Drug Development Research 22:331-337 (1991)

Reversal of Stress-Induced Arousal in Sleeping Rats by Some lmidazopyri m id i ne Benzod iazepi ne Receptor Ligands Vernon James and Colin R. Gardner

Roussel Laboratories, Covingham, Swindon, Wiltshire, England

ABSTRACT

James, V., and C.R. Gardner: Reversal of stress-induced arousal in sleeping rats by some imidazopyrimidine benzodiazepine receptor ligands. Drug Dev. Res. 22:331-337, 1991.

Five selected imidazopyrimidine ligands for benzodiazepine receptors which show non- sedative anxiolytic behavioral profiles, were tested for their ability to reverse arousal in- duced by intermittent footshock stress in sleeping rats. RU32698, RU32759, RU33203, and RU33543 showed dose-related inhibition of stress-induced arousal in the dose range 2-1 0 mg/kg orally, as did chlordiazepoxide. After 14 daily doses there was little or no decrease in the magnitude of this effect with RU32759 and RU32698, respectively. In contrast, RU32514 only weakly reversed stress-induced arousal at 10 mg/kg, a dose which is max- imally active in a conflict procedure in rats. It is suggested that RU32514 possesses only weak agonist activity at benzodiazepine receptors and this is insufficient for it to be effective in this test. RU32698 and RU32759 show potential for development as non-sedative anx- iolytics and the sub-acute dosing experiment suggests that they may not show marked tolerance.

Key words: stress, arousal, imidazopyrimidines, rat

INTRODUCTION

The stress-induced arousal model based on electrocorticogram (ECoG) recordings was originally developed to select potential hypnotic agents [James and Piper, 19781. Following

Received final version December 10, 1990; accepted December 20, 1990.

Address reprint requests to Dr. C.R. Gardner, Roussel Laboratories, Kingfisher Drive, Covingham, Swindon, SN3 5BZ, Wiltshire, England.

0 1991 Wiley-Liss, Inc.

332 James and Gardner

the development of benzodiazepine (BDZ) receptor agonists which do not possess sedative or muscle relaxant properties the predictivity of this model for sedative drug effects was ques- tioned. Non-sedative BDZ receptor agonists such as ZK91296, CGS9896, and RU43028 [Gardner, 1988al were able to reduce arousal evoked by intermittent footshock stress [Gardner and James, 19871. This raised the possibility that this response was related to the anxiolytic effects of these drugs. The intermittent nature of the footshock stress may result in uncertainty and thus a degree of anxiety. This would in turn lead to increased arousal. Whether or not reduction of stress-induced arousal results from an anxiolytic drug effect it is reversed by compounds with only weak agonist efficacy on BDZ receptors.

For this reason, some imidazopyrimidines which interact with BDZ receptors [Clem- ents-Jeweq et al., 19881 and which show different degrees of separation of anxiolytic and sedative/myorelaxant effects [Gardner, 1988a,b], were tested in this model. In addition, as sedative and myorelaxant effects of BDZs show far more rapid tolerance than their anxiolytic effects [File, 19851, two of the imidazopyrimidines with ideal profiles for potential clinical use (RU32698 and RU32759) were tested after their sub-acute administration.

MATERIALS AND METHODS

Male Wistar CFHB rats (200-275 g) were prepared for chronic electrocorticographic and electromyographic recording with implantation of four silver/silver chloride epidural elec- trodes and two disc nuchal electrodes. A minimum of 10 days was allowed for recovery from surgery. The animals were introduced into the sound-attenuated recording chambers, the temperature of which was controlled to 21°C. The chambers measured 24 X 24 X 20 inches and the light cycle was adjusted to 8 am-6 pm which was identical to that of the holding animal rooms. Food and water were available ad lib. The frontal-occipital ECoG and electromyogram (EMG) were recorded via nonrestraining recording leads on an eight channel polygraph (Mi- nograf EEG 8) and a four channel FM tape recorder (Racal Store 4). Analysis of different sleep stages was performed by the method of Johns et al. [1977]. Sleep was separated into its different phases mainly on the basis of frequency of the ECoG and amplitude of the EMG. Arousal was identified by very high amplitude EMG with low amplitude ECoG. This was distinguished from paradoxical sleep (PS) in that both ECoG and EMG amplitudes are low. In addition, under these recording conditions, a 5.5-8.5 Hz rhythm is detected during PS which allows definitive identification using a filter set with this range. Any slow wave activity associated with high EMG is artifactual, due to eating or grooming for example, and was included in arousal. Threshold amplitude levels for ECoG and EMG required by the automated analysis were varied for each recording until there was >95% agreement between the analysis of the computed (Digital MINC) and a visual analysis of a 2 hr sample of the recording, by the experimenter. The full experimental recording was then analyzed.

A 24-hr acclimatization period in the recording chamber then elapsed before any ex- perimental data were recorded. On the following test day rats were placed in the recording chambers at 8.30 am and allowed to sleep for 2.5 hr whilst ECoG and EMG recordings were taken. After this period the rats were dosed orally with either test drug or vehicle (deminer- alised water 2 ml/kg) and replaced in the chambers. Thirty minutes later a 30 sec train of electrical footshock pulses (0.5 mA, 15 msec width at 1 Hz) was delivered via the grid floor of the chambers. During the footshock period the ECoG and EMG recording circuits were automatically interrupted by a two-way relay switch system. The stress was then automatically repeated by a timer every 30 min for the remaining 5 hr recording.

All drugs were administered orally as sonicated and continuously stirred solutions or suspensions. The starting dose for each test compound was the lowest dose showing a maximal effect in a licking conflict procedure in rats in our laboratories [Gardner, 1988a,b]. Stressed and non-stressed control observations were interspersed with drug observations and each drug effect was compared to control data which were obtained over the same time period.

Imidazopyrimidines and Stressed Arousal 333

R 2 R 5

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R U 3 2 7 5 9 M e

R 6

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Fig. 1. Structures of the imidazopyrimidines chosen for study.

In experiments involving sub-acute dosing, rats were dosed daily at the usual dose time for 13 days and recordings with drug administration were performed on the 14th day. Parallel control groups were given vehicle under the same dose regime and tested either with or without stress. Parallel groups demonstrating the acute effects of the test compounds were given vehicle for 13 days and test drug on day 14.

RESULTS

Four of the five structures tested (Fig. 1) inhibited stress-induced arousal markedly and this effect continued throughout the 5 hr recording period. Such an effect of 10 mg/kg

334 James and Gardner

AROUSAL minl3omin

30

20

10

0

STRESS (S)

; S +RU32698

NONS

4 1 2 3 4 5 8 7

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Fig. 2. Effect of RU32698 and RU32514 (both at 10 mgikg orally) on stress-induced arousal in sleeping rats. The mean arousal stabilized at low levels as the rats began to sleep in the control period. The rats were dosed and the stress schedule commenced as indicated by the arrow. Stressed (S) rats (A) showed high levels of arousal throughout the recording period while non-stressed control rats (H) returned to sleep with low levels of arousal. In stressed rats RU32698 (*) reduced arousal to similar levels to non-stressed rats but RU32514 (+) showed only a weak and transient effect. n = 4 for all groups.

RU32698 is shown in Figure 2. In comparison, the equivalent chosen starting dose of RU32514 (10 mg/kg) showed only a slight reversal of stress-induced arousal over the first 3 hr of recording (Fig. 2). By taking the difference between the arousal in the stressed and non-stressed controls over the 5 hr recording after drug administration as loo%, mean per- centage reversal of stress-induced arousal was calculated for each drug group. This indicates that the effects of the four active imidazopyrimidines RU32698, RU32759, RU33203, and RU33543, were dose-related, as was the effect of chlordiazepoxide (CDZP) tested in com- parison (Fig. 3).

In rats dosed sub-acutely (14 days) levels of arousal in stressed or non-stressed controls were similar to those in single dose experiments. Similarly, the effects of the acute adminis- tration of 10 mglkg RU32759 were in the same range of magnitude as in single dose exper- iments, although RU32698 appeared to be less effective. In rats dosed sub-acutely with RU32698 (10 mg/kg) the reversal of stress-induced arousal on day 14 was similar to that in acutely-dosed rats (Fig. 4). In rats dosed sub-acutely with RU32759 (10 mglkg) there was still marked inhibition of stress-induced arousal although it was less than after acute dosing, particularly in the last 2 hr of recording (Fig. 4).

DISCUSSION

All the imidazopyrimidines tested showed maximal release of punished drinking in a rat conflict model at doses tested in these experiments [Gardner, 1988a,b]. However, although

Imidazopyrimidines and Stressed Arousal 335

--D- cow + RU33P03 --ti RU32608 + RU32750 -0- RU33543 - RU32514

mg/kg p.0.

Fig. 3. Dose-response curves for selected imidazopyrimidines showing mean percentage reversal of stress-induced arousal over the 5 hr stressed recording period, in comparison with the effects of CDZP. n = 4 for all groups.

RU32698, RU32759, RU33203, and RU33543 showed marked, dose-related inhibition of stress-induced arousal in the same dose range as the anticonflict effects, RU32.514 showed little reversal of stress-induced arousal at a dose which was fully effective in the conflict test [Gardner, 1988al. RU325 14 does not exhibit anticonvulsant, sedative, or myorelaxant effects and this behavioral profile suggests that it has only weak agonist efficacy which is weaker than that of the other compounds tested [Gardner, 1988a1. The ability of RU325 14 to antagonize the anticonvulsant action of diazepam in leptazol seizures in mice suggests that it is a weak partial agonist at BDZ receptors [Gardner and Guy, 19871. Therefore, although stress-induced arousal in this protocol can be reversed by non-sedative BDZ receptor ligands, either the other imidazopyrimidines in this study or other chemical classes [Gardner and James, 19871, it is not sensitive to partial agonists with very low efficacy. This stress-induced arousal model is sensitive to efficacies of BDZ receptor ligands detected by other behavioral models proposed as models of anxiety (e.g., some licking conflict protocols) and detects compounds claimed to be non-sedative anxiolytics [Gardner and James, 1987; Gardner, 1988~1. This does not con- stitute proof that it is a model of clinical anxiety but the possible induction of uncertainty due to the intermittent nature of the stress could lead to an anxiety-like state in the rats.

RU32698 is a partial agonist at BDZ receptors in that it can occupy virtually all BDZ binding sites in vivo without producing a sedative/myorelaxant effect as measured with a rotating drum in mice [Gardner, 1988~1. RU32759 has a very similar behavioral profile to RU32698 [Clements-Jewery et al., 19881. Full agonists at BDZ receptors (classical BDZs) show more rapid tolerance to their sedative and myorelaxant effects than to their effects associated with less net efficacy at BDZ receptors [Gardner, 1988~1 such as anticonvulsant or particularly anxiolytic effects [File, 19851. It may take several weeks of daily dosing in rodents

336 James and Gardner

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307

- 20-

-

10-

-

RU 32698

10

1 '

2 3 4 5 6 7 T I M E h

Fig. 4. Comparison of the effects of acute or sub-acute administration of RU32698 or RU32759 (both at 10 mgikg orally) on stress-induced arousal. Upper panel: control rats (stressed [Al, non-stressed [ +]) dosed sub-acutely with vehicle. Lower panels: The effects of each imidazopyrimidine after acute (0) or sub-acute (w) administration in stressed rats are shown. Animals were dosed and the stress schedule commenced as indicated by the arrow. n = 4 for all groups.

to demonstrate any tolerance in models of anxiety. Of particular interest is the recent finding that partial agonists at BDZ receptors do not show tolerance in their anticonvulsant effects against leptazol seizures, in contrast to classical BDZs when they are tested over the same period of time [Haigh et al., 1986; Haigh and Feely, 19881. RU32698 similarly does not show tolerance under these conditions [Feely et al., 19891. Neither RU32698 nor RU327.59 showed marked tolerance to their reversal of stress-induced arousal in this model. This could result

Imidazopyrimidines and Stressed Arousal 337

from either the fact that this model is associated with the detection of low BDZ receptor efficacy and/or that these compounds are partial agonists at BDZ receptors. In either case, it is unlikely that these compounds would show tolerance to their potential anxiolytic effects in clinical use.

REFERENCES

Clements-Jewery, S . , Danswan, G., Gardner, C.R., Matharu, S.S . , Murdoch, R., Tully, W.R., and Westwood, R.: (Imidazo [ 1,2-a]pyrimidin-2-yI)phenylmethanones and related compounds as po- tential non-sedative anxiolytics. J. Med. Chem. 31:1220-1226, 1988.

Feely, M., Boyland, P., Picardo, A., Cox, A., and Gent, J.P.: Lack of anticonvulsant tolerance with RU32698 and Ro17-1812. Eur. J. Phamacol. 164:377-380, 1989.

File, S.E.: Tolerance to the behavioral actions of benzodiazepines. Neurosci. Biobehav. Rev. 9: 113-121, 1985.

Gardner, C.R.: Pharmacological profiles in vivo of benzodiazepine receptor ligands. Drug Dev. Res. 12:l-28, 1988a.

Gardner, C.R.: Imidazoquinolines and pyrimidines: Benzodiazepine receptor ligands with different sep- arations of anxiolytic and CNS depressant effects. Psychopharmacology 96:(suppl):S16, 1988b.

Gardner, C.R.: Functional in vivo correlates of the benzodiazepine agonist-inverse agonist continuum. Prog. Neurobiol. 31:425-476, 1988c.

Gardner, C.R., and Guy, A.P.: Different effects on leptazol seizures in rats or mice of RU32514, a new benzodiazepine receptor ligand. Br. J. Pharmacol. 92:656P, 1987.

Gardner, C.R., and James, V.: Activity of some benzodiazepine receptor ligands with reduced sedative and muscle relaxant properties on stress-induced electrocorticogram arousal in sleeping rats. J. Pharmacol. Methods 18:47-55, 1987.

Haigh, J.R.M, and Feely, M.: Ro16-6028, a benzodiazepine receptor partial agonist, does not exhibit anticonvulsant tolerance in mice. Eur. J . Pharmacol. 147:283-285, 1988.

Haigh, J.R.M., Feely, M., and Gent, J.P.: Tolerance to the anticonvulsant effect of clonazepam in mice: No concurrent change in plasma concentration. J . Pharm. Pharmacol. 38:93 1-934, 1986.

James, V., and Piper, D.C.: A method for evaluating potential hypnotic compounds in rats. J. Pharmacol. Methods 1:145-154, 1978.

Johns, T.G., Piper, D.C., James, G. W.L., Birtley, R.D.N., and Fischer, M.: Automated analysis of sleep in the rat. Electroencephalogr. Clin. Neurophysiol. 43:103-105, 1977.