Dilip J. Upadhyay

Ciprofloxacin-Resistant Neisseria meningitidis, Delhi, India

Decreased susceptibility of Neisseria meningitidis isolates to ciprofloxacin emerged from an outbreak in Delhi, India. Results of antimicrobial susceptibility testing of the meningococcal isolates to ciprofloxacin and further sequencing of DNA gyrase A quinolone-resistance–determining region confirmed the emergence of ciprofloxacin resistance in the outbreak.

Mode of Action of Ranbezolid against Staphylococci and Structural Modeling Studies of Its Interaction with Ribosomes

ABSTRACT Oxazolidinones are known to inhibit protein biosynthesis and act against a wide spectrum of gram-positive bacteria. A new investigational oxazolidinone, ranbezolid, inhibited bacterial protein synthesis in Staphylococcus aureus and Staphylococcus epidermidis. In S. epidermidis, ranbezolid showed inhibition of cell wall and lipid synthesis and a dose-dependent effect on membrane integrity. A kill-kinetics study showed that ranbezolid was bactericidal against S. epidermidis. In vitro translation of the luciferase gene done using bacterial and mammalian ribosomes indicated that ranbezolid specifically inhibited the bacterial ribosome. Molecular modeling studies revealed that both linezolid and ranbezolid fit in similar manners the active site of ribosomes, with total scores, i.e., theoretical binding affinities after consensus, of 5.2 and 6.9, respectively. The nitrofuran ring in ranbezolid is extended toward C2507, G2583, and U2584, and the nitro group forms a hydrogen bond from the base of G2583. The interaction of ranbezolid with the bacterial ribosomes clearly helps to elucidate its potent activity against the target pathogen.

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.

Design, Synthesis, and Identification of Silicon Incorporated Oxazolidinone Antibiotics with Improved Brain Exposure

Therapeutic options for brain infections caused by pathogens with a reduced sensitivity to drugs are limited. Recent reports on the potential use of linezolid in treating brain infections prompted us to design novel compounds around this scaffold. Herein, we describe the design and synthesis of various oxazolidinone antibiotics with the incorporation of silicon. Our findings in preclinical species suggest that silicon incorporation is highly useful in improving brain exposures. Interestingly, three compounds from this series demonstrated up to a 30-fold higher brain/plasma ratio when compared to linezolid thereby indicating their therapeutic potential in brain associated disorders.

Non invasive real-time monitoring of bacterial infection & therapeutic effect of anti-microbials in five mouse models

Background & objectives: In vivo imaging system has contributed significantly to the understanding of bacterial infection and efficacy of drugs in animal model. We report five rapid, reproducible, and non invasive murine pulmonary infection, skin and soft tissue infection, sepsis, and meningitis models using Xenogen bioluminescent strains and specialized in vivo imaging system (IVIS). Methods: The progression of bacterial infection in different target organs was evaluated by the photon intensity and target organ bacterial counts. Genetically engineered bioluminescent bacterial strains viz. Staphylococcus aureus Xen 8.1, 29 and 31; Streptococcus pneumoniae Xen 9 and 10 and Pseudomonas aeruginosa Xen-5 were used to induce different target organs infection and were validated with commercially available antibiotics. Results: The lower limit of detection of colony forming unit (cfu) was 1.7-log10 whereas the lower limit of detection of relative light unit (RLU) was 4.2-log10. Recovery of live bacteria from different target organs showed that the bioluminescent signal correlated to the live bacterial count. Interpretation & conclusions: This study demonstrated the real time monitoring and non-invasive analysis of progression of infection and pharmacological efficacy of drugs. These models may be useful for pre-clinical discovery of new antibiotics.

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.

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.

Anti-anaerobic potential of ranbezolid: insight into its mechanism of action against Bacteroides fragilis

This study reports the anti-anaerobic properties of ranbezolid, a new investigational oxazolidinone. A time-kill kinetics study against anaerobes showed that ranbezolid was superior to linezolid and killed the anaerobic pathogens at 4-8h, except for Bacteroides fragilis where killing was observed at 24h. In addition, the time-kill kinetics study showed a concentration-dependent bactericidal potential of ranbezolid against anaerobes. Ranbezolid showed 5.39log(10) reduction and linezolid showed 1.15log(10) reduction in murine disk implant infection with B. fragilis ATCC 25285. Ranbezolid was very potent and showed fast protein synthesis inhibition against B. fragilis, a Gram-negative anaerobe. In addition, non-specific cell wall synthesis inhibition was also observed with ranbezolid. The potent and fast protein synthesis inhibition along with an additional mode of action of cell wall synthesis inhibition could be responsible for the cidal effect of ranbezolid against Gram-negative anaerobes.

In Vitro and In Vivo Activities of the Novel Ketolide RBx 14255 against Clostridium difficile

ABSTRACT The MIC90 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.

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.