β-lactamase

β-Lactamase assays [1]
Table 1 shows the different properties of enzyme inhibitory activity. Against TEM-1, AVE1330A was five- and 16-fold more active than tazobactam and clavulanic acid in terms of IC50. Against P99, AVE1330A showed stronger inhibition than tazobactam, with an IC50 >60-fold lower than that of tazobactam; clavulanic acid was inactive.
The Tn for AVE1330A was 2, compared with 214 for clavulanic acid against TEM-1. Maximal TEM-1 inhibition was observed at 10 min for all AVE1330A/enzyme ratios (data not shown). Complete inactivation of P99 by AVE1330A occurred at a ratio of 5, compared with 55 for tazobactam. On the other hand, maximal P99 inhibition occurred after 60 min for AVE1330A and tazobactam (data not shown).
For TEM-1, 50% deacylation was observed over 7 days of incubation with AVE1330A, compared with 7 min with clavulanic acid. In the case of P99, the enzyme recovered 50% of its activity over 7 days, compared with 290 min with tazobactam.

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In vitro antibacterial activity against β-lactamase overproducers [1]
Table 2 shows the MICs of ceftazidime alone compared with ceftazidime in combination with AVE1330A or clavulanic acid against the three strains tested. MICs of AVE1330A and clavulanic acid alone were ≥16 mg/L. MICs of ceftazidime for the SHV-4 and AmpC producers clearly increased from 1–2 to >32 mg/L, when β-lactamase production was increased by L-arabinose induction. Unlike clavulanic acid, AVE1330A maintained MICs at ≤1 mg/L of ceftazidime for all the strains tested, including the AmpC producer, whatever the level of β-lactamase induction.

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Comparative in vitro antibacterial activity of ceftazidime combined with AVE1330A at a 4:1 ratio [1]
Comparative MICs of ceftazidime alone, ceftazidime/AVE1330A and ceftazidime/clavulanic acid at a ratio of 4:1 for known β-lactamase-producing strains are reported in Table 3. MICs of AVE1330A and clavulanic acid alone were ≥8 mg/L for the strains tested. Most of them lacked susceptibility to ceftazidime. For strains producing class A plasmid-encoded enzymes, AVE1330A restored susceptibility to ceftazidime, as did clavulanic acid, both combinations being up to 64-fold more active than ceftazidime alone. Ceftazidime/AVE1330A was also active against class C plasmid-encoded enzymes, MICs ranging from 0.5 to 4 mg/L. Conversely, no synergy was observed with clavulanic acid against ceftazidime-resistant isolates.
More extended data are reported in Table 4. All the E. coli isolates were inhibited by 4 mg/L ceftazidime/AVE1330A. No other comparator was as active in terms of reduction in MICs. Against ceftazidime-resistant isolates, the MIC90 of ceftazidime decreased from >64 to 4 and 16 mg/L in the presence of AVE1330A and clavulanic acid, respectively. Both inhibitors had similar behaviour against class A enzyme producers. In contrast to clavulanic acid, AVE1330A restored high ceftazidime activity against class C enzyme producers (MIC90 of 16 or 2 mg/L, respectively). Unlike other species, few E. coli isolates were inhibited in the presence of 4 mg/L AVE1330A alone, but the MIC50 and MIC90 still remained ≥8 mg/L.
For ceftazidime-resistant Klebsiella, most of them producing class A enzymes, ceftazidime/AVE1330A lowered the MIC90 from >64 to 2 mg/L, i.e. to values similar to those of ceftazidime/clavulanic acid.
Ceftazidime/AVE1330A was two to four times more potent than ceftazidime alone against ceftazidime-susceptible Enterobacter strains. All the ceftazidime-resistant isolates were re-classified as susceptible in the presence of AVE1330A, while ceftazidime/clavulanic acid, co-amoxiclav and piperacillin–tazobactam were inactive.
Against the indole-positive Proteeae and Serratia representatives, the activity of ceftazidime/AVE1330A was most noticeable with regard to maximum MIC, as no ceftazidime-resistant isolate was tested, except Morganella morganii, for which the MIC90 was 16 times lower in the presence of AVE1330A (1 mg/L). Combination with clavulanic acid was less active (MIC90 32 mg/L). Ceftazidime/AVE1330A produced the highest activity against Proteus mirabilis, all isolates being inhibited at 4 mg/L. As shown by the maximum MICs, clear antagonism (at least an eight-fold increase in the MIC of ceftazidime) was observed with clavulanic acid against several isolates of ceftazidime-susceptible E. cloacae, ceftazidime-susceptible Citrobacter and M. morganii. In contrast, AVE1330A did not produce any such antagonism.

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Effect of inoculum size [1]
As shown in Table 5, the MIC ranges of ceftazidime and ceftazidime/AVE1330A were 0.25–256 mg/L and 0.25–8 mg/L, respectively. For the ceftazidime/AVE1330A combination only, a two- to four-fold increase in MIC was observed when the inoculum size was increased from 5.3 to 9.5 log10 cfu/mL.

Isolation of β-lactamases [1]
P99 β-lactamase from Enterobacter cloacae 293HT6 was prepared from crude extract obtained after lysis by French press, and purified by phenyl boronic acid affinity chromatography, as described previously.10 Activities of enzymes were stabilized at 37°C in buffer (50 mM phosphate pH 7.0, 2% glycerol and 0.1 mg/mL bovine serum albumin).

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Inhibition [1]
Inhibition was determined spectrophotometrically at 37°C after 5 min pre-incubation, in the presence of 100 μM nitrocefin as substrate (extinction coefficient: 20 500 M−1 cm−1), and 1 nM TEM-1 or 0.42 nM P99 in a final volume of 0.2 mL. The concentration of inhibitor needed to reduce the initial rate of hydrolysis of substrate by 50% (IC50) was recorded as the residual activity of β-lactamase at 485 nm. Data were processed using GraFit.

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Turnover [1]
The turnover number (Tn) was the number of inhibitor molecules required to inactivate one enzyme molecule. It was determined at 37°C, using different molar enzyme/inhibitor ratios at 10 and 60 min for TEM-1 and P99, respectively.11 These defined times corresponded to the minimal period of time taken to obtain maximal inhibition. Residual activity was measured with 400 μM nitrocefin as substrate. The Tn values were deduced from the extrapolated value for 99% inactivation from the plot of the residual activity versus inhibitor/enzyme ratios.

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Deacylation of the acylenzyme intermediate [1]
Enzyme (84 nM P99 or 200 nM TEM-1) was saturated by inhibitor at the turnover concentration for 10 min and 60 min in the case of TEM-1 and P99, respectively. Gel filtration using Sephadex G-50 micro-column was used to eliminate free inhibitor. Recovery of β-lactamase activity was measured at 485 nm in the presence of 100 μM nitrocefin at 37°C after appropriate enzyme dilutions and expressed as a percentage versus enzyme activity in the absence of inhibitor.

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In vitro antibacterial activity against β-lactamase-regulated producers [1]
In order to study the effect of variable amounts of β-lactamase on the inhibitory efficacy of AVE1330A, an isogenic panel of SHV-4- and AmpC-enzyme-producing E. coli was constructed in the common host XL-1 Blue strain {genotype: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′proAB lacIqZΔM15 Tn10 (Tetr)]} under an inducible arabinose promoter. The expression vector was pBAD18-kan with ArabinosePBAD promoter.12 Overexpression from pBAD was modulated using L-arabinose inducer concentrations ranging from 0.02% to 0.2%. MICs were determined by microdilution in Luria–Bertani broth in the presence of 1–2 × 107 bacterial cfu/mL after incubation at 37°C for 18 h.

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Susceptibility testing [1]
Standard MICs were determined by a two-fold agar dilution method in Mueller–Hinton medium. An inoculum of 104 cfu/spot was used throughout the study. All plates were incubated at 37°C for 24 h. The MIC was defined as the lowest concentration at which no visible growth could be detected on agar plates. According to the NCCLS breakpoints, MICs for ceftazidime-resistant and -susceptible strains were ≥32 and ≤8 mg/L, respectively.

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Effect of inoculum size [1]
A two-fold agar dilution method in Mueller–Hinton medium was used to evaluate the effect of five inoculum sizes (∼5–9 log10 cfu/mL) on the antibacterial activity of the combination ceftazidime/AVE1330A 4:1 against four strains which produced ESBL or AmpC enzymes. Bacterial enumerations were carried out by using a Spiral counter system to ascertain the inoculum sizes, obtained by appropriate dilutions from an overnight culture in Mueller–Hinton broth.

[1]. In vitro activity of AVE1330A, an innovative broad-spectrum non-beta-lactam beta-lactamase inhibitor. J Antimicrob Chemother. 2004 Aug;54(2):410-7.

Avibactam sodium is an organic sodium salt, the monosodium salt of avibactam. It is used in combination with ceftazidime pentahydrate to treat complicated urinary tract infections, including pyelonephritis. It is an EC 3.5.2.6 (β-lactamase) inhibitor with antibacterial and antimicrobial activity. It contains an avibactam (1-) domain. Ceftazidime has been shown to induce the expression of AmpC cephalosporinase, and some stable de-inhibiting bacterial mutants have been screened out, against which ceftazidime is ineffective. In fact, we found that AVE1330A inhibits the expression of these enzymes, preventing a sufficient number of enzymes from being induced, thus allowing ceftazidime to exert its activity. Furthermore, unlike ceftazidime/clavulanic acid, no antagonistic effect of ceftazidime/AVE1330A was observed in MIC studies of all tested strains, including chromosome-inducible AmpC enzyme-producing bacteria such as Citrobacter, Enterobacter, and Serratia. The absence of this antagonistic effect may indirectly indicate that the combination is unlikely to induce or screen for drug-resistant mutants. This is obviously a result of the non-β-lactam structure of AVE1330A. More specially designed studies are currently underway to confirm this observation. There is currently no extensive therapeutic experience published on the combination of cephalosporins and β-lactamase inhibitors. However, AVE1330A may be valuable as an alternative to carbapenem monotherapy. In summary, AVE1330A represents a new class of β-lactamase inhibitors that do not have significant antibacterial activity on their own, but are effective against both class A and class C β-lactamases, thus extending the antibacterial spectrum of ceftazidime to most drug-resistant bacteria. The combination of AVE1330A with β-lactam antibiotics is expected to become a highly promising drug in our antibacterial drug library and deserves further study. [1] Objective: The production of β-lactamases is the main mechanism by which Gram-negative bacteria develop resistance to β-lactam antibiotics. Despite the widespread use of clavulanic acid, sulbactam, and tazobactam, the prevalence of class A and class C enzymes continues to rise globally, necessitating the development of novel β-lactamase inhibitors. This article reports the antibacterial activity of a novel bridging bicyclic [3.2.1]diazabicyclic octanone compound, AVE1330A, in combination with ceftazidime. Materials and Methods: The inhibitory effect of AVE1330A on β-lactamases was characterized using IC50 values and hydrolysis kinetic parameters. The MIC values of over 600 bacterial strains were determined using the ceftazidime/AVE1330A combination at a fixed ratio of 4:1. Results: The IC50 values of AVE1330A against TEM-1 and P99 enzymes were 0.0023 mg/L (8 nM) and 0.023 mg/L (80 nM), respectively, while the IC50 values of clavulanic acid were 0.027 mg/L (130 nM) and 205.1 mg/L (1 × 10⁶ nM), respectively, and the IC50 values of tazobactam were 0.013 mg/L (40 nM) and 1.6 mg/L (5000 nM), respectively. The highly stable covalent complex resulted in a lower turnover rate of AVE1330A. The MIC value of ceftazidime/AVE1330A against Enterobacteriaceae was at least 8 times lower than that of ceftazidime alone. All strains of Escherichia coli, Klebsiella pneumoniae, Citrobacter and Proteus mirabilis, including ceftazidime-resistant strains, were inhibited at concentrations of 4-8 mg/L. Other Proteobacteriaceae, Enterobacteriaceae, Salmonellaceae and Serratiaceae bacteria were inhibited at concentrations of only 2 mg/L. Conclusion: Ceftazidime combined with AVE1330A has broad-spectrum antibacterial activity against Ambler A and C Enterobacteriaceae. [1]

C7H10N3NAO6S

287.22

287.018

C, 29.27; H, 3.51; N, 14.63; Na, 8.00; O, 33.42; S, 11.16

396731-20-7

1192491-61-4

24944097

Typically exists as solid at room temperature

1

6

3

18

462

2

S(=O)(=O)([O-])ON1C(N2[C@@H](C(N)=O)CC[C@H]1C2)=O.[Na+]

RTCIKUMODPANKX-UYXJWNHNSA-M

1S/C7H11N3O6S.Na/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)/q+1/p-1/t4-,5+/m0./s1

1,6-Diazabicyclo(3.2.1)octane-2-carboxamide, 7-oxo-6-(sulfooxy)-, monosodium salt, (1R,2S,5R)-rel-

ent-Avibactam sodium; Avibactam sodium; 1192491-61-4; Avibactam Sodium Salt; Avibactam sodium [USAN]; 9V824P8TAI; CHEBI:85982; AVIBACTAM SODIUM [JAN]; ...; 396731-20-7;

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.)