psychopharmacological effects of an imino-thiazolidine derivative antidepressant candidate,...

Drug Development Research 22:385-399 (1991)

Psychopharmacological Effects of an Imi no-Th iazol idine Derivative Antidepressant Candidate, EGYT-4201 lstvan Gyertyan, Lujza Petocz, Istvan Gacsalyi, Marton I.K. Fekete, Kornelia Tekes, and Laszlo Kapolnai

EGIS Pharmaceuticals, Division of Pharmacology (I. G., L.P., I.G., M.I.K. F., L. K.), and Department of Pharmacodynamics, Semmelweis Medical School (K. T.), Budapest, Hungary

ABSTRACT

Gyertyan, I., L. Petocz, 1. Gacsalyi, M.I.K. Fekete, K. Tekes, and L. Kapolnai: Psycho- pharmacological effects of an imino-thiazolidine derivative antidepressant candidate, EGYT-4201. Drug Dev. Res. 22:385-399, 1991.

The imino-thiazolidine derivative EGYT-4201 exhibited marked activity in pharmacological tests characteristic for antidepressants. In the behavioral despair model and in antagonizing the effect of tetrabenazine its efficacy was considerably higher than that of several tricyclic and second generation antidepressant drugs. The compound also proved to be active in inhibiting the hypothermic effect of apomorphine. It potentiated the lethal effect of yohimbine, the hypermotility provoked by L-dopa, and the stereotypy induced by D-amphetamine. In contrast to amitriptyline and nomifensine, EGYT-4201 showed neither sedative nor stimulant actions on spontaneous and conditioned behavior. The compound, similar to amitriptyline and viloxazine, exerted a moderate anticonvulsive effect against pentylenetetrazole, bicuculline, and picrotoxin. EGYT-4201 did not exhibit significant anticholinergic activity on physostigmine induced lethality, oxotremorine evoked tremor, and isolated guinea pig ileum. In acute toxicity studies it proved to be more favourable than the reference compounds. EGYT-4201 did not inhibit the enzymatic activity of the monoamino-oxidase (MAO) enzyme, nor did it block the uptake of monoamines. The molecule was found to be inactive in binding studies of noradrenergic, dopaminergic, and serotonergic receptor types.

Key words: psychopharmacology, tetrabenzine, D-amphetamine

Received final version August 6, 1990; accepted September 6, 1990.

Address reprint requests to Istvin Gyertyin, EGIS Pharmaceuticals, Department of Pharmacology, Budapest, P.O.B. 100, Hungary, H-1475.

0 1991 Wiley-Liss, Inc.

386 Gyertyan et al.

CI

NH2 I

N A ti 2 ETS03 H

Fig. 1. Chemical structure of EGYT-4201: 2-imino-3-(2’-amino-4’-chloro-phenyl)-thiazo~idine diesilate.

INTRODUCTION

Tricyclic antidepressant drugs have been used for more than three decades in the therapy of affective disorders. In the last 15 years several representatives of the so called “second generation” antidepressants have appeared in the literature and in the therapy. These drugs are of various types of chemical structures, possess less adverse effects and greater safety, and often act by a new or even unknown mechanism; nevertheless their clinical efficacy does not necessarily surpass that of the traditional drugs [Shopsin et al., 1981; Coccaro et al., 1985; Leonard, 1985; Hollister, 19861. In 1986 Lempert et al. synthesized imino-thiazolidine derivative molecules. Out of more than 70 compounds tested up to now in our laboratory 2-imino-3-(2’-amino-4‘-chloro-phenyl)-thiazolidine diesilate (EGYT-4201, Fig. 1) has been chosen for further investigation because of its remarkable psychopharmacological effects. The drug has been considered as a potential atypical antidepressant. The present paper contains the basic psychopharmacology of EGYT-4201.

MATERIALS AND METHODS

The following drugs were used: EGYT-4201 (the compound [molecular weight: 4491 is a white powder, is stable at room temperature, has a melting point of 134”C, and has very good solubility in water), amitriptyline HC1, imipramine HCl, desmethylimipramine HCI, L-dopa, nialamide (all produced and supplied by EGIS, Budapest, Hungary), viloxazine HCI (ICI, Macclesfield, U.K.), trazodone HC1 (Boehringer Inc., Ingleheim, FRG), nomifensine maleate (Hoechst, Frankfurt, FRG), yohimbine HCI, pentylenetetrazole (Serva, Heidelberg, FRG), D-amphetamine sulphate (Eryfarm Italiana, Milan, Italy), apomorphine HCl, pargyline HCI, physostigmine hemisulphate, isoproterenol HCl, benztropine methansulphonate (Sigma, St. Louis, MO), tetrabenazine, oxotremorine, picrotoxin, bicuculline, Tris-HC1 (Fluka, Buchs, Switzerland), and chlorimipramine HCI (CIBA-GEIGY, Basle, Switzerland). Radioactive materials such as 3H-spiperone (22.9 Cilmmol), 3H-prazosine (19.8 Ci/mmol), 3H-5-HT (30 Ci/mmol), and 3H-ketanserine (61.8 Ci/mmol) were obtained from New England Nuclear (Bad Homburg, FRG), 3H-dihidroalprenolol (74 Ci/mmol) from the Biological Research Center of the Hungarian Academy of Sciences (Budapest, Hungary), 3H-clonidine (24 Ci/mmol), 3H- noradrenaline (32.97 Ci/mmol), 3H-dopamine (47 Ci/mmol), 3H-serotonin (14.51 Ci/mmol), and I4C-tyramine (56 mCi/mmol) from Amersham (Buckinghamshire, U.K.).

Drugs were dissolved in distilled water except L-dopa and nialamide which were given in suspension of 0.1% polysorbate (Tween) 80. To promote solution a small amount of 1 M hydrochloride acid was added in the case of nomifensine, bicuculline, and tetrabenazine. Apomorphine was dissolved adding 1 mg ascorbic acid for each milliliter of the final solution. Volume of injections to mice was 20 ml/kg. Rats were administered 5 ml/kg solution.

All the doses referred in the study relate to the salt. The following animals were used: mice of the CFLP strain, weighing 20-25 g, rats of

the Wistar (140-180 g), Long-Evans (120-160 g), and OFA (120-200 g) strains, and guinea

EGYT-4201, A Potential Antidepressant 387

pigs. Animals were obtained from LATI (Laboratory Animal Breeding Institute, Godollo, Hungary) and kept in conditions of 22-24”C, 12 hr light-dark cycle, and free access to food and water. Mice and rats were housed in groups of 10 and 5, respectively. Where not stated otherwise animals of both sexes were used in the experiments.

Tests for Antidepressant Activity

Tetrabenazine induced ptosis. Groups of 10 mice were treated orally with doses of the test compounds or saline. Thirty minutes later animals were administered 50 mg/kg tetrabenazine intraperitoneally (i.p.). The number of animals with ptosis was recorded at 30-60-90-120 min. Mean ptosis frequency was calculated for each group.

Behavioral despair. The experiments were carried out in male Long-Evans rats according to Porsolt [1981]. The test compounds were administered orally. Percent of the time spent in the characteristic immobile posture by the rats was measured.

Yohimbine induced lethality. The experiment was performed in mice according to Quinton [1963]. Sixty rnin after oral administration of the test drug 20 mg/kg yohimbine was injected i.p. Twenty-four hours after yohimbine injection lethality was recorded.

Apomorphine induced hypothermia. The experiment was performed in male mice according to the modified method of Puech et al. [1981]. EGYT-4201 or saline was administered i.p. 30 min prior to the subcutaneous (s.c.) injection of apomorphine (16 mg/kg). Rectal temperature of mice was measured 30 min after the application of the hypothermic agent.

D-amphetamine elicited stereotyped behavior. Male Wistar rats received oral doses of EGYT-4201 or saline and were placed into metal wire cages for a 30 rnin habituation period. This was followed by the S.C. injection of 2 mg/kg D-amphetamine. Animals were replaced into the cages and were rated for stereotypy every 15 min for 210 min. The scale of Costall and Naylor [1972] was used.

L-dopa induced hypermotility. The experiment was based on the method of Everett [1967] with a modification: instead of using a scoring system animals were tested in an 8-channel activity meter (Omnitech Electronics Inc., Colombus, OH) working with infrared photobeams. Male mice were treated with pargyline 50 mg/kg i.p. I hr before oral administration of the test compounds or saline. L-dopa was given in a dose of 100 mg/kg i.p. 30 min following the test compounds. After another 30 min motor activity (i.e., number of lightbeam interruptions) was recorded in the activity meter for 30 min.

Tests for Sedative-Stimulant Activity and for Activity on Learning Performance

Spontaneous motor activity of mice. Male mice were injected with the test compounds or saline p.0. then 60 rnin later were placed into a 10-channel activity meter. Three mice per channel were used. Number of infrared lightbeam interruptions was registered for 30 min .

Potentiation of ethanol induced narcosis. One hour after oral drug treatment 15% (v/v) ethanol was given to mice intravenously (i.v.) in a volume of 10 ml/kg. The reaction was considered positive if the sleeping time of the drug treated animals was at least 2.5 fold longer than the mean value of the control group.

Stereotyped behavior in rats. After the oral administration of the test compounds animals were placed into metal wire cages for 90 min. The observation period began 30 rnin later. Stereotyped behavior was scored every 10 min for 60 min according to Costall and Naylor [ 19721.

GO - NO GO avoidance conditioning. The conditioning paradigm was based on the method of RosiC and Bignami [1970]. Male Wistar rats of 120-150 g at the beginning of the experiment were conditioned to perform active avoidance response in a two way Shuttle-box (VKI, Hungary) with 96 trials a day. The conditioned stimulus (CS), i.e., the “GO” signal was flashing (1 Hz) white light with a duration of 15 sec. The unconditioned stimulus (US)

388 Gyertyan et al.

was scrambled footshock of 0.6 mA given in the last 5 sec of the CS period. CS was terminated by an avoidance response; CS and US were terminated by an escape response. The length of intertrial interval was 15 sec. Intertrial responses were not punished. Having reached a stable response level of at least 75% performance animals were entered into the GCL-NO GO conditioning schedule: passive trials were introduced, pairs of which were alternately given with pairs of active trials, 48 trials a day of each. In the passive trials the “NO GO” signal (CS) was constant red light with a duration of 15 sec, the US was scrambled footshock of 0.6 mA given simultaneously with the CS in the other compartment of the Shuttle-box. Animals were required to refrain from crossing in order to avoid shock. After stabilization of the performance level drugs were administered to the animals. The rats received the compounds orally 1 hr before the beginning of the daily session, once a week. Learning performance of each animal under drug treatment was compared to the performance of the same animal on the previous day (drug-free condition). Anticholinergic Activity

Oxotremorine induced tremor. The experiments were carried out in groups of 10-30 mice according to the method of Spencer [1966]. Oral pretreatment with the test compounds or saline was performed 30 min before the S.C. injection of 0.5 mg/kg oxotremorine. Forty-five minutes after the administration of the tremorogenic agent tremor was scored as follows: 2, spontaneous tremor; 1 , provoked tremor; and 0, absence of tremors.

Physostigmine induced lethality. The experiments were performed in groups of 10-30 mice according to the method of Nose and Kojima [ 19701. One hour after oral treatment with the test compounds or saline standard physostigmine dose ( 2 mg/kg i.p.) was administered to the animals and the survivors were counted after 60 min.

Isolated guinea pig ileum. Segment of terminal ileum was isolated from decapitated male guinea pigs (365-615 g). A 35°C Tyrode’s solution saturated with carbogen was used in a 4 ml organ bath. Isotonic recording (B-396 transducer; Hugo Sachs Elektronik) was done with 0.5 g preload. Dose-response curves were obtained with acetylcholine in the absence and in the presence of the test compounds (incubation period 15 min). pA2 values were calculated according to the method of Arunlakshana [ 19591. Anticonvulsant Action

Convulsions evoked by chemical agents. Mice were treated orally with the test compounds or saline 60 min before the administration of the convulsant. Picrotoxin and bicuculline were injected S.C. in a dose of 10 and 3 mglkg, respectively. Pentylenetetrazole was given in a 125 mg/kg i.p. dose. Number of animals showing tonic extensor convulsions in the hindlegs were registered. The observation period was 30 min in the case of picrotoxin and bicuculline, and 20 min for pentylenetetrazole.

Convulsions induced by maximal electroshock. Studies were carried out according to Swinyard et al. [1952]. Sixty minutes after oral administration of the test compounds or saline a 50 Hz, 45 mA, 0.4 sec electroshock was applied through corneal electrodes to mice. The complete inhibition of the tonic extensor convulsions of the hindlegs was considered as the criterium of anticonvulsant effect. Frequency of non responding animals was recorded. Group size was 10. Inhibition of the Activity of the Monoamino-Oxidase (MAO) Enzyme

Effect on M A 0 activity in vitro. P, fractions of crude mitochondria from the liver and the brain of male OFA rats were prepared according to Whittaker [ 19651. Protein content was measured according to Bradford [1976]. MA0 activity was assayed in phosphate buffer in 0.75 ml final volume with 14C-tyramine (0.014 mmol/liter). The enzymatic reaction was started after 20 min preincubation of the enzyme with the test compounds by adding the substrate to the medium. Following 20 rnin incubation at 37”C, 0.03 ml of 3 moliliter HC1 was added to terminate the reaction. Radioactivity was measured by liquid scintillation (Beckman


LS 7500) in toluol cocktail containing 0.3% 2,5 diphenyl-oxazol (PPO) and 0.015% 1,4- di-(2-(5-phenyl)oxaziol)beurol (POPOP).

Effect on M A 0 activity ex vivo. Rats were treated orally with 25 mg/kg EGYT-4201 or saline. Two hours later animals were killed and MA0 activity was determined as described above.

Potentiation of the lethal effect of tryptamine. The experiments were performed according to the method of Maxwell et al. [1961] in groups of 10-30 mice. One hour after oral treatment with the test compounds or vehicle 250 mgikg tryptamine was administered S.C.

Lethality was recorded after 3, 6, and 24 hr. Receptor binding studies. Male OFA rats of 120-200 g were used. Membrane prep-

arations were made from the cortex for alpha-1 [Greengrass and Bremner, 19791, beta [Bylund and Snyder, 19761, and 5-HT-2 [Leysen, 19811 binding studies; from whole brain [Pasternak et al., 19751 for alpha-2; from the striatum [Leff et al., 19811 for D-2; and from the hippocampus [Nelson et al., 19781 for 5-HT-1 measurements. Protein content was measured according to Bradford [1979]. The incubation of the membrane with the radioligands in the presence or absence of the test compounds was carried out in Tris-HC1 buffer in 1 ml volume at 25°C for 20 rnin in the case of alpha-1, D-2, and 5-HT-2 binding determination (0.2 nmol/liter 3H-prazosin, 0.4 nmol/liter 3H-spiperone, and 1 nmol/liter 3H-ketanserine, respectively); for 30 min in the case of alpha-2 (2 nmol/liter 3H-clonidine); or in 0.25 ml volume for measuring beta and 5-HT-1 receptor binding (1 nmol/liter 3H-dihidroalprenolol and 1.5 nmol/ liter 3H-serotonin, respectively). Incubation was terminated by rapid vacuum filtration. The radioactivity was measured in dioxane based scintillation fluid. The results were calculated by “Ligand” computer fitting program [Munson and Rodbard, 19801.

Inhibition of monoamine uptake in vitro. Hypothalamus, striatum, and hippocampus of decapitated male Wistar rats weighing 130-150 g were prepared as described by Glowinski and Iversen [ 19661. The cell free homogenate was used for the uptake studies according to the modified method of Snyder and Coyle [1969], in a final volume of 1 ml. The uptake of 3H-noradrenaline was measured in the hypothalamus, that of 3H-dopamine in the striatum, and the uptake of 3H-serotonin in the hippocampus. After 5 rnin preincubation at 37°C the reaction was started by adding the 3H-biogenic amine. After 5 rnin incubation reaction was terminated by 5.0 ml of ice-cold buffer, then the reaction mixture was quickly filtered on Whatman GF/C glass fiber filters under vacuum. The radioactivity of the filters was measured by liquid scintillation (Beckman LS 9000) in toluol, containing 0.5% PPO and 0.01% POPOP. The values were corrected for the counts of blank samples, incubated at 0°C. Protein content was measured according to Peterson [ 19771.

Inhibition of monoamine uptake ex vivo. Groups of 10 rats were treated i.p. with 5 and 25 mg/kg EGYT-4201 and were decapitated after 12 and 24 hr. Uptake of noradrenaline, dopamine, and serotonin was measured as described above.

Acute toxicity. Frequency of lethality after oral treatment with the test compounds was registered for 7 days in mice and rats. Ten to 40 animals were used per dose group.

Statistical evaluation. Basic data of groups in Tables 1-9 and Figures 1-6 are of three types: frequency, mean 2 SEM, and median with interquartile halfrange values. The latter was calculated for any type of scores. For determination of statistical significance analysis of variance (ANOVA), Student’s t-test, and Mann-Whitney U-test were used. ED50 and LD50 values (with confidence intervals in brackets) were determined by probit analysis, and ID50 and IC50 values were calculated by linear regression.

RESULTS Antidepressant Activity

itriptyline and viloxazine (Fig. 2). EGYT-4201 was more effective in antagonizing the effect of tetrabenazine than am-

390 Gyertyan et al.

0.5 1 2.0 5.0 6.25 10 12.5 2 0 25 & m w b p.0.

I , Fig. 2. Dose-effect curves of EGYT-4201 (V) and reference antidepressants on tetrabenazine (50 mgikg i .p . ) induced ptosis in mice. Mean ptosis frequency of the saline treated group was 8.2; n = 10 in each group. The ED50 values are 1.5 (0.91-2.36) mglkg for EGYT-4201,3.5 (1.7-7.0) mglkg for viloxazine (0) and 12.0 (7.6-18.9) mgikg for amitriptyline (0).

In the behavioral despair model (Fig. 3 ) the compound dose-dependently inhibited the time spent in immobile posture by the rats. Comparing approximately equiactive doses of the different compounds tested it can be concluded that EGYT-4201 proved to be about twice as potent as the tricyclic drugs and half as effective as nomifensine. Trazodone showed weak effect in this model.

EGYT-4201 potentiated the lethal effect of yohimbine (Table 1). Its efficacy was com- parable to that of amitriptyline and it surpassed those of imipramine and viloxazine. The compound dose-dependently inhibited the hypothermic action of apomorphine (Table 2). However, EGYT-4201 exhibited significant inhibition only in relatively high doses and of moderate magnitude. The drug potentiated the amphetamine induced stereotyped behavior in rats (Fig. 4). Its effect, however, was not dose-dependent.

EGYT-4201 potentiated the L-dopa induced increase in motor activity of mice (Table 3 ) . In lower doses the moderate and not significant effect of the compound was like that of amitriptyline, while at 50 mg/kg the potentiation by EGYT-4201 reached the statistically significant level and a higher magnitude, in contrast to amitriptyline which showed no potentiation at all at this dose.

Tests for Sedative-Stimulant Properties and for Activity on Learning Performance

EGYT-4201 did not affect the spontaneous motor activity of mice either in sedative or in stimulant direction (Table 4). Amitriptyline, viloxazine, and trazodone exhibited considerable sedative effect (in the above order of potency), whereas desipramine caused only a mild and non dose-dependent decrease in motility. In contrast to these drugs, nomifensine increased the locomotor activity of mice.

In a wide dose-range EGYT-4201 had no influence on the duration of narcosis provoked by ethanol (Table 5). Amitriptyline and trazodone displayed notable sedative liability in this model as well, but viloxazine was inactive.


%

EGYT-4201

80

f 70

**

dose 125 25 50 100 50 50 50 50 125 50 m q k g

Pa

Fig. 3. Effect of EGYT-4201 and some antidepressant drugs on the behavioral despair (forced swim- ming) model. Ordinate: percent inhibition of the time spent in the characteristic immobile posture by rats. Abbreviations: AMI, amitriptyline; IMI, imipramine; DMI, desipramine; VIL, viloxazine; NOM, nomifensine; TRA, trazodone. The number of animals tested are shown in the appropriate column. * and ** sign difference of P < 0.05 and P < 0.01 statistical significance, respectively (Student’s t-test).

TABLE 1. Potentiation of Yohimbine (20 mg/kg i.p.) Induced Lethality in Mice Compound EDSO(mg/kg p.0.) Therapeutic index” EGYT-4201 13.9 (7.6-25.4) m:89.9; f:48.9 Amitriptyline 12.5 (8.3-18.7) 18.0 Imipramine 21.0 (12.2-35.7) 21.4 Viloxazine 20.0 (10.3-39.0) 5 .5

“LD50iED50

TABLE 2. Effect of EGYT-4201 on Aoomorohine Induced Hypothermia in Mice

Apomorphine S.C. 16 mg/kg Dose (mg/kg i.p.)” Change (“C) Inhibition (70) 0 (saline) -3.10 - 1 -2.64 15.0 2.5 -2.91 6.6 5 -2.44 21.5 12.5 - 1.12** 63.9 25 -0.94** 69.8

an = 20 in all groups. *P < 0.01 Student’s t-test.

EGYT-4201 showed negligible stimulant action on spontaneous behavior of rats up to 100 mg/kg (Fig. 5). In contrast to it, 50 and 100 mg/kg of nomifensine caused moderate stereotyped behavior. In these doses rats displayed increased locomotor activity with contin- uous sniffing and lateral head movements. This degree of stereotypy sustained until the end of the observation period.

EGYT-4201 elicited a mild though significant improvement in the active avoidance

392 Gyertyan et al.

score

2

1

AmFh 4 2 0 1 dore 2 125 25 50 100 rm&

p c o 0 5 MannMdney U-test

t . .

I

Fig. 4. Potentiation by EGYT-4201 of D-amphetamine (2 mg/kg s.c.) induced stereotyped behavior in rats. Curves: saline (a), 12.5 mgikg ( O ) , 25 mg/kg (A), 50 mg/kg (O), and 100 mg/kg (V) EGYT-4201 p.0. and D-amphetamine s.c., respectively. Ordinate: medians of stereotypy scores according to Costall and Naylor [ 19721. Abscissa: time following amphetamine injection. Interquartile halfrange values were not greater than 0.72. Asterisks sign differences of P < 0.05 statistical significance (Mann-Whitney U-test) from the saline treated group at the corresponding time point. n = 8 for each dose group. The insert shows the medians of the maximal scores in each group.

responding in a GO-NO GO conditioning paradigm (Fig. 6). Nomifensine considerably aug- mented the active learning performance of rats, but at the same time it caused a similarly considerable impairment in the response level of the NO GO phase. This bidirectional effect indicates that the observed changes are not learning specific, they rather reflect a generalized stimulant action of the compound. Unlike nomifensine, EGYT-420 1 only slightly decreased the performance in the passive trials, thus exhibiting a more balanced influence on the overall avoidance response. Amitriptyline remarkably and significantly reduced the percentage of correct responses in the GO trials without the simultaneous disruption of the passive performance of animals. This effect can be attributed to its sedative properties.

Anticholinergic Activity

EGYT-4201 proved to be inactive in the oxotremorine induced tremor test in a dose- range of 10-160 mg/kg p.0. Amitriptyline and imipramine blocked the effect of oxotremorine giving ID50 values of 6.6 mg/kg and 31.5 mg/kg, respectively. EGYT-4201 also failed to block the lethal effect of physostigmine up to 80 mg/kg. At the 160 mg/kg dose it exhibited 30% inhibition only.

On isolated guinea pig ileum in lop5 mol/liter concentration the compound was completely ineffective in blocking the contractions induced by acetylcholine. Amitriptyline had a PA, value of 7.8; atropine, used as standard reference drug, gave 8.9.


TABLE 3. Effect on L-dopa Induced Hypermotility

Compound mdkg time (min) counts ( k SEM) Effect (%) Dosea Pretreatment No. motility

Pargylin i . p . 50 120 1,773.6( k 208.1) -

Pargylin i . p . 50 120

Pargylin i.p. 50 120

+ saline p.0 60 + vehicle i.p. 30

+ Saline p.0. 60 + L-dopa i.p. 100 30 6,382.2(+ 1,437.6) 259.9*1-

+ L-dopa i.p. 100 30 + EGYT-4201 P.O. 12.5 60 9,473.6(* 1,479.9) 48.4

25.0 60 8,703.1(21,619.5) 36.4 50.0 60 11,571.6(+ 1,873.1) 81.3*

Pargylin i.p. + Saline p.0. + Vehicle i.p

+ saline p.0. + L-dopa i.p.

+ L-dopa i.p. + Amitnptyline p.0.

Pargylin i.p.

Pargylin i.p.

50

50

100 50

100 12.5 25 so

120 1,192.8( 2 232.9) -

60 30

120 60 30 7,22 1.8( *2,0 19.5) 505.5*/-

120 30 60 11,041.4(+1,433.7) 52.9 60 10,894.4(+ 1,262.2) 50.9 60 6,650.7(? 1,945.8) -7.9

an = 9 in all groups. *P < 0.05 Student’s t-test.

TABLE 4. Effect of Antidevressant Compounds on Spontaneous Activitv of Mice % Changea Dose

(mg/kg p.0.) A M I ~ VIL TRA DMI 420 1 NOM 12.5 - 43 -38 - 20 - 24 + 29 25 -78 -43 - 44 -31 -5 + 42 50 -83 -7 1 - 63 - 29 + 3 + 44 100 - 87 - 77 - 77 - 35 + 6 200 - 24

- - - ID50 (mglkg) 11.2 25 .O 34.1

aRelated to the mean number of photobeam interruptions of the control group. n = 10 in all groups. bFor abbreviations of compounds see Figure 3.

Anticonvulsant Profile

EGYT-4201 showed moderate anticonvulsive activity against chemical convulsants (Table 6). Against picrotoxin and pentylenetetrazole its potency was somewhat weaker than that of amitriptyline and viloxazine, but it was stronger than that of imipramine and desipramine. The compound proved to be the most effective antagonist of bicuculline among the tested drugs. EGYT-4201, unlike the other molecules, yielded ED50 values in a very narrow range against three different convulsant agents. The compound was less active in inhibiting seizures induced by electroshock exhibiting equal efficacy with imipramine and being considerably weaker than amitriptyline or viloxazine.

394 Gyertyan et al.

TABLE 5. Influence of EGYT-4201 and Other Antidepressants on the Duration of Ethanol Induced Narcosis in Mice

EGYT-4201 Amitripty line Trazodone Viloxazine

Dose Positive Positive Positive Positive mg/kg p.0. treated' (%b) treated (%) treated (%) treated (%)

6.25 0/6 (0) 1/6 (16.7) 12.5 0/10 (0) 6/12 (50) 016 (0) 016 (0) 25 0110 (0) 8/12 (75) 1/6 (16.7) 016 (0) 50 0112 (0) 8/10 (80) 216 (33.3) 016 (0) 100 016 (0) 016 (0) 5/6 (83.3) 200 016 (0) ED50 - 20.1 55.8 - (mg/kg) ( 13.6-29.7) (32.5-96.0) "Potentiation of narcosis was considered to be positive if the sleeping time of the drug treated animal was at least 2.5 fold longer than the mean value of the control group. The mean duration of ethanol narcosis in the control groups varied between 50.8 and 59.2 sec. bPercent potentiation.

NOMlFENSlK EGY T - 4201 score

Fig. 5. Stereotyped behavior in rats after nomifensine and EGYT-4201 treatment. Medians of scores against time following oral drug administration are plotted. Each point of the curves represent median score of 10 animals. Interquartile halfrange values were not greater than 0.67. Symbols, 0, 12.5 mglkg; 0, 25 mglkg, 0, 50 mg/kg, and A, 100 mg/kg for both compound. Data points with a score value greater than 0.75 represent statistically significant difference of P < 0.05 (Mann-Whitney U-test) from controls.

Mechanism of Action Studies

Compared to well known M A 0 inhibitors EGYT-4201 practically did not possess MA0 inhibiting effect in vitro (Table 7). Measuring ex vivo after 2 hr pretreatment time 25 mg/kg oral dose of the molecule brought about 20.6% and 0% inhibition of M A 0 activity in rat brain and liver, respectively (n = 3). Lacking of in vivo M A 0 inhibiting activity is also supported by the fact that EGYT-4201 did not potentiate the lethal effect of tryptamine in mice (Table 7).

EGYT-4201 was a very weak inhibitor of the synaptosomal uptake of monoamine neurotransmitters in vitro (Table 8) being 2-3 orders of magnitude less potent than the refer-


z

a -40

w -30. U z I

x

Fig. 6. Effect of EGYT-4201, nomifensine, and amitriptyline on the learning performance of rats in a GO-NO GO avoidance conditioning paradigm (for details see Methods). On the ordinate absolute change in learning performance (expressed as percentage of correct responses) is scaled. Number of rats tested are shown in each column. * and ** sign P < 0.05 and P < 0.01 statistical significance, respectively (paired t-test).

TABLE 6. Anticonvulsant Profile (ED50 Values) of Some Antidepressant Drugs and EGYT-4201 Convulsive agent

Picrotoxin Bicuculline Pentetrazole Maximal Compound (I0 mglkg s.c.) (3 mg/kg s.c.) (140 mg/kg i.p.) electroshock EGYT-420 1 14.6 9.5 14.0 103.0

(1 0.2-20.9) (6.2-14.5) (7.9-24.9) (81.6-129.8)

tyline (4.4-10.1) (9.6-1 7.2) (3.3 - 8.2) (23.3-53.1) Amitrip- 6.6 12.9 5.2 35.4

Imipramine 27.7 61.6 31.0 122.3 (17.7-43.4) (47.1-80.6) (17.4-55.1) (87.6-170.8)

Desipramine 24.6 >180 45.0 >200 ( 12.4-48.6) (1 3.5 - 149)

Viloxazine 7.7 29.2 7.0 31.3 (5.1-1 1.6) (1 9.5 -43.5) (4.7-10.4) (18.1-54.2)

TABLE 7. Effect of EGYT-4201 and Two MAo Inhibitors on MA0 Activity In Vitro and In Vivo

Enzyme activity IC50 (mol/liter) Tryptamine potentiation

Compound Brain Liver ED50 (mg/kg) EGYT-4201 > > 10-4 > 160 Nialamide 4 x 10-7 1.8 x 10-7 11.3 (9.4-13.6) Tranylcipromine 1.7 X lo-* 4 x 10-7 1.75 ( 1.54-1.99)

ence compounds. Administered ex vivo the compound showed no inhibition of the monoamine uptake in the applied doses.

molil EGYT-4201 produced less than 50% displacement on the following receptors (Ki values of the appropriate reference drugs are given in parentheses in nmol/liter): alpha-1 (prazosine, 7); alpha-2 (yohimbine, -100, amitriptyline, -10 000); beta

In a concentration of

396 Gyertyan et al.

TABLE 8. Effect of EGYT-4201 and Reference Drugs on the Uptake of Monoamine Neurotransmitters in Rat Brain Regions In Vitro

Serotonin Dopamine Noradrenaline Compound (cortex) (striaturn) (hypothalamus) EGYT-4201 3.45 x 4.36 x 1 0 ~ Clomipramine 4.15 x Imipramine 4.20 x 1 0 - ~ 8.1 x 10-8 Desipramine 2.6 x 10-9 Nomifensine 1.1 x 10-7 Benztropine 2.1 x lop7

"Data are IC50 values in moliliter.

(dl-isoproterenol, 68; amitriptyline, 15 000); dopamine-2 (chlorpromazine, 50.5); 5-HT1 and 5-HT2 (cyproheptadine, 0.85; amitriptyline, 19.9).

Acute Toxicity

Compared to other antidepressant drugs EGYT-4201 is a drug with relatively low toxicity (Table 9). The compound showed sexual divergence in mice. This phenomenon was not observed in rats and cats in which the obtained LD50 values are 1,050 (796-1,384) mg/kg and 180 (126-257) mg/kg, respectively.

DISCUSSION

The imino-thiazolidine derivative EGYT-4201 showed considerable activity in models used for screening antidepressant drugs, such as tetrabenazine antagonism [Howard et al., 19811, behavioral despair [Porsolt, 1981; Bizikre et al., 19851, or yohimbine potentiation [Malick, 1981; Bizikre et al., 19851. Efficacy of the compound in these tests was higher than or at least equal to the reference antidepressant drugs. Inhibition of hypothennia induced by high dose apomorphine is also a frequently used method for detecting antidepressant activity and EGYT-4201 proved to be effective in this model as well. However, its potency did not reach that of other antidepressants cited in the literature [Puech et al., 1981; Pawlowski and Mazela, 19861. The compound was also active in potentiation of the effect of L-dopa and D-amphetamine, although it showed only moderate activity. Several known antidepressant molecules are usually effective in these tests, however the two models are not specific for this type of psychopharmacons [Howard et al., 1981; Willner, 1984; Dall'Olio et al., 19861.

In animal experiments EGYT-4201 seemed to lack unwanted side-effects. It showed neither sedative nor stimulant action on spontaneous and learned behavior. Amitriptyline, viloxazine, trazodone and, to a lesser extent, desipramine exerted sedative effects in our tests, while nomifensine showed a stimulant character. These results are in good agreement with the literature [Braestrup and Scheel-Krtiger, 1976; LuEki and Nobler, 1985; Tucker and File, 19861.

In contrast to amitriptyline and imipramine, EGYT-4201 had no anticholinergic activity tested up to high doses. This effect is more or less a characteristic feature of the tricyclic compounds [Richelson, 198 11. Second generation antidepressants usually had no or minimal anticholinergic action [Shopsin et al., 1981; Coccaro and Siever, 1985; Hollister, 19861.

The drug exhibited moderate inhibiting effect in various seizure models. Its profile was most similar to that of amitriptyline and viloxazine. Anticonvulsive activity of viloxazine [Greenwood, 19751, amitriptyline, and imipramine [Vernier, 19611 has already been described in rodents, despite their proconvulsant action in humans [Trimble, 19781.

EGYT-4201 showed minimal MA0 inhibiting action both in in vitro and in vivo ex-


TABLE 9. LD50 Values of EGYT-4201 and Some Reference Antidemessant Comoounds in Mice Drug EGYT-420 1 male EGYT-4201 female Amitriptyline Imipramine Desipramine Viloxazine Trazodone EGYT-4201 Amitriptyline

Amitriptyline EGYT-4201

Species LD50 (mg/kg p.0.) Mice 1,250 (906-1,723) Mice 680 (509-908) Mice 225 (196-258) Mice 450 (382-530) Mice 610 (500-746) Mice 480 (443-520) Mice 584 (487-701) Rats 1,050 (796-1,384) Rats 433 (354-532) Cats 180 (126-257) Cats 37 (24-57)

periments. It can be assumed that the compound does not exert its antidepressant effects through this mode of action.

In addition to the tricyclics, the majority of the second generation antidepressants have high potency in inhibiting the uptake of one or more monoamine neurotransmitters, and developing new compounds results in more selective inhibitors of this process [Delini-Stula, 1986; Tucker and File, 19861. However, EGYT-4201 proved to be inactive as a monoamine uptake blocker, as well.

Several antidepressant substances have relatively high affinity to one or few monoamine receptors. Effective 5-HT-2 and alpha-1 receptor ligands are, e.g., amitriptyline, mianserine, and trazodone [Clements-Jewery et al., 1980; Peroutka and Snyder, 19811. Mianserine is also a potent alpha-2 antagonist [Clements-Jewery et al., 19801, while several tricyclics possess moderate affinity to D-2 receptors [Peroutka and Snyder, 19811. In contrast to these molecules, EGYT-4201 exhibited very weak affinity to several types of neurotransmitter receptors.

The above findings suggest that the compound may act by a completely alternative mechanism, which is different from those of the presently marketed drugs.

Summarizing our results we can conclude that EGYT-4201 is a potent compound of antidepressant property with a favourable psychopharmacological activity-profile, i.e., without those side-effects which could limit its use in human therapy. Its chemical structure differs either from that of the tri- or tetracyclics or those of the “atypical” ones [Richelson, 1981; Shopsin et al., 1981; Hollister, 19861. EGYT-4201 likely exerts its antidepressant action through nonmonoaminergic neurotransmitter systems.

REFERENCES

Arunlakshana, O., and Schild, H.O.: Some quantitative uses of drug antagonists. Br. J. Pharmacol.

Bizikre, K., Worms, P., Kan, J.-P., Mandel, P., Garattini, S . , and Roncucci, R.: Minaprine, a new drug

Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein

Braestrup, C., and Scheel-Kriiger, J.: Methylphenidate-like effects of the new antidepressant drug

Bylund, D.B., and Snyder, S.H.: Beta adrenergic receptor binding in membrane preparations from

Clements-Jewery, S . , Robson, P.A., and Chindley, L.J.: Biochemical investigations into the mode of

Coccaro, E.F., and Siever, L.J.: Second generation antidepressants: a comparative review. J. Clin.

14:48-58, 1959.

with antidepressant properties. Drug Exp. Clin. Res. 11:831-840, 1985.

utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254, 1976.

nomifensine (HOE 984). Eur. J. Pharmacol. 38:305-312, 1976.

mammalian brain. Mol. Pharmacol. 12568-580, 1976.

action of trazodone. Neuropharmacology 19:1165-1173, 1980.

Pharmacol. 25241-260, 1985.

398 Gyertyan et al.

Costall, B., and Naylor, R.J.: Possible involvement of a noradrenergic area of the amygdala with stereotyped behavior. Life Sci. 11:1135-1146, 1972.

Dall’Olio, R., Vaccheri, A., Gandolfi, O., and Montanaro, N.: Behavioral differentiation between pharmacokinetic and pharmacodynamic components of the interaction of antidepressants or neuroleptics with metamphetamine. Psychopharmacology 90: 18-23, 1986.

Delini-Stula, A.: New pharmacological findings in depression. Psychopathology 19(S2):94-102, 1986. Everett, G.M.: The dopa response potentiation test and its use in screening for antidepressant drugs. In

Garattini, S . , and Dukes, M. (eds.): “Antidepressant Drugs.” Amsterdam: Excerpta Medica Foundation, 1967, pp. 164-167.

Glowinski, J., and Iversen, L.L.: Catechol amines in rat brain I. Disposition of norepinephrine-3H, dopamine-3H, and dopa-3H in various regions of the brain. J. Neurochem. 13:655-669, 1966.

Greengrass, P., and Bremner, R.: Binding characteristics of 3H-prazosin to rat alpha-adrenergic receptors. Eur. J . Pharmacol. 55323-326, 1979.

Greenwood, D.T.: Animal pharmacology of viloxazine (Vivalan). J. Int. Med. Res. 3(S3):18-28, 1975. Hollister, L.E.: Current antidepressants. Ann. Rev. Pharmacol. Toxicol. 26:23-37, 1986. Howard, J.L., Soroko, F.E., and Cooper, B.R.: Empirical behavioral models of depression, with em-

phasis on tetrabenazine antagonism. In Enna, S.J., Malick, J.B., and Richelson, E. (eds.): “An- tidepressants: Neurochemical, Behavioral, and Clinical Perspectives. ” New York: Raven Press, 1981, pp. 107-120.

Leff, S . , Sibley, D.R., Hamblin, M., and Creese, I.: Ascorbic acid enables reversible dopamine receptor 3H-agonist binding. Life Sci. 29:2081-2090, 1981.

Lempert, K., Hornyak, G., Bartha, F., Doleschall, G., Fetter, J . , Nyitrai, J., Simig, G., Zauer, K . , Huszthy, P.: Iminothiazolidine derivatives and their therapeutic use. Chemical Abstracts 104:658 (CAS numher:88531y), 1986.

Leonard, B.E.: New antidepressants and the biology of depression. Stress Med. 1:9--16, 1985. Leysen, J.E. : Serotoninergic receptors in brain tissue: properties and identification of various 3H-ligand

binding sites in vitro. J. Physiol. 77:351-362, 1981. LuEki, I . , and Nobler, M.S.: The acute effects of antidepressant drugs on the performance of conditioned

avoidance behavior in rats. Pharmacol. Biochem. Behav. 22:261-264, 1985. Malick, J.B.: Yohimbine potentiation as a predictor of antidepressant action. In Enna, S.J., Malick, J.B.,

and Richelson, E. (eds.): “Antidepressants: Neurochemical, Behavioral, and Clinical Perspec- tives.” New York: Raven Press, 1981, pp. 141-155.

Maxwell, D.R., Gray, W.R., and Taylor, E.M.: Relative activity of some inhibitors of mono-amine oxidase in potentiating the action of tryptamine in vitro and in vivo. Br. J. Pharmacol. 17:

Munson, P.J., and Rodbard, D.: Ligand: A versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107:220-239, 1980.

Nelson, D.L., Herbet, A,, Bourgoin, S . , Glowinski, J., and Hamon, M.: Characteristics of central 5-HT receptors and their adaptive changes following intracerebral 5,7-dihydroxytryptamine administration in the rat. Mol. Pharmacol. 15983-995, 1978.

Nose, T., and Kojima, M.: A simple screening method for antiparkinsonian drugs in mice. Eur. J. Pharmacol. 10533-86, 1970.

Pasternak, G.W., Wilson, H.A., and Snyder, S.H.: Differential effects of protein-modifying reagents on receptor binding of opiate agonists and antagonists. Mol. Pharmacol. 11:340-351, 1975.

Pawlowski, L. , and Mazela, H.: Effects of antidepressant drugs, selective noradrenaline- or 5-hydroxy- tryptamine uptake inhibitors, on apomorphine-induced hypothermia in mice. Psychopharmacology

Peroutka, S.J., and Snyder, S.H.: Interactions of antidepressants with neurotransmitter receptor sites. In Enna, S.J., Malick, J.B., and Richelson, E. (eds.): “Antidepressants: Neurochemical, Behav- ioral, and Clinical Perspectives.” New York: Raven Press, 1981, pp. 75-90.

Peterson, G.L.: A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83:346-356, 1977.

Porsolt, R.D.: Behavioral despair. In Enna, S.J., Malick, J.B., and Richelson, E. (eds.): “Antidepres- sants: Neurochemical, Behavioral, and Clinical Perspectives.” New York: Raven Press, 1981, pp. 121-139.

Puech, A.J., Chermat, R., Poncelet, M., Doar2, L., and Simon, P.: Antagonism of hypothemia and

310-320, 1961.

88:240-246, 1 986.


behavioral response to apomorphine: a simple, rapid and discriminating test for screening antidepressants and neuroleptics. Psychopharmacology 75:84-91, 1981.

Quinton, R.M.: The increase in the toxicity of yohimbine induced by imipramine and other drugs in mice. Br. J. Pharmacol. 2 1 5 - 6 6 , 1963.

Richelson, E.: Tricyclic antidepressants: interactions with histamine and muscarinic acetylcholine receptors. In Enna, S.J., Malick, J.B., and Richelson, E. (eds.): “Antidepressants: Neurochemical, Behavioral, and Clinical Perspectives.” New York: Raven Press, 1981, pp. 53-73.

RosiC, N., and Bignami, G.: Scopolamine effects on go-no go avoidance discriminations: influence of stimulus factors and primacy of training. Psychopharmacology 17:203-2 15, 1970.

Shopsin, B., Cassano, G.B., and Conti, L.: An overview of new “second generation” antidepressant compounds: research and treatment implications. In Enna, S.J. , Malick, J.B., and Richelson, E. (eds.): “Antidepressants: Neurochemical, Behavioral, and Clinical Perspectives.” New York: Raven Press, 1981, pp. 219-251.

Snyder, S.H., and Coyle, J.T.: Regionai differences in 3H-norepinephrine and 3H-dopamine uptake into rat brain homogenates. J . Pharmacol. Exp. Ther. 16578-86, 1969.

Spencer, P.S.J.: The antagonism of oxotremorine effects in the mouse by thymoleptics. Life Sci. 5: 1015-1023, 1966.

Swinyard, E.A., Brown, W.C., and Goodman, L.S.: Comparative assays of antiepileptic drugs in mice and rats. J. Pharmacol. 106:319-330, 1952.

Trimble, M. : Non-monoamine oxidase inhibitor antidepressants and epilepsy: a review. Epilepsia 19: 241-250, 1978.

Tucker, J.C., and File, S.E.: The effects of tricyclic and “atypical” antidepressants on spontaneous locomotor activity in rodents. Neurosci. Biobehav. Rev. 10:115-121, 1986.

Vernier, V.G.: The pharmacology of antidepressant agents. Dis. Nervous System 22(S5):7-13, 1961. Whittaker, V.P.: The application of subcellular fractionation techniques to the study of brain function.

Willner, P.: The validity of animal models of depression. Psychopharmacology 83:1-16, 1984. Prog. Biophys. Mol. Biol. 1539-96, 1965.

psychopharmacological effects of an imino-thiazolidine derivative antidepressant candidate,...

Documents