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 by intravenous (iv) and oral (po) administration were evaluated in the rat at multiple doses (Table 14). The systemic exposure by iv administration, as shown by the Cmax and AUC values, increased in a dose proportional manner. Overall the parameters are similar to those of most β-lactam antibiotics, evidencing high Cmax and AUC, short half-life, and low volume of distribution. Plasma protein binding in the rat is 85%. QPX7728/Compound 35 also displays oral bioavailability (F) in fasted rats, with values ranging between 43% and 53% at doses of 30–100 mg/kg, whereas it declined to 24–28% at higher doses. Given its polarity (log D7.4 = −2.85), we speculate that active transport may be involved in oral uptake, which may be saturated at higher doses. The compound was well tolerated at all doses.

Safety [2]
Compound 35/QPX7728 was studied in a 7-day pilot toxicology study at daily doses of 30, 100, and 300 mg/kg in rats (five males, five females per dose level) administered by iv infusion. No changes were observed (tissue histology 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 major advances in the β-lactamase inhibitor field, certain enzymes remain refractory to inhibition by agents recently introduced. Most important among these are the class B (metallo) enzyme NDM-1 of Enterobacteriaceae and the class D (OXA) enzymes of Acinetobacter baumannii. Continuing the boronic acid program that led to vaborbactam, efforts were directed toward expanding the spectrum to allow treatment of a wider range of organisms. Through key structural modifications of a bicyclic lead, stepwise gains in spectrum of inhibition were achieved, ultimately resulting in QPX7728 (35). This compound displays a remarkably broad spectrum of inhibition, including class B and class D enzymes, and is little affected by porin modifications and efflux. Compound 35 is a promising agent for use in combination with a β-lactam antibiotic for the treatment of a wide range of multidrug resistant Gram-negative bacterial infections, by both intravenous and oral administration.[1]

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QPX7728 is an ultrabroad-spectrum boronic acid beta-lactamase inhibitor that demonstrates inhibition of key serine and metallo-beta-lactamases at a nanomolar concentration range in biochemical assays with purified enzymes. The broad-spectrum inhibitory activity of QPX7728 observed in biochemical experiments translates into enhancement of the potency of many beta-lactams against strains of target pathogens producing beta-lactamases. The impacts of bacterial efflux and permeability on inhibitory potency were determined using isogenic panels of KPC-3-producing isogenic strains of Klebsiella pneumoniae and Pseudomonas aeruginosa and OXA-23-producing strains of Acinetobacter baumannii with various combinations of efflux and porin mutations. QPX7728 was minimally affected by multidrug resistance efflux pumps either in Enterobacteriaceae or in nonfermenters, such as P. aeruginosa or A. baumannii Against P. aeruginosa, the potency of QPX7728 was further enhanced when the outer membrane was permeabilized. The potency of QPX7728 against P. aeruginosa was not affected by inactivation of the carbapenem porin OprD. While changes in OmpK36 (but not OmpK35) reduced the potency of QPX7728 (8- to 16-fold), QPX7728 (4 μg/ml) nevertheless completely reversed the KPC-mediated meropenem resistance in strains with porin mutations, consistent with the lesser effect of these mutations on the potency of QPX7728 compared to that of other agents. The ultrabroad-spectrum beta-lactamase inhibition profile, combined with enhancement of the activity of multiple beta-lactam antibiotics with various sensitivities to the intrinsic resistance mechanisms of efflux and permeability, indicates that QPX7728 is a useful inhibitor for use with multiple beta-lactam antibiotics.[1]

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QPX7728 is a new boronate BLI with potent inhibitory activity against both serine and metallo-beta-lactamases. The broad-spectrum inhibitory activity of QPX7728 previously observed in cell-free biochemical experiments using purified enzymes translates into enhancement of the activity of many beta-lactams against strains of target pathogens producing beta-lactamases.[1]
The potent inhibitory activity of QPX7728 in whole cells is driven in part by a lack of efflux by major transporters from Gram-negative bacteria at concentrations that are relevant for beta-lactamase inhibition. A lack of efflux of QPX7728 is particularly important for inhibitory activity in P. aeruginosa and represents a significant improvement over the earlier boronate BLI vaborbactam. Mutations in outer membrane porin proteins of Enterobacteriaceae are associated with the reduced potency of many antibiotics and beta-lactamase inhibitors. The potency of QPX7728 in Enterobacteriaceae is affected by the inactivation of the major general porins OmpK35/OmpF and OmpK36/OmpC much less than the boronate inhibitor vaborbactam is.[1]
The potent, ultrabroad-spectrum inhibitory activity of QPX7728 shown with multiple beta-lactam antibiotics with various sensitivities to beta-lactamases as well as intrinsic resistance mechanisms makes it an ideal candidate for multiple product development strategies. Conventional approaches for product configurations include the development of a fixed-combination beta-lactam–beta-lactamase inhibitor for which there is a well-established regulatory path. An important limitation of this strategy is identifying a partner beta-lactam that, in combination with the BLI, has the best overall activity against most but perhaps not all target pathogens with different mixtures of resistance mechanisms. Another approach would be the development of QPX7728 as a stand-alone drug product that could be coadministered with different existing beta-lactam antibiotics, depending on the mechanisms present in the specific pathogen. This approach has several clinical and regulatory implications but could be an important step toward individualized treatment of infections caused by drug-resistant pathogens by taking into account local epidemiology, patient factors, and antibiotic stewardship. The multiple benefits of this strategy should encourage the establishment of a defined path for future regulatory approval.[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.)