Durlobactam in the Treatment of Multidrug-Resistant Acinetobacter baumannii Infections: A Systematic Review

A. baumannii is a frequent cause of difficult-to-treat healthcare-associated infections. The use of a novel beta-lactamase inhibitor, durlobactam, has been proposed against multidrug-resistant A. baumannii. A systematic review of studies assessing the efficacy and safety of durlobactam in the treatment of multidrug-resistant A. baumannii infections was carried out. The study protocol was pre-registered on PROSPERO (CRD42022311723). Published articles on durlobactam were identified through computerized literature searches with the search terms “durlobactam” and “ETX2514” using PubMed. PubMed was searched until 15 February 2022. Articles providing data on the main characteristics of durlobactam and on the efficacy and safety of durlobactam in the treatment of A. baumannii infections were included in this systematic review. Attempt was made to obtain information about unpublished studies. English language restriction was applied. The risk of bias in the included studies was not assessed. Both quantitative and qualitative information were summarized by means of textual descriptions. Thirty studies on durlobactam were identified, published from June 2017 to November 2020. Sixteen studies met the inclusion criteria. Durlobactam is effective against A. baumannii when used in combination with sulbactam. Future clinical trials are needed to confirm the possibility to treat infections caused by multidrug-resistant A. baumannii with this combination.


Introduction
Worldwide, the rise of multidrug-resistant (MDR) bacteria is an increasing threat to human health [1,2]. Among the most worrisome MDR bacteria, the World Health Organization (WHO) recently recognized Acinetobacter baumannii as a critical pathogen, frequently causing healthcare-associated infections (www.who.int accessed on 1 May 2022).
A. baumannii is a ubiquitous, non-fermenting, rod-shaped Gram-negative coccobacillus. It can be considered an opportunistic pathogen, because predominantly affects immunecompromised and critically ill patients [3,4]. In this particularly frail population, A. baumannii can cause ventilator-associated pneumonia and bloodstream infections, with an overall reported mortality up to 40% [5,6]. A. baumannii possesses the pernicious, innate ability to evade the commonly used antibiotic therapy.
Generally, A. baumannii resistance mechanisms of intrinsic and acquired antibiotic resistance are categorized into three groups. First, resistance can be achieved by increasing efflux of the antibiotic from the bacteria and thus preventing access to the target, i.e., overexpression of drug efflux pumps. Second, A. baumannii can protect its antibiotic target through genetic mutations or post-translational modifications, i.e., mutations in antibiotic binding targets via genetic insertion sequences. Third, antibiotics can be directly inactivated by The study protocol was pre-registered on PROSPERO (CRD42022311723) (Tables S1 and S2). Published articles (from June 2017 to November 2020) assessing the main characteristics of durlobactam and the efficacy and safety of durlobactam in the treatment of A. baumannii infections were identified through computerized literature searches using MEDLINE (National Library of Medicine Bethesda MD) and by reviewing the references of retrieved articles. PubMed was searched until 15 February 2022. Combinations of the following search terms were applied: ((durlobactam) OR (ETX2514)). Attempt was made to obtain information about unpublished studies. English language restriction was applied. Studies published only in abstract form, correction articles, reviews, case-reports, editorials, guidance articles or guidelines and clinical trial protocols were excluded from further assessment. Reviewed articles were maintained in a master log and any reason for exclusion from analysis was documented in the rejected log.

Eligibility Criteria
Studies of any design which reported data on durlobactam were eligible for inclusion in this systematic review.

Study Selection and Data Extraction
Eligibility assessment and extraction of data were performed independently by two investigators. Each investigator was blinded to the other investigator's data extraction. In case of disagreement between the two reviewers, a third reviewer was consulted. Data from each study were verified for consistency and accuracy, and then entered into a computerized database. Abstracted information included: author, year of publication, country in which the study was conducted; study design, start and end date of study, health-care setting, sample size; criteria for the diagnosis of bacterial infection, if applicable; proportion of animals or patients receiving antibiotics, if applicable; minimum inhibitory concentration; pharmacokinetics and pharmacodynamics data; animals or patients' outcome data. For the syntheses, studies were grouped in two groups. Group I: in vitro studies on general characteristics and pharmacokinetic of durlobactam and on the activity of durlobactam against A. baumannii isolates. Group II: clinical studies on the efficacy of durlobactam against infections due to A. baumannii.

Data Synthesis
Both quantitative and qualitative information were summarized by means of textual descriptions.

Assessment of Bias
A formal assessment for risk of bias was deemed to have limited utility given the lack of an appropriate assessment tool. Although a risk-of-bias tool has been developed for systematic reviews, many aspects of the tool are not directly relevant to our research question. Figure 1 shows the selection process of studies included in the systematic review. Through a PubMed search with the search terms "durlobactam" and "ETX2514", we identified 29 studies published from June 2017 to November 2020. Among the 29, five studies that represented review articles, four studies that did not report data on durlobactam, one study representing a "correction article", one representing an "expert opinion article", and two representing case reports were excluded. Of the 16 studies included in this systematic review ( Figure 1) [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28], six were clinical trials: four phase I trials [16,17,19,27], one phase II clinical trial [18] and one ongoing phase III clinical trial [28]. applicable; proportion of animals or patients receiving antibiotics, if applicable; minimum inhibitory concentration; pharmacokinetics and pharmacodynamics data; animals or patients' outcome data. For the syntheses, studies were grouped in two groups. Group I: in vitro studies on general characteristics and pharmacokinetic of durlobactam and on the activity of durlobactam against A. baumannii isolates. Group II: clinical studies on the efficacy of durlobactam against infections due to A. baumannii.

Data Synthesis
Both quantitative and qualitative information were summarized by means of textual descriptions.

Assessment of Bias
A formal assessment for risk of bias was deemed to have limited utility given the lack of an appropriate assessment tool. Although a risk-of-bias tool has been developed for systematic reviews, many aspects of the tool are not directly relevant to our research question. Figure 1 shows the selection process of studies included in the systematic review. Through a PubMed search with the search terms "durlobactam" and "ETX2514", we identified 29 studies published from June 2017 to November 2020. Among the 29, five studies that represented review articles, four studies that did not report data on durlobactam, one study representing a "correction article", one representing an "expert opinion article", and two representing case reports were excluded. Of the 16 studies included in this systematic review ( Figure 1) [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28], six were clinical trials: four phase I trials [16,17,19,27], one phase II clinical trial [18] and one ongoing phase III clinical trial [28].  Of the 16 studies included in the systematic review, in vitro studies and phase I and phase II studies produce data on general characteristics, pharmacokinetic and pharmacodynamic of durlobactam; seven studies assess the in vitro activity of durlobactam against A. baumannii from patients; one was an ongoing phase III trial study on the efficacy of durlobactam against A. baumannii. The included studies were separated in two different tables with a summary description of their characteristics: Table 1, in vitro studies and  Table 2, Phase I, phase II or phase III studies.

Durlobactam Molecular Structure and Pharmacodynamics
Durlobactam, formerly known as ETX2514, is a novel beta-lactamase inhibitor belonging to the diazabicyclooctanone, boronic acid and pyridine-2-carboxylic acid classes {[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo [3.2.1]oct-3-en-6-yl] hydrogen sul-fate} [13]. Durlobactam molecular structure differs from other beta-lactamases for its endocyclic double bond and its methyl substituent (Figures 2 and 3). Durlobactam is a polar compound, therefore it is able to penetrate into Gram-negative cells through outer membrane porins, i.e., OmpA [26]. Once into the bacterial cell, durlobactam is carbamoylated on its active, nucleophile serine site. This covalent bond is reversible because the sulfated amine is able to recyclize onto the carbamate [15]. These molecular characteristics confer a favorable binding kinetic and extend durlobactam inhibitory effect to a broad range of beta-lactamases, including Amber class A, C and D beta-lactamases [15]. Durlobactam is able to bind penicillin-binding proteins in a rapid and reversible manner, resulting in an efficient and long-lasting inhibition [15].

Durlobactam Pharmacokinetics
Durlobactam is suitable for intravenous administration, with stability for more than 6 h at room temperature and a water solubility higher than 200 mg/mL [13].
The pharmacokinetics and pharmacodynamics of durlobactam in combination with sulbactam have been studied in a phase I trial enrolling 30 healthy adults (ClinicalTrials registration no. NCT03303924) [17].

Durlobactam Pharmacokinetics
Durlobactam is suitable for intravenous administration, with stability for more than 6 h at room temperature and a water solubility higher than 200 mg/mL [13].
The pharmacokinetics and pharmacodynamics of durlobactam in combination with sulbactam have been studied in a phase I trial enrolling 30 healthy adults (ClinicalTrials registration no. NCT03303924) [17].
In this trial, 1 g of durlobactam with 1 g of sulbactam were infused as 3 h intravenous infusion every 6 h, for three consecutive doses. At these regimens, the durlobactam/sulbactam combination was safe and generally well tolerated. In this study the concentration of durlobactam and sulbactam were determined in plasma and epithelial lining fluid. Generally, the total plasma concentrations of each agent resulted higher than those observed in the epithelial lining fluid. The times of the observed maximum plasma concentration (Tmax) were similar for durlobactam and sulbactam, with arithmetic means of 2.62 and 2.56 h, respectively. The means of the minimum plasma concentrations (C min) of durlobactam at 6 h after the first, second, and third infusions were 5.71 ± 1.77, 6.63 ± 1.97, and 5.79 ± 2.08 mg/L, respectively [17].
Of note, following the third infusion, durlobactam showed values of area under the concentration-time curves from 0 to 6 h (AUC0-6) of 109.05 ± 23.44 mg/h/L, the half-life of 1.40 ± 0.18 h and a volume of distribution at steady state of 16.7 ± 3.0 L [17].
Moreover, regarding durlobactam distribution to the epithelial fluid, the AUC0-6 based on mean epithelial lining fluid concentrations resulted 40.1 mg/h/L. The ratio of epithelial lining fluid to total plasma durlobactam concentrations based on the mean AUC0-6 values was 0.37. These results support the use of the durlobactam/sulbactam combination in the treatment of lower respiratory tract bacterial infections [17].
A subsequent, phase II clinical trial performed on 80 patients with complicated urinary tract infection confirmed these findings. In this study, patients were randomized to receive imipenem plus the combination of durlobactam/sulbactam at a regimen of 1 plus 1 g, infused over 3 h every 6 h for 7 days (n: 53), or imipenem plus placebo (n: 27) [18]. The durlobactam/sulbactam combination was generally well tolerated, with 37.7% patients reporting drug-related moderate adverse events such as headache, diarrhea, nausea, and phlebitis. One patient had self-limiting urticaria on day 3 of administration. In one patient, renal function decrease was observed on day 3 of administration. In this phase II trial, durlobactam and sulbactam mean elimination half-lives were of 2.2 and 1.6 h, respectively. The mean steady-state clearance and volume of distribution of durlobactam were 10.3 L/h and 31.6 L, respectively. These values were similar to the mean clearance and volume of distribution estimates for sulbactam (13.4 L/h and 36.0 L, respectively) [18]. On these findings, the authors proposed a durlobactam/sulbactam dose of 1 g of each component to be administered every 6 h via a 3 h infusion to achieve the optimal concentrations [18].
To consider, although the urinary excretion of durlobactam over 48 h is only 66% of the systemic clearance, durlobactam exposure doubled in patients with renal impairment with creatinine clearance lower than 30 mL/min/1.73 m 2 [21]. Therefore, changes in durlobactam pharmacokinetics should be considered in patients with severely compromised renal function and hemodialysis should be considered as a factor that can remove nearly half a dose of durlobactam [19].

In Vitro Studies on the Activity of Durlobactam/Sulbactam against A. baumannii Durlobactam and Sulbactam Synergistic Bactericidal Activity
The sole durlobactam possesses minimal antibacterial activity against A. baumannii, mainly due to durlobactam ability to bind A. baumannii PBPs [13].
However, in the last decades, MDR A. baumannii strains emerged with a reduced expression of PBP2 and produced beta-lactamases able to degrade sulbactam, such as TEM-1, ADC-30, AmpC and a number of OXAs. Unfortunately, these resistance mechanisms narrowed the clinical utility of the sole sulbactam in MDR A. baumannii infections [23,25].
Therefore, considering that the sole durlobactam does not have significant activity against A. baumannii, its use in combination with sulbactam has been proposed. Exploiting inhibition of beta-lactamases by durlobactam, sulbactam is free to exert its intrinsic A. baumannii bactericidal activity by binding PBPs [23,25].
To date, the frequency of A. baumannii spontaneous resistance to the durlobactam/sulbactam combination seems low, even if resistance has been recently reported due to the metallobeta-lactamase NDM−1 or PBP3 substitutions [20].
In a recent study, the in vitro antibacterial activities of the durlobactam/sulbactam combination were assessed by broth microdilution against 1722 A. baumannii clinical isolates, collected across the five continents in 2016 and 2017 [14]. Over 50% of these samples resulted resistant to carbapenems. Against this strain collection, durlobactam/sulbactam showed MIC 50 and MIC 90 values of 1 and 2 g/mL, respectively. These MICs were lower than that observed for the sole sulbactam, with a MIC 50 of 8 g/mL and a MIC 90 of 64 g/mL. This level of activity was found to be consistent across regions, sources of infection, and subsets of resistance phenotypes, including MDR isolates. Of note, in this study colistin was the only traditional antimicrobial with activity similar to durlobactam/sulbactam. Moreover, genome sequencing of the 39 specimens (2.3%) with a durlobactam/sulbactam MIC of 4 g/mL confirmed that these strains encoded either the metallo-beta-lactamase NDM-1, not inhibited by durlobactam, or single amino acid substitutions near the active site of PBP3, i.e., the primary target of sulbactam [14].
Similar results were reported in a study on 246 patients infected by A. baumannii collected between 2012 and 2016 from 94 hospitals in 37 different countries. Antimicrobial susceptibility testing was performed by broth microdilution. The observed durlobactam/sulbactam MIC 50 and MIC 90 values were 0.25 and 0.5 mg/L, respectively. Sulbactam MIC was lowered 16-fold to 64-fold by the addition of durlobactam. Conversely, the addition of imipenem to the durlobactam/sulbactam combination did not improve activity, with only nine isolates showing higher MIC. These samples encoded the metallo-beta-lactamase NDM-1 [21].
In an in vitro study on 982 samples of A. baumannii infection in China between 2016 and 2018, the observed MIC 90 of durlobactam/sulbactam was 2 mg/L [22]. In this study, 84.6% of the collected strains was imipenem resistant. The authors of this study propose a concentration of 4 mg/L as a reasonable preliminary cut-off for evaluation of durlobactam/sulbactam susceptibility in A. baumannii.
Moreover, a recent study used whole-genome sequencing and molecular characterization to relate the presence of carbapenemase genes in 28 A. baumannii clinical isolates from India. The authors found that 93% of the collected samples expressed carbapenemases, including OXA-23, OXA-58 and NDM genes, with over a third expressing dual carbapenemase genes. The presence of these carbapenemase genes resulted in sulbactam resistance (MIC: 16-256 mg/L) in all of the studied isolates [23].
The authors then assessed the efficacy of durlobactam against these strains through in silico intermolecular interaction analysis. Several nonsynonymous single nucleotide polymorphisms were identified in PBP2 and PBP3 sequences, but minimal variations were recorded in the protein backbone dynamics in active-site motifs of wild-type and mutants, which correlated with negligible binding energy fluctuations for the PBP2-durlobactam complex. The authors suggested that the stable interaction profiles of durlobactam with carbapenemases can possibly restore sulbactam activity against both wild type and PBP mutant examinee [23].
Finally, a recent study evaluated the antimicrobial activity of durlobactam/sulbactam against a collection of 112 Brazilian MDR A. baumannii patients [24]. The in vitro activity of durlobactam/sulbactam was evaluated by the broth microdilution method using durlobactam at a fixed concentration of 4 mg/L. In this study, the samples presented a variety of beta-lactamases encoding genes, including several OXAs. Despite the high resistance rates to most antimicrobial agents tested, the authors reported in vitro activity of durlobactam/sulbactam, with MIC 90 values of 4 mg/L [24].

In Vivo Studies on the Efficacy of Durlobactam/Sulbactam to Treat A. baumannii Infections
A study on the efficacy of durlobactam/sulbactam against MDR A. baumannii was performed in the animal model. In thigh and lung murine infection models, durlobactam/sulbactam showed a dose dependent reduction in A. baumannii counts. Bactericidal activity of durlobactam/sulbactam greater than one-log kill was achieved when sulbactam concentrations exceeded the combination MIC of 0.5 mg/L. In this study, no bactericidal activity was observed when sulbactam was administered alone, at the dosage of 15 mg/kg every three hours. The addition of durlobactam to sulbactam increased its activity in a dose-dependent manner [25].
A phase II trial evaluated the tolerability and pharmacokinetic of durlobactam/sulbactam in patients with complicated urinary tract infections, including acute pyelonephritis. In this study all patients received background therapy with imipenem, in addition to either durlobactam/sulbactam or placebo. The microbiological intent-to-treat population were similar in the two groups, 36 (76.6%) and 17 (81.0%) patients in the durlobactam/sulbactam and in the placebo group, respectively [18].
Currently, a phase III trial, i.e., the ATTACK trial, is evaluating the efficacy and safety of the durlobactam/sulbactam combination in patients with bloodstream infections or hospital-associated bacterial pneumonia due to A. baumannii-calcoaceticus complex (NCT03894046) [28]. The first part of this multinational, pathogen-targeted, randomized, active-controlled, comparator-controlled trial compared two treatment arms: durlobactam/sulbactam (1 g/1 g qid) plus imipenem/cilastin (1 g/1 g qid) versus colistin (2.5 mg/kg bid) plus imipenem/cilastin (1 g/1 g qid). This trial concluded the patients' recruitment, with a total of 207 enrolled patients, and recently an update on the study results has been released [28].

Discussion
Globally, severe infections due to MDR A. baumannii give rise to high mortality rates and remain a great challenge for clinicians [6,7].
Among the reasons to explain this increased mortality, there are the potential toxicity and the suboptimal pharmacokinetics and efficacy of the currently available therapeutic approaches [29,30].
During the last decades, polymyxins represented the most used antimicrobial options against MDR A. baumannii. Polymyxins have been considered as a "last resort" to fight MDR infections, often representing the only antimicrobial to achieve adequate serum levels and MICs. Therefore, the reports of colistin-resistant A. baumannii isolates raises concern, considering the further limitations of antimicrobial options and the high mortality rate associated with these infections [4].
The clinical studies demonstrated that durlobactam is safe and generally well tolerated when administered in combination with sulbactam [16,27]. Durlobactam showed a half-life of 1.40 ± 0.18 h and a mean steady-state clearance and volume of distribution of 10.3 L/h and 31.6 L, respectively [17,18]. The predominant clearance mechanism is through renal excretion [16]. Decreasing renal function increases peak plasma concentration and AUC in a generally linear manner and hemodialysis is effective at removing both durlobactam and sulbactam from plasma [19]. In an ongoing phase III clinical trial comparing durlobactam/sulbactam and colistin for the treatment of carbapenem-resistant A. baumannii infection, durlobactam/sulbactam met the primary efficacy endpoint of all-cause mortality at 28 days (durlobactam/sulbactam mortality was 19.0% versus 32.3% of the colistin arm, treatment difference of −13.2%; 95% CI: −30.0, 3.5) [28].
In our systematic review, we included 16 studies dealing with the possible use of durlobactam in the treatment of MDR A. baumannii infections. The included studies were extremely heterogeneous, being studies performed in different settings with different designs.
Moreover, we could identify only one phase III clinical trial, providing data on the efficacy of durlobactam and sulbactam in the treatment of infections due to A. baumannii.
The results coming from the first studies evaluating the use of durlobactam against A. baumannii placed great hope in this novel beta-lactam inhibitor. The combination of durlobactam and sulbactam shows excellent in vitro potency against A. baumannii isolates, including MDR isolates that were resistant to carbapenems, aminoglycosides, tetracyclines and polymyxins.
Moreover, durlobactam pharmacokinetic and pharmacodynamic profiles suggest that it may be considered in the treatment of the most common A. baumanni infections, such as blood stream infections and pneumonia.
To consider, among the weaknesses of this novel compound, durlobactam does not inhibit class B beta-lactamases, i.e., NDM-1. This may halt durlobactam future usefulness, although fortunately the most recent surveillance studies report that infection of A. baumannii producing B metallo-beta-lactamases are globally rare [20].
Another factor that may halt the benefit of durlobactam/sulbactam use against infection due to A. baumannii is the potential development of resistance.

Conclusions
Certainly, data from randomized clinical trials will be required to confirm the effectiveness of durlobactam/sulbactam to treat infections caused by A. baumannii. In this regard, we are awaiting with interest the definitive results from the ongoing phase III open label clinical trial ATTACK (Acinetobacter Treatment Trial Against Colistin; NCT03894046). In the ATTACK trial, patients were randomized to receive either durlobactam/sulbactam plus imipenem/cilastatin or colistin plus imipenem/cilastatin. Probably, this therapy regimen is more reflective of real-world practice with MDR A. baumannii than monotherapy regimens employed in previous trials. However, this may lead to difficulty in interpreting the stand-alone efficacy of the durlobactam/sulbactam combination.
Positive results from this ongoing trial and from future clinical trials would enable again the possibility to treat infections caused by MDR A. baumannii with this unusual, dual beta-lactamase inhibitor combination.
Funding: This research received no external funding.