Avibactam sodium (NXL-104) is a non-β-lactam β-lactamase inhibitor that inhibits TEM-1, P99, and KPC-2 β-lactamases (IC50=8/80/38 nM) covalently and reversibly. Avibactam sodium has antimicrobial activity and can be used to treat urinary tract infections.

TEM1:8 nM, P99:80 nM, KPC2:38 nM

方法：产 KPC 的肺炎克雷伯菌用 Avibactam sodium (1-64 mg/L) 和 Ceftazidime (0.0625-512 mg/L) 处理，通过 broth microdilution method 测定 MICs。
结果：CAZ-AVI 易感菌株和 CAZ-AVI 耐药菌株的 Ceftazidime MIC 值与 Avibactam 浓度的增加几乎呈对数线性下降。[1]

方法：为检测体内抗感染活性，将 Avibactam sodium (4-24 mg/kg，每 2 h 到每 12 h) 和 Ceftazidime 皮下注射给绿脓杆菌感染的 CD-1 小鼠。
结果：给药 Avibactam q2h 明显更有效，将 Avibactam 静效的日剂量降低了 2.7 和 10.1 倍，而两种方案的给药间隔中游离药物浓度保持在 1 mg/L 阈值浓度以上产生抑菌作用的平均百分比相似，平均值为 21.6 (q2h) 和 18.5 (q8h)。[2]

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 is diluted with 180 μL assay buffer to restore the original enzyme concentration. After two UFC treatments, the amount of free Avibactam is quantified by LC/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 a non-UFC-treated enzyme, and loss is found to be <5%[1].

Avibactam is prepared in sterile water[2].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].

Avibactam is reconstituted in sterile water to a stock solution of 5,120 mg/L and the further solution is prepared in Mueller-Hinton broth. Outbred female CD-1 mice, 7 to 8 weeks old and weighing 20 to 25 g, are used in the experiments. Eight dose combinations are used. For the thigh-infected animals, the combinations of Ceftazidime and Avibactam are 16/4, 8/1, 64/32, and 2/128 mg/kg. For the Lung-infected mice, combinations of 32/16, 4/2, 128/8, and 1/64 mg/kg of the respective constituents are used. These combinations are chosen in order to detect possible pharmacokinetic interactions between the two compounds (Ceftazidime and Avibactam) and to cover a wide range of doses of each compound.