Quantitative analyses of antagonism: combinations of midazolam and either flunitrazepam or pregnanolone in rhesus monkeys discriminating midazolam

Abstract

Adverse effects of benzodiazepines limit their clinical use; these effects might be reduced without altering therapeutic effects by administering other positive GABA(A) modulators (i.e., neuroactive steroids) with benzodiazepines. One concern with this strategy involves reversing these combined effects in case of overdose. The current study examined whether flumazenil can attenuate the combined effects of two benzodiazepines, midazolam and flunitrazepam, and the combined effects of midazolam and the neuroactive steroid pregnanolone, in four monkeys discriminating midazolam. Each positive modulator produced ≥80% midazolam-lever responding. Interactions between midazolam and either flunitrazepam or pregnanolone were additive. Flumazenil antagonized the benzodiazepines when they were administered alone or in combination. Schild analyses yielded slopes that did not deviate from unity, regardless of whether benzodiazepines were administered alone or together; the pA(2) value for flumazenil was 7.58. In contrast, flumazenil enhanced the effects of pregnanolone with 0.32 mg/kg flumazenil shifting the pregnanolone dose-effect curve 2-fold leftward. Flumazenil attenuated the combined effects of midazolam and pregnanolone, although antagonism was not dose-dependent. Thus, the interaction between two benzodiazepines was similar to that of a benzodiazepine and a neuroactive steroid; however, flumazenil more efficiently attenuated a combination of two benzodiazepines compared with a combination of a benzodiazepine and a neuroactive steroid. Although the magnitude of antagonism of a benzodiazepine combined with a neuroactive steroid was reduced, these results support continued exploration of the use of combinations of positive modulators to enhance therapeutic effects while reducing adverse effects.

Fig. 1.

Discriminative stimulus effects of midazolam, flunitrazepam, and pregnanolone administered alone (left) and midazolam administered in combination with flunitrazepam or pregnanolone (right). Each dose-effect curve represents data from four monkeys; dose-effect curves shown in this figure for midazolam, flunitrazepam, and a combination of midazolam and flunitrazepam were determined in subjects RO, SA, LI, and NI, and dose-effect curves for pregnanolone and a combination of midazolam and pregnanolone were determined in subjects HE, SA, LI, and NI. Abscissa, left: dose in milligrams per kilogram body weight; right: dose expressed as multiples of ED₅₀ values, which are denoted by ×. In monkeys that received combinations of midazolam and flunitrazepam, the ED₅₀ value for midazolam when it was administered alone was 0.097 mg/kg and the ED₅₀ for flunitrazepam alone was 0.12 mg/kg. In monkeys that received combinations of midazolam and pregnanolone, the ED₅₀ value for midazolam when it was studied alone was 0.051 mg/kg and the ED₅₀ for pregnanolone alone was 3.45 mg/kg. Data above V represent the effects of vehicle. Ordinates, mean (±1 S.E.M.) percentage of total responding that occurred on the midazolam lever.

Fig. 2.

Discriminative stimulus effects of flunitrazepam administered alone (○) or after administration of various doses of flumazenil in three monkeys (subjects SA, LI, and NI). Abscissa, dose in milligrams per kilogram body weight; data above V represent the effects of vehicle. Ordinate, mean (±1 S.E.M.) percentage of total responding that occurred on the midazolam lever.

Fig. 3.

Discriminative stimulus effects of a combination of midazolam and flunitrazepam after administration of vehicle (○) or various doses of flumazenil in four monkeys (subjects RO, SA, LI, and NI). Abscissa, dose in milligrams per kilogram body weight with the top label on the abscissa indicating the dose of midazolam and the bottom label indicating the dose of flunitrazepam; data above V represent the effects of vehicle. Doses of midazolam and flunitrazepam were multiples of ED₅₀ values obtained when that drug was studied alone; those values were 0.097 mg/kg for midazolam and 0.12 mg/kg for flunitrazepam. Ordinate, mean (±1 S.E.M.) percentage of total responding that occurred on the midazolam lever.

Fig. 4.

Schild plots for flumazenil studied with midazolam, flunitrazepam, or a combination of the two benzodiazepines. Raw data are shown in Fig. 2 (flumazenil with flunitrazepam) and Fig. 3 (flumazenil with the combination of midazolam and flunitrazepam) or are not shown (flumazenil with midazolam). Abscissa, negative log of the molar dose of flumazenil. Ordinate, log(dose ratio − 1). DR, dose ratio.

Fig. 5.

Discriminative stimulus effects of pregnanolone administered alone (○) or after administration of various doses of flumazenil in four monkeys (subjects HE, SA, LI, and NI). Abscissa, dose in milligrams per kilogram body weight; data above V represent the effects of vehicle. Ordinate, mean (±1 S.E.M.) percentage of total responding that occurred on the midazolam lever.

Fig. 6.

Discriminative stimulus effects of a combination of midazolam and pregnanolone after administration of vehicle (○) or various doses of flumazenil in four monkeys (subjects HE, SA, LI, and NI). Abscissa, dose in milligrams per kilogram body weight with the top label on the abscissa indicating the dose of midazolam and the bottom label indicating the dose of pregnanolone; data above V represent the effects of vehicle. Doses of midazolam and pregnanolone were multiples of ED₅₀ values obtained when that drug was studied alone; those values were 0.051 mg/kg for midazolam and 3.45 mg/kg for pregnanolone. Ordinate, mean (±1 S.E.M.) percentage of total responding that occurred on the midazolam lever.