Class A KPC-2 (IC50 = 4 nM); Class C AmpC (IC50 = 14 nM); D OXA-24 (IC50 = 190 nM)

Durlobactam Is a More Potent and Efficient BlaC Inhibitor Compared to the Other DBOs Avibactam and Relebactam; Durlobactam Is a Potent Inhibitor of Several Peptidoglycan Transpeptidases of Mtb; Durlobactam Restores the Susceptibility of M. tuberculosis Isolates to β-Lactams [3].

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ETX2514 is an antiotic with intrinsic antibacterial activity, and can enhance its ability to restore carbapenem activity against CRE strains. Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of β-lactamases, enzymes that inactivate β-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new β-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine β-lactamase inhibitors that potently inhibit clinically relevant class A, C and D β-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of β-lactam activity in a broad range of MDR Gram-negative pathogens. One of the most promising combinations is sulbactam–ETX2514, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need [1].

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Combinations of the β-lactam/β-lactamase inhibitor sulbactam-durlobactam and seventeen antimicrobial agents were tested against strains of Acinetobacter baumannii in checkerboard assays. Most combinations resulted in indifference with no instances of antagonism. These results suggest sulbactam-durlobactam antibacterial activity against A. baumannii is unlikely to be affected if co-dosed with other antimicrobial agents [2].

Sulbactam–ETX2514 exhibited in vivo efficacy in MDR A. baumannii infection mouse models [1].

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In vivo neutropenic lung and thigh infection model studies with sulbactam alone [4]
In thigh studies with sulbactam alone vs. A. baumannii ARC2058, %fT>MIC magnitudes associated with 1-log10 CFU/g reduction, 2-log10 CFU/g reduction, and the EC80 were 20.5, 31.5, and 47.0, respectively (Table 3). In the lung model, the mean %fT>MIC magnitudes associated with 1-log10 CFU/g reduction, 2-log10 CFU/g reduction, and the EC80 were 37.8, 50.1, and 68.5, respectively.
In vivo neutropenic thigh and lung infection model studies with sulbactam in combination with durlobactam vs. CRAB strains [4]
Individual strain %fT>MIC estimates for sulbactam to achieve PK/PD endpoints of 1-log10 CFU/g reduction, 2-log10 CFU/g reduction, and the EC80 vs. CRAB strains are summarized in Table 3 for thigh and lung infection models utilizing a 4:1 dose titration of sulbactam:durlobactam. Co-modeling of the %fT>MIC sulbactam exposure response data (when administered in combination with durlobactam) across multiple CRAB strains and the sulbactam susceptible strain ARC2058 is shown in Fig. 1 for thigh and lung models. Sulbactam %fT>MIC magnitudes associated with 1-log10 CFU/g reduction, 2-log10 CFU/g reduction, and the EC80 of the co-modeled data are summarized in Table 3. Magnitudes of %fT>MIC were required for 1-log10, and 2-log10 CFU reduction was nearly identical between the mean of the individual PK/PD endpoints determined across all the strains compared with the PK/PD endpoints determined from co-modeling the data.

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Sulbactam-durlobactam is a β-lactam/β-lactamase inhibitor combination currently in development for the treatment of infections caused by Acinetobacter, including multidrug-resistant (MDR) isolates. Although sulbactam is a β-lactamase inhibitor of a subset of Ambler class A enzymes, it also demonstrates intrinsic antibacterial activity against a limited number of bacterial species, including Acinetobacter, and has been used effectively in the treatment of susceptible Acinetobacter-associated infections. Increasing prevalence of β-lactamase–mediated resistance, however, has eroded the effectiveness of sulbactam in the treatment of this pathogen. Durlobactam is a rationally designed β-lactamase inhibitor within the diazabicyclooctane (DBO) class. The compound demonstrates a broad spectrum of inhibition of serine β-lactamase activity with particularly potent activity against class D enzymes, an attribute which differentiates it from other DBO inhibitors. When combined with sulbactam, durlobactam effectively restores the susceptibility of resistant isolates through β-lactamase inhibition. The present review describes the pharmacokinetic/pharmacodynamic (PK/PD) relationship associated with the activity of sulbactam and durlobactam established in nonclinical infection models with MDR Acinetobacter baumannii isolates. This information aids in the determination of PK/PD targets for efficacy, which can be used to forecast efficacious dose regimens of the combination in humans.[5]

Inhibition Kinetics of PonA1 and Ldts[3]
The reaction scheme of eq 1 and the Henri–Michaelis–Menten equation (eq 2) were also applied to characterize the inhibition kinetic values for PonA1 and the Ldts of Mtb. Kinetic assays with purified enzyme were performed using a BioTek Synergy2 Multi-Mode Microplate Reader and Gen5 analysis software with a 0.25 cm path at 30 °C. Reactions were conducted in 50 mM Tris-HCl (pH 7.5) and 300 mM sodium chloride, and nitrocefin was the reporter substrate for all assays. To obtain KmNCF for each enzyme, fixed concentration of enzyme with increasing concentrations of nitrocefin were monitored for the change in absorbance at λ of 482 nm over 15 min. The data was fitted by nonlinear least-squares fit to eq 2 using Origin 8.1. Subsequently, KI app was determined for each enzyme–inhibitor combination using a fixed concentration of enzyme with nitrocefin concentration of 5 × KmNCF and increasing concentrations of inhibitor. Changes in absorbance at λ of 482 nm of each reaction were measured over 30 min. Dixon plots of 1/Δabsorbance vs [inhibitor] linearized data and corrected KI app were determined using eq 3 as with BlaC.

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Inhibition Kinetics of BlaC[3]
Inhibition kinetics were determined with purified BlaC enzyme as previously described. In brief, the reaction scheme is represented in eq 1, where E is the enzyme, S is the substrate, E:S is the Michaelis–Menten complex, E–S is the acyl-enzyme complex, and P is the reaction product. Kinetic parameters for BlaC were determined using an Agilent 8453 diode array spectrophotometer). Reactions were conducted in 100 mM morpholineethanesulfonic acid (MES) (pH 6.4) at room temperature in a 1 cm-path-length cuvette. First, the parameters of nitrocefin (Δε = 17 400 M–1 cm–1) hydrolysis by BlaC were determined. KmNCF, the Michaelis constant of nitrocefin is the concentration of nitrocefin, [S], where the reaction rate, v, is equal to half the maximal reaction rate, Vmax. Initial reaction rates were measured for increasing concentrations of nitrocefin mixed with 0.28 μg/mL BlaC, and nonlinear least-squares fit of the data (Henri–Michaelis–Menten equation) was performed using Origin 8.1 according to eq 2...

In Vitro Susceptibility Testing[3]
ATCC Mtb H37Ra, H37Rv, and nine contemporary clinical Mtb isolates were subjected to antimicrobial susceptibility testing by broth microdilution. Antibiotic compounds were purchased from commercial sources except for durlobactam, which was generously provided by Entasis Therapeutics. Middlebrook 7H9 broth supplemented with 10% (v/v) oleic albumin dextrose catalase (OADC), 0.05% (v/v) Tween 80, and 0.5% (v/v) glycerol served as the culture media. Serial 2-fold dilutions of drugs were performed using a 96-well microplate. For combinations, clavulanate was added in a fixed concentration of 2.5 μg/mL; all other combinations (β-lactam/durlobactam or dual β-lactam) were combined in a 1:1 mass/vol ratio. Microplate wells were inoculated with approximately 5 × 105 colony-forming units (CFU)/mL. Following 14–18 days of incubation at 37 °C, the lowest antibiotic concentration of drug that prevented visible growth was recorded as the MIC. Visual growth was confirmed with the resazurin-based reagent alamarBlue HS.

In vivo neutropenic lung and thigh infection models [4]
In vivo infection models of pneumonia and thigh tissue abscess were performed in CD-1 mice (15–18 g) rendered neutropenic by cyclophosphamide treatment prior to infection as previously described. In the lung model, mice were infected with A. baumannii isolates in 1% agar slurry via direct intratracheal instillation. In the thigh model, mice were infected intramuscularly in the right thigh. Infected mice were treated with either sulbactam alone or sulbactam combined with durlobactam at a constant 4:1 ratio. Dosing was initiated 2 hours after infection and administered as eight subcutaneous (SC) injections administered q3h or four SC injections administered q6h. Vehicle groups received a single SC injection, and positive control groups received either a single SC injection of colistin sulphate (maximum tolerated dose of 40 mg/kg) or two oral doses of levofloxacin (200 mg/kg bid) 2 hours after infection. Colistin sulphate administered as a single 40-mg/kg dose achieved 0.25 to 2.6 log10 CFU/g reduction vs. MDR A. baumannii strains, and levofloxacin achieved −2.4 log10 CFU vs. A. baumannii ARC2058 in the thigh model. In the lung model, colistin administration resulted in 1-log10 CFU/g growth to 2.1 log10 CFU/g reduction vs. MDR A. baumannii strains and levofloxacin administration resulted in 1.8 log10 CFU/g reduction vs. A. baumannii ARC2058. The overall performance of positive controls was acceptable relative to isolate susceptibility. Efficacy was determined by harvesting infected tissue and determining viable bacterial counts 24 hours after the start of treatment. A full dose response study utilized meropenem to validate the model system, where meropenem was administered SC on a q6h regimen from 1 to 300 mg/kg following uranyl nitrate pre-treatment to extend drug exposure. Subcutaneous doses of sulbactam and durlobactam were administered at a 4:1 ratio at doses of 20:5, 40:10, 60:15, 80:20, and 160:40 mg/kg (SUL:DUR) with plasma PK sampling (n = 3 samples/timepoint) at 0.5, 1, 2, 3, 4, 6, 8, 10, and 12 hours post dose. These studies were completed in neutropenic CD-1 mice with active thigh infections. An additional PK and ELF distribution study was completed at a SUL:DUR dose of 100:25 mg/kg in neutropenic lung-infected animals with PK timepoints sampled at 0, 1, 3, 6, and 12 hours post dose. Blood was processed for plasma using microcontainer tubes containing ethylenediamine tetraacetic acid (EDTA, Beckton Dickenson) and centrifugation for 5 minutes at 13,200 rpm. Plasma samples were treated 1:1 with a SigmaFAST protease inhibitor cocktail prepared from one tablet dissolved in 10 mL of deionized water. The samples were then stored at −80°C until bioanalysis.

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Pharmacokinetic-pharmacodynamic analysis[4]
As the plasma PK of neutropenic lung-infected animals matched that of thigh-infected animals, the thigh-infected PK was used for the development of the population PK model and subsequent exposure estimates for doses used in the PK/PD exposure-response analyses. Pharmacokinetic models were fit to time vs. drug concentration profiles generated in infected mice using Phoenix6.2 WinNonLin and NLME. For the purposes of estimating exposures across all doses used in dose response studies, population PK models were developed for sulbactam alone, for sulbactam when co-administered with durlobactam, and for durlobactam co-administered with sulbactam (dose ratio 4:1, sulbactam:durlobactam). Population PK parameter estimates were derived from a two-compartment PK model incorporating a log-additive error model constructed from fitting of sulbactam and durlobactam concentration vs. time data (Table S4). Representative model fit concentration vs. time profiles vs. observed data are shown in Fig. S1 for sulbactam at 320 and 40 mg/kg and durlobactam at 80 and 10 mg/kg. Predicted exposures were utilized in the PK/PD analyses as all dosing solutions tested were within the variability of the bioanalytical method (20%).
For pharmacokinetic-pharmacodynamic evaluation sulbactam %fT>MIC and durlobactam fAUC0-24/MIC were simulated for each dose using the population PK model. A Hill-type model was fit to the pharmacodynamic data generated from the dose response studies with linear least squares regression analysis of the relationships between change in log10 CFU from baseline at 24 hours and sulbactam %fT>MIC and durlobactam fAUC/MIC. Magnitudes associated with 1-log10 and 2-log10 CFU reduction from baseline at 24 hours as well as the exposure required for an 80% reduction in bacterial burden (EC80) were determined from the fitted function.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Sulbactam produces low levels in milk that are not expected to cause adverse effects in breastfed infants. It is likely that durlobactam produces similar levels in milk. Occasionally, disruption of the infant's gastrointestinal flora, resulting in diarrhea or thrush, have been reported with penicillins, but these effects have not been adequately evaluated. Sulbactam-durlobactam is acceptable in nursing mothers.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

[1]ETX2514 is a broad-spectrum \u03b2-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria includingAcinetobacter baumannii. Nat Microbiol. 2017 Jun 30;2:17104.

[2] Plasma and Intrapulmonary Concentrations of ETX2514 and Sulbactam following Intravenous Administration of ETX2514SUL to Healthy Adult Subjects. Antimicrob Agents Chemother. 2018 Aug 20. pii: AAC.01089-18.

[2]. In vitro antibacterial activity of sulbactam-durlobactam in combination with other antimicrobial agents against Acinetobacter baumannii-calcoaceticus complex. Diagn Microbiol Infect Dis . 2024 May 9;109(3):116344.
[3]. Durlobactam, a Diazabicyclooctane β-Lactamase Inhibitor, Inhibits BlaC and Peptidoglycan Transpeptidases of Mycobacterium tubercul. ACS Infect Dis . 2024 May 10;10(5):1767-1779.
[4]. In vivo dose response and efficacy of the β-lactamase inhibitor, durlobactam, in combination with sulbactam against the Acinetobacter baumannii-calcoaceticus complex. Antimicrob Agents Chemother. 2024 Jan; 68(1): e00800-23.
[5]. he Pharmacokinetics/Pharmacodynamic Relationship of Durlobactam in Combination With Sulbactam in In Vitro and In Vivo Infection Model Systems Versus Acinetobacter baumannii-calcoaceticus Complex. Clin Infect Dis . 2023 May 1;76(Suppl 2):S202-S209.

Durlobactam sodium is an organic sodium salt that is the monosodium salt of durlobactam. It has a role as an EC 3.5.2.6 (beta-lactamase) inhibitor and an antibacterial drug. It contains a durlobactam(1-).
See also: Durlobactam (has active moiety) ... View More ...

C8H10N3NAO6S

299.2363

277.04

C, 32.11; H, 3.37; N, 14.04; Na, 7.68; O, 32.08; S, 10.71

1467157-21-6

1467829-71-5 (free acid);1467157-21-6 (sodium);

89851851

White to light yellow solid

1

6

3

19

541

2

CC1=C[C@@H]2CN([C@@H]1C(=O)N)C(=O)N2OS(=O)(=O)[O-].[Na+]

WHHNOICWPZIYKI-IBTYICNHSA-M

InChI=1S/C8H11N3O6S.Na/c1-4-2-5-3-10(6(4)7(9)12)8(13)11(5)17-18(14,15)16/h2,5-6H,3H2,1H3,(H2,9,12)(H,14,15,16)/q+1/p-1/t5-,6+/m1./s1

sodium (2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl sulfate

ETX2514 ETX-2514 ETX 2514 ETX2514 sodium Durlobactam sodium

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ~250 mg/mL (835 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)