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.

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.

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.

Approaches towards the development of chimeric DPP4/ACE inhibitors for treating metabolic syndrome.

Designing drug candidates exhibiting polypharmacology is one of the strategies adopted by medicinal chemists to address multifactorial diseases. Metabolic disease is one such multifactorial disorder characterized by hyperglycaemia, hypertension and dyslipidaemia among others. In this paper we report a new class of molecular framework combining the pharmacophoric features of DPP4 inhibitors with those of ACE inhibitors to afford potent dual inhibitors of DPP4 and ACE.

Cissampelos pareira Linn: Natural Source of Potent Antiviral Activity against All Four Dengue Virus Serotypes.

BACKGROUND: Dengue, a mosquito-borne viral disease, poses a significant global public health risk. In tropical countries such as India where periodic dengue outbreaks can be correlated to the high prevalence of the mosquito vector, circulation of all four dengue viruses (DENVs) and the high population density, a drug for dengue is being increasingly recognized as an unmet public health need. METHODOLOGY/PRINCIPAL FINDINGS: Using the knowledge of traditional Indian medicine, Ayurveda, we developed a systematic bioassay-guided screening approach to explore the indigenous herbal bio-resource to identify plants with pan-DENV inhibitory activity. Our results show that the alcoholic extract of Cissampelos pariera Linn (Cipa extract) was a potent inhibitor of all four DENVs in cell-based assays, assessed in terms of viral NS1 antigen secretion using ELISA, as well as viral replication, based on plaque assays. Virus yield reduction assays showed that Cipa extract could decrease viral titers by an order of magnitude. The extract conferred statistically significant protection against DENV infection using the AG129 mouse model. A preliminary evaluation of the clinical relevance of Cipa extract showed that it had no adverse effects on platelet counts and RBC viability. In addition to inherent antipyretic activity in Wistar rats, it possessed the ability to down-regulate the production of TNF-α, a cytokine implicated in severe dengue disease. Importantly, it showed no evidence of toxicity in Wistar rats, when administered at doses as high as 2g/Kg body weight for up to 1 week. CONCLUSIONS/SIGNIFICANCE: Our findings above, taken in the context of the human safety of Cipa, based on its use in Indian traditional medicine, warrant further work to explore Cipa as a source for the development of an inexpensive herbal formulation for dengue therapy. This may be of practical relevance to a dengue-endemic resource-poor country such as India.

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.

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.

In vitro and in vivo activities of the novel Ketolide RBx 14255 against Clostridium difficile.

The MIC(90) of RBx 14255, a novel ketolide, against Clostridium difficile was 4 µg/ml (MIC range, 0.125 to 8 µg/ml), and this drug was found to be more potent than comparator drugs. An in vitro time-kill kinetics study of RBx 14255 showed time-dependent bacterial killing for C. difficile. Furthermore, in the hamster model of C. difficile infection, RBx 14255 demonstrated greater efficacy than metronidazole and vancomycin, making it a promising candidate for C. difficile treatment.

Antihyperglycemic effect of Annona squamosa hexane extract in type 2 diabetes animal model: PTP1B inhibition, a possible mechanism of action?

AIM: The mechanism of action of Annona squamosa hexane extract in mediating antihyperglycemic and antitriglyceridimic effect were investigated in this study. MATERIALS AND METHODS: The effects of extract on glucose uptake, insulin receptor-ß (IR-ß), insulin receptor substrate-1 (IRS-1) phosphorylation and glucose transporter type 4 (GLUT4) and phosphoinositide 3-kinase (PI3 kinase) mRNA expression were studied in L6 myotubes. The in vitro mechanism of action was tested in protein-tyrosine phosphatase 1B (PTP1B), G-protein-coupled receptor 40 (GPR40), silent mating type information regulation 2 homolog 1 (SIRT1) and dipeptidyl peptidase-IV (DPP-IV) assays. The in vivo efficacy was characterized in ob/ob mice after an oral administration of the extract for 21 days. RESULTS: The effect of extract promoted glucose uptake, IR-ß and IRS-1 phosphorylation and GLUT4 and PI3 kinase mRNA upregulation in L6 myotubes. The extract inhibited PTP1B with an IC(50) 17.4 µg/ml and did not modulate GPR40, SIRT1 or DPP-IV activities. An oral administration of extract in ob/ob mice for 21 days improved random blood glucose, triglyceride and oral glucose tolerance. Further, the extract did not result in body weight gain before and after treatment (29.3 vs. 33.6 g) compared to rosiglitazone where significant body weight gain was observed (28.4 vs. 44.5 g; *P<0.05 after treatment compared to before treatment). CONCLUSION: The results suggest that Annona squamosa hexane extract exerts its action by modulating insulin signaling through inhibition of PTP1B.

Dual epidermal growth factor receptor (EGFR)/insulin-like growth factor-1 receptor (IGF-1R) inhibitor: a novel approach for overcoming resistance in anticancer treatment.

Small molecule inhibitors of epidermal growth factor receptors (EGFR) have been found to show a good initial response in cancer patients but during the course of treatment, patients develop resistance after a few weeks of time. Development of secondary mutations or over-activation of insulin like growth factor (IGF-1R) pathway are a few of the several mechanisms proposed to explain the resistance. To study the effect of dual inhibition of EGFR and IGF-1R in overcoming the resistance, three strategies were envisaged and are reported in this manuscript: 1) a virtual predictive tumor model, 2) in vitro experimental data using a combination of EGFR and IGF-1R inhibitors and 3) in vitro experimental data using in house dual inhibitors. Findings reported in this manuscript suggest that simultaneous inhibition of IGF-1R and EGFR either by combination of two inhibitors or by dual kinase inhibitors is more efficacious compared to single agents. In vitro cell based experiments conducted using epidermoid cancer cell line, A431 and an EGFR mutant cell line, H1975 along with virtual predictions reported here suggests that dual inhibition of EGFR and IGF-1R is a viable approach to overcome EGFR resistance.

Activity of RBx 11760, a novel biaryl oxazolidinone, against Clostridium difficile.

OBJECTIVES: RBx 11760, a novel oxazolidinone, was investigated for in vitro and in vivo activity against Clostridium difficile. METHODS: The in vitro activity of RBx 11760 and three other agents against 50 diverse C. difficile clinical isolates and other obligate anaerobic bacteria was determined. The effect of RBx 11760 on sporulation and toxin production was determined against different C. difficile isolates. We used a hamster infection model to investigate the efficacy of RBx 11760, vancomycin and metronidazole. The mechanism of action of RBx 11760 against C. difficile ATCC 43255 was determined by macromolecular synthesis inhibition. RESULTS: RBx 11760 MICs were in the range of 0.5-1 mg/L for C. difficile isolates, and it demonstrated concentration-dependent killing of C. difficile ATCC 43255 and C. difficile 6387 up to 2-4× MIC (1-2 mg/L). RBx 11760, at concentrations as low as 0.25-0.5 mg/L, resulted in a significant reduction in de novo toxin production as well as sporulation in different C. difficile isolates. In contrast, vancomycin, metronidazole and linezolid had little or no effect on toxin production and appeared to promote the formation of spores. In the hamster infection model, treatment with RBx 11760 resulted in prolonged survival of animals as compared with vancomycin or metronidazole, which correlated well with the histopathology results. Macromolecular labelling results suggest that RBx 11760 is a potent inhibitor of bacterial protein synthesis. CONCLUSIONS: RBx 11760 showed excellent in vitro and in vivo activity against C. difficile, and it could be a promising novel candidate for future drug development against C. difficile infection.

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.

RBx-0597, a potent, selective and slow-binding inhibitor of dipeptidyl peptidase-IV for the treatment of type 2 diabetes.

Dipeptidyl peptidase IV (DPP-IV) inhibiton is a well recognized approach to treat Type 2 diabetes. RBx-0597 is a novel DPP-IV inhibitor discovered in our laboratory. The aim of the present study was to characterize the pharmacological profiles of RBx-0597 in vitro and in vivo as an anti-diabetic agent. RBx-0597 inhibited human, mouse and rat plasma DPP-IV activity with IC(50) values of 32, 31 and 39nM respectively. RBx-0597 exhibited significant selectivity over dipeptidyl peptidase8 (DPP-8), dipeptidyl peptidase9 (DPP-9) (150-300 fold) and other proline-specific proteases (>200-2000 fold). Kinetic analysis revealed that RBx-0597 is a competitive and slow binding DPP-IV inhibitor. In ob/ob mice, RBx-0597 (10mg/kg) inhibited plasma DPP-IV activity upto 50% 8h post-dose and showed a dose-dependent glucose excursion. RBx-0597 (10mg/kg) showed a significant glucose lowering effect (∼25% AUC of △ blood glucose) which was sustained till 12h, significantly increased the active glucagon-like peptide-1(GLP-1) and insulin levels. It showed a favourable pharmacokinetic profile (plasma clearance:174ml/min/kg; C(max) 292ng/ml; T(1/2) 0.28h; T(max) 0.75h and V(ss) 4.13L/kg) in Wistar rats with the oral bioavailability (F(oral)) of 65%. In summary, the present studies indicate that RBx-0597 is a novel DPP-IV inhibitor with anti-hyperglycemic effect and a promising candidate for further development as a drug for the treatment of type 2 diabetes.

Activity of a novel series of acylides active against community-acquired respiratory pathogens.

Resistance to macrolides and beta-lactams has increased sharply amongst key respiratory pathogens, leading to major concern. A novel series of acylides was designed to overcome this resistance and was evaluated for in vitro and in vivo activity. This series of acylides was designed starting from clarithromycin by changing the substitution on the desosamine nitrogen, followed by conversion to 3-O-acyl and 11,12-carbamate. Minimum inhibitory concentrations (MICs) of acylides were determined against susceptible as well as macrolide-lincosamide-streptogramin B (MLS(B))--and penicillin-resistant Streptococcus pneumoniae, Streptococcus pyogenes and Moraxella catarrhalis by the agar dilution method. Microbroth MICs for Haemophilus influenzae were determined according to Clinical and Laboratory Standards Institute guidelines. In vivo efficacy was determined by target organ load reduction against S. pneumoniae 3579 (ermB). The bactericidal potential of promising acylides was also determined. MICs of these compounds against S. pneumoniae, S. pyogenes, H. influenzae and M. catarrhalis were in the range of 0.06-2, 0.125-1, 1-16 and 0.015-0.5 microg/mL, respectively, irrespective of their resistance pattern. Mycoplasma pneumoniae and Legionella pneumophila showed MIC ranges of 0.004-0.125 microg/mL and 0.004-0.03 microg/mL, respectively. The acylides also showed better activity against telithromycin-resistant S. pneumoniae strains. Compounds with a 4-furan-2-yl-1H-imidazolyl side chain on the carbamate (RBx 10000296) showed a target organ load reduction of >3 log(10) colony-forming units/mL and concentration-dependent bactericidal potential against S. pneumoniae 994 mefA and H. influenzae strains. This novel and potent series of acylides active against antibiotic-resistant respiratory pathogens should be further investigated.

An orally active calcium-sensing receptor antagonist that transiently increases plasma concentrations of PTH and stimulates bone formation.

Daily subcutaneous administration of exogenous parathyroid hormone (PTH) promotes bone formation in patients with osteoporosis. Here we describe two novel, short-acting calcium-sensing receptor antagonists (SB-423562 and its orally bioavailable precursor, SB-423557) that elicit transient PTH release from the parathyroid gland in several preclinical species and in humans. In an ovariectomized rat model of bone loss, daily oral administration of SB-423557 promoted bone formation and improved parameters of bone strength at lumbar spine, proximal tibia and midshaft femur. Chronic administration of SB-423557 did not increase parathyroid cell proliferation in rats. In healthy human volunteers, single doses of intravenous SB-423562 and oral SB-423557 elicited transient elevations of endogenous PTH concentrations in a profile similar to that observed with subcutaneously administered PTH. Both agents were well tolerated in humans. Transient increases in serum calcium, an expected effect of increased parathyroid hormone concentrations, were observed post-dose at the higher doses of SB-423557 studied. These data constitute an early proof of principle in humans and provide the basis for further development of this class of compound as a novel, orally administered bone-forming treatment for osteoporosis.

Antagonists of the calcium receptor. 2. Amino alcohol-based parathyroid hormone secretagogues.

When administered as a single agent to rats, the previously reported calcium receptor antagonist 3 elicited a sustained elevation of plasma PTH resulting in no increase in overall bone mineral density. The lack of a bone building effect for analogue 3 was attributed to the large volume of distribution (V(dss)(rat) = 11 L/kg), producing a protracted plasma PTH profile. Incorporation of a carboxylic acid functionality into the amino alcohol template led to the identification of 12 with a lower volume of distribution (V(dss)(12) = 1.18 L/kg) and a shorter half-life. The zwitterionic nature of antagonist 12 necessitated the utility of an ester prodrug approach to increase overall permeability. Antagonist 12 elicited a rapid and transient increase in circulating levels of PTH following oral dosing of the ester prodrug 11 in the dog. The magnitude and duration of the increases in plasma levels of PTH would be expected to stimulate new bone formation.

Antagonists of the calcium receptor I. Amino alcohol-based parathyroid hormone secretagogues.

Functional screening of the former SmithKline Beecham compound collection against the human calcium receptor (CaR) resulted in the identification of the amino alcohol-based hit 2 (IC(50) = 11 microM). Structure-activity studies of 2 focused on the optimization of the right- and left-hand side aromatic moieties as well as the amino alcohol linker region. Critical to the optimization of this antagonist template was the discovery that the chirality of the C-2 secondary alcohol played a key role in enhancing both CaR potency as well as selectivity over the beta-adrenergic receptor subtypes. These SAR studies ultimately led to the identification of 38 (NPS 2143; SB-262470A), a potent and orally active CaR antagonist. Pharmacokinetic characterization of 38 in the rat revealed that this molecule had a large volume of distribution (11 L/kg), which resulted in a prolonged systemic exposure, protracted increases in the plasma levels of PTH, and an overall lack of net bone formation effect in a rodent model of osteoporosis.

Novel biaryl oxazolidinones: in vitro and in vivo activities with pharmacokinetics in an animal model.

RBx 1000075 and RBx 1000276, the new investigational oxazolidinones, have an extended spectrum of in vitro activity against Gram-positive pathogens and showed minimum inhibitory concentrations (MICs) lower than comparator drugs. MIC for 90% of the organisms (MIC(90)) values of RBx 1000075, RBx 1000276 and linezolid against the isolates tested were, respectively: methicillin-sensitive Staphylococcus aureus, 0.25, 1 and 4 microg/mL; methicillin-resistant S. aureus (MRSA), 0.5, 2 and 4 microg/mL; methicillin-sensitive Staphylococcus epidermidis, 0.25, 1 and 2 microg/mL; methicillin-resistant S. epidermidis, 0.5, 1 and 2 microg/mL; and enterococci, 0.25, 1 and 4 microg/mL. Against respiratory pathogens, MIC(90) values were: Streptococcus pneumoniae, 0.125, 0.5 and 2 microg/mL; Streptococcus pyogenes, 1, 0.5 and 2 microg/mL; and Moraxella catarrhalis, 0.5, 2 and 4 microg/mL. In vivo efficacies of RBx 1000075 and RBx 1000276 were evaluated in murine systemic infection against S. aureus (MRSA 562) and in a pulmonary infection model against S. pneumoniae ATCC 6303. In murine systemic infection, RBx 1000075 and RBx 1000276 showed efficacy against MRSA 562, exhibiting a 50% effective dose (ED(50)) of 6.25 and 9.92 mg/kg body weight for once-daily administration and 4.96 and 5.56 mg/kg body weight for twice-daily administration, respectively, whereas for linezolid the corresponding ED(50) values were 9.9 and 5.56 mg/kg body weight. In pulmonary infection with S. pneumoniae ATCC 6303, 50% survival was observed with RBx 1000075 at 50mg/kg once daily, whereas 60% survival was observed with RBx 1000276 at 50mg/kg thrice daily. The absolute oral bioavailabilities of RBx 1000075 and RBx 1000276 were 48% and 73%, with half-lives of 13.5 and 3.2h, respectively. RBx 1000075 and RBx 1000276 are promising investigational oxazolidinones against Gram-positive pathogens.

From a glucocentric to a lipocentric approach towards metabolic syndrome.

Insulin resistance, the essential component of metabolic syndrome, has traditionally been defined from a glucocentric viewpoint, with glucotoxicity playing a lead role. However, as overabundant circulating fatty acids are now known to be overt contributors, there is a paradigm shift in the understanding of metabolic syndrome acknowledging the importance of lipotoxicity as a major perpetuator of insulin resistance. Ectopic accumulation of fat in liver, adipose, muscle and pancreatic islets, provokes insulin resistance through various mechanisms. Chronic inflammation/adipocytokine generation, endoplasmic reticulum stress and mitochondrial dysfunction/oxidative stress also contribute significantly towards insulin resistance. Targets that can act as counter regulators/master switches at the converging point of all these metabolic pathways are currently under intense development.