Antagonism by neuroleptics of neurotransmitter receptors of normal human brain in vitro

European Journal of Pharmacology, 103 (1984 ) 197 - 204

197

Elsevier

A N T A G O N I S M BY N E U R O L E P T I C S HUMAN BRAIN IN VITRO

OF NEUROTRANSMITTER

RECEPTORS

OF NORMAL

ELLIOTI" RICHELSON * and ALBERT NELSON

Departments of P,~vchiati~vand Pharmacology, Mayo Clinic and Foundation, Rochester. MN 55905, U.S.A. Received 28 February 1984, revised MS received 17 April 1984, accepted 17 May 1984

E. R I C H E L S O N a n d A. N E L S O N , Antagonism t~v neuroleptics of neurotransmitter receptors o f normal human brain in

vitro, European J. Pharmacol. 103 (1984) 197 204. Using radioligand binding techniques, we determined the equilibrium dissociation constants (Ko's) for a series of neuroleptics at the dopamine (D-2), muscarinic, histamine H~, off- and a2-adrenergic receptors of normal human brain tissue obtained at autopsy. Seventeen different compounds were studied at the D-2 receptor and 15 compounds at the remaining receptors. At the D-2 receptor of caudate nucleus, spiperone was the most potent compound (K D = 0.16 nM); clozapine the least potent ( K o = 180 nM). The KD's for six compounds at the D-2 receptor of nucleus accumbens were not significantly different from their respective KD's in the caudate nucleus. The most potent and least potent compounds at the other receptors were clozapine and molindone at the muscarinic receptor, mesoridazine and molindone at the H t receptor, spiperone and molindone at the at-receptor, and clozapine and haloperidol at the ~2-receptor, respectively. Dopamine (D-2) receptor Antipsychotics

Muscarinic receptor

H i s t a m i n e H~ r e c e p t o r

1. Introduction The a n t a g o n i s m by n e u r o l e p t i c s of certain neur o t r a n s m i t t e r receptors m a y explain their thera p e u t i c effects, some of their adverse effects a n d s o m e of their d r u g - d r u g interactions. Thus, the affinity of a n e u r o l e p t i c for a p a r t i c u l a r receptor, for example, m a y be predictive of the likelihood that the drug will cause certain adverse effects in patients. D r u g - r e c e p t o r affinities (or their inverse, e q u i l i b r i u m dissociation constants) have comm o n l y been d e t e r m i n e d by r a d i o l i g a n d b i n d i n g assays. However, most studies have used receptors from a n i m a l tissue which has an u n k n o w n relationship to h u m a n s . Indeed, there is evidence for differences between a n i m a l s a n d h u m a n s in the w a y that their receptors b i n d drugs (for example, K a n b a a n d Richelson, 1984). Therefore, our radio-

* To whom all correspondence should be addressed. 0014-2999/84/$03.00 ~;~ 1984 Elsevier Science Publishers B.V.

a-Adrenergic receptors

ligand b i n d i n g assays have used n o r m a l h u m a n b r a i n tissue, o b t a i n e d at the time of autopsy, as the source of the several p u t a t i v e n e u r o t r a n s m i t t e r receptors from which e q u i l i b r i u m dissociation constants of the neuroleptics were determined. Here we report the results of these studies for 17 neuroleptics at the d o p a m i n e (D-2) r e c e p t o r (6 of which were studied at the D-2 r e c e p t o r of both the c a u d a t e nucleus a n d the nucleus a c c u m b e n s ) a n d for 15 neuroleptics at m u s c a r i n i c acetylcholine, histamine H i, c~I- a n d a2-adrenergic receptors.

2. Materials and methods 2.1. Tissue preparation N o r m a l h u m a n b r a i n (see Section 2.3) tissue was o b t a i n e d at the time of a u t o p s y a n d was stored in a liquid nitrogen refrigerator until it was h o m o g e n i z e d in 10 vol. of ice-cold 50 m M T r i s - H C l

198

buffer (pH 7.7 at 25°C) using a Brinkman Polytron PT-45 (45 s, setting 8). The homogenate was centrifuged at 38000 × g for 10 min. The pellets were resuspended in fresh Tris-HC1 buffer and centrifuged again at 38000 × g. The final pellets were resuspended in 50 mM Na-K phosphate buffer (pH 7.4 at 25°C) and the suspension was diluted to a volume which would provide the appropriate amount in 0,75 ml aliquots (table 1). The homogenate was stored at - 3 3 ° C. 2.2. Receptor assays

Into glass culture tubes (12 × 75 ram) was placed the tritiated ligand (0.05 ml), drug solution (0.2 ml) and buffer (0.2 ml). The solution was stirred using a vortex mixer. Homogenate (0.75 ml) was added and the suspension was again stirred using a vortex mixer. The final concentrations of labelled ligands and of homogenates are given in table 1. Duplicate samples were incubated at 37 ° C for 1 h and then the suspensions were filtered under vacuum using Whatman G F / B filters (24 mm diameter). The tubes and filters were rinsed with 3 x 5 ml of ice-cold 50 mM Na-K phosphate buffer. The filters were placed in 6 ml plastic mini-vials (Research Products International) and 4 ml scintillation fluid (Safety Solve, Research Products International) was added. After the samples stood 5 h, the radioactivity was measured using either an Isocap 300 or a Beckman LS 7800 liquid

scintillation counter at an efficiency of 30% or 35%, respectively. The specific bound was defined as the total amount bound (zero unlabelled ligand) minus the nonspecific amount bound (1 10 ttM ligand, table 1). With the use of a Hewlett-Packard 9845B computer connected by a telephone modem to a Cyber computer (Control Data Corp., Minneapolis), we analyzed the data using the computer program L I G A N D (Munson and Rodbard, 1980) to provide values for the equilibrium dissociation constant, KD. We have modified this program to give us the Hill coefficient (nn) as well. The mean K D and n n values ( +_the standard error of the mean, S.E.M.) in tables 2 through 6 arc from at least 3 independent experiments each determined in duplicate. 2.3. Normal human brain tissue

Parts of brains from 19 individuals (5 females, 14 males) were used to complete this study over about two and one-half years. Each piece of tissue was homogenized separately and used in a series of experiments. The neuropathologist grossly judged the brains to be normal except for arteriosclerotic changes in the older ones. We requested and received brains from individuals who had no neuropsychiatric disease prior to death. This was possible since our institution does about 800 atopsies per year. Autopsies were performed (mean _+ S.E.M.) 4.8 _+ 0.4 h (range 2.3-10.7 h) after

TABLE 1 P a r a m e t e r s used in r a d i o l i g a n d b i n d i n g assays. ~' Creese et al. (1977); t, W a s t e k and Y a m a m u r a (1978): " K a n b a and Richelson (1984); a H o r n u n g et al. (1979); e Perry and U ' P r i c h a r d (1981). Receptor

Labelling of receptor 3 H - C o m p o u n d Specific

Final

activity Conc. ( C i / m m o l ) (nM)

Range of E s t i m a t i o n of

H u m a n brain

specific bound

Non-specific b o u n d

tissue used

Compound

Region

(cpm)

Final Range cone (/zM) (cpm)

D o p a m i n e ( D - 2 ) Spirerone ~ Muscarinic QNB b H i s t a m i n e H~ Doxepin"

27.6 26.8 76.8

0.16 0.18 0.11

ot1

Prazosin a

20,2

0.13

260-399 d-Butaclamol 1 748--1285 A t r o p i n e 10 561-881 d - C h l o r p h e n - 10 iramine 353-645 P h e n t o l a m i n e 10

~x

R a u w o l s c i n e e 87.4

0.37

394 1009 Y o h i m b i n e

1

(Total

volume)/ (tube (rag wet w t ) / (tube) (mid

5 0 - 1 1 8 C a u d a t e 1.5 2 5 - 7 2 C a u d a t e 0.7 108-211 Frontal 15.0 cortex 46-91 Frontal 7.5 cortex 2 0 7 - 4 3 2 Frontal 10.0 cortex

1.00 1.00 1.00 1.00 1.00

199

death. The m e a n _ S.E.M. age of the individuals was 61 + 4 years (range 2-82 years). The causes of death were as follows: myocardial infarction (4); respiratory distress (4); hemorrhage (4); secondary to surgical procedures (3); trauma (2); and carcinomatosis (2). Most were on some medications prior to death. Most commonly, these were cardiovascular drugs (6 were on diuretics, antihypertensives, digoxin a n d / o r nitroglycerin). The psychotropic drugs used were opiates and benzodiazepines. In addition, one patient had been on the histamine H 1 antagonist hydroxyzine but the brain parts from this individual were used for studying dopamine receptors.

2.4. Drugs [3H]Prazosin was from Amersham (Arlington Heights, IL); [3H]rauwolscine, [3H]doxepin, [3H]quinuclidinyl benzilate ([3H]QNB) and [ 3 H ] spiperone from New England Nuclear (Boston, MA); prazosin and thiothixene from Pfizer Inc. (New York, NY); phentolamine from Ciba Geigy (Summit, N J); spiperone from Janssen Pharmaceutica Inc. (New Brunswick, N J); mesoridazine from Boehringer Ingelheim Ltd. (Ridgefield, CT); chlorpromazine from Sigma Chemical Co. (St. Louis, MO); thioridazine and clozapine from Sandoz Pharmaceuticals (East Hanover, N J); promazine from Wyeth Laboratories (Philadelphia, PA); haloperidol from McNeil Pharmaceutical (Spring House, PA); fluphenazine from E.R. Squibb & Sons, Inc. (Princeton, N J); perphenazine from Schering Corp. (Kenilworth, N J); prochlorperazine and trifluoperazine from Smith Kline & French Laboratories (Philadelphia, PA); loxapine from Lederle Laboratories (Wayne, N J); dbutaclamol from Ayerst Laboratories (New York, NY) and molindone from Dupont Pharmaceuticals (Wilmington, DE).

3. Results

3.1. Dopamine (D-2) receptor In human brain caudate nucleus, spiperone was the most potent drug studied at the D-2 receptor

~o

0.09 /f o.o7

~ ~ '•

x Spiperone ' r , w o Perphenazine Trifluoperazine Prochlorperazine Mesoridazine

'

' i

8

7

0.03 I

O.Ol 11

10

9

t

I

1

6

5

4

I

-Log[total ligand ](molarity) Fig. 1. Competition by neuroleptics for [3H]spiperone binding to the dopamine (D-2) receptor of human brain caudate nucleus. This graph which presents the results of one experiment was generated by computer with the use of the program LIGAND (Munson and Rodbard, 1980). The final concentration of radioligand was 0.16 nM and the concentrations of unlabelled compounds were varied as indicated. with a K D = 0.16 + 0.02 (n = 27) and a Hill coefficient essentially equal to unity. Thus, spiperone bound with high-affinity to a single class of sites in this part of the human brain. Competition for

TABLE 2 Neuroleptics: equilibrium dissociation constants (KD'S) for the dopamine (D-2) receptor of h u m a n brain caudate nucleus. "-+ S.E.M. Neuroleptics

K D (nM) *

Hill coefficient *

Spiperone cis-Thiothixene Fluphenazine d-Butaclamol Perphenazine Trifluoperazine Triflupromazine Haloperidol Prochlorperazine Chlorprothixene Mesoridazine Chlorpromazine Thioridazine Loxapine Molindone Promazine Clozapine

0.16+ 0.02 0.45 _+ 0.08 0.8 + 0.1 0.86_+ 0.06 1.4 + 0.2 2.6 -+ 0.3 2.8 -+ 0.5 4 _+ 1 7 _+ 1 8 ± 2 19 -+ 3 19 + 2 26 + 8 70 -+ 10 120 -+ 40 160 _+ 5 180 _+ 5

0.98_+0.03 0.89_+ 0.07 0.88_+0.07 1.1 _+0.2 1.0 _+0.1 0.80-+0.09 0.9 _+0.1 1.0 _+0.1 0.91 -+0.03 1.03_ 0.05 0.82-+0.09 1.0 -t-0.2 1.10_+0.04 0.87 -+ 0.04 0.97 __+0.05 0.94__+0.02 0.96_+0.03

200 ........ r=O 8 0 ~" ~'O ~

100

I

........

I

P=O 0 0 0 5 SJope=O 9 ± 0 2

........

I

'

.......

07

1

06

CLOZAPJNE • • MOLINDON~ PROMAZINE LOX APINE I*

~ ~.)~) ~)E ~ ~

10

D ~ E

10

__

MESQRIDA

THIORIOAZINE

E/

• C~tLORPROMAZINE~ C

PROCHLORPERAZIN HALOPERIOOL t

CHLORPROTHIXENE

TRIFLUOPERAZlNE d BUTACLAMOL •

I

il~ ~ \ ~

O5

P. o4 -~

• TRIFLUPROMAZINE

• PERPHENAZINE • FLUPHENAZINE

I'

I

1

[

* QNB o Thioridazine o Uesoridazine * Promazine

~, L~,~ ~ ; r i f r l ; 2 2 2 r a i z ' 2

~o3

e

o

~3

o2 01

.E I

01

, . I SP,IIPIIERONE . . . . . . I 01

10

........

,

........

10

,

1

1130

Average clinical daily dose for treating schizophrenia (/.~nol/kg) Fig. 2. Relationship between neuroleptic K I3'S at the dopamine (D-2) receptor of human brain caudate nucleus and average daily dose for treating schizophrenia. The equation for this regression is y = 0.48 _+_0.92x (r = 0.80: P = 0.0005).

[-~H]spiperone binding sites by other neuroleptics (for example, fig. 1), yielded a broad range of K l~'s (table 2). Hill coefficients for all compounds were essentially equal to one. Spiperone was more than 1000-fold more potent than the weakest compound, clozapine. For the seventeen compounds, there was a significant correlation (r = 0.80, P = 0.0005) between the log of the KD's and the log of the average daily doses (/~mol/kg) for treating schizophrenia (fig. 2). There was no significant difference in their respective K D'S for 6 compounds (spiperone, thiothixene, fluphenazine, haloperidol, thioridazine and molindone) studied at the D-2 receptor in both the caudate nucleus and the nucleus accumbens (data not shown). The assays with the nucleus accumbens required up to 3 times as much tissue per tube as in the assays with caudate nucleus to compensate for the lower D-2 receptor concentration (about one-third that of the caudate) in this area of the brain.

3.2. Muscarinic acetyleholine receptor The antimuscarinic radioligand [3H]QNB was by far the most potent c o m p o u n d studied with a K D = 44 4-_ 5 pM (n = 23). Competition between [ 3 H ] Q N B and 15 neuroleptics (for example, fig. 3),

10

I

~

7 6 5 4 Log [total ligand] (molarity) 9

8

3

2

Fig. 3. Competition by neuroleptics for [~H]quinuclidinyl henzilate ([3QNB]) binding lo the muscarinic acetylcholine receptor of human brain caudate nucleus. This graph presents the results of one experiment analyzed as described in tile legend to fig. 1. The concentration of [)H]QNB was 0.18 nM and the concentrations of the neuroleptics were varied as indicated. Although ( + ) Q N B was used to compete with [)H]QNB, we assumed that only the (-)-form was active.

showed that the most potent neuroleptic at muscarinic receptors of human brain caudate nucleus was clozapine, the weakest molindone (ta-

TABLE 3 Neuroleptics: equilibrium dissociation constants (K D's) for thc muscarinic acetylcholine receptor of human caudate nucleus. * +S.E.M. K ~)(nM)

*

Hill coefficient *

Neuroleptics Clozapine Thioridazine Mesoridazine Chlorpromazine Promazine Loxapine Prochlorperazine Trifluoperazine Perphenazine Fluphenazine Spiperone cis-Thiothixene d-Butaclamol Haloperidol Molindone Antimuscarinics QN g Atropine

12 + 4 18 + 1 69 +2 70 +_ 6 150 +30 450_+ 80 540+ 120 660 + 40 1,500 + 30 1,900 ± 5(10 2,700 + 800 2,900_+ 100 12,000+ 3,000 24,000_+ 9,000 390,000+90,000

0.95 + 0.06

0.044 _+0.005 2.4 +_0.6

1.02 + 0.04 1.1 +0.1

1.05 + 0.01 1.0 +_0.2 1.1 +-0.2 1.32 ± 0.08 1.01 +- 0.09 1.2 _+0.1 1.4 +0.2 0.7 +-0.1 1.3 +_0.1

201

ble 3). The Hill coefficients (where obtained) for these compounds were about equal to unity for all but the weakest compounds (table 3). The meaning of n ~ ' s significantly different from one for compounds that have KD'S greater than 105 times that of the radioligand is uncertain. The correlation between the log of the K D'S and the log of the average daily doses showed a significant negative correlation ( r = - 0 . 5 9 , P = 0.019 for all 15 compounds; r = - 0 . 8 4 , P = 0.0005 when molindone was omitted from the list).

3. 3. Histamine Hj receptor Initially, we used [3H]pyrilamine (Hill et al., 1978) to study the histamine H~ receptor of human brain frontal cortex, but later we used [3H]doxepin because of its higher affinity (Kanba and Richelson, 1984) and specific activity. For pyrilamine (mepyramine) the K D and n H determined in 11 independent experiments were 5.0_+ 0.9 nM and 0.70 ± 0.03, respectively; for doxepin in 27 experiments, these values were 0.24 ± 0.02 nM and 0.99 __+0.04, respectively. Neuroleptics competitively antagonized the binding of these radioligands (for example, fig. 4) and many were found to be potent histamine H 1 receptor antagonists (table 4).

I

r

I

I

~X,

013

\

'k~ 0.11

"0

~

r

I

x Doxepin _ o Trifuoperazine o d-Butaclamol A Promazine

kk.

0.09

t-'l

mO 0.07

005

10

l

1

9

8

7

l

I

6

5

Neuroleptics: equilibrium dissociation constants (K ~)*s) for the histamine H~ receptor of human brain frontal cortex. * _+S.E,M.

Neuroleptics Mesoridazine Promazine Clozapine Loxapine cis-Thiothixene Perphenazine Chlorpromazine Thioridazine Prochlorperazine Fluphenazine Trifluoperazine d-Butaclamol Spiperone Haloperidol Molindone Am±histamines d-Chlorpheniramine

K D (nM) *

Hill coefficient *

1.8_+ 0.1 2.0_+ 0.1 2.8 ± 0.8 4.9+ 0.8 6 _42 8 ± 1 9 ± 3 16 ± 3 19,0± 0.2 21 ± 4 62 _+ 7 390 ± 70 480 i 70 1 900 -+ 300 124000 -+12000

0,66_+0.09 0,7 +0.2 1.03 _+0.04 0.71 ±0.03 0.9 4__0.1 1.2 -+ 0.2 1.0 ±0.2 0.97±0.06 0.8 0 . 1 0.9 _+0.2 1.3 _+0.3 1.00-_t__0,07 1.08 ±0.05 0.77±0.05 0.53 ± 0.07

15

_+

2

0.85 ±0.06

Mesoridazine was the most potent of this class of drugs. This compound and promazine were two potent compounds with n H values less than one, suggesting heterogeneity of binding or negative cooperativity in binding. The correlation between the log of the K D'S and the log of the daily doses was not significant (r = -0.41, P = 0.124) except when molindone was removed from the list; then the negative correlation was significant (r = - 0.74, P = 0.003).

3. 4. %-Adrenergic receptor

o

0.03

TABLE 4

4

-LogEtotal ligand'l(molarity) Fig. 4. Competition by neuroleptics for [3HJdoxepin binding to the histamine H I receptor of human brain frontal cortex. This graph presents the results of one experiment which was analyzed as described in the legend to fig. 1. The concentration of [3Hldoxepin was 0.11 nM and the concentrations of unlabelled compounds were varied as indicated.

The radioligand [3H]prazosin used for a~-receptor binding to human brain frontal cortex had a Kt) = 0.09_+ 0.01 nM (n = 31) and a n , = 0.98 + 0.02. Neuroleptics were competitive antagonists of this radioligand (for example, fig. 5) and most were relatively potent (table 5). Hill coefficients for all compounds studied were essentially equal to unity (table 5). Spiperone which was most potent as a D-2 antagonist (table 2) was also most potent as an aj-receptor antagonist. Molindone was

202 03

l

I

I

. • ~ ~ \, ~x " ~ ~ " ~

0,2

I

[

TABLE 6

x Prazosin ,, Mesoridazine o Clozapine • Promazine " " Prochlorperazine

Neuroleptics: equilibrium dissociation constants (K i)'s) for the a2-adrenergic receptor of human brain frontal cortex. * ± S,E.M. KD (nM) *

Hill coefficient *

160 200 310 510 640 660 750 800 900 1 550 1 600 1 700 2400 2 600 3 800

0.98+_0.02 1.1 +0.2 (/.96_+0.06 1.10+_0.08 0.91 ±0.05 0.89+0.04 1.33-+0.02 1.23 + 0.06 1.00 ± 0.02 0.86 ±0.04 0.70-+0.03 0.97 + 0.05 0.91 +0.05 1.33 _+0.07 1.23 ± 0.07

"0 t--

2~

O m

O'

0

I

10

[

1

I

I

9 8 7 6 5 -Log[total ligand](rnolarity)

4

Fig. 5. Competition by neuroleptics for [3H]prazosin binding to the al-adrenergic receptor of human brain frontal cortex. This graph presents the results of one experiment which was analyzed as described in the legend to fig. 1. The concentration of [3H]prazosin was 0.13 nM and the concentrations of unlabelled drugs were varied as indicated.

w e a k e s t . T h e r e w a s n o c o r r e l a t i o n b e t w e e n t h e log o f t h e K ~ ' s a n d t h e log o f t h e a v e r a g e d a i l y d o s e s

Neuroleptics Clozapine cis-Thiothixene d-Butaclamol Perphenazine Molindone Spiperone Chlorpromazine Thioridazine Promazine Fluphenazine Mesoridazine Prochlorperazine Loxapine Trifluoperazine Haloperidol Antiadrenergic Yohimbine Rauwolscine

+ 20 ± 20 + 40 + 20 .+ 100 ± 20 ± 50 4 100 ± 100 + 20 -+ 100 + 100 -+600 + 200 + 400

1.6 +_ 0.2 2.7+ 0.2

1.04 ± 0.08 0.96-+_:0.03

(r = - 0.005, P = 0.99).

TABLE 5

3.5. a:-Adrenergic receptor

Neuroleptics: equilibrium dissociation constants (KD's) for al-adrenergic receptor of human brain frontal cortex. * -+ S.E.M. K D(nM) * Neuroleptics Spiperone Mesoridazine Chlorpromazine Thioridazine Promazine Haloperido[ Clozapine Fluphenazine Perphenazine cis-Thiothixene Prochlorperazine Trifluoperazine Loxapine d-Butaclamol Molindone Ant±hypertensives Prazosin Phentolamine

Hill coefficient *

In h u m a n b r a i n f r o n t a l c o r t e x , [ 3 H ] r a u w o l s c i n e h a d a K D = 2.7 + 0.2 (n = 27) a n d a nH = 0 . 9 6 ± 0.03. T h i s r a d i o l i g a n d w a s c o m p e t i t i v e l y a n t a g o n i z e d b y n e u r o l e p t i c s , H o w e v e r , in g e n e r a l t h e s e r compounds

1.2 2.0 2.6 5 6 6.1 9 9 10 11 24 24 28 56 2500

.+ 0.2 ± 0.5 +- 0.3 _+ 1 ± 2 -+ 0.8 -+ 3 .+ 2 ± 2 +± 1 -+ 7 ± 3 ± 6 _+ 8 -+600

0.09 ± 15 +

0.01 4

0.82.+0.03 1.1 ±0.1 0.97 ± 0.08 1.1 .+0,l 0.82 .+ 0.04 0.81 .+0.07 0.90 ± 0.05 1.02 _+0.06 1.10 ± 0.04 0.9 -+0.1 1.10.+0.05 1,0 +0.1 0.9 _+0.1 0.9 ±0.1 0.71 _+0.07 0.98 _+0.02 0.82-+0.03

were

relatively

weak

a2-receptor

a n t a g o n i s t s ( t a b l e 6). In a d d i t i o n , t h e K D ' s for t h e s e d r u g s fell w i t h i n a r e l a t i v e l y n a r r o w r a n g e w i t h less t h a n a 2 5 - f o l d d i f f e r e n c e in K D ' s b e tween the drug with the highest affinity (clozapine) and the one with the lowest affinity (haloperidol). T h e r e w a s n o c o r r e l a t i o n b e t w e e n t h e log o f t h e K D ' s a n d t h e log o f t h e d a i l y d o s e s ( r = P = 0.81),

-0.07,

4. Discussion

This p a p e r presents the data of the b i n d i n g p o t e n c i e s o f a l a r g e series o f n e u r o l e p t i c s at 5 d i f f e r e n t r e c e p t o r sites in h u m a n b r a i n tissue, namely, d o p a m i n e (D-2), m u s c a r i n i c acetylcholine,

203 histamine H~, a~- and a2-adrenergic receptors. As a group their most potent interaction was at D-2 receptor sites. In confirmation of results from others (Reynolds et al., 1982; Seeman and Ulpian, 1983) who used human brain tissue, we found no difference in the respective affinities of these drugs for the D-2 receptor of the nucleus accumbens compared to the D-2 receptor of caudate nucleus. Thus, there is no site specificity for these drugs at the D-2 receptor. The equilibrium dissociation constants for neuroleptics and the D-2 receptor of human caudate nucleus were significantly correlated with their average daily dose for treating schizophrenia (fig. 2), a finding which has been reported by others (Creese et al., 1976: Seeman et al., 1976) for neuroleptics and the D-2 receptor from animal brains. We also found correlations, although negative, between the daily doses and the K D's for the muscarinic receptor and the K t)'s for the histamine H~ receptor, especially after the outlier molindone was excluded from the list. These correlations most likely reflect the fact that the potencies of neuroleptics at blocking the D-2 receptor were inversely correlated with their potencies at blocking both the muscarinic receptor (r = - 0 . 7 5 , P = 0.002, n = 14) and the histamine H~ receptor (r = - 0.61, P = 0.02, n = 14). A corollary of these facts is that the antimuscarinic and antihistaminic potencies were significantly correlated for all 15 neuroleptics studied ( r = 0 . 8 4 , P=0.0001). It is well known that antihistaminic drugs frequently have antimuscarinic properties too (Marshall, 1955). We could compare our KD's for 10 compounds at human brain D-2, histamine H~ and a~-adrenergic receptors with those for rat brain obtained by another group of workers (Peroutka and Snyder, 1980). There was a significant correlation between our data and those of Peroutka and Snyder (1980) for the D-2 (r = 0.96, P = 0.0001) and histamine H~ (r = 0.97, P = 0.0001) receptors but not for the a~-adrenergic receptor (r = 0.58, P = 0.08). Despite these excellent correlations for neuroleptics at the D-2 and histamine H t receptors, the values were not identical since we consistently obtained lower KD's (i.e. higher affinities) for all 10 compounds in common at these two receptor sites. The lack of

a significant correlation between our data for the a~-receptor of human brain and those of rat brain could be due to a number of different factors, including the use in our studies of an apparently more selective radioligand ([-~H]prazosin) for the o~-receptor. There have been few reports of K t)'s for neuroleptics at muscarinic receptors of animal tissue (Miller and Hiley, 1974; Richelson and DivinetzRomero, 1977). Once again, our data obtained using human brain tissue provided, in general, higher affinities for these drugs than those reported previously for muscarinic receptors from animal tissue. We could find no report of K D's for neuroleptics at a2-receptors. However, after deriving inhibitor constants from the ICso's reported by Perry et al. (1983) for neuroleptics at a2-receptors of bovine caudate nucleus, we found similar affinities and a significant correlation ( r = 0.76; P = 0.006) for the 11 compounds in common between these studies. The antagonism by drugs of D-2, muscarinic, histamine H~ and a~-adrenergic receptors can cause a number of different adverse effects in patients (Richelson, 1981). For example, D-2 receptor blockade can lead to extrapyramidal side effects such as a parkinsonian-like state and tardive dyskinesia; muscarinic receptor blockade can cause dry mouth and constipation and can reverse some of the extrapyramidal side effects of neuroleptics (Miller and Hiley, 1974); histamine H~ receptor blockade can cause sedation; and al-receptor antagonism can cause postural hypotension. A side effect associated with az-receptor blockade is not known but this antagonism can reduce the effectiveness of antihypertensive drugs (for example, clonidine) which are thought to act by stimulating these receptors. The rankings of these drugs (tables 2-6) as antagonists of these receptors therefore can be used clinically to predict the likelihood that these drugs will cause certain adverse effects in patients.

Acknowledgements We thank Doctor H. Okazaki, Department of Neuropathology, Mayo Clinic, for supplying us with the human brain tissue and the many drug companies for supplying us with their

204 compounds. This work was supported by a grant from Dupont Pharmaceuticals and by the Mayo Foundation.

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