A comparison of the behavioural effects of some hypotensive imidazoline derivatives in rats

EUROPEAN JOURNAL OF PHARMACOLOGY 9 (1969) 163-169. NORTH-HOLLAND PUBLISHING COMP., AMSTERDAM

A C O M P A R I S O N O F T H E B E H A V I O U R A L E F F E C T S O F SOME H Y P O T E N S I V E I M I D A Z O L I N E D E R I V A T I V E S IN R A T S R. LAVERTY Psychopharmacology Unit, Department of Pharmacology, WellcomeMedical Research Institute, University of Otago Medical School, Dunedin, New Zealand

Received 29 August 1969

Accepted 23 October 1969

R. LAVERTY, A comparison of the behavioural effects of some hypotensive imidazoline derivatives in rats, European J. Pharmacol. 9 (1969) 16~--169. Six related analogues of 2-(2,6-dichlorophenylamino)-2-imidazoline (clonidine, St 155) have been studied in rats for their hypotensive potency and for their effects on measures of rat behaviour including duration of chloral hydrate hypnosis, Y-runway activity, spontaneous cage activity, and conditioned avoidance response. Two compounds, St 600 (5-fluoro-2-methylphenylamino) and St 608 (2-chloro-3-methylphenylamino) had slightly less hypotensive activity than clonidine, but very much less action on the behavioural measures. Imidazolines

Hypotensive drugs

1. INTRODUCTION Clonidine ( 2-(2,6-dichlorophenylamino)-2-imidazoline HC1; St 155, Catapres, Boehringer Ingelheim) is a potent hypotensive drug of potential use clinically in the control of high blood pressure. Its potency and unique site of action in the central nervous system make it extremely interesting pharmacologically. However, clonidine has been reported to have other central nervous system effects in humans, (Ng et al., 1967; Iisalo and Laurila, 1967) and in animals (Laverty and Taylor, 1969; Morpurgo 1968). For this reason, a group of analogues of St 155 (clonidine) have been studied for their effects on rat behaviour and blood pressure, in order to determine whether the behavioural and hypotensive properties of these drugs are separable, and perhaps to find compounds having suitable hypotensive properties without marked central nervous system effects, that might warrant further investigation with a view to clinical use.

Rat behaviour

Clonidine (St 155)

2. METHODS Young adult rats were used throughout. Albino rats, randomly bred, were used for most behavioural and all brain noradrenaline studies. Albino rats from the New Zealand strain with genetic hypertension (Phelan, 1968) were used for the blood pressure studies, and randomly-bred black rats for most of the conditioned avoidance response experiments. All drugs were injected subcutaneously dissolved in saline solution and are expressed in terms of the weights of their salts, which were hydrochlorides except for St 375,612 and 678 which were nitrates; control rats received an equal injection of saline solution. Drugs were given chronically by adding the drug to a drinking fluid consisting of 5% glucose and 2.5% sodium chloride in tap water. Control animals received the same drinking fluid without the drug. Behavioural tests used were as follows: sleeping time after chloral hydrate (300 mg/kg i.p.) injection (Fastier, Speden and Waal, 1957); exploratory activ-

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R.LAVERTY

ity in a Y-runway (Steinberg, Rushton and Tinson, 1961); spontaneous nocturnal activity (Laverty and Meek, unpublished). The combined activity of rats, grouped normally 6 or 8 to a cage, was measured using a pen recorder at 3 intervals each of 1 hr duration during the night, usually at 1800, 2300 and 0500 hr, by detecting the capacitance changes due to slight movements in a flexible metal false floor of the cage; conditioned avoidance (pole-climbing)response (Cook and Weidley, 1957). Systolic blood pressures of lightly anaesthetized rats were measured using a tail-cuff technique (Phelan, 1968). The content of noradrenaline in rat brains was determined fluorometrically using an iodine oxidation technique (Laverty and Taylor, 1968).

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BEHAVIOURAL EFFECTS OF SOME HYPOTENSIVE IMIDAZOLINE DERIVATIVES

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3.2. Duration of chloral hydrate hypnosis In the doses used, only compounds St 464, and to a lesser degree, St 678 and 612 were like St 155 in causing a significant prolongation of the sleeping time after chloral hydrate administration (fig. 2). The drugs were administered subcutaneously 10 min prior to the intraperitoneal injection of chloral hydrate (300 mg/kg) and the animals kept in a room temperature of 29°C during the experiment. The other St compounds all caused a slight prolongation of sleeping time, but these sleeping times were not significantly different from that of their saline-injected control groups.

3.3. Y-runway activity All compounds reduced the exploratory rearing and entries of rats placed in the Y-runway, though St 155 and 464 were by far the most potent (fig. 3). All drugs reduced the number of faecal pellets dropped by the rats in the Y-runway. However, whether this is an effect of the drug on fear induced by the strange environment (Broadhurst, 1957) or a direct constipating effect of the drug (Ng et al., 1967; Laverty and Taylor, 1969) has yet to be established.

3.4. Effects on conditioned avoidance response As has already been shown (Laverty and Taylor, 1969), St 155 caused a marked inhibition of conditioned avoidance response (CAR) behaviour. This was also shown by St 464 given in approximately 5 times greater dosage, but all other compounds were only effective in doses of at least 20 times those of St 155 (fig. 4). It has also been shown that there is a marked intrastrain difference between white and black rats in the dose levels of St 155 required to inhibit CAR behaviour (Laverty and Taylor, 1969). The results in fig. 4 were obtained using black rats, which are easier to train, more consistent in their CAR behaviour and more sensitive to these drugs. A similar relationship between length of training and sensitivity to drugs has been reported recently (Latz, Bain and Kornetsky, 1969) within one strain. However, as can be seen in table 2, some analogues of St 155 are effective on CAR behaviour in white rats, though, as found earlier, approximately 5 times the dose was required to achieve a response comparable to that observed in black rats. Shortages of drug precluded extensive comparisons between the effectiveness of analogues in black and white rats.

BEHAVIOURAL EFFECTS OF SOME HYPOTENSIVE IMIDAZOLINE DERIVATIVES

167

Table 2 The effects of St 155 and analogues on CAR behaviour in white rats. The effect of the drug is measured as the number of CAR responses in 10 trials following the drug expressed as a percentage of the number of CAR responses in 10 trials obtained the day prior to drug treatment; the mean of these percentages was then calculated. Groups of 9 rats were used in all cases. Drug St 155 464

600

Dose (mg/kg) 2.5 0.5 1.0 2.5 5.0 1 2 10 25 50

Mean percentage effect (+ S.E.M.) 43 _+ 5 91+ 7 77 + 10 32 + 7 40 + 5 96+ 3 104+ 8 87 + 7 88 ± 10 68 ± 7

Fig. 5. Fighting behaviour induced in rats by chronic oral administration of St 464 (approximately 0.5 mg/rat/day).

3.5. Chronic administration of drugs Drugs were added for periods of 7 days or more to the drinking fluid of cages containing groups of 6 - 8 rats to observe chronic effects of these compounds. Drug-treated animals were often quieter than controls but measurement of nocturnal activity failed to reveal a significant difference with any analogue. Only St 464 produced a consistently reproducible and sustained fighting behaviour similar to that seen after chronic St 155 treatment (Laverty and Taylor,

1969), though a higher dose (15 mg/1 drinking fluid equivalent to approximately 0.5 mg/rat/day) was necessary (fig. 5). Fighting was also observed occasionally with St 375 (2 and 4 mg/rat/day) and with St 678 (4 mg/rat/day) but was not detected with St 600 (maximum dose taken 2 mg/rat/day), St 608 (4 mg/rat/day) or St 612 (2 mg/rat/day). Control animals drank approximately twice as much as the drug-treated animals; all treated groups drank the same amount (approx. 200 ml/day/6 rats) regardless of drug or dosage.

Table 3 A comparison of approximately equi-effective doses of St analogues. Doses are given as mg/kg.

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3.6. Effects on brain noradrenaline content Of the compounds only St 464 caused an increase in the levels of noradrenaline in the rat brain. In this respect it was similar to St 155 (Laverty and Taylor, 1969). Two hours after the injection of 5 mg/kg s.c., the mean whole brain noradrenaline content (+- S.E.M.) from a group of 5 rats was 0.41 -+ 0.03/1g/g, whereas the mean of 6 salineinjected rats was 0.30 -+ 0.06/~g/g; this difference was significant at p < 0 . 0 0 I. No other analogue tested 2 hr after doses of up to 50 mg/kg produced any significant change in brain noradrenaline content.

4. DISCUSSION Of the analogues of St 155 tested, St 464 most resembles St 155 in its spectrum of activities, though its potency is reduced by a factor of approximately 4. This similarity of action is probably related to the similarity in chemical structure, the change being the direct substitution of bromines for chlorines in the benzene ring. However, there are difficulties in relating chemical structure to pharmacological activity in the other members of the series. The hypotensive potency seems to follow the electro-negativity of the phenyl ring and substituents, the 2,6-dichloro derivative being the most potent, and the 2-bromo-4-methyl the least; on this basis one might expect St 678, the 2-bromo-4-chloro derivative to be more potent than, for example, the 2-chloro-4-methyl derivative, but this was not observed. Similarly the effects of the compounds on chloral hydrate sleeping time and Y-runway activity, being approximately in the same order as the hypotensive potency (table 3) and the CAR effects, do not seem to correlate with structure. The fighting behaviour does not appear in these experiments to correlate with the hypotensive or any of the other behavioural properties, nor with the effects on brain noradrenaline storage. Hence at this stage it does not seem possible to predict the pharmacological potency or spectrum of actions on the basis of chemical structure. All analogues shared the ability to produce piloerection, exophthalmos and perhaps constipation. This study has shown that it has been possible in this series of compounds to separate the hypotensive action o f an analogue from its central nervous effects

and also some aspects of drug-induced behaviour from other central nervous effects. In particular, St 600 and 608, while having less potency that St 155 as a hypotensive drug, have in rats very much less activity on central nervous system effects such as activity and CAR behaviour (table 3). If a similar differentiation of actions should occur in other animal species and in man, and the central nervous side effects o f St 155 should prove troublesome in clinical practice, then it is possible that St 600 or 608 may prove to be a useful drug for clinical use. Of these two analogues, St 608 may have less central nervous system depressant activity for an equal hypotensive activity, but more extensive quantitative studies would be needed to confirm this.

ACKNOWLEDGEMENTS Samples of the St compounds were kindly supplied through Dr. K. Higgins, Boehringer Ingelheim Pty Ltd., Australia. Capable technical asistance was provided by Mr. P.J. Arnott and Mrs. D. Farmer. This work was supported by grants from the Golden Kiwi Medical Research Distribution Committee and the Medical Research Council of New Zealand. Research on the hypertensive rat colony is supported by United States Public Health Service Grant no. HE 10942.

REFERENCES Broadhurst, P.L., 1957, Determinants of emotionality in the rat. I. Situational factors, Brit. J. Psychol. 48, 1. Cook, L. and E. Weidley, 1957, Behavioural effects of some psychopharmacological agents, Ann. N.Y. Acad. Sci. 66, 740. Fastier, F.N., R.N. Speden and H. Waal, 1957, Prolongation of chloral hydrate sleeping time by 5-hydroxytryptamine and by certain other drugs, Brit. J. Pharmacol. 12, 251. lisalo, E. and S. Laurila, 1967, A clinical trial with a new antihypertensive drug, St 155 (Catapresan), Current Therap. Res. 9,358. Latz, A., G.T. Bain and C. Kornetsky, 1969, Attenuated effect of chlorpromazine on conditioned avoidance as a function of rapid acquisition, Psychopharmacologia 14, 13. Laverty, R. and K.M. Taylor, 1968, The fluorometric assay of catecholamines and related compounds: improvements and extensions to the hydroxyindole technique, Anal. Biochem. 22,269.

BEHAVIOURAL EFFECTS OF SOME HYPOTENSIVE IMIDAZOLINE DERIVATIVES Laverty, R. and K.M. Taylor, 1969, Behavioural and biochemical effect of 2-(2,6-dichlorophenylamino)-2-imidazoline hydroehloride (St 155) on the central nervous system, Brit. J. Pharmacol. 35,253. Morpurgo, C., 1968, Aggressive behaviour induced by large doses of 2-(2,6-dichlorophenylamino)-2-imidazoline hydrochloride (St 155) in mice, Europ. J. Pharmacol. 3, 374.

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Ng, J., E.L. Phelan, D.D. McGregor, R. Laverty, K.M. Taylor and Sir Horace Smirk, 1967, Properties of Catapres, a new hypotensive durg: a preliminary report, New Zealand Med. J. 66,864. Phelan, E.L., 1968, The New Zealand strain of rats with genetic hypertension, New Zealand Med. J. 67,334. Steinberg, H., R. Rushton and C. Tinson, 1961, Modification of the effects of an amphetamine-barbiturate mixture by the past experience of rats, Nature 192, 533.