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JL13, A PYRIDOBENZOXAZEPINE COMPOUND WITH POTENTIAL ATYPICAL ANTIPSYCHOTIC ACTIVITY: A REVIEW OF ITS BEHAVIOURAL PROPERTIES
Pharmacological Research, Vol. 36, No. 4, 1997
JL13, A PYRIDOBENZOXAZEPINE COMPOUND WITH POTENTIAL ATYPICAL ANTIPSYCHOTIC ACTIVITY: A REVIEW OF ITS BEHAVIOURAL PROPERTIES ´ 5 , JACK BERGMAN‡, JACQUES BRUHWYLERU ,1, JEAN-FRANC ¸ OIS LIEGEOIS† GALEN CAREY‡, ANDREW GOUDIE§, ANITA TAYLOR§, HERBERT MELTZER¶, U ´ JACQUES DELARGE† and JOSEPH GECZY U
Therabel Research s.a., Rue Van Ophem 110, 1180 Brussels, Belgium, †Laboratory of Medicinal Chemistry, Uni®ersity of Liege, ` rue Fusch 3-5, 4000 Liege, ` Belgium, 5 Laboratory of Physiology, Uni®ersity of Liege, ` Place Delcour, 17, 4020 Liege, ` Belgium, ‡Har®ard Medical School, Alcohol and Drug Addiction Research Center, McLean Hospital, 115 Mill Street, Belmont MA 02178, USA and §Department of Psychology, Li®erpool Uni®ersity, P.O. Box 147, Li®erpool L69 3BX, United Kingdom and ¶Psychiatric Hospital of Vanderbilt, Vanderbilt Uni®ersity Medical Center, 1601 23rd A®enue South, Suite 306, Nash®ille, TN 37212, USA Accepted 5 September 1997
The search for an improved clozapine-like compound has resulted in the selection of a new molecule: JL13 Ž5-Ž4-methylpiperazin-1-yl.-8-chloro-pyridow2,3-b xw1,5xbenzoxazepine fumarate.. Like clozapine, JL13 did not antagonize apomorphine-induced stereotypy and did not produce catalepsy but antagonized apomorphine-induced climbing in rodents ŽID50 s 3.9 mg kgy1 s.c... It was inactive against d-amphetamine-induced stereotypy but antagonized d-amphetamine-induced hyperactivity in the mouse ŽID50 s 4.4 mg kgy1 i.p... JL13, like clozapine, was able to antagonize Ž".-DOI-induced head-twitches in the mouse ŽID50 s 2.0 mg kgy1 i.p... In the open-field test in the rat and forced swimming test in the mouse a high similarity was noted between the two drugs in the same range of doses. In a complex temporal regulation schedule in the dog, JL13 showed a high resemblance with clozapine without inducing sialorrhea, palpebral ptosis or any significant motor side effects. In rats trained to discriminate clozapine, JL13 Ž10 mg kgy1 i.p.. induced a high level of generalization Ž70%. to clozapine. In a drug discrimination procedure in the squirrel monkey, JL13 Ž3]10 mg kgy1 i.m.. produced a full substitution of clozapine. On the basis of these preclinical data, it is thus predicted that JL13 would be a promising atypical antipsychotic drug. Q 1997 The Italian Pharmacological Society KEY
WORDS:
atypical antipsychotic, clozapine, JL13, behavioural pharmacology.
INTRODUCTION Schizophrenia remains a major therapeutic challenge, not only because it is a common, severe and chronic condition, but also, because currently available antipsychotic drugs have a number of limitations. Firstly, they are not always effective, as 15]30% of patients derive little benefit from treatment with these agents. Secondly, even when patients do respond, some aspects of psychopathology show little improvement. In many patients, positive symp-
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Corresponding author.
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toms may be alleviated while negative or deficit symptoms remain unresponsive. Thirdly, currently available drugs are associated with a variety of side effects, particularly neurological adverse reactions such as drug-induced parkinsonism, akathisia, tardive dyskinesia and acute or tardive dystonia w1x. The demonstration that clozapine possesses both increased efficacy, particularly in treatment-resistant patients w2x, and reduced neurotoxicity, however, has forever altered the conventional wisdom which held all antipsychotics were equally efficacious and uniformly neurotoxic w3x. Nevertheless, the prescription of clozapine is restricted due to a 1]2% incidence of agranulocytosis w4, 5x and only the patients who are Q1997 The Italian Pharmacological Society
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resistant to, or intolerant of, conventional antipsychotic drugs can benefit from its use w6x. Together with haematological adverse reactions, the most common side effects occurring with clozapine are orthostatic hypotension, drowsinessrsedation, hypersalivation, tachycardia, dizzinessrvertigo, constipation, nausearvomiting and seizures w7]9x. So there remains a need for improved atypical antipsychotic agents. This challenge is currently the focus of the most active efforts in contemporary CNS pharmacology w10]12x. The dibenzazepine pharmacophore constitutes an interesting basis for the chemical development of new psychotropic compounds and for the understanding of the mechanism of action of antipsychotic drugs. Indeed, a slight modification of its tricyclic nucleus leads to a profound alteration of its pharmacological and clinical activities. Clothiapine is a typical neuroleptic with strong propensity to induce extrapyramidal side effects ŽEPS. w13x, amoxapine is a mixed antidepressant w14x with significant neuroleptic activity while clozapine and fluperlapine show an atypical antipsychotic profile, producing only minimal EPS w15, 16x. In this context, a series of N-methylpiperazinopyridobenzazepine derivatives have been synthesized and evaluated using several psychopharmacological models w17, 18x. These researches have resulted in the selection of an interesting compound with an atypical antipsychotic profile, JL13: 5-Ž4-methylpiperazin-1-yl.-8-chloro-pyridow2,3-b xw1,5xbenoxazepine Žfumarate. ŽFig. 1.. The aim of this paper is to describe the psychopharmacological characteristics, currently available, of this original compound and to compare them with that of clozapine.
BEHAVIOURAL PROPERTIES OF JL13 COMPARED TO CLOZAPINE Rem: JL13 is used as a fumarate salt but the doses are also reported in free base equivalent. 1. Catalepsy: One and 2 h after s.c. vehicle, JL13 or clozapine administration, male Wistar rats were examined for catalepsy in a 6-component trial Žgrid, block-standing, paw extension, back position, buccal rod and ro-
Fig. 1. Chemical structure of JL13.
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tating tube.. The different scores obtained 1 and 2 h post-administration were added. The ED50 Ždose inducing an increase in total score by 50%. of clozapine and JL13 was calculated using non-linear regression analysis. The ED50 of clozapine was equal to 108 mg kgy1 s.c. while that of JL13 was superior to 117 mg kgy1 s.c. Ž) 88 mg kgy1 s.c. free base equivalent. ŽTable I.. 2. Apomorphine-induced stereotypy: The methodology used for this test was inspired by that of Janssen et al. w19x. Briefly, male Wistar rats were placed in a cage from which behaviour could easily be observed. Clozapine or JL13 was administered at 20 mg kgy1 Žs.c.. and 60 min later apomorphine HCl was administered at 2.5 mg kgy1 Žs.c... The animals were observed for 60 s at 10-min intervals for a 1-h period and the different stereotypies were recorded Žsniffing, gnawing, rearing and grooming. and summed to give a total score. Neither clozapine, nor JL13 did antagonize apomorphine-induced stereotypy at 20 mg kgy1 s.c. Ž15 mg kgy1 s.c. free base equivalent. w18x ŽTable I.. 3. Apomorphine-induced climbing: Male MF1 mice ŽHarlan Olac Ltd., Bicester, UK. weighing 25]35 g were used in this experiment. Clozapine, JL13 or vehicle was injected s.c. 30 min before s.c. treatment with 0.75 mg kgy1 apomorphine HCl. Immediately after apomorphine injection, the animals were placed, individually, in wire mesh circular cages. Climbing behaviour, as described by Protais et al. w20x was scored 10 and 20 min after apomorphine administration. The ED50 Ždose inhibiting climbing behaviour at 50%. of drugs was calculated using non-linear regression analysis. The ED50 of clozapine was equal to 2.0 mg kgy1 s.c. while that of JL13 was equal to 3.9 mg kgy1 s.c. Ž2.9 mg kgy1 s.c. free base equivalent. ŽTable I.. 4. d-Amphetamine-induced stereotypy: Male NMRI mice, aged 5]6 weeks, were used. Stereotyped behaviour was induced by s.c. administration of d-amphetamine sulphate, at a dose of 12 mg kgy1 . Clozapine or JL13 was administered i.p. 30 min prior to damphetamine. After the injections, the mice were individually placed into wire mesh cages and were observed during a 2-h period. Stereotypy was scored according to the following criteria : 0 s absence of stereotypy, 1 s slight stereotypy of the head with intermittent sniffing, 2 s intense stereotypy of the head with licking andror gnawing. The minimal effective dose, defined as the lowest
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Table I Psychopharmacological properties of clozapine and JL 13 in several animal models Clozapine Induction of catalepsy in the rat
a
y1
ED50 s 108 mg kg
s.c.
JL 13 (free base equi®alent) ED50 ) 117 mg kgy1 s.c. Ž) 88 mg kgy1 .
Apomorphine-induced stereotypy antagonism in the rat b
Inactive at 20 mg kgy1 s.c.
Inactive at 20 mg kgy1 s.c. Ž15 mg kgy1 .
Apomorphine-induced climbing antagonism in the mouse a
ED50 s 2.0 mg kgy1 s.c.
ED50 s 3.9 mg kgy1 s.c. Ž2.9 mg kgy1 .
d-Amphetamine-induced stereotypy in the mouse c
Inactive up to 18.3 mg kgy1 i.p. Min active doses 32.7 mg kgy1 i.p.
Inactive up to 24.9 mg kgy1 i.p. Ž18.7 mg kgy1 . Min active dose ) 33 mg kgy1 i.p. Ž24.8 mg kgy1 .
d-Amphetamine-induced hyperactivity in the mouse c
Min active doses 3.3 mg kgy1 i.p.
Min active doses 4.4 mg kgy1 i.p. Ž3.3 mg kgy1 .
Ž".-DOI-induced head-twitches in the mouse c
ED50 s 0.5 mg kgy1 i.p.
ED50 s 2.0 mg kgy1 i.p. Ž1.5 mg kgy1 .
Open-field test in the rat Žclozapine and JL13 8]16]24 mg kgy1 i.p.. ŽJL13 base 6]12]18 mg kgy1 i.p.. d
}No effect on the ambulation score }No effect on the entry score }Significant decrease in the rearing score }Significant decrease in the defecation score
}Significant decrease in the ambulation score }No effect on the entry score }Significant decrease in the rearing score }Significant decrease in the defecation score
Forced swimming test in the mouse Žclozapine and JL13 4]8]16 mg kgy1 i.p.. ŽJL13 base 3]6]12 mg kgy1 i.p.. d
Significant dose-dependent increase in the immobility time
Significant dose-dependent increase in the immobility time
Temporal regulation schedule in the dog e ŽClozapine 5]10]15 mg kgy1 p.o.; JL13 5]10]20 mg kgy1 p.o.. ŽJL13 base 3.8]7.6]15.2 mg kgy1 .
}Dose-dependent decrease in total response rate Ž P) 0.05. and correct response rate Ž P- 0.01. }Extension of the temporal distribution of response duration to the right and left Ž P- 0.01. }Incomplete responses Ž P- 0.05. }Sialorrhea Ž P- 0.05. }Palpebral ptosis Ž P- 0.05. }Ataxia Ž P - 0.01. }Catalepsy Ž P) 0.05. }Akinesia Ž P- 0.01. }Tremor Ž P - 0.01. }Hyperkinesia Ž P ) 0.05.
}Dose-dependent decrease in total response rate Ž P) 0.05. and correct response rate Ž P) 0.05. }Extension of the temporal distribution of response duration to the right and the left Ž P- 0.01. }Incomplete responses Ž P) 0.05. }Sialorrhea: absent }Palpebral ptosis Ž P) 0.05. }Ataxia Ž P) 0.05. }Catalepsy Ž P ) 0.05. }Akinesia Ž P) 0.05. }Tremor: absent }Hyperkinesia Ž P) 0.05.
Drug discrimination in the rat f ŽClozapine 0.625]5 mg kgy1 i.p.; JL13 2.5]20 mg kgy1 i.p.. ŽJL13 base 1.9]15.2 mg kgy1 i.p..
95% clozapine lever selection at 5 mg kgy1 ED50 s 2.1 mg kgy1 No decrease in response rate
70% generalization to clozapine at 10 mg kgy1 ED50 s 7.6 mg kgy1 s 5.7 mg kgy1 free base Dose-related decrease in response rate
Drug discrimination in the Squirrel monkey g ŽClozapine 0.1]3.0 mg kgy1 i.m.; JL13 0.1]10 mg kgy1 i.m.. ŽJL13 base 0.08]7.6 mg kgy1 i.m..
More than 90% clozapine lever selection between 0.3 and 1.0 mg kgy1. Dose-related decrease in response rate
More than 90% clozapine lever selection between 3 and 10 mg kgy1 Ž2.25]7.50 mg kgy1 free base.. Dose-related decrease in response rate
a
Original results obtained by Connick et al.; Liegeois et al. w18x; ´ c Original results obtained by Matagne et al.; d Bruhwyler et al. w33x; e Bruhwyler et al. w26x; f Taylor et al. w38x; g Carey and Bergman w39x. b
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dose which significantly Ž P- 0.05. inhibited d-amphetamine stereotypy, was calculated using the Kolmogorov]Smirnov 2-sample test. JL13 was completely inactive against d-amphetamine-induced stereotypy, up to a dose of 24.9 mg kgy1 i.p. Ž18.7 mg kgy1 i.p. free base equivalent. and the minimal active dose was at least superior to 33 mg kgy1 i.p. Ž24.8 mg kgy1 i.p. free base equivalent.. Clozapine was inactive up to 18.3 mg kgy1 i.p. while a dose of 32.7 mg kgy1 i.p. produced a decrease of 48% Ž P- 0.05. ŽTable I.. 5. d-Amphetamine-induced hyperacti©ity: Male NMRI mice, aged 5]6 weeks, were used. Hyperactivity was induced by s.c. administration of d-amphetamine sulphate, at a dose of 4 mg kgy1 , 30 min before testing. Clozapine or JL13 was administered i.p. 30 min prior to d-amphetamine. For testing, each mouse was placed into an open-field cage and locomotor activity was recorded for 10 min. The minimal active dose, defined as the lowest dose w h ic h s ig n ifi c a n t ly in h ib it e d d amphetamine-induced hyperactivity, was calculated using the Mann-Whitney U-test Ž2tailed.. JL13 decreased d-amphetamine-induced hyperactivity by 45% Ž P- 0.05. from a dose of 4.4 mg kgy1 i.p. Ž3.3 mg kgy1 i.p. free base equivalent. and clozapine by 46% Ž P- 0.01. from a dose of 3.3 mg kgy1 i.p. ŽTable I.. 6. (")-DOI-induced head-twitches: Male NMRI mice, aged 5]6 weeks were used. Head-twitch behaviour was induced by i.p. administration of Ž " . -1- Ž 2,5-dimethoxy-4-iodophenyl . 2-aminopropane HCl ŽDOI., at a dose of 1.6 mg kgy1 . Clozapine or JL13 was administered i.p. 30 min prior to DOI. After DOI injection, the mice were individually placed into glass cylinders and the number of headtwitches was counted 5 min after DOI administration for a duration of 5 min. The ED50 values were calculated by non-linear regression analysis. JL13 produced a dose-related inhibition of DOI-induced headtwitches, with an ED50 value of 2.0 mg kgy1 i.p. Ž1.5 mg kgy1 i.p. free base equivalent.. For clozapine the ED50 was equal to 0.5 mg kgy1 i.p. ŽTable I.. 7. Open-field test: The procedure has been described in previous studies w21]23x. Male Wistar rats, 100]120 days old and weighing from 350 to 400 g were used. The apparatus consisted of a square wooden open-field Ž96 g = 96 cm., surrounded by a 28-cm high wooden wall. Clozapine, JL13 or vehicle was administered 30 min before the test. Thirty minutes after the administration the subject
was carefully placed in a particular square next to the wall and left in the open-field for 10 min. During this period, the total ambulation score, the entry score, the number of rears and the number of faecal boluses were measured, always by the same observer who was unaware of the drug injection. Clozapine had no significant effect on the total ambulation score while JL13 significantly Ž P- 0.05. decreased it from 8 mg kgy1 i.p. Ž6 mg kgy1 i.p. free base equivalent.. Both drugs had no effect on the entry score and significantly Ž P- 0.05. decreased rearing Žfrom 8 mg kgy1 i.p w6 mg kgy1 i.p. JL13 free base equivalentx. and defecation Žfrom 8 mg kgy1 i.p. and 16 mg kgy1 i.p. w12 mg kgy1 i.p. free base equivalentx respectively for clozapine and JL13. scores w33x ŽTable I.. 8. Forced swimming test: The procedure used in this study corresponded to that described by Porsolt et al. w24x with minor modifications. Outbred male mice ŽNMRI., of mean weight 35 g were used. The apparatus consisted of a glass cylinder Žheight 25 cm, diameter 10 cm. containing water Žliquid height 9 cm. at a constant temperature of 238C. Clozapine, JL13 or vehicle was administered 60 min before the test. Sixty min after drug administration, the animal was placed in the cylinder and the time of immobility was measured during the final 4 min of a 6-min test, always by the same observer who was unaware of the drug administered. Both clozapine and JL13 produced a dose-dependent increase in the immobility time, significant Ž P- 0.05. at the highest dose Ž16 mg kgy1 i.p. w12 mg kgy1 i.p. JL13 free base equivalentx. w33x ŽTable I.. 9. Temporal regulation in the dog: The procedure used in this study has previously been described as a complex multidimensional Žphysiological, motor and operant. approach of the behavioural effects of psychotropic drugs w25x. It was effective in discriminating barbiturates, 1,4- and 1,5-benzodiazepines, antidepressants, and typical and atypical antipsychotic drugs w25]28x. Naive male Beagle dogs Ž2]4 years old. were used. The program was a Differential Reinforcement of Response Duration ŽDRRD. schedule of 9 s with Limited Hold ŽLH 1.5 s. and positive and negative external cues. It consisted, in the random alternation, of two kinds of trials. In the first type of trial, a maintenance response lasting 9 s on a board was required for obtaining reinforcement. At the end of this time delay, an auditory stimulus of 1.5 s was given to the animal. Every time the dog left the board between 9 and 10.5 s and then
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jumped on to a food dispenser, it received a piece of meat Ž5 g.. The second type of trial differed from the first by the addition of the same auditory stimulus, randomly presented between the 3rd and the 6th s of the time delay. The auditory stimuli presented between 3 and 6 s and at 9 s were physically identical and had the same duration Ž1.5 s.; the animal could only discriminate between them according to their location in time. Both kinds of trials were distributed randomly during the session. Thus, the added stimulus was doubly random, first, because it was not given on each trial and, second, because it was given at random between the 3rd and the 6th s. In every case, the only reinforced response was that to the stimulus at 9 s; any anticipated Ž- 9 s. or delayed Ž) 10.5 s. response was not reinforced. Clozapine or JL13 was administered in capsule form in a random order at 2-week intervals. The experimental session began 4 h following drug administration. On the day before drug administration, the subjects received a placebo 4 h before a control session which was considered as baseline. All measures were taken by the same rater working blind. Operant performance was scored for total and correct response rates Žresponses per min., temporal distribution of response durations, and number of subjects producing incomplete responses. Effects on involuntary movements were recorded as the frequency of ataxic movements, cataleptic positions w29x, akinetic movements, trembling positions, dystonic symptoms. The number of subjects showing sialorrhea or palpebral ptosis was also noted. Clozapine Ž5]15 mg kgy1 p.o.. and JL13 Ž5]20 mg kgy1 p.o. or 3.8]15.2 mg kgy1 p.o. free base equivalent. produced a dose-dependent non-significant Ž P ) 0.05. decrease in the total response rate and a dose-dependent decrease in the correct response rate that was significant Ž P- 0.01. for clozapine but not for JL13. Both compounds induced a complete and significant Ž P- 0.01. temporal disorganization with a shift towards shorter and longer response durations. Clozapine also produced significant levels of incomplete responses Ž P0.05., sialorrhea Ž P- 0.05., palpebral ptosis Ž P- 0.05., ataxia Ž P- 0.01., akinesia Ž P0.01. and tremor Ž P- 0.01.. With JL13, these effects were either non-significant Ž P) 0.05. or even completely absent in the case of sialorrhea and tremor w26x ŽTable I.. 10. Drug discrimination in the rat: Twenty individually housed female Wistar rats were trained, in sound attenuated, computer con-
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trolled Colbourne Instruments ŽUSA. Skinner boxes, to discriminate clozapine at 5 mg kgy1 Ži.p.. from vehicle in a standard Fixed Ratio 30 quantal food rewarded two lever operant drug discrimination assay. This procedure was described in detail in previous papers w30, 31x. Clozapine and JL13 were administered i.p. On any experimental day rats received only one injection; either of clozapine or vehicle. All injections were administered 30 min before operant sessions of 15 min duration. On any individual trial accuracy of lever selection was assessed in terms of the total number of responses made on both levers prior to the presentation of the first reward. This index was termed the FRF, the total number of responses that were made prior to the First Reinforcement. The group as a whole was trained over 60 initial daily training sessions, by which time all individual animals were reliably selecting the correct lever on most experimental days, and the group was consistently showing greater than 85% correct lever selections on all training days. Subsequently, generalisation and substitution studies were initiated wsee 30 for detailsx. In these tests a series of doses of clozapine and JL13 were administered in counterbalanced orders. For the group as a whole it was thus possible to define, for each test dose, the percentage of tested animals selecting the drug Žclozapine. lever. In addition, on test days it was possible to assess any intrinsic actions of the drugs on operant response rate. In the test sessions clozapine induced dose-related generalization, as expected, with 95% drug lever selection being seen at the training dose of 5 mg kgy1 . Thus, as reported in previous studies w34]37x, clozapine, unlike typical neuroleptics such as haloperidol, is reliably discriminated by rats ŽED50 s 2.1 mg kgy1 i.p... This dose of clozapine had minimal effects on response rate in drug experienced rats. JL13 induced a marked Žmaximum 70%. generalization to clozapine. The ED50 for JL13 Ž7.6 mg kgy1 i.p.s 5.7 mg kgy1 i.p. free base equivalent. was calculated by logrlinear regression analysis of the linear portion of the dosereffect curve. The calculated regression line had an r 2 value of 0.61, thus it provided a reasonable, although not perfect fit to the data. JL13 had a clear dose-related suppressant effect on response rate, with substantial Žapprox. 40%. suppression of response being seen at the highest dose tested w38x.
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11.
Drug discrimination in the squirrel monkey: The aim of this test was to determine whether JL13 would substitute for clozapine in squirrel monkeys Ž Saimiri sciureus, 700]1250 g; n s 3. trained to discriminate i.m. injections of clozapine from vehicle in a standard drug discrimination procedure. During experimental sessions, monkeys were seated in chairs similar to the one described by Spealman et al. w32x and enclosed in ventilated, sound-attenuating chambers provided with white noise to mask extraneous sound. Two response levers were mounted in front of the monkey on a transparent wall of the chair. The tail of the monkey was placed in a small stock fitted with brass electrodes, which rested on, a shaved portion of the tail; electrode paste ensured a low-resistance contact between the electrodes and the tail. Electric shock of 5 mA intensity could be delivered to the tail for 200 ms from a 650-V a.c. transformer. Coloured lamps could be illuminated to serve as visual stimuli. Subjects were trained to press the lever to turn off visual stimuli associated with impending electric shock Žstimulus-shock termination schedule. under a 10-response fixed-ratio ŽFR. schedule under terminal conditions. Responding on one lever was operative after injection of clozapine and responding on the second lever was operative after injection of saline. Responding on the inoperative lever was counted but had no scheduled consequences. Once a criterion of at least 90% of responses on the injection-appropriate lever for ten consecutive sessions was reached, substitution testing began. Both clozapine and JL13 produced dose-dependent increases in responding on the clozapine-associated lever. Doses of clozapine between 0.3 and 1.0 mg kgy1 i.m. produced G 90% responding on the clozapine-associated lever Žfull substitution . in each of the three subjects. Although one monkey Žsubject 95]87. was more sensitive to the clozapine-like effects of JL13, doses of 3.0]10.0 mg kgy1 i.m. Ž2.25]7.50 mg kgy1 i.m. free base equivalent. of the novel diarylazepine produced G 90% responding on the clozapine-associated lever in all monkeys. Like clozapine, doses of JL13 that resulted in responding on the drug-associated lever had variable effects on rates of FR responding. However, the highest doses of JL13 Ž5.6]10.0 mg kgy1 i.m. s 4.2]7.5 mg kgy1 i.m. free base equivalent. markedly reduced response rates in both monkeys in which they were studied w39x.
DISCUSSION
All the results obtained in the different behavioural models attest that the psychopharmacological profile of JL13 possesses a high level of resemblance with that of clozapine. Like clozapine, JL13 presents a wide separation Žat least a factor 10. between the doses that inhibit apomorphine- or amphetamine-induced stereotypy and the doses that inhibit apomorphine-induced climbing or amphetamine-induced hyperactivity. Catalepsy is only detected at very high doses. These observations contrast with that made with traditional neuroleptics, such as haloperidol or chlorpromazine, for which all these effects are measured approximately in the same dose range. Catalepsy being a characteristic response to neostriatal dopamine receptor blockade w40x and apomorphine or amphetamine-induced stereotypy being a response dependent on the integrity of the dopaminergic input to the neostriatum w41x, it seems that JL13 like clozapine w42]45x only minimally interferes with this neural system. However, apomorphine-induced climbing and amphetamine-induced hyperactivity, which are mediated by enhanced dopaminergic neurotransmission in the mesolimbic system w46x, are antagonized both by clozapine w43]45x and JL13. Both drugs are able to antagonize Ž".-DOI-induced head twitches, a behaviour that is considered as mediated through an agonist effect on serotonergic 5-HT2A andror 5-HT2C receptors w47, 48x. Clozapine is three times more potent than JL13 Žbase equivalent.. Neurochemical studies have shown that clozapine has a high affinity both for 5-HT2A Ž K i " 5.0 nM. w17, 18, 49, 50x and 5-HT2C Ž K i " 7.5 nM. w50, 51x receptors while JL13 binds to 5-HT2A receptors Ž K i " 60 nM. w18, unpublished results of Meltzer and coll.x but is completely inactive on 5HT2C receptors Ž K i ) 10 m M. Žunpublished results of Meltzer and colleagues.. These results corroborate those obtained by Darmani et al. w48x in the shrew and by Schreiber et al. w52x in the rat tending to demonstrate that the effects of Ž".-DOI are mediated by 5-HT2A and not by 5-HT2C receptors. The antagonism of apomorphine or amphetamine effects is generally considered as related to the binding of drugs on dopaminergic D 2 receptors. Moreover, it has also been demonstrated that induction of climbing behaviour required both D1 and D 2 receptor activation w53x. Clozapine binds to the D 2 receptors with a medium affinity Ž K i " 100 nM. w17, 18, 49, 50, 54x while JL13 has only a negligible affinity for these receptors Ž K i s 1.2 m M. w18x, insufficient to explain its ability to antagonize apomorphine-induced climbing or amphetamine-induced hyperactivity in approximately the same dose range
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as clozapine. Other types of receptor interactions have thus to be implicated, such as the antagonism on D1 Ž K i clozapine " 140 nM w17, 18, 49, 54x; K i JL13 s 302 nM w18x., D4 Ž K i clozapine "25 nM w54]56x, K i JL13 s 164 nM wunpublished results of Van Tol and colleaguesx. or 5-HT2A receptors, to take into account the behavioural observations. Selective 5-HT2A antagonists, such as amperozide and MDL 100,907, which are virtually devoid of D 2 antagonist activity, are also able to reduce amphetamine-induced hyperactivity without affecting amphetamine-induced stereotypy and without producing catalepsy w57x. According to Schmidt et al. w58x, these results support the idea that 5-HT2A antagonists can dampen behavioural states associated with excessive dopaminergic activity. The reasons for looking for eventual anxiolytic andror antidepressant effects with clozapine and JL13 were triple. Firstly, like it has been mentioned in the introduction, the tricyclic azaarylaazepine structure constitutes a crossroads between typical Žclothiapine. or atypical Žclozapine. antipsychotics and antidepressant drugs Žamoxapine.. Secondly, anxiolytic properties have been recognized to clozapine in some animal models w59]61x and these properties have also been noted and even amplified with olanzapine, a thienobenzodiazepine structurally close to clozapine w61,62x. Thirdly, the fact that clozapine and JL13 present a higher affinity for 5-HT2A than for D 2 receptors could orient the pharmacological profile towards anxiolytics such as is the case for ritanserin, a potent 5-HT2Ar5-HT2C antagonist w63]65x. Two rodent models were used for this purpose: the open-field and forced swimming tests respectively for anxiolytic andror antidepressant properties. A complex temporal regulation ŽDRRD. schedule was also used in the dog because it had been shown to differentiate anxiolytics, antidepressants and typical or atypical antipsychotics w25]28x like other temporal regulation schedules such as DRL w66]70x. In the three models, clozapine and JL13 behaved as antipsychotics, decreasing locomotion and rearing without effect on the entry score in the open-field test w33x, increasing immobility time in the forced swimming test w33x and decreasing both total and correct response rates while disturbing the distribution of response duration in the temporal regulation procedure w26x. Of course, it is well known that the effects observed are largely dependent of the animal model itself. For example, ritanserin appears as a recognized anxiolytic in some w63]65x but not in all models w61, 71x. Therefore, it is not impossible that clozapine and JL13 give totally different results when evaluated in other procedures. The complete absence of catalepsy and stereotypyrhyperkinesia winterpreted as akathisia see 25x while tranquillization was significant with both compounds in the DRRD model confirmed their ‘atypicality’ w26x. As far as physiological and motor side effects
261
are concerned, some differences between clozapine and JL13 could be highlighted. Clozapine induced incomplete responses Žanhedonia., sialorrhea, palpebral ptosis, ataxia, akinesia and tremor at a significant level. With JL13, these effects were either completely absent Žsialorrhea, tremor. or produced only at a non-significant level Žincomplete responses, ataxia, palpebral ptosis and akinesia. in the same dose range as clozapine w26x. In drug discrimination procedures, JL13 substituted for clozapine, confirming that the compound has discriminative properties similar to that of clozapine. In the three squirrel monkeys, generalization to clozapine was complete Ž) 90%. respectively for 0.3, 5.6 and 10 mg kgy1 i.m. of JL13 Ž0.225, 4.2 and 7.5 mg kgy1 i.m. free base equivalent.. Reduction in response rate were measured from 5.6 mg kgy1 i.m. of JL13 Ž4.2 mg kgy1 i.m. free base equivalent. and 3 mg kgy1 i.m. of clozapine. Classical neuroleptics such as haloperidol, chlorpromazine and loxapine but also atypical antipsychotics such as olanzapine, risperidone, sertindole and remoxipride did not gerenalize to clozapine Žno more than 45% of subtitution. w39x. In the rat, maximal generalization Ž70%. was observed with JL13 at 10 mg kgy1 Ž7.5 mg kgy1 free base equivalent.. It is just possible that more complete generalization between clozapine and JL13 would have been seen at even higher doses of the drug, although this seems unlikely in that, surprisingly, slightly less generalization Ž55%. was seen at the highest dose tested Ž20 mg kgy1 i.p. salt s 15 mg kgy1 i.p. free base equivalent., despite the fact that this dose of JL13 had the most marked rate suppressant effect. It means that the discrimination is not simply based on a general tranquillizing or sedative effect of JL13. In the same procedure, typical neuroleptics but also risperidone, olanzapine, sertindole and amisulpiride did not generalize to clozapine Žno more than 50% generalization to clozapine. w38x. In conclusion, JL13 is a compound with multiple receptor targets ŽD4 , 5-HT2A, a 1, H1, .... and with a negligible affinity for D 2 receptors presenting a profile of activity similar to that of the atypical agent clozapine with some advantages such as the absence of sialorrhea and tremor. It would therefore be predicted that JL13 will have an atypical profile and be less likely to induce undesirable extrapyramidal symptoms in the clinic than currently available antipsychotic drugs.
ACKNOWLEDGEMENTS Dr JF Liegeois is Senior Research Associate of the ´ FNRS ŽFonds National de la Recherche Scientifique. Belgium. This study was supported in part by U.S. Public Health Service Grants DA 03774, MH 07658, and RR 00168. We are grateful to Dr Connick and Tonnaer from Akzo Nobel Organon, to Dr
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Matagne and Klitgaard from UCB and to C. Lejeune from the University of Namur for their collaboration to some of these experiments. The collaboration of Dr Van Tol from the University of Toronto for the determination of D4 binding is gratefully acknowledged.
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JL13, A PYRIDOBENZOXAZEPINE COMPOUND WITH POTENTIAL ATYPICAL ANTIPSYCHOTIC ACTIVITY: A REVIEW OF ITS BEHAVIOURAL PROPERTIES ´ 5 , JACK BERGMAN‡, JACQUES BRUHWYLERU ,1, JEAN-FRANC ¸ OIS LIEGEOIS† GALEN CAREY‡, ANDREW GOUDIE§, ANITA TAYLOR§, HERBERT MELTZER¶, U ´ JACQUES DELARGE† and JOSEPH GECZY U
Therabel Research s.a., Rue Van Ophem 110, 1180 Brussels, Belgium, †Laboratory of Medicinal Chemistry, Uni®ersity of Liege, ` rue Fusch 3-5, 4000 Liege, ` Belgium, 5 Laboratory of Physiology, Uni®ersity of Liege, ` Place Delcour, 17, 4020 Liege, ` Belgium, ‡Har®ard Medical School, Alcohol and Drug Addiction Research Center, McLean Hospital, 115 Mill Street, Belmont MA 02178, USA and §Department of Psychology, Li®erpool Uni®ersity, P.O. Box 147, Li®erpool L69 3BX, United Kingdom and ¶Psychiatric Hospital of Vanderbilt, Vanderbilt Uni®ersity Medical Center, 1601 23rd A®enue South, Suite 306, Nash®ille, TN 37212, USA Accepted 5 September 1997
The search for an improved clozapine-like compound has resulted in the selection of a new molecule: JL13 Ž5-Ž4-methylpiperazin-1-yl.-8-chloro-pyridow2,3-b xw1,5xbenzoxazepine fumarate.. Like clozapine, JL13 did not antagonize apomorphine-induced stereotypy and did not produce catalepsy but antagonized apomorphine-induced climbing in rodents ŽID50 s 3.9 mg kgy1 s.c... It was inactive against d-amphetamine-induced stereotypy but antagonized d-amphetamine-induced hyperactivity in the mouse ŽID50 s 4.4 mg kgy1 i.p... JL13, like clozapine, was able to antagonize Ž".-DOI-induced head-twitches in the mouse ŽID50 s 2.0 mg kgy1 i.p... In the open-field test in the rat and forced swimming test in the mouse a high similarity was noted between the two drugs in the same range of doses. In a complex temporal regulation schedule in the dog, JL13 showed a high resemblance with clozapine without inducing sialorrhea, palpebral ptosis or any significant motor side effects. In rats trained to discriminate clozapine, JL13 Ž10 mg kgy1 i.p.. induced a high level of generalization Ž70%. to clozapine. In a drug discrimination procedure in the squirrel monkey, JL13 Ž3]10 mg kgy1 i.m.. produced a full substitution of clozapine. On the basis of these preclinical data, it is thus predicted that JL13 would be a promising atypical antipsychotic drug. Q 1997 The Italian Pharmacological Society KEY
WORDS:
atypical antipsychotic, clozapine, JL13, behavioural pharmacology.
INTRODUCTION Schizophrenia remains a major therapeutic challenge, not only because it is a common, severe and chronic condition, but also, because currently available antipsychotic drugs have a number of limitations. Firstly, they are not always effective, as 15]30% of patients derive little benefit from treatment with these agents. Secondly, even when patients do respond, some aspects of psychopathology show little improvement. In many patients, positive symp-
1
Corresponding author.
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toms may be alleviated while negative or deficit symptoms remain unresponsive. Thirdly, currently available drugs are associated with a variety of side effects, particularly neurological adverse reactions such as drug-induced parkinsonism, akathisia, tardive dyskinesia and acute or tardive dystonia w1x. The demonstration that clozapine possesses both increased efficacy, particularly in treatment-resistant patients w2x, and reduced neurotoxicity, however, has forever altered the conventional wisdom which held all antipsychotics were equally efficacious and uniformly neurotoxic w3x. Nevertheless, the prescription of clozapine is restricted due to a 1]2% incidence of agranulocytosis w4, 5x and only the patients who are Q1997 The Italian Pharmacological Society
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resistant to, or intolerant of, conventional antipsychotic drugs can benefit from its use w6x. Together with haematological adverse reactions, the most common side effects occurring with clozapine are orthostatic hypotension, drowsinessrsedation, hypersalivation, tachycardia, dizzinessrvertigo, constipation, nausearvomiting and seizures w7]9x. So there remains a need for improved atypical antipsychotic agents. This challenge is currently the focus of the most active efforts in contemporary CNS pharmacology w10]12x. The dibenzazepine pharmacophore constitutes an interesting basis for the chemical development of new psychotropic compounds and for the understanding of the mechanism of action of antipsychotic drugs. Indeed, a slight modification of its tricyclic nucleus leads to a profound alteration of its pharmacological and clinical activities. Clothiapine is a typical neuroleptic with strong propensity to induce extrapyramidal side effects ŽEPS. w13x, amoxapine is a mixed antidepressant w14x with significant neuroleptic activity while clozapine and fluperlapine show an atypical antipsychotic profile, producing only minimal EPS w15, 16x. In this context, a series of N-methylpiperazinopyridobenzazepine derivatives have been synthesized and evaluated using several psychopharmacological models w17, 18x. These researches have resulted in the selection of an interesting compound with an atypical antipsychotic profile, JL13: 5-Ž4-methylpiperazin-1-yl.-8-chloro-pyridow2,3-b xw1,5xbenoxazepine Žfumarate. ŽFig. 1.. The aim of this paper is to describe the psychopharmacological characteristics, currently available, of this original compound and to compare them with that of clozapine.
BEHAVIOURAL PROPERTIES OF JL13 COMPARED TO CLOZAPINE Rem: JL13 is used as a fumarate salt but the doses are also reported in free base equivalent. 1. Catalepsy: One and 2 h after s.c. vehicle, JL13 or clozapine administration, male Wistar rats were examined for catalepsy in a 6-component trial Žgrid, block-standing, paw extension, back position, buccal rod and ro-
Fig. 1. Chemical structure of JL13.
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tating tube.. The different scores obtained 1 and 2 h post-administration were added. The ED50 Ždose inducing an increase in total score by 50%. of clozapine and JL13 was calculated using non-linear regression analysis. The ED50 of clozapine was equal to 108 mg kgy1 s.c. while that of JL13 was superior to 117 mg kgy1 s.c. Ž) 88 mg kgy1 s.c. free base equivalent. ŽTable I.. 2. Apomorphine-induced stereotypy: The methodology used for this test was inspired by that of Janssen et al. w19x. Briefly, male Wistar rats were placed in a cage from which behaviour could easily be observed. Clozapine or JL13 was administered at 20 mg kgy1 Žs.c.. and 60 min later apomorphine HCl was administered at 2.5 mg kgy1 Žs.c... The animals were observed for 60 s at 10-min intervals for a 1-h period and the different stereotypies were recorded Žsniffing, gnawing, rearing and grooming. and summed to give a total score. Neither clozapine, nor JL13 did antagonize apomorphine-induced stereotypy at 20 mg kgy1 s.c. Ž15 mg kgy1 s.c. free base equivalent. w18x ŽTable I.. 3. Apomorphine-induced climbing: Male MF1 mice ŽHarlan Olac Ltd., Bicester, UK. weighing 25]35 g were used in this experiment. Clozapine, JL13 or vehicle was injected s.c. 30 min before s.c. treatment with 0.75 mg kgy1 apomorphine HCl. Immediately after apomorphine injection, the animals were placed, individually, in wire mesh circular cages. Climbing behaviour, as described by Protais et al. w20x was scored 10 and 20 min after apomorphine administration. The ED50 Ždose inhibiting climbing behaviour at 50%. of drugs was calculated using non-linear regression analysis. The ED50 of clozapine was equal to 2.0 mg kgy1 s.c. while that of JL13 was equal to 3.9 mg kgy1 s.c. Ž2.9 mg kgy1 s.c. free base equivalent. ŽTable I.. 4. d-Amphetamine-induced stereotypy: Male NMRI mice, aged 5]6 weeks, were used. Stereotyped behaviour was induced by s.c. administration of d-amphetamine sulphate, at a dose of 12 mg kgy1 . Clozapine or JL13 was administered i.p. 30 min prior to damphetamine. After the injections, the mice were individually placed into wire mesh cages and were observed during a 2-h period. Stereotypy was scored according to the following criteria : 0 s absence of stereotypy, 1 s slight stereotypy of the head with intermittent sniffing, 2 s intense stereotypy of the head with licking andror gnawing. The minimal effective dose, defined as the lowest
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Table I Psychopharmacological properties of clozapine and JL 13 in several animal models Clozapine Induction of catalepsy in the rat
a
y1
ED50 s 108 mg kg
s.c.
JL 13 (free base equi®alent) ED50 ) 117 mg kgy1 s.c. Ž) 88 mg kgy1 .
Apomorphine-induced stereotypy antagonism in the rat b
Inactive at 20 mg kgy1 s.c.
Inactive at 20 mg kgy1 s.c. Ž15 mg kgy1 .
Apomorphine-induced climbing antagonism in the mouse a
ED50 s 2.0 mg kgy1 s.c.
ED50 s 3.9 mg kgy1 s.c. Ž2.9 mg kgy1 .
d-Amphetamine-induced stereotypy in the mouse c
Inactive up to 18.3 mg kgy1 i.p. Min active doses 32.7 mg kgy1 i.p.
Inactive up to 24.9 mg kgy1 i.p. Ž18.7 mg kgy1 . Min active dose ) 33 mg kgy1 i.p. Ž24.8 mg kgy1 .
d-Amphetamine-induced hyperactivity in the mouse c
Min active doses 3.3 mg kgy1 i.p.
Min active doses 4.4 mg kgy1 i.p. Ž3.3 mg kgy1 .
Ž".-DOI-induced head-twitches in the mouse c
ED50 s 0.5 mg kgy1 i.p.
ED50 s 2.0 mg kgy1 i.p. Ž1.5 mg kgy1 .
Open-field test in the rat Žclozapine and JL13 8]16]24 mg kgy1 i.p.. ŽJL13 base 6]12]18 mg kgy1 i.p.. d
}No effect on the ambulation score }No effect on the entry score }Significant decrease in the rearing score }Significant decrease in the defecation score
}Significant decrease in the ambulation score }No effect on the entry score }Significant decrease in the rearing score }Significant decrease in the defecation score
Forced swimming test in the mouse Žclozapine and JL13 4]8]16 mg kgy1 i.p.. ŽJL13 base 3]6]12 mg kgy1 i.p.. d
Significant dose-dependent increase in the immobility time
Significant dose-dependent increase in the immobility time
Temporal regulation schedule in the dog e ŽClozapine 5]10]15 mg kgy1 p.o.; JL13 5]10]20 mg kgy1 p.o.. ŽJL13 base 3.8]7.6]15.2 mg kgy1 .
}Dose-dependent decrease in total response rate Ž P) 0.05. and correct response rate Ž P- 0.01. }Extension of the temporal distribution of response duration to the right and left Ž P- 0.01. }Incomplete responses Ž P- 0.05. }Sialorrhea Ž P- 0.05. }Palpebral ptosis Ž P- 0.05. }Ataxia Ž P - 0.01. }Catalepsy Ž P) 0.05. }Akinesia Ž P- 0.01. }Tremor Ž P - 0.01. }Hyperkinesia Ž P ) 0.05.
}Dose-dependent decrease in total response rate Ž P) 0.05. and correct response rate Ž P) 0.05. }Extension of the temporal distribution of response duration to the right and the left Ž P- 0.01. }Incomplete responses Ž P) 0.05. }Sialorrhea: absent }Palpebral ptosis Ž P) 0.05. }Ataxia Ž P) 0.05. }Catalepsy Ž P ) 0.05. }Akinesia Ž P) 0.05. }Tremor: absent }Hyperkinesia Ž P) 0.05.
Drug discrimination in the rat f ŽClozapine 0.625]5 mg kgy1 i.p.; JL13 2.5]20 mg kgy1 i.p.. ŽJL13 base 1.9]15.2 mg kgy1 i.p..
95% clozapine lever selection at 5 mg kgy1 ED50 s 2.1 mg kgy1 No decrease in response rate
70% generalization to clozapine at 10 mg kgy1 ED50 s 7.6 mg kgy1 s 5.7 mg kgy1 free base Dose-related decrease in response rate
Drug discrimination in the Squirrel monkey g ŽClozapine 0.1]3.0 mg kgy1 i.m.; JL13 0.1]10 mg kgy1 i.m.. ŽJL13 base 0.08]7.6 mg kgy1 i.m..
More than 90% clozapine lever selection between 0.3 and 1.0 mg kgy1. Dose-related decrease in response rate
More than 90% clozapine lever selection between 3 and 10 mg kgy1 Ž2.25]7.50 mg kgy1 free base.. Dose-related decrease in response rate
a
Original results obtained by Connick et al.; Liegeois et al. w18x; ´ c Original results obtained by Matagne et al.; d Bruhwyler et al. w33x; e Bruhwyler et al. w26x; f Taylor et al. w38x; g Carey and Bergman w39x. b
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dose which significantly Ž P- 0.05. inhibited d-amphetamine stereotypy, was calculated using the Kolmogorov]Smirnov 2-sample test. JL13 was completely inactive against d-amphetamine-induced stereotypy, up to a dose of 24.9 mg kgy1 i.p. Ž18.7 mg kgy1 i.p. free base equivalent. and the minimal active dose was at least superior to 33 mg kgy1 i.p. Ž24.8 mg kgy1 i.p. free base equivalent.. Clozapine was inactive up to 18.3 mg kgy1 i.p. while a dose of 32.7 mg kgy1 i.p. produced a decrease of 48% Ž P- 0.05. ŽTable I.. 5. d-Amphetamine-induced hyperacti©ity: Male NMRI mice, aged 5]6 weeks, were used. Hyperactivity was induced by s.c. administration of d-amphetamine sulphate, at a dose of 4 mg kgy1 , 30 min before testing. Clozapine or JL13 was administered i.p. 30 min prior to d-amphetamine. For testing, each mouse was placed into an open-field cage and locomotor activity was recorded for 10 min. The minimal active dose, defined as the lowest dose w h ic h s ig n ifi c a n t ly in h ib it e d d amphetamine-induced hyperactivity, was calculated using the Mann-Whitney U-test Ž2tailed.. JL13 decreased d-amphetamine-induced hyperactivity by 45% Ž P- 0.05. from a dose of 4.4 mg kgy1 i.p. Ž3.3 mg kgy1 i.p. free base equivalent. and clozapine by 46% Ž P- 0.01. from a dose of 3.3 mg kgy1 i.p. ŽTable I.. 6. (")-DOI-induced head-twitches: Male NMRI mice, aged 5]6 weeks were used. Head-twitch behaviour was induced by i.p. administration of Ž " . -1- Ž 2,5-dimethoxy-4-iodophenyl . 2-aminopropane HCl ŽDOI., at a dose of 1.6 mg kgy1 . Clozapine or JL13 was administered i.p. 30 min prior to DOI. After DOI injection, the mice were individually placed into glass cylinders and the number of headtwitches was counted 5 min after DOI administration for a duration of 5 min. The ED50 values were calculated by non-linear regression analysis. JL13 produced a dose-related inhibition of DOI-induced headtwitches, with an ED50 value of 2.0 mg kgy1 i.p. Ž1.5 mg kgy1 i.p. free base equivalent.. For clozapine the ED50 was equal to 0.5 mg kgy1 i.p. ŽTable I.. 7. Open-field test: The procedure has been described in previous studies w21]23x. Male Wistar rats, 100]120 days old and weighing from 350 to 400 g were used. The apparatus consisted of a square wooden open-field Ž96 g = 96 cm., surrounded by a 28-cm high wooden wall. Clozapine, JL13 or vehicle was administered 30 min before the test. Thirty minutes after the administration the subject
was carefully placed in a particular square next to the wall and left in the open-field for 10 min. During this period, the total ambulation score, the entry score, the number of rears and the number of faecal boluses were measured, always by the same observer who was unaware of the drug injection. Clozapine had no significant effect on the total ambulation score while JL13 significantly Ž P- 0.05. decreased it from 8 mg kgy1 i.p. Ž6 mg kgy1 i.p. free base equivalent.. Both drugs had no effect on the entry score and significantly Ž P- 0.05. decreased rearing Žfrom 8 mg kgy1 i.p w6 mg kgy1 i.p. JL13 free base equivalentx. and defecation Žfrom 8 mg kgy1 i.p. and 16 mg kgy1 i.p. w12 mg kgy1 i.p. free base equivalentx respectively for clozapine and JL13. scores w33x ŽTable I.. 8. Forced swimming test: The procedure used in this study corresponded to that described by Porsolt et al. w24x with minor modifications. Outbred male mice ŽNMRI., of mean weight 35 g were used. The apparatus consisted of a glass cylinder Žheight 25 cm, diameter 10 cm. containing water Žliquid height 9 cm. at a constant temperature of 238C. Clozapine, JL13 or vehicle was administered 60 min before the test. Sixty min after drug administration, the animal was placed in the cylinder and the time of immobility was measured during the final 4 min of a 6-min test, always by the same observer who was unaware of the drug administered. Both clozapine and JL13 produced a dose-dependent increase in the immobility time, significant Ž P- 0.05. at the highest dose Ž16 mg kgy1 i.p. w12 mg kgy1 i.p. JL13 free base equivalentx. w33x ŽTable I.. 9. Temporal regulation in the dog: The procedure used in this study has previously been described as a complex multidimensional Žphysiological, motor and operant. approach of the behavioural effects of psychotropic drugs w25x. It was effective in discriminating barbiturates, 1,4- and 1,5-benzodiazepines, antidepressants, and typical and atypical antipsychotic drugs w25]28x. Naive male Beagle dogs Ž2]4 years old. were used. The program was a Differential Reinforcement of Response Duration ŽDRRD. schedule of 9 s with Limited Hold ŽLH 1.5 s. and positive and negative external cues. It consisted, in the random alternation, of two kinds of trials. In the first type of trial, a maintenance response lasting 9 s on a board was required for obtaining reinforcement. At the end of this time delay, an auditory stimulus of 1.5 s was given to the animal. Every time the dog left the board between 9 and 10.5 s and then
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jumped on to a food dispenser, it received a piece of meat Ž5 g.. The second type of trial differed from the first by the addition of the same auditory stimulus, randomly presented between the 3rd and the 6th s of the time delay. The auditory stimuli presented between 3 and 6 s and at 9 s were physically identical and had the same duration Ž1.5 s.; the animal could only discriminate between them according to their location in time. Both kinds of trials were distributed randomly during the session. Thus, the added stimulus was doubly random, first, because it was not given on each trial and, second, because it was given at random between the 3rd and the 6th s. In every case, the only reinforced response was that to the stimulus at 9 s; any anticipated Ž- 9 s. or delayed Ž) 10.5 s. response was not reinforced. Clozapine or JL13 was administered in capsule form in a random order at 2-week intervals. The experimental session began 4 h following drug administration. On the day before drug administration, the subjects received a placebo 4 h before a control session which was considered as baseline. All measures were taken by the same rater working blind. Operant performance was scored for total and correct response rates Žresponses per min., temporal distribution of response durations, and number of subjects producing incomplete responses. Effects on involuntary movements were recorded as the frequency of ataxic movements, cataleptic positions w29x, akinetic movements, trembling positions, dystonic symptoms. The number of subjects showing sialorrhea or palpebral ptosis was also noted. Clozapine Ž5]15 mg kgy1 p.o.. and JL13 Ž5]20 mg kgy1 p.o. or 3.8]15.2 mg kgy1 p.o. free base equivalent. produced a dose-dependent non-significant Ž P ) 0.05. decrease in the total response rate and a dose-dependent decrease in the correct response rate that was significant Ž P- 0.01. for clozapine but not for JL13. Both compounds induced a complete and significant Ž P- 0.01. temporal disorganization with a shift towards shorter and longer response durations. Clozapine also produced significant levels of incomplete responses Ž P0.05., sialorrhea Ž P- 0.05., palpebral ptosis Ž P- 0.05., ataxia Ž P- 0.01., akinesia Ž P0.01. and tremor Ž P- 0.01.. With JL13, these effects were either non-significant Ž P) 0.05. or even completely absent in the case of sialorrhea and tremor w26x ŽTable I.. 10. Drug discrimination in the rat: Twenty individually housed female Wistar rats were trained, in sound attenuated, computer con-
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trolled Colbourne Instruments ŽUSA. Skinner boxes, to discriminate clozapine at 5 mg kgy1 Ži.p.. from vehicle in a standard Fixed Ratio 30 quantal food rewarded two lever operant drug discrimination assay. This procedure was described in detail in previous papers w30, 31x. Clozapine and JL13 were administered i.p. On any experimental day rats received only one injection; either of clozapine or vehicle. All injections were administered 30 min before operant sessions of 15 min duration. On any individual trial accuracy of lever selection was assessed in terms of the total number of responses made on both levers prior to the presentation of the first reward. This index was termed the FRF, the total number of responses that were made prior to the First Reinforcement. The group as a whole was trained over 60 initial daily training sessions, by which time all individual animals were reliably selecting the correct lever on most experimental days, and the group was consistently showing greater than 85% correct lever selections on all training days. Subsequently, generalisation and substitution studies were initiated wsee 30 for detailsx. In these tests a series of doses of clozapine and JL13 were administered in counterbalanced orders. For the group as a whole it was thus possible to define, for each test dose, the percentage of tested animals selecting the drug Žclozapine. lever. In addition, on test days it was possible to assess any intrinsic actions of the drugs on operant response rate. In the test sessions clozapine induced dose-related generalization, as expected, with 95% drug lever selection being seen at the training dose of 5 mg kgy1 . Thus, as reported in previous studies w34]37x, clozapine, unlike typical neuroleptics such as haloperidol, is reliably discriminated by rats ŽED50 s 2.1 mg kgy1 i.p... This dose of clozapine had minimal effects on response rate in drug experienced rats. JL13 induced a marked Žmaximum 70%. generalization to clozapine. The ED50 for JL13 Ž7.6 mg kgy1 i.p.s 5.7 mg kgy1 i.p. free base equivalent. was calculated by logrlinear regression analysis of the linear portion of the dosereffect curve. The calculated regression line had an r 2 value of 0.61, thus it provided a reasonable, although not perfect fit to the data. JL13 had a clear dose-related suppressant effect on response rate, with substantial Žapprox. 40%. suppression of response being seen at the highest dose tested w38x.
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11.
Drug discrimination in the squirrel monkey: The aim of this test was to determine whether JL13 would substitute for clozapine in squirrel monkeys Ž Saimiri sciureus, 700]1250 g; n s 3. trained to discriminate i.m. injections of clozapine from vehicle in a standard drug discrimination procedure. During experimental sessions, monkeys were seated in chairs similar to the one described by Spealman et al. w32x and enclosed in ventilated, sound-attenuating chambers provided with white noise to mask extraneous sound. Two response levers were mounted in front of the monkey on a transparent wall of the chair. The tail of the monkey was placed in a small stock fitted with brass electrodes, which rested on, a shaved portion of the tail; electrode paste ensured a low-resistance contact between the electrodes and the tail. Electric shock of 5 mA intensity could be delivered to the tail for 200 ms from a 650-V a.c. transformer. Coloured lamps could be illuminated to serve as visual stimuli. Subjects were trained to press the lever to turn off visual stimuli associated with impending electric shock Žstimulus-shock termination schedule. under a 10-response fixed-ratio ŽFR. schedule under terminal conditions. Responding on one lever was operative after injection of clozapine and responding on the second lever was operative after injection of saline. Responding on the inoperative lever was counted but had no scheduled consequences. Once a criterion of at least 90% of responses on the injection-appropriate lever for ten consecutive sessions was reached, substitution testing began. Both clozapine and JL13 produced dose-dependent increases in responding on the clozapine-associated lever. Doses of clozapine between 0.3 and 1.0 mg kgy1 i.m. produced G 90% responding on the clozapine-associated lever Žfull substitution . in each of the three subjects. Although one monkey Žsubject 95]87. was more sensitive to the clozapine-like effects of JL13, doses of 3.0]10.0 mg kgy1 i.m. Ž2.25]7.50 mg kgy1 i.m. free base equivalent. of the novel diarylazepine produced G 90% responding on the clozapine-associated lever in all monkeys. Like clozapine, doses of JL13 that resulted in responding on the drug-associated lever had variable effects on rates of FR responding. However, the highest doses of JL13 Ž5.6]10.0 mg kgy1 i.m. s 4.2]7.5 mg kgy1 i.m. free base equivalent. markedly reduced response rates in both monkeys in which they were studied w39x.
DISCUSSION
All the results obtained in the different behavioural models attest that the psychopharmacological profile of JL13 possesses a high level of resemblance with that of clozapine. Like clozapine, JL13 presents a wide separation Žat least a factor 10. between the doses that inhibit apomorphine- or amphetamine-induced stereotypy and the doses that inhibit apomorphine-induced climbing or amphetamine-induced hyperactivity. Catalepsy is only detected at very high doses. These observations contrast with that made with traditional neuroleptics, such as haloperidol or chlorpromazine, for which all these effects are measured approximately in the same dose range. Catalepsy being a characteristic response to neostriatal dopamine receptor blockade w40x and apomorphine or amphetamine-induced stereotypy being a response dependent on the integrity of the dopaminergic input to the neostriatum w41x, it seems that JL13 like clozapine w42]45x only minimally interferes with this neural system. However, apomorphine-induced climbing and amphetamine-induced hyperactivity, which are mediated by enhanced dopaminergic neurotransmission in the mesolimbic system w46x, are antagonized both by clozapine w43]45x and JL13. Both drugs are able to antagonize Ž".-DOI-induced head twitches, a behaviour that is considered as mediated through an agonist effect on serotonergic 5-HT2A andror 5-HT2C receptors w47, 48x. Clozapine is three times more potent than JL13 Žbase equivalent.. Neurochemical studies have shown that clozapine has a high affinity both for 5-HT2A Ž K i " 5.0 nM. w17, 18, 49, 50x and 5-HT2C Ž K i " 7.5 nM. w50, 51x receptors while JL13 binds to 5-HT2A receptors Ž K i " 60 nM. w18, unpublished results of Meltzer and coll.x but is completely inactive on 5HT2C receptors Ž K i ) 10 m M. Žunpublished results of Meltzer and colleagues.. These results corroborate those obtained by Darmani et al. w48x in the shrew and by Schreiber et al. w52x in the rat tending to demonstrate that the effects of Ž".-DOI are mediated by 5-HT2A and not by 5-HT2C receptors. The antagonism of apomorphine or amphetamine effects is generally considered as related to the binding of drugs on dopaminergic D 2 receptors. Moreover, it has also been demonstrated that induction of climbing behaviour required both D1 and D 2 receptor activation w53x. Clozapine binds to the D 2 receptors with a medium affinity Ž K i " 100 nM. w17, 18, 49, 50, 54x while JL13 has only a negligible affinity for these receptors Ž K i s 1.2 m M. w18x, insufficient to explain its ability to antagonize apomorphine-induced climbing or amphetamine-induced hyperactivity in approximately the same dose range
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as clozapine. Other types of receptor interactions have thus to be implicated, such as the antagonism on D1 Ž K i clozapine " 140 nM w17, 18, 49, 54x; K i JL13 s 302 nM w18x., D4 Ž K i clozapine "25 nM w54]56x, K i JL13 s 164 nM wunpublished results of Van Tol and colleaguesx. or 5-HT2A receptors, to take into account the behavioural observations. Selective 5-HT2A antagonists, such as amperozide and MDL 100,907, which are virtually devoid of D 2 antagonist activity, are also able to reduce amphetamine-induced hyperactivity without affecting amphetamine-induced stereotypy and without producing catalepsy w57x. According to Schmidt et al. w58x, these results support the idea that 5-HT2A antagonists can dampen behavioural states associated with excessive dopaminergic activity. The reasons for looking for eventual anxiolytic andror antidepressant effects with clozapine and JL13 were triple. Firstly, like it has been mentioned in the introduction, the tricyclic azaarylaazepine structure constitutes a crossroads between typical Žclothiapine. or atypical Žclozapine. antipsychotics and antidepressant drugs Žamoxapine.. Secondly, anxiolytic properties have been recognized to clozapine in some animal models w59]61x and these properties have also been noted and even amplified with olanzapine, a thienobenzodiazepine structurally close to clozapine w61,62x. Thirdly, the fact that clozapine and JL13 present a higher affinity for 5-HT2A than for D 2 receptors could orient the pharmacological profile towards anxiolytics such as is the case for ritanserin, a potent 5-HT2Ar5-HT2C antagonist w63]65x. Two rodent models were used for this purpose: the open-field and forced swimming tests respectively for anxiolytic andror antidepressant properties. A complex temporal regulation ŽDRRD. schedule was also used in the dog because it had been shown to differentiate anxiolytics, antidepressants and typical or atypical antipsychotics w25]28x like other temporal regulation schedules such as DRL w66]70x. In the three models, clozapine and JL13 behaved as antipsychotics, decreasing locomotion and rearing without effect on the entry score in the open-field test w33x, increasing immobility time in the forced swimming test w33x and decreasing both total and correct response rates while disturbing the distribution of response duration in the temporal regulation procedure w26x. Of course, it is well known that the effects observed are largely dependent of the animal model itself. For example, ritanserin appears as a recognized anxiolytic in some w63]65x but not in all models w61, 71x. Therefore, it is not impossible that clozapine and JL13 give totally different results when evaluated in other procedures. The complete absence of catalepsy and stereotypyrhyperkinesia winterpreted as akathisia see 25x while tranquillization was significant with both compounds in the DRRD model confirmed their ‘atypicality’ w26x. As far as physiological and motor side effects
261
are concerned, some differences between clozapine and JL13 could be highlighted. Clozapine induced incomplete responses Žanhedonia., sialorrhea, palpebral ptosis, ataxia, akinesia and tremor at a significant level. With JL13, these effects were either completely absent Žsialorrhea, tremor. or produced only at a non-significant level Žincomplete responses, ataxia, palpebral ptosis and akinesia. in the same dose range as clozapine w26x. In drug discrimination procedures, JL13 substituted for clozapine, confirming that the compound has discriminative properties similar to that of clozapine. In the three squirrel monkeys, generalization to clozapine was complete Ž) 90%. respectively for 0.3, 5.6 and 10 mg kgy1 i.m. of JL13 Ž0.225, 4.2 and 7.5 mg kgy1 i.m. free base equivalent.. Reduction in response rate were measured from 5.6 mg kgy1 i.m. of JL13 Ž4.2 mg kgy1 i.m. free base equivalent. and 3 mg kgy1 i.m. of clozapine. Classical neuroleptics such as haloperidol, chlorpromazine and loxapine but also atypical antipsychotics such as olanzapine, risperidone, sertindole and remoxipride did not gerenalize to clozapine Žno more than 45% of subtitution. w39x. In the rat, maximal generalization Ž70%. was observed with JL13 at 10 mg kgy1 Ž7.5 mg kgy1 free base equivalent.. It is just possible that more complete generalization between clozapine and JL13 would have been seen at even higher doses of the drug, although this seems unlikely in that, surprisingly, slightly less generalization Ž55%. was seen at the highest dose tested Ž20 mg kgy1 i.p. salt s 15 mg kgy1 i.p. free base equivalent., despite the fact that this dose of JL13 had the most marked rate suppressant effect. It means that the discrimination is not simply based on a general tranquillizing or sedative effect of JL13. In the same procedure, typical neuroleptics but also risperidone, olanzapine, sertindole and amisulpiride did not generalize to clozapine Žno more than 50% generalization to clozapine. w38x. In conclusion, JL13 is a compound with multiple receptor targets ŽD4 , 5-HT2A, a 1, H1, .... and with a negligible affinity for D 2 receptors presenting a profile of activity similar to that of the atypical agent clozapine with some advantages such as the absence of sialorrhea and tremor. It would therefore be predicted that JL13 will have an atypical profile and be less likely to induce undesirable extrapyramidal symptoms in the clinic than currently available antipsychotic drugs.
ACKNOWLEDGEMENTS Dr JF Liegeois is Senior Research Associate of the ´ FNRS ŽFonds National de la Recherche Scientifique. Belgium. This study was supported in part by U.S. Public Health Service Grants DA 03774, MH 07658, and RR 00168. We are grateful to Dr Connick and Tonnaer from Akzo Nobel Organon, to Dr
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Matagne and Klitgaard from UCB and to C. Lejeune from the University of Namur for their collaboration to some of these experiments. The collaboration of Dr Van Tol from the University of Toronto for the determination of D4 binding is gratefully acknowledged.
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