GABAA/a1 RECEPTOR SITE INVOLVEMENT IN THE HYPERPHAGIC EFFECT 0}: BENZODIAZEPINE ADMINISTRATION IN SQUIRREL MONKEYS
A Thesis Presented to the Faculty of the College of Science and Technology Morehead State University
In Partial Fulfillment of the Requirements for the Degree Master of Arts
by Angela Nicole Duke August2002 />1 s 1,/ r/f£S£S t'ol5.,~ D'877j
Accepted by the faculty 'of the College of Science and Technology, Morehead State University, in partial fulfillment of the requirements for the Master of Arts degree.
Director of Thesis
Master's Committee: ------'-L-A~-4~11----=/----'' Chair CJ=l /4_,f'!s+) j 2oOL- Date GABAA/a1 RECEPTOR SITE INVOLVEMENT IN THE HYPERPHAGIC EFFECT OF BENZODIAZEPINE ADMINISTRATION IN SQUIRREL MONKEYS Angela Nicole Duke, M.A. Morehead State University, 2002 Director ofThesis:.______.L____(;.L--=...=L._A"---+---.J%~~J-=.1---- ABSTRACT Benzodiazepines are effective pharmaceutical agents used to alleviate insomnia, anxiety, muscle tension and mild epileptic seizures; however, side effects such as increased appetite often deter long-term use of these compounds. Benzodiazepines act at multiple receptor sites and it has been suggested that the effects of these compounds may be related to their activity at specific receptor subtypes. An important focus of pharmacological research is to elucidate behavioral properties mediated by each receptor subtype in order to enable the development of compounds that retain therapeutic properties but have reduced unwanted side effects. The present study sought to determine the involvement of GABAA receptors containing the a 1 subunit (i.e., GABAA/a1 receptor) in the appetite-enhancing effects (i.e., hyperphagia) ofbenzodiazepines. Zolpidem, a GABAA/a1-preferring agonist, and t.riazolam, a nonselective benzodiazepine agonist, were evaluated for their abi lity to induce a hyperphagic effect. Five squirrel monkeys received var yi ng doses of uiazolam (0.003 - 0.3 mg/kg) and zolpidem (0.3 - 17.8 mg/kg). All monkeys demonstrated an increase in feeding behavior compared to baseline intake. To further assess the role of GABAA/a.1 receptors in the hyperphagic effects of benzodiazepines, the effects of triazolam and zolpi dem were re-determined following pre-treatments with varying doses of a nonselective benzodi azepine antagoni st, fl umazenil (0.003 - 10.0 mg/kg), and a GABAA/a. 1-preferring antagonist, ~-CCt (0.03 - 10.0 mg/kg). Flumazeni l antagonized tri azolam- and zolpidem-induced hyperphagia, suggesting this effect is mediated by benzodiazepine receptors. ~-CCt also blocked the increase in food intake induced by tri azolam and zolpidem, suggesting GABAA/a, 1 receptors play an important role in mediating benzodiazepine-induced hyperphagia. F uture pharmaceutical development of benzodiazepine compounds potentially should concentrate efforts to synthesize compounds with reduced activity at GABAA/a,1 receptors, in order to reduce the unwanted side effect of hyperphagia. Accepted by: ACKNOWLEDGEMENTS I would like to thank the members ofmy committee for their endless support, guidance, and patience. Thanks to Drs. Bruce Mattingly and James Rowlett for the opportunity to learn and work under their supervision and for the support I received throughout the completion of this thesis. I would also like to thank Dr. Dale Dickson for his continued guidance and support throughout my education. Thanks to Dr. Donna Platt who provided significant assistance and direction with the design and progression of this project. Special thanks to Dr. Annemarie Duggan and Bethann Johnson who assisted in the data collection and to Dr. Jim Cook who provided ~-CC!. This thesis was supported by the following NIH grants: DAI 1792, DA13591, and RR00168. TABLE OF CONTENTS Page Introduction...... 1 Methods...... 12 Results...... 17 Discussion...... 31 References...... 39 Endnotes...... 49 Appendix A: ED5o values for each subject under each condition...... 51 Appendix B: ANOVA summary tables...... 55 LIST OF FIGURES Figure Page 1. Illustration of a GABAA receptor with binding sites for GABA, benzodiazepines, and barbiturates ...... : ...... 5 2. The observation chamber equipped with chains, perches and foraging substrate...... 13 3. Food intake expressed as a percentage of control intake (Mean± SEM) plotted as a function oftriazolam dose...... 20 4. Food intake expressed as a percentage of control intake (Mean± SEM) plotted as a function ofzolpidem dose...... 21 5. Food intake expressed as a percentage of control intake (Mean ± SEM) plotted as a function of flumazenil dose ...... 23 6. Food intake expressed as a percentage of control intake (Mean± SEM) plotted as a function of ~-CCt dose ...... 24 7. Food intake (Mean± SEM) following the co-administration of triazolam with 0.03 mg/kg and 0.3 mg/kg offlumazenil...... 26 8. Food intake (Mean± SEM) following the co-administration of triazolam with 1.0 mg/kg and 3.0 mg/kg of ~-CCt...... 27 9. Food intake (Mean± SEM) following the co-administration of zolpidem with 0.03 mg/kg and 0.3 mg/kg offlumazenil...... 28 ii Figure 10. Food intake (Mean± SEM) following the co-administration of zolpidem with 1.0 mg/kg and 3.0 mg/kg !3-CCt...... 29 111 LIST OF TABLES Table Page 1. Differential effects ofbenzodiazepine-type drugs on food intake across several species...... 11 2. One-sample t-tests assessing significant increases in food intake compared to control intake following a range of doses of triazolam and zolpidem...... 22 3. One-sample t-tests assessing significant increases in food intake compared to control intake following a range of doses of flumazenil and ~-CCt...... 25 4. EDso values (Mean± SEM) and dose ratios calculated for each drug condition (includes 95% confidence intervals)...... 30 IV 1 I. Early Development of Se~ative-Anxiolytics In 1864, Adolf von Baeyer first synthesized barbituric acid from malonic acid and urea, which subsequently led to the development of a class of drugs now known as barbiturates (Palfai & Jankiewicz, 1997). This new class of drugs displayed 1 2 sedative , hypnotic , anticonvulsant, and anesthetic behavioral properties and were consequently prescribed for over seventy psychiatric disorders throughout the twentieth century. The first barbiturate synthesized was barbital in 1882, followed by the commonly-used compounds phenobarbital, pentobarbital and nearly 2,500 other barbiturates (McKim, 1997). Barbiturates exhibit effects similar to those of ethanol and were prescribed as sedative-anxiolytics3 (Pies, 1998; Snyder, 1986). Short-acting barbiturates were used as general anesthetics and in the treatment of insomnia, while long-acting compounds were used to treat anxiety disorders, such as generalized anxiety disorder and panic disorder. While barbiturates are safer than alcohol, they can be lethal at high doses or when combined with alcohol, and were found to produce dependence and withdrawal. When used to treat insomnia, barbiturates can interrupt REM (rapid eye movement) sleep and produce residual effects manifested as daytime drowsiness, clumsiness, and loss of motivation (Carvey, 1998; Palfai & Jankiewicz, 1997). Virtually all sedative anxiolytics have the ability to alleviate anxiety; however, the dose required to achieve this effect usually induces sedation. Consequently, intensive efforts to identify compounds that reduce anxiety without inducing sedation have been a major focus of pharmaceutical development since the 19th century. 2 Based on the development and use of chlorpromazine as a drug to treat psychiatric disorders in the mid-l 950s, Leo Stembach, a chemist at Hoffman LaRoche Laboratories, synthesized approximately 40 compounds referred to as quinazolines (Perrine, 1996). All but one of these compounds were evaluated using behavioral measures and were shown to be devoid of relevant behavioral effects. Consequently, the project was abandoned. However, in 1957, the last compound was submitted for behavioral testing as a result of routine laboratory cleaning (Palfai & Jankiewicz, 1997). The compound was shown to have anxiolytic, anticonvulsant, muscle relaxant, sedative and appetite-stimulating effects (Randall, Schallek, Heise, Keith, & Bagdon, 1960). The latter compound was named chlordiazepoxide and after additional behavioral testing, was marketed as Librium®, the first benzodiazepine (Palfai & Jankiewicz, 1997). Stembach followed this success with the development of diazepam (Valium®), which became the most prescribed drug of any kind after its introduction in 1963 (Palfai & Jankiewicz, 1997). Since their discovery, over 3,000 benzodiazepines have been synthesized and approximately 15 benzodiazepine compounds are currently in clinical use in the United States (Grilly, I 998; Palfai & Jankiewicz, 1997). Alprazolam (Xanax®) has recently replaced diazepam as the most prescribed anxiolytic (Grilly, 1998). Benzodiazepines used as sedative-anxiolytics are safe, long-acting drugs that are useful clinically as hypnotics, anxiolytics, muscle relaxants and anticonvulsants. However, acute and chronic administration ofbenzodiazepines can result in harmful 3 side effects that have hindered their usefulness as therapeutic agents. In this regard, benzodiazepine administration is associated with the potential for tolerance, dependence, withdrawal effects, and memory impairment (Ashton, 1991; Griffiths & Weerts, 1997; Lader, 1995; Marin, Salvatierra, & Ramirez, 1996; Mintzer & Griffiths, 1999): In addition, a number of clinically useful benzodiazepines have been reported to induce hyperphagia (see Table I). This increase in food consumption can be detrimental to an individual's treatment, potentially causing health problems related to weight gain and hindering compliance (Menkes, 1992; Schenck, Hurwitz, Bundlie, & Mahowald, 1991). II. GABAA Receptors and Benzodiazepine Action While benzodiazepines have been used extensively since their introduction in the 1960s, their specific action at the molecular level was, at the time, unknown. In the l 970's, researchers discovered that the mechanisms of action ofbenzodiazepines involved the neurotransmitter gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the nervous system (Snyder, 1986). In 1977, specific benzodiazepine receptor sites were found on GABA receptors. This discovery of benzodiazepine receptor sites gave rise to a neurochemical basis for anxiety, as well as the effects of benzodiazepines in the central nervous system. Subsequent research found that benzodiazepines do not compete with GABA, but rather enhance the action of GABA at the specific recognition sites (Gallager, Thomas, & Tallman, 1978). 4 While GABAA, GABAa and GABAc receptors have been identified in the mammalian nervous system, benzodiazepines act only at GABAA receptors (Sieghart, 1995). GABAA receptors are pentameric membrane proteins composed of five subunits that constitute a chloride ion channel (see Figure 1). When stimulated by GABA, the chloride channel opens to allow ,an inflow of chloride into the neuron resulting in hyperpolarization. In the presence of GABA, benzodiazepine agonists exert their action by binding to a specific recognition site on the GABAA receptor and allosterically modulating5 the efficiency of GABA (Luddens, Korpi, & Seeburg, 1995; Sanger et al., I 994). By increasing the frequency in which the chloride ionophore opens, benzodiazepines enhance the amplitude of miniature inhibitory postsynaptic currents (mIPSC) caused by the increase in chloride concentration in the neuron (Mohler, Fritschy & Rudolph, 2002). The enhancement ofmIPSCs decreases the likelihood that the postsynaptic cell will fire, resulting in greater inhibition of the postsynaptic neuron. Because benzodiazepines do not produce pharmacological action without the presence of GABA, they are a safer class of compounds compared to barbiturates, which have the ability to stimulate GABAA receptors at high doses without the presence of GABA (Sieghart, 1995). 5 Figure I GABA, benzodiazepines, and barbiturates bind to the GABA,1 receptor, a pentamer of five subunit proteins constituting a chloride ion channel. GABAA Receptor Of the subunit classes that have been identified, GABAA receptors typically consist of two a, two ~, and one y subunit (Pritchett et al., 1989; Rudolph, Crestani, & Mohler, 2001). Receptors containing these subunits are sensitive to benzodiazepines; however, the a subunit seems to determine the benzodiazepine pharmacology of the receptor (Doble & Martin, 1992; Pritchett et al., 1989). For example, receptors containing the a1 subunit (GABAA/a1 receptors) are sensitive to 6 zolpidem, zaleplon and CL 218,872; .however, these compounds have low or no affinity6 for GABAA receptors containing a:2, a:3, or as subunits (Atack, Smith, Emms, & McKernan, 1999; Doble & Martin, 1992; Noguchi, Kitazumi, Mori, & Shiba, 2002; Sieghart, 1995). While GABAA receptor subunits are ubiquitous throughout the central nervous system, particular subunits tend to cluster in certain regions (McKernan & Whiting, 1996). GABAA/a:1 receptors account for approximately 43% of all GABAA receptors and are localized to interneurons in most brain regions. GABAA/a:2 receptors are found in the spinal cord and limbic system and constitute approximately 26% of GABAA receptors. While GABAA/a:3 receptors are found predominately in the cerebellum and spinal cord (I 7%), GABAA/a:5 receptors only account for 4% of GABAA receptors and are found primarily in the hippocampus. This differential distribution suggests that distinct subtypes may mediate various actions of benzodiazepines. For example, a high concentration of GABAA/a:1 receptors in the cerebellar cortex suggests these receptors are involved in the motor-impairing effects ofbenzodiazepines (Rudolph et al., 2001). Likewise, a high concentration of GABAA/a:2 receptors in the limbic system suggests these receptors may be linked to the addictive properties ofbenzodiazepines, while GABAA/a:5 receptors in the hippocampus may mediate the anterograde amnesic effects. Because the hypothalamus is involved in eating behavior, the GABAA subunits occupying this brain region may be involved in the hyperphagic effect ofbenzodiazepines. 7 III. Mediation of Benzodiazepine Effects by GABAA Receptor Subtypes The diverse behavioral effects of benzodiazepines are thought to be mediated through varying affinities and stimulation of specific GABAA receptor subunits (Mohler et al., 2002; Rudolph et al., 200 I). Recently, genetically altered mice have been used to evaluate the behavioral effects associated with GABAA receptor subtypes. By rendering specific a subunits insensitive to benzodiazepine receptor ligands, evidence has been obtained suggesting GABAA/a1 receptors mediate the 7 sedative, motor-impairing and anticonvulsant actions ofbenzodiazepine agonists , while GABAA/a2 receptors mediate the anxiolytic and myorelaxant properties of these drugs (Crestani, Martin, Mohler, & Rudolph, 2000; Low et al., 2000; McKernan et al., 2000; Rudolph et al., 2001). While research using geneticalfy altered mice provides important behavioral information concerning receptor subtypes, this strategy also presents complicating factors (Rudolph et al., 2001). For example, genetic mutation potentially can cause adaptive changes in neuronal function and can alter the expression in neighboring genes that may confound research results and interpretations. An alternate strategy for studying the behavioral effects associated with different GABAA receptor subtypes involves the use of subtype selective compounds in non-genetically altered subjects. Investigations based upon this methodology provide consistent evidence ofa differential role ofGABAA/a1 and GABAA/a2 receptors. P-carboline-3-carboxylate-t-butyl ester (P-CCt) is a selective 8 8 benzodiazepine antagonist displaying a 21- and 26-fold selectivity for GABAA/a1 versus GABAA/a2 and GABAA/a3 receptors, respectively, and 154-fold selectivity for GABAA/a1 compared to GABAAl (Rowlett, Tornatzky, Cook, Ma, & Miczek, 2001). Moreover, /3-CCt antagonized anticonvulsant effects thought to be mediated by GABAA/a1 receptor activity, but failed to inhibit the myorelaxant effects ofbenzodiazepine agonists which are believed to be associated with GABAA/a2 receptors (Griebel et al., I 999; Shannon, Guzman, & Cook, I 984). Through the use of non-genetically altered subjects, these conclusions strengthen the interpretations obtained from transgenic mice and provide a more comprehensive analysis of GABAA receptor subunit function. N. Hyperphagic Effect ofBenzodiazepines As noted previously, benzodiazepine administration is associated with pronounced appetite enhancing effects. In several experimental studies, diazepam, alprazolam, and triazolam increased eating behavior in both naive human volunteers and volunteers reporting a history of drng abuse (e.g., Haney, Comer, Fischman, & Fol tin, 1997; Kelly, Foltin, King, & Fischman, I 992). Similarly, a cessation of nocturnal bingeing was observed in two case studies following a reduction of triazolam administration, suggesting the hyperphagic effect was due to the pharmacological effects oftriazolam (Menkes, 1992; Schenck et al., 1991). 9 Consistent with research documenting this enhancement of food intake in people, several benzodiazepines have been shown to produce a similar hyperphagic effect in animals (see Table 1). However, the mechanisms of action underlying the hyperphagic effects of benzodiazepines are not understood completely. The nonselective benzodiazepine antagonist flumazenil attenuated the hyperphagic effect produced by diazepam, chlordiazepoxde, and midazolam (Cooper & Yerbury, 1986; Foltin, Ellis, & Schuster, 1985; Mansbach, Stanley, & Barret, 1984; Sanger & Zivkovic, 1988). In addition, benzodiazepine inverse agonists9 that bind to the benzodiazepine receptor site but decrease the effects of GABA have been shown to induce dose-dependent reductions in food intake (Cooper, Barber, Gilbert, & Moores, 1985; Cooper & Yerbury, 1986). Because benzodiazepine-induced hyperphagia can be attenuated through the co-administration of a benzodiazepine antagonist, and inverse agonists suppress food consumption, research thus far suggests benzodiazepine receptors are involved in the appetite enhancing effects observed with the use ofbenzodiazepines. Recent research has revealed that benzodiazepine agonists with preferential action at GABAA/a1 receptors also produce a hyperphagic effect (see Table I). Although these studies are consistent with the involvement of the GABAA/a1 receptor in the hyperphagic effect of benzodiazepines, the available research is not entirely consistent. For example, several studies reported no increase in food consumption after administration of zolpidem in deprived and non-deprived rats (Sanger & 10 Zivkovic, 1988; Yerbury & Cooper, 1989; Davies et al., 1994). However, others have documented a hyperphagic effect following zolpidem administration in baboons (Griffiths, Sannerud, Ator, & Brady, 1992; Weerts, Ator, Grach, & Griffiths, 1998). Thus, at present, the involvement of specific GABAA subunit(s) in mediating the hyperphagic effect ofbenzodiazepine agonists is not clear. V. Purpose of the Present Study The purpose of this thesis was to further explore the role ofGABAA/a1 receptors in the hyperphagic effect ofbenzodiazepine agonists. In the present study, food intake following administration of triazolam, a prototypical nonselective benzodiazepine agonist, was compared to that ofzolpidem, a GABAA/a1-preferring agonist. To further characterize the role of GABAA/a1 receptors in the hyperphagic effects of benzodiazepine agonists, the effects of triazolam and zolpidem were evaluated in the presence of the GABAA/a1-preferring antagonist ~-CCt in comparison with antagonism produced by the reference nonselective antagonist flumazenil. Due to similarities in genotypes, the use of nonhuman primates will facilitate the generalization of these conclusions to humans. Thus, this study sought to document the hyperphagic effect in squirrel monkeys following the administration of 1) a nonselective benzodiazepine agonisi (triazolam), 2) a GABAJa1-preferring agonist (zolpidem), and 3) the combination of each of the agonists with a nonselective antagonist, flumazenil, and a GABAA/a,-preferring antagonist, ~-CC!, to examine the role of the GABAA/a1 receptors in the hyperphagic effect ofbenzodiazepine agonists. 11 Table I Differential Effects of Benzodiazepine-Type Drugs on Food Intake Across Several Species Effect on Nonselective Agonists Food Intake S2ecies Reference Triazolam + baboons 1, 2 Midazolam + n.d. rats 3,4,5 Clonazepam + n.d. rats 4 Chlordiazepoxide + rats, rhesus monkeys, 4, 6, 7, I 5 squirrel monkeys Diazepam + rhesus monkeys, squirrel 7,8,9,10 monkeys, dwarf goats, baboons, rabbits Brotizolam + dwarf goats, mice 8, 11 Climazolam + dwarf goats 8 Flunitrazepam + n.d. rats 12 Lorazepam + rhesus monkeys 7 CGS 9896 + n.d. rats 12 GABAA/o:l-2referring agonists CL 218,872 + n.d. rats, squirrel monkeys 4, 16 Zaleplon + baboons 2 Quazepam + mice I I Zolpidem +/0 baboons, mice, rats I, 6, 11, 12, 13, 14 Antagonists and Inverse Agonists Flumazenil 0 baboons 3, 10 f3-CCE 0 rabbits IO FG 7142 n.d. rats 3, 5 DMCM n.d. rats 3 +, increase; 0, no effect;-, decrease; n.d., non-deprived 1Griffiths et al., 1992; 2Ator, Weerts, Kaminski, Kautz, & Griffiths, 2000; 3Cooper et al., 1985; 4Cooper & Moores, 1985; 5Cooper & Yerbury, 1986; 6Sanger & Zivkovic, 1988; 7Kumar, Palit, Singh, & Dhawan, 1999; 8 Van Miert, Koot, & Van Duin, 1989; 9Foltin, Fischman, & Byrne, 1989; IOMansbach et al., 1984; 11 Perrault, Morel, Sanger, & Zivkovic, 1990; 12Davies et al., 1994; 13Weerts, Ator et al., 1998; 14Yerbury & Cooper, I 989; 15Weerts, Macey, & Miczek, I 998; 16Wettstein & Spealman, 1986 12 Methods Subjects Five adult male squirrel monkeys (Saimiri sciureus), weighing 750 to 950g, served as subjects in this study. Sessions were conducted daily between 9 a.m. and 2 p.m. Monday through Saturday. Between sessions, monkeys were individually housed with unrestricted access to food (Teklad Monkey Diet supplemented with fresh fruit) and water. Three monkeys had participated in an observational study in which the behavioral effects of dopamine receptor ligands were assessed. The remaining two subjects were experimentally naive at the start of the present study. All animals were maintained in accordance with the guidelines of the Standing Committee on Animals of the Harvard Medical School and the "Guide for Care and Use of Laboratory Animals" of the Institute of Laboratory Animal Resources, National Research Council, Department of Health, Education and Welfare Publication No. (NIH) 85-23, revised 1996. Research protocols were approved by the Harvard Medical School Institutional Animal Care and Use Committee. Drugs Triazolam base (Sigma/RBI; St. Louis, MO), zolpidem base (G. Dawson, Merck, Sharp & Dohme, Harlow, UK), flumazenil (Hoffman-La Roche, Inc., Nutley, NJ) and B-CCt base (J. Cook, University of Wisconsin - Milwaukee) were dissolved in small amounts of95% ethanol and 0.IN HCl as required and diluted to the desired concentrations with a 50% propylene glycol/50% saline solution. When possible, injection volumes were 0.1 ml/kg of body weight. The solubility limitations of 13 zolpidem precluded using the above iajection volume and higher doses ofzolpidem ( e.g., 10.0 or 17.8 mg/kg) were administered in the smallest volume possible depending on the size of the monkey (e.g., approximately 0.4 ml/kg ofbody weight). Apparatus Studies were conducted in a ventilated, transparent Plexiglas chamber (114 cm X 122 cm X 213 cm) located in a lighted room isolated from other animals (see Figure 2). The chamber was equipped with perches, suspended plastic chains, and a wood chip foraging substrate. Figure 2 The observation chamber was isolated from other monkeys and was equipped with chains. perches and foraging substrate. video camera 'it plastic chain perch bar foraging substrate metal bowl 14 Procedure The monkeys were initially habituated to the observation chamber and the haudling and injection procedures described below for a period of approximately one month. During this phase, saline was administered five minutes prior to the session, and each monkey was placed in the chamber with access to 100 food pellets (Noyes Precision Food Pellets, sucrose with fruit punch, 190 mg, Lancaster, NH) for ten minutes per day in order to establish baseline levels of food intake. Feeding between sessions was not restricted. Drug test sessions were conducted three times per week with saline control sessions on intervening days. A full rauge of doses oftriazolam (0.003 - 0.3 mg/kg), zolpidem (0.3 - 17.8 mg/kg), flumazeni! (0.003 - I 0.0 mg/kg), and P-CCt (0.03 - 10.0 mg/kg) were tested. Pretreatment intervals were chosen on the basis ofresults from preliminary studies (triazolam: 60 min, zolpidem: 10 min, tlui:nazenil and P-CCt: 40 min, Platt et al., in press). Antagoriism studies were also conducted in which selected doses offlumazenil (0.03 and 0.3 mg/kg) and P-CCt (1.0 and 3.0 mg/kg) were studied in combination with a range of doses oftriazolam and zolpidem. All drugs, including saline controls, were administered via intramuscular injections in the calf or thigh. Saline and drug test sessions both consisted often minute sessions in which I 00 food pellets were available as described above. Data Analysis Food intake was determined by measuring the number of pellets remaining in the food dish and bedding at the completion of the test session. Due to a small 15 number of subjects in the study and subsequent variability, food intake was converted to a percentage of control intake for each animal. The number of pellets consumed during drug test sessions was divided by the number of pellets consumed during preceding saline sessions and multiplied by 100. The percentage of control intake, averaged across subjects, was plotted as a function of dose for each drug and drug combination (mean±SEM). A one-way repeated measures analysis of variance (ANOVA) was computed for each agonist to determine differences between doses. To assess observed increases in intake, one-sample !-tests were used to compare the mean intake for each dose of agonist and antagonist to 100, which represented control intake. A significant difference between the mean intake and control intake indicated the corresponding dose produced a reliable change in eating. Doses corresponding to 50% of the maximum effect (ED5o) were calculated for each animal under all doses of agonists and antagonists using log-linear regression analysis. For this analysis, the percentage of control intake that corresponded to half of the maximum increase in food intake (Iso) was determined by adding 100 to each animal's peak percentage intake (EMAX) for each dose and dividing by two [Iso = (EMAX + 100)/2]. This value was used in the generated regression line equation to calculate the ED50 for an individual monkey. EDso values for some doses of antagonists could not be calculated if the amount of intake corresponding to 50% of the maximum effect of the agonist (Iso) was not reached.· EDso values were then converted to dose ratios to assess the magnitude of the shift of dose-response curves. Dose ratios for each drug condition were calculated by dividing the combined EDso 16 values of the agonist and antagonist by the ED50 value of the agonist alone. The magnitude of the shift between each agonist and the two corresponding doses of antagonists were assessed by calculating 95% confidence intervals for the dose ratios. If the lower and upper confidence interval limits of the corresponding antagonists did not overlap, the dose ratios were presumed significantly different indicating the antagonist reliably shifted the agonist dose-response curve. 17 Results The average food intake for each dose of triazolam was plotted as a function of triazolam dose and revealed a peak intake of 197% at a dose of O.03 mg/kg ( see Figure 3). One-sample t-tests were performed to determine whether increases in food intake were significantly different from the control intake percentage of I 00. As seen in Table 2, the 0.03 mg/kg dose oftriazolam was reliably different from control levels (! (4)=2.197, g < .05). A one-way repeated measures analysis of variance revealed no significant difference among doses oftriazolam (E (4, 16)=1.22, g > 0.5). Average food intake following doses of zolpidem was also plotted as a function ofzolpidem dose and revealed a peak intake of292% at a dose of3.0 mg/kg (see Figure 4). One-sample t-tests revealed doses of zolpidem ranging from 1.0 mg/kg to 10.0 mg/kg produced significant increases in intake relative to baseline (! (4)=3.274, IL< .05; 1 (4)=3.479, g,< .05; 1 (4)=3.302, g < .05, respectively). A one way repeated measures analysis of variance revealed a difference between doses of .zolpidem (E (4, 16)=7.24, g < .05). Fisher's post hoc analysis revealed that 1.0 mg/kg, 3.0 mg/kg, and I 0.0 mg/kg doses of zolpidem yielded a significantly greater intake than the 0.03 mg/kg dose. Similarly, the 3.0 mg/kg and 10.0 mg/kg doses yielded a significantly greater intake than 17.8 mg/kg ofzolpidem. The average food intake for each dose of flumazenil and P-CCt were plotted as a function of dose and did not reveal a clear trend for an increase or decrease in intake as dose increased (see Figures 5 & 6). One-sample !-tests confirmed that doses of flumazenil ranging from 0.003 mg/kg to I 0.0 mg/kg and doses of P-CCt ranging 18 from 0.03 mg/kg to I 0.0 mg/kg did not yield a reliable difference from control levels (see Table 3). The average food intake following administration of each agonist alone and when combined with two doses of each antagonist were also plotted. Pre-treatment with both doses of flumazenil as well as both doses of ~-CCt with each agonist yielded dose-response functions that were shifted to the right in a largely parallel fashion (see Figures 7-10). To further assess these rightward shifts, ED50 values were generated for each animal under each drug condition (see Appendix). The mean ED50 values for each agonist increased as doses of the antagonist increased (see Table 4). Dose ratios revealed the magnitude in which dose-response functions of agonists were shifted when combined with an antagonist (see Table 4). The co administration of0.03 mg/kg offlumazenil with various doses oftriazolam yielded a. dose ratio of3.9 (see Table 4, Figure 7). This dose ratio indicates that the ED50 of triazolam when in the presence of 0.03 mg/kg of flumazenil is approximately four times greater than the ED so of triazolam when administered alone. Triazolam co administered with 0.3 mg/kg offlumazenil yielded a dose ratio of 11.7, indicating that a 0.3 mg/kg dose offlumazenil shifted the dose-response function oftriazolam 11.7- fold to the right. Similarly, 1.0 mg/kg of~-CCt engendered a 7.8-fold rightward shift when combined with triazolam (see Figure 8). The co-administration of flumazenil and ~-CCt with zolpidem produced comparable increases in dose ratios (Figures 9 & I 0). A dose of 0.03 mg/kg of flumazenil shifted the dose-response function ofzolpidem 5.0-fold to the right, while 19 0.3 mg/kg offlumazenil produced an 8.9-fold increase. The zolpidem dose-response function was shifted to the right by a magnitude of 4.6- and 5.4-fold in the presence of 1.0 mg/kg and 3.0 mg/kg of ~-CCt, respectively. All dose ratios described above were significant by comparison of 95% confidence intervals (see Table 4). 20 Figure 3 Food intake (Mean+ SEM) expressed as a percentage of control food intake plotted as a function oftriazolam dose. A one-sample t-test confirmed the 0.03 mg/kg dose of triazolam produced a significant increase in intake compared to control intake, indicated by the asterisk(*). 400 '<) .,. 300 "' .....-" 0... * -"0 ,_u 200 -0 "...u ~" 100 0+------~------~------, 0.001 0.01 0.1 Triazolam Dose (mg/kg, i.m.) 21 Figure 4 Food intake (Mean + SEM) expressed as a percentage of control food intake plotted as a function of zolpidem dose. * Denotes significant increases in food intake compared to control intake, assessed using one-sample t-tests. 400 * .,. ""' 300 * ...... -" * 0.... E 0 ....u 200 0 E "....'-' ~" 100 0+------,------.------i 0.1 1 10 100 Zolpidem Dose (mg/kg, i.m.) 22 Table 2 Doses of triazolam and zolpidem produced significant increases in food intake compared to control food intake as assessed by one-sample !-tests. All analyses have 4 degrees of freedom (critical !-value= 2.132, alpha= .05). Triazolam Dose (mg/kg) Mean SEM t p .003 81.408 47.478 -0.392 ns .01 155.403 37.551 1.475 ns .03 197.230 44.249 2.197 <.05 .I 192.735 63.781 1.454 ns .3 108.678 58.693 0.148 ns Zolpidem Dose (mg/kg) Mean SEM t p .3 114.838 22.460 0.661 ns 1.0 238.087 42.175 3.274 <.05 3.0 292.198 55.238 3.479 <.05 10.0 254.670 46.845 3.302 <.05 17.8 175.558 38.753 1.949 ns 23 Figure 5 Food intake (Mean+ SEM) expressed as a percentage of control food intake plotted as a function of flumazenil dose. 400 ..s:" 300 ..sEl g C 0 u 200 '- 0 ~ C "~ p.." 100 0+------~-----~------y------~~ 0.001 0.01 0.1 10 Flumazenil Dose (mg/kg, i.m.) 24 Figure 6 Food intake {Mean+ SEM) expressed as a percentage of control food intake plotted as a function of B-CCt dose. 400 0) ~ 300 ....." ] 0" u 200 '-0 ~ 0) "g 0) p.. 100 0+------~------~------~-' 0.01 0.1 ~-CCt Dose (mg/kg, i.m.) 25 Table 3 Flumazenil and B-CCt did not reliably affect food intake when compared to control food intake as assessed by one-sample !-tests. All a_nalyses have 4 degrees of freedom (critical t-value = 2.776, alpha= .05). Flumazenil Dose (mg/kg) Mean SEM t p .003 91.511 23.103 -0.367 ns .01 93.973 41.867 -0. 144 ns .03 77.748 35.714 -0.623 ns .1 112.955 18.119 0.715 ns .3 151.465 33.389 1.541 ns 1.0 112.494 27.743 0.450 ns 3.0 150.329 22.699 2.217 ns 10.0 117.815 37.291 0.478 ns ~-CCt Dose (mg/kg) Mean SEM t p .03 83.063 6.646 -2.548 , ns .1 113.028 17.998 0.724 ns .3 81.761 31.410 -0.581 ns 1.0 67.265 31.539 -1.038 ns 3.0 118.435 15.433 1.195 ns 10.0 54.082 45.103 -1.018 ns 26 Figure 7 Food intake (Mean+ SEM) following the co-administration oftriazolam with 0.03 mg/kg and 0.3 mg/kg offlumazenil. 400 ...... Triazolam -0- + 0.03 Flumazenil ..../:r- + 0.3 Flumazenil .,.," 300 ..s-" e C -0 u 200 '- 0 -C "8 " "" 100 0-1------~~------~------~----....J 0.001 0.01 0.1 Triazolam Dose (mg/kg, i.m.) 27 Figure 8 Food intake (Mean+ SEM) following the co-administration oftriazolam with 1.0 mg/kg and 3.0 mg/kg of B-CCt. 400 -a- Triazolam -0- + 1.0 j3-CCt -1:::r- + 3.0 j3-CCt j 300 ..=i 8 ~ "0 .....u 200 0 ~ "'" p..~ 100 0-1------~------~------, 0.001 0.01 0.1 1 Triazolam Dose (mg/kg, i.m.) 28 Figure 9 Food intake (Mean+ SEM) following the co-administration of zolpidem with 0.03 mg/kg and 0.3 mg/kg offlumazenil. 400 .... Zolpidem -0- + 0.03 Flumazenil -b- + 0.3Flumazenil 3" 300 ..s 8 C -0 u <,... 200 0 -C "8 ~ 100 o+------.------,------0.1 1 10 100 Zolpidem Dose (mg/kg, i.m.) 29 Figure 10 Food intake (Mean+ SEM) following the co-administration ofzolpidem with 1.0 mg/kg and 3 .0 mg/kg of B-CCt. 400 ♦ Zolpidem -0- + 1.0 j3-CCt -£:r- + 3.0 j3-CCt ~" 300 ..s- ] i:: 0 u 200 ....0 ~ i:: "~ 11." 100 0+------,------~------; 0.1 IO l00 Zolpidem Dose (mg/kg, i.m.) 30 Table 4 ED22 values (Mean±SEM) and dose ratios" calculated for each drug condition. The upper and lower limits of95% confidence intervals (CI) confirm doses offlumazenil and B-CCt reliably shifted the agonist dose-response function to the right. EDso SEM Dose Ratio Lower CI Upper CI Triazolam 0.014 0.002 1.0 1.0 1.0 0.03 Flumazenil 0.042 0.009 3.9 2.9 4.9 0 .3 Fl umazenil 0.146 0.038 11.7 8.9 14.4 1.0 P-CCt 0.100 0.068 7.8 2.4 13.2 Zolpidem 0.910 0.167 1.0 1.0 1.0 0.03 Flumazenil 2.777 0.964 5.0 1.2 8.8 0.3 Flumazenil 6.264 1.424 8.9 3.4 14.4 1.0 P-CCt 3.893 1.680 4.6 3.6 5.6 3.0 P-CCt 3.882 1.361 5.4 3.2 7.6 • Dose ratios were calculated by dividing the ED so values of the agonist combined with the antagonist by the ED so value of the agonist alone. 31 Discussion Previous research has documented an increase in food intake following the administration of several nonselective benzodiazepine agonists (i.e., chlordiazepoxide, diazepam, midazolam) involving various species (see Table I). Consistent with these findings, the prototypical nonselective benzodiazepine agonist triazolam significantly increased the pellet intake of squirrel monkeys. An increase in food-maintained operant behavior has been reported for various benzodiazepine agonists in squirrel monkeys (Wettstein & Spealman, 1986); however, the present study measured hyperphagic effects more directly by evaluating pellet intake in non deprived monkeys, increasing external validity. The results of this thesis are also consistent with reports of nocturnal bingeing after triazolam use in people (Menkes, 1992; Schenck et al., 1991 ). While triazolam nonselectively binds to benzodiazepine sensitive GABAA receptors, it may have greater efficacy than other conventional benzodiazepines (Ducic, Puia, Vicini, & Costa, I 993). Altogether, these findings suggest the possibility for increased side effects of triazolam compared to other benzodiazepines, perhaps due to relatively high effica~y at GABAA receptors. A dose-dependent increase in food intake was also found following administration of the GABAA/a.,-preferring agonist zolpidem. Other compounds with selectivity for GABAA/a, 1 receptors, such as CL 218,872, zaleplon and quazepam, similarly increased food intake in rats and baboons (see Table !). However, research assessing the appetite stimulating effects ofzolpidem is inconsistent. Although an 32 increase in the number of pellets obtained under operant procedures used to evaluate the development of dependence and tolerance to zolpidem was documented (Griffiths et al., 1992; Weerts, Ator et al., 1998), other studies assessing the feeding behavior induced by zolpidem have reported no significant increase in food intake (Davies et al., 1994; Perrault et al., 1990; Sanger & Zivkovic, 1988; Y erbury & Cooper, 1989). It is important to note that the two studies reporting zolpidem-induced hyperphagia utilized non-human primates (Griffiths et al., 1992; Weerts, Ator et al., 1998), while others reporting no increase in intake following zolpidem administration involved the use ofrodents (Davies et al., 1994; Perrault et al., 1990; Sanger & Zivkovic, 1988; Yerbury & Cooper, I 989) suggesting that species differences may play a role in the inconsistent results with regard to zolpidem-induced hyperphagia. Species differences between non-human primates and rodents have previously been reported when assessing the behavioral effects of zolpidem. For example, research using baboons reported similarities between the discriminative stimulus effects of zolpidem and other benzodiazepines, while other studies using rats suggested discriminative stimulus effects of zolpidem were different from other benzodiazepines (reviewed in Griffiths et al., 1992; Rowlett & Woolverton, 1997). Further, tolerance to zolpidem has been documented in baboons, contrasting previous reports in which rats develop relatively less tolerance to zolpidem than other benzodiazepines. The nonselective benzodiazepine antagonist flumazenil did not significantly alter food intake when administered alone, consistent with previous reports (Cooper et al., 1985; Mansbach et al., 1984). However, flumazenil antagonized the • 33 hyperphagic effects oftriazolam and zolpidem in a surmountable fashion. Flumazenil has been found to antagonize hyperphagia and other behavioral effects associated with benzodiazepines when co-administered with triazolarn and zolpidem (Ator et al., 2000; Davies et al., 1994; Reddy & Kulkarni, 1998; Rowlett & Woolverton, 1996; Sanger & Zivkovic, 1988). Similarly, diazepam- and CL 218,872-induced increases in food-maintained operant responding in squirrel monkeys were antagonized by flumazenil (Wettstein & Spealman, 1986). The surmountable antagonism of hyperphagia by flumazenil suggests that this effect is mediated by GABAA/benzodiazepine receptors. Because flumazenil is nonselective, it is unclear the extent to which binding to a single or multiple GABAA receptor subtypes are responsible for this effect. ~-CC!, a GABAA/a1-preferring antagonist, did not significantly alter food intake when administered alone, consistent with previous research reporting no effects of this antagonism (Belzung, Le Guisquet, & Griebel, 2000; Lelas et al., 2002). However, ~-CCt antagonized triazolam- and zolpidem-induced hyperphagia in a surmountable fashion. Because ~-CC! is approximate 20-fold selective for GABAA/a1 receptors compared to other GABAA receptor subtypes, these findings suggest that hyperphagia observed following zolpidem and triazolarn administration involved mechanisms mediated by GABAA/a, receptors. ~-CCt has not been shown previously to antagonize benzodiazepine-induced hyperphagia, but has reportedly blocked behavioral effects induced by zolpidem, as well as nonselective agonists. For 34 example, P-CCt antagonized zolpidem-induced, but not triazolam-induced distress vocalizations in mouse pups (Rowlett, et al., 2001). P-CCt also blocked the anticonvulsant and locomotor effects of zolpidem and diazepam (Griebel et al., 1999; Shannon et al., 1984). Researchers originally suggested the hyperphagic effect following administration ofbenzodiazepines was a consequence of reduced anxiety (reviewed in Pecina & Berridge, 1996). This theory was supported by studies reporting an increase in the intake of novel food compared to familiar chow (Johnson, 1978) and increased food intake in an unfamiliar environment (Britton & Britton, 198 I). That is, benzodiazepines purportedly relieved stress and anxiety produced by an unfamiliar environment and/or foods that normally would inhibit eating. However, subjects do not exhibit behaviors indicative of agitation or distress during saline control tests (Weerts, Macey, & Miczek, 1998). Moreover, benzodiazepine agonists have produced hyperphagia in non-stressful conditions (i.e., tested in homecages) and increased the intake of standard chow as well as novel food (Cooper & McClelland, 1980; Griffiths et al., 1992; Kumar et al., 1999). Finally; agonists preferring GABAA/a.1 receptors, such as zolpidem and CL 218,872, relieve anxiety to a lesser degree than typical benzodiazepines, but show an increase in feeding, suggesting that a reduction of anxiety is not likely responsible for the hyperphagic effect (Mohler et al., 2002; Table 1). 35 Another hypothesis suggests that benzodiazepines activate mechanisms · related to feeding, possibly increasing appetite. Researchers supported this idea by reporting an increase in operant responding maintained by food delivery (i.e., Faltin et al., I 989). For example, benzodiazepines induced an increase in the rates of lever pressing to obtain food. In addition, benzodiazepine-induced hyperphagia has been documented in non-deprived, satiated animals (present results; see Table I). Taken together, these findings suggest that benzodiazepines increase appetite via stimulation ofGABAA receptors. As an alternative to changes in anxiety or appetite, Pecina and Berridge ( I 996) have suggested that benzodiazepines enhance the palatability10 of food. Support for this hypothesis is provided by reports of a higher increase in average intake of preferred foods as well as bitter, non-preferred food engendered by benzodiazepines (Cooper & Moores, 1985; Berridge & Treit, 1986). Using a procedure that quantified species-typical reactions to aversive and pleasurable types of food, Berridge and Treit (1986) found that administration of chlordiazepoxide increased "favorable" responses to preferred food. However, negative responses to aversive foods were not decreased, suggesting benzodiazepines may only enhance the "positive evaluation" of preferred foods. Human research provides further evidence that benzodiazepines alter palatability. Compared to triazolam, subjects given the benzodiazepine agonist zopiclone reported an altered perception of taste, a side effect that subsided after administration was discontinued (Fleming, McClure, Mayes, Phillips, & Bourgouin, 1990). 36 While GABAA receptors are ubiquitous throughout the central nervous system, the receptors located in the brainstem may be responsible for the increase in palatability stimulated by benzodiazepines. Microinjections of diazepam delivered to the brainstem (via the fourth ventricle) ofrats significantly enhanced the palatability of food compared to microinjections of diazepam in the forebrain (via the lateral ventricles), suggesting GABAA receptors located in the brainstem may play a significant role in the observed hyperphagia (Pecina & Berridge, 1996). However, due to the widespread distribution of GABAA receptors in the nervous system, other brain regions may play a role in benzodiazepine-induced hyperphagia. The hypothalamus is a region in the mammalian brain directly related to feeding behavior and appetite (Kandel, Schwartz, & Jessel!, 2000). However, the distribution and density of GABAA/a, receptors in the hypothalamus are relatively low to moderate compared to other brain regions (Fritschy & Mohler, 1995). A low concentration of these receptors in the hypothalamus suggests an increase in palatability, rather than appetite per se, may contribute to benzodiazepine-induced hyperphagia. The sensation of taste originates in the taste buds, is projected through the medulla oblongata, where the sensation is consolidated in the solitary nuclear complex, projected to the thalamus and on to the cerebral cortex (Kandel et al., 1991 ). Consequently, GABAA/a1 receptors are found in the thalamus and throughout the cortex (Mohler et al., 2002). Diazepam injected into the fourth ventricle may have stimulated GABAA/a, receptors in the medulla oblongata affecting, altering 37 neurotransmission to the thalamus and cortex, resulting in an increase in palatability. Instead of direct infusions in specific brain regions, the present study administered benzodiazepines via systemic injections, activating benzodiazepine receptors in many brain regions, including the cerebral cortex. GABAA/a1 receptors in the cerebral cortex, in addition to the brainstem, may be related to the taste pathway and capable of altering palatability when stimulated. Other brain regions should be investigated for further evidence of this theory. Benzodiazepine-induced hyperphagia can potentially facilitate the understanding of the neural mechanisms of appetite and feeding behavior as well as mechanisms involved in the behavioral effects of benzodiazepines. A better understanding of this effect can lead to benzodiazepines that either avoid this side effect, or new compounds that use hyperphagia as a treatment in eating disorders. For example, compounds used to relieve anxiety may be developed that are devoid of this side effect via decreased selectivity and/or efficacy at GABAA/a1 receptors. In addition, terminally ill patients with decreased appetite or an altered sense of taste may benefit from benzodiazepines used to stimulate feeding behavior. Benzodiazepines selective for other GABAA receptor subunits should be assessed in similar experiments to further characterize the role of GABAA/a1 receptors in benzodiazepine-induced hyperphagia. For example, L-838,417 and SL 65 I ,498 are benzodiazepine agonists with selectivity for GABAA receptors containing the a 2, a 3, and as subunits, while QH-ii-66 is an agonist with selectivity for GABAA/a5 38 receptors (Greibel et al., 2001; Huang et al., 2000; McKeman et al., 2000). 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Pharmacology Biochemistry and Behavior, 33, 303-307. 49 Endnotes 1 "Sedative" refers to an agent that induces a state of reduced alertness, decreased motor activity and relaxation; a state of consciousness during which the individual experiences short bursts of sleep-)ike electrical activity in the brain but is neither asleep or aroused. Sedative-hypnotic drugs are used to promote sleep. 2 "Hypnotic" drugs induce a state of drowsiness leading to sleep from which the individual can be aroused. 3 "Anxiolytics" are compounds used to relieve anxiety. Anxiety is defined as an unpleasant emotional state consisting of psychophysiological responses to anticipation of unreal or imagined danger and is applied to a cluster of symptoms: increased heart rate, alter_ed respiration rate, sweating, trembling, weakness and fatigue, feeling of impending danger, powerlessness, apprehension and tension. 4 "Hyperphagia" is an increase in food intake. 5An allosteric modulator is a compound ( e.g. benzodiazepine) that binds to a site on_ a receptor other than the neurotransmitter binding site. The compound can modify the activity of a receptor, or its affinity for its neurotransmitter. Allosteric modulators often do not have activity on their own. For example, the GABAA receptor is coupled with a chloride ion channel and has a binding site for GABA. However, it also has a separate binding site in which benzodiazepines bind. A benzodiazepine has no action alone, but in the presence of GABA, the agonist increases the frequency of which the er ion channel is opened, thus increasing the effect of GABA. 50 6 Affinity refers to the degree of attraction for a drug at a n,ceptor site, while efficacy refers to the ability of a drug to produce the desired therapeutic effect. 7 Agonist generally refers to a drug with the ability to stimulate physiological activity at these receptors, similar to that produced by naturally occurring substances. A benzodiazepine agonist is a compound that binds to the benzodiazepine receptor site and enhances the electrophysiological effect of GABA. 8 Antagonist generally refers to a drug that blocks or inhibits the effects of an agonist. Benzodiazepine antagonists are compounds that bind to the benzodiazepine receptor site with presumably no pharmacological effects when administered alone, but block the binding of pharmacologically active benzodiazepines to the receptor. 9 Benzodiazepine inverse agonists are compounds that bind to the benzodiazepine site and decrease the frequency of chloride ion channel opening. Inverse agonists decrease the effects of GABA and can produce opposite effects of benzodiazepine agonists, such as convulsions, insomnia and anxiety. 10 "Palatability" refers to a property or characteristic of food, denotes an agreeable or pleasurable taste. 51 Appendix A EDsa values were calculated for each animal in all drug conditions using the dose that produced the peak response. The EMAX value represents the maximum effect for the dose listed that produced the greatest increase in intake and is represented as a percentage of control intake. Isa is the percentage that corresponds to 50% of the maximum effect. ED so is the dose that corresponds to half of the maximum effect. Triazolam Subject Dose (mg/kg) EMAX¾ Isa% EDsa (mg/kg) 89-94 0.03 209 154 0.02 430-95 0.03 250 175 0.02 450-95 0.1 432 266 0.02 324-97 0.01 215 157 0.01 89-99 0.01 130 115 0.01 Triazolam + 0.03 Flumazenil Subject Dose (mg/kg) EMAx¾ Isa% EDsa (mg/kg) 89-94 0.1 150 154 0.1 430-95 0.1 241 175 0.1 450-95 0.1 324 266 0.03 324-97 0.03 168 157 0.02 89-99 0.1 105 115 52 Triazolam + 0.3 Flumazenil Subject Dose (mg/kg) EMAX¾ Iso % EDso (mg/kg) 430-95 0.3 219 175 0.2 450-95 0.3 432 266 0.2 I 324-97 0.1 192 157 0.1 ~/ 89-99 0.1 105 115 Triazolam + 1.0 P-CCt Subject Dose (mg/kg) EMAX¾ Iso % EDso (mg/kg) 430-95 0.3 206 175 0.2 ' ' t~ 450~95 0.3 194 266 324-97 0.1 376 157 0.01 _.,,..,.....,, ,89-99 0.1 151 115 0.1 Triazolam + 3.0 P-CCt Subject Dose (mg/kg) EMAX¾ lso % EDso (mg/kg) 430-95 0.3 156 175 450-95 0.3 209 266 324-97 0.3 145 157 89-99 0.1 86. -115 53 Zo,lpidem Subject Dose (mg/kg) EMAX¾ lso % EDso (mgikg) 430-95 3.0 -236 168 1.0 450-95 3.0 490 295 0.7 324-97 3.0 330 215 0.6 , 89-99 . 10.0 228 164 1.4 Zolpidem + 0.03 Flumazenil Subject Dose (mg/kg) EMAX¾ Iso % EDso (mg/kg) 430-95 17.8 175 168 450-95 17.8 533" 295 2.6 324-97 10.0 468'' 215 4 ..5 89-99 3.0 217 164 1.2 Zolpidem + 0.3 Flumazenil Subject Dose (mg/kg) EMAX¾ Iso % EDso (mg/kg) 430-95 10.0 241 168 4.9 450-95 10.0 374 295 7.7 324-97 10.0 430 215 89-99 3.0 163 164 54 Zolpidem + 1.0 P-CCt Subject Dose (mg/kg) EMAX¾ lso % EDso (mg/kg) 430-95 17.8 181 168 450-95 10.0 432 295 2.2 324-97 17.8 430 215 89-99 10.0 203 164 5.6 Zolpidem + 3.0 P-CCt Subject Dose (mg/kg) EMAx¾ lso % EDso (mg/kg) 430-95 17.8 164 168 450-95 3.0 403 295 1.6 324-97 10.0 399 215 3.7 89-99 10.0 198 164 6.3