PD study
Neutropenic mice were infected intramuscularly with 100 µL of inoculum bacteria suspension at a concentration of 1×106 to 107 CFU mL-1 in the left thigh 2 hrs before drug administration. A combination of nacubactam (0, 1.2, 3.6, 12, 36, 120, 360, 1,200 mg kg-1 day-1) and aztreonam (0, 1,200, 2,400, 4,800 mg kg-1 day-1) were subcutaneously administered from 2 to 26 hrs after bacterial inoculation (n = 3 per dose). In the dose fractionation study, aztreonam was administered every 2 hrs (q2h). In contrast, nacubactam was administered at a variable interval schedule (q2h, q4h, and q8h) from 2 to 26 hrs after bacterial inoculation (Fig. S1). In a dose-ranging study, each drug was administered in 12 divided doses (q2h) from 2 to 26 hrs after bacterial inoculation (Fig. S1). The control mice were euthanised by cervical dislocation at 0 h (2 h after bacterial inoculation). Other neutropenic mice were euthanised 24 h after initial drug dosing (26 h after bacterial inoculation). Then, the left thigh was aseptically collected and homogenised (1,800 rpm, 120 s, 18 °C) using Multi-Beads Shocker (Yasui Kikai Corp., Osaka, Japan). Serial dilution series of each homogenate were prepared, and an aliquot of each suspension was applied to Mueller-Hinton agar plates. After incubation at 37 °C for about 20 hrs, the number of colonies that grew was measured. The lowest detection limit in this method was 2.20 log10 CFU thigh-1.
PK/PD analysis withfT>MICi
The time-course aztreonam MICi after nacubactam administration was calculated by applying the time-course free plasma nacubactam concentration to the dose-response relationship equation. The percentage of time that the free plasma aztreonam concentration exceeded the MICi was defined as %f T>MICi (Fig. 1). The mean change in the bacterial count of each group obtained by in vivo PD study was plotted based on %f T>MICi and analysed using the Inhibitory Effect Sigmoid I maxModel [equation (2)].
E = E 0I max×(f T>MICi)ɤ ×{(f T>MICi)ɤ+ IC50ɤ }-1Eq.(2)
where E is the changes in the bacterial count,E 0 is bacterial counts atf T>MICi = 0%,I max is the maximum inhibitory effect, IC50 is the f T>MICiat which 50% of the maximum inhibition and ɤ is the sigmoid coefficient.