In Vitro Activity of Ceftibuten/VNRX-5236 against Urinary Tract Infection Isolates of Antimicrobial-Resistant Enterobacterales

ABSTRACT Ceftibuten/VNRX-7145 is a cephalosporin/boronate β-lactamase inhibitor combination under development as an oral treatment for complicated urinary tract infections caused by Enterobacterales producing serine β-lactamases (Ambler class A, C, and D). In vivo, VNRX-7145 (VNRX-5236 etzadroxil) is cleaved to the active inhibitor, VNRX-5236. We assessed the in vitro activity of ceftibuten/VNRX-5236 against 1,066 urinary isolates of Enterobacterales from a 2014–2016 global culture collection. Each isolate tested was preselected to possess a multidrug-resistant (MDR) phenotype that included nonsusceptibility to amoxicillin-clavulanate and resistance to levofloxacin. MICs were determined by CLSI broth microdilution. VNRX-5236 was tested at a fixed concentration of 4 μg/ml. Ceftibuten/VNRX-5236 inhibited 90% of all isolates tested (MIC90) at 2 μg/ml; MIC90s for ESBL- (n = 566), serine carbapenemase- (n = 116), and acquired AmpC-positive (n = 58) isolate subsets were ≤0.25, >32, and 8 μg/ml, respectively. At concentrations of ≤1, ≤2, and ≤4 μg/ml, ceftibuten/VNRX-5236 inhibited 89.1, 91.7, and 93.1% of all isolates tested; 96.5, 97.7, and 98.4% of ESBL-positive isolates; 75.9, 81.9, and 81.9% of serine carbapenemase-positive isolates; and 70.7, 81.0, and 87.9% of acquired AmpC-positive isolates. Ceftibuten/VNRX-5236 at concentrations of ≤1, ≤2, and ≤4 μg/ml inhibited 85-89, 89-91, and 91-92% of isolates that were not susceptible (defined by CLSI and EUCAST breakpoint criteria) to nitrofurantoin, trimethoprim-sulfamethoxazole, and/or fosfomycin, (as part of their MDR phenotype), oral agents commonly prescribed to treat uncomplicated urinary tract infections. The potency of ceftibuten/VNRX-5236 (MIC90, 2 μg/ml) was similar (within one doubling-dilution) to intravenous-only agents ceftazidime-avibactam (MIC90 2 μg/ml) and meropenem-vaborbactam (MIC90 1 μg/ml). Continued investigation of ceftibuten/VNRX-5236 is warranted.

amoxicillin-clavulanate in the 1980s. Today, resistance to amoxicillin-clavulanate among Enterobacterales is a major concern, because increasing numbers of clinical isolates carry ESBLs as well as KPC and OXA-48-like carbapenemases. These enzymes were not a clinical concern when amoxicillin-clavulanate was first introduced. Clavulanic acid possesses inhibitory activity against only certain Ambler class A ESBLs but demonstrates essentially no activity against class C cephalosporinases (AmpCs) or class A (KPC) or class D (OXA-48-like) carbapenem hydrolyzing enzymes. New oral antimicrobial agents to treat outpatients with urinary tract infections caused by Enterobacterales carrying serine b-lactamases, including ESBLs, AmpC enzymes and carbapenemases, represents an important contemporary unmet medical need (6).
Ceftibuten/VNRX-7145 is currently in phase 1 clinical trials that involve first-inhuman dose-ranging studies to evaluate the safety and pharmacokinetics of escalating doses of VNRX-7145 (ClinicalTrials.gov identifier: NCT04243863). In this study ceftibuten/VNRX-5236 and 16 comparators were tested against 1,066 MDR urinary isolates of Enterobacterales chosen from a 2014-2016 global culture collection. The MDR phenotype of each isolate tested was preselected to include nonsusceptibility to amoxicillin-clavulanate, resistance to levofloxacin, and nonsusceptibility to one or more additional oral and parenteral agents from other structural categories where agents have potential for use in treating patients with complicated or uncomplicated urinary tract infections (13). The objective of this study was to determine the ability of VNRX-5236 to restore the activity of ceftibuten against this challenge set of antimicrobial-resistant isolates.

DISCUSSION
Ceftibuten in combination with VNRX-7145 (VNRX-5236 etzadroxil) is under development as an oral treatment for complicated urinary tract infections caused by serine TABLE 3 In vitro activity of ceftibuten/VNRX-5236 at ceftibuten concentrations of 1, 2, and 4 mg/ml against urinary isolates of Enterobacterales preselected to possess a MDR phenotype that included nonsusceptibility to amoxicillin-clavulanate and resistance to levofloxacin; isolates are stratified by phenotypic nonsusceptibility to agents other than amoxicillin-clavulanate and levofloxacin that were associated with MDR phenotypes b-lactamase-producing Enterobacterales, including isolates carrying ESBLs and carbapenemases (10). In the current study we challenged ceftibuten/VNRX-5236 using a recent collection of 1,066 urinary isolates of Enterobacterales preselected to possess a MDR phenotype that included nonsusceptibility to amoxicillin-clavulanate and resistance to levofloxacin. We preselected isolates that were MDR and possessed resistance to potential empirical first-line agents (amoxicillin-clavulanate and levofloxacin) used in the treatment of patients with complicated urinary tract infections, including acute pyelonephritis, and included phenotypes identified as CDC and WHO priority pathogens (i.e., carbapenem-resistant and/or third-generation cephalosporin-resistant Enterobacterales) (2, 3). CDC and WHO priority pathogens necessitate the development of new agents, particularly oral agents, for outpatient treatment to reduce duration of hospitalization or avoid admission altogether (2,3). The current study determined that the addition of VNRX-5236 (4 mg/ ml) to ceftibuten restored the activity of ceftibuten in many resistant isolates. Increasing resistance among agents commonly prescribed to treat urinary tract infections (trimethoprim-sulfamethoxazole, fluoroquinolones, oral b-lactams including amoxicillin-clavulanate) and for agents with pharmacokinetic (nitrofurantoin) and spectrum limitations (nitrofurantoin, fosfomycin) indicate that new oral agents are urgently needed to address the inadequacies of current agents (6,15). In the current study, ceftibuten/VNRX-5236 (MIC 90 2 mg/ml) was significantly more potent in vitro than .32 mg/ml), and it inhibited a majority of isolates that were not susceptible to each of these agents in addition to their nonsusceptibility to amoxicillin-clavulanate and resistance to levofloxacin. Ceftibuten/VNRX-5236 may therefore also have promise in patients where resistant pathogens are suspected or in some patients with complicated infection where hospital avoidance and oral therapy is reasonable.
Ceftibuten/VNRX-5236 demonstrated an in vitro potency that is similar to established and newer parenteral therapies. MIC 90 s for ceftibuten/VNRX-5236, ceftazidimeavibactam, and meropenem-vaborbactam were all 1-2 mg/ml. In contrast, ceftolozanetazobactam was less active than other b-lactam-b-lactamase inhibitor combinations, with an MIC 90 of .8 mg/ml and only 55.9% of isolates categorized as susceptible. Ceftibuten/VNRX-5236, therefore, may also have potential as a step-down oral agent from a broad-spectrum empirical or directed parenteral agent (e.g., ceftazidime-avibactam or meropenem-vaborbactam) where Enterobacterales producing serine b-lactamases, including carbapenemases, is confirmed or highly suspected. Against MDR Enterobacterales, ceftibuten/VNRX-5236 has been reported to demonstrate similar potency in vitro to carbapenems and current b-lactam/b-lactamase inhibitors such as meropenem, meropenem-vaborbactam, and ceftazidime-avibactam (16). These results are consistent with those in the current study and suggest that ceftibuten/ VNRX-5236 should be further developed as a potential carbapenem-sparing oral agent to treat patients with complicated urinary tract infections due to MDR Enterobacterales.
In the current study, ceftibuten/VNRX-5236 demonstrated potent in vitro activity against ESBL-producing isolates of Enterobacterales that did not co-produce a class C or a class D b-lactamase (Table 4). VNRX-5236 restored ceftibuten MICs to #1 mg/ml for 86.7% of isolates expressing CMY, whether alone or in combination with an ESBL, consistent with its spectrum of inhibition against this enzyme family (8, 10). However, DHA-producing Enterobacterales, whether alone or in combination with an ESBL (but without serine carbapenemases or metallo-b-lactamases), generated higher ceftibuten/VNRX-5236 MICs than did isolates without these enzymes present. We also observed that although ceftibuten/VNRX-5236 is active against OXA-48/OXA-48-like producers in isolation or in isolates also producing an ESBL, MICs are elevated against isolates co-producing OXA-48/OXA-48-like and CMY or OXA-48/OXA-48-like and DHA (again, without serine carbapenemases or metallo-b-lactamases). While the epidemiology of b-lactam resistance may change across time and geographies, the proportion of isolates expressing DHA alone (with or without ESBLs) or OXA-48/OXA-48-like and an acquired AmpC enzyme (with or without ESBLs) was limited to 2.4 and 1.4% of isolates in the current challenge set, respectively. In the current study, ceftibuten activity was not restored by VNRX-5236 for metallob-lactamase-producing isolates. This observation is consistent with the spectrum of inhibitory activity of VNRX-5236 as determined in biochemical assays (8,10) and is similar to the activities of other tested agents in the current study including the carbapenemboronate b-lactamase inhibitor combination, meropenem-vaborbactam. Currently no approved b-lactam-b-lactamase inhibitor combination covers metallo-b-lactamaseproducing isolates. There are only two b-lactam-b-lactamase inhibitor combinations in development that possess a spectrum that covers metallo-b-lactamases; cefepimetaniborbactam and aztreonam-avibactam. Cefepime-taniborbactam was evaluated in the present study; taniborbactam restored the activity of cefepime to #8 mg/ml for 20 of the 28 isolates that expressed NDM or VIM and any combination of ESBL, acquired AmpC and/or serine carbapenemases.
Uehara et al. demonstrated that over-expression of an ESBL (CTX-M-15) or KPC (KPC-2, KPC-3) in isogenic strains of E. coli did not significantly increase ceftibuten-VNRX-5236 (4 mg/ml) MICs while the over-expression of an AmpC b-lactamase (P99, CMY-42) increased MICs from 0.25 mg/ml (control) to 4 mg/ml (17). This observation suggests that the overexpression of certain b-lactamases may account for the relatively reduced activity of ceftibuten/VNRX-5236 observed in the current study compared with earlier studies, as described above, with the caveat that analysis of gene expression would be needed to definitively identify the reason for the discordance.
There is currently discordance between CLSI and EUCAST MIC breakpoints for ceftibuten. CLSI investigational MIC breakpoints for ceftibuten tested against Enterobacterales are susceptible, #8 mg/ml; intermediate, 16 mg/ml; and resistant, $32 mg/ml; these breakpoints are for testing and reporting of urinary tract isolates of Enterobacterales only (18). EUCAST MIC breakpoints for ceftibuten tested against Enterobacterales are: susceptible, #1 mg/ml and resistant, .1 mg/ml; these breakpoints apply to infections originating in the urinary tract (14). Initial pharmacokinetic/pharmacodynamic data for VNRX-5236 suggests that the free area under the concentration-time curve to MIC ratio (fAUC 0-24 /MIC) is the parameter that best correlates with in vivo activity of VNRX-5236 (i.e., dosing frequency of VNRX-5236 does not impact the extent of VNRX-5236 potentiation of ceftibuten activity) and that based upon a hypothetical clinical dose of 300 mg of ceftibuten given every 8 h that the susceptible MIC breakpoint for ceftibuten/VNRX-5236 may be in the range of 1 to 2 mg/ml (i.e., closer to the current EUCAST breakpoint than the current CLSI breakpoint) The. First, each isolate of Enterobacterales tested was preselected to possess a MDR phenotype that current study has at least three important limitations included nonsusceptibility to amoxicillin-clavulanate and resistance to levofloxacin. Such an isolate collection, enriched for resistant phenotypes, invalidates comparisons with prevalence-based studies. Second, isolates in the current study were not characterized for non-b-lactamase-mediated resistance mechanisms (e.g., porin mutation/expression and efflux pump expression), which are known to affect the activity of cephalosporins, including ceftibuten, and b-lactam-b-lactamase inhibitor combinations   (20). Third, no data are included regarding isolate background, including clinical syndrome and underlying host comorbidities. Based on the data generated in the current study, ceftibuten/VNRX-5236 appears to have potential as an oral treatment option for complicated urinary tract infections caused by serine b-lactamase-expressing Enterobacterales (ESBL, KPC, OXA-48/OXA-48like) for which there are currently few oral treatment options available. Ceftibuten/VNRX-5236 also exhibited potent in vitro activity against isolates that were not susceptible to current, frequently prescribed oral (fosfomycin, nitrofurantoin, and trimethoprim-sulfamethoxazole) and parenteral (ceftriaxone, piperacillin-tazobactam, and ceftolozane-tazobactam) agents. Further clinical development of ceftibuten-VNRX-7145 is warranted.
Antimicrobial susceptibility testing. MICs were determined using the CLSI reference broth microdilution method (23). Broth microdilution panels were prepared at IHMA using cation-adjusted Mueller-Hinton broth (CAMHB) (Becton, Dickinson, Sparks, MD) and stored at 280°C until the day of testing. CAMHB with TES (TREK Diagnostic Systems, Independence, OH) was used for inoculum preparation. Tryptic soy agar (TSA) plates containing 5% sheep blood (Liofilchem, Waltham, MA) were used to subculture isolates.