In vitro and in vivo evaluation of metal-chelating agents as novel metallo beta-lactamase inhibitors against carbapenem-resistant enterobacteriaceae.

Abstract

Infectious diseases remain one of the leading causes of death worldwide, despite the discovery of new and improvements on existing antibiotics. Bacteria are constantly developing sophisticated mechanisms of resisting the effects of antibiotics, this in turn has increased their pathogenicity and virulence. Drugs belonging to the beta-lactam class of antibiotics are most commonly prescribed as they display a broad-spectrum activity against both gram-positive and gramnegative bacteria. Carbapenems which are a member of this class is regarded as the last line of defence against bacterial infections. Resistance to carbapenems is on the increase especially by bacterial strains that are capable of producing metallo-beta lactamase enzymes. Infections caused by carbapenem resistant Enterobacteriaceae are deadly especially those mediated by metallo beta-lactamases. Efforts are being made to synthesize compounds that can inhibit these enzymes. Thus far little progress has been made as a clinically available metallo beta-lactamase inhibitor has not yet emerged, hence the scourge of carbapenem resistant infections rages on. Therefore, the main aim of this study was to evaluate the in vitro and in vivo activities of metal chelating agents NO3PY and NOTA as potential metallo beta-lactamase inhibitors against carbapenem resistant Enterobacteriaceae. The metal-chelating agents used in this study were NOTA and NO3PY. In vitro analysis was performed to determine the minimum inhibitory concentrations by broth microdilution of meropenem alone and when co-administered with the chelators against resistant bacterial strains. The strains used in this study were Escherichia coli NDM-1, Klebsiella pneumoniae 449, Escherichia coli IMP-1 and Enterobacter cloacae NDM-1. Time kill kinetics was also evaluated at graded concentrations of MIC, 1*MIC, 2*MIC, 4*MIC, 8*MIC and 16*MIC. For the in vivo pharmacokinetics were determined using LC-MS/MS analysis. Forty-eight healthy male Balb/c mice were divided into two groups; meropenem+NO3PY group and meropenem+NOTA group. Both groups received intraperitoneal doses at 10 mg/kg of meropenem and the MBLIs. Thereafter, the in vivo efficacy of meropenem co-administered with NOTA (100 mg/kg each) in a murine thigh infection was determined. Both chelators were able to restore the efficacy of meropenem to a concentration as low as 0.06 µg/ml. The time kill kinetics also showed that both compounds were able to significantly extend the killing time of meropenem. In vivo pharmacokinetic analysis revealed that NO3PY may not xiv be a suitable candidate for in vivo efficacy study as the MBLI was not bioavailable in plasma at 10mg/kg. NOTA on the other hand was bioavailable at the same concentration as NO3PY. The former was able to potentiate the effect of meropenem in vivo in a murine thigh infection model. It was evident by a significant reduction of colony forming unit counts in groups treated with meropenem co-administered with NOTA when compared to infected controls Further preclinical work such as in vitro and in vivo cytotoxicity tests, post beta-lactamase inhibitor effects among others are recommended for NOTA to further ascertain its suitability as a potential clinical metallo beta-lactamase inhibitor.