Potential of Indoleamides for Tuberculosis.

Indole-2-carboxamide derivatives are inhibitors of MmpL3, the cell wall-associated mycolic acid transporter of Mycobacterium tuberculosis In the present study, we characterized indoleamide effects on bacterial cell morphology and reevaluated pharmacokinetics and in vivo efficacy using an optimized oral formulation. Morphologically, indoleamide-treated M. tuberculosis cells demonstrated significantly higher numbers of dimples near the poles or septum, which may serve as the mechanism of cell death for this bactericidal scaffold. Using the optimized formulation, an expanded-spectrum indoleamide, compound 2, showed significantly improved pharmacokinetic (PK) parameters and in vivo efficacy in mouse infection models. In a comparative study, compound 2 showed superior efficacy over compound 3 (NITD-304) in a high-dose aerosol mouse infection model. Since indoleamides are equally active on drug-resistant M. tuberculosis, these findings demonstrate the therapeutic potential of this novel scaffold for the treatment of both drug-susceptible and drug-resistant tuberculosis.

Potential of the fluoroketolide RBx 14255 against Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae in an experimental murine meningitis model.

BACKGROUND: RBx 14255 is a fluoroketolide in pre-clinical evaluation with potent activity against MDR Gram-positive pathogens. OBJECTIVES: To investigate the efficacy of RBx 14255 against bacterial meningitis caused by Streptococcus pneumoniae, Neisseria meningitidis or Haemophilus influenzae in an experimental murine meningitis model. METHODS: In vitro activity of RBx 14255 was evaluated against clinical isolates of S. pneumoniae, N. meningitidis and H. influenzae. The in vivo efficacy of RBx 14255 was evaluated against bacterial meningitis, induced with S. pneumoniae 3579 erm(B), S. pneumoniae MA 80 erm(B), N. meningitidis 1852 and H. influenzae B1414 in a murine meningitis model. RESULTS: RBx 14255 showed strong in vitro bactericidal potential against S. pneumoniae, N. meningitidis and H. influenzae with MIC ranges of 0.004-0.1, 0.03-0.5 and 1-4 mg/L, respectively. In a murine meningitis model, a 50 mg/kg dose of RBx 14255, q12h, resulted in significant reduction of bacterial counts in the brain compared with the pretreatment control. The concentration of RBx 14255 in brain tissue correlated well with the efficacy in this mouse model. CONCLUSIONS: RBx 14255 showed superior bactericidal activity in time-kill assays in vitro and in vivo in an experimental murine meningitis model. RBx 14255 could be a promising candidate for future drug development against bacterial meningitis.

In Vitro and In Vivo Activities of DS86760016, a Novel Leucyl-tRNA Synthetase Inhibitor for Gram-Negative Pathogens.

The emergence of multidrug-resistant (MDR) Gram-negative bacilli is a major concern in the treatment of nosocomial infections. Antibacterial agents with novel modes of action can be useful, as these pathogens have become resistant to almost all existing standard-of-care agents. GSK2251052, a leucyl-tRNA synthetase inhibitor, has a novel mode of action against Gram-negative bacteria. However, the phase 2 studies with this drug were terminated due to microbiological failures based on the rapid emergence of drug resistance during the treatment of complicated urinary tract infections. DS86760016 is a novel leucyl-tRNA synthetase inhibitor active against MDR Gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, with an improved pharmacokinetic profile. DS86760016 showed lower plasma clearance, longer plasma half-life, and higher renal excretion than GSK2251052 did in mice, rats, monkeys and dogs. DS86760016 also showed lower mutant prevention concentrations against P. aeruginosa than did GSK2251052. No resistant bacteria were observed in murine urinary tract infection models at a dose that maintained urinary concentrations above the mutant prevention concentration. DS86760016 also showed a lower risk of resistance development than did GSK2251052 in comparative in vivo studies with murine urinary tract infection models. These results suggest that DS86760016 has potential as a new drug for the treatment of MDR Gram-negative bacterial infections, with a lower risk of drug resistance development than that of GSK2251052.

In Vitro and In Vivo Activities of DS-2969b, a Novel GyrB Inhibitor, and Its Water-Soluble Prodrug, DS11960558, against Methicillin-Resistant Staphylococcus aureus.

DS-2969b is a novel GyrB inhibitor under clinical development. In this study, the in vitro activity of DS-2969b and the in vivo activities of DS-2969b and its water-soluble prodrug, DS11960558, against methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. DS-2969b inhibited the supercoiling activity of S. aureus DNA gyrase and the decatenation activity of its topoisomerase IV. DS-2969b showed antibacterial activity against Gram-positive aerobes but not against Gram-negative aerobes, except for Moraxella catarrhalis and Haemophilus influenzae DS-2969b was active against MRSA with an MIC90 of 0.25 µg/ml, which was 8-fold lower than that of linezolid. The presence of a pulmonary surfactant did not affect the MIC of DS-2969b. DS-2969b showed time-dependent slow killing against MRSA. The frequency of spontaneous resistance development was less than 6.2 × 10-10 in all four S. aureus isolates at 4× MIC of DS-2969b. In a neutropenic MRSA-induced murine muscle infection model, DS-2969b was more efficacious than linezolid by both the subcutaneous and oral routes. DS-2969b and DS11960558 showed efficacy in a neutropenic murine MRSA lung infection model. The pharmacokinetics and pharmacodynamics of DS-2969b and DS11960558 against MRSA were characterized in a neutropenic murine thigh infection model; the percentage of time during the dosing period in which the free drug concentration exceeded the MIC (fTMIC) correlated best with in vivo efficacy, and the static percent fTMIC was 43 to 49%. A sufficient fTMIC was observed in a phase 1 multiple-ascending-dose study of DS-2969b given orally at 400 mg once a day. These results suggest that DS11960558 and DS-2969b have potential for use as intravenous-to-oral step-down therapy for treating MRSA infections with a higher efficacy than linezolid.

Synthesis and evaluation of thiomannosides, potent and orally active FimH inhibitors.

FimH is a type I fimbrial lectin located at the tip of type-1 pili of Gram-negative uropathogenic Escherichia coli (UPEC) guiding its ability to adhere and infect urothelial cells. Accordingly, blocking FimH with small molecule inhibitor is considered as a promising new therapeutic alternative to treat urinary tract infections caused by UPEC. Herein, we report that compounds having the S-glycosidic bond (thiomannosides) had improved metabolic stability and plasma exposures when dosed orally. Especially compound 5h showed the potential to inhibit biofilm formation and also to disrupt the preformed biofilm. And compound 5h showed prophylactic effect in UTI model in mice.

In vivo efficacy and pharmacokinetics of bi-aryl oxazolidinone RBx 11760 loaded polylactic acid-polyethylene glycol nanoparticles in mouse hematogenous bronchopneumonia and rat groin abscess caused by Staphylococcus aureus.

RBx 11760 is a bi-aryl oxazolidinone antibacterial agent active against Staphylococcus aureus but has poor solubility. Here we have encapsulated RBx 11760 in PLA-PEG NPs with an aim to improve physicochemical, pharmacokinetics and in vivo efficacy. The average size and zeta potential of RBx 11760 loaded NPs were found to be 106.4 nm and -22.2 mV, respectively. The absolute size of nanoparticles by HRTEM was found to be approximately 80 nm. In vitro antibacterial agar well diffusion assay showed clear zone of inhibition of bacterial growth. In pharmacokinetic study, nanoparticle showed 4.6-fold and 7-fold increase in AUCinf and half-life, respectively, as compared to free drug. RBx 11760 nanoparticle significantly reduced bacterial counts in lungs and improved the survival rate of immunocompromised mice as compared to free drugs. Thus, RBx 11760 loaded nanoparticles have strong potential to be used as nanomedicine against sensitive and drug resistant Staphylococcus aureus infections.

In Vitro and In Vivo Activities of a Bi-Aryl Oxazolidinone, RBx 11760, against Gram-Positive Bacteria.

RBx 11760, a bi-aryl oxazolidinone, was investigated for antibacterial activity against Gram-positive bacteria. The MIC90s of RBx 11760 and linezolid against Staphylococcus aureus were 2 and 4 mg/liter, against Staphylococcus epidermidis were 0.5 and 2 mg/liter, and against Enterococcus were 1 and 4 mg/liter, respectively. Similarly, against Streptococcus pneumoniae the MIC90s of RBx 11760 and linezolid were 0.5 and 2 mg/liter, respectively. In time-kill studies, RBx 11760, tedizolid, and linezolid exhibited bacteriostatic effect against all tested strains except S. pneumoniae RBx 11760 showed 2-log10 kill at 4× MIC while tedizolid and linezolid showed 2-log10 and 1.4-log10 kill at 16× MIC, respectively, against methicillin-resistant S. aureus (MRSA) H-29. Against S. pneumoniae 5051, RBx 11760 showed bactericidal activity, with 4.6-log10 kill at 4× MIC compared to 2.42-log10 and 1.95-log10 kill for tedizolid and linezolid, respectively, at 16× MIC. RBx 11760 showed postantibiotic effects (PAE) at 3 h at 4 mg/liter against MRSA H-29, and linezolid showed the same effect at 16 mg/liter. RBx 11760 inhibited biofilm production against methicillin-resistant S. epidermidis (MRSE) ATCC 35984 in a concentration-dependent manner. In a foreign-body model, linezolid and rifampin resulted in no advantage over stasis, while the same dose of RBx 11760 demonstrated a significant killing compared to the initial control against S. aureus (P < 0.05) and MRSE (P < 0.01). The difference in killing was statistically significant for the lower dose of RBx 11760 (P < 0.05) versus the higher dose of linezolid (P > 0.05 [not significant]) in a groin abscess model. In neutropenic mouse thigh infection, RBx 11760 showed stasis at 20 mg/kg of body weight, whereas tedizolid showed the same effect at 40 mg/kg. These data support RBx 11760 as a promising investigational candidate.

A novel ketolide, RBx 14255, with activity against multidrug-resistant Streptococcus pneumoniae.

We present here the novel ketolide RBx 14255, a semisynthetic macrolide derivative obtained by the derivatization of clarithromycin, for its in vitro and in vivo activities against sensitive and macrolide-resistant Streptococcus pneumoniae. RBx 14255 showed excellent in vitro activity against macrolide-resistant S. pneumoniae, including an in-house-generated telithromycin-resistant strain (S. pneumoniae 3390 NDDR). RBx 14255 also showed potent protein synthesis inhibition against telithromycin-resistant S. pneumoniae 3390 NDDR. The binding affinity of RBx 14255 toward ribosomes was found to be more than that for other tested drugs. The in vivo efficacy of RBx 14255 was determined in murine pulmonary infection induced by intranasal inoculation of S. pneumoniae ATCC 6303 and systemic infection with S. pneumoniae 3390 NDDR strains. The 50% effective dose (ED50) of RBx 14255 against S. pneumoniae ATCC 6303 in a murine pulmonary infection model was 3.12 mg/kg of body weight. In addition, RBx 14255 resulted in 100% survival of mice with systemic infection caused by macrolide-resistant S. pneumoniae 3390 NDDR at 100 mg/kg four times daily (QID) and at 50 mg/kg QID. RBx 14255 showed favorable pharmacokinetic properties that were comparable to those of telithromycin.

High content screening strategies for large-scale compound libraries with a focus on high-containment viruses.

A majority of viral diseases do not have FDA-approved drugs. The recent outbreaks caused by SARS-CoV-2, monkeypox, and Sudan ebolavirus have exposed the critical need for rapid screening and identification of antiviral compounds against emerging/re-emerging viral pathogens. A high-content screening (HCS) platform is becoming an essential part of the drug discovery process, thanks to developments in image acquisition and analysis. While HCS has several advantages, its full potential has not been realized in antiviral drug discovery compared to conventional drug screening approaches, such as fluorescence or luminescence-based microplate assays. Therefore, this review aims to summarize HCS workflow, strategies, and developments in image-based drug screening, focusing on high-containment viruses.

Correlation of in vitro and in vivo plasma protein binding using ultracentrifugation and UPLC-tandem mass spectrometry.

The aim of the present study is to develop and demonstrate the correlation between in vitro and in vivo Plasma Protein Binding (PPB) using the ultracentrifugation method for its validation by using marketed compounds like atenolol, theophylline and phenytoin. In this study, in vitro PPB is carried out using ultracentrifugation, by spiking the selected marketed compounds at concentrations of 5 and 15 µM in plasma. In an in vivo study, rats (n = 3) were given a single oral dose (10 mg kg(-1)) and post-dose samples were subjected to ultracentrifugation to obtain the protein-free fraction. A rapid and highly sensitive method was developed and validated for determining the free fraction of marketed compounds in rat plasma using protein precipitation and analysis using an ultra performance liquid chromatography electrospray ionization (ESI) tandem mass spectrometer system (UPLC-MS/MS). The in vitro free fraction (fup) values were 0.93 ± 0.07 for atenolol, 0.31 ± 0.03 for theophylline and 0.09 ± 0.02 for phenytoin which correlated well with the corresponding in vivo values of 0.91 ± 0.03 for atenolol, 0.25 ± 0.02 for theophylline and 0.09 ± 0.01 for phenytoin with a coefficient of variation less than 11.06%, 11.45% and 13.67%, respectively. Therefore the validated high-throughput in vitro PPB study is expected to have a powerful impact on reducing the cost as well as time in the drug discovery process.

Synthesis and evaluation of 4,5-dihydro-5-methylisoxazolin-5-carboxamide derivatives as VLA-4 antagonists.

A novel set of compounds containing a 4,5-dihydro-5-methylisoxazoline have been successfully designed as VLA-4 receptor antagonists. Compound (14p) had a high receptor binding affinity of 4 nM and also found to be metabolically stable in vitro.

ADME, Pharmacokinetic Scaling, Pharmacodynamic and Prediction of Human Dose and Regimen of Novel Antiviral Drugs.

The search for new drugs is an extremely time-consuming and expensive endeavour. Much of that time and money go into generating predictive human pharmacokinetic profiles from preclinical efficacy and safety animal data. These pharmacokinetic profiles are used to prioritize or minimize the attrition at later stages of the drug discovery process. In the area of antiviral drug research, these pharmacokinetic profiles are equally important for the optimization, estimation of half-life, determination of effective dose, and dosing regimen, in humans. In this article we have highlighted three important aspects of these profiles. First, the impact of plasma protein binding on two primary pharmacokinetic parameters-volume of distribution and clearance. Second, interdependence of primary parameters on unbound fraction of the drug. Third, the ability to extrapolate human pharmacokinetic parameters and concentration time profiles from animal profiles.

Novel fluorobenzothiazole as a dual inhibitor of gyrase B and topoisomerase IV against Gram-positive pathogens.

Aim: The development of a novel inhibitor targeting gyrase B and topoisomerase IV offers an opportunity to combat multidrug resistance. Methods: We investigated the activity of RBx 10080758 against Gram-positive bacteria in vitro and in vivo. Results: RBx 10080758 showed a potent 50% inhibitory concentration of 0.13 µM and 0.25 µM against gyrase B and topoisomerase IV, respectively, and exhibited strong whole-cell in vitro activity with MIC ranges of 0.015-0.06 and 0.015-0.03 µg/ml against Staphylococcus aureus and Streptococcus pneumoniae, respectively. In a rat thigh infection model with methicillin-resistant S. aureus, RBx 10080758 at 45 mg/kg exhibited a >3 log10 CFU reduction in thigh muscles. Conclusion: RBx 10080758 displayed potent activity against multiple multidrug-resistant Gram-positive bacteria with a dual-targeting mechanism of action.

Azithromycin Exhibits Activity Against Pseudomonas aeruginosa in Chronic Rat Lung Infection Model.

Pseudomonas aeruginosa forms biofilms in the lungs of chronically infected cystic fibrosis patients, which are tolerant to both the treatment of antibiotics and the host immune system. Normally, antibiotics are less effective against bacteria growing in biofilms; azithromycin has shown a potent efficacy in cystic fibrosis patients chronically infected with P. aeruginosa and improved their lung function. The present study was conducted to evaluate the effect of azithromycin on P. aeruginosa biofilm. We show that azithromycin exhibited a potent activity against P. aeruginosa biofilm, and microscopic observation revealed that azithromycin substantially inhibited the formation of solid surface biofilms. Interestingly, we observed that azithromycin restricted P. aeruginosa biofilm formation by inhibiting the expression of pel genes, which has been previously shown to play an essential role in bacterial attachment to solid-surface biofilm. In a rat model of chronic P. aeruginosa lung infection, we show that azithromycin treatment resulted in the suppression of quorum sensing-regulated virulence factors, significantly improving the clearance of P. aeruginosa biofilms compared to that in the placebo control. We conclude that azithromycin attenuates P. aeruginosa biofilm formation, impairs its ability to produce extracellular biofilm matrix, and increases its sensitivity to the immune system, which may explain the clinical efficacy of azithromycin in cystic fibrosis patients.

Encapsulating Rifampicin into SLNs: A Viable Option for Managing its Bioavailability Issues Upon Co-Delivery with Isoniazid.

BACKGROUND: Rifampicin is known to degrade at the acidic pH of the stomach, especially in the presence of isoniazid. Although isoniazid also degrades partially, its degradation is reversible. OBJECTIVE: Presently, we provide a proof of the fact that the simultaneous oral administration of rifampicin (RIF), upon incorporation into solid lipid nanoparticles (RIF-SLNs), with isoniazid (INH) overcomes its INH-induced degradation and improves its oral bioavailability in rats. METHODS: Solid lipid nanoparticles of RIF (RIF-SLNs) were prepared using a novel and patented method. The effect of INH was investigated on in vivo bioavailability of RIF both in its free and encapsulated (RIF-SLNs) form, after oral administration to rats. RESULTS: Cmax and AUC0-∞ of RIF increased 158 % and 125 %, respectively, upon incorporation into SLNs versus free RIF when combined with INH. The Tmax decreased from 5.67 h to 3.3 h, and the plasma concentration of RIF remained above its MIC (8 µg/ml) at all the tested time points starting with 15 min, when administered as RIF-SLNs in combination with INH. CONCLUSION: The results confirm the scope of combining RIF-SLNs with INH to overcome the bioavailability of free RIF when combined with INH, especially in fixed dose combinations.

Targeting Mycolic Acid Transport by Indole-2-carboxamides for the Treatment of Mycobacterium abscessus Infections.

Mycobacterium abscessus is a fast-growing, multidrug-resistant organism that has emerged as a clinically significant pathogen in cystic fibrosis (CF) patients. The intrinsic resistance of M. abscessus to most commonly available antibiotics seriously restricts chemotherapeutic options. Herein, we report the potent activity of a series of indolecarboxamides against M. abscessus. The lead compounds, 6 and 12, exhibited strong activity in vitro against a wide panel of M. abscessus isolates and in infected macrophages. High resistance levels to the indolecarboxamides appear to be associated with an A309P mutation in the mycolic acid transporter MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis remained unaffected, the indolecarboxamides strongly inhibited the transport of trehalose monomycolate, resulting in the loss of trehalose dimycolate production and abrogating mycolylation of arabinogalactan. Our data introduce a hereto unexploited chemical structure class active against M. abscessus infections with promising translational development possibilities for the treatment of CF patients.

RBx10080307, a dual EGFR/IGF-1R inhibitor for anticancer therapy.

Pharmacological intervention of epidermal growth factor receptor (EGFR) family members by antibodies or small molecule inhibitors has been one of the most successful approaches for anticancer therapy. However this therapy has its own limitations due to the development of resistance, over a period of time. One of the possible causes of the development of resistance to the therapy with EGFR inhibitors could be the simultaneous activation of parallel pathways. Both EGFR and insulin like growth factor-1 receptor (IGF-1R) pathways are reported to act reciprocal to each other and converge into the mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. Inhibiting one pathway alone may therefore not be sufficient and could be a cause of development of resistance. The other cause could be mutations of EGFR which would be less sensitive to the inhibitors. We, therefore, suggest that co-targeting IGF-1R and EGFR kinases by dual inhibitors can lead to improved efficacy and address the problems of resistance. In the present manuscript, we report the identification of a novel, small molecule dual EGFR/IGF-1R inhibitor, RBx10080307 which displayed in vitro activity at the molecular level and oral efficacy in mouse xenograft model. The compound also showed in vitro activity in an EGFR mutant cell line and may thus have the potential to show activity in resistant conditions. Additional efficacy studies are needed in EGFR resistant mouse cancer model and if found efficacious, this can be a major advantage over standalone erlotinib and other existing therapies.

Pharmacodynamic and pharmacokinetic profile of RBx 343E48F0: a novel, long acting muscarinic receptor antagonist.

RBx 343E48F0 is a novel, potent, selective and long acting muscarinic receptor antagonist with a potential for use in the treatment of Chronic Obstructive Pulmonary Disease (COPD). The aim of the present study was to describe the in vitro and in vivo profile of RBx 343E48F0 and to compare the results with the present day benchmark therapy, tiotropium. Radioligand binding and isolated tissue based functional assays were used to evaluate the affinity, potency and receptor subtype selectivity of RBx 343E48F0. Inhibition of carbachol-induced bronchoconstriction in the anaesthetized rat and acetylcholine-induced bronchoconstriction in the conscious rat were used to assess the extent and duration of the bronchospasmolytic activity of RBx 343E48F0. In vitro and in vivo pharmacokinetic studies were conducted to evaluate the pharmacokinetic and lung retention properties of the compound. In vitro radioligand binding studies using human recombinant muscarinic receptors showed that RBx 343E48F0 had a pKi of 9.6 at the M(3) receptor and a 60-fold selectivity for the M(3) receptor over the M(2) receptor. In isolated tissue bioassays, it exhibited surmountable antagonism at the guinea pig trachea with a pK(B) of 9.5. Intratracheal administration to anaesthetized rats demonstrated a dose-dependent inhibition of carbachol-induced bronchoconstriction with an ED(50) value of 110 ng/kg. RBx 343E48F0 also exhibited a fast onset of action and long duration of action of greater 24h.