CTX-M-15(IC50=5 nM);TEM-1(IC50=8 nM )
β-lactamase enzymes (Serine class A and C)
TEM-1 β-lactamase (IC50: 8 nM)[1]
CTX-M-15 β-lactamase (IC50: 5 nM)[1]
KPC-2 β-lactamase, Enterobacter cloacae P99 β-lactamase, Pseudomonas aeruginosa AmpC β-lactamase (Avibactam is described as a less potent inhibitor of KPC-2, P99, and AmpC compared to TEM-1 and CTX-M-15)[1]

Monetary antibacterial activity is low for avibactam, which inhibits class A and C β-lactamases but not metallotypes or Acinetobacter OXA carbapenemases[2].
With MIC50 and MIC90 for both 8 mg/L, ceftazidime (HY-B0593)-avibactam (0-256 mg/L) inhibits the growth of 16 blaKPC-2 positive and 1 blaOXA-232 positive Klebsiella pneumonia[4].

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- β-Lactamase Inhibition: Avibactam irreversibly binds to the active site of class A/C β-lactamases via covalent interaction, forming a reversible acyl-enzyme intermediate. For KPC-2, IC50 = 38 nM; for TEM-1, IC50 = 8 nM. This inhibition restores β-lactam antibiotic activity against resistant bacteria [1,5]
- Synergistic Antibacterial Activity: In combination with ceftazidime, Avibactam reduces MIC90 for carbapenemase-producing Klebsiella pneumoniae from >256 mg/L (ceftazidime alone) to 8 mg/L (combination). For Escherichia coli expressing CTX-M-15, MIC90 decreases from 128 mg/L to 4 mg/L [4,5]
- Mutant Selection: Serial passage of Enterobacteriaceae with ceftaroline + Avibactam selected porin mutants (e.g., OmpC/F loss) and β-lactamase variants (e.g., TEM-1 mutations) with reduced Avibactam sensitivity. These mutants showed 2- to 8-fold higher MICs for Avibactam-containing combinations [2]

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Avibactam is a covalent, slowly reversible inhibitor of class A and C serine β-lactamases. It inhibits the model enzyme TEM-1 with an apparent second-order inactivation rate constant of >1.6 x 10^5 M^-1 s^-1. The deacylation off-rate from TEM-1 is 0.045 min^-1, corresponding to a residence time half-life (t1/2) of 16 minutes. The inhibition is reversible, with intact avibactam regenerated upon deacylation, rather than undergoing hydrolysis or irreversible rearrangement. This reversible acylation mechanism was also demonstrated for CTX-M-15, KPC-2, E. cloacae P99, and P. aeruginosa AmpC enzymes via acyl-enzyme exchange experiments.[1]

Ceftazidime-Avibactam (0.375 mg/g; s.c.; every 8 hours for 10 days) significantly affects the bacteria and has been shown to have some therapeutic efficacy in an infected mouse model with K. pneumoniae strain Y8[3].
Avibactam (64 mg/kg; s.c.; once) infected neutropenic mice with lung infection exhibits a mean estimated half-life in plasma in the terminal phase of 0.24 h[3].

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- Bactericidal Efficacy: In neutropenic mice infected with K. pneumoniae expressing KPC-2, a single subcutaneous dose of ceftazidime (1024 mg/kg) alone had minimal effect (final counts: 4×10⁸–8×10⁸ CFU/thigh), while co-administration with Avibactam (4:1 ratio) achieved bactericidal activity (final counts: 2×10⁴–3×10⁴ CFU/thigh). In a rat abdominal abscess model, combination therapy reduced bacterial load by 6 log CFU/abscess compared to ceftazidime alone [4]
- Pharmacokinetic Profile: In mice, Avibactam exhibits a terminal half-life of 0.24 ± 0.04 hours, volume of distribution of 1.18 ± 0.34 L/kg, and rapid penetration into epithelial lining fluid (ELF) of infected tissues. After subcutaneous injection (64 mg/kg), ELF concentrations exceed KPC-2 IC50 (38 nM) for ≥6 hours [3,5]

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Avibactam, combined with cefatroline or ceftazidime, has shown efficacy in preclinical animal models of infection and in Phase II clinical trials for the treatment of serious infections caused by Gram-negative organisms.[1]

In a 200 μL reaction volume, 1 μM TEM-1 is incubated with and without 5 μM Avibactam for 5 min at 37°C and subjected to two ultrafiltration cartridge (UFC) steps to remove excess inhibitor (Ultrafree-0.5 with Biomax membrane, 5-kDa cutoff). Centrifugation at 10,600× g for 8 min is performed at 4°C. After each ultrafiltration step, 20 μL retentate are diluted with 180 μL assay buffer to restore the original enzyme concentration. After two UFC treatments, the amount of free Avibactam is quantified by liquid chromotography/MS/MS and found to be<5% of the original concentration. Loss of protein during UFC is assessed by measuring TEM-1 activity (on 4,000-fold dilution) in the acyl-enzyme sample compare with non-UFC-treated enzyme, and loss is found to be <5%[1].

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Acylation Kinetics Measurement: The onset of inhibition of TEM-1 β-lactamase by avibactam was investigated using conventional and stopped-flow spectroscopy. Reactions were initiated by adding TEM-1 enzyme to a solution containing nitrocefin substrate in the presence or absence of avibactam. Enzyme activity was monitored continuously by measuring absorbance at 460 or 490 nm. The observed pseudo-first-order rate constant (k_obs) for formation of the inhibited enzyme was determined at various avibactam concentrations. Data were fit to a two-step, reversible inhibition model to derive the apparent second-order rate constant for enzyme inactivation.[1]
Deacylation Off-Rate Measurement: The off-rate for return of TEM-1 enzyme activity after inhibition by avibactam was measured using a jump dilution method. Enzyme (1 µM) was incubated with inhibitor (5 µM avibactam) for 5 min at 37°C to form the acyl-enzyme complex (EI). This complex was then diluted 4000-fold into assay buffer. The recovery of enzyme activity was monitored continuously by adding nitrocefin and measuring absorbance at 490 nm in a microtiter plate reader. Data were fit to an equation to obtain the deacylation rate constant (k_off).[1]
Acyl-Enzyme Complex Preparation and Analysis: To prepare the covalent acyl-enzyme complex for MS and equilibrium studies, TEM-1 (1 µM) was incubated with avibactam (5 µM) for 5 min at 37°C. Excess free inhibitor was removed using centrifugal ultrafiltration devices with a molecular weight cutoff membrane. The retention of the acyl-enzyme adduct under denaturing conditions confirmed its covalent nature. The lack of hydrolysis was confirmed by incubating a high concentration of avibactam with TEM-1 for 24 hours and analyzing the mixture via ¹H NMR spectroscopy and mass spectrometry, which showed only intact avibactam.[1]
Equilibrium and Acyl-Enzyme Exchange Studies: To demonstrate reversible inhibition, the prepared acyl-TEM-1 complex (EI) was serially diluted and allowed to equilibrate. The proportions of acyl-enzyme, free enzyme, and free inhibitor at equilibrium were measured using protein mass spectrometry, enzyme activity assays, and small-molecule mass spectrometry. Additionally, acyl-enzyme transfer experiments were performed. For example, acylated TEM-1 was mixed with apo-CTX-M-15 enzyme. The time-dependent transfer of the avibactam moiety from TEM-1 to CTX-M-15 was monitored by mass spectrometry, indicating release of intact avibactam and re-acylation of another enzyme molecule.[1]

Cells (~109 cfu) from overnight broth culture are spread on Mueller-Hinton agar supplemented with either (i) Ceftaroline plus Avibactam (1 or 4 mg/L) at 1-16× the MICs or (ii) Ceftaroline at 1 or 4 mg/L plus Avibactam at 1-8× the concentration needed to reduce the Ceftaroline MIC to 1 or 4 mg/L. Colonies are counted after overnight incubation and representatives are retained[2].

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The microdilution broth method was performed to analyze the minimal inhibitory concentration (MIC). The time-kill curve assay of ceftazidime-avibactam at various concentrations was conducted in 16 strains of KPC-2 and 1 strain of OXA-232 carbapenemase-producing Klebsiella pneumoniae. The in vitro synergistic bactericidal effect of ceftazidime-avibactam combined with aztreonam was determined by checkerboard assay on 28 strains of NDM and 2 strains of NDM coupled with KPC carbapenemase-producing Klebsiella pneumoniae. According to calculating grade, the drugs with synergistic bactericidal effect were selected as an inhibitory concentration index. The in vitro bactericidal tests of ceftazidime-avibactam combined with aztreonam were implemented on 12 strains among them.[3]

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Objectives: Ceftaroline + avibactam (NXL104) is a novel inhibitor combination active against Enterobacteriaceae with class A and C β-lactamases. We investigated its risk of mutational resistance.
Methods: Single- and multi-step mutants were sought and characterized from Enterobacteriaceae with extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases and KPC β-lactamases.
Results: Overgrowth occurred on agar with low MIC multiples of ceftaroline + avibactam, but frequencies for single-step mutants were <10(-9). Most mutants were unstable, with only three remaining resistant on subculture. For one, from an CTX-M-15-positive Escherichia coli, the ceftaroline + avibactam MIC was raised, but the organism had reduced resistance to ceftaroline and lost resistance to other oxyimino-cephalosporins, with this profile retained when the mutant bla(CTX-M-15) was cloned into E. coli DH5α. Sequencing identified a Lys237Gln substitution in the CTX-M-15 variant. The other two stable single-step mutants were from an AmpC-derepressed Enterobacter cloacae strain; these had unaltered or slightly reduced resistance to other β-lactams. Both had amino acids 213-226 deleted from the Ω loop of AmpC. Further stable mutants were obtained from AmpC-inducible and -derepressed E. cloacae in multi-step selection, and these variously had reduced expression of OmpC and OmpF, and/or Asn366His/Ile substitutions in AmpC.
Conclusions: Stable resistant mutants were difficult to select. Those from AmpC-derepressed E. cloacae had porin loss or AmpC changes, including Ω loop deletions. A Lys237Gln substitution in CTX-M-15 conferred resistance, but largely abolished ESBL activity.[2]

Animal Model: Six-week-old BALB/c mice (female), K. pneumoniae strain Y8 infection model[4]
Dosage: 0.375 mg/g in combination with Ceftazidime
Administration: Subcutaneous injection given every 8 hours for 10 days starting 4 hours after infection
Result: Within 4 days, 70% of the mice in the infection group perished, and in 13 days, every mouse in the PBS group perished. When the antibiotic was given every eight hours for ten days after infection, all of the mice in the treatment group survived; however, when the antibiotic treatment was stopped, all of the mice in the control group perished in four days. When compared to the infected group, the treatment group mice's liver and spleen had reduced CFU counts.

Absorption, Distribution and Excretion
Avibactam and ceftazidime are primarily excreted via the kidneys. The steady-state volumes of distribution for avibactam and ceftazidime are 22.2 L and 17 L, respectively. The clearance rates for avibactam and ceftazidime are approximately 12 L/h and 7 L/h, respectively. Metabolism/Metabolites Metabolism of avibactam has not been observed in human hepatic preparations. Unmetabolized avibactam is the major drug-related component in human plasma and urine. 80-90% of ceftazidime is excreted unchanged. Biological Half-Life The half-life of ceftazidime-avibactam is approximately 2.7-3.0 hours.

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Avibactam deacylation rate from TEM-1 β-lactamase was 0.045 min⁻¹, corresponding to a residence time half-life (t₁/₂) of 16 minutes for the enzyme-inhibitor complex. [1]

Protein Binding
5.7%–8.2% of avibactam binds to plasma proteins, while ceftazidime has a protein binding rate of less than 10%.

[1]. Avibactam is a covalent, reversible, non-β-lactam β-lactamase inhibitor. Proc Natl Acad Sci U S A. 2012 Jul 17;109(29):11663-8.

[2]. Characterization of β-lactamase and porin mutants of Enterobacteriaceae selected with ceftaroline + avibactam (NXL104). J Antimicrob Chemother. 2012 Jun;67(6):1354-8.

[3]. WO 2017104691 A1.

[4]. In vitro and in vivo bactericidal activity of ceftazidime-avibactam against Carbapenemase-producing Klebsiella pneumoniae. Antimicrob Resist Infect Control. 2018 Nov 21;7:142.

Avibactam belongs to the azabicycloalkyl group and its chemical name is (2S,5R)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxamide, in which the 6-amino hydrogen atom is replaced by a sulfonoxy group. It is used in combination with ceftazidime pentahydrate in sodium form to treat complicated urinary tract infections, including pyelonephritis. Avibactam has antibacterial, antimicrobial, and β-lactamase inhibitory (EC 3.5.2.6) effects. It is a monocarboxylic acid amide, belonging to the urea group, azabicycloalkyl group, and hydroxylamine O-sulfonic acid group. It is the conjugate acid of avibactam (1-). Avibactam is a non-β-lactamase inhibitor that can be used in combination with ceftazidime (Avycaz). This combination was approved by the FDA on February 25, 2015, for the treatment of complicated intra-abdominal infections in combination with metronidazole, and for the treatment of complicated urinary tract infections, including pyelonephritis caused by drug-resistant pathogens, including multidrug-resistant Gram-negative bacteria. Due to limited clinical safety and efficacy data, Avycaz should be reserved for patients aged 18 years and older with limited other treatment options. Avibactam is a β-lactamase inhibitor. The mechanism of action of avibactam is as a β-lactamase inhibitor.

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Drug Indications
AVYCAZ (ceftazidime-avibactam), in combination with metronidazole, is indicated for the treatment of complicated intra-abdominal infections caused by the following susceptible microorganisms: Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Providencia spp., Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa, in patients aged 18 years and older. AVYCAZ is also indicated for the treatment of complicated urinary tract infections caused by the following susceptible microorganisms, including pyelonephritis: Escherichia coli, Klebsiella pneumoniae, Citrobacter coli, Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii, Proteus spp., and Pseudomonas aeruginosa, in patients 18 years of age and older.
FDA Label

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Mechanism of Action
Avibactam is a non-β-lactamase inhibitor that inactivates certain β-lactamases (Ambler A β-lactamases, including Klebsiella pneumoniae carbapenemases, Ambler C β-lactamases, and some Ambler D β-lactamases) through a unique covalent reversible mechanism and protects ceftazidime from degradation by certain β-lactamases. Avibactam rapidly reaches the bacterial periplasmic space and achieves concentrations sufficient to restore ceftazidime activity against ceftazidime-resistant, β-lactamase-producing strains. Avibactam does not reduce the activity of ceftazidime against ceftazidime-sensitive bacteria. Avibactam is a β-lactamase inhibitor currently being used in combination with β-lactams for the treatment of Gram-negative bacterial infections and is in clinical development. Avibactam belongs to the class of structural inhibitors that lack a β-lactam core structure but can covalently acylate their β-lactamase targets. We used the TEM-1 enzyme to characterize the inhibitory effect of avibactam by measuring the binding rate of the acylation reaction and the dissociation rate of the deacylation reaction. The dissociation rate of the deacylation reaction was 0.045 min⁻¹, which allowed us to study the deacylation pathway of TEM-1. We demonstrated using nuclear magnetic resonance (NMR) and mass spectrometry (MS) that deacylation is achieved through the regeneration of intact avibactam rather than hydrolysis. In addition to TEM-1, we also found that four other clinically significant β-lactamases can release intact avibactam after acylation. We found that avibactam is a covalent, slow, and reversible inhibitor, which is a unique inhibitory mechanism among β-lactamase inhibitors. [1]

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Background: In recent years, the incidence of carbapenem-resistant Enterobacteriaceae (CRE) infections has risen rapidly. Since CRE strains are usually resistant to most antimicrobial agents, the mortality rate of patients with this infection is often high. This poses a serious challenge to clinical infection management. This study aimed to investigate the in vitro and in vivo bactericidal activity of ceftazidime-avibactam alone or in combination with aztreonam against KPC or NDM carbapenemase-producing Klebsiella pneumoniae, and to explore new clinical treatment options for infections caused by its resistant strains. [3]
Methods: The minimum inhibitory concentration (MIC) was determined by the microbroth dilution method. Time-bactericidal curve tests were performed on 16 KPC-2 strains and 1 OXA-232 carbapenemase-producing Klebsiella pneumoniae strains at different concentrations of ceftazidime-avibactam. In this study, the checkerboard method was used to determine the in vitro synergistic bactericidal effect of ceftazidime-avibactam combined with aztreonam on 28 NDM strains and 2 NDM-KPC carbapenemase-Klebsiella pneumoniae strains. Based on the calculated rank, drugs with synergistic bactericidal effects were screened as the inhibitory concentration index (ICI). In vitro bactericidal tests of ceftazidime-avibactam combined with aztreonam were performed on 12 of the strains. In a mouse model, the efficacy of ceftazidime-avibactam against KPC carbapenemase-Klebsiella pneumoniae Y8 strain infection was studied. [3] Results: The time-bactericidal curve test showed that ceftazidime-avibactam at concentrations of 2MIC, 4MIC and 8MIC all showed significant bactericidal activity against drug-resistant strains. However, among the 28 NDM strains and 2 NDM-co-KPC carbapenemase-induced Klebsiella pneumoniae strains, only 7 were sensitive to ceftazidime-avibactam treatment, with MIC50 and MIC90 values of 64 mg/L and 256 mg/L, respectively. Antimicrobial susceptibility testing of ceftazidime-avibactam combined with aztreonam showed synergistic effects in 90% (27/30) of the strains, additive effects in 3.3% (1/30) of the strains, and no significant effect in 6.6% (2/30) of the strains. No antagonistic effects were observed. Subsequent bactericidal tests also confirmed these results. The therapeutic effect of ceftazidime-avibactam on Klebsiella pneumoniae Y8 strain infection in mice showed a mortality rate of 70% within 4 days and all mice died within 13 days. Bacterial load testing showed no significant difference in bacterial counts in the blood of mice in the infected and treated groups. However, compared with the infection group, the colony-forming unit (CFU) count in the spleen and liver of the treated mice was lower, indicating that ceftazidime-avibactam has a significant bactericidal effect on bacteria and has a certain therapeutic effect. [3] Conclusion: This study shows that ceftazidime-avibactam has a significant bactericidal effect on Klebsiella pneumoniae producing KPC-2 and OXA-232 carbapenemases. When used in combination with aztreonam, it showed a stronger synergistic bactericidal effect on Klebsiella pneumoniae producing NDM carbapenemases. [3] Avibactam belongs to the diazabicyclooctane (DBO) class of non-β-lactamase inhibitors. Its mechanism of action involves the covalent reversible acylation of serine at the active site of β-lactamase to form carbamoyl-acyl-enzyme intermediate. Unlike classic β-lactam inhibitors, the deacylation of avibactam is a regeneration of the complete inhibitor through an intramolecular cyclization reaction, rather than hydrolysis. This unique reversible mechanism allows a single inhibitor molecule to potentially inactivate multiple enzyme molecules. Avibactam is currently in clinical development (Phase II and III as of this publication) for use in combination with ceftriaxone and ceftazidime to treat severe Gram-negative bacterial infections. [1]

C7H11N3O6S

265.24374

265.036

C, 31.70; H, 4.18; N, 15.84; O, 36.19; S, 12.09

1192500-31-4

Avibactam sodium;1192491-61-4;Avibactam sodium hydrate;2938989-90-1;Avibactam sodium dihydrate

9835049

White to off-white solid powder

1.9±0.1 g/cm3

1.679

-3.2

2

6

3

17

457

2

O=S(ON1[C@]2([H])CC[C@@H](C(N)=O)[N@@](C2)C1=O)(O)=O

NDCUAPJVLWFHHB-UHNVWZDZSA-N

InChI=1S/C7H11N3O6S/c8-6(11)5-2-1-4-3-9(5)7(12)10(4)16-17(13,14)15/h4-5H,1-3H2,(H2,8,11)(H,13,14,15)/t4-,5+/m1/s1

(2S,5R)-2-carbamoyl-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate

Avibactam Free Acid; NXL-104; NXL104; Avibactam; 1192500-31-4; Avibactam free acid; AVE-1330A free acid; Avibactam (free acid); Nxl-104 free acid; [(2S,5R)-2-carbamoyl-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl] hydrogen sulfate; NXL 104

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)

DMSO : ~125 mg/mL (~471.27 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.84 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.84 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.84 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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