D1-receptor antagonists: Comparison of [3H]SCH39166 to [3H]SCH23390

Journal of Chemical Neuroanatomy, gol 5:357 366 (1992)

Dl-Receptor Antagonists: Comparison of [SHlSCH39166 to [3H]SCH23390 Mario E. Alburges, MaryAnne E. Hunt, Robert D. McQuade* and James K. Wamsley Neuropsychiatric Research Institute, 700 First Avenue South, Fargo, ND, and the Departments of Pharmacology and Neuroscience, The University of North Dakota School of Medicine, Grand Forks and Fargo, ND, USA *Schering-PIough Research, 60 Orange Street, Bloomfield, N J, USA

ABSTRACT A radiolabeled form of the benzonaphthazephine, SCH39166 was used to characterize the binding of this D I antagonist in cortex, and an autoradiographic comparison of the localization of [3H]SCH39166 to [3H]SCH23390 (D 1 antagonist and forerunner of SCH39166) binding was performed. The Kd for [3H]SCH39166, calculated from dissociation and association rate constants (1.09 riM), was comparable to the Ka value derived from Scatchard analyses of saturation data (1.74 nM). [3H]SCH 39166 binds to brain tissue in a saturable manner with high affinity and low non-specific binding. Inhibition o[" [3H]SCH39166 binding by dopaminergic and serotonergic agents supports the hypothesis that this is indeed a D:specific compound with little overlap onto serotonin (5-HT) receptors. The affinity of [3H]SCH39166 for 5-HT 2and 5-HTic receptors is at least an order of magnitude lower than the affinity of [3H]SCH23390 tbr these same receptor sites. Quantitative autoradiographic analysis ot"[3H]SCH39166 and [3H]SCH23390 binding indicates high D:receptor density in the caudate-putamen, nucleus accumbens, olfactory tubercle, substantia nigra and entopeduncular nucleus. Low levels of binding (not significantly above background) were detected with [3H]SCH39166 in lamina IV of the cortex and in choroid plexus: areas which had significant [3H]SCH23390 binding and are known to have a high density of 5-HT (5-HT 2 and 5-HT~ respectively) receptors. KEYWORDS:

Dopamine receptors Dl-receptor localization Serotonin receptors

INTRODUCTION The first selective dopamine type 1 (D~)-receptor antagonist described was a benzazepine derivative [(R)-( + )-8-chloro-2,3,4,5-tetrahydro-3-methyl5-phenyl-lH-3-benzazepine-7-ol],better known as SCH23390. Its properties included the ability to inhibit dopamine-stimulated adenylate cyclase activity (a D:receptor-mediated effect) without increasing serum prolactin levels (a D2-receptor effect), and the inability to compete for D2-receptor binding (Apud et al., 1985; Hyttel, 1983: Iorio et al., 1983: Navarra et al., 1988; Rovescalli et al., 1987: Stoofand Kebabian, 1984). The use of [3H]SCH23390 in binding studies with membrane preparations or tissue slices for subsequent autoradiographic analyses, has provided a wealth of information on the characteristics and localization of D Ereceptors in experimental animals and human tissues (Barnett et al., 1986; Beart, 1989; Dawson et al., 1986; Gehlert et al., 1986; Address correspondence to: James K. Wamsley, Neuropsychiatric Research Inslitute, 700 First Avenue South, Fargo, ND 58103, USA

0891-0618/92/050357 l0 $10.00 © 1992 by John Wiley and Sons Ltd

Waddington and O'Boyle, 1989). Pharmacological and biochemical studies of SCH23390 have shown the selectivity of this compound for the D : r e c e p t o r subtype and supplied evidence of somewhat lower affinity binding to serotonin 2 (5-HT 2) and 5-HT~, receptors. Schering-Plough Research has introduced a new Dl-receptor antagonist (Chipkin et al., 1988) with potential antipsychotic activity, SCH39166 [( - ) - t r a n s - 6 , 7 , 7 a , 8 , 9 , 1 3 b - h e x a h y d r o - 3 - c h l o r o 2 - hydroxy - N - methyl - 5 H - benzo[d]n aph th o - [2,1b] azepine]. This second-generation benzonaphthazepine compound has high selectivity for D t receptor sites with little binding to 5-HT receptor subtypes (McQuade et al., 1991 a, b). Previous investigations performed with [3H]SCH39166 focused on the binding of this ligand to brain membranes (McQuade et al., 1991a) or slice preparations ot" caudate-putamen (Wamsley et al., 1991). In the present study, we verify the binding characteristics in preparations of the cerebral cortex and extend the localization studies to other brain regions. Also, we make a direct comparison between [3H]SCH23390 and [3H]SCH39166 binding to demonstrate the superior selectivity of the latter compound.

358 (~

M.E. Alburges et al. DISSOCIATION CURVE ['H]SCH3 g 1 66

ASSOCIATION CURVE ['H]SCH39 166 4000

4000

•

•

I\

3000

! a. r~

®

Tolal

.•

3000

•

Total

•

Spealfl©

•

Non - Spl~Dif|©

•

2000

r~

~

2000

•

•

• •

1000

1000

0 0

4

II

12

16

20

0

24

10

DI88OCIATION KINETICS ['HISCH39166

®

-

0.0189

40

50

60

ASSOCIATION KINETICS [ ' ~ " P l a 9 1 6 6 3.00

0,00 K.:

30

INCUBATION TIME (MINUTES)

RINSE TiME (MINUTES)

®

20

K.,

-

0.0173

min'tnM "'

min -1

-0.20 rn m f-

:'l

2.25

m

1.50

-0.40

m

-0,00

=:

0.75

-0,80 0.00

- 1.00 0

6

12

18

24

30

RINSE TIME (MINUTES) Fig. 1. Dissociation time-course and kinetic analysis of specifically bound [sH]SCH39166. The rate of dissociation of [sH]SCH39166 (A), was determined in slide-mounted sections of rat cortices tissue incubated for 30 min at 25°C, followed by two rinses in cold 50 I~M-Tris-HCI buffer (pH 7.4) for periods of time ranging from 1 to 20 min. The dissociation rate constant, K ~(B), was determined from the dissociation experiment and was equal to the slope of the regression line in the plot of In (BJBo) versus time, where B. is the specific binding at time zero and B, is the binding at time t. Non-specific binding was defined with 1 ltM of the fluphenazine ( 18-28 % of the total binding). Values represent the means of two separate experiments incubated in sextuplieate with SEM less than 10% (Key: A, total; O, specific; B, nonspecific).

MATERIALS AND METHODS Timae preparation Male, Long-Evans rats, weighing 180-220 g, were purchased from Simonsen Laboratories (Gilroy, CA) and were maintained on a 12/12 h light/dark cycle with food and water available ad libitum. Animals were deeply anesthetized by intraperitoneal injection of sodium pentobarbital (Abbott Labs, North Chicago, IL) (100 mg/kg) prior to intracardial perfusion with an ice-cold isotonic saline solution (NaCI, 0.9% w/v) and the brains removed. For receptor-binding experiments, cortices were dissected, pooled and gently homogenized by making two strokes with a Teflon-coated pestle in a Potter-Elvehjem homogenizer. This tissue mash was

" 0

' 10

20

80

' 40

' 50

' 60

INCUBATION TIME (MINUTES) Fig. 2. Association time-course and kinetic analysis of specificaUy bound [3H]SCH39166. An association time-course (A) was established by incubating slides of cortical mash in 1.5 nM[3H]SCH39166 in buffer at 25°C for varying periods of time, ranging from 5 to 60 min. The association rate constant, K÷ ] (B), was calculated from the equation K÷ ~= (Ko~- K_ ~)/[radio~nd], where Ko, is the slope of the regression line in the plot ofln (B,q/ B~q- Bt) versus time, B, is the specific radioligand binding at time t and B~ is the specific radioligand binding at the equilibrium. Values represent the means of two separate experiments incubated in sextuplieate with SEM less than 10%. (Key: A, total; e~ specific; I , non-specific).

centrifuged at 500 rpm for 5 min in a Sorvall RT 6000 B table-top centrifuge (Du Pont, Wilmington, DE), in order to remove any air bubbles which might be present as the result of the homogenization process. The mash was then frozen by placing the tubes upright in a - 7 0 ° C freezer and stored until needed. Frozen cylinders of brain tissue were expelled from the test tubes and 10 Hm thick tissue sections were cryostat-cut and thaw-mounted onto chrom-alum gelatin-coated, glass microscope slides. For quantitative autoradiographic analyses, the brains were rapidly removed and frozen by immersion in 2-methylbutane (isopentane) at - 7 0 ° C for approximately 30 s and stored at - 7 0 ° C until needed. Sagittal sections of 10 I~m thickness were cryostat-cut from these brains and mounted onto chrome-alum gelatin-coated microscope slides.

D~-Receptor A n t a g o n i s t , [3H]SCH39166

®

SATURATION

ISOTHERM

['H]SCH3g

166

®

50

/

.72':..

•

(.3 Z

•

75

HiiIop.

Z

40

/

30

E 20 o Z

COMPETITION STUDIES ['HISCH39166 Against D o p a m i n e - L i k e Compounds 100

60 01

359

10

L~O tad QtO

50

25

O 0D

0

0

0

2

4

6

8

10

-12

['H]SCH39166 [nM]

®

SCATCHARD

ANALYSIS

a

-8

-6

-4

•

K, =

['H]SCH3g

1.74

166

®

C O M P E T I T I O N S T U D I E S ['H]SCH3g 1 6 6 Against S s r o t o n i n - L i k e Compounds

nM mol/mg

30

-2

COMPETITIVE LICAND, log[M]

100

40

Z

75 Z on

w OE i,

a z

L

-10

(.3

ft.

20

•

MMul.

•

Ketan.

•

8erot.

•

Matin.

*

Cysno.

5o

L,J 0-

o

~

10

.

0

.

10

.

.

20

.

.

30

.

40

50

60

70

BOUND

Fig. 3. Saturation and Scatchard analyses of [3H]SCH39166 binding to the dopamine receptor. Saturation studies (A) were conducted in slide-mounted sections of tissue incubated at 25c'C for 30 min with various concentrations of [3H]SCH39166 (from 0.125 to 10.0nM) in the presence and in the absence of 1 laM of fluphenazine, followed by 5-min rinses in ice-cold buffer; only specific binding is reported. Scatchard analysis (B) showed a K~) of 1.74_+0.20 nMand B,,,a ~ of 62.13 + 4.58 fmol/mg tissue. Values represent the means _+SEM of two separate experiments.

Radioligand binding analyses Dissociation, association, s a t u r a t i o n a n d competition experiments were c o n d u c t e d with the slidem o u n t e d cortical m a s h sections. A n association time-course was established by i n c u b a t i n g sextuplicate slides of cortical m a s h in the presence of i .5 nM[3H]SCH39166 in a 50 mM-Tris-HC1 buffer (pH 7.4) c o n t a i n i n g 120 mM-NaC1, 5 mM-KC1, 2 mM-CaC12, I mM-MgCI 2 ( G o t t b e r g e t a l . , 1989) at 25°C for v a r y i n g periods o f time r a n g i n g from 5 to 60 min. Non-specific b i n d i n g was defined as the percentage of [3H]SCH39166 that r e m a i n e d b o u n d in the presence of 1 pM-fluphenazine, a n d constituted 18-28% of the total binding. T o determine the rate of dissociation o f specifically b o u n d [3H]SCH39166, tissues were i n c u b a t e d for 30 m i n at 25°C followed by two rinses in cold buffer for periods of time r a n g i n g from 1 to 22 min. S a t u r a t i o n experiments were performed by varying

25 0 -12

i

-10

i

i

-8

-6

-4

-2

COMPETITIVE LICAND, log[M]

Fig. 4. Inhibition of specific [3H]SCH39166 binding in cortical receptor preparation by dopaminergic and serotonergic compounds. Competition curves with a fixed concentration of [3H]SCH39166 (1.0 riM)were generated for D, and D2antagonists (A) and 5-HT agonists and antagonists (B) at concentrations ranging from 1 x 10 -'M to 1 x 10 ,2M for the competing nonradioactive ligands in the incubation medium. The IC50was equal to the negative reciprocal of the slope of the regressionline in the plot of B versus B x [/], where B is the amount of specifically bound ligand and [I] the concentration of the inhibitor. The K, was obtained by the equation K.= IC50/ 1+ [L] over KD,where [L] is the concentration of labeled ligand and K~)is the dissociation constant of the labeled ligand. The K, values_+SEM were determined in sextuplicate sections of cortical tissue preparations from a group of four animals and each experiment was repeated twice. (Key for (A): e , sulpiride; &, SCH23390; B, haloperidol. Key for (B): &, mesulergine; e , ketanserin: T, 5-HT: II, mianserin: O, cyanopindolol).

the c o n c e n t r a t i o n of radioactive ligand in the incub a t i o n m e d i u m using c o n d i t i o n s dictated by the results o f the previously described experiments. Tissues were i n c u b a t e d in [3H]SCH39166 at concentrations r a n g i n g from 0.125 nM to 10 nM for 30 m i n at room t e m p e r a t u r e followed by two 5-min rinses in ice-cold buffer w i t h o u t added radioactivity. C o m p e t i t i o n curves were generated for D~ a n d D 2 a n t a g o n i s t s a n d 5 - H T agonists a n d a n t a g o n i s t s at c o n c e n t r a t i o n s r a n g i n g from 1 x 10 .... M to l x l 0 - J 2 M for the c o m p e t i n g n o n - r a d i o a c t i v e ligands in the i n c u b a t i o n m e d i u m .

360

M.E. Alburges et al. [=H]SCHS9166 and [=H]SCH23590 S t u d l e a Againat Ketun3erin

Drugs

Compefifion

100, (3 Z

_z m

--

so

O

u~

2s

0

-' 2

'

'

-10

,

'

-8

.

'

-6

"-C.-

-4

-2

KETANSERIN, log[M]

Fig, 5. Inhibition of specific [3H]SCH39166 and [3H]SCH23390 binding to dopamine receptor by ketanserin. Slide-mounted sections of cortices tissue were prepared and assayed as described in the text, and incubated in the presence of various concentrations (from l x 10-2M to l x 10 -t2) of ketanserin. The IC~0 and Ki values were calculated as described before. The K~ values of ketanserin for [3H]SCH23390 and [3HlSCH39166 were 324.03 + 40.21 nm and 5350.91 + 70t.30 riM, respectively. Values represent the means_+ SEM of two separate experiments using sextuplicate slides. (Key: O, SCH23390; I , SCH39166).

Autoradiography and image analysis For quantitative autoradiographic analyses, animals were perfused, the brains removed and sagittal sections obtained as previously described. Autoradiograms were generated from sections incubated at 25°C for 30 min, in a 50 mM-Tris-HC1 buffer (pH 7.4) containing 120 mM-NaCl, 5 mM-KC1, 2 mMCaCl 2, 1 mM-MgCl 2 plus 1.0 nM of [3H]SCH39166 or 1.0 nM of [3H]SCH23390. Incubation was followed by two 5-rain rinses in ice-cold buffer. Non-specific binding was defined with a 1 I~M concentration of fluphenazine. Following the final rinse, slides were dipped in H20 at 4°C to remove salts, then dried by blowing cool, desiccated air over the tissue surface. These dried tissue sections were apposed to sheets of tritium-sensitive film (Hyperfilm, Amersham, Arlington Heights, IL) in light-tight X-ray cassettes. A set of commercially prepared tritium standards ([3H]-microscales, Amersham, Arlington Heights, IL) was included in each cassette. After 35 days of exposure, the films were developed with Kodak D-19 developer for 5 min at 18°C, and fixed in Kodak rapid fixer with hardener for 5 min. Quantitative analyses of the autoradiograms were performed using a Hewlett Packard computerbased, Leitz MPV-Combi microscope, photometer system (Wild Leitz, Rockleigh, N J). The optical density (OD) of the film area exposed to each section of the tritiated microscale was read and its natural log (In) plotted against the In of the known nCi/g calibration values of the microscale. The resultant linear regression equation was used to determine the nCi/g values associated with the ODs for specific brain regions. These values were then converted to fmol/mg tissue.

The drugs used in these experiments were supplied by the following sources: [3H]SCH39166 (specific activity 76.0 Ci/mmol) was supplied by Dr Robert McQuade of Schering-Plough Research, Bloomfield, New Jersey; [3H]SCH23390 (specific activity 70.7 Ci/mmol) was purchased from DuPont-NEN, Boston, Massachusetts; SCH23390, ketanserin tartrate, mianserin hydrochloride and fluphenazine hydrochloride were purchased from Research Biochemicals Inc., Natick, Massachusetts; sulpiride and haloperidol were purchased from Sigma Chemical Company, St Louis, Missouri; mesulergine and cyanopindolol were kindly donated by Dr D. R6mer from Sandoz Pharmad AG, CH4002 Basel, Switzerland.

RESULTS

Association and dissociation timo-course of

specificany bound131.qSCH39t66 Preliminary experiments were performed to determine conditions which maximized the specific to non-specific ratio of [3H]SCH39166 binding in cortical tissue mash and to attempt to duplicate information available from binding in striatum. The [3H]SCH39166 binding to cortical mash was readily reversible (Fig. 1). Specific binding remained at a relatively high percentage (72-82%) throughout the entire experiment. A rinse time of 10 min (2 x 5 min) was selected for utilization in subsequent experiments. Specific binding reached equilibrium within 30 min and did not change signficantly during an additional 60 min of incubation (Fig. 2). Consequently, a 30-min incubation period was used in all subsequent experiments. The dissociation binding constant (Kd), in cortex, was calculated from the association and dissociation rate constants ( K s = K j/K+ ~) as determined previously (Wamsley et al.. 1991). Using a dissociation rate constant (K_ 1) of 0.0189 min- ~ and an association rate constant (K,l), calculated according to the equation described by Bennett and Yamamura (1985), of 0.0173 min-i nM-~, gives a Kd of 1.09 nM. This figure is comparable to the K s value derived from saturation experiments.

Saturation and Scatcimrd analysis of [~HISCH39166 Wadingto the depamine receptor Specific binding began to saturate at approximately 2.0 nM and the dopamine receptor population was fully occupied at a 4 nM concentration of [3H]SCH39166 (Fig. 3A). Scatchard analyses of these data revealed a linear relationship (r= 0.94925) (Fig. 3B), with a Kd of 1.74+0.204 n i and a Bmax of 62.13+4.58fmol/mg tissue, as previously reported (Wamsley eta/., 1991).

D 1 - R e c e p t o r A n t a g o n i s t , [3H]SCH39166

361

Table I. Regional distribution of [3H]SCH39166 and [3H]SCH23390 binding sites in rat brain Bound (fmol/mg tissue + SEM) Brain area Frontoparietal cortex (FrP) Laminae I-III Laminae IV Laminae V V1 Occipital cortex (Oc2Mm) Laminae I Ill Laminae 1V Laminae V VI Retrosplenial agranular cortex (RSA) Caudate-putamen (CPu) Accumbens nucleus (Acb) Globus pallidus (GP) Ventral pallidus (VP) Basal nucleus of Meynert (B) Entopeduncular nucleus (EP) Olfactory tubercle (Tu) Anterior olfactory nucleus, lateral part (AOL) Dentate gyrus (DG) Field CA, of Ammon's horn (CA 0 Laterodorsal and lateral posterior thalamic nucleus (LD,LP) Ventral posterolateral and ventral posteromedial thalamic nucleus (VPL,VPM) Substantia nigra pars reticulata (SNR) Superior gray layer of the superior colliculus (SuG) External cortex of the inferior colliculus (ECIC) Primary fissure of the cerebellum (PrF) Prepyramidal fissure of the cerebellum (PPF) Lateral recess of the 4th ventricle (LR4V)

[3H]SCH3 9 1 6 6

[3H]SCH23390

16.10±0.66 "15.80_+0.48" "32.60 i 0.96

22.77__+0.74 58.86_+ 1.25 49.39 ± 8.32

19.54 ± 1.01 q3.96 ±0.77" ~38.24 ± 0.98

25.34_+0.98 49.02_+0.81 45.62 ± 1.39

36.83 4-4.19 "252.82 ± 23.65* "198.44± 10.36"* 68.45 i 10.96 84.92 -+8.54 75.64± 10,70 "117.50 ± 12.49 195.32 ± 31.57

29.97 -+2.65 338.62 ± 11.69 269.59_+ 11.06 68.65 ± 3.78 88.33 ± 6.95 106.90-+ 13.19 202.70± 59.47 240.55 ± 38.08

34.87+ 1.48 26.87+ 1.27 22.25 ± 1.55

43.64_+3.31 24.49-+ 1.02 22.11 ± 6.35

23.05 ± 2.03

25.31 -+2.77

19.36_+2.71

21.07+ 1.60

"274.64 4- 2.60*

330.24 ± 13.30

38.93 ±2.67

40.13 _+0.62

16.81 +_2.61

15.15±2.47

23.48_+3.15

23.72±5.88

3.78 ± 0.80

3.42 ± 0.50

0.09 ± 0.002***

38.42 4-6.41

Values are expressed as the mean+standard error of the mean determined from densitometric measurements made on autoradiograms generated from sagittal sections. The experiments were repeated twice, each representing 12 sections/condition from four animals. Significant differences between groups are indicated by *, P < 0.05; **, P < 0.001 ***. "Data from Wamsley et al., (1991).

Inhibition of specific [3HISCH39166 binding to cortical receptor preparation by dopaminergic and serotonergic compounds In o r d e r to d e t e r m i n e the relative selectivity o f [~H]SCH39166 b i n d i n g to the D r r e c e p t o r subtype, c o m p e t i t i o n e x p e r i m e n t s were p e r f o r m e d in cortical tissue p r e p a r a t i o n s . T h e results o f these c o m p e t i t i o n e x p e r i m e n t s are s u m m a r i z e d in Fig. 4 (A a n d B). T h e K, values are: 0.779_+0.1 1 nM for SCH23390; 438 178 4-_50 304 nM for sulpiride; 120.65 +_ 15.42 nM for h a l o p e r i d o l ; 5350.41 +- 701.30 nM for k e t a n s e r i n a n d 551.85 +- 89.69 nM for mianserin; 12 700 4-_ 1832 nM for 5-HT; 1168.47_+ 144.98 nM for mesulergine and 95256+10847nM for c y a n o p i n d o l o l . T h e relative abilities o f these d r u g s to c o m p e t e with [3H]SCH39166 for D i - r e c e p t o r sites are as follows: S C H 2 3 3 9 0 > h a l o p e r i d o l > sulpiride > m i a n s e r i n > mesulergine > k e t a n s e r i n > 5 - H T > c y a n o p i n d o l o l .

These results also indicate t h a t the p o t e n c y o f the original Schering c o m p o u n d (SCH23390) is a p p r o x i m a t e l y twice that o f the new c o m p o u n d (SCH39166). These d a t a closely a p p r o x i m a t e those f o u n d by C h i p k i n e t al. (1988), using rat striatal tissue h o m o g e n a t e a n d [3H]SCH23390 as a r a d i o ligand. C o m p e t i t i o n studies with fixed c o n c e n t r a t i o n s o f [3H]SCH23390 (1.0riM) and [3H]SCH39166 ( l . 0 n M ) b u t v a r y i n g c o n c e n t r a t i o n s (1 × I 0 - 2 M to 1 × 1 0 - ' 2 M ) o f k e t a n s e r i n were p e r f o r m e d in o r d e r to depict the degree o f interaction o f these D~-receptor a n t a g o n i s t s with the 5 - H T 2 r e c e p t o r site. The results o f these c o m p e t i t i o n e x p e r i m e n t s are shown in Fig. 5. The i n h i b i t o r y p o t e n c y o f k e t a n s e r i n for [3H]SCH23390 a n d [3H]SCH39166 presented as K i values o f 324.03+_40.21 nM a n d 5350.41 +701.30riM, respectively. These results

362

M . E . A l b u r g e s et al.

Fig. 6. Photomicrographs of autoradiograms showing the binding of[~H]SCH39166 versus [3H]SCH23390. Images of sagittal seclions of the rat brain were generated and photographed from the MCID CRT screen. In sections labeled with 1 nM of [3H]SCH39166 (A), total binding was high in the caudate-putamen (CPu), nucleus accumbens (Acb), olfactory tubercle (Tu), entopeduncular nucleus (EP) and substantia nigra (SN). A lower density of labeling was found in the dentate gyrus (DG), and lamina IV of the cerebral cortex (VI). In sections labeled with I nM of[3H]SCH23390 (B), total binding was similar in all the areas mentioned above, but additionally appeared high in the choroid plexus (ChP). Note also the binding is higher in the anterior cerebellum (Ant Cb) than the posterior lobule (Post Cb) with both ligands. See Table 1 for the quantification of specific areas.

Di-Receptor Antagonist, [3H]SCH39166 363

V

Fig. 7. Photomicrographs of autoradiograms showing the binding of [3H]SCH39166 vs [3H]SCH23390 in rat cerebral cortex. Images were generated and photographed as previously described. In sections labeled with [3H]SCH39166 (A), lamina IV of the frontoparietal cortex (IV) shows less binding than lamina VI; whereas sections labeled with [3H]SCH23390 (B) indicate similar labeling in both regions. See Table I for quantitated information. Scale = 1000 ~tm.

support previous findings that [3H]SCH23390 labels 5-HT 2 receptors in cortex (Billard et al., 1984; Dawson et al., 1986, 1987; Hyttel, 1983; McQuade et al., 1988) In contrast, the overlap of this new selective Drreceptor antagonist (SCH39166) on the 5-HT receptor sites is minimal.

Quantitative autoradiographyof [3H]SCH39166 and [3H]SCH23390 bindingsites Tissue sections were prepared and labeled, and quantitation was performed according to the procedures described in the Methods section. The regional distribution of [3H]SCH39166 and [3H]SCH23390 binding sites is shown in Table 1 and Fig. 6 (A and B). D~ receptors, as labeled with [3H]SCH39166, were heterogeneously distributed in the rat brain. The highest concentrations ( > 100 fmol/mg tissue) of D l receptors were found

in the caudate-putamen, nucleus accumbens, entopeduncular nucleus, olfactory tubercle and the substantia nigra reticulata. Moderate densities (> 50 fmol/mg tissue) of D~ receptors were found in the globus pallidus, ventral palladium and basal nucleus of Meynert. Low densities of D t receptors were found in the frontal and occipital cortices (laminae I lIl and IV showing the lower densities of binding, Fig. 7), retrosplenial agranular cortex, hippocampal formation, anterior olfactory nucleus (lateral part), superficial gray layer of the superior colliculus, and several thalamic and hypothalamic areas. Low binding densities were also found in the cerebellum (Fig. 6A, bottom). Interestingly, there was significantly higher binding in the region most rostral to the primary fissure than in the more caudal aspects of the cerebellum. Binding in the lateral recess of the fourth ventricle dorsal to the cerebellum did not differ significantly from

364

M . E . Alburges et al.

Fig. 8. Photomicrographs showing the binding of [3H]SCH39166in the cerebellum. Images were generated and photographed as previouslydescribed. Sectionslabeled with [3H]SCH39166(A) show a very low density of binding in the choroid plexus(ClaP);a region where[3H]SCH23390is known to overlaponto 5HTtcsites. SeeTable I for quantitativeinformation.A cresylviolet-stainedsectionof the cerebellum(B) is included to show the presenceof the choroid plexus (ChP) in the section used to generate the autoradiogramshown in (A). Scale= 500 ~m. background (Table 1; Fig. 8). Significant differences in binding between [3H]SCH39166 and [3H]SCH23390 were found in lamina IV of the frontoparietal cortex, and laminae IV of the occipital cortex (Table 1; Fig. 7). In some areas of basal ganglia, such as the caudate-putamen, binding obtained with [3H]SCH39166 was significantly lower than that obtained with [3H]SCH23390. Other regions exhibiting a significant difference between [3H]SCH39166 a and [ H]SCH23390 binding include the substantia nigra pars reticulata and the lateral recess o f the 4th ventricle (which is known to harbor the choroid plexus). The latter region shows high binding with [3H]SCH23390 (Fig. 6B) but virtually no binding with [3H]SCH39166, even though cresyl violet staining confirmed the presence of choroid plexus (Table 1; Fig. 8A and B).

DISCUSSION Schering's selective D~ antagonist, SCH39166, is now in clinical trials (Chipkin, 1990). This compound has different characteristics from its predecessor SCH23390. SCH39166 belongs to a different chemical class (benzonaphthazepine); it has a longer action in non-human primates (Chipkin et aL, 1988); and it has more selectivity for D t receptors than SCH23390 (McQuade et al., 1991 a,b; Wamsley et al., 1991). In addition, this new compound (SCH39166) has not shown any limitation in the toxicity test when evaluated in rodents and nonhuman primates (Chipkin et al., 1988). One of the primary aims of the present investigation was the characterization of [ 3 H ~ H 3 9 1 6 6 binding to Dr-receptor sites in sections of rat

D~-Receptor Antagonist, [3H]SCH39166 cortical tissue. Under the conditions described herein, [3H]SCH39166 bound to D I receptors in a reversible and saturable manner, and represented 72-82% of the total binding. The K d derived from Scatchard analyses of saturation data closely approximated the K d value calculated from kinetics experiments; 1.74 nM and t.09 nM respectively. These findings are consistent with those reported by McQuade et al. (1991a) using striatal membrane homogenates, and Wamsley et al. ( 1991) using striatal tissue mash. The results of competition experiments using dopaminergic and serotonergic drugs indicated that [3H]SCH39166 binds with high selectivity to D~-receptor sites. The only compound analysed which was capable of displacing 50% of the specifically bound [3H]SCH39166 at a low concentration was SCH23390. Other dopamine-specific compounds were relatively weak inhibitors of [3H]SCH39166, exhibiting ICs0s ranging from 100 to 100 000 times that of SCH23390. Furthermore, serotonergic compounds (Peroutka, 1988), such as ketanserin, mianserin, mesulergine and cyanopindolol, showed less overlap on the D~-receptor sites labeled with [3H]SCH39166 than those sites labeled with [3H]SCH23390, suggesting a lesser degree of occupancy of 5-HT,, 5 - H T I o 5-HTIB and 5-HT~A receptor sites with-[3H]SCH39166 as opposed to [3H]SCH23390. Most of the classical stereotypies associated with dopaminergic drugs in animal studies have been attributed to D2-receptor activity. Only a few very discrete behavioral properties have been ascribed to D~ activity; however, D~ receptors appear to be involved in regulation and modulation of D~ receptors (Arnt, 1987; Clark and White, 1987i Waddington and O'Boyle, 1989) Non-subtypeselective and D,-selective dopamine antagonists have been used in the treatment of schizophrenia. A highly selective D~ antagonist with virtually no 5-HT overlap, such as SCH39166, may prove an effective treatment for certain neuropsychiatric disorders. The interaction of these receptors with other dopamine receptors which may be involved in behavior remains to be determined (Sokoloff et al., 1990; Tiberi et al., 1991, Van Tol et al., 1991 ). Findings in this study establish the utility of [sH]SCH39166 for localization of Dl-receptor binding sites in rat brain. The DI-receptor population, as labeled with [3H]SCH39166, was heterogeneously distributed and qualitatively similar to that reported previously for [3H]SCH23390 by Dawson et al. ( 1985, 1986) and Savasta et al. (1986). However, the number of D~-binding sites as defined by these compounds differed significantly in the areas under study. This difference can be accounted for by the superior selectivity of [3H]SCH39166. Binding in areas known to be replete with 5-HT receptors, such as lamina IV of the cerebral cortex and choroid plexus (Dawson et al., 1987; McQuade et al., 1988; Nicklaus et al., 1988), were significantly lower with [3H]SCH39166 than with [3H]SCH23390.

365

In summary, this new benzonaphthazepine compound possesses a high affinity and is highly selective for Dl-receptor sites. As previously noted, compounds with little or no affinity for D I receptors do not interfere with the binding of[3H]SCH39166. The introduction of SCH39166 and characterization of its binding and pharmacological properties in the central nervous system indicate that this is the compound of choice where antagonism of D~-receptormediated effects is desired. Research with this drug may be regarded as a step toward the development of a treatment for diseases where D I receptors may be involved. ACKNOWLEDGEMENTS The authors wish to thank Cindy Belohlavek, Lisa Longlet and Candy Johnson for expert technical assistance, Karen Johnson for outstanding secretarial help in preparing this manuscript, and Diane Nordeng, librarian, for invaluable assistance in procuring relevant literature. M.E.A. is an Aggregate Professor from the University of Zulia, School of Medicine in Maracaibo. Zulia, Venezuela. This research was supported by a grant from the Public Health Service(DA05167). REFERENCES Apud, J. A., Masotto, C., Ongini, E. and Racagni, G. (1985). Interaction by SCH2339(1, a D~ selective antagonist, with the anterior pituitary D e receptors and prolactin secretion in the rat. Eur. J. Pharmacol. 112, 187 193. Arnt, J. (1987). Behavioral studies ofdopamine receptors: Evidence for regional selectivity and receptor multiplicity. In Dopamine Receptors (eds Creese, I. and Fraser, C. M.), pp. 199--231. Alan R. Liss, Inc., New York. Barnett, A., lorio, L. C., McQuade, R. D, and Chipkin, R. E. (1986). Pharmacological and behavioral effects of D~ dopamine antagonist. In Central D; Dopamine Receptors(edsGoldstein, M., Fuxe, K. and Tabachnick, I.), pp. 137- 144. Plenum Press, New York. Beart, P. M. (1989). Dopamine receptors: Classification, properties and drug development. Clin. E.vp. Pharmacol. Physiol. 16, 511 -515. Bennett, J. P. and Yamamura, H. 1. (1985). Neurotransmitter, hormone, or drug receptor binding methods. In Neurotransmitter Receptor Bindin~ (eds Yamamura, H. O., Enna, S. J. and Kuhar, M. J.), pp, 61 89. Raven Press, New York. Billard, W., Ruperto, V., Crosby, G., lorio, L. C. and Barnett, A. (1984). Characterization of the binding of 3H-SCH23390, a selective D~ receptor antagonist ligand, in rat striatum. L(tO Sci. 35, 1885 1893. Chipkin, R. E. (1990). Dopamine D~ receptor antagonists and schizophrenia: D~ antagonist in clinical trial. TiPS I1,185. Chipkin, R. E., Iorio, L. C., Coffin, V. L., McQuade, R. D., Berger, J. G. and Barnett, A. (1988). Pharmacological profile of SCH39166: A dopaminc D~ selective benzonaphthazepine with potential antipsychotic activity. J. Pharmacol. Exp. Ther. 347, 1093 11(12. Clark, D. and White, F. J. (1987). Review: D Ldopamine receptor The search for a function: A critical evaluation of the Dt/D z dopamine receptor classification and its functional implications. Synapse 1,347 388.

366

M . E . Alburgeset al.

Dawson, T. M., Gehlert, D. R., Yamamura, H. 1., Barnett, A. and Wamsley, J. K. (1985). D~ dopamine receptors in the rat brain: Autoradiographic localization using [3H]SCH23390. Eur. J. Pharmacol. 108, 323-325. Dawson, T. M., Gehlert, D. R,. McCabe, R. T., Barnett, A. and Wamsley, J. K. (1986). D~ dopamine receptors in the rat brain: A quantitative autoradiographic analysis. J. Neurosci. 6, 2352-2365. Dawson, T. M., Gehlert, D. R. and Wamsley, J. K. (1986). Quantitative autoradiographic localization of central dopamine D-I and D-2 receptors. In Neurobiology oJ Central Dl-Doparnine Receptors (eds Breese, G. and Creese I.), pp. 93-118. Plenum Press, New York. Dawson, T. M., McCabe, R. T., Stensaas, S. S. and Wamsley, J. K. (1987). Autoradiographic evidence of [3H]SCH23390 binding sites in human prefrontal cortex (Brodmann's area 9). J. Neurochem. 49, 789--796. Gehlert, D. R., Dawson, T. M., Barnett, A., Yamamura, H. 1. and Wamsley, J. K. (1986). Autoradiographic localization ofdopamine type- I receptors after labeling with [3H]SCH23390 in the rat and human brain: Comparison with the distribution of D 2 receptors. In Modulation ~]" Central and Peripheral Transmitter Function,

Symposia in Neuroscience Ill (eds Biggio, G., Spano, P. F., Toffano, G. and Gessa, G. L.), pp. 27 35. FIDIA Research Series, Liviana Press, Padova, Italy. Gottberg, E., Diop, L., Montrevil, B. and Reader, A. (1989). Effects of sodium, lithium, and magnesium on in vitro binding of [3H]SCH23390 in rat neostriatum and cerebral cortex. Neurochem. Res. 14, 419-426. Hyttel, J. (1983). SCH23390~the first selective dopamine D~ antagonist. Eur. J. Pharmacol. 91, 153--154. Iorio, L. C., Barnett, A., Leitz, F. ft., Houser, V. P. and Korduba, C. Z. (1983). SCH23390, a potential benzazepine antipsychotic with unique interactions on dopamine systems. J. Pharmacol. Exp. Ther. 226, 462-468. McQuade, R. D., Ford, D., Chipkin, R. E., lorio, L. C. and Barnett, A. (1988). Serotonergic component of SCH23390: In vitro and in vivo binding analyses. Life Sci. 43, 1861-1869. McQuade, R. D., Duffy, R. A., Anderson, C. C., Coffin, V. L., Chipkin, R. E. and Barnett, A. (1991a). 3HSCH39166, A new D-I selective radioligand: In vitro and in vivo binding analyses. J. Neurochem. 57, 2001-2010. McQuade, R. D., Duffy, R. A., Coffin, V. L., Chipkin, R. E. and Barnett, A. (1991b). In vivo binding of SCH39166: A D-1 selective antagonist. J. Pharmacol. Exp. Ther. 257, 1 8.

Navarra, P., Carmine, R., Ragazzoni, E., Vacca, M. and PreziosL P. (1988). Effects of the benzazepine SCH23390 on prolactin release from isolated pituitary. Pharmacol. Toxicol. 63, 135-136. Nicklaus, K. J., McGonigle, P. and Molinoff, P. B. (1988). [3H]SCH23390 labels both dopamine-1 and 5hydroxytryptaminej~ receptors in the choroid plexus. J. Pharmacol. Exp. Ther. 247, 343-348. Peroutka, S. J. (1988). 5-Hydroxytryptamine receptor subtypes: Molecular, biochemical and physiological characterization. TINS 11,496-500. Rovescalli, A. C., Brunello, N., Monopoli, A., Ongini, E. and Racagri, G. (1987). Absence of [3H]SCH23390 specific binding sites in anterior pituitary: Dissociation from effects on prolactin synthesis. Eur. J. Pharmacol. 135, 129-136. Savasta, M., Dubois, A. and Scatton, B. ~1986). Autoradiographic localization of Dj dopamine receptors in the rat brain with [3H]SCH23390. Brah7 Res. 375, 291-30 I. Sokoloff, P., Giros, B., Martres, M. P., Bouthenet, M. L. and Schwartz, J. C. (1990). Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature 347, 146-151. Stoof, J. C. and Kebabian, J. W. (1984). Two dopamine receptors: Biochemistry, physiology and pharmacology. Li['e Sei. 35, 2281-2296. Tiberi, M., Jarvie, K. R., Silvia, C., Falardeau, P., Gingrich, J. A., Godinot, N., Bertrand, L., Yang-Feng, T. L., Fremeau, R. T., Jr and Caron, M. G. (1991). Cloning, molecular characterization, and chromosomal assignment of a gene encoding a second D~ dopamine receptor subtype: Differential expression pattern in rat brain compared with the D~Areceptor. Proc. Natl. Acad. Sci. USA 88, 7491-7495. Van Tol, H. H. M,, Bunzow, J. R., Guan, H. C., Sunahara, R. K., Seeman, P., Niznik, H. B. and Civelli, O. (1991). Cloning of the gene for a human dopamine4 receptor with high affinity for the antipsychotic clozapine. Nature 350, 610-615. Waddington, J. L. and O'Boyle, K. M. (1989). Drugs acting on brain dopamine receptors: A conceptual reevaluation five years after the first selective D~ antagonist. Pharmacol. Ther. 43, 1-52. Wamsley, J. K., Hunt, M. A., McQuade, R. D. and Aiburges, M. E. (1991). [3H]SCH39166, a D~ dopamine receptor antagonist: binding characteristics and localization. Exp. Neurol. 111,145-151.

A c c e p t e d 19 February 1992