Life Sciences, Vol. Printed in the USA
BMY-14802
Patricia
51, pp. 1145-1149
PROTECTS AGAINST NEURONAL DAMAGE IN
C. Contreras,
CNS D i s e a s e s
N a n c y M. Gray, H. L a n t h o r n
Research,
G. D. Searle 63198
Pergamon Press
ISCHEMIA-INDUCED THE GERBIL
Diane M. R a g a n
& Co.,
a n d Thomas
Chesterfield,
MO
(Received in final form July 24, 1992) Summarv BMY-14802, a s e l e c t i v e sigma l i g a n d c u r r e n t l y u n d e r i n v e s t i g a t i o n as an a t y p i c a l a n t i p s y c h o t i c agent, was t e s t e d for p o t e n t i a l a n t i - i s c h e m i c activity. BMY-14802 (i0, 30 a n d 50 mg/kg) d i d not p r o d u c e a n y s t e r e o t y p e d behavior, ataxia or seizures. When gerbils were p r e t r e a t e d w i t h i0, 30 or 50 m g / k g of B M Y - 1 4 8 0 2 30 m i n p r i o r to b i l a t e r a l o c c l u s i o n of c a r o t i d a r t e r i e s for 5 min, B M Y - 1 4 8 0 2 s i g n i f i c a n t l y p r o t e c t e d a g a i n s t i s c h e m i a i n d u c e d n e u r o n a l loss in the h i p p o c a m p u s . Thus, BMY14802 m a y also be u s e f u l as an a n t i - i s c h e m i c agent that does not p r o d u c e p s y c h o t o m i m e t i c effects. An e x t e n s i v e s e a r c h for a n t i - i s c h e m i c a g e n t s has i n c l u d e d a n t a g o n i s t s of e x c i t a t o r y a m i n o a c i d t r a n s m i s s i o n . However, the s e a r c h has y i e l d e d few b i o a v a i l a b l e , p o t e n t a g e n t s that p r o t e c t a g a i n s t i s c h e m i a - i n d u c e d n e u r o n a l i n j u r y w i t h o u t p r o d u c i n g gross behavioral or c a r d i o v a s c u l a r effects. The dose of nonc o m p e t i t i v e a n t a g o n i s t s of N - m e t h y l - D - a s p a r t a t e (NMDA), such as MK-801 or p h e n c y c l i d i n e (PCP) required to p r o t e c t against ischemia-induced neurotoxicity also produces gross behavioral e f f e c t s such as a t a x i a and s t e r e o t y p e d b e h a v i o r (i) . An a l t e r n a t i v e class of a n t i - i s c h e m i c agents might be sigma ligands. Ifenprodil, a polyamine antagonist, is a l s o a v e r y potent sigma ligand (2,3) that is n e u r o p r o t e c t i v e (4) . If an interaction with sigma receptors mediate the n e u r o p r o t e c t i v e p r o p e r t y of i f e n p r o d i l , then o t h e r sigma l i g a n d s s h o u l d p r o t e c t a g a i n s t i s c h e m i a - i n d u c e d n e u r o n a l damage. Consistent with this h y p o t h e s i s is the f i n d i n g that d e x t r o m e t h o r p h a n , w h i c h b i n d s w i t h high affinity to s i g m a sites and with low affinity to PCP r e c e p t o r s (5), is n e u r o p r o t e c t i v e (6). However dextromethorphan is q u i c k l y m e t a b o l i z e d to d e x t r o r p h a n , a potent PCP ligand, w h i c h could explain both its neuroprotective properties and the observation that dextromethorphan produces severe ataxia and s t e r e o t y p y (7). M o r e s e l e c t i v e sigma ligands are a n a l o g s of U5 0 , 4 8 8 H w h i c h i n t e r a c t s e l e c t i v e l y with sigma, not k a p p a o p i o i d receptors (8). The U - 5 0 , 4 8 8 H a n a l o g s that d i d not p r o d u c e any a t a x i a or s t e r e o t y p y were also neuroprotective in the g e r b i l m o d e l of i s c h e m i a (9). A n o t h e r s e l e c t i v e sigma l i g a n d that is chemically unrelated to U-50,488H is BMY-14802, which is c u r r e n t l y in c l i n i c a l trials as a novel a n t i p s y c h o t i c agent. The 0024-3205/92 $5.00 + .00 All rights reserved.
Copyright © 1992 Pergamon Press Ltd
1146
BMY-14802
is Neuroprotective
Vol.
p u r p o s e of this study was to a s s e s s w h e t h e r B M Y - 1 4 8 0 2 p r o t e c t a g a i n s t i s c h e m i a - i n d u c e d n e u r o n a l damage.
51, No.
could
14, 1992
also
Methods I s c h e m i a was i n d u c e d and m e a s u r e d in M o n g o l i a n g e r b i l s as d e s c r i b e d by C o n t r e r a s et al. (9). Briefly, Mongolian gerbils ( T u m b l e b r o o k Farms) weighing 50-70 g were injected ip. w i t h v a r y i n g doses of (±) B M Y - 1 4 8 0 2 or v e h i c l e (50%DMSO) 30 min p r i o r to o c c l u s i o n of the c a r o t i d a r t e r i e s . Gerbils were lightly a n e s t h e t i z e d w i t h h a l o t h a n e and p l a c e d on a h e a t e d p a d w i t h t h e i r snout in a n o s e c o n e . N i t r o u s oxide (70%) : o x y g e n (30%) p l u s 0.5% halothane was circulated through the nosecone to provide c o n t i n u o u s a n e s t h e s i a d u r i n g the s u r g i c a l p r o c e d u r e . A midline i n c i s i o n was m a d e in the n e c k and a l e n g t h of s u t u r e was p l a c e d around each carotid artery. The suture was tightened and p r e s s u r e a p p l i e d to the t h r e a d to i n s u r e t h a t b l o o d f l o w was o c c l u d e d for 5 minutes. Then the sutures were r e m o v e d a n d r e f l o w t h r o u g h the c a r o t i d a r t e r i e s was v i s u a l l y c o n f i r m e d . The w o u n d was c l o s e d w i t h a u t o c l i p s and the g e r b i l s a l l o w e d to r e c o v e r in egg i n c u b a t o r s m a i n t a i n e d at 30 0 C, The egg i n c u b a t o r has b e e n found to p r e v e n t h y p o t h e r m i a i n d u c e d by c o m p o u n d s such as M K - 8 0 1 (i0) . S e v e n days later, the g e r b i l s were a n e s t h e t i z e d w i t h i00 m g / k g of s o d i u m p e n t o b a r b i t a l and p e r f u s e d t r a n s c a r d i a l l y w i t h s a l i n e (with heparin) f o l l o w e d by b u f f e r e d formalin. The b r a i n s w e r e r e m o v e d and c o r o n a l s e c t i o n s t h r o u g h the h i p p o c a m p u s were p r e p a r e d and s t a i n e d w i t h t h i o n i n The d e g r e e of n e u r o n a l d a m a g e was a s s e s s e d in the CA1 region of the h i p p o c a m p u s by q u a n t i f y i n g the n u m b e r of p y r a m i d a l cell b o d i e s in a 0.5 m m l e n g t h of CA1 on the s e c t i o n c o r r e s p o n d i n g to P 1.7 m m in the atlas of L o s k o t a et al (ii) . The e f f e c t of B M Y - 1 4 8 0 2 was a s s e s s e d by c o m p a r i n g the n u m b e r of n e u r o n s f o u n d in the B M Y - 1 4 8 0 2 - t r e a t e d g r o u p to the control groups using non-parametric W i l c o x o n R a n k S u m s test. Seven g e r b i l s w e r e in e a c h group. For s t a t i s t i c a l analysis, e a c h h i p p o c a m p u s was t r e a t e d separately. Results
B M Y - 1 4 8 0 2 , at a d o s e of I0, 30 and 50 m g / k g alone, did not produce any obvious behavioral effects s u c h as s t e r e o t y p e d b e h a v i o r , ataxia, s e i z u r e s or a n e s t h e s i a in the gerbils, but did produce significant protection against ischemia-induced neuronal c e l l loss. A 5 m i n u t e b i l a t e r a l o c c l u s i o n of the c a r o t i d a r t e r i e s r e s u l t e d in a s i g n i f i c a n t d e c r e a s e in the n u m b e r of cell b o d i e s in the CA1 of the h i p p o c a m p u s (fig. i) . P r e t r e a t m e n t of g e r b i l s w i t h 50 m g / k g of B M Y - 1 4 8 0 2 s i g n i f i c a n t l y r e d u c e d the loss of n e u r o n s in the CA1 as c o m p a r e d to t h a t f o u n d in the v e h i c l e - t r e a t e d gerbils. The n u m b e r of n e u r o n s r e m a i n i n g in the h i p p o c a m p i of the g e r b i l s p r e t r e a t e d w i t h 50 m g / k g of B M Y - 1 4 8 0 2 was s i g n i f i c a n t l y g r e a t e r t h a n t h a t of v e h i c l e - t r e a t e d g e r b i l s . Lower doses of B M Y - 1 4 8 0 2 , I0 a n d 30 m g / k g , also produced significant neuroprotection.
Vol.
51, NO. 14, 1992
BMY-14802 is Neuroprotective
1147
160-
L 140-
120 -
(n
100-
_[
T .J
uj
60
40-
20-
"*°°#
.e~y EXP. B
FIG.
1
Effect of BMY-14802-pretreatment on i s c h e m i a - i n d u c e d cell loss in t h e C A 1 a r e a of t h e h i p p o c a m p u s . The number of n e u r o n s in t h e CA1 a r e a w a s d e t e r m i n e d in untreated gerbils (n=10) or gerbils pretreated with vehicle (50% DMSO in water) (ischemia control) ( n = 7 / g r o u p ) or v a r y i n g d o s e s of B M Y - 1 4 8 0 2 (n = 7 / g r o u p ) administered ip. 30 m i n b e f o r e occlusion of carotid arteries. R e s u l t s a r e s h o w n as m e a n = S . E . M . *Value significantly different from unoperated gerbil or ischemia-control (p
BMY-14802
Patricia
51, pp. 1145-1149
PROTECTS AGAINST NEURONAL DAMAGE IN
C. Contreras,
CNS D i s e a s e s
N a n c y M. Gray, H. L a n t h o r n
Research,
G. D. Searle 63198
Pergamon Press
ISCHEMIA-INDUCED THE GERBIL
Diane M. R a g a n
& Co.,
a n d Thomas
Chesterfield,
MO
(Received in final form July 24, 1992) Summarv BMY-14802, a s e l e c t i v e sigma l i g a n d c u r r e n t l y u n d e r i n v e s t i g a t i o n as an a t y p i c a l a n t i p s y c h o t i c agent, was t e s t e d for p o t e n t i a l a n t i - i s c h e m i c activity. BMY-14802 (i0, 30 a n d 50 mg/kg) d i d not p r o d u c e a n y s t e r e o t y p e d behavior, ataxia or seizures. When gerbils were p r e t r e a t e d w i t h i0, 30 or 50 m g / k g of B M Y - 1 4 8 0 2 30 m i n p r i o r to b i l a t e r a l o c c l u s i o n of c a r o t i d a r t e r i e s for 5 min, B M Y - 1 4 8 0 2 s i g n i f i c a n t l y p r o t e c t e d a g a i n s t i s c h e m i a i n d u c e d n e u r o n a l loss in the h i p p o c a m p u s . Thus, BMY14802 m a y also be u s e f u l as an a n t i - i s c h e m i c agent that does not p r o d u c e p s y c h o t o m i m e t i c effects. An e x t e n s i v e s e a r c h for a n t i - i s c h e m i c a g e n t s has i n c l u d e d a n t a g o n i s t s of e x c i t a t o r y a m i n o a c i d t r a n s m i s s i o n . However, the s e a r c h has y i e l d e d few b i o a v a i l a b l e , p o t e n t a g e n t s that p r o t e c t a g a i n s t i s c h e m i a - i n d u c e d n e u r o n a l i n j u r y w i t h o u t p r o d u c i n g gross behavioral or c a r d i o v a s c u l a r effects. The dose of nonc o m p e t i t i v e a n t a g o n i s t s of N - m e t h y l - D - a s p a r t a t e (NMDA), such as MK-801 or p h e n c y c l i d i n e (PCP) required to p r o t e c t against ischemia-induced neurotoxicity also produces gross behavioral e f f e c t s such as a t a x i a and s t e r e o t y p e d b e h a v i o r (i) . An a l t e r n a t i v e class of a n t i - i s c h e m i c agents might be sigma ligands. Ifenprodil, a polyamine antagonist, is a l s o a v e r y potent sigma ligand (2,3) that is n e u r o p r o t e c t i v e (4) . If an interaction with sigma receptors mediate the n e u r o p r o t e c t i v e p r o p e r t y of i f e n p r o d i l , then o t h e r sigma l i g a n d s s h o u l d p r o t e c t a g a i n s t i s c h e m i a - i n d u c e d n e u r o n a l damage. Consistent with this h y p o t h e s i s is the f i n d i n g that d e x t r o m e t h o r p h a n , w h i c h b i n d s w i t h high affinity to s i g m a sites and with low affinity to PCP r e c e p t o r s (5), is n e u r o p r o t e c t i v e (6). However dextromethorphan is q u i c k l y m e t a b o l i z e d to d e x t r o r p h a n , a potent PCP ligand, w h i c h could explain both its neuroprotective properties and the observation that dextromethorphan produces severe ataxia and s t e r e o t y p y (7). M o r e s e l e c t i v e sigma ligands are a n a l o g s of U5 0 , 4 8 8 H w h i c h i n t e r a c t s e l e c t i v e l y with sigma, not k a p p a o p i o i d receptors (8). The U - 5 0 , 4 8 8 H a n a l o g s that d i d not p r o d u c e any a t a x i a or s t e r e o t y p y were also neuroprotective in the g e r b i l m o d e l of i s c h e m i a (9). A n o t h e r s e l e c t i v e sigma l i g a n d that is chemically unrelated to U-50,488H is BMY-14802, which is c u r r e n t l y in c l i n i c a l trials as a novel a n t i p s y c h o t i c agent. The 0024-3205/92 $5.00 + .00 All rights reserved.
Copyright © 1992 Pergamon Press Ltd
1146
BMY-14802
is Neuroprotective
Vol.
p u r p o s e of this study was to a s s e s s w h e t h e r B M Y - 1 4 8 0 2 p r o t e c t a g a i n s t i s c h e m i a - i n d u c e d n e u r o n a l damage.
51, No.
could
14, 1992
also
Methods I s c h e m i a was i n d u c e d and m e a s u r e d in M o n g o l i a n g e r b i l s as d e s c r i b e d by C o n t r e r a s et al. (9). Briefly, Mongolian gerbils ( T u m b l e b r o o k Farms) weighing 50-70 g were injected ip. w i t h v a r y i n g doses of (±) B M Y - 1 4 8 0 2 or v e h i c l e (50%DMSO) 30 min p r i o r to o c c l u s i o n of the c a r o t i d a r t e r i e s . Gerbils were lightly a n e s t h e t i z e d w i t h h a l o t h a n e and p l a c e d on a h e a t e d p a d w i t h t h e i r snout in a n o s e c o n e . N i t r o u s oxide (70%) : o x y g e n (30%) p l u s 0.5% halothane was circulated through the nosecone to provide c o n t i n u o u s a n e s t h e s i a d u r i n g the s u r g i c a l p r o c e d u r e . A midline i n c i s i o n was m a d e in the n e c k and a l e n g t h of s u t u r e was p l a c e d around each carotid artery. The suture was tightened and p r e s s u r e a p p l i e d to the t h r e a d to i n s u r e t h a t b l o o d f l o w was o c c l u d e d for 5 minutes. Then the sutures were r e m o v e d a n d r e f l o w t h r o u g h the c a r o t i d a r t e r i e s was v i s u a l l y c o n f i r m e d . The w o u n d was c l o s e d w i t h a u t o c l i p s and the g e r b i l s a l l o w e d to r e c o v e r in egg i n c u b a t o r s m a i n t a i n e d at 30 0 C, The egg i n c u b a t o r has b e e n found to p r e v e n t h y p o t h e r m i a i n d u c e d by c o m p o u n d s such as M K - 8 0 1 (i0) . S e v e n days later, the g e r b i l s were a n e s t h e t i z e d w i t h i00 m g / k g of s o d i u m p e n t o b a r b i t a l and p e r f u s e d t r a n s c a r d i a l l y w i t h s a l i n e (with heparin) f o l l o w e d by b u f f e r e d formalin. The b r a i n s w e r e r e m o v e d and c o r o n a l s e c t i o n s t h r o u g h the h i p p o c a m p u s were p r e p a r e d and s t a i n e d w i t h t h i o n i n The d e g r e e of n e u r o n a l d a m a g e was a s s e s s e d in the CA1 region of the h i p p o c a m p u s by q u a n t i f y i n g the n u m b e r of p y r a m i d a l cell b o d i e s in a 0.5 m m l e n g t h of CA1 on the s e c t i o n c o r r e s p o n d i n g to P 1.7 m m in the atlas of L o s k o t a et al (ii) . The e f f e c t of B M Y - 1 4 8 0 2 was a s s e s s e d by c o m p a r i n g the n u m b e r of n e u r o n s f o u n d in the B M Y - 1 4 8 0 2 - t r e a t e d g r o u p to the control groups using non-parametric W i l c o x o n R a n k S u m s test. Seven g e r b i l s w e r e in e a c h group. For s t a t i s t i c a l analysis, e a c h h i p p o c a m p u s was t r e a t e d separately. Results
B M Y - 1 4 8 0 2 , at a d o s e of I0, 30 and 50 m g / k g alone, did not produce any obvious behavioral effects s u c h as s t e r e o t y p e d b e h a v i o r , ataxia, s e i z u r e s or a n e s t h e s i a in the gerbils, but did produce significant protection against ischemia-induced neuronal c e l l loss. A 5 m i n u t e b i l a t e r a l o c c l u s i o n of the c a r o t i d a r t e r i e s r e s u l t e d in a s i g n i f i c a n t d e c r e a s e in the n u m b e r of cell b o d i e s in the CA1 of the h i p p o c a m p u s (fig. i) . P r e t r e a t m e n t of g e r b i l s w i t h 50 m g / k g of B M Y - 1 4 8 0 2 s i g n i f i c a n t l y r e d u c e d the loss of n e u r o n s in the CA1 as c o m p a r e d to t h a t f o u n d in the v e h i c l e - t r e a t e d gerbils. The n u m b e r of n e u r o n s r e m a i n i n g in the h i p p o c a m p i of the g e r b i l s p r e t r e a t e d w i t h 50 m g / k g of B M Y - 1 4 8 0 2 was s i g n i f i c a n t l y g r e a t e r t h a n t h a t of v e h i c l e - t r e a t e d g e r b i l s . Lower doses of B M Y - 1 4 8 0 2 , I0 a n d 30 m g / k g , also produced significant neuroprotection.
Vol.
51, NO. 14, 1992
BMY-14802 is Neuroprotective
1147
160-
L 140-
120 -
(n
100-
_[
T .J
uj
60
40-
20-
"*°°#
.e~y EXP. B
FIG.
1
Effect of BMY-14802-pretreatment on i s c h e m i a - i n d u c e d cell loss in t h e C A 1 a r e a of t h e h i p p o c a m p u s . The number of n e u r o n s in t h e CA1 a r e a w a s d e t e r m i n e d in untreated gerbils (n=10) or gerbils pretreated with vehicle (50% DMSO in water) (ischemia control) ( n = 7 / g r o u p ) or v a r y i n g d o s e s of B M Y - 1 4 8 0 2 (n = 7 / g r o u p ) administered ip. 30 m i n b e f o r e occlusion of carotid arteries. R e s u l t s a r e s h o w n as m e a n = S . E . M . *Value significantly different from unoperated gerbil or ischemia-control (p
