A Pharmacokinetic-Pharmacodynamic Study of Intravenous Midazolam and Flumazenil in Adult New Zealand White-Californian Rabbits (Oryctolagus cuniculus)

Abstract

Flumazenil, a competitive GABA_A receptor antagonist, is commonly used in rabbits to shorten sedation or postanesthetic recovery after benzodiazepine administration. However, no combined pharmacokinetic (PK) and pharmacodynamic (PD) data are available to guide its administration in this species. In a prospective, randomized, blinded, crossover study design, the efficacy of IV flumazenil (FLU; 0.05 mg/kg) or saline control (SAL; equal volume) to reverse the loss of righting reflex (LORR) induced by IV midazolam (1.2 mg/kg) was investigated in 15 New Zealand white rabbits (2.73 to 4.65 kg, 1 y old). Rabbits were instrumented with arterial (central auricular artery) and venous (marginal auricular vein) catheters. After baseline blood sampling, IV midazolam was injected (T0). Flumazenil or saline (FLU/SAL) was injected 30 s after LORR. Arterial blood samples were collected at 1 and 3 min after midazolam injection, and at 1, 3, 6, 10, 15, 21, 28, 36, 45 and 60 min after injection with flumazenil. Plasma samples for midazolam, 1-OH-midazolam and flumazenil were analyzed using high performance liquid chromatography-high-resolution mass spectrometry and the time to return of righting reflex (ReRR) was compared between groups (Wilcoxon test). FLU terminal half-life, plasma clearance and volume of distribution were 26.3 min [95%CI: 23.3 to 29.3], 18.74 mL/min/kg [16.47 to 21.00] and 0.63 L/kg [0.55 to 0.71], respectively. ReRR was 25 times faster in rabbits treated with FLU (23 [8 to 44] s) compared with SAL (576 [130 to 1141] s; 95%CI [425 to 914 s]). Return of sedation (lateral recumbency) occurred in both groups (7/13 in FLU; 12/13 in SAL) with return of LORR in a few animals (4/13 in FLU; 7/13 in SAL) at 1540 [858 to 2328] s. In the population and anesthesia protocol studied, flumazenil quickly and reliably reversed sedation induced by midazolam injection. However, the potential return of sedation after flumazenil administration warrants careful monitoring in the recovery period.

Figure 1.

Experimental timeline. Randomized, controlled, cross-over study design with each rabbit receiving flumazenil or saline, separated by 2 wk between treatments. Arterial blood samples collected and analyzed immediately after collection. n = 15 in each treatment group.

Figure 2.

Sedation levels assessed using a modified version of the sedation scale.

Figure 3.

Example of observed plasma midazolam concentrations over time following an intravenous bolus of midazolam (1.2 mg/kg) in a New-Zealand white-Californian cross rabbit (ID: rabbit1arm1). Circles indicate arterial sampling times.

Figure 4.

Example of observed plasma 1-OH-midazolam concentrations over time following an intravenous bolus of midazolam (1.2 mg/kg) in a New-Zealand white-Californian cross rabbit (ID: rabbit1arm1). Circles indicate arterial sampling times.

Figure 5.

Example of observed plasma flumazenil concentrations over time following an intravenous bolus of flumazenil (0.05 mg/kg) in a New-Zealand white-Californian cross rabbit (ID: rabbit1arm1). Circles indicate arterial sampling times.

Figure 6.

Time to return of righting reflex (ReRR) in 13 rabbits administered intravenous midazolam (1.2 mg/kg) followed by intravenous flumazenil (FLU, 0.05 mg/kg) or saline control (SAL, equal volume). Treatment (FLU/SAL) was administered 30 s after loss of righting reflex. Time to ReRR measured from FLU/SAL injection. Data are median (horizontal line) superimposed over individual data points. Time to ReRR significantly shorter in flumazenil group. (B) Scatter plot of sedation scale scores of rabbits administered intravenous midazolam (1.2 mg/kg) followed by intravenous flumazenil (FLU; 0.05 mg/kg) or saline control (SAL; equal volume). Treatment (FLU/SAL) was administered 30 s after loss of righting reflex and time of injection is represented by vertical broken line. Horizontal lines represent median. Sedation scores were significantly lower in FLU group at the 10 min postinjection time point.

Figure 7.

Mean arterial blood pressure (MAP) of rabbits administered intravenous midazolam (1.2 mg/kg) followed by intravenous flumazenil (0.05 mg/kg). Flumazenil was administered 30 s after loss of righting reflex and is represented by vertical broken line. Data are mean ± SD. MAP was lower than baseline (BL) at each timepoint, beginning 15 min post treatment injection (P < 0.001).