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CGS8216 noncompetitively antagonizes the discriminative effects of diazepam in rats
Life Sciences, Vol. 34, pp. 2589-2596 Printed in the U.S.A.
Pergamon Press
CGS8216 NONCOMPETITIVELY ANTAGONIZES THE DISCRIMINATIVE EFFECTS OF DIAZEPAM IN RATS Harlan E. Shannon and Sue L. Davis National Institute on Drug Abuse Addiction Research Center P.O. Box 12390 Lexington, KY 40583 U.S. Department of Health and Human Services Public Health Service Alcohol, Drug Abuse and Mental Health Administration (Received in final form April 16, 1984) Summary The benzodiazepine antagonist properties of CG88216 were evaluated in rats trained to discriminate between saline and 1.0 mg/kg of diazepam in a two-choice, stimulus-shock termination procedure. CGS8216 (0.3 to 100 mg/kg) administered alone, either s.c., p.o. or i.p., occasioned only saline-appropriate responding. When administered concomitantly with a constant 1.0 mg/kg dose of diazepam, CGS8216 produced dose-related decreases in drug-appropriate responding. CGS8216 was most potent by the i.p. route, and approximately tenfold less potent by the oral route. CGS8216 was dermatotoxic after s.c. administration. CGS8216 i.p. had a long duration of action. A dose of 30 mg/kg completely antagonized the discriminative effects of the 1.0 mg/kg training dose of diazepam when the antagonist was administered 8 hr before the start of the test session. In order to determine the type of antagonism by CGS8216, the dose-effect curve for diazepam was redetermined in the presence of varying doses of CGS8216 (0.3 to 3.0 mg/kg, i.p.). CGS8216 produced a dose-related rightward shift in the diazepam dose-effect curve, but also decreased the slope and appeared to decrease the maximal effect. These results are consistent with the interpretation that CGS8216 antagonizes diazepam in a noncompetitive manner. It may do so because either it interacts with a subpopulation of benzodiazepine receptors, it functions as a pseudoirreversible antagonist due to its high affinity, or because it is an antagonist with agonist properties. CGS8216 is a pyrazoloquinoline that potently inhibited [3H]flunitrazepam and [3H]diazepam binding to rat brain membranes in vitro and in vivo (1,2). [3H]CGS8216 specific binding was inhibited by benzodiazepine agonists and antagonists with an order of potencies similar to that observed with [3H]flunitrazepam or [3H]diazepam. However, CGS8216 exhibited a mixed-type inhibition of [3H]flunitrazepam binding and the calculated total number of binding sites for [3H]CGS8216 was less than the number for [3H]flunitrazepam ( i ) . These latter results suggested that CGS8216 may interact with a subpopulation of flunitrazepam binding sites ( i ) . Pharmacologically, CGS8216 is a benzodiazepine antagonist. It has been reported to antagonize most of the effects of benzodiazepine-like drugs, includin6 their anticonvulsant, anticonflict and ataxia-produeing properties (2,3,4). 0024-3205/84 $3.00 + .000
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Antagonism of Diazepam by CGS8216
Vol. 34, No. 26, 1984
The purpose of the present study was to evaluate the effectiveness of CGS8216 in antagonizing the discriminative stimulus properties of diazepam in rats. Firstly, the effectiveness of CGS8216 as an antagonist was compared when administered subcutaneously, orally or intraperitoneally. Secondly, the duration of action of CGS8216 was determined by administering graded doses of the antagonist i.p. at 0.5, 2, ~ or 8 hr before the start of the test session; a standard dose of diazepam (1.0 mg/kg) was administered 0.5 hr before the start of the test session. Finally, the type of antagonism was evaluated by determining dose-effect curves for diazepam in the presence of varying doses of CGS8216 and analyzing the results by the method of Schild (e.g., 5). With this method of analysis, a plot of the ratios of the ED50s of the agonist in the presence of varying doses of the antagonist to the ED50 of the agonist alone yields a straight line with a slope of -1.0 if the antagonism is of the competitive type. Methods Subjects: The subjects were 21 Fischer-derived F3~4 male rats (Harlan Industries, Indianapolis, IN) weighing 250 to 300 g at the start of discrimination training. Between experimental sessions, the rats were housed three per cage in a large colony room. Food and water were freely available. The lights in the colony room were illuminated between 6:00 a.m. and 6:00 p.m. Apparatus: Two-lever rat chambers (model ll01-L, Grason-Stadler Co., Inc., Bolton, MA) were enclosed in sound- and light-attenuating, ventilated enclosures. A clear Plexiglas partition separated the levers. Scrambled electric shock was delivered to the grid floor of the chamber by a constant current shock generator (model 700, Grason-Stadler). White noise was presented continuously throughout the session. Schedule contingencies were controlled and data recorded by a SCAT 3002/PDP8E system (BKP Scientific, Berlin, MA). Discrimination Training: The rats were trained under a FR5 schedule of stimulus-shock termination to respond on one lever after an injection of saline and on the other lever after an injection of 1.0 mg/kg of diazepam. In the presence of the houselight, the rats were required to emit 5 consecutive responses on the lever appropriate for the presession injection in order to terminate the houselight and shock presentation. Beginning 4.0 sec after the illumination of the houselight, shock (1.0 mA) was presented as 1.0-sec pulses with ~.0 sec between pulses until the consecutive response requirement was met. Completion of the FR requirement immediately extinguished the houselight, terminated the stimulus-shock complex and initiated a 45-sec timeout period during which the chamber was dimly illuminated by a red stimulus light. Responses on the inappropriate choice lever reset the ratio requirement. Sessions ended after 20 fixed-ratio components or 30 min, whichever occurred first. Drug Testing: Training sessions were conducted 5 days/week until the rats completed at least 90% of the total responses on the correct choice lever during eight consecutive sessions. The next two sessions (one diazepam and one saline session) were conducted as test sessions under nondifferential reinforcement of choice responding (i.e., completion of the consecutive response requirement on either choice lever terminated the stimulus-shock complex). A rat was considered to have acquired the discrimination if at least 90% of the responses during both test sessions were emitted on the choice lever appropriate for the presession injection. After the rats had met this criterion for acquisition of the discrimination, training sessions continued to be conducted on the first, second and fourth session of each week. Saline and diazepam were administered in single alternation across
Vol. 34, No. 26, 1984
Antagonism of Diazepam by CGS216
training sessions. A test session where choice lever terminated the stimulus-shock a n d f i f t h sessions of each week if the rat responses on the appropriate choice lever sessions; otherwise, an additional training
2591
5 consecutive responses on either complex was conducted on the third completed at least 90% of the total during the previous two training session was conducted.
Dru~s: Diazepam (a generous gift from Hoffmann-LaRoche, Nutley, NJ) was dissolved in saline pH 1.5. CGS8216 (2-phenylpyrazolo[4,3-c] quinolin-3(SH)one) (a generous gift from CIBA/Geigy, Summit, NJ) was dissolved in saline pH ll. Drug or drug vehicle was administered s.c., p.o., or i.p. typically 30 min before the start of the session in a volume of 1.0 ml/kg. In antagonism experiments, CGS8216 was injected a few seconds, 1.5 hr, 3.5 hr or 7.5 hr before diazepam (1.0 mg/kg), and the latter drug was always administered 0.5 hr before the start of the session. When two injections were given, they were administered by separate routes or at different s.c. sites. Doses of each drug, or the appropriate vehicle, were administered in a different mixed sequence in each rat. A given animal received all doses of a given doseeffect curve. Data Analysis: Discrimination data are expressed as the precentage of the total responses emitted on the diazepam-appropriate choice lever. The rats always completed all 20 FH components of each test session unless otherwise noted. The Schild analysis for CGS8216 against diazepam was done as described in detail previously for in vivo data (6,7). Briefly, an EDs0 was determined from linear regression for each curve, and the ratios of the ED50 for diazepam in the presence of each dose of CGS8216 to the ED50 of diazepam alone were calculated (i.e., dose ratio, or DR). The log(DR-l) was plotted as a function of the negative log(dose of CGS8216 in moles/kg). The line of best fit and its slope were determined by linear regression. Results Route of Administration: Administered alone, CGS8216 occasioned only saline-appropriate responding by the subcutaneous (Fig. l, left panel), oral (Fig. i, middle panel) and intraperitoneal (Fig. i, right panel) routes. However, doses of 30 and 100 mg/kg s.c. were dermatotoxic and produced purulent skin lesions. In each of the three groups, diazepam (1.0 mg/kg, s.c.) occasioned greater than 95% diazepam~apprcpriate responding. CGS8216 administered by each of the three routes concomitantly with 1.0 mg/kg of diazepam produced a dose-related reversal of the effects of diazepam. CGS8216 was most potent by the i.p. route: a dose of only 3.0 mg/kg was required to completely reverse the effects of diazepam. Orally, a dose of 30 mg/kg reduced the effects of diazepam to less than 30% drug-appropriate responding. Subcutaneously, a dose of l0 mg/kg of CGS8216 reduced the effects of diazepam to approximately 40% drug-appropriate responding; higher doses of CGS8216 were not tested s.c. because of its dermatotoxicity. Duration of Action: CGS8216 i.p. produced a dose-dependent as well as time-dependent antagonism of the discriminative effects of 1.0 mg/kg of diazepam (Fig. 2). Consistent with the findings reported above, a dose of 3.0 mg/kg was required to completely antagonize the effects of diazepam s.c. when both drugs were administered 0.5 hr before the start of the test session. As the time between CGS8216 and diazepam administration was increased, the dose of CGS8216 required to antagonize the effects of diazepam also increased. When administered 2 hr before the start of test sessions, 3.0 mg/kg of CGS8216 reduced the effects of diazepam to approximately 25% drug-appropriate responding. Doses of l0 and 30 mg/kg of CGS8216 administered ~ and 8 hr, respectively, before the start of test sessions completely antagonized the effects of 1.0 mg/kg of diazepam administered 0.5 hr before the start of test sessions.
2592
Antagonism of Diazepam by CGS8216
I: CGS 8216 (s.c.) + Diazepam (I.0 mg/kg)
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Vol. 34, No. 26, 1984
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FIG. 1 Dose-related antagonism by CGS8216 administered s.c., p.o. or i.p. of the discriminative effects of 1.0 mg/kg of diazepam s.c. Each point represents the mean of one observation in each of four or five rats. Abscissae: dose of CGS8216 in mg/kg. Ordinates: % diazepam-appropriate responses. Points above V represent the effects of CGS8216 vehicle alone (open symbols) or vehicle plus 1.0 mg/kg of diazepam (closed symbols). Vertical lines indicate ± 1 S.E.M. Schild Analysis of Antagonism: Diazepam administered alone occasioned dose-related increases in the percentage of responses emitted on the diazepamappropriate choice lever over the dose-range of 0.1 to 3.0 mg/kg (Fig. 3). Graded doses of CGS8216 (0.3 to 3.0 mg/kg) produced dose-related shifts in the dose-effect curve for diazepam. However, only the 0.56 mg/kg dose of CGS8216 produced a significant, parallel shift in the diazepam dose-effect curve. The slopes of the diazepam dose-effect curves decreased as the dose of CGS8216 was increased from 1.0 to 1.75 to 3.0 mg/kg, and each of these three curves were significantly nonparallel to the curve for diazepam alone. Further, the peak effect also appeared to decrease as the dose of CGS8216 increased. Doses of diazepam higher than those presented in Figure 3 disrupted the ability of the rats to respond, even in the presence of CGS8216, and could not be tested. The ratios of the ED50s in the presence of CGS8216 compared to the absence of CGS8216 are plotted according to the method of Schild in Figure 4. The slope of the regression line was -2.22, which was significantly different from the theoretical value of -1.0 which would be expected if CGS8216 interacted with diazepam in a competitive manner.
Vol. 34, No. 26, 1984
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FIG. 2 Time-related antagonism by CGS8216 i.p. of the discriminative effects of 1.0 mg/kg of diazepam s.c. CGS8216 was administered at the indicated times before the start of the test session; diazepam was always administered 0.5 hr before the start of the test session. Each point represents the mean of one observation in each of the same five rats. Abscissa: dose of CGS8216 in mg/kg. Ordinate: % diazepam-appropriate responses. Vertical lines indicate + 1 S.E.M. Discussion CGS8216 antagonized the discriminative effects of diazepam when the former drug was administered either subcutaneously, orally or intraperitoneally. CG88216 was most potent by the i.p. route; it was approximately tenfold less potent by the oral route. Although CGS8216 was at least somewhat effective as an antagonist by the s.c. route, its dermatotoxic effects would seem to contraindicate this route of administration. In addition, CG88216 had a long duration of action. A dose of 30 mg/kg of CGS8216 antagonized the effects of the 1.0 mg/kg training dose of diazepam when administered 8 hr before the test session. These findings are in agreement with previous reports that C088216 is a long-acting benzodiazepine antagonist devoid of typical benzodiazepine-like actions (2,3,4,8,9). CGS8216 exhibited a mixed-type of antagonism: it produced both a ri6htward shift in the diazepam dose-effect curve and also decreased the slope of the curve as well as the magnitude of the effect of diazepam. A Schild analysis of the antagonism also indicated CGS8216 is not a competitive antagonist of diazepam. In contrast, the benzodiazepine antagonist Ro15-1788 antagonized the discriminative effects of diazepam in a competitive manner (10). The present results in vivo are in excellent agreement with results in
2594
Antagonism of Diazepam by CGS8216
Vol. 34, No. 26, 1984
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FIG. 3 Dose-related shifts by CGS8216 i.p. in the diazepam dose-effect curve in rats trained to discriminate between saline and 1.0 mg/kg diazepam s.c. Each point represents the mean of one observation in each of five, six or twelve (diazepam) rats. Abscissa: dose of diazepam in mg/kg. Ordinate: % diazepam-appropriate responding. Vertical lines indicate + 1 S.E.M. Points above V + V represent the effects of diazepam vehicle plus CGS8216 vehicle. Points above V + C represent the effects of diazepam vehicle plus the indicated doses of CGS8216. vitro that CGS8216 exhibited a mixed-type inhibition of [3H]flunitrazepam binding (I). The latter authors interpreted their findings with CGS8216 as indicating that this antagonist may interact with a subpopulation of [3H]flunitrazepam binding sites. However, other interpretations are also possible. The noncompetitive antagonism of diazepam by CGS8216 may also be due to the high affinity and slow dissociation rate of the latter drug observed under some assay conditions (1). Drugs which dissociate slowly from binding sites can function as pseudoirreversible compounds. Irreversible blocking agents, by reducing the number of binding sites, would produce a family of curves similar to those for noncompetitive antagonists (ll). Indeed, the alkylatina benzodiazepine receptor ligands kenazepine and irazepine exhibited noncompetitive inhibition of [3H]diazepam binding in vitro (12,13). The initial parallel shift, or competitive type antagonism, observed with CGS8216 in the present study would be expected with a pseudoirrever~ible antagonist if there were a receptor reserve (ll). It has been estimated that for a half-maximal anticonvulsant effect of benzodiazepines, only 20-25% of the receptors are occupied (14). These latter findings are consistent with a receptor reserve for at least some of the pharmacologic effects of benzodiazepines. However,
Vol. 34, No. 26, 1984
Antagonism of Diazepam by CGS8216
2595
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~ ' ~ 4.5 5.0 5.5 6.0 6.5 -Log Dose of C G S 8 2 1 6 ( m o l e s / k g ) FIG. 4 Schild plot of the ratios of the ED50 doses of diazepam obtained in the presence and absence of the graded doses of CGS8216 from data presented in Figure 3. Abscissa: negative log of the dose of CGS8216 in moles/kg. Ordinate: log of the dose ratio minus 1. The line of best fit determined by linear regression had a slope of -2.22 and intercept on the abscissa of 5.74. the slow dissociation rate of CG88216 in vitro was observed only at 0oc; at 37oc, CGS8216 completely dissociated within 1 min. Moreover, the potency of CGS8216 steadily decreased with time, whereas a pseudoirreversible antagonist would be expected to maintain its potency for an extended period of time. Thus, it is doubtful that CGS8216 functions as a pseudoirreversible antagonist due to slow dissociation from benzodiazepine receptors in vivo. Within the theoretical framework of Ariens and colleagues (e.g., ii), mixed-type antagonists interact with two receptors. Within this context, diazepam would be an agonist at one receptor and CGB8216 a competitive antagonist at this same receptor. CG88216 also would act at a second receptor to noncompetltively inhibit the actions of diazepam at the first receptor. The empirical dose-effect curves obtained in the present study are in good agreement with theoretical dose-effect curves derived from this two-receptor model by Ariens et al. (ll). However, evidence for multiple binding sites for CGS8216 were not obtained by Czernik et al. (1).
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Antagonism of Diazepam by CGS8216
Vol. 34, No. 26, 1984
Another possible explanation for the noncompetitive type of antagonism by diazepam by CCS8216 is that this represents the interaction between an a~onist and an antagonist with agonist properties. CGS8216 has been reported to disrupt punished responding in rats (3,15), potentiate the convulsant effects of pentylenetetrazole and picrotexin in mice (16), and reduce social interactions in rats (17). These effects of CGS8216 are also produced by B-carboline inverse agonists (e.g., 18,19). However, the inverse agonist effects of B-carbolines are reversed by the benzodiazepine antagonist Re 15-1788 (18,19). In contrast, Ro 15-1788 reversed the effects of CGS8216 on pentylenetetrazoleinduced, but not picrotoxin-induced, seizures (16). Further, Ro 15-1788 did not reverse the disruption of punished responding by CGS8216 in rats (Katzman and Shannon, unpublished). Thus, CGS8216 may have decreased the slope of the dose-effect curves for diazepam in the present study because the former drug has agonist properties which do not appear to be mediated entirely by benzodiazepine receptors. References I.
2. 3. 4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17. 18. 19.
A.J. CZERNIK, B. PETRACK, H.J. KALINSKY, S. PSYCHOYOS, W.D. CASH, C. TSAI, R.K. RINEHART, F.R. GRANAT, R.A. LOVELL, D.E. BRUNDISH, and R. WADE, Life Sci. 30 363-372 (1982). N. YOKOYAMA, B. R!TTER, and A.D. NEUBERT, J. Ned. Chem. 25 337-339 (1982). P. BERNARD, K. BERGEN, R. SOBISKI, and R.D. ROBSON, The Pharmacologist 23 150 (1981). J.B. PATEL, C. MARTIN, and J.B. MALICK, Eur. J. Pharmacol. 86 295-298 (1983). 0. ARUNLAKSHANA and H.0. SCHILD, Br. J. Pharmacol. 14 48-58 (1959). S.E. SMITS and A.E. TAKEMORI, Br. J. Pharmaeol. 39627-638 (1970). R.J. TALLARIDA, A. COWAN and M.W. ADLER, Life Sci. 25 637-654 (1979). H.E. SHANNON and S. HERLING, Eur. J. Pharmacol. 92 155-157 (1983). H.E. SHANNON and S. HERLING, J. Pharmacol. Exp. Ther. 227 160-166 (1983). S. HERLING and H.E. SHANNON, Life Sci. 31 2105-2112 (1982). E.J. ARIENS, A.M. SIMONIS, and J.M. vanROSSUM, Molecular Pharmacology, Vol. I, E.J. AR!ENS (ed.), 287-393, Academic Press, New York (1964). P. SKOLNICK, M. SCHWERI, E. KUTTER, E. WILLIAMS, and S.M. PAUL, J. Neurochem. 39 1142-1146 (1982). E.F. WILLIAMS, K.C. RICE, S.M. PAUL, and P. SKOLNICK, J. Neurochem. 35 591-597 (1980). T. DUKA, V. HOELLT, and A. HERZ, Brain Res. 179 147-156 (1979). W.B. MENDELSON, T. DAVIS, S.M. PAUL, and P. SKOLNICK, Life Sci. 32 2241-2246 (1983). S.E. FILE, Neurosci. Lett. 35 317-320 (1983). S.E. FILE and R.G. LISTER, Pharmacol. Biochem. Behav. 18 185-188 (1983). M.O. CORDA, W.D. BLAKER, W.B. MENDELSON, A. GUIDOTTI, and E. COSTA, Proc. Natl. Acad. Sci. 80 2072-2076 (1983). S.S. TENEN and J.D. HIRSCH, Nature 288 609-610 (1980).
Pergamon Press
CGS8216 NONCOMPETITIVELY ANTAGONIZES THE DISCRIMINATIVE EFFECTS OF DIAZEPAM IN RATS Harlan E. Shannon and Sue L. Davis National Institute on Drug Abuse Addiction Research Center P.O. Box 12390 Lexington, KY 40583 U.S. Department of Health and Human Services Public Health Service Alcohol, Drug Abuse and Mental Health Administration (Received in final form April 16, 1984) Summary The benzodiazepine antagonist properties of CG88216 were evaluated in rats trained to discriminate between saline and 1.0 mg/kg of diazepam in a two-choice, stimulus-shock termination procedure. CGS8216 (0.3 to 100 mg/kg) administered alone, either s.c., p.o. or i.p., occasioned only saline-appropriate responding. When administered concomitantly with a constant 1.0 mg/kg dose of diazepam, CGS8216 produced dose-related decreases in drug-appropriate responding. CGS8216 was most potent by the i.p. route, and approximately tenfold less potent by the oral route. CGS8216 was dermatotoxic after s.c. administration. CGS8216 i.p. had a long duration of action. A dose of 30 mg/kg completely antagonized the discriminative effects of the 1.0 mg/kg training dose of diazepam when the antagonist was administered 8 hr before the start of the test session. In order to determine the type of antagonism by CGS8216, the dose-effect curve for diazepam was redetermined in the presence of varying doses of CGS8216 (0.3 to 3.0 mg/kg, i.p.). CGS8216 produced a dose-related rightward shift in the diazepam dose-effect curve, but also decreased the slope and appeared to decrease the maximal effect. These results are consistent with the interpretation that CGS8216 antagonizes diazepam in a noncompetitive manner. It may do so because either it interacts with a subpopulation of benzodiazepine receptors, it functions as a pseudoirreversible antagonist due to its high affinity, or because it is an antagonist with agonist properties. CGS8216 is a pyrazoloquinoline that potently inhibited [3H]flunitrazepam and [3H]diazepam binding to rat brain membranes in vitro and in vivo (1,2). [3H]CGS8216 specific binding was inhibited by benzodiazepine agonists and antagonists with an order of potencies similar to that observed with [3H]flunitrazepam or [3H]diazepam. However, CGS8216 exhibited a mixed-type inhibition of [3H]flunitrazepam binding and the calculated total number of binding sites for [3H]CGS8216 was less than the number for [3H]flunitrazepam ( i ) . These latter results suggested that CGS8216 may interact with a subpopulation of flunitrazepam binding sites ( i ) . Pharmacologically, CGS8216 is a benzodiazepine antagonist. It has been reported to antagonize most of the effects of benzodiazepine-like drugs, includin6 their anticonvulsant, anticonflict and ataxia-produeing properties (2,3,4). 0024-3205/84 $3.00 + .000
2590
Antagonism of Diazepam by CGS8216
Vol. 34, No. 26, 1984
The purpose of the present study was to evaluate the effectiveness of CGS8216 in antagonizing the discriminative stimulus properties of diazepam in rats. Firstly, the effectiveness of CGS8216 as an antagonist was compared when administered subcutaneously, orally or intraperitoneally. Secondly, the duration of action of CGS8216 was determined by administering graded doses of the antagonist i.p. at 0.5, 2, ~ or 8 hr before the start of the test session; a standard dose of diazepam (1.0 mg/kg) was administered 0.5 hr before the start of the test session. Finally, the type of antagonism was evaluated by determining dose-effect curves for diazepam in the presence of varying doses of CGS8216 and analyzing the results by the method of Schild (e.g., 5). With this method of analysis, a plot of the ratios of the ED50s of the agonist in the presence of varying doses of the antagonist to the ED50 of the agonist alone yields a straight line with a slope of -1.0 if the antagonism is of the competitive type. Methods Subjects: The subjects were 21 Fischer-derived F3~4 male rats (Harlan Industries, Indianapolis, IN) weighing 250 to 300 g at the start of discrimination training. Between experimental sessions, the rats were housed three per cage in a large colony room. Food and water were freely available. The lights in the colony room were illuminated between 6:00 a.m. and 6:00 p.m. Apparatus: Two-lever rat chambers (model ll01-L, Grason-Stadler Co., Inc., Bolton, MA) were enclosed in sound- and light-attenuating, ventilated enclosures. A clear Plexiglas partition separated the levers. Scrambled electric shock was delivered to the grid floor of the chamber by a constant current shock generator (model 700, Grason-Stadler). White noise was presented continuously throughout the session. Schedule contingencies were controlled and data recorded by a SCAT 3002/PDP8E system (BKP Scientific, Berlin, MA). Discrimination Training: The rats were trained under a FR5 schedule of stimulus-shock termination to respond on one lever after an injection of saline and on the other lever after an injection of 1.0 mg/kg of diazepam. In the presence of the houselight, the rats were required to emit 5 consecutive responses on the lever appropriate for the presession injection in order to terminate the houselight and shock presentation. Beginning 4.0 sec after the illumination of the houselight, shock (1.0 mA) was presented as 1.0-sec pulses with ~.0 sec between pulses until the consecutive response requirement was met. Completion of the FR requirement immediately extinguished the houselight, terminated the stimulus-shock complex and initiated a 45-sec timeout period during which the chamber was dimly illuminated by a red stimulus light. Responses on the inappropriate choice lever reset the ratio requirement. Sessions ended after 20 fixed-ratio components or 30 min, whichever occurred first. Drug Testing: Training sessions were conducted 5 days/week until the rats completed at least 90% of the total responses on the correct choice lever during eight consecutive sessions. The next two sessions (one diazepam and one saline session) were conducted as test sessions under nondifferential reinforcement of choice responding (i.e., completion of the consecutive response requirement on either choice lever terminated the stimulus-shock complex). A rat was considered to have acquired the discrimination if at least 90% of the responses during both test sessions were emitted on the choice lever appropriate for the presession injection. After the rats had met this criterion for acquisition of the discrimination, training sessions continued to be conducted on the first, second and fourth session of each week. Saline and diazepam were administered in single alternation across
Vol. 34, No. 26, 1984
Antagonism of Diazepam by CGS216
training sessions. A test session where choice lever terminated the stimulus-shock a n d f i f t h sessions of each week if the rat responses on the appropriate choice lever sessions; otherwise, an additional training
2591
5 consecutive responses on either complex was conducted on the third completed at least 90% of the total during the previous two training session was conducted.
Dru~s: Diazepam (a generous gift from Hoffmann-LaRoche, Nutley, NJ) was dissolved in saline pH 1.5. CGS8216 (2-phenylpyrazolo[4,3-c] quinolin-3(SH)one) (a generous gift from CIBA/Geigy, Summit, NJ) was dissolved in saline pH ll. Drug or drug vehicle was administered s.c., p.o., or i.p. typically 30 min before the start of the session in a volume of 1.0 ml/kg. In antagonism experiments, CGS8216 was injected a few seconds, 1.5 hr, 3.5 hr or 7.5 hr before diazepam (1.0 mg/kg), and the latter drug was always administered 0.5 hr before the start of the session. When two injections were given, they were administered by separate routes or at different s.c. sites. Doses of each drug, or the appropriate vehicle, were administered in a different mixed sequence in each rat. A given animal received all doses of a given doseeffect curve. Data Analysis: Discrimination data are expressed as the precentage of the total responses emitted on the diazepam-appropriate choice lever. The rats always completed all 20 FH components of each test session unless otherwise noted. The Schild analysis for CGS8216 against diazepam was done as described in detail previously for in vivo data (6,7). Briefly, an EDs0 was determined from linear regression for each curve, and the ratios of the ED50 for diazepam in the presence of each dose of CGS8216 to the ED50 of diazepam alone were calculated (i.e., dose ratio, or DR). The log(DR-l) was plotted as a function of the negative log(dose of CGS8216 in moles/kg). The line of best fit and its slope were determined by linear regression. Results Route of Administration: Administered alone, CGS8216 occasioned only saline-appropriate responding by the subcutaneous (Fig. l, left panel), oral (Fig. i, middle panel) and intraperitoneal (Fig. i, right panel) routes. However, doses of 30 and 100 mg/kg s.c. were dermatotoxic and produced purulent skin lesions. In each of the three groups, diazepam (1.0 mg/kg, s.c.) occasioned greater than 95% diazepam~apprcpriate responding. CGS8216 administered by each of the three routes concomitantly with 1.0 mg/kg of diazepam produced a dose-related reversal of the effects of diazepam. CGS8216 was most potent by the i.p. route: a dose of only 3.0 mg/kg was required to completely reverse the effects of diazepam. Orally, a dose of 30 mg/kg reduced the effects of diazepam to less than 30% drug-appropriate responding. Subcutaneously, a dose of l0 mg/kg of CGS8216 reduced the effects of diazepam to approximately 40% drug-appropriate responding; higher doses of CGS8216 were not tested s.c. because of its dermatotoxicity. Duration of Action: CGS8216 i.p. produced a dose-dependent as well as time-dependent antagonism of the discriminative effects of 1.0 mg/kg of diazepam (Fig. 2). Consistent with the findings reported above, a dose of 3.0 mg/kg was required to completely antagonize the effects of diazepam s.c. when both drugs were administered 0.5 hr before the start of the test session. As the time between CGS8216 and diazepam administration was increased, the dose of CGS8216 required to antagonize the effects of diazepam also increased. When administered 2 hr before the start of test sessions, 3.0 mg/kg of CGS8216 reduced the effects of diazepam to approximately 25% drug-appropriate responding. Doses of l0 and 30 mg/kg of CGS8216 administered ~ and 8 hr, respectively, before the start of test sessions completely antagonized the effects of 1.0 mg/kg of diazepam administered 0.5 hr before the start of test sessions.
2592
Antagonism of Diazepam by CGS8216
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Vol. 34, No. 26, 1984
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Vol. 34, No. 26, 1984
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FIG. 2 Time-related antagonism by CGS8216 i.p. of the discriminative effects of 1.0 mg/kg of diazepam s.c. CGS8216 was administered at the indicated times before the start of the test session; diazepam was always administered 0.5 hr before the start of the test session. Each point represents the mean of one observation in each of the same five rats. Abscissa: dose of CGS8216 in mg/kg. Ordinate: % diazepam-appropriate responses. Vertical lines indicate + 1 S.E.M. Discussion CGS8216 antagonized the discriminative effects of diazepam when the former drug was administered either subcutaneously, orally or intraperitoneally. CG88216 was most potent by the i.p. route; it was approximately tenfold less potent by the oral route. Although CGS8216 was at least somewhat effective as an antagonist by the s.c. route, its dermatotoxic effects would seem to contraindicate this route of administration. In addition, CG88216 had a long duration of action. A dose of 30 mg/kg of CGS8216 antagonized the effects of the 1.0 mg/kg training dose of diazepam when administered 8 hr before the test session. These findings are in agreement with previous reports that C088216 is a long-acting benzodiazepine antagonist devoid of typical benzodiazepine-like actions (2,3,4,8,9). CGS8216 exhibited a mixed-type of antagonism: it produced both a ri6htward shift in the diazepam dose-effect curve and also decreased the slope of the curve as well as the magnitude of the effect of diazepam. A Schild analysis of the antagonism also indicated CGS8216 is not a competitive antagonist of diazepam. In contrast, the benzodiazepine antagonist Ro15-1788 antagonized the discriminative effects of diazepam in a competitive manner (10). The present results in vivo are in excellent agreement with results in
2594
Antagonism of Diazepam by CGS8216
Vol. 34, No. 26, 1984
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FIG. 3 Dose-related shifts by CGS8216 i.p. in the diazepam dose-effect curve in rats trained to discriminate between saline and 1.0 mg/kg diazepam s.c. Each point represents the mean of one observation in each of five, six or twelve (diazepam) rats. Abscissa: dose of diazepam in mg/kg. Ordinate: % diazepam-appropriate responding. Vertical lines indicate + 1 S.E.M. Points above V + V represent the effects of diazepam vehicle plus CGS8216 vehicle. Points above V + C represent the effects of diazepam vehicle plus the indicated doses of CGS8216. vitro that CGS8216 exhibited a mixed-type inhibition of [3H]flunitrazepam binding (I). The latter authors interpreted their findings with CGS8216 as indicating that this antagonist may interact with a subpopulation of [3H]flunitrazepam binding sites. However, other interpretations are also possible. The noncompetitive antagonism of diazepam by CGS8216 may also be due to the high affinity and slow dissociation rate of the latter drug observed under some assay conditions (1). Drugs which dissociate slowly from binding sites can function as pseudoirreversible compounds. Irreversible blocking agents, by reducing the number of binding sites, would produce a family of curves similar to those for noncompetitive antagonists (ll). Indeed, the alkylatina benzodiazepine receptor ligands kenazepine and irazepine exhibited noncompetitive inhibition of [3H]diazepam binding in vitro (12,13). The initial parallel shift, or competitive type antagonism, observed with CGS8216 in the present study would be expected with a pseudoirrever~ible antagonist if there were a receptor reserve (ll). It has been estimated that for a half-maximal anticonvulsant effect of benzodiazepines, only 20-25% of the receptors are occupied (14). These latter findings are consistent with a receptor reserve for at least some of the pharmacologic effects of benzodiazepines. However,
Vol. 34, No. 26, 1984
Antagonism of Diazepam by CGS8216
2595
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~ ' ~ 4.5 5.0 5.5 6.0 6.5 -Log Dose of C G S 8 2 1 6 ( m o l e s / k g ) FIG. 4 Schild plot of the ratios of the ED50 doses of diazepam obtained in the presence and absence of the graded doses of CGS8216 from data presented in Figure 3. Abscissa: negative log of the dose of CGS8216 in moles/kg. Ordinate: log of the dose ratio minus 1. The line of best fit determined by linear regression had a slope of -2.22 and intercept on the abscissa of 5.74. the slow dissociation rate of CG88216 in vitro was observed only at 0oc; at 37oc, CGS8216 completely dissociated within 1 min. Moreover, the potency of CGS8216 steadily decreased with time, whereas a pseudoirreversible antagonist would be expected to maintain its potency for an extended period of time. Thus, it is doubtful that CGS8216 functions as a pseudoirreversible antagonist due to slow dissociation from benzodiazepine receptors in vivo. Within the theoretical framework of Ariens and colleagues (e.g., ii), mixed-type antagonists interact with two receptors. Within this context, diazepam would be an agonist at one receptor and CGB8216 a competitive antagonist at this same receptor. CG88216 also would act at a second receptor to noncompetltively inhibit the actions of diazepam at the first receptor. The empirical dose-effect curves obtained in the present study are in good agreement with theoretical dose-effect curves derived from this two-receptor model by Ariens et al. (ll). However, evidence for multiple binding sites for CGS8216 were not obtained by Czernik et al. (1).
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Antagonism of Diazepam by CGS8216
Vol. 34, No. 26, 1984
Another possible explanation for the noncompetitive type of antagonism by diazepam by CCS8216 is that this represents the interaction between an a~onist and an antagonist with agonist properties. CGS8216 has been reported to disrupt punished responding in rats (3,15), potentiate the convulsant effects of pentylenetetrazole and picrotexin in mice (16), and reduce social interactions in rats (17). These effects of CGS8216 are also produced by B-carboline inverse agonists (e.g., 18,19). However, the inverse agonist effects of B-carbolines are reversed by the benzodiazepine antagonist Re 15-1788 (18,19). In contrast, Ro 15-1788 reversed the effects of CGS8216 on pentylenetetrazoleinduced, but not picrotoxin-induced, seizures (16). Further, Ro 15-1788 did not reverse the disruption of punished responding by CGS8216 in rats (Katzman and Shannon, unpublished). Thus, CGS8216 may have decreased the slope of the dose-effect curves for diazepam in the present study because the former drug has agonist properties which do not appear to be mediated entirely by benzodiazepine receptors. References I.
2. 3. 4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17. 18. 19.
A.J. CZERNIK, B. PETRACK, H.J. KALINSKY, S. PSYCHOYOS, W.D. CASH, C. TSAI, R.K. RINEHART, F.R. GRANAT, R.A. LOVELL, D.E. BRUNDISH, and R. WADE, Life Sci. 30 363-372 (1982). N. YOKOYAMA, B. R!TTER, and A.D. NEUBERT, J. Ned. Chem. 25 337-339 (1982). P. BERNARD, K. BERGEN, R. SOBISKI, and R.D. ROBSON, The Pharmacologist 23 150 (1981). J.B. PATEL, C. MARTIN, and J.B. MALICK, Eur. J. Pharmacol. 86 295-298 (1983). 0. ARUNLAKSHANA and H.0. SCHILD, Br. J. Pharmacol. 14 48-58 (1959). S.E. SMITS and A.E. TAKEMORI, Br. J. Pharmaeol. 39627-638 (1970). R.J. TALLARIDA, A. COWAN and M.W. ADLER, Life Sci. 25 637-654 (1979). H.E. SHANNON and S. HERLING, Eur. J. Pharmacol. 92 155-157 (1983). H.E. SHANNON and S. HERLING, J. Pharmacol. Exp. Ther. 227 160-166 (1983). S. HERLING and H.E. SHANNON, Life Sci. 31 2105-2112 (1982). E.J. ARIENS, A.M. SIMONIS, and J.M. vanROSSUM, Molecular Pharmacology, Vol. I, E.J. AR!ENS (ed.), 287-393, Academic Press, New York (1964). P. SKOLNICK, M. SCHWERI, E. KUTTER, E. WILLIAMS, and S.M. PAUL, J. Neurochem. 39 1142-1146 (1982). E.F. WILLIAMS, K.C. RICE, S.M. PAUL, and P. SKOLNICK, J. Neurochem. 35 591-597 (1980). T. DUKA, V. HOELLT, and A. HERZ, Brain Res. 179 147-156 (1979). W.B. MENDELSON, T. DAVIS, S.M. PAUL, and P. SKOLNICK, Life Sci. 32 2241-2246 (1983). S.E. FILE, Neurosci. Lett. 35 317-320 (1983). S.E. FILE and R.G. LISTER, Pharmacol. Biochem. Behav. 18 185-188 (1983). M.O. CORDA, W.D. BLAKER, W.B. MENDELSON, A. GUIDOTTI, and E. COSTA, Proc. Natl. Acad. Sci. 80 2072-2076 (1983). S.S. TENEN and J.D. HIRSCH, Nature 288 609-610 (1980).