Microbiological Characterization of VNRX-5236, a Broad-Spectrum β-Lactamase Inhibitor for Rescue of the Orally Bioavailable Cephalosporin Ceftibuten as a Carbapenem-Sparing Agent against Strains of Enterobacterales Expressing Extended-Spectrum β-Lactamases and Serine Carbapenemases

Abstract

There is an urgent need for oral agents to combat resistant Gram-negative pathogens. Here, we describe the characterization of VNRX-5236, a broad-spectrum boronic acid β-lactamase inhibitor (BLI), and its orally bioavailable etzadroxil prodrug, VNRX-7145. VNRX-7145 is being developed in combination with ceftibuten, an oral cephalosporin, to combat strains of Enterobacterales expressing extended-spectrum β-lactamases (ESBLs) and serine carbapenemases. VNRX-5236 is a reversible covalent inhibitor of serine β-lactamases, with inactivation efficiencies on the order of 10⁴ M^-1 · sec^-1, and prolonged active site residence times (t_1/2, 5 to 46 min). The spectrum of inhibition includes Ambler class A ESBLs, class C cephalosporinases, and class A and D carbapenemases (KPC and OXA-48, respectively). Rescue of ceftibuten by VNRX-5236 (fixed at 4 μg/ml) in isogenic strains of Escherichia coli expressing class A, C, or D β-lactamases demonstrated an expanded spectrum of activity relative to oral comparators, including investigational penems, sulopenem, and tebipenem. VNRX-5236 rescued ceftibuten activity in clinical isolates of Enterobacterales expressing ESBLs (MIC₉₀, 0.25 μg/ml), KPCs (MIC₉₀, 1 μg/ml), class C cephalosporinases (MIC₉₀, 1 μg/ml), and OXA-48-type carbapenemases (MIC₉₀, 1 μg/ml). Frequency of resistance studies demonstrated a low propensity for recovery of resistant variants at 4× the MIC of the ceftibuten/VNRX-5236 combination. In vivo, whereas ceftibuten alone was ineffective (50% effective dose [ED₅₀], >128 mg/kg), ceftibuten/VNRX-7145 administered orally protected mice from lethal septicemia caused by Klebsiella pneumoniae producing KPC carbapenemase (ED₅₀, 12.9 mg/kg). The data demonstrate potent, broad-spectrum rescue of ceftibuten activity by VNRX-5236 in clinical isolates of cephalosporin-resistant and carbapenem-resistant Enterobacterales.

Keywords: Enterobacterales; VNRX-5236 etzadroxil; VNRX-7145; ceftibuten; oral antibiotics.

FIG 1

Structures of orally bioavailable prodrug VNRX-7145 (VNRX-5236 etzadroxil) (left) and active BLI, VNRX-5236 (right).

FIG 2

Partner β-lactam activity in combination with VNRX-5236 against strains of Enterobacterales expressing serine β-lactamases. All testing was conducted according to the CLSI broth microdilution method, with VNRX-5236 fixed at 4 μg/ml and partner β-lactam titrated (26). Clinical isolates tested included (A) 12 E. coli and 13 K. pneumoniae; (B) 19 K. pneumoniae, 1 E. coli, 3 E. cloacae, and 2 Klebsiella oxytoca; (C) 13 K. pneumoniae and 12 E. coli; (D) 7 E. coli, 7 K. pneumoniae, 6 Serratia marcescens, 3 Klebsiella aerogenes, 1 Citrobacter freundii, and 1 Proteus mirabilis.

FIG 3

Time-kill assay of CTB/VNRX-5236 against strains of Enterbacterales with defined resistance mechanisms. Hexagon, growth control; square, CTB alone at 8 μg/ml; open circle, CTB at 2 μg/ml with VNRX-5236 fixed at 4 μg/ml; × inside a circle, CTB at 2 μg/ml with clavulanic acid fixed at 4 μg/ml; upside-down triangle, amoxicillin at 8 μg/ml with clavulanic acid at 4 μg/ml (a 2:1 ratio); triangle, tebipenem at 2 μg/ml; diamond, sulopenem at 2 μg/ml.