Neurochem. Int. Vol. 20, Suppl.,pp. 119S-122S,1992 Printed in Great Britain.All fights reserved
0197-0186/92$5.00+0.00 Copyright © 1992PergamonPresspie
HIGHLIGHTS OF D1 DOPAMINE RECEPTOR ANTAGONIST RESEARCH ALLEN BARNETT, ROBERTD. McQuADE and CLARK TEDFORD Schering-Plough Research, 60 Orange Street, Bloomfield, NJ 07003, U.S.A.
Abstract-- SCH 39166 is now undergoing clinical trials in schizophrenics as a selective Dt dopamine receptor antagonist. It differs from SCH 23390, the prototype D~ receptor antagonist, by having reduced affinity for scrotonin receptors and a longer duration of action in primates, as measured in the squirrel monkey conditioned avoidance paradigm. Further studies on this difference in primates indicates that it may be attributable to reduced affinity of SCH 39166 compared to SCH 23390 for the hepatic glucuronosyltransferase system. Their affinities are similar in rats, a species in which both have similar duration of action. These and other studies presented are consistent with the novel profile of SCH 39166. The discovery and development of D~ specific dopamine antagonists has been strongly tied to the progression of the benzazepines as a chemical class. The initial discovery of the unique behavioral effects of the benzazepine SCH 12679 (Barnett et al., 1974), was followed by SCH 23390 (Iorio et al., 1981; 1983), which has become the prototype specific D~ antagonist. However the poor oral absorption and short duration of action of this c o m p o u n d in primates precluded its further development (Barnett et al., 1986). It is now clear that SCH 39166, a benzonaphthazepine, has the appropriate pharmacologic spectrum for further progression (Chipkin et al., 1988) and it is now in early clinical trials as the first D, specific dopamine antagonist to be studied for therapeutic purposes.
EXPERIMENTAL PROCEDURES In vitro receptor binding studies In vitro bindingstudies of Dr, D2 and 5HT receptor affinities were done according to methods described previously (Chipkin et al., 1988).
CH30~ ~ / N CH3 CH30 /
V
In vivo D~ receptor binding study in rats
The effect of SCH 39166 on Dt receptor binding was studied in vivo using 3H-SCH 39166 as a ligand, according to the method described by McQuade and colleagues (1989). Conditioned avoidance responding ( C A R ) in rats and monkeys
The effect of SCH 39166 and haloperidol on CAR in rats and monkeys utilized methods described in the work of Chipkin and colleagues (1988). In vitro glucuronosyltransferase activity in hepatic microsomes from rats and monkeys
The affinity of SCH 23390 and SCH 39166 for glucuronosyltransferase activity was studied in microsomal preparations from rat and squirrel monkey livers. The assays for ~H-SCH 23390 and 3H-SCH 39166-glucuronidationwere described previously by Tedford et al., (1990). Briefly, buffer containing 1.5 mM UDPGA and 8.0 nM 3H-SCH 39166 or 3H-SCH 23390 was added to 100 ml of the liver microsomal preparation (25-50 mg prot/ml). Samples were incubated for 30 rain at 37°C. The reaction was stopped and unchanged 3HSCH 39166 or 3H-SCH 23390 was separated from the aqueous phase by extraction with 3-heptanone. A 100 ml aliquot of the aqueous phase was removed and 3Hglucuronide formation was determined.
Clan
CI CH 3
I]
~l
tN
, ~
SCH 12679
SCtt 23390
SCH 39166
Fig. 1. Chemical structures of the benzazepines, SCH 12679 and SCH 23390, and the benzonaphthazepine, SCH 39166. 119S
CH 3
[)(~paminc90
120S RESULTS AND D I S C U S S I O N
Since SCH 39166 is the first D~ antagonist to be studied clinically, a brief summary of its major properties is worthwhile. Its structure is shown in figure 1, which also includes the benzazepines SCH 12679 and SCH 23390. Its in vitro properties include potent and selective displacement of Dt receptor binding with a K~vs3H-SCH 23390 of 1.9 nM vs a K~of 514 nM for displacement of ~H-spiperone as a D: ligand (both in rat striatum) vs a K~of 151 nM for displacement of 3H-ketanserin as a 5HT~ ligand in rat cortex (Chipkin et al., 1988). The biggest difference between SCH 39166 and SCH 23390 in these studies is that SCH 39166 has approximately 12-fold lower affinity for 5HT2 receptors. This latter finding has recently been corroborated by in vitro autoradiography studies (Wamsley et al., 1990) and by in vivo data using an EEDQ receptor protection paradigm (McQuade et al., 1990). Other in vivo binding studies support the D kvs D 2selectivity of SCH 39166 (McQuade et al., 1989; 1990). With respect to potential clinical indications for D t antagonists, schizophrenia is the primary target. Animal data which support this indication include inhibition of conditioned avoidance responding
~
•
/~\~o
{3HJ-SCIq39166IN vlvo BINDI,'J,:
o 1.0mg/kg,scSCH39166 CA;!
80-
i
b 6o
4o
TIME AFTER INJECTION (H)
Fig. 2. Time-course of the effect of SCH 39166 mg/kg s.c. for the displacement of in vivo binding of 3H-SCH 39166 and for inhibition of CAR in rats. Both r e s p o n s e s are plotted as a percent of their own maximum response at peak time postdosing. (CAR) in rats and squirrel monkeys. The correlation between inhibition of CAR and in vivo displacement of
CAR dose - response in monkeys (A)
100
E
1 h
--
Post-treatment []
(B) o
100
~
5O
/SCH
39166
6 h Post-treatment
5o
~
H 39166
0 0.1 0.2 0.3
1 1.78 3
Dose of drug (mg/kg po)
5.6
0.6 1
3
8.9
17.8
Dose of drug (mg/kg po)
Fig. 3. Dose-response for inhibition of CAR in squirrel monkeys at 1 and 6 hr post-dosing for SCH 39166 (O --- O) and haloperidol (D .... []).
Dopamine 90
121S
120
11o 10o
ICSO
90. g3300 131U
80.
: :
¢47 v l l i ~ . S 6 ) 10.0 e1~4.:1-16.7)
70.
i
60. 50. 40. 30 -
SCtl ~ I S O
0
SCH 10-
o: -10
.......
.01
| •1
........
i 1
........
I 10
........
l 1 O0
........
| 1000
CONC (uM) 12o-
11o: lOO"
IC$0
oo:
i8
SCH 39166 : 0.37 uM(.O&.66) SCH 3916S = 4S.1 uM(30.1-60.1)
7o: 6O
5O 40
¸
so: 2o: IO
•
SCH m l M
o
SCt13916S
o -1o .oi
.1
1
10
100
1000
CONC (uM)
Fig. 4. Effects of SCH 23390 and SCH 39166 and their corresponding less potent stereoisomers, SCH 23388 and SCH 39165, respectively, on glucuronidation of 3H-SCH 23390 in rat liver homogcnates. In vitro concentration of test drug ~M) is plotted vs. % of control formation of 3H-SCH 23390-glucuronide. IC~0s and 95% confidence limits are indicated for each. Dt receptor binding is illustrated by the matching time course data for the two activities in rats (Fig. 2). A major potential advantage of Dt antagonists for this indication is lack of acute extrapyramidal side effects (EPS). This is predicted from studies in cebus monkeys which show that neither SCH 23390 nor SCH 39166 produce signs of E I ~ after chronic dosing, whereas haloperidol shows a typical profile of ab-
normal movements (Coffin et al., 1989). These studies are covered in greater depth by Dr. Coffin, elsewhere in this publication. As indicated previously, the problem of short duration of action in squirrel monkeys after oral dosing limited the further progression of SCH 23390. SCH 39166 clearly has a longer duration of action in primates as it is still effective vs. CAR at 6 hours post-
122S
Dopamine 90
(A) Rat
sV
¢-
.g
4
3 2
E 0
E
1
SCH 23390
SCH 39166
(8) [ ~
Monkey
0.12 t--
0.10
•~
c
0.08
2 o.
0.06
liver agrees with their similar long duration of pharmacological action in this species (Chipkin et aL. 1988}. In contrast, when squirrel monkey liver homogenates are studied, there are two key differences. As shown in figure 5 the absolute level of glucuronidation was substantially lower for both drugs in the monkey. This is consistent with the greater pharmacologic potency of both drugs in this species. Moreover, SCH 39166 is glucuronidated to a lesser extent than SCH 23390 a finding which is consistent with the longer duration of SCH 39166. Even though these arc only in vitro studies which require corroboration by in vivo pharmacokinetics, the results appear to explain some of the aforementioned pharmacological results. Thus SCH 39166 appears to be a suitable candidate with which to test the efficacy and utility of specific D, dopamine receptor antagonists for the treatment of schizophrenia and other behavioral and neurological disorders. REFERENCES
E
~,0
E o.
0.04
0.02
0 9CH 23390 SGH 39166 Fig. 5. In vitro formation of 3H-SCH 23390-glucuronideand 3H-SCH 39166-glucuronide in rat (upper bar graph) and squirrel monkey (lower bar graph) liver homogenates. dosing (Fig. 3) whereas SCH 23390 effects only lasted 1-2 hours at comparable doses (Barnett et al., 1986). To study this difference further, studies were undertaken on the in vitro glucuronidation of key benzazepines. It is clear that glucuronidation is a major pathway for inactivation of benzazepines in vivo and that it is also possible to study glucuronyl transferase activity in vitro in liver homogenates. These studies (Tedford et al., 1990) show that in rat liver homogenates both SCH 23390 and SCH 39166 are reasonable substrates for glucuronyl transferase, using formation of 3H-SCH 23390-glucuronide as a measure. Thus in competition experiments the IC50 for cold SCH 23390 is 0.47 btM and for SCH 39166 is 0,37/~M (Fig. 4). Another interesting finding in this glucuronyl transferase competition experiment is that there is enantioselectivity, with the inactive S enantiomers being 21-fold (SCH 23388) and 122-fold (SCH 39165) less potent than their corresponding R-enantiomer: The similar ICs0s for SCH 39166 and SCH 23390 in rat
Barnett, A., Taber, R.J. and Steiner, S. (1974). The behavioral pharmacology of SCH 12679, a new psychoactive agent. Psychopharmacol. 36, 281-290. Barnett, A., Iorio, L.C., McQuade, R: and Chipkin, R.E. (1986). Pharmacological and behavioral effects of D~ dopamine antagonists. In Central D, Dopamine Receptors. (eds M. Goldstein, K. Fuxe, and I. Tabachnick). Plenum Press, New York. Chipkin, R.E., lorio, L.C., Coffin, V.L., McQuade, R.D., Berger, J.G. and Barnett, A. (1988); Pharmacological profile of SCH 39166: a dopamine D~ selective benzonaphthazepine with potential antipsychotic activity. J. Pharmacol. Exp. Ther. 247, 1093-1102. Coffin, V.L., Latranyi, M.B. and Chipkin, R.E. (1989). Acute extrapyramidal syndrome in monkeys: Development mediated by dopamine D2 but not D, receptors. J. Pharmacol. Exp. Ther., 249, 769-774. lorio, L.C., Houser, V.P., Korduba, C.A., Leitz, F.H. and Barnett, A. (1981). SCH 23390,a benzazepinewith a typical effects on dopaminergicsystems.Pharmacologist,23, 1137. Iorio, L.C., Barnett, A., Leitz, F.H., Houser, V.P. and Korduba, C.A. (1983). SCH 23390, a potential benzazepine antipsychoticwith unique interactions on dopaminergicsystems. J. Pharmacol. Exp. Ther., 226, 462--468. McQuade; R.D., Crosby, G.C., Duffy, R.A. and Chipkin, R.E. (1989). 3H-SCH 39166, a new D-I specific radioligand: in vitro and in vivo binding properties. Soc. Neurosci. Abs. 15, 429. McQuade, R.D., Duffy, R.A., Anderson, C., Chipkin, R.E. and Barnett, A. (1990). Protection of receptors from EEDQ inactivation by SCH 39 I66: evidencefor increased Dt selectivity.FASEB J., 4,A601. Tedford, C.E., Ruperto, V.B. and Barnett, A. (1990). In vitro glucuronidation of specific D~ receptor antagonists; SCH 23390, SCH 39166, and related benzazepine analogs. FASEB J., 4, A888. Wamsley, J.K., Hunt, M.E,, McQuade, R.D. and Alburges, M.E.(1990). 3H-SCH 39166, a D~ dopamine receptor antagonist: binding characteristics and localization. Exper. Neurol. In press.
0197-0186/92$5.00+0.00 Copyright © 1992PergamonPresspie
HIGHLIGHTS OF D1 DOPAMINE RECEPTOR ANTAGONIST RESEARCH ALLEN BARNETT, ROBERTD. McQuADE and CLARK TEDFORD Schering-Plough Research, 60 Orange Street, Bloomfield, NJ 07003, U.S.A.
Abstract-- SCH 39166 is now undergoing clinical trials in schizophrenics as a selective Dt dopamine receptor antagonist. It differs from SCH 23390, the prototype D~ receptor antagonist, by having reduced affinity for scrotonin receptors and a longer duration of action in primates, as measured in the squirrel monkey conditioned avoidance paradigm. Further studies on this difference in primates indicates that it may be attributable to reduced affinity of SCH 39166 compared to SCH 23390 for the hepatic glucuronosyltransferase system. Their affinities are similar in rats, a species in which both have similar duration of action. These and other studies presented are consistent with the novel profile of SCH 39166. The discovery and development of D~ specific dopamine antagonists has been strongly tied to the progression of the benzazepines as a chemical class. The initial discovery of the unique behavioral effects of the benzazepine SCH 12679 (Barnett et al., 1974), was followed by SCH 23390 (Iorio et al., 1981; 1983), which has become the prototype specific D~ antagonist. However the poor oral absorption and short duration of action of this c o m p o u n d in primates precluded its further development (Barnett et al., 1986). It is now clear that SCH 39166, a benzonaphthazepine, has the appropriate pharmacologic spectrum for further progression (Chipkin et al., 1988) and it is now in early clinical trials as the first D, specific dopamine antagonist to be studied for therapeutic purposes.
EXPERIMENTAL PROCEDURES In vitro receptor binding studies In vitro bindingstudies of Dr, D2 and 5HT receptor affinities were done according to methods described previously (Chipkin et al., 1988).
CH30~ ~ / N CH3 CH30 /
V
In vivo D~ receptor binding study in rats
The effect of SCH 39166 on Dt receptor binding was studied in vivo using 3H-SCH 39166 as a ligand, according to the method described by McQuade and colleagues (1989). Conditioned avoidance responding ( C A R ) in rats and monkeys
The effect of SCH 39166 and haloperidol on CAR in rats and monkeys utilized methods described in the work of Chipkin and colleagues (1988). In vitro glucuronosyltransferase activity in hepatic microsomes from rats and monkeys
The affinity of SCH 23390 and SCH 39166 for glucuronosyltransferase activity was studied in microsomal preparations from rat and squirrel monkey livers. The assays for ~H-SCH 23390 and 3H-SCH 39166-glucuronidationwere described previously by Tedford et al., (1990). Briefly, buffer containing 1.5 mM UDPGA and 8.0 nM 3H-SCH 39166 or 3H-SCH 23390 was added to 100 ml of the liver microsomal preparation (25-50 mg prot/ml). Samples were incubated for 30 rain at 37°C. The reaction was stopped and unchanged 3HSCH 39166 or 3H-SCH 23390 was separated from the aqueous phase by extraction with 3-heptanone. A 100 ml aliquot of the aqueous phase was removed and 3Hglucuronide formation was determined.
Clan
CI CH 3
I]
~l
tN
, ~
SCH 12679
SCtt 23390
SCH 39166
Fig. 1. Chemical structures of the benzazepines, SCH 12679 and SCH 23390, and the benzonaphthazepine, SCH 39166. 119S
CH 3
[)(~paminc90
120S RESULTS AND D I S C U S S I O N
Since SCH 39166 is the first D~ antagonist to be studied clinically, a brief summary of its major properties is worthwhile. Its structure is shown in figure 1, which also includes the benzazepines SCH 12679 and SCH 23390. Its in vitro properties include potent and selective displacement of Dt receptor binding with a K~vs3H-SCH 23390 of 1.9 nM vs a K~of 514 nM for displacement of ~H-spiperone as a D: ligand (both in rat striatum) vs a K~of 151 nM for displacement of 3H-ketanserin as a 5HT~ ligand in rat cortex (Chipkin et al., 1988). The biggest difference between SCH 39166 and SCH 23390 in these studies is that SCH 39166 has approximately 12-fold lower affinity for 5HT2 receptors. This latter finding has recently been corroborated by in vitro autoradiography studies (Wamsley et al., 1990) and by in vivo data using an EEDQ receptor protection paradigm (McQuade et al., 1990). Other in vivo binding studies support the D kvs D 2selectivity of SCH 39166 (McQuade et al., 1989; 1990). With respect to potential clinical indications for D t antagonists, schizophrenia is the primary target. Animal data which support this indication include inhibition of conditioned avoidance responding
~
•
/~\~o
{3HJ-SCIq39166IN vlvo BINDI,'J,:
o 1.0mg/kg,scSCH39166 CA;!
80-
i
b 6o
4o
TIME AFTER INJECTION (H)
Fig. 2. Time-course of the effect of SCH 39166 mg/kg s.c. for the displacement of in vivo binding of 3H-SCH 39166 and for inhibition of CAR in rats. Both r e s p o n s e s are plotted as a percent of their own maximum response at peak time postdosing. (CAR) in rats and squirrel monkeys. The correlation between inhibition of CAR and in vivo displacement of
CAR dose - response in monkeys (A)
100
E
1 h
--
Post-treatment []
(B) o
100
~
5O
/SCH
39166
6 h Post-treatment
5o
~
H 39166
0 0.1 0.2 0.3
1 1.78 3
Dose of drug (mg/kg po)
5.6
0.6 1
3
8.9
17.8
Dose of drug (mg/kg po)
Fig. 3. Dose-response for inhibition of CAR in squirrel monkeys at 1 and 6 hr post-dosing for SCH 39166 (O --- O) and haloperidol (D .... []).
Dopamine 90
121S
120
11o 10o
ICSO
90. g3300 131U
80.
: :
¢47 v l l i ~ . S 6 ) 10.0 e1~4.:1-16.7)
70.
i
60. 50. 40. 30 -
SCtl ~ I S O
0
SCH 10-
o: -10
.......
.01
| •1
........
i 1
........
I 10
........
l 1 O0
........
| 1000
CONC (uM) 12o-
11o: lOO"
IC$0
oo:
i8
SCH 39166 : 0.37 uM(.O&.66) SCH 3916S = 4S.1 uM(30.1-60.1)
7o: 6O
5O 40
¸
so: 2o: IO
•
SCH m l M
o
SCt13916S
o -1o .oi
.1
1
10
100
1000
CONC (uM)
Fig. 4. Effects of SCH 23390 and SCH 39166 and their corresponding less potent stereoisomers, SCH 23388 and SCH 39165, respectively, on glucuronidation of 3H-SCH 23390 in rat liver homogcnates. In vitro concentration of test drug ~M) is plotted vs. % of control formation of 3H-SCH 23390-glucuronide. IC~0s and 95% confidence limits are indicated for each. Dt receptor binding is illustrated by the matching time course data for the two activities in rats (Fig. 2). A major potential advantage of Dt antagonists for this indication is lack of acute extrapyramidal side effects (EPS). This is predicted from studies in cebus monkeys which show that neither SCH 23390 nor SCH 39166 produce signs of E I ~ after chronic dosing, whereas haloperidol shows a typical profile of ab-
normal movements (Coffin et al., 1989). These studies are covered in greater depth by Dr. Coffin, elsewhere in this publication. As indicated previously, the problem of short duration of action in squirrel monkeys after oral dosing limited the further progression of SCH 23390. SCH 39166 clearly has a longer duration of action in primates as it is still effective vs. CAR at 6 hours post-
122S
Dopamine 90
(A) Rat
sV
¢-
.g
4
3 2
E 0
E
1
SCH 23390
SCH 39166
(8) [ ~
Monkey
0.12 t--
0.10
•~
c
0.08
2 o.
0.06
liver agrees with their similar long duration of pharmacological action in this species (Chipkin et aL. 1988}. In contrast, when squirrel monkey liver homogenates are studied, there are two key differences. As shown in figure 5 the absolute level of glucuronidation was substantially lower for both drugs in the monkey. This is consistent with the greater pharmacologic potency of both drugs in this species. Moreover, SCH 39166 is glucuronidated to a lesser extent than SCH 23390 a finding which is consistent with the longer duration of SCH 39166. Even though these arc only in vitro studies which require corroboration by in vivo pharmacokinetics, the results appear to explain some of the aforementioned pharmacological results. Thus SCH 39166 appears to be a suitable candidate with which to test the efficacy and utility of specific D, dopamine receptor antagonists for the treatment of schizophrenia and other behavioral and neurological disorders. REFERENCES
E
~,0
E o.
0.04
0.02
0 9CH 23390 SGH 39166 Fig. 5. In vitro formation of 3H-SCH 23390-glucuronideand 3H-SCH 39166-glucuronide in rat (upper bar graph) and squirrel monkey (lower bar graph) liver homogenates. dosing (Fig. 3) whereas SCH 23390 effects only lasted 1-2 hours at comparable doses (Barnett et al., 1986). To study this difference further, studies were undertaken on the in vitro glucuronidation of key benzazepines. It is clear that glucuronidation is a major pathway for inactivation of benzazepines in vivo and that it is also possible to study glucuronyl transferase activity in vitro in liver homogenates. These studies (Tedford et al., 1990) show that in rat liver homogenates both SCH 23390 and SCH 39166 are reasonable substrates for glucuronyl transferase, using formation of 3H-SCH 23390-glucuronide as a measure. Thus in competition experiments the IC50 for cold SCH 23390 is 0.47 btM and for SCH 39166 is 0,37/~M (Fig. 4). Another interesting finding in this glucuronyl transferase competition experiment is that there is enantioselectivity, with the inactive S enantiomers being 21-fold (SCH 23388) and 122-fold (SCH 39165) less potent than their corresponding R-enantiomer: The similar ICs0s for SCH 39166 and SCH 23390 in rat
Barnett, A., Taber, R.J. and Steiner, S. (1974). The behavioral pharmacology of SCH 12679, a new psychoactive agent. Psychopharmacol. 36, 281-290. Barnett, A., Iorio, L.C., McQuade, R: and Chipkin, R.E. (1986). Pharmacological and behavioral effects of D~ dopamine antagonists. In Central D, Dopamine Receptors. (eds M. Goldstein, K. Fuxe, and I. Tabachnick). Plenum Press, New York. Chipkin, R.E., lorio, L.C., Coffin, V.L., McQuade, R.D., Berger, J.G. and Barnett, A. (1988); Pharmacological profile of SCH 39166: a dopamine D~ selective benzonaphthazepine with potential antipsychotic activity. J. Pharmacol. Exp. Ther. 247, 1093-1102. Coffin, V.L., Latranyi, M.B. and Chipkin, R.E. (1989). Acute extrapyramidal syndrome in monkeys: Development mediated by dopamine D2 but not D, receptors. J. Pharmacol. Exp. Ther., 249, 769-774. lorio, L.C., Houser, V.P., Korduba, C.A., Leitz, F.H. and Barnett, A. (1981). SCH 23390,a benzazepinewith a typical effects on dopaminergicsystems.Pharmacologist,23, 1137. Iorio, L.C., Barnett, A., Leitz, F.H., Houser, V.P. and Korduba, C.A. (1983). SCH 23390, a potential benzazepine antipsychoticwith unique interactions on dopaminergicsystems. J. Pharmacol. Exp. Ther., 226, 462--468. McQuade; R.D., Crosby, G.C., Duffy, R.A. and Chipkin, R.E. (1989). 3H-SCH 39166, a new D-I specific radioligand: in vitro and in vivo binding properties. Soc. Neurosci. Abs. 15, 429. McQuade, R.D., Duffy, R.A., Anderson, C., Chipkin, R.E. and Barnett, A. (1990). Protection of receptors from EEDQ inactivation by SCH 39 I66: evidencefor increased Dt selectivity.FASEB J., 4,A601. Tedford, C.E., Ruperto, V.B. and Barnett, A. (1990). In vitro glucuronidation of specific D~ receptor antagonists; SCH 23390, SCH 39166, and related benzazepine analogs. FASEB J., 4, A888. Wamsley, J.K., Hunt, M.E,, McQuade, R.D. and Alburges, M.E.(1990). 3H-SCH 39166, a D~ dopamine receptor antagonist: binding characteristics and localization. Exper. Neurol. In press.
