β-Lactamases

Xeruborbactam is a strong carbapenemase class D inhibitor of Acinetobacter baumannii [1]. The activity of Pseudomonas aeruginosa's principal MDR efflux pump had no effect on Xeruborbactam, which is a major advancement over Vaborbactam, a boronic acid beta-lactamase inhibitor (BLI) from the previous generation [1]. Xeruborbactam is a multi-lactam antibiotic that is useful when combined with lactam antibiotics due to its extended-spectrum β-lactamase inhibitory properties and enhanced activity of multiple β-lactam antibiotics with differential susceptibility to intrinsic resistance mechanisms of efflux and permeability [1].

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QPX7728 (compound 35) displays a remarkably broad spectrum of inhibition, including class B and class D enzymes, and is little affected by porin modifications and efflux [2].

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Biochemical Evaluation [2]
QPX7728 displays “slow-binding” kinetics with serine β-lactamases, whereas it exhibits “fast on–fast off” kinetics with metalloenzymes. Inhibition constants (Ki) for various β-lactamases purified from overexpressing recombinant E. coli strains, using nitrocefin (NCF) or imipenem (IMI) as substrate, are shown in Table 11. Ki values were <100 nM for all enzymes except IMP-1. Compound 35 was clearly superior to the comparators vaborbactam and avibactam against the class D enzymes OXA-48 and OXA-23 of Acinetobacter baumannii and against the class B metalloenzymes NDM-1, VIM-1, and IMP-1.

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Microbiology [2]
QPX7728/Compound 35 (at a fixed concentration of 8 μg/mL) was evaluated in combination with cefepime, ceftolozane, and meropenem against panels of Enterobacterales, including Klebsiella pneumoniae (n = 511), E. coli (n = 297), Enterobacter cloacae complex (n = 88), and other organisms (n = 119); of these, 507 isolates expressed ESBL enzymes and 508 were carbapenem-resistant isolates. Compound 35 was also tested against carbapenem-resistant Acinetobacter baumannii (n = 503) and Pseudomonas aeruginosa (n = 500). The MIC50/MIC90 values are shown in Table 12. Comparative data with the marketed agents ceftazidime–avibactam and meropenem–vaborbactam are also shown. All three combinations with 35 were highly effective against Enterobacterales producing ESBLs as well as strains expressing the KPC and OXA-48 carbapenemases. In strains expressing metalloenzymes (NDM and VIM), cefepime and meropenem potencies were enhanced to MIC90 values of 1 and 2 μg/mL, respectively, whereas ceftolozane achieved little benefit. This is attributed to a high ceftolozane hydrolyzing activity of MBLs combined with a lower (compared to serine enzymes) inhibition of MBLs by 35. Against carbapenem-resistant A. baumannii, only meropenem potency was adequately enhanced, whereas against P. aeruginosa, all three combinations achieved an MIC90 ≤ 8 μg/mL. Studies in strains overexpressing and/or deficient in efflux pumps and porin modifications showed that 35 is largely unaffected by these common resistance mechanisms (data not shown). Interestingly, 35 displays weak antibacterial activity of its own, with a modal MIC of 16 μg/mL against Enterobacterales (range of <1 to ≥32 μg/mL); in general this activity is at much higher concentration than the potentiated MICs and therefore is unlikely to be a major contributor to the activity of the combinations. Overall, the results of these studies demonstrate the broad utility of 35 and that there may be benefit to more than one combination being available to the clinician.

Efficacy in Infection Models [2]
Compound 35 (QPX7728) was evaluated in a 24 h neutropenic mouse thigh infection model against Klebsiella pneumoniae strain KP1244 (Figure 6). This strain expresses KPC-3, SHV-11, and SHV-12 and is resistant to carbapenems, with an MIC to meropenem of >64 μg/mL; in the presence of 8 μg/mL of 35, the MIC to meropenem is 0.25 μg/mL. The doses shown of both meropenem and QPX7728 were administered every 2 h for 24 h (12 doses of the designated amount per day). In this study, meropenem at a dose of 300 mg/kg given every 2 h fails to control the infection, whereas the coadministration of 0.5–1 mg/kg of 35 affords a static effect, and 10 mg/kg achieves a >1 log reduction in CFU/thigh (relative to the start of treatment).

Determination of Ki Values of Inhibition of β-Lactamases [2]
Ki values of inhibition of β-lactamases purified from overexpressing recombinant E. coli strains were determined spectrophotometrically using nitrocefin (NCF) or imipenem as reporter substrates. Enzymes were mixed with inhibitors at varying concentrations in reaction buffer (50 mM Na-phosphate, pH 7.0, 0.1 mg/mL BSA, plus 20 μM ZnCl2 for metalloenzymes) and incubated for 10 min at 37 °C. 50 μM NCF was added, and substrate cleavage profiles were recorded at 490 nm every 10 s for 10 min on a SpectraMax plate reader for NCF. Substrate cleavage profiles were recorded at 294 nm every 30 s for 30 min on a SpectraMax plate reader for imipenem. Ki values were calculated by the method of Waley.

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Effect of QPX7728 on Serine Proteases [2]
All enzymes and substrates were from commercial sources, and testing was performed according to manufacturer’s protocols with some modifications. Briefly, 50 μL of the diluted enzyme was mixed with 50 μL of an inhibitor at various concentrations and 50 μL of a corresponding buffer (Table 15). Reaction mixtures were incubated for 10 min at 37C. Subsequently, 50 μL of corresponding substrate was added and absorbance or fluorescence was monitored for 30 min on SpectraMax M2 plate reader. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and leupeptin were used as positive controls. Rates of reaction were calculated and presented relative to “no treatment” control. IC50 values were calculated based on inhibitor concentration producing 50% of enzyme inhibition.

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Panels of engineered bacterial strains containing various combinations of porin and efflux mutations. [1]
The efflux/porin isogenic panels of K. pneumoniae, P. aeruginosa, and A. baumannii strains were constructed to evaluate the impact of various molecular determinants on the whole-cell antibiotic potentiation activity of QPX7728. The construction of a panel of isogenic KPC-3-producing strains (in which KPC-3 was carried on a naturally occurring plasmid, pKpQIL) of K. pneumoniae with various combinations of porin (ompK35 and ompK36) and efflux (acrAB-tolC) mutations was described earlier (22). The panel of isogenic KPC-2-producing strains of P. aeruginosa overexpressing or lacking MDR RND efflux pumps MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM and producing or lacking the carbapenem porin OprD was constructed by transforming plasmid pUCP24-KPC-2 into various mutants. The panel of isogenic OXA-23-producing strains of A. baumannii overexpressing MDR RND efflux pumps AdeABC and AdeIJK was constructed by conjugating the natural plasmid that carries OXA-23 from clinical isolate AB1177 into various efflux mutants.

Antimicrobial susceptibility testing. [1]
Bacterial isolates were subjected to broth microdilution susceptibility testing, performed according to Clinical and Laboratory Standards Institute (CLSI) methods, using panels prepared in-house. A checkerboard assay conforming to the Moody procedures in the Clinical Microbiology Procedures Handbook was used to evaluate the effect of various concentrations of QPX7728 or vaborbactam on the MICs of various antibiotics. PV50 and the maximal potentiation value (PVmax) were used to define the potencies of the beta-lactamase inhibitors. PV50 was defined as the minimal concentration of a BLI required to achieve 50% of the antibiotic potentiation effect or a concentration of a BLI to reduce the antibiotic MIC to the middle point of the MIC range between the MIC for the beta-lactamase-producing strain and the MIC for the corresponding the beta-lactamase-lacking strain. The MIC middle point is the geometric mean of the antibiotic MIC values for the beta-lactamase-producing strain and the beta-lactamase-lacking strain and is calculated as the square root of the product of the antibiotic MIC values for the beta-lactamase-producing and the beta-lactamase-lacking strain. PVmax was defined as the maximal potentiating value, which was the concentration of the BLI required to reduce the antibiotic MIC to the level seen in the parent strain that lacks beta-lactamase (KPC) (corresponding to the complete inhibition of KPC).

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Determination of Monomer Formation [2]
Test compounds were weighed out into a 1.5 mL Eppendorf tube. A solution of water was prepared in 1.00, 10.0, or 100 mg/mL concentrations of the compound based on active fraction. A volume of 20 μL was subtracted to allow room for addition of acid or base to adjust the pH. The solution pH was measured. The pH was then adjusted through addition of acid or base as necessary until the reading was in the 7.6–8 range. A final pH measurement was made after ensuring all the compound was in solution. The solution was injected onto LC–UV in 0.1–5 μL volume within less than half an hour after the time the sample was prepared. Elution was done using 0.1% TFA in water for mobile phase A and 0.085% TFA in methanol for mobile phase B on an Excel ACE 5 Super C18 2.1 mm × 100 mm column at 0.6 mL/min flow rate with 9 min gradient, 3.9 min hold at 90% B, and 18 min run time. The absorption was measured at 220, 254, and 300 nm with a 4 nm bandwidth.

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Susceptibility Testing [2]
MICs were determined using Clinical and Laboratory Standards Institute (CLSI) broth microdilution methods as described in CLSI document M07-A11 (2018).

Pharmacokinetic Studies in Rats [2]
After acclimation, rats (n = 3/dose level) were administered either single intravenous infusions (in 0.9% saline) of QPX7728 at 30, 100, or 300 mg/kg or at 30, 100, 300, or 1000 mg/kg via the oral route (in water). Intravenous doses were infused over 0.5 h via an indwelling femoral vein cannula, while oral doses were administered via a bead tipped oral gavage. Plasma (∼0.3 mL) samples were collected from each rat at designated time points up to 24 h. Blood samples were centrifuged within 5 min of collection at 12 000g for 5 min to obtain plasma. The plasma samples were analyzed using an HPLC–MS method. PK analysis was performed using WinNonlin.

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Mouse Efficacy Studies [2]
Swiss-Webster mice were rendered neutropenic by the administration of cyclophosphamide and were infected under isoflurane anesthesia by intramuscular injection of K. pneumoniae KP1244 (inoculum 1 × 106) in both thighs (meropenem MIC of >64 μg/mL; meropenem MIC in the presence of 8 μg/mL QPX7728 was 0.25 μg/mL). Treatments (formulated in water) were administered every 2 h by the intraperitoneal route, starting 2 h postinfection. Animals were sacrificed 24 h after the start of treatment, and the thighs were removed, homogenized, and plated to determine bacterial counts.

Pharmacokinetics [1]
The pharmacokinetics of QPX7728 administered intravenously (iv) and orally (po) in rats were evaluated at various doses (Table 14). Systemic exposure (expressed as Cmax and AUC values) increased proportionally with dose after intravenous administration. Overall, its parameters were similar to those of most β-lactam antibiotics, exhibiting high Cmax and AUC, short half-life, and low volume of distribution. Protein binding in rat plasma was 85%. Oral bioavailability (F) of QPX7728/compound 35 was also observed in fasting rats, ranging from 43% to 53% at doses of 30–100 mg/kg, decreasing to 24–28% at higher doses. Given its polarity (log D7.4 = −2.85), we speculate that oral absorption may involve active transport and may reach saturation at high doses. The compound was well tolerated at all doses.

Safety [2] Compound 35/QPX7728 was studied in a preliminary toxicology study over a 7-day period at daily doses of 30, 100, and 300 mg/kg in rats (5 males and 5 females at each dose level) via intravenous infusion. No changes were observed (histological and clinical chemistry).

[1]. The Impact of Intrinsic Resistance Mechanisms on Potency of QPX7728, a New Ultra-Broad-Spectrum Beta-lactamase Inhibitor of Serine and Metallo Beta-Lactamases in Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii.Antimicrob Agents Chemother. 2020 May 21;64(6):e00552-20.
[2]. Discovery of Cyclic Boronic Acid QPX7728, an Ultrabroad-Spectrum Inhibitor of Serine and Metallo-β-lactamases. J Med Chem. 2020;63(14):7491-7507.

Despite significant progress in the field of β-lactamase inhibitors, some enzymes remain resistant to recently introduced drugs. Among the most important are the class B (metalloid) enzyme NDM-1 of Enterobacteriaceae and the class D (OXA) enzyme of Acinetobacter baumannii. Building on the development of vaborbactam, borate drugs, researchers have worked to broaden their inhibitory spectrum to treat a wider range of microorganisms. By making key structural modifications to a bicyclic lead compound, their inhibitory spectrum was gradually broadened, resulting in QPX7728 (35). This compound exhibits an extremely broad inhibitory spectrum, including both class B and class D enzymes, and is almost unaffected by porin modifications and efflux. Compound 35 is a promising drug for use in combination with β-lactam antibiotics, administered intravenously and orally, to treat infections caused by a variety of drug-resistant Gram-negative bacteria. [1]

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QPX7728 is an ultra-broad-spectrum borate β-lactamase inhibitor that, in biochemical analysis of the purified enzyme, inhibits key serine and metalloid β-lactamases in the nanomolar concentration range. The broad-spectrum inhibitory activity of QPX7728 observed in biochemical experiments can be translated into enhanced antibacterial activity against various β-lactam antibiotics targeting β-lactamase-producing pathogens. This study constructed a homologous strain library using Klebsiella pneumoniae and Pseudomonas aeruginosa homologous strains (both producing KPC-3) and Acinetobacter baumannii OXA-23-producing strains (with different combinations of efflux pumps and porin mutations) to determine the effects of bacterial efflux pumps and permeability on antibacterial efficacy. The results showed that the antibacterial efficacy of QPX7728 was almost unaffected by multidrug-resistant efflux pumps in Enterobacteriaceae or non-fermenting bacteria (such as Pseudomonas aeruginosa or Acinetobacter baumannii). When the outer membrane permeability of Pseudomonas aeruginosa increased, the antibacterial efficacy of QPX7728 against Pseudomonas aeruginosa was further enhanced. Inactivation of the carbapenem porin OprD did not affect the antibacterial efficacy of QPX7728 against Pseudomonas aeruginosa. Although the change in OmpK36 (instead of OmpK35) reduced the potency of QPX7728 (8 to 16 times), QPX7728 (4 μg/ml) was still able to completely reverse KPC-mediated meropenem resistance in porin mutant strains, which is consistent with the fact that these mutations have less effect on the potency of QPX7728 than on other drugs. QPX7728 has broad-spectrum β-lactamase inhibitory properties and can enhance the activity of a variety of β-lactam antibiotics sensitive to different resistance mechanisms (such as efflux and permeability), indicating that QPX7728 is an effective inhibitor that can be used for a variety of β-lactam antibiotics. [1] QPX7728 is a novel borate β-lactamase inhibitor (BLI) with potent inhibitory activity against both serine and metallo-β-lactamases. The broad-spectrum inhibitory activity of QPX7728 previously observed in cell-free biochemical experiments using purified enzymes can be translated into enhanced activity of various β-lactam antibiotics against target pathogens that produce β-lactamases. [1]
The potent inhibitory activity of QPX7728 in intact cells is partly due to the inhibition of efflux by major transport proteins in Gram-negative bacteria, and the concentration of this inhibition is related to β-lactamase inhibition. The efflux inhibition of QPX7728 is particularly important for its inhibitory activity in Pseudomonas aeruginosa and is a significant improvement compared to the earlier borate β-lactamase inhibitor vaborbactam. Mutations in the outer membrane porins of Enterobacteriaceae are associated with reduced efficacy of many antibiotics and β-lactamase inhibitors. The efficacy of QPX7728 in Enterobacteriaceae is affected much less by the inactivation of the major universal porins OmpK35/OmpF and OmpK36/OmpC than by the borate inhibitor vaborbactam. [1]
QPX7728 exhibits potent, broad-spectrum inhibitory activity against a variety of β-lactam antibiotics with varying sensitivities to β-lactamases and inherent resistance mechanisms, making it an ideal candidate for various product development strategies. Traditional product formulation approaches involve developing fixed-combination β-lactam antibiotic-β-lactamase inhibitors, for which well-established regulatory pathways exist. A significant limitation of this strategy is finding a β-lactam antibiotic that, when used in combination with a β-lactamase inhibitor (BLI), produces optimal overall activity against most (but perhaps not all) mixtures of target pathogens with different resistance mechanisms. Another approach is to develop QPX7728 as a standalone drug product that can be used in combination with different existing β-lactam antibiotics, depending on the specific pathogen's mechanism of action. This approach has multiple clinical and regulatory implications, but considering local epidemiology, patient factors, and antibiotic administration, it could be an important step toward personalized treatment of infections caused by drug-resistant pathogens. The multiple advantages of this strategy should contribute to the development of a clear future regulatory approval pathway. [1]

C10H8BFO4

221.9775

222.049

C, 54.11; H, 3.63; B, 4.87; F, 8.56; O, 28.83

2170834-63-4

Xeruborbactam disodium;2170848-99-2;(1R,2S)-Xeruborbactam disodium;2170836-14-1

140830474

Typically exists as solid at room temperature

2

5

1

16

326

2

B1([C@@H]2C[C@@H]2C3=C(O1)C(=C(C=C3)F)C(=O)O)O

KOHUFVUIYUCFNG-PHDIDXHHSA-N

InChI=1S/C10H8BFO4/c12-7-2-1-4-5-3-6(5)11(15)16-9(4)8(7)10(13)14/h1-2,5-6,15H,3H2,(H,13,14)/t5-,6-/m1/s1

(1aR,7bS)-5-fluoro-2-hydroxy-1a,7b-dihydro-1H-cyclopropa[c][1,2]benzoxaborinine-4-carboxylic acid

QPX7728; 2170834-63-4; Xeruborbactam [INN]; DE79L822UY; UNII-DE79L822UY; CHEMBL4633785; QPX-7728;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)