Meropenem-ANT3310, a unique β-lactam-β-lactamase inhibitor combination with expanded antibacterial spectrum against Gram-negative pathogens including carbapenem-resistant Acinetobacter baumannii

ABSTRACT ANT3310 is a novel broad-spectrum diazabicyclooctane serine β-lactamase inhibitor being developed in combination with meropenem (MEM) for the treatment of serious infections in hospitalized patients where carbapenem-resistant Gram-negative pathogens are expected. In this study, we evaluated the in vitro antibacterial activity of MEM in the presence of ANT3310 at 8 µg/mL against global clinical isolates that included Acinetobacter baumannii (n = 905), carbapenem-resistant Enterobacterales (CRE), carrying either oxacillinase (OXA) (n = 252) or Klebsiella pneumoniae carbapenemase (KPC) (n = 180) carbapenemases, and Pseudomonas aeruginosa (n = 502). MEM was poorly active against A. baumannii, as were MEM-vaborbactam, ceftazidime-avibactam, aztreonam-avibactam, cefepime-taniborbactam, cefepime-zidebactam, and imipenem-relebactam (MIC90 values of ≥32 µg/mL). On the other hand, MEM-ANT3310 displayed an MIC90 value of 4 µg/mL, similar to that observed with sulbactam-durlobactam, a drug developed to specifically treat A. baumannii infections. ANT3310 (8 µg/mL) additionally restored the activity of MEM against OXA- and KPC-producing CREs decreasing MEM MIC90 values from >32 µg/mL to 0.25 and 0.5 µg/mL, respectively. The combination of 8 µg/mL of both MEM and ANT3310 prevented growth of 97.5% of A. baumannii and 100% of OXA- and KPC-positive CREs, with ~90% of P. aeruginosa isolates also displaying MEM MICs ≤8 µg/mL. Furthermore, MEM-ANT3310 was efficacious in both thigh and lung murine infection models with OXA-23 A. baumannii. This study demonstrates the potent in vitro activity of the MEM-ANT3310 combination against both carbapenem-resistant A. baumannii and Enterobacterales clinical isolates, a key differentiator to other β-lactam/β-lactamase combinations.

1 pathogen by both organizations.CRAB was responsible for ~8,500 infections in hospitalized patients in the USA during 2017 (5).Nearly half (43.6%) of the Acineto bacter spp.isolates reported by 30 European Union/European Economic Area (EU/EEA) countries to the European Antimicrobial Resistance Surveillance Network (EARS-Net) for 2019 were resistant to all three antibiotic groups under surveillance (fluoroquinolones, aminoglycosides, and carbapenems) (7) and levels of multi-drug-resistant A. baumannii are over four times higher than for Klebsiella pneumoniae and Pseudomonas aeruginosa (8).Production of class D β-lactamases (oxacillinase; OXA) is one of the most important mechanisms of carbapenem resistance in A. baumannii (9).
Treatment options for carbapenem-resistant Gram-negative pathogens, and particularly CRAB, are limited and the current pharmaceutical development pipeline includes very few novel anti-infective agents which are effective against these organisms (10)(11)(12).ANT3310 is a novel broad-spectrum serine-β-lactamase (SBL) inhibitor of the diazabicyclooctane (DBO) class which strongly inhibits both K. pneumoniae carbapene mase (KPC) and OXA carbapenemases and potentiates the activity of meropenem (MEM) against CRAB as well as carbapenem-resistant Enterobacterales (CRE) (13).
MEM-ANT3310 is being developed for the treatment of hospital-acquired infec tions, such as complicated urinary tract infections (cUTI), hospital-acquired bacterial pneumonia (HABP), ventilator-associated bacterial pneumonia (VABP), and complicated intra-abdominal infections caused by Gram-negative pathogens, including those that are carbapenem-resistant, notably CRE and CRAB, and received Qualified Infectious Disease Product designation from the US Federal Drugs Administration (FDA) in January 2020 for the treatment of these serious diseases.As part of the MEM-vaborbactam clinical trials, it has already been shown that 2 g of MEM dosed every 8 h (q8h) as a 3-h IV infusion is generally safe and well tolerated (14) and achieves >90% probability of target attainment for MEM MICs ≤8 µg/mL (15,16).This dosing regimen is being evaluated in combination with ANT3310 in Phase 1 clinical trials (ClinicalTrials.govID NCT05905913; EudraCT#: 2022-002258 -18).
Here, we describe the inhibitory activity of ANT3310 against different OXA-type enzymes, as well as its potentiation effect on MEM activity both in vitro, against 905 randomly selected A. baumannii collected in 2018 and 2019, and in vivo, in thigh and lung murine infection models.In addition, this combination was also evaluated against CRE and P. aeruginosa isolates.

Inhibitory activity of ANT3310 and other SBL inhibitors against OXA carbape nemases
The in vitro activities of ANT3310 and other SBL inhibitors were evaluated against OXA carbapenemases typically present in CRE (OXA-48) and CRAB (OXA-23, -24/40, -51, and -58), with KPC-2 included as a non-OXA control (Table 1).ANT3310 showed inhibitory activity against all enzymes tested, including all the OXA enzymes from CRAB, with half-maximal inhibitory concentration (IC 50 ) values ranging from 8 to 602 nM.In this respect, ANT3310 demonstrated a wider spectrum of activity than avibactam, relebactam, zidebactam, nacubactam, taniborbactam, or vaborbactam.Only durlobac tam showed an equivalent spectrum, although there were noticeable differences, with durlobactam being more active against OXA-48 and ANT3310 being more active against OXA-23 and OXA-51.

Determination of appropriate β-lactam antibiotic partner
An initial small study was performed against a set of clinical isolates including four CRAB and five SBL-positive CRE to determine the most appropriate β-lactam antibiotic partner for ANT3310.Different β-lactam antimicrobial agents, including carbapenems MEM and imipenem, and cephalosporins ceftazidime, cefepime, cefotaxime, and ceftriaxone were used in this study (data not shown).ANT3310 potentiated the activity of all β-lactams similarly against CRE; however, against A. baumannii isolates, it was only effective at decreasing the MICs of the two carbapenems (data not shown).In general, addition of ANT3310 to either MEM or imipenem resulted in very similar MICs.Although both MEM and imipenem could be partnered with ANT3310 to deliver a broad-spectrum combination therapy against CRE and CRAB isolates, a decision to use MEM over imipenem as the β-lactam partner was made based on (i) the lack of requirement for cilastatin (an inhibitor of imipenem metabolism) and (ii) the fact that high doses of MEM have already been shown to be safe and tolerated in a previous combination (14).3).The % susceptible and resistant have been assigned using the Clinical and Laboratory Standards Institute (CLSI) (17) and the European Committee on Antimicrobial Susceptibil ity Testing (EUCAST) breakpoints (BP) (18) (Table 3).Overall, 703 (78%) of isolates were resistant to MEM (MEM R ) (CLSI, MIC ≥8 µg/mL), and can thus be designated as CRAB, with MEM R rates ranging between 74% (Asia/Pacific) and 91% (Middle East) in all regions except North America where the rate was lower (40%).

Potentiation of MEM activity at different ANT3310 concentrations
Initially, for the first set of 500 clinical isolates (from 2018), MEM-ANT3310 combinations were tested with ANT3310 at 4 and 8 µg/mL to determine the optimal concentration for potentiation of MEM.The data showed that while addition of ANT3310 at 4 µg/mL reduced the MEM MIC 90 from >32 µg/mL to 16 µg/mL, increasing the concentration to 8 µg/mL resulted in an additional fourfold reduction of the MEM MIC 90 to 4 µg/mL (Table 3).Therefore, subsequent studies and comparisons versus other agents were performed using MEM-ANT3310 (8 µg/mL).
The robust activity of MEM-ANT3310 against the CRAB subset further highlighted the difference between this and other BL/BLI combinations, as only sulbactam-durlobactam had comparable activity (Table 3; Fig. 2C and D)

Activity of MEM-ANT3310 (8 μg/mL) and comparator antibacterial agents against P. aeruginosa clinical isolates
Susceptibility testing of MEM-ANT3310 and comparator antibacterial agents was also performed against 502 P. aeruginosa isolates randomly selected from the 2019 IHMA global collection.Addition of ANT3310 to MEM had a small but positive effect against strains at the lower end of the MEM MIC spectrum.This resulted in a twofold decrease in the MIC 50 of the combination, from 0.5 to 0.25 µg/mL, although the MIC 90 remained the same, 8 µg/mL.The activity of MEM-ANT3310 was comparable to that of imipenem-rele bactam and cefepime-taniborbactam (Table 6; Fig. 4), but cefiderocol and colistin were more potent, with MIC 90 values of 1 µg/mL.

In vivo efficacy of MEM and MEM-ANT3310 against OXA-23 A. baumannii in thigh and lung murine infection models
The ability of ANT3310 to restore MEM activity in vivo was evaluated in both murine thigh and lung infection models with A. baumannii ACC00445 (OXA-23).Due to its short half-life in mice, it is necessary to dose MEM frequently to maintain plasma levels above the MIC for 40% of the dosing interval.Consequently, efficacy studies in the thigh model were performed dosing MEM IV at 300 mg/kg every 2 h, as this results in a %T above 8 µg/mL equivalent to that achieved by 2 g of MEM administered q8h by 3 h IV infusion in humans (20), which is the dosing regimen proposed for the MEM-ANT3310 combina tion.In  units (CFUs) versus MEM alone (P < 0.0001) (Fig. 5A).The combination of MEM-ANT3310 resulted in stasis at 300/50 mg/kg and had a killing effect at 300/100 mg/kg.Lower doses of MEM were necessary to achieve efficacy in the 14-h respiratory tract infection model.No efficacy was observed when MEM was dosed IV at 30 mg/kg but addition of ANT3310 at 25, 50, and 100 mg/kg demonstrated a statistically significant reduction of 4.3, 4.6, and 4.2 log 10 CFUs, respectively, versus MEM alone (P < 0.0001), with the combination of MEM-ANT3310 at 30/25 mg/kg reducing bacterial counts 3-log 10 below stasis (Fig. 5B).limited by nephrotoxicity (21), provided adequate coverage against A. baumannii.In fact, the 2023 Infectious Diseases Society of America (IDSA) guidelines recommend against the use of colistin to treat CRE infections (22), whereas the FDA does not recognize the CLSI BP for either CRE or A. baumannii and also recommends the use of alternative agents (23).
In addition, although cefiderocol has activity against CRE and A. baumannii, including CRAB, and is approved in the USA for the treatment of cUTI, HABP, and VABP, and in the European Medicines Agency (EMA) for the treatment of infections due to aero bic Gram-negative organisms, the recent publication of lower susceptibility BPs than originally proposed for Enterobacterales [EUCAST susceptible BP of ≤2 µg/mL (18) instead of CLSI BP of ≤4 µg/mL] and for A. baumannii [FDA susceptible BP of ≤1 µg/mL (24) instead of CLSI BP of ≤4 µg/mL] limits its clinical utility.
Of the BL/BLI combinations tested, all showed similarly strong inhibitory activity against KPC-CRE, whereas only MEM-ANT3310, ceftazidime-avibactam, and cefepimezidebactam demonstrated potent activity against OXA-CRE.Furthermore, only MEM-ANT3310 and sulbactam-durlobactam, which was recently approved by the FDA for the treatment of HABP and VABP due to Acinetobacter ABC complex (under the trade name Xacduro), showed potent activity against A. baumannii isolates.Therefore, no antibacte rial agent, either marketed or in late clinical development, possesses clinical coverage of both CRE and CRAB isolates and MEM-ANT3310 was the only agent tested that showed potent activity against both these important pathogen classes.
We have previously reported the in vivo efficacy of MEM-ANT3310 against CRE strains in a murine thigh infection model (13); here, we have demonstrated that MEM-ANT3310, delivered IV, is equally efficacious against an OXA-23 CRAB isolate in a similar mouse thigh infection model at equivalent doses (25, 50, and 100 mg/kg).Furthermore, we have demonstrated that ANT3310 shows effective potentiation of MEM against the same CRAB strain in a mouse lung infection, and at even lower doses than for the thigh infection model, indicating good distribution and retention of activity of ANT3310 in lung tissues, and supporting the potential for MEM-ANT3310 as an effective combination to treat respiratory infections.
In summary, MEM-ANT3310 has excellent in vitro and in vivo activities against key carbapenem-resistant Gram-negative pathogens, including KPC-CRE, OXA-CRE, and CRAB, and shows broad in vitro coverage of P. aeruginosa.MEM-ANT3310 is thus clearly differentiated from other BL/BLI combinations, all of which display limited activity against one or more key pathogen groups, and shows strong potential for development as a best-in-class treatment for serious Gram-negative nosocomial infections.

Bacterial isolates
All isolates were randomly selected from the frozen culture collection of IHMA collec ted in 2018 and 2019.Matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to confirm the identity of the organisms tested (Bruker Daltonics, Bremen, Germany).

Antimicrobial susceptibility testing
MICs for all antimicrobial agents were determined at IHMA by broth microdilution in accordance with both CLSI (17) and EUCAST (18) guidelines.Microtiter panels were prepared by IHMA and stored at −70°C in cation-adjusted Mueller Hinton broth (CAMHB) (Becton Dickinson).Iron-depleted CAMHB was used to test cefiderocol.Panels contain ing ANT3310 were prepared fresh on the day of testing.The panels were incubated at 35°C for 16 to 20 h in ambient air before MIC endpoints were read visually.MIC values corresponded to the first well with no visible growth.Quality control testing was performed on each day of testing with E. coli ATCC 25922, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 700603 and BAA 1705, and A. baumannii NCTC 13304.MICs were interpreted using 2023 CLSI (17) and EUCAST (18) BPs.

Animals
Specific pathogen-free male CD-1 mice (11-15 g on receipt) (Charles River Laboratories, Margate, Kent, UK) were allowed to acclimatize for ~10 days then rendered neutro penic by immunosuppression with cyclophosphamide by intraperitoneal injection at 150 mg/kg 4 days before infection and 100 mg/kg 1 day before infection.The immuno suppression regime leads to neutropenia starting 24 h post administration of the first injection continuing throughout the study.Efficacy studies were performed at Evotec, Alderley Park, Cheshire, UK.
Vehicle (phosphatebuffered saline, PBS), MEM, and MEM-ANT3310 were administered IV at 1, 3, 5, and 7 h post-infection at 10 mL/kg.Tigecycline, dosed once IV at 40 mg/kg, was used as a positive control.One group of animals was humanely euthanized using pentobarbitone overdose 1 h post-infection to provide a pre-treatment control group.All animals in the additional groups were euthanized at the end of the study, 9 h postinfection.Thigh samples were homogenized in ice-cold sterile PBS; the homogenates were quantitatively cultured onto cystinelactoseelectrolytedeficient (CLED) agar in triplicate and incubated at 37°C for 18-24 h before colonies were counted.The data from the culture burdens were analyzed using appropriate non-parametric statistical models (Kruskal-Wallis using Conover-Inman to make all pairwise comparisons between groups) with StatsDirect software v. 3.2.7,and compared to vehicle control.For all calculations, the thighs from each animal were treated as two separate data points even though they are not completely independent samples.

Murine lung infection model
Mice (six per group) were infected with 0.05 mL of a suspension of A. baumannii ACC00445 (OXA-23) (1.23 × 10 7 CFU/mouse) by oropharyngeal administration under temporary inhaled anaesthesia (2.5% isofluorane in 87.5% oxygen for 3-4 min).Vehicle (PBS), MEM, and MEM-ANT3310 were administered IV at 2, 4, 6, 8, 10, and 12 h postinfection at 10 mL/kg.Tigecycline (30 mg/kg), dosed IV at 2 and 8 h post-infection, was used as a positive control.One group of animals was humanely euthanized using pentobarbitone overdose 2 h post-infection to provide a pre-treatment control group.All animals in the additional groups were euthanized at the end of the study, 14 h postinfection.Lung samples were homogenized in ice-cold sterile PBS; the homogenates were quantitatively cultured onto CLED agar in triplicate and incubated at 37°C for 18-24 h before colonies were counted.The data from the culture burdens were analyzed using appropriate non-parametric statistical models (Kruskal-Wallis using Conover-Inman to make all pairwise comparisons between groups) with StatsDirect software v. 3.2.7,and compared to vehicle control.

5 TABLE 3 340 ( 6 TABLE 3 a
In vitro susceptibility data for MEM, MEM-ANT3310, and comparator antibiotics against A. baumannii and CRAB isolates from 2018 and 2019 global collections (Continued) Antimicrobial agent Number of isolates and cumulative % inhibited at MIC (µg/mL) of: Continued on next page) Full-Length Text Antimicrobial Agents and Chemotherapy March 2024 Volume 68 Issue 3 10.1128/aac.01120-23In vitro susceptibility data for MEM, MEM-ANT3310, and comparator antibiotics against A. baumannii and CRAB isolates from 2018 and 2019 global collections (Continued)Antimicrobial agent Number of isolates and cumulative % inhibited at MIC (µg/mL) of:For comparative purposes only, percent susceptible and percent resistant for MEM-ANT3310 correspond to the percentage of isolates inhibited at ≤8 µg/mL and ≥16 µg/mL, respectively.% of isolates with MEM and MEM-ANT3310 of ≤8 µg/mL MICs have been highlighted in gray.b MEM R , MICs >4 µg/mL (CLSI BP).c Number of isolates and cumulative % inhibited at MIC of >16 μg/mL.d Number of isolates and cumulative % inhibited at MIC of ≤0.25 μg/mL.e -, Breakpoints not defined.Full-Length Text Antimicrobial Agents and Chemotherapy March 2024 Volume 68 Issue 3 10.1128/aac.01120-237

TABLE 1
Inhibitory activity of ANT3310 and other SBL inhibitors b

Inhibitory activity of MEM-ANT3310 and comparator agents against clinical isolates of A. baumannii
To determine whether it has intrinsic antibacterial activity, ANT3310 was tested alone against 300 OXA-or KPC-positive Enterobacterales, 502 P. aeruginosa, and 405 A. baumannii clinical isolates.No clinically significant antibacterial activity was observed for ANT3310, with MIC 50/90 values of >64/>64 µg/mL against Enterobacterales and P. aeruginosa and 64/>64 µg/mL against A. baumannii (Table2).Only 6 of the 1,207 isolates tested had ANT3310 MICs ≤8 µg/mL.This confirms that the role of ANT3310 in the MEM-ANT3310 combination is to potentiate MEM activity to clinically relevant levels.

TABLE 2
In vitro susceptibility data for ANT3310 against Enterobacterales, A. baumannii, and P.

TABLE 3
In vitro susceptibility data for MEM, MEM-ANT3310, and comparator antibiotics against A. baumannii and CRAB isolates from 2018 and 2019 global collections

TABLE 5
Summary MIC and susceptibility data for MEM, MEM-ANT3310, and relevant comparators, against 180 KPC-producing Enterobacterales b For comparative purposes only, percent susceptible and percent resistant for MEM-ANT3310 correspond to the percentage of isolates inhibited at ≤8 µg/mL and ≥16 µg/mL, respectively.

TABLE 6
In vitro susceptibility data for MEM, MEM-ANT3310, and comparator antibiotics against 502 P. aeruginosa a % of isolates at MIC 50 and MIC 90 values have been highlighted in light and dark gray, respectively. a