Determination of minimal steady-state plasma level of diazepam causing seizure threshold elevation in rats

Abstract

Objective: Diazepam, administered by the intravenous, oral, or rectal routes, is widely used for the management of acute seizures. Dosage forms for delivery of diazepam by other routes of administration, including intranasal, intramuscular, and transbuccal, are under investigation. In predicting what dosages are necessary to terminate seizures, the minimal exposure required to confer seizure protection must be known. Here we administered diazepam by continuous intravenous infusion to obtain near-steady-state levels, which allowed an assessment of the minimal levels that elevate seizure threshold.

Methods: The thresholds for various behavioral seizure signs (myoclonic jerk, clonus, and tonus) were determined with the timed intravenous pentylenetetrazol seizure threshold test in rats. Diazepam was administered to freely moving animals by continuous intravenous infusion via an indwelling jugular vein cannula. Blood samples for assay of plasma levels of diazepam and metabolites were recovered via an indwelling cannula in the contralateral jugular vein.

Results: The pharmacokinetic parameters of diazepam following a single 80-μg/kg intravenous bolus injection were determined using a noncompartmental pharmacokinetic approach. The derived parameters V_d , CL, t_1/2α (distribution half-life) and t_1/2β (terminal half-life) for diazepam were, respectively, 608 mL, 22.1 mL/min, 13.7 minutes, and 76.8 minutes, respectively. Various doses of diazepam were continuously infused without or with an initial loading dose. At the end of the infusions, the thresholds for various behavioral seizure signs were determined. The minimal plasma diazepam concentration associated with threshold elevations was estimated at approximately 70 ng/mL. The active metabolites nordiazepam, oxazepam, and temazepam achieved levels that are expected to make only minor contributions to the threshold elevations.

Significance: Diazepam elevates seizure threshold at steady-state plasma concentrations lower than previously recognized. The minimally effective plasma concentration provides a reference that may be considered when estimating the diazepam exposure required for acute seizure treatment.

Keywords: continuous infusion; diazepam; pharmacokinetics; seizure threshold; time intravenous pentylenetetrazol seizure test.

Figure 1

Plasma levels of diazepam and its metabolites nordiazepam, temazepam and oxazepam after an acute i.v. bolus injection of diazepam (80 µg/kg). Blood samples were collected 5, 10, 30, 60, 90, 120 and 240 min after the diazepam bolus. Data point represent mean ± S.E.M. of measurements in 8 animals. The levels of nordiazepam and temazepam were below the limit of detection in all animals at 90 and 240 min, respectively, and therefore no data values are shown at or after these time points.

Figure 2

Plasma levels of diazepam and its metabolites nordiazepam, temazepam, and oxazepam at various time points during continuous i.v. administration of diazepam at the indicated rates. Blood samples were collected at intervals equal to multiples of the terminal elimination half-life (t_½β) value (77, 154, 231 and 308 min). Data point represent mean ± S.E.M. of measurements in 6 animals. Diazepam area under the curve for time zero to last sampling time (AUC_0–308) increased linearly with dose [slope, 5,818 ± 471 (ng/ml • min) (mg/kg)⁻¹].

Figure 3

Effect of continuous intravenous infusion of diazepam (2, 4.5 and 9 µg/min for 4 half-lives or 308 min) on the threhold for myoclonic jerks, generalized clonus, and tonus in the timed intravenous PTZ test in rats. PTZ threshold was measured at the end of continuous diazepam infusion. The values indicate mean ± S.E.M. of measurements in 6 to 8 rats normalized with respect to the thresholds in the vehicle-treated groups, which for myoclonic jerks, clonus, and tonus were, respectively, 26.5 ± 2.6, 68.7 ± 10.9 and 111.7 ± 9.5 mg/kg. To assess the effect of the vehicle, threshold values were determined following infusion of a 24% cyclodextrin solution for 5 h in 3 rats, which resulted in threshold values of 24.1 ± 6.2, 29.1 ± 7.2 and 63.7 ± 22.7 mg/kg. *p < 0.05; ***p < 0.001; ****p < 0.0001 compared with the vehicle control group (ANOVA followed by Tukey’s Multiple Comparison test).

Figure 4

Plasma levels of diazepam and its metabolites nordiazepam, temazepam, and oxazepam at various time points after an intravenous loading dose and during continuous intravenous administration of diazepam (maintenance dose). The legend shows the dosing schemes. Blood samples were collected at 15, 30 and 77 min after the loading dose. The first 2 blood collections were during the maintenance phase and the last blood collection was at the end of the maintenance phase, which equals the mean t_½β value (Table 1). Data point represent mean ± S.E.M. of measurements in 6 animals. LD, loading dose; MD, maintenance dose. The levels of nordiazepam at 15 and 30 min were below the assay limit of detection. Diazepam area under the curve for first to last sampling time (AUC_15–77) increased linearly with dose [slope, 1,164 ± 101 (ng/ml • min) (MD mg/kg)⁻¹].

Figure 5

Effect of continuous intravenous infusion of diazepam at different loading and maintenance doses on myoclonic jerk, generalized clonus, and tonic extension in response to intravenous PTZ infusion in rats. PTZ threshold was measured at the end of continuous diazepam infusion. Bars indicate mean ± S.E.M. of values from 6 to 8 rats normalized with respect to the thresholds in the vehicle-treated groups (V), which (in milligrams per kilogram) for myoclonic jerk, clonus, and tonic extension, respectively, were 26.49 ± 1.65, 36.88 ± 3.48, 92.82 ± 6.97. **p < 0.001; ****p < 0.0001 compared with the vehicle control group (ANOVA followed by Tukey’s test).