Specific in vivo binding of neuroleptic drugs in rat brain

BiochemicalPharmacology. Vol. 26, pp. 1003 1007. PergamonPress, 1977, Printedin Great Britain.

PRELIMINARY COMMUNICATION

SPECIFIC

IN VIVO

BINDING

OF

NEUROLEPTIG

DRUGS

IN RAT

BRAIN

P. Laduron and J. Leysen Department of Biochemical Pharmacology, Janssen Pharmaceutica, B-2340 Beerse, Belgium (Received 24 February 1977; accepted 24 February 1977}

Neuroleptic

d r u g s a r e k n o w n to i n c r e a s e

ganglia i and mesolimbic have been interpreted

system

in t e r m s

dopamine

2 b u t a l s o in t h e m e s o c o r t i c a l

of b l o c k a d e o f d o p a m i n e

drugs can inhibit dopamine-sensitive

f i c a l l y b i n d to r e c e p t o r s

in d o p a m i n e r g i c

the enzyme cellular

the hypothesis

regions

from rat striatum

9,

investigations macological

w a s to e x a m i n e

prototype

rats

o r 3H p i m o z i d e

travenous

injection.

removed

and various

(250 g) w e r e

areas

were

dissected.

was measured

B o t h d r u g s w e r e f o u n d to be m o r e

as two possible targets of t h e p r e s e n t

i n v o l v e d in t h e p h a r -

derivative

and pimozide,

earlier

A s s h o w n i n t a b l e 1, t h e d i f f e r e n c e

areas

k n o w n to c o n t a i n d o p a m i n e r g i c and brain remainder)

instance,

the cerebellum.

contain more

the

dopamine

higher retention

a n d of 3H p i m o z i d e

closely parallels

in in v i t r o c o n d i t i o n s .

r e g i o n is d i r e c t l y 3H s p i p e r o n e

pronounced 3H s p i p e r o n e

a higher retention

than the parietal

has recently

related

oxidi-

in t h e r a t b r a i n . areas

of t h e r a t

a n d 3H p i m o -

side,

drugs.

in t h e n u c l e u s a c c u m b e n s

treatment

4

The substantia

11, a l s o a p p e a r e d distribution

receptor

areas

sites

displaced

to p o s s e s s

nigra the

of 3H s p i p e r o n e

binding sites when

that the different retention

1003

ce-

t h a n in

c a p a c i t y in t h e d o p a m i n e r g i c

The regional

of r e c e p t o r

w a s a l s o f o u n d to b e s p e c i f i c a l l y

cortex,

than with pimozide.

and this may explain the much higher HVA

t h a t of t h e n e u r o l e p t i c

This suggests

parietal

with spiperone

t h a t t h e f r o n t a l c o r t e x w a s f o u n d to

been demonstrated

to t h e n u m b e r

were

spectrometer.

and the other ones (e.g.

It is n o t e w o r t h y

capacity for neuroleptic

by in-

Brains

in a t i s s u e s a m p l e

a n d 3H p i m o z i d e

t h a n in the l a t t e r a f t e r h a l o p e r i d o l

release

9 Ci/

in t h e l a b e l l e d d r u g c o n t e n t b e t w e e n t h e b r a i n

terminals

u s i n g 3H p i m o z i d e .

3H s p i p e r o n e

combusted

t a k e n u p in t h e d o p a m i n e r g i c

was much more

Although less marked,

in t h e f o r m e r

Pharmaceutica)

f i n d i n g s 10 in t h e d o g w h e n u s i n g 3H h a l o p e r i d o l

t h e r e w a s a b o u t 10 t i m e s m o r e

was also observed

Janssen

w e r e k i l l e d by d e c a p i t a t i o n .

They were

specifically

zide.

measured

direct

Since

in d i f f e r e n t s u b -

The purpose

are really

in a l i q u i d s c i n t i l l a t i o n

thus confirming

where

receptors.

( 0 . 0 0 5 m g . k g "l s p e c . a c t .

13 G l / m m o l

of 3H s p i p e r o n e

brain,

increase

6 and speci-

a n in v i v o a s s a y w a s d e v e l o p e d u s i n g

g i v e n 3H s p i p e r o n e

the a n i m a l s

Table 1 shows the regional distribution

For

effects.

a butyrophenone

( 0 . 0 2 m g . k g -1 s p e c . a c t . Two hours later,

zer and the radioactivity

rebellum

Therefore,

; spiperone,

The recent

cyclase

of t h e d i p h e n y l b u t y l p i p e r i d i n e s . Male Wistar

mmol

drugs

drugs.

r e f . 5).

to be l o c a l i z e d

to w h a t e x t e n t t h e s e r e c e p t o r s

a c t i v i t y of n e u r o l e p t i c

two different neuroleptic

and behavioral

These effects

have prov!ded more

t h e y m i g h t be c o n s i d e r e d

c a p a b l e of e l i c i t i n g d i f f e r e n t b i o c h e m i c a l

3,4

drugs block dopamine

and the specific binding sites have been reported

structures

(cfr.

adenylate

of t h e b r a i n 7 , 8 ,

that neuroleptic

n o t o n l y in b o t h t h e b a s a l

system

receptors

findings that neuroleptic

e v i d e n c e to s u p p o r t

turnover

c a p a c i t y of a g i v e n

involved. by a l a r g e r

dose.of unlabelled

corn-

Preliminary Communication

IO04 T a b l e 1.

R e g i o n a l d i s t r i b u t i o n of l a b e l l e d d r u g s in r a t b r a i n 2 h o u r s a f t e r i . v .

i n j e c t i o n of

3H s p i p e r o n e ( 0 . 0 0 5 m g . k g -1) a n d 3H p i m o z i d e ( 0 . 0 2 m g . k g - 1 ) .

p g . m g -I Jr S E M

p g . m g -1 +_ S E M (n=5)

(n=6)

Pimozide

Brain r e g i o n

Spiperone

Nucleus a c c u m b e n s

2.79_+ 0.38

Striatum

2.50 _+ 0.15

7.63 4- 0.49

Substantia nigra

1.71 +_ 0.30

4.11 _+ 0.19

Tuberculum

i. 59 _+ 0.08

5.06 +_ 0.37

olfactorium

6.36 + 0.19

Frontal cortex

1.02 + 0.05

4.27 +_ 0.19

Parietal cortex

0.74 _+ 0.06

4.05 _+ 0.19

Cerebellum

0.29 _+ 0.02

2.65 + 0.25

Rest of the brain

0.77 _+ 0.06

3.35 + 0.21

pound in the d o p a m i n e containing areas. much

more

Indeed fig.1 shows that labelled spiperone decreased

rapidly in the striatum w h e n unlabelled spiperone was injected one hour after the

radioactive drug.

In contrast to this, no displacement w a s detectable in the cerebellum.

other dopaminergic areas such as the nucleus a c c u m b e n s ,

the tuberculum olfactorium and

STRIATUM I

2

~

8

16

HOURS AFTER 3H SPIPERONE

Fig. I.

In vivo displacement of 3 H spiperone (0.005 m g . k g -I i.v.) in rat

striatum and cerebellum. removed

In the first group ( H

), rat brains w e r e

at different time intervals after injection of labelled spiperone.

In the second group ( O----
The hatched part

E a c h point is the m e a n

of 4 deter-

Significant differences f r o m control values are indicated by ~

p~.001.

(P : student t-test)

In

Preliminary Communicati0n e v e n , but to a l e s s e r

extent,

the f r o n t a l c o r t e x ,

1005

we have o b s e r v e d a s i m i l a r

displacement

a f t e r a l a r g e d o s e of u n l a b e l l e d d r u g u s i n g e i t h e r 3H s p i p e r o n e o r 3H p i m o z i d e (in p r e p a r a tion).

Thus,

dopaminergic

only those molecules structures

bound aspecifically,

w h i c h a r e s p e c i f i c a l l y b o u n d to the r e c e p t o r s

can be d i s p l a c e d f r o m t h e i r b i n d i n g s i t e s w h e r e a s

of the

those which are

cannot.

A t h i r d l i n e of e v i d e n c e f o r s p e c i f i c in v i v o b i n d i n g o f n e u r o l e p t i c d r u g s w a s p r o v i d e d by the s t u d y o f the s u b c e l l u l a r l o c a l i z a t i o n of 3H s p i p e r o n e in r a t s t r i a t u m samples

and cerebellum.

Brain

w e r e f r a c t i o n a t e d b y d i f f e r e n t i a l c e n t r i f u g a t i o n a c c o r d i n g to the a n a l y t i c a l f i v e -

fraction procedure thus obtained,

12, 13

In o r d e r to a s s e s s

several marker

t h e c o m p o s i t i o n of t h e s u b c e l l u l a r f r a c t i o n s

enzymes were determined

a c c o r d i n g to m e t h o d s p r e v i o u s l y

described 12, 13 Fig.2 shows the distribution pattern of 3 H spiperone in fractions obtained f r o m rat striatum and cerebellum homogenates.

Ninety percent of the radioactivity w a s recovered

in particulate fractions in the striatum against only 49 percent in the cerebellum. r o n e w a s m a i n l y f o u n d to h a v e b e e n e n r i c h e d striatum

but n o t in t h e c e r e b e l l u m

the s t r i a t u m when assayed

closely parallels

(7 %).

in the P ( m i c r o s o m a l )

in in v i t r o c o n d i t i o n s .

f r a c t i o n (42 ~o) in the

It is n o t e w o r t h y t h a t the 3H s p i p e r o n e p r o f i l e in

t h a t of t h e n e u r o l e p t i c

receptor

In c o n t r a s t to t h i s ,

l y d i f f e r e d f r o m t h a t of c y t o c h r o m e

3 H spipe-

as previously reported

in b o t h r e g i o n s ,

its p r o f i l e m a r k e d

o x i d a s e ( m i t o c h o n d r i o n ) of l a c t a t e d e h y d r o g e n a s e

n a p t o s o m e ) o f N - a c e t y l - ~ -D g l u c o s a m i n i d a s e adenylate cyclase as already described

Consequently,

n o n e of t h e s e s t r u c t u r e s

CEREBELLUM CYTOCHROME OXPDASE

+[ 'I

LACTATE DEHYD~OGE NASE

i:

2!i

N. ACE "~VE-j3- O . GLUCOSAMINIDASE

,t'1

~o

$'-NUCLEOTIOASE

,I RADIOACtWlTy 2

~ o N o

M 20

L ~o

P ~

s 80 1oo

0

N o

2o

M

LP

s

~o

6o

~o

io~

PERCENTAGE OF TOTAL RECOVEPED PI~OTEINS

Fig. 2.

Distribution pattern of radioactivity and m a r k e r e n z y m e s in sub-

cellular fractions of rat striatum and cerebellum obtained by differential centrifugation, kg-l).

two hours after i.v. injection of 3 H spiperone (0.005 rag.

The absolute a m o u n t of 3 H spiperone in the starting material was

2.9 ng g-i in the striatum and 0.4 ng g-I in the cerebellum, while their distribution in percent w a s respectively 6 and 17 in N (nuclear fraction), 28 and 19 in M (heavy mitochondrial fraction), 14 and 6 in L (light mitochondrial fraction), 4Z and 7 in P ( m i c r o s o m a l fraction), I0 and 51 in S (supernatant).

(sy-

( l y s o s o m e ) a n d e v e n of d o p a m i n e - s e n s i t i v e

13.

STRtATUM

9,

seems

1006

Preliminary Communication

to be the m a i n target in vivo for neuroleptic drug activity.

In control experiments in which

labelled spiperone w a s a d d e d to the starting h o m o g e n a t e at a concentration corresponding to that n o r m a l l y found in the h o m o g e n a t e under in vivo conditions, only 18 % and 40 % of 3 H spiperone w e r e respectively r e c o v e r e d in the P a n d S fractions in the striatum.

O n the

contrary, th+
Therefore,

one m a y

conclude that the 3 H spiperone e n r i c h m e n t in the P fraction corresponds to a specific in vivo binding on neuroleptic receptors.

Moreover,

although w e cannot quite rule out that a small

a m o u n t of spiperone should also be associated with structures bearing dopamine-sensitive adenylate cyclase, the inhibition of this e n z y m e

does not s e e m to be of great importance for

the antipsychotic activity of neuroleptic drugs.

W e believe that the inhibition of the d o p a m i -

he-sensitive adenylate cyclase is only involved in the m e c h a n i s m

of action of neuroleptic

drugs e n d o w e d with sedative effects. T h r o u g h o u t the present work,

radioactivity w a s always considered either as u n c h a n g e d spi-

perone or as u n c h a n g e d pimozide.

Metabolic studies have s h o w n that 2 hours after intrave-

nous injection 90 percent of 3 H spiperone and 3 H pimozide w e r e found in the rat brain as unchanged drug 14.

M o r e o v e r the a m o u n t of pimozide metabolites per mg.tissue w a s similar

in all the brain regions for 24 hours after injection,

T h e s e metabolites w e r e never found

to have been.particulate-bound but well contained in the supernatant fraction (J. Heykants ; unpublished results). Spiperone s e e m s to be particularly appropriate for such in vivo studies ; indeed, recent in vitro binding experiments have s h o w n that, w h e n c o m p a r e d

to haloperidol, spiperone has a

slower dissociation rate, a higher affinity for neuroleptic receptor but a lower one for aspecific sites (unpublished results).

This is also true for pimozide except that its aspecific bin-

ding w a s relatively higher, a fact w h i c h can explain w h y the retention of 3 H pimozide in the c e r e b e l l u m w a s relatively m o r e

p r o n o u n c e d than that of 3 H spiperone (table i).

In the present study, three lines of evidence w e r e provided to demonstrate a specific in vivo binding for neuroleptic drugs.

First, these drugs w e r e specifically taken up in

the d o p a m i n e r g i c areas of the brain so that the higher retention capacity in these regions, w h e n c o m p a r e d to that in non d o p a m i n e r g i c areas, an index for specific in vivo binding.

However,

(e.g. the cerebellum), might be taken as

specific distributions are only possible if a

very low dose of the drug is u s e d in a w a y that the high affinity specific binding is p r e d o m i nant, thus not m a s k e d

by the low affinity aspecific binding 15.

Secondly, larger doses of un-

labelled drug could displace the labelled drug in vivo only in the d o p a m i n e r g i c areas.

Recent

results have s h o w n that a p o m o r p h i n e a n d aminotetralin derivatives can also be u s e d in these displacement experiments, a fact w h i c h provides further evidence concerning the d o p a m i n e r gic nature of the neuroleptic receptor.

Thirdly, specificity for this in vivo binding w a s fur-

ther a s s e s s e d at the subcellular level since the labelled neuroleptic drug w a s found to be specifically enriched in the m i c r o s o m a l

fraction of the d o p a m i n e containing areas.

T h e present results support the view that, in vivo, the neuroleptic receptor m a y a more

important target for neuroleptic drugs than dopamine-sensitive adenylate cyclase.

This work was technical

partially

assistance.

supported

by a grant

from

IRSIA.

We t h a n k P . F . M .

Janssen

for

be

Preliminary Communication

1007

REFERENCES

1.

A. C a r l s s o n and M. L i n d q v i s t , A c t a P h a r m a c o l .

(Kbh)ZO, 140-144 (1963).

2.

N.E. And4n, J.Pharm. P h a r m a c .

3.

B. Scatton, J. Glowinskiand L. Julou, Brain Res. I09, 184-189 (1976).

4.

P. Laduron, K. De Bie and J. Leysen, Naunyn-Schmiedeberg's Arch, Pharmacol.

Z44, 905-906 (1972).

z96, 183-185 (1977). 5.

L.L.

I v e r s e n , S c i e n c e 188, 1084-1089 (1975).

6.

J . W . Kebabian, G . L . P e t z o l d a n d P .

7.

I. C r e e s e , D . R . B u r t and S . H . S n y d e r , L i f e Sci. 17, 993-1002 (1975).

8.

P. S e e m a n ,

Greengard,

Proc,n.atn. Acad,Sci. U.S.A.

69, 2145-2149 (1972). M. C h a n - W o n g , J. T e d e s c o and K. Wong, P r o c , n a t n , A c a d . Sci.

U . S . A . 72, 4376-4380 (1975). 9.

J . L e y s e n and P. L a d u r o n ,

10.

P.A.J.

Janssen,

Life Sci. Z_0, 281-Z88 (1977).

W. Soudijn, I. Van W i j n g a r d e n and A. D r e s s e ,

Arzneim,-Forsch.

(Drug R e s . ) 18, 261-279 (1967). 11.

L . B . Geffen, I . M . J e s s e l l , A . C . C u e l l o and L . L .

12.

P. L a d u r o n ,

M. V e r w i m p , P . F . M .

Janssen andW.

I v e r s e n , N a t u r e 260, 258-Z60. G o m m e r e n , B i o c h i m i e 57,

253-Z60 (1975). 13.

P. L a d u r o n ,

M. V e r w i m p ,

P.F.M.

J a n s s e n and J . L e y s e n , Life Sci. 18, 433-440

(1976). 14.

J. Heykants, Biological R e s e a r c h Report,

15.

J. L e y s e n , in M e m b r a n o u s e l e m e n t s and m o v e m e n t s of m o l e c u l e : t e c h n i q u e V o l . 6 ( C e l l u l a r b i o c h e m i s t r y ) (Ed. E , Reid).

Janssen Pharmaceutica,

Horwood, C h i c h e s t e r U . K .

May (1974).

in p r e s s (1977)