A comparison between astemizole and other antihistamines on sleep-wakefulness cycles in dogs

Nuur,lphorm~r,,llr,g, Vol Primed m Great Br~la!n

20. pp X53 10 859. 1’481 All rights rraerted

CopyrIght

028-3908 81/090853-0710200/O 0 1981 Perpamon Press Ltd

A COMPARISON BETWEEN ASTEMIZOLE AND OTHER ANTIHISTAMINES ON SLEEP-WAKEFULNESS CYCLES IN DOGS* A. WAUQUIER, W. A. E. VAN VEN BROECK, F. AWOUTERS and P. A. J. JANSSEN Department

of Pharmacology.

Janssen

Pharmaceutics (Accepfed

Research

30 Murch

Laboratories,

B-2340 Beerse, Belgium

1981)

SummaryThe effects of four reference antihistamines: ketotifen, diphenhydramine, chlorpheniramine and pyrilamine and of astemizole, a new potent and long-acting antihistamine. were studied on 16 hr sleepwakefulness patterns in the same dogs. Using a computer-based on-line analysis and automatic stage classification, a differentiation was made between wakefulness, transition to sleep (drowsiness), slow wave sleep and rapid eye movement (REM) sleep. The reference antihistamines significantly increased non-REM sleep. The reference antihistamines significantly increased non-REM sleep: diphenhydramine and chlorpheniramine increased drowsiness, ketotifen increased slow-wave sleep and pyrilamine increased both. All, but astemizole, significantly prolonged the latency to the first REM period, prolonged the interval between successive REM periods and suppressed the total amount of REM sleep. With all antihistamines, the effects were most pronounced for the first 4 hr and there was no within-night rebound. Chlorpheniramine had long-lasting effects throughout the night, especially on REM sleep. The effects on non-REM sleep might be due to a blockage of brain histamine HI receptors, whereas the effects on REM sleep might be due to the anticholinergic properties of the antihistamines. Astemizole was devoid of any significant effect on the sleepwakefulness pattern, in spite of its long-lasting antihistamine effects, suggesting that the common clinical side-effects of antihistamines. such as sedation and dryness of mucosal surfaces. will be lacking

One of the major clinical side-effects associated with the use of antihistamines is sedation (Goldstein, Murphree and Pfeiffer, 1968). This effect may be related to a blockade of central histamine HI receptors (Uzan, Le Fur and Malgouris, 1979). However, the central sedative action, specifically as far as nonphenothiazines is concerned. is poorly documented and is mainly based on clinical impressions. Further, only indirect evidence exists for a role of histamine as a central transmitter and its function is presently unclear (Schwartz, 1977). Antihistamines also possess central and peripheral effects which do not relate to their antihistaminic activity. Some antihistamines for instance, are active in tests for antidepressant activity (Barnett, Taber and Roth, 1969), alter metrazol-produced convulsions (Dashputra. Sharma, Jagtap, Khapre and Rajapurkar, 1966) or enhance the effects of intracranial self-stimulation (Wauquier and Niemegeers, 1981). In the present study, the effects of four reference antihistamines as compared with the new, potent and long-acting antihistamine astemizole (Van Wauwe.

Awouters, Niemegeers, Janssen, Van Nueten and Janssen, 1981) were assessed on the sleepwakefulness pattern in dogs. METHODS Subjects

The subjects were 5 adult male beagles approx. 2 years old at the time of surgery and weighing 10.5-15 kg. Surgq

* Preliminary results presented at the Fifth European Cmyrrs,s of S/rep Resrcrrch, Amsterdam (Wauquier et al., 1980a). t Hypnorm: 10 mg fluanisone (R 2028) and 0.315 mg fentanyl dihydrogen citrate and 0.2 mg fentanyl base (R 4263) per ml. Key words: antihistamines, astemizole, sleep patterns dogs, brain histamine receptors. 853

Dogs were premeditated with 5 ml Hypnormt subcutaneously followed by 5 ml Nembutal (pentobarbital sodium, 60 mg/ml) intravenously. They were then intubated and artificially ventilated from a N20 blender (Mark 4, Bird). If required, the dogs were given an additional 2 ml of Nembutal to ensure an adequate depth of anaesthesia during surgery. After surgery the dogs were placed in a recovery room and treated daily with Dicastrepton 1500 intramuscularly for at least 5 days. Stainless-steel screws were fixed in the skull bone over the frontal, temporal and occipital cortex (positioned according to anatomic landmarks, Klemm, 1969). Subcortical electrodes consisted of a stainlesssteel needle (0.5 mm dia.) with a stainless-steel wire inserted so as to protrude 1 mm beneath its tip. All electrodes were insulated except for 4 mm at the tip. The subcortical electrodes were stereotaxically im-

854

A. WALQUIER rt ul.

planted in the basolateral amygdala, dorsal hippocampus. lateral geniculate and oral pontine reticular formation. One circular platinum wire (0.2 mm) was placed dorsally and one laterally to an eye in order to record eye movements. Two circular platinum wires (0.2 mm) were sutured in the posterior neck muscles to record muscle activity. Finally, two stainless-steel screws were fixed, one close to the nasal bone and one posterior to the midline of the skull, to serve as indifferent or ground electrodes. The coordinates of the depth electrodes were determined according the stereotaxic atlas of Dua-Sharma. Sharma and Jacobs (1970) and corrected for individual skull dimensions (Sadowski. Wauquier. Jageneau and Janssen, 1979). The dogs have not yet been sacrificed for histological control since they are scheduled for further drug studies, Recording

Beginning 2 weeks after surgery, the dogs were frequently transferred to the experimental room for adaptation to the environment. Recordings were not made until a minimum of 4 weeks had elapsed since surgery. The dogs were placed in a relatively small cage in a room where the laboratory illumination was continuously on. Recordings and on-line computer analysis were made in a separate room. However. the dogs could be indirectly observed. The dogs had an indication of the time of day, from common laboratory noise. Dogs were run for repeated 16 hr recording, from 3 a.m. to 7 p.m., but at least 1 week elapsed between each drug experiment. Several 16 hr recordings were taken for each dog. The first 3-5 recordings were disregarded. The first 3 hr were recorded on paper; the whole 16 hr period was analysed by the computer. The following derivations were recorded on a Elema-Schonander mingograph: frontal-occipital cortex (left and right). frontal-frontal cortex. temporal-temporal cortex, occipital-occipital cortex, basolateral amygdala, dorsal hippocampus, lateral geniculate. pontine reticular formation, electromyogram (EMG) and electro-oculogram (EOG). Other channels served to indicate time triggers. The paper speed used was 15 mmisec, occasionally 30 mmjsec. Filters and time constants were. for the EEG. respectively 30 Hz and 0.3 sec. for the EMG respectively 700 Hz and 0.015 sec. Attn[ysis

Visual and computer-based analysis was performed on 30sec epochs. These epochs were classified as specified previously (Wauquier, Verheyen, Van Den Broeck and Janssen. 1979) in 5 stages: wakefulness, transition to sleep, light slow-wave sleep. deep slowwave sleep and REM sleep. For drug comparisons light and deep slow-wave sleep were combined. One cortical derivation, the hippocampal derivation. the EMG and EOG were analysed on-line by

a PDP 1l:ElO computer system. Power spectral Transformation was analysis, using Fast Fourier made on the frontal-occipital cortex derivation each 30 set epoch for 25.6 set (2048 data points) which left 4.4 set for computations. The power contained in the frequency bands, 6 (0.553.5 Hz), 0 (3.5-7.5 Hz), a (7.5513.0 Hz) and (/I (13.CL25.0 Hz) was calculated. The power in the 0 band was also calculated for the hippocampal derivation. The EMG and EOG mean amplitude was taken and a special algorithm detected spindle activity. These data were analysed on-line (real time): and an off-line automatic sleep stage classification was done as based on the data using a minimal distance approach. Drugs

A dose of 10 mgjkg of each of the following drugs, contained in a capsule, was given orally to non-food deprived dogs: astemizole, chlorpheniramine, diphenhydramine, pyrilamine and ketotifen. Astemizole, 1-(4-fluoro-phenylmethyl)-N-[1-[2-4(methoxyphenyl)ethyl]-4-piperidinyll-lH-benzimidazol-2-amine, is a new histamine HI receptor antagonist (Van Wauwe, Awouters. Niemegeers, Janssens, Van Nueten and Janssen, 1981) originally developed in this laboratory. All drugs were given to the same dogs immediately preceding the start of the recording. RESULTS

Figure 1 shows the hypnograms obtained in I dog during a control recording and following drug administration. Figure 2 shows the mean time (in min) spent in the different sleep-wakefulness stages for the 5-dogs as a group during control recordings and following drug administration. Baseline

sleep-wkIfir/ne.s.s

As illustrated in Figure 1, during control recording, a regular cyclic variation occurred, slow wave sleep (stage 2 or 3) was often followed by a period of REM. For the total 16 hr control periods in 5 dogs the following mean (+ SEM) percentage partition was obtained: wakefulness: 32.6 k 1.2. transition stage: 9.3 f 0.84. slow wave sleep (stage 2 and 3 combined): 42.5 _+ 0.72 and REM sleep: 15.7 + 0.79. The mean (+ SEM) duration of each stage in min was as follows: wakefulness, movement, 6.0 + 0.8; wakefulness. rest. 3.6 f 0.2; transition to sleep (stage 1). 1.8 + 0.1; light slow wave sleep (stage 2). 3.3 + 0.3; deep slow wave sleep (stage 3). 5.9 rt 0.2; and REM sleep. 5.8 + 0.3. The mean ( f SEM) latency in min to stage 3 (time elapsing between the start of the recording and the first period of at least 1 min) was 48.2 k 16.3. The mean (+ SEM) REM latency in min (time elapsing between the start of the recording and the first REM period of at least I min was 56.7 Ifr 12.2). S/eep partition All reference

of the tofu/

I6 hr recording

antihrstamines.

except

(Fig. 2)

astemizole.

in-

Effects of astemizole and antihistamines

on sleep in dogs

855

CONTROL R W

I 2 3

ASTEMIZOLE R W

REM:15 5 W :30. 4 1 7.5 2+3 146.7

I 2 3

CHLORPHEZNIRAMINE

R W

REM:1 3. 3 W :27* 3 1 :10.7 2t3 :48.8

I 2 3

PYRILAMINE

KETOTIFEN

REM: W 1 2t3

1

0

I

I

2%0

1

8

4&o

1

1

720

,

,

9.9 :34.1 :10.6 :45.4

1

960

Nin Fig. I. Computer-printed hypnograms of beagle B 2497 in a control session and after IOmg/kg orally given at time 0 of the indicated antihistamines; on the right-hand side the percentage partition of the different stages. Abbreviations: R--REM sleep; W-wakefulness (thick lines indicating movement periods); l-stage 1, transitional stage; Z-stage 2, light slow wave sleep: 3-stage 3. deep slow wave sleep.

A. WAUQUER et al.

TRANSITION

WAKEFULNESS

SLOW

WAVE

SLEEP

REM

TO SLEEP CONTROL

ASTEMIZOLE

KETOTIFEN

DlPHENHVDRAMlNE

PYRILAMINE

MIN

I 0

I I I IO0 200 300

I LOO

I

I

0

I

I

0

100 200

I

I

I

100 200 300

I

I

LOO 500

(+ SEMI time in mln spent in the various stages of sleepwakefulness during contra! and after IO mgjkg oraify given antihistamines for a group of 5 dogs. Significant difference as compared to controi estimated with the Wilcoxon matched pairs signed ranks test, *P < 0.05. one-tailed probability,

Fig. 2. Mean

creased non-REM sleep and decreased REM sleep. None of the antihistamines significantly changed the total time spent in wakefulness. As compared to the control (percentage vs control equalized at 100, between brackets): diphenhydramine (12%), pyrilarnine (141%) and ~b~orpheniramine (170%) significantly increased the transition stage; ketotifen (114%) and pyrilamine (i 22%) significantly increased slow wave sleep; diphenhydramine (66.4%), ketotifen (65.4%), pyrilamine (57.37;) and chlorpheniramine (21.6%) significantly decreased REM sleep. Sleep partt’tion per 4 hr period The s~g~~fi~a~t changes obtained in the total 16 hr period were mainly due to significant effects obtained during the first 4 hr, except for chlorpheniramine which had long Iasting effects. The mean (+ SEM) percentages compared to control for the 4 consecutive 4 hr periods obtained with chlorpheniramine were:

for the transition to sleep: 314 ( & 77.9)*, 236 (-E_73.8)*, 129 (L- 27.8), 157 (+ 47.3) and for REM sleep: 0.4 (2 0.3)*, 14.8 (+ 6.5)*, 32.4 (+ 12.1)*, 43.0 (+ 19.2) (* indicating P < 0.05). As seen here, but evident with aI1 the compounds is the fact that there was no data supporting a withinthe-night rebound effect, but instead a gradual return ta control levels was seen Duration und number

of epochs

The increased time spent in the transition stage following diphenhydramine, pyrilamine and chlorpheniramine was not due to an increase in the number but to a significantly increased duration of the periods transition; in percentage compared to control (= 100) respectively 122, 140 and 144:~;. The increased slow wave sleep following ketotifen was due to an increase in both the number (126Y<) and duration (1077;) of stage 3 periods. The increased

Table 1. Mean (-& SEM) REM latency (time between start recording and first and second REM period af at least 1mm). mean f+ SEM) stage 3 latency (time between start recording and first and second stage 3 period of at least 1 min) of 5 dogs during contra1 and after 10 mg/kg of ~tihistam~~~ orally given at the start of the recording REM latency (min) First Control Astemizole Ketotifen ~~~henh~dram~ne Chlor~h~~~ram~ne Fyrilamine

56.7 (+ 12.2) 101 (& 17.5) 146 (+ 38.6)* 165 (rt: 40.6)* 390 (+44)” 237 fi: 49.2)*

Stage 3 latency (mm)

Second 91.9( f 138 (& 208 ( f 249 (2 512 Ii: 336 (+

13.1) 16.6) 27.2)* 35,2)* 49.1)* 31.9)*

+ P < 0.05. Wilcoxon matched-pairs signed-ranks test one-tailed probability.

First 49.2 ( + 16.3) 68.8 ( + 8.2) 62.3 ( & 10.4) 51.4{f 7.6) 77.3 ( + 27.7) 44.0 ( &- 14.5)

Second

Effects of astemizole and antihistamines on sleep in dogs slow wave sleep following pyrilamine was mainly due to a significantly increased number of the periods of stage 3 (121%). The decreased REM sleep with all reference antihistamines was due to both a decrease in the number of REM periods, and a shortening of the duration of the REM periods. The percentage vs control (= 100) for respectively the number and the duration of the REM periods was for ketotifen 79.5 and 86.2?;, for diphenhydramine 87.4 and 77.4”/;;, for chlorpheniramine 36.2 and 56.99, and for pyrilamine 70.1 and 84.84:. Stuge 3 nnd REM iatenq Table 1 depicts the mean latency to stage 3 and REM sleep. With astemizoie, ketotifen, diphenhydramine and chlorpheniramine there was a tendency towards a postponement of the first and second period of stage 3, these effects were however, not significant. From Table 1 and also from Figure 1 it is obvious that the REM latency is significantly increased following ketotifen, diphenhydramine, chlorpheniramine and pyrilamine. In addition, the second REM period came at a significantly longer interval after the first REM period with diphenhydramine, chlorpheniramine and pyrilamine. DISCUSSION

Astemizole is the prototype of a new series of compounds with potent antihistaminic properties (Van Wauwe et al., 1981). In the rat, using compound 48/80-induced lethal shock (Niemegeers, Awouters, Van Nueten, De Nollin and Janssen, 1978) the lowest EDSo of astemizole after oral administration was 0.097 mg/kg (Table 2). Orally in rats, astemizole was 6 times more potent than ketotifen (lowest EDSO = 0.59 mg/kg), 386 times more potent than chlorpheniramine and diphenhydramine (lowest EDSo = 31.4mg/kg) and 582 times more potent than pyrilamine (lowest EDso = 56.6 mg/kg). The antihistaminic activity of astemizole was also found to be very long-lasting, a dose 3 times the lowest ED5o afforded protection from shock for 24 hr. At a corresponding dose level the most potent reference drug, ketotifen, had a duration of action of 4 hr only. Oral absorption of astemizole is also much better than that of the reference compounds of which the oral activity was invariably lower than the subcutaneous activity: for chlorpheniramine 1.7, for diphenhydramine 4.6, for pyrilamine 10.5 and for ketotifen 49.2 times. Astemizole at time of peak effect was equipotent with both oral and subcutaneous administration (Table 2). The therapeutic use of antihistamines has revealed two types of common side-effects, one of the sedative type most frequently reported as somnolence, and one of the anticholinergic type, mostly reported as dryness of the mouth and other mucosal surfaces. Side-effects of the sedative type have been invariably associated with all histamine H,-antagonists. This property may thus be unavoidable with substances that block fir

857

8%

A. WAUQUIER rt cd.

central receptors, although the reported central action of Hi-antagonists in man has been based mainly upon subjective assessment. The anticholinergic effect of compounds can be assessed pharmacologically by measuring the pupil diameter, mydriasis being an expression of peripheral anticholinergic activity. Central anticholinergic activity can be evaluated using the physostigmineinduced lethality test. Central anticholinergics are able to block physostigmine-induced lethality (Niemegeers, unpublished). In an oral dose of up to 40 mg/kg, i.e. a dose at least 400 times the antihistaminic dose, astemizole did not induce mydriasis and was devoid of central anticholinergic activity. Given subcutaneously, chlorpheniramine induced mydriasis at 19.2 mg/kg whereas physostigmine-antagonism was (EDso) obtained with 28.8 mg/kg (EDs,,). With subcutaneous injection of diphenhydramine these values were 7.12 and 9.38 mg/kg and with pyrilamine, given subcutaneously, 9.38 and 28.3 mg/kg respectively. The central effects of antihistamines are much more difficult to evaluate pharmacologically (Green, Garland and Hodson, 1979). However, sedative as well as excitatory effects of these drugs have been detected using intracranial selfstimulation in rats (Wauquier, 1976). In this test astemizole was found completely inactive, whereas with pyrilamine there was a doserelated decrease, and with chlorpheniramine a doserelated increase in the number of lever pressings resulting in intracranial stimulation (Wauquier and Niemegeers, 1981). Nevertheless, instead of measuring the direct effect of the compounds, the potential for sedative or central side-effects can be evaluated more easily in animal models in which the interaction with other agents, affecting CNS activity, is studied. Astemizole at an oral dose of 40mg/kg or more, thus at least 400 times the antihistaminic dose, was found to be inactive in the ATN-test (Niemegeers, Lenaerts, Artois and Janssen, 1977). as a central dopamine and serotonin antagonist. Moreover the duration of hypnosis in rats and mice treated with either methohexital, ethanol or chlordiazepoxide was not statistically different with or without astemizole pretreatment. Furthermore, astemizole had no effect on the inhibitory action of haloperidol in the apomorphine test or of fentanyl in the tail-withdrawal reaction test, nor on the potentiating effect of tranylcypromine in the tryptamine test (Awouters and Niemegeers, unpublished). All these studies indicate indirectly that astemizole up to very high dose levels, is devoid of effects, which are not related to reactions mediated by exogenous or endogenous histamine. A more direct measurement of the central effects of antihistamines has been provided in the past using EEG recording. The sedative action was found to occur at therapeutic doses and was particularly pronounced with phenothiazine-like drugs (Goldstein et al., 1968; Kugler, Thurmayer and Rode, 1972). Power spectral analysis and other forms of statistical EEG

analysis revealed that in man, antihistamines increased the slow frequencies (below 7 Hz), decreased the fast frequencies above 25 Hz (Fink and Erwin, 1979) increased the variability of the signal (Goldstein et al., 1968) and decreased the vigilance (Kugler et al., 1972). In the present studies, administration of clinically used Hi-antagonists also led to significant changes in the sleep-wakefulness pattern as evaluated using EEG recordings. This suggests that the study of the EEG in dogs is a suitable method for evaluating the central action of compounds of this pharmacological class. The type of central effect which may be produced may also be directly related to the changes observed in the dog EEG. The increase in slow wave sleep, as observed with ketotifen and pyrilamine, and the increase in time spent in the transition to sleep (drowsiness) as observed with diphenhydramine, chlorpheniramine and pyrilamine probably reflects a central action of the sedative type. The results obtained are of significance for several reasons, Firstly, all compounds were given to the same dogs, thus the differences between the compounds were not related to differences between the dogs. Secondly, the effects were specifically related to the stage of sleep and not due to a “balance’‘-effect, since for instance slow wave sleep increased following ketotifen without affecting the time of wakefulness. Thirdly, though significant changes were predominantly due to changes obtained during the first 4 hr of the recording, they were even evident when considering the effects on the total 16 hr period. In contrast, astemizole was devoid of signilicant effects on the sleepwakefulness pattern. The importance of this differentiation is strengthened by the fact that the reference drugs elicited these effects at a dose near to the antihistamine dose, whereas the dose of astemizole was much greater than the antihistamine dose. For example, the smallest effective oral dose of astemizole in the dog Ascuris allergy test (Awouters and Niemegeers. unpublished) was 0.16 mg/kg and maximal effects lasting for at least 10 days. were achieved with 10 mg/kg of astemizole. The sedative action of the reference antihistamines is possibly a consequence of the blockade of central histamine receptors. Histamine is present in non-mast cells in the brain, and is unevenly but probably distinctly distributed and localized in nerve terminals Feger and Schwartz. 1974: (Garbarg, Barbin, Schwartz, 1977). Histamine is known to produce an arousing or stimulating effect (Monnier, Sauer and Hatt, 1970; Nistico, Rotiroti, De Sarro, Waccari and Stephenson, 1980). Increase in slow wave sleep and/or transition to sleep after administration of H,-antagonists is consistent with an antagonism of these central histamine effects. All four reference antihistamines tested also decreased REM sleep, the effects being long-lasting for ketotifen, whereas a non-significant decrease was obtained during the first 4 hr of the recording after

Effects of astemizole

and antihistamines

astemizole treatment. This type of EEG effect is seen with a variety of drugs, more specifically with stimulants and antidepressants (Kales, Heuser, Kales, Rickles, Rubin, Scharf. Ungerleider and Winters, 19693, and could therefore be associated with excitatory effects, though drugs belonging to different classes may also have REM suppressant effects (e.g. Wauquier, Van Den Broeck and Janssen, 1980b). The central excitatory effects of antihistamines may well be the result of anti-~holinergi~ properties (Barnett et ul., 1969; Colpaert, Lenaerts. Niemegeers and Janssen, 1975). Experiments in normal volunteers, provided evidence that cholinergic drugs (arecoline) are REM sleep inducers, whereas anticholinergic drugs (scopolamine) are REM sleep suppressors (Sitaram, Moore and Gillin, 1978; Sitaram, Nurnberger and Gershon, 1980). The authors suggested that cholinergic systems regulate the timing of REM sleep since the drugs did not alter primarily the amount of REM sleep. Thus, the sleep cycle oscillation would be under the excitatory control of cholinergic neurones. In the present experiments, the REM latency was increased and also the cyclic variation. since the interval between the successive REM periods was also significantly changed. This might be the reason why the total amount of REM sleep was decreased. In conclusion: the new antihistamine astemizole did not alter the slee~wakefulness pattern in dogs, suggesting that it has no central effects, whereas the reference antihistamines increased non-REM sleep and decreased REM sleep. The changes in the non-REM sleep might be due to a blockage of brain histamine H, receptors; the decreased REM sleep might be due to anti~holiner~ic properties. Acitnowlr~~mlenrs-We sincerely thank D. Ashton help in the preparation of the manuscript. Part study was supported by a grant from I.W.O.N.L.

for his of this

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