Apparent pA 2 Values of Benzodiazepine Antagonists and Partial Agonists in Monkeys 1

Drugs that bind to benzodiazepine recognition sites of g-aminobutyric acid type A receptor complexes may function as agonists in some behavioral assays and as antagonists in other behavioral assays. The present studies compared the effects of the benzodiazepines midazolam, flumazenil, bretazenil, Ro 417812, and Ro 42-8773 and the b-carboline, b-carboline-3carboxylate-t-butyl ester (b-CCt) under two different types of schedule-controlled responding in squirrel monkeys. One group of monkeys responded under a fixed-ratio schedule of stimulus-shock termination, and a second group of monkeys responded under a multiple fixed-ratio schedule of food presentation involving suppressed and nonsuppressed behavior. Under the schedule of stimulus-shock termination, midazolam produced dose-related decreases in response rate, and these effects were surmountably antagonized by flumazenil, bretazenil, Ro 41-7812, Ro 42-8773, and b-CCt. Schild plot analysis of these data revealed the following mean pA2 values: flumazenil, 7.18; bretazenil, 7.62; Ro 41-7812, 7.06; Ro 42-8773, 6.95. Apparent pA2 values were not calculated for b-CCt because the CL of the slope of the Schild plot included positive values. Under the multiple schedule, midazolam, bretazenil, and Ro 42-8773 dose-dependently increased rates of suppressed responding, whereas flumazenil, Ro 41-7812, and b-CCt had no significant rate-altering effects. Flumazenil antagonized the antisuppressant effects of midazolam and bretazenil; however, individual variability in these effects prohibited the determination of apparent pA2 values. These results indicate that in vivo pA2 values may be determined for benzodiazepine-site ligands. These results further demonstrate that some benzodiazepinesite ligands, e.g., bretazenil and Ro 42-8773, may function as both agonists and as competitive antagonists in vivo. Dose-ratio, or Schild, analysis can be used to identify competitive interactions between drugs, both in vitro and in vivo. If an antagonist produces dose-related, parallel, rightward displacements of an agonist dose-effect function, and if the slope of a Schild regression does not differ from unity, the relationship between drugs is presumed to be a competitive interaction at a homogeneous receptor population, and the resultant pA2 value provides an estimate of the KB of the antagonist. In studies with opioid drugs, the determination of in vivo, or apparent, pA2 values under different conditions have proven useful in characterizing receptor mechanisms that underlie specific opioid actions in whole animals. For example, Schild analysis of studies with the opioid antagonist quadazocine yields similar apparent pA2 values, approximately 7.4 to 7.5 for both the ventilatory depressant and the analgesic effects of alfentanil, supporting the idea that both effects are mediated by m-opioid receptors in nonhuman primates (Butelman et al., 1993). In contrast, Schild analysis of the ventilatory depressant and analgesic effects of ethylketocyclazocine (EKC) results in different apparent pA2 values (8.0 and 6.1, respectively), consistent with the hypothesis that m-opioid receptors mediate the ventilatory effects of EKC, whereas k-opioid receptor mechanisms mediate the analgesic effects of EKC (Dykstra et al., 1987; Butelman et al., 1993). Despite the success of Schild analysis in identifying receptor mechanisms that mediate the behavioral effects of opioids, apparent pA2 values have been infrequently calculated for benzodiazepine-receptor ligands. This is surprising, in view of the proposed involvement of multiple subtypes of g-aminobutyric acid type A (GABAA) receptors in mediating the effects of benzodiazepines (Lippa et al., 1979; Shannon et al., 1984b; Sanger, 1995). A possible explanation for the infrequent use of Schild analysis with benzodiazepine agonists and antagonists is that benzodiazepine-site ligands do not conform to the requirements of Schild analysis. Unlike opioid agonists, benzodiazepine-site agonists do not produce Received for publication February 19, 1999. 1 This work was supported by National Institutes of Health Grants DA11453 and DA03774. Preliminary results of this work were presented at the annual meetings of the American Society for Pharmacology and Experimental Therapeutics, San Diego, CA, 1997 and the College on Problems of Drug Dependence, Nashville, TN, 1997. ABBREVIATIONS: b-CCt, b-carboline-3-carboxylate-t-butyl ester; FR, fixed-ratio; GABA, g-aminobutyric acid; EKC, ethylketocyclazocine. 0022-3565/99/2903-1222$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 290, No. 3 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 290:1222–1229, 1999 1222 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals. org D ow nladed from direct cellular responses; rather, they modulate the effects of GABA at the GABAA receptor-channel complex. Thus, most measurable effects of benzodiazepines are indirect effects. Nonetheless, quantitative analysis has been applied to some data obtained with benzodiazepine-site ligands. For example, in one study of the antagonism of the anticonvulsant effects of diazepam in rats, an apparent pA2 value of 5.28 for flumazenil was reported (Kunchandy and Kulkarni, 1986), whereas in a subsequent study of the pentobarbital-like discriminative stimulus effects of benzodiazepines in rhesus monkeys, a mean apparent pA2 value of 6.56 for flumazenil was obtained with diazepam (Woolverton and Nader, 1995). More recently, studies of flumazenil in combination with midazolam in rhesus monkeys trained to discriminate midazolam from vehicle yielded two different apparent pA2 values; a value of 7.55 was obtained for the discriminative stimulus effects of midazolam in the presence of flumazenil, whereas an apparent pA2 value of 7.16 was calculated for response rate-decreasing effects (Lelas et al., 1999). Taken together, these results might suggest that different receptor subtypes mediate the anticonvulsant, discriminative stimulus and response rate-decreasing effects of benzodiazepine agonists. In a recent review, apparent pA2 and pKB values were calculated post hoc in an effort to quantitatively analyze data obtained with benzodiazepine-site ligands across different studies (Rowlett and Woolverton, 1996). The results of these analyses also suggested that the population of receptors that mediate the effects of benzodiazepine-site ligands was heterogeneous. However, the data from these studies often resulted in Schild plots with slope values that differed from unity or that contained both positive and negative values within the 95% CL. A rigorous experimental examination of the applicability of in vivo Schild analysis for benzodiazepine-site ligands has, to date, not been fully explored. The present studies were conducted, in part, to identify suitable conditions for quantitatively evaluating the behavioral effects of benzodiazepine-site antagonists. Using midazolam as a benzodiazepine-site full agonist, we compared the effects of flumazenil, the prototypical benzodiazepine antagonist, with the effects of b-carboline-3-carboxylate-t-butyl ester (b-CCt), an antagonist that may selectively bind GABAA complexes that contain a1 subunits, and bretazenil, Ro 428773, and Ro 41-7812, three drugs previously described as benzodiazepine-site partial agonists, on different types of schedule-controlled responding. The effects of all drugs on rates of lever-press responding were determined under a simple fixed-ratio (FR) schedule of stimulus-shock termination, and, in a separate group of monkeys, under a multiple schedule involving suppressed and nonsuppressed foodmaintained behavior. The antagonist effects of drugs thatdid not decrease response rates were next studied by determining how pretreatment modified the dose-effect function for midazolam under the schedule of stimulus shock-termination. Finally, the antagonist effects of flumazenil were studied by comparing its modification of the effectsof midazolam and bretazenil under the multiple schedule of suppressed and nonsuppressed behavior. The results of these pretreatment studies were then evaluated by dose-ratio analysis and, when meeting the assumptions of Schild analysis, the calculation of apparent pA2 values. Materials and Methods