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NE-100, a novel sigma receptor ligand: In vivo tests
Life Sciences,Vol. 53, pp. PL 285-290 Printed in the USA
Pergamon Press
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NE-100, A NOVEL SIGMA RECEPTOR LIGAND : IN VIVO TESTS Shigeru Okuyama1, Yasuko Imagawa1, Shin-ichi Ogawa 1, Hiroaki Araki 1, Asako Ajima1, Makoto Tanaka 1, Makoto Muramatsu 1, Atsuro Nakazato 1, Kazumasa Yamaguchi2, Masumi Yoshida2 and Susumu Otomo 1, 1 Research Center, Taisho Pharmaceutical Co., Ltd., 1.403, Yoshino-cho, Ohmiya, Saitama 330, Japan 2 Nihon Bioresearch Center Inc. 6-104, Fukujucho, Majima, Hashima, Gifu 501-62, Japan (Submitted June 23, 1993; acceptedJuly6, 1993; received in finalformAugust24, 1993) Abstract: It has been suggested that sigma receptor antagonists may be useful as antipsychotic drugs. N, N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) is a novel compound with high affinity for the sigma receptor (IC50 = 4.16 nM), but low affinity (IC50 > 10000 nM) for D1, D2, 5-HTIA, 5-HT2 and phencyclidine (PCP) receptors. The head-weaving behavior induced by either (+)SKF10047 or PCP was dose-dependently antagonized by NE-100 with oral ED50 at 0.27 and 0.12 mg/kg, respectively. NE-100 did not affect dopamine agonists-induced stereotyped behavior and/or hyperactivity. NE-100 failed to induce catalepsy in rats. These findings indicate that NE-100 may have antipsychotic activity without the liability of motor side effects typical of neuroleptics. Introduction N-allylnormetazocine ((+)SKFI0047) and related benzomorphans induced psychotomimetic effects in animals and humans (I), and bind a unique, non-opioid receptor, denoted as the sigma site (2). Sigma receptors have been found in cortical and limbic structures in human postmortem brain (3), whereas their distribution is different from phencyclidine (PCP) sites (4). Weissman et al. (5) reported the selective loss of cerebral cortical sigma, but not PCP binding sites in schizophrenia. A more compelling role for sigma sites in schizophrenia is indicated by the high affinity of some neuroleptic drugs, including haloperidol, for sigma sites (6). Rimcazole, a weak but selective sigma ligand, partially reduced schizophrenic symptomatology in a majority of patients (7). Remoxipride has a high affinity for sigma sites (8), has very good clinical efficacy as a neuroleptic agent and has few side effects (9). Thus, sigma ligands may be useful in the treatment of schizophrenia. The present study describes the receptor-binding and neuropharmacological activity of a novel sigma receptor ligand, NE-100 (N, N-dipropyl-2-[4-methoxy-3-(2-phenyl ethoxy)phenyl]-ethylaminemonohydrochloride) (Fig.l).
/ ~
sCH2CH2CH3 ,HCl
CH30---~~CH2CH2N~cH2CH2CH3 OCH2CH2--~
Chemical
Fig. 1 structure
0024-3205/93 $6.00 + .00 Copyright ©1993PergamonPress Ltd All rights reserved.
of
NE-IO0
PL-286
NE-100: A Novel Sigma Receptor Ligand
Vol. 53, No. 18, 1993
Methods Radioligand bindin~ studv Male Wistar rats (200-250 g, J a p a n SLC, Japan) were decapitated and their brains were rapidly removed. Whole brain or striatum (D2 receptor binding) was homogenized with 20 volumes of 50 mM Tris/HC! (pH 7.7) buffer (Tris buffer) using a Physcotoron homogenizer. The homogenate was centrifuged at 50,000 x g for 10 min, and the pellet was rehomogenized with Tris buffer and recentrifuged. This procedure was repeated twice. The final pellet was resuspended with Tris buffer and was used for radioligand binding assay. Sigma (2 nM[3H](+)-3-PPP), D1 (1 nM[3H]SCH23390), D2 (15 nM[3H]sulpiride), PCP (5 nM[3H]PCP), 5-HT1A (0.2 nM[3H]8-OH-DPAT) and 5-HT2 (5 nM[3H]ketanserin) receptor sites were determined according to the methods of Tanaka et al. (10) and Billard et al. (11). Briefly, 1 ml of m e m b r a n e suspension and radiolabeled ligand was incubated with various concentrations (at least 9) of unlabeled drugs. Incubations were terminated by rapid filtration through a Whatman GF/B glass fiber filter that had been soaked for at least 4 hr in a solution of 0.5% polyethyleneimine and washed with Tris buffer. Then, filter-bound radioactivity was measured using a liquid scintillation spectrometer. IC50 values from competitive inhibition experiments were determined using the Marquardt-Levenberg nonlinear curve fitting procedure of the RS/1 program (BBN Research System) running on a VAX/VMS system. Effects on (+)SKF10047- or PCP-induced head-weaviw, behavior in rats Male Wistar rats (80 - 120 g, Charles River, Japan) were placed individually into a clear acrylic cage (31 x 36 x 17.5 cm) and allowed a minimum of 45 min to acclimatize to the new environment. Ten min after oral administration of NE-100 and 30 rain after oral administration of Dup734, XJ448, BMY14802, haloperidol and sulpiride, (+)SKF10047 (20 mg/kg, i.p.) or PCP (7.5 mg/kg, i.p.) was administered. The n u m b e r of head-weaving (animals made slow, side-to-side or lateral head movement) was counted until 40 min post injection (12). Ten rats, at vehicle and each of 3 dosages of drugs, were used to generate dose-response reactions. The total count of the control group was defined as 100%, and the percent of inhibition of each t r e a t m e n t group was calculated, and ED50 values were determined. Effects on donamine a~onists-induced stereotvned behavior in rats Male Wistar rats (180 - 220 g, Charles River, Japan) were placed individually into a clear acrylic cage (37 x 24 x 25 cm) and allowed a minimum of 60 min to acclimatize to the new environment. The drugs were given as follows (time of dose before dopamine agonists & route): NE-100 (10 rain & p.o.), BMY14802 (30 min & p.o.), rimcazole (30 min & p.o.), haloperidol (30 min & i.p.) and sulpiride (60 min & i.p.), respectively, followed by methamphetamine (MAP) at 10 mg/kg (i.p.) and apomorphine (APO) at 5 mg/kg (s.c). Ten rain later, the stereotyped behavior was scored every 10 minutes for 80 minutes using the following scoring system: 0, normal behavior; 1, exploratory behavior or discontinuous sniffing; 2, continuous sniffing; 3, continuous sniffing, discontinuous licking, biting or gnawing; 4, continuous licking, biting and gnawing. Six rats, at vehicle and each of 3 dosages of drugs, were used to generate dose-response reactions. The total score of the control group was defined as 100%, the percent of inhibition of each treatment group was calculated, and ED50 values were determined. Induction of catalensv in rats Male Wistar rats (180 - 220 g, Charles River, Japan) were used. The drugs were given as follows (Time of dose before test & route): NE-100 (10 min & p.o.), BMY14802 (30 rain & p.o.), rimcazole (30 min & p.o.), haloperidol (30 min & i.p.) and suipiride (60 rain & i.p.),respectively. The animals were watched for catalepsy on 3 occasions, once after the
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NE-100: A Novel Sigma Receptor Ligand
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administration of each drug, and then twice every 30 min until 60 min. Catalepsy was scored using the following scoring system: 0.5 (maximum score: 1.0), a posture with the right and left fore-Umbs on the right and left platforms 3 cm high was kept for 10 seconds, 1.0 ( m a x i m u m score: 2.0), a posture with the right and left fore-limbs on the right and left platforms 9 cm high, kept for 10 seconds (2.0 points were given only when the posture with both forelimbs on the platforms was kept for 30 seconds). Six rats, at vehicle and each of 3 dosages of drugs, were used to generate dose-response reactions. The m a x i m u m score (3.0 points) was defined as 100%, the percent of induction of catalepsy of each treatment group was calculated, and ED50 values were calculated at the peak effect of each drug. Effects on snontaneous locomotor activitv in mice Male ICR mice (20 - 30 g, Charles River, Japan) were used. The drugs were given as follows (time of dose before test & route): NE-100 (10 min & p.o.), BMY14802 (30 min & p.o.), rimcazole (30 rain & p.o.), haloperidol (30 rain & i.p.) and sulpiride (60 rain & i.p.),respectively. The animals were housed in a s o u n d - p r o o f box, and spontaneous locomotor activity was recorded every 5 min for 30 min using a SCANET a p p a r a t u s (Neuroscience Inc.). Six groups of 5 mice, at vehicle and each of 3 dosages of drugs, were used to generate dose-response reactions. The total count of the control group was defined as 100%, the percent of inhibition of each treatment group was calculated, and ED50 values were determined. Effects on methamnhetamine (MAP).induced hvneractivitv in mice Male ICR mice (20 - 30 g, Charles River, Japan) were used. The animals were housed individually in transparent acrylic cages (37 x 24 x 25 cm), and acclimatized with a SCANET apparatus placed in a sound-proof box for 60 rain. The drugs were given as follows (time of dose before test & route): NE-100 (10 rain & p.o.), BMY14802 (30 min & p.o.), rimcazole (30 rain & p.o.), haloperidol (30 rain & i.p.) and sulpiride (60 rain & i.p.), respectively, and then MAP (1 mg/kg, i.p.) was given. Fifteen min later, locomotor activity was recorded every 5 min for 30 rain using a SCANET a p p a r a t u s placed in a sound proof box. Six groups of 5 mice, at vehicle and each of 3 dosages of drugs, were used to generate dose-response reactions. The total count of the control group was defined as 100%, the percent of inhibition of each treatment group was calculated, and ED50 values were determined.
Dxaga NE-100, PCP, Dup734, XJ448, BMY14802 rimcazole and YM-09151-2 were synthesized in the laboratories of Taisho Pharmaceutical Co. Ltd. Haloperidol and snipiride were purchased from Dainippon Pharmaceutical Co. Ltd. and Fujisawa Pharmaceutical Co. Ltd., respectively. (+)SKF10047 was purchased from Research Biochemical Inc. In the in vitro study, all drugs were dissolved in 100 % dimethyl sulfoxide (DMSO). The final concentration of DMSO was less than 1 % and did not affect the binding assay. In the in vivo study, NE-100, Dup734 and XJ448 were dissolved in distilled water. P C P and (+)SKF10047 were dissolved in saline solution. Haloperidol and sulpiride were used for Serenase and Dogmatyl injections respectively, and the volume was made up with distilled water. BMY14802 and rimcazole were suspended in 5 % arabic gum. The time of dose of subject compounds before initiation of test was decided according to the observation results of the peak drug effect times of each subject compound.
Results Radioli~and bindin2 studv Competition binding experiments showed that NE-100 has a high affinity for the
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NE-100: A Novel Sigma Receptor Ligand
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sigma binding sites in rat brain membranes (TABLE I). The IC50 value of NE-100 was 4.2 nM, which is comparable to that for baloperidol (3.1 nM) and Dup734 (2.4 nM). The order of potency to displace bound [3H](+)-3-PPP was: Dup734 = haloperidol = NE-100 > XJ448 = YM-09151-2 > BMY14802 > rimcazole. The selectively of NE-100 for the sigma sites was also examined. NE-100 had no affinity for dopamine D1, dopamine D2, 5-HTIA, 5-HT2 or PCP (IC50 values are over 10000 nM). Effects on (+)SKF10047- or PCP-induced head-weavin~ behavior in rats In the control group, the number of head-weaving induced by (+)SKF10047 was 148+9 (n=10) to 155+10 (n=10), and that by PCP was 145+8 (n=10) to 157+_12 (n=10). NE100 was orally active in inhibiting the head-weaving behavior induced by either (+)SKF10047 or PCP (TABLE II). NE-100 was more potent than standards in inhibiting both types of head-weaving behavior. The order of potency of inhibition of (+)SKF10047induced head-weaving behavior was: NE-100 > haloperidol > BMY14802 > Dup734 > XJ448, and that of PCP-induced head-weaving behavior was: NE-100 > haloperidol > Dup734 > BMY14802 > XJ448. Sulpiride had no effect on either type of head-weaving behavior. TABLEI In vitro receptor binding affinity IC50 (nM) Compounds NE-100 Dup734 XJ448 BMY14802 Rimcazole Haloperidoi Sulpiride YM-09151-2
sigma
D1
4.2 2.4 16.5 158 2355 3.1 >10000 17.7
>10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000
D2
5-HT1A
5-HT2
PCP
>10000 660 >10000 6030 >10000 1.1 17.6 0.1
>10000 >10000 >10000 1381 >10000 >10000 >10000 >10000
>10000 974 >10000 >10000 >10000 1820 >10000 649
>10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000
Average of 2 to 4 determinations. TABLE II Effects of NE-100 and standards on (+)SKF10047- and PCP-induced head-weaving behavior in rats ED50 (95% Confidence Limit) mg/kg, p.o. Compounds (+)SKF10047 . . . .NE-100 .................. "0~27. . . . . Dup734 37.8 XJ448 111.0 BMY14802 6.1 Haloperidol 0.68 Sulpiride >300
PCP
"(-0~4-'-. . . . -0-'3-23. . . . . . . . . . . . . 0~]'2"---?'0:11-'-. . . . . "0--{'33. . . . (33.9 42.0 ) 3.4 ( 2.4 4.8 ) (92.4 133.3 ) 95.2 (71.9 - 126.2 ) ( 5.6 6.7 ) 6.7 ( 6.2 7.2 ) (0.65 0.72) 1.3 ( 1.2 1.4 ) >300
Effects on donamine a2onists-induced stereotvned behavior, and induction of catalepsy in rats In the control group, the total score of APO-induced stereotyped behavior was 27.2+_1.2 (n=6) to 31.7+_0.2 (n=6), and that of MAP-induced stereotyped behavior was 24.0i-0.9 (n=6) to 28.8+_1.0 (n=6). NE-100, BMYI4802, rimcazole and sulpiride did not affect either APO- or MAP-induced stereotyped behavior, and did not induce catalepsy (TABLE
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III). Haloperidol strongly blocked both stereotyped behavior, and induced catalepsy (TABLE III). The peak effect time of catalepsy induced by haloperidoi was observed after 2 hr administration. Effect on snontaneous locomotor activitv and methamnhetamine (MAP) hvneractivitv in mice As shown in Table III, NE-100 and suipiride did not inhibit spontaneous locomotor activity of MAP induced hyperactivity. BMY14802 and rimcazole caused little suppression of spontaneous locomotion and MAP hyperactivity even at the higher doses tested. Haloperidol strongly inhibited both spontaneous locomotion and MAP hyperactivity. TABLE III Effects of NE.100 and standards on apomorphine (APO)- and Methamphetamine (MAP)induced stereotyped behavior, induction of catalepsy in rats, and spontaneous locomotor activity and MAP hyperactivity in mice ED50 (mg/kg) Rat
Mice
Drugs Stereotypies
NE-100 BMY14802 Rimcazole Haloperidoi Sulpiride
APO
MAP
>10 >50 >100 0.4 >100
>10 >50 >100 0.1 >100
Catalepsy
Locomotor Activities Spontaneous MAP-hyperactivity
>10 >50 >100 0.5 >100
>10 42 99 0.3 >100
>10 8.3 60.1 0.2 >100
The present studies have demonstrated that NE-100 is a potent and selective ligand of the central sigma receptor. Competition experiments of [3H](+)3-PPP binding in rat brain membrane indicated that NE-100 has a high affinity for the sigma sites, with almost the same potency as Dup734 and haloperidol. Although, haloperidol has been found to be a most active compound for the sigma site, it also has an affinity for the dopamine D2 receptor. The affinity of BMY14802 for the 5-HTIA receptor is micromolar. This is not significant activity, particularly when the affinity for the sigma site is ten fold higher. Likewise, Dup734 is only weakly active at the dopamine D2 and 5-HT2 receptors. Dup734's separation versus dopamine D2 and 5-HT2 is probably sufficient to assure in vivo selectivity. These observations are in keeping with those of previous reports (13-15, 20). Thus, NE-100 is one of the most potent and selective sigma ligands in comparison to the more recently reported sigma ligands (21, 22). PCP and (+)SKFI0047 produced head-weaving behavior in rats (12), and sigma ligands reduced the head-weaving induced by (+)SKFI0047 or PCP (16). Microinjection of PCP into the frontal cortex produced the most intensive head-weaving behavior compared to microinjection into other structures (19). The frontal cortex is an important structure for generating schizophrenia (17, 18). In the present study, the head-weaving behavior induced by (+)SKFI0047, a prototypic sigma receptor agonist (I), was antagonized by NE-100 and sigma ligands. This behavior was not affected by sulpiride, a pure dopamine D2 antagonist. Thus, NE-100 might be an antagonist for the sigma receptor resulting from the inhibition of (+)SKF10047-induced head-weaving behavior. NE-100 is also active in blocking PCP induced head-weaving behavior in rats. Therefore, to the extent that sigma receptor ligands (antagonists) might be useful as antipsychotic drugs, NE-100 may be an effective therapeutic agent for the treatment of schizophrenia.
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In addition, higher doses of NE-100 (1-10 mg/kg, p.o.) did not cause abnormal behavior such as sedation, hypothermia, salivation and lacrimation (unpublished observation). No antagonism of the behavior induced by the dopamine agonists and lack of induction of catalepsy following NE-100 suggesting a reduced liability for producing extrapyramidai side effects. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
W.R. MARTIN, C.E. EADES, J.A. THOMPSON and R.E. HUPPLER, J. Pharmacol. Exp. Ther. 197 517-532 (1976). J.M. WALKER, W.D. BOWEN, F.O. WALKER, R.R. MATSUMOTO, B.DE COSTA and K.C. RICE, Pharmacol. Rev. 42 355-402 (1990). A.D. WEISSMAN, T-P. SU, J.C. HEDREEN and E.D. LONDON, J. Pharmacol. Exp. Ther. 247 29-33 (1988). B.L. LARGENT, A.L. GUNDLACH and S.H. SYNDER, J. Pharmacol. Exp. Ther. 238 739-748 (1986). A.D. WEISSMAN, M.F. CASANOVA, J.E. KLEINMAN, E.D. LONDON and E.B. DE SOUZA, Biol. Psychiatry ~ 41-54 (1991). B.L. LARGENT, H. WILKSTROM, A.M. SNOWMAN and S.H. SNYDER, Eur. J. Pharmacol. 155 345-347 (1988). G. SCHWARCZ, A. HALARIS, A. DREN and P. MANBERG, Drug Develo. Res. 5 387-393 (1985). H. ERICSON and S.B. ROSS, Eur. J. Pharmacol. 226 157-161 (1992). J.A. DEN BOER, D.P. RAVELLI, J. HUISMAN, J. OHRVIK W.M.A. VERHOEVEN and H.G.M. WESTENBERG, Pschopharmacol. 102 76-84 (1990) M. TANAKA, S. CHAKI, Y. IMAGAWA, S. OKUYAMA, M. MURAMATSU and S. OTOMO, Eur. J. Pharmacol. (1993, in press). W. BILLARD, V. RUPERTO, G. CROSDY, L.C. LORIO and A. BARNETT, Life Sci. 35 1885-1893 (1984) M. HIRAMATSU, T. NABESHIMA, H. FURUKAWA and T. KAMEYAMA, Pharmacol. Biochem. Behav. 28 489-494 (1987). S.W. TAM, G.F. STEINFELS, P.J. GILLIGAN, W.K. SCHMIDT and L. COOK, J. Pharmacol. Exp. Ther. 263 1167-1174 (1992). L.J. BRISTOW, L. BAUCUTT, L. THORN, P.H. HUTSON, A. NOBLE, M. BEER, D.N. MIDDLEMISS and M.D. TRICKLEBANK, Eur. J. Pharmacol. 204 21-28 (1991). C.M. KRAUSE, C.M. ROMINGER, S.W. TAM and R. ZACZEK, Sci. Neursci. Abs. 18 547 (1992). S. SAGRATELLA, A. S. CAROLIS, A. PEZZOLA and P. POPOLI, Psychopharmacol. 105 485-491 (1991). E.D. LONDON, M. DAM and A.D. WEISSMAN, Sigma and Phencyclidine-like Compounds as Molecular Probes in Biology, Edward F. Domino and Jean-Marc Kamenka (Eds), 297-307, NPP Books, Ann Arbor, MI (1988). N.D. VOLKOW, A.P. WOLF and P.VAN GELDER, Am. J. Psychiatry. 144 151-158 (1987). K. YAMAGUCHI, T. NABESHIMA and T. KAMEYAMA, J. Pharmcobio-Dyn. 9 987996 (1986). L.COOK, S. W. TAM and K. W. ROHRBACH, J. Pharmacoi. Exp. Ther. 263 11591166 (1992). E.W. KARBON, M. E. ABREU, R. H. ERICKSON, C. KAISER, K. J. NATALIE, Jr., D. B. CLISSOLD, S. BOROSKY, M. BAILEY, L. A. MARTIN, M. J. PONTECORVO, S. J. ENNA and J. W. FERKANY, J. Pharmacol. Exp. Ther. 265 866-875 (1993). M. PONCELET, V. SANTUCCI, R. PAUL, C. GUEUDET, S. LAVASTRE, J. GUITARD, R. STEINBERG, J. P. TERRANOVA, J. C. BRELIERE, P. SOUBRIE and G. LE FUR, Neuropharmacol. 32 605-615 (1993).
Pergamon Press
PHARMACOLOGYLETTERS Accelerated Communication
NE-100, A NOVEL SIGMA RECEPTOR LIGAND : IN VIVO TESTS Shigeru Okuyama1, Yasuko Imagawa1, Shin-ichi Ogawa 1, Hiroaki Araki 1, Asako Ajima1, Makoto Tanaka 1, Makoto Muramatsu 1, Atsuro Nakazato 1, Kazumasa Yamaguchi2, Masumi Yoshida2 and Susumu Otomo 1, 1 Research Center, Taisho Pharmaceutical Co., Ltd., 1.403, Yoshino-cho, Ohmiya, Saitama 330, Japan 2 Nihon Bioresearch Center Inc. 6-104, Fukujucho, Majima, Hashima, Gifu 501-62, Japan (Submitted June 23, 1993; acceptedJuly6, 1993; received in finalformAugust24, 1993) Abstract: It has been suggested that sigma receptor antagonists may be useful as antipsychotic drugs. N, N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) is a novel compound with high affinity for the sigma receptor (IC50 = 4.16 nM), but low affinity (IC50 > 10000 nM) for D1, D2, 5-HTIA, 5-HT2 and phencyclidine (PCP) receptors. The head-weaving behavior induced by either (+)SKF10047 or PCP was dose-dependently antagonized by NE-100 with oral ED50 at 0.27 and 0.12 mg/kg, respectively. NE-100 did not affect dopamine agonists-induced stereotyped behavior and/or hyperactivity. NE-100 failed to induce catalepsy in rats. These findings indicate that NE-100 may have antipsychotic activity without the liability of motor side effects typical of neuroleptics. Introduction N-allylnormetazocine ((+)SKFI0047) and related benzomorphans induced psychotomimetic effects in animals and humans (I), and bind a unique, non-opioid receptor, denoted as the sigma site (2). Sigma receptors have been found in cortical and limbic structures in human postmortem brain (3), whereas their distribution is different from phencyclidine (PCP) sites (4). Weissman et al. (5) reported the selective loss of cerebral cortical sigma, but not PCP binding sites in schizophrenia. A more compelling role for sigma sites in schizophrenia is indicated by the high affinity of some neuroleptic drugs, including haloperidol, for sigma sites (6). Rimcazole, a weak but selective sigma ligand, partially reduced schizophrenic symptomatology in a majority of patients (7). Remoxipride has a high affinity for sigma sites (8), has very good clinical efficacy as a neuroleptic agent and has few side effects (9). Thus, sigma ligands may be useful in the treatment of schizophrenia. The present study describes the receptor-binding and neuropharmacological activity of a novel sigma receptor ligand, NE-100 (N, N-dipropyl-2-[4-methoxy-3-(2-phenyl ethoxy)phenyl]-ethylaminemonohydrochloride) (Fig.l).
/ ~
sCH2CH2CH3 ,HCl
CH30---~~CH2CH2N~cH2CH2CH3 OCH2CH2--~
Chemical
Fig. 1 structure
0024-3205/93 $6.00 + .00 Copyright ©1993PergamonPress Ltd All rights reserved.
of
NE-IO0
PL-286
NE-100: A Novel Sigma Receptor Ligand
Vol. 53, No. 18, 1993
Methods Radioligand bindin~ studv Male Wistar rats (200-250 g, J a p a n SLC, Japan) were decapitated and their brains were rapidly removed. Whole brain or striatum (D2 receptor binding) was homogenized with 20 volumes of 50 mM Tris/HC! (pH 7.7) buffer (Tris buffer) using a Physcotoron homogenizer. The homogenate was centrifuged at 50,000 x g for 10 min, and the pellet was rehomogenized with Tris buffer and recentrifuged. This procedure was repeated twice. The final pellet was resuspended with Tris buffer and was used for radioligand binding assay. Sigma (2 nM[3H](+)-3-PPP), D1 (1 nM[3H]SCH23390), D2 (15 nM[3H]sulpiride), PCP (5 nM[3H]PCP), 5-HT1A (0.2 nM[3H]8-OH-DPAT) and 5-HT2 (5 nM[3H]ketanserin) receptor sites were determined according to the methods of Tanaka et al. (10) and Billard et al. (11). Briefly, 1 ml of m e m b r a n e suspension and radiolabeled ligand was incubated with various concentrations (at least 9) of unlabeled drugs. Incubations were terminated by rapid filtration through a Whatman GF/B glass fiber filter that had been soaked for at least 4 hr in a solution of 0.5% polyethyleneimine and washed with Tris buffer. Then, filter-bound radioactivity was measured using a liquid scintillation spectrometer. IC50 values from competitive inhibition experiments were determined using the Marquardt-Levenberg nonlinear curve fitting procedure of the RS/1 program (BBN Research System) running on a VAX/VMS system. Effects on (+)SKF10047- or PCP-induced head-weaviw, behavior in rats Male Wistar rats (80 - 120 g, Charles River, Japan) were placed individually into a clear acrylic cage (31 x 36 x 17.5 cm) and allowed a minimum of 45 min to acclimatize to the new environment. Ten min after oral administration of NE-100 and 30 rain after oral administration of Dup734, XJ448, BMY14802, haloperidol and sulpiride, (+)SKF10047 (20 mg/kg, i.p.) or PCP (7.5 mg/kg, i.p.) was administered. The n u m b e r of head-weaving (animals made slow, side-to-side or lateral head movement) was counted until 40 min post injection (12). Ten rats, at vehicle and each of 3 dosages of drugs, were used to generate dose-response reactions. The total count of the control group was defined as 100%, and the percent of inhibition of each t r e a t m e n t group was calculated, and ED50 values were determined. Effects on donamine a~onists-induced stereotvned behavior in rats Male Wistar rats (180 - 220 g, Charles River, Japan) were placed individually into a clear acrylic cage (37 x 24 x 25 cm) and allowed a minimum of 60 min to acclimatize to the new environment. The drugs were given as follows (time of dose before dopamine agonists & route): NE-100 (10 rain & p.o.), BMY14802 (30 min & p.o.), rimcazole (30 min & p.o.), haloperidol (30 min & i.p.) and sulpiride (60 min & i.p.), respectively, followed by methamphetamine (MAP) at 10 mg/kg (i.p.) and apomorphine (APO) at 5 mg/kg (s.c). Ten rain later, the stereotyped behavior was scored every 10 minutes for 80 minutes using the following scoring system: 0, normal behavior; 1, exploratory behavior or discontinuous sniffing; 2, continuous sniffing; 3, continuous sniffing, discontinuous licking, biting or gnawing; 4, continuous licking, biting and gnawing. Six rats, at vehicle and each of 3 dosages of drugs, were used to generate dose-response reactions. The total score of the control group was defined as 100%, the percent of inhibition of each treatment group was calculated, and ED50 values were determined. Induction of catalensv in rats Male Wistar rats (180 - 220 g, Charles River, Japan) were used. The drugs were given as follows (Time of dose before test & route): NE-100 (10 min & p.o.), BMY14802 (30 rain & p.o.), rimcazole (30 min & p.o.), haloperidol (30 min & i.p.) and suipiride (60 rain & i.p.),respectively. The animals were watched for catalepsy on 3 occasions, once after the
Vol. 53, No. 18, 1993
NE-100: A Novel Sigma Receptor Ligand
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administration of each drug, and then twice every 30 min until 60 min. Catalepsy was scored using the following scoring system: 0.5 (maximum score: 1.0), a posture with the right and left fore-Umbs on the right and left platforms 3 cm high was kept for 10 seconds, 1.0 ( m a x i m u m score: 2.0), a posture with the right and left fore-limbs on the right and left platforms 9 cm high, kept for 10 seconds (2.0 points were given only when the posture with both forelimbs on the platforms was kept for 30 seconds). Six rats, at vehicle and each of 3 dosages of drugs, were used to generate dose-response reactions. The m a x i m u m score (3.0 points) was defined as 100%, the percent of induction of catalepsy of each treatment group was calculated, and ED50 values were calculated at the peak effect of each drug. Effects on snontaneous locomotor activitv in mice Male ICR mice (20 - 30 g, Charles River, Japan) were used. The drugs were given as follows (time of dose before test & route): NE-100 (10 min & p.o.), BMY14802 (30 min & p.o.), rimcazole (30 rain & p.o.), haloperidol (30 rain & i.p.) and sulpiride (60 rain & i.p.),respectively. The animals were housed in a s o u n d - p r o o f box, and spontaneous locomotor activity was recorded every 5 min for 30 min using a SCANET a p p a r a t u s (Neuroscience Inc.). Six groups of 5 mice, at vehicle and each of 3 dosages of drugs, were used to generate dose-response reactions. The total count of the control group was defined as 100%, the percent of inhibition of each treatment group was calculated, and ED50 values were determined. Effects on methamnhetamine (MAP).induced hvneractivitv in mice Male ICR mice (20 - 30 g, Charles River, Japan) were used. The animals were housed individually in transparent acrylic cages (37 x 24 x 25 cm), and acclimatized with a SCANET apparatus placed in a sound-proof box for 60 rain. The drugs were given as follows (time of dose before test & route): NE-100 (10 rain & p.o.), BMY14802 (30 min & p.o.), rimcazole (30 rain & p.o.), haloperidol (30 rain & i.p.) and sulpiride (60 rain & i.p.), respectively, and then MAP (1 mg/kg, i.p.) was given. Fifteen min later, locomotor activity was recorded every 5 min for 30 rain using a SCANET a p p a r a t u s placed in a sound proof box. Six groups of 5 mice, at vehicle and each of 3 dosages of drugs, were used to generate dose-response reactions. The total count of the control group was defined as 100%, the percent of inhibition of each treatment group was calculated, and ED50 values were determined.
Dxaga NE-100, PCP, Dup734, XJ448, BMY14802 rimcazole and YM-09151-2 were synthesized in the laboratories of Taisho Pharmaceutical Co. Ltd. Haloperidol and snipiride were purchased from Dainippon Pharmaceutical Co. Ltd. and Fujisawa Pharmaceutical Co. Ltd., respectively. (+)SKF10047 was purchased from Research Biochemical Inc. In the in vitro study, all drugs were dissolved in 100 % dimethyl sulfoxide (DMSO). The final concentration of DMSO was less than 1 % and did not affect the binding assay. In the in vivo study, NE-100, Dup734 and XJ448 were dissolved in distilled water. P C P and (+)SKF10047 were dissolved in saline solution. Haloperidol and sulpiride were used for Serenase and Dogmatyl injections respectively, and the volume was made up with distilled water. BMY14802 and rimcazole were suspended in 5 % arabic gum. The time of dose of subject compounds before initiation of test was decided according to the observation results of the peak drug effect times of each subject compound.
Results Radioli~and bindin2 studv Competition binding experiments showed that NE-100 has a high affinity for the
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NE-100: A Novel Sigma Receptor Ligand
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sigma binding sites in rat brain membranes (TABLE I). The IC50 value of NE-100 was 4.2 nM, which is comparable to that for baloperidol (3.1 nM) and Dup734 (2.4 nM). The order of potency to displace bound [3H](+)-3-PPP was: Dup734 = haloperidol = NE-100 > XJ448 = YM-09151-2 > BMY14802 > rimcazole. The selectively of NE-100 for the sigma sites was also examined. NE-100 had no affinity for dopamine D1, dopamine D2, 5-HTIA, 5-HT2 or PCP (IC50 values are over 10000 nM). Effects on (+)SKF10047- or PCP-induced head-weavin~ behavior in rats In the control group, the number of head-weaving induced by (+)SKF10047 was 148+9 (n=10) to 155+10 (n=10), and that by PCP was 145+8 (n=10) to 157+_12 (n=10). NE100 was orally active in inhibiting the head-weaving behavior induced by either (+)SKF10047 or PCP (TABLE II). NE-100 was more potent than standards in inhibiting both types of head-weaving behavior. The order of potency of inhibition of (+)SKF10047induced head-weaving behavior was: NE-100 > haloperidol > BMY14802 > Dup734 > XJ448, and that of PCP-induced head-weaving behavior was: NE-100 > haloperidol > Dup734 > BMY14802 > XJ448. Sulpiride had no effect on either type of head-weaving behavior. TABLEI In vitro receptor binding affinity IC50 (nM) Compounds NE-100 Dup734 XJ448 BMY14802 Rimcazole Haloperidoi Sulpiride YM-09151-2
sigma
D1
4.2 2.4 16.5 158 2355 3.1 >10000 17.7
>10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000
D2
5-HT1A
5-HT2
PCP
>10000 660 >10000 6030 >10000 1.1 17.6 0.1
>10000 >10000 >10000 1381 >10000 >10000 >10000 >10000
>10000 974 >10000 >10000 >10000 1820 >10000 649
>10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000
Average of 2 to 4 determinations. TABLE II Effects of NE-100 and standards on (+)SKF10047- and PCP-induced head-weaving behavior in rats ED50 (95% Confidence Limit) mg/kg, p.o. Compounds (+)SKF10047 . . . .NE-100 .................. "0~27. . . . . Dup734 37.8 XJ448 111.0 BMY14802 6.1 Haloperidol 0.68 Sulpiride >300
PCP
"(-0~4-'-. . . . -0-'3-23. . . . . . . . . . . . . 0~]'2"---?'0:11-'-. . . . . "0--{'33. . . . (33.9 42.0 ) 3.4 ( 2.4 4.8 ) (92.4 133.3 ) 95.2 (71.9 - 126.2 ) ( 5.6 6.7 ) 6.7 ( 6.2 7.2 ) (0.65 0.72) 1.3 ( 1.2 1.4 ) >300
Effects on donamine a2onists-induced stereotvned behavior, and induction of catalepsy in rats In the control group, the total score of APO-induced stereotyped behavior was 27.2+_1.2 (n=6) to 31.7+_0.2 (n=6), and that of MAP-induced stereotyped behavior was 24.0i-0.9 (n=6) to 28.8+_1.0 (n=6). NE-100, BMYI4802, rimcazole and sulpiride did not affect either APO- or MAP-induced stereotyped behavior, and did not induce catalepsy (TABLE
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NE-IO0: A NovelSigma ReceptorLigand
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III). Haloperidol strongly blocked both stereotyped behavior, and induced catalepsy (TABLE III). The peak effect time of catalepsy induced by haloperidoi was observed after 2 hr administration. Effect on snontaneous locomotor activitv and methamnhetamine (MAP) hvneractivitv in mice As shown in Table III, NE-100 and suipiride did not inhibit spontaneous locomotor activity of MAP induced hyperactivity. BMY14802 and rimcazole caused little suppression of spontaneous locomotion and MAP hyperactivity even at the higher doses tested. Haloperidol strongly inhibited both spontaneous locomotion and MAP hyperactivity. TABLE III Effects of NE.100 and standards on apomorphine (APO)- and Methamphetamine (MAP)induced stereotyped behavior, induction of catalepsy in rats, and spontaneous locomotor activity and MAP hyperactivity in mice ED50 (mg/kg) Rat
Mice
Drugs Stereotypies
NE-100 BMY14802 Rimcazole Haloperidoi Sulpiride
APO
MAP
>10 >50 >100 0.4 >100
>10 >50 >100 0.1 >100
Catalepsy
Locomotor Activities Spontaneous MAP-hyperactivity
>10 >50 >100 0.5 >100
>10 42 99 0.3 >100
>10 8.3 60.1 0.2 >100
The present studies have demonstrated that NE-100 is a potent and selective ligand of the central sigma receptor. Competition experiments of [3H](+)3-PPP binding in rat brain membrane indicated that NE-100 has a high affinity for the sigma sites, with almost the same potency as Dup734 and haloperidol. Although, haloperidol has been found to be a most active compound for the sigma site, it also has an affinity for the dopamine D2 receptor. The affinity of BMY14802 for the 5-HTIA receptor is micromolar. This is not significant activity, particularly when the affinity for the sigma site is ten fold higher. Likewise, Dup734 is only weakly active at the dopamine D2 and 5-HT2 receptors. Dup734's separation versus dopamine D2 and 5-HT2 is probably sufficient to assure in vivo selectivity. These observations are in keeping with those of previous reports (13-15, 20). Thus, NE-100 is one of the most potent and selective sigma ligands in comparison to the more recently reported sigma ligands (21, 22). PCP and (+)SKFI0047 produced head-weaving behavior in rats (12), and sigma ligands reduced the head-weaving induced by (+)SKFI0047 or PCP (16). Microinjection of PCP into the frontal cortex produced the most intensive head-weaving behavior compared to microinjection into other structures (19). The frontal cortex is an important structure for generating schizophrenia (17, 18). In the present study, the head-weaving behavior induced by (+)SKFI0047, a prototypic sigma receptor agonist (I), was antagonized by NE-100 and sigma ligands. This behavior was not affected by sulpiride, a pure dopamine D2 antagonist. Thus, NE-100 might be an antagonist for the sigma receptor resulting from the inhibition of (+)SKF10047-induced head-weaving behavior. NE-100 is also active in blocking PCP induced head-weaving behavior in rats. Therefore, to the extent that sigma receptor ligands (antagonists) might be useful as antipsychotic drugs, NE-100 may be an effective therapeutic agent for the treatment of schizophrenia.
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In addition, higher doses of NE-100 (1-10 mg/kg, p.o.) did not cause abnormal behavior such as sedation, hypothermia, salivation and lacrimation (unpublished observation). No antagonism of the behavior induced by the dopamine agonists and lack of induction of catalepsy following NE-100 suggesting a reduced liability for producing extrapyramidai side effects. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
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