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Systemic and intracerebroventricular effects of opioid peptides in withdrawn morphine-dependent rhesus monkeys
Life Sciences, Vol. 33, Sup. I, 1983, pp. 361-364 Printed in the U.S.A.
Pergamon Press
SYSTEMIC AND INTRACEREBROVENTRICULAR EFFECTS OF OPIOID PEPTIDES IN WITHDRAWN MORPHINE-DEPENDENT RHESUS MONKEYS Debra E. Gmerek, Jonathan L. Katz, Charles P. France and James H. Woods Department of Pharmacology, University of Michigan Ann Arbor, Michigan 48109 (Received in final form June 26, 1983) Summary The effects of the degradation-resistant enkephalin analogs FK 33-824 and metkephamid were determined after systemic and intracerebroventricular (i.c.v.) administration in withdrawn morphine-dependent rhesus monkeys. Both peptides suppressed completely signs of 12-hr morphine deprivation, as does the prototype mu-receptor agonist morphine. The peptides were 100 and 2000 times more potent, respectively, after i.c.v. than s.c. injection. Thus, although peptidaseresistant, these compounds have restricted entrance into the central nervous system after systemic administration. The i.c.v, administration of compounds in rhesus monkeys should prove to be a valuable tool in the study of peptide ligands for opiate receptors. Centrally acting compounds which are large molecular weight and poorly soluble in lipids have difficulty penetrating the blood-brain barrier (BBB). Some compounds (e.g., peptides) are in addition rapidly metabolized in peripheral tissues. Thus, these compounds may be more readily and economically studied by injecting them directly into the cerebroventricular system. This technique is particularly useful when investigating activities of endogenous opioid peptides and their analogs. The single-dose suppression (SDS) test in morphinedependent rhesus monkeys is a well-established method for determining various activities of opioids in non-human primates. In this procedure, dependent monkeys (3 mg/kg/6 hr) are deprived of two consecutive maintenance doses of morphine. Single doses of test agents are then administered to the withdrawn animals and their behavior is monitored. Compounds which act at mureceptors suppress signs of abstinence in morphine-deprived dependent monkeys ( i ) . Very few peptides have been studied, in part, perhaps, due to the presumed rapid systemic degradation and poor central nervous system penetration. We have now established a procedure for intracerebroventricular (i.c.v.) administration of opioids in rhesus monkeys. In preparation for 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
362
Intraventricular
Peptides in Monkeys
Vol. 33, Sup. I, 1983
studies w i t h rapidly d e g r a d e d (systemically inactive) compounds, we have characterized the effects of two enzyme-resistant peptides, FK 33-824 and m e t k e p h a m i d , in the SDS test after systemic and i.c.v, administration. The r e l a t i v e systemic and i.c.v, potencies for suppressing morphine-abstinence were compared. MATERIALS
AND M E T H O D S
Cannulation Mature m o r p h i n e - d e p e n d e n t rhesus m o n k e y s w e i g h i n g 4-6 kg w e r e a n e s t h e t i z e d w i t h k e t a m i n e and p e n t o b a r b i t a l . They were i m p l a n t e d w i t h stainless steel c a n n u l a (Plastic Products) d i r e c t e d towards the lateral cerebral v e n t r i c l e a c c o r d i n g to the atlas of Snider and Lee (2). S t a n d a r d aseptic and stereotaxic techniques were used. Cannula placements were verified by r a d i o g r a p h y i m m e d i a t e l y after surgery and every 6 weeks thereafter. Anesthetized monkeys were x-rayed before and after i n j e c t i o n of m e t r i z a m i d e (15 mg; Winthrop). Only those animals w i t h e v i d e n c e of dye in the c e r e b r o v e n t r i c u l a r space w e r e used. SDS P r o c e d u r e Rhesus m o n k e y s m a i n t a i n e d on 3 mg/kg m o r p h i n e sulfate (s.c.) every 6 hr for at least 90 days w e r e d e p r i v e d of m o r p h i n e for 12-16 hr. Single doses of test agents w e r e then a d m i n i s t e r e d by s.c. or i.c.v, routes. The severity of withdrawal was rated at regular intervals by two e x p e r i e n c e d observers, a c c o r d i n g to the p r o c e d u r e s o u t l i n e d by D e n e a u and Seevers (3) and V i l l a r r e a l (I). Three to five animals were observed simultaneously. Monkeys were tested at w e e k l y intervals. Each test agent was given to at least 3 m o n k e y s per dose. The w i t h d r a w a l severity scores at each time point were averaged. C o m p o u n d s and Injections FK 33-824 (Sandoz), m e t k e p h a m i d (LY 127623, Eli Lilly) and m o r p h i n e sulfate (Mallinckrodt) were d i s s o l v e d in sterile water. Doses refer to the p a r t i c u l a r salt. The m o n k e y s are t r a i n e d to come out of their cages for s.c. injections (0.i ml/kg). They are p l a c e d in r e s t r a i n i n g chairs for i.c.v, injections. Sterile or aseptic solutions are injected slowly i.c.v, in v o l u m e s of 100-500 ul. RESULTS
and D I S C U S S I O N
M e t k e p h a m i d and FK 33-824 s u p p r e s s e d m o r p h i n e - a b s t i n e n c e in a dose-related m a n n e r following b o t h routes of a d m i n i s t r a t i o n (FIG 1 and 2). This is in a g r e e m e n t w i t h p r e v i o u s w o r k indicating that m e t k e p h a m i d and FK 33-824 have m o r p h i n e - l i k e effects (4,5,6,7,8,9). The m o n k e y s w e i g h an a v e r a g e of 5 kg. Thus, FK 33-824 is a p p r o x i m a t e l y 100 times more potent [(5x5.6)/0.3] in c o m p l e t e l y s u p p r e s s i n g a b s t i n e n c e w h e n given by central c o m p a r e d to systemic routes. Metkephamid is a p p r o x i m a t e l y 2000 times [(5xi00)/0.3] m o r e p o t e n t after i.c.v, i n j e c t i o n (assuming that a systemic dose of 100 m g / k g w o u l d c o m p l e t e l y suppress abstinence). This is in c o m p a r i s o n to morphine, w h i c h is only 5 times more potent after i.c.v, administration (unpublished observations). Unlike morphine, the h i g h e s t i.c.v, doses of FK 33-824 and m e t k e p h a m i d caused severe ataxia and apnea, such that n a l o x o n e had to be a d m i n i s t e r e d as an antidote. The e f f e c t of
Vol. 33, Sup. I, 1 9 8 3
Intraventricular Peptidesin Monkeys
SUBCUTANEOUS
363
INTRACEREBROVENTRICULAR
iI
control~ 1.7 mg/kg
>,
0.3 mg 3 0.
~
o
mg/kg
,~js.6 mg/kg
\
~ I 2 ,3 time (hours)
P.I.
4
5
P.I.
2
3
4
5
time
6
7
I~
22
24
28
(hour:~)
FIG. 1 Effect of FK 33-824 on 12-hr morphine abstinence.
INTRACEREBROVENTRICULAR
SUBCUTANEOUS
controls
1
0 mg/kg :30 m g / k g
>.
"c_~,
~
~770.i
mcj
/
,), 0.3 mcj
mg/kg £
P,I,
~
2
{.ime (hours)
3
4
S
P,I.
1
2 time
FIG. 2 Effect of metkephamid on 12-hr morphine abstinence.
'~ (heur~)
4
5
364
Intraventricular
Peptides in Monkeys
Vol. 33, Sup. I, 1983
the p e p t i d e s lasted longer than naloxone, however. W h e r e a s an i.c.v, dose of m o r p h i n e that suppresses abstinence completely lasts for 5 hours (unpublished observations), FK 33-824 comp l e t e l y s u p p r e s s e d w i t h d r a w a l signs for more than 13 hours after i.c.v, injection. The d r a m a t i c increase in p o t e n c y and d u r a t i o n of action of FK 33-824 and m e t k e p h a m i d after i.c.v, i n j e c t i o n i l l u s t r a t e s the e f f e c t i v e n e s s of the BBB. Therefore, e n z y m a t i c s t a b i l i z a t i o n is not always s u f f i c i e n t for a c c u r a t e a s s e s s m e n t of p e p t i d e efficacy. We p r e v i o u s l y found that w h e r e a s FK 33-824 and metk e p h a m i d share d i s c r i m i n a t i v e stimulus p r o p e r t i e s w i t h m o r p h i n e like compounds, they were r e l a t i v e l y i n e f f e c t i v e as r e i n f o r c e r s after systemic a d m i n i s t r a t i o n in m o n k e y s (5,7). At that time it was s u g g e s t e d that the BBB was r e s p o n s i b l e for the d e l a y e d onset of the d i s c r i m i n a t i v e stimulus effect. It is now a p p a r e n t that the BBB does inhibit entry of FK 33-824 and m e t k e p h a m i d into the central n e r v o u s system, w h i c h is r e q u i r e d for drug d i s c r i m i n a tion (i0). This p r o b a b l y also a c c o u n t s for the limited reinforcing effects of systemically administered morphine-like peptides. Assessment of o p i o i d p e p t i d e e f f i c a c y may be best d e t e r m i n e d by central a d m i n i s t r a t i o n . The i.c.v, a d m i n i s t r a t i o n of c o m p o u n d s in rhesus m o n k e y s should prove to be a v a l u a b l e tool in the study of p e p t i d e ligands for opiate receptors. ACKNOWLEDGEMENTS This study was s u p p o r t e d by USPHS grant DA-00254. Metk e p h a m i d and FK 33-824 w e r e g e n e r o u s l y supplied by Drs. R.C.A. F r e d r i c k s o n and E. S m i t h w i c k (Eli Lilly, Co.) and Dr. D. Roemer (Sandoz, Ltd.), respectively. We also thank J. G o o d r i c h and F. Adams for their t e c h n i c a l assistance. REFERENCES I.
J.E. VILLARREAL, A g o n i s t and A n t a g o n i s t A c t i o n s of N a r c o t i c A n a l g e s i c Drugs, H.W. Kosterlitz, H.O.J. C o l l i e r and J.E. Villarreal, eds. p. 73-93, U n i v e r s i t y Park Press, B a l t i m o r e (1973). 2. R.S. SNIDER and J.C. LEE, A S t e r e o t a x i c Atlas of the M o n k e y Brain, U n i v e r s i t y of Chicago Press, Chicago (1961). 3. G.A. D E N E A U and M.H. SEEVERS, Proc. 25th Ann Meet, CPDD, NAS (1963) A d d e n d u m 25. 4. ROEMER, D., H.H. BUESCHER, R.C. HILL, J. PLESS, W. BAUER, F. CARDINAUX, A. CLOSSE, D. H A U S E R and R. HUGUENIN, Nature 268 547-549 (1977). 5. J.H. WOODS, D.W. HEIN, S. HERLING, A.M. YOUNG and R.J. VALENTINO, E n d o g e n o u s and E x o g e n o u s Opiate A g o n i s t s and Antagonists, E.L. Way, ed. p. 443-445, P e r g a m o n Press, New York (1979). 6. HERLING, S., E.H. COALE, JR, R.J. VALENTINO, D.W. HEIN and J.H. WOODS, J. Pharmacol. Exp. Ther. 214 139-146 (1980). 7. J.H. WOODS, S. H E R L I N G and A.M. YOUNG, N e u r o p e p t i d e s 1 409-419 (1981). 8. COWAN, A., F.C. T O R T E L L A and M.W. ADLER, Eur. J. Pharmacol. 71 117-121 (1981). 9. LEANDER, J.D. and C.R. WOOD, Peptides 3 771-773 (1982). I0. R.J. VALENTINO, S. HERLING, J.H. WOODS, F. M E D Z I H R A D S K Y and H. MERZ, J. Pharmacol. Exp. Ther. 217 652-659 (1981).
Pergamon Press
SYSTEMIC AND INTRACEREBROVENTRICULAR EFFECTS OF OPIOID PEPTIDES IN WITHDRAWN MORPHINE-DEPENDENT RHESUS MONKEYS Debra E. Gmerek, Jonathan L. Katz, Charles P. France and James H. Woods Department of Pharmacology, University of Michigan Ann Arbor, Michigan 48109 (Received in final form June 26, 1983) Summary The effects of the degradation-resistant enkephalin analogs FK 33-824 and metkephamid were determined after systemic and intracerebroventricular (i.c.v.) administration in withdrawn morphine-dependent rhesus monkeys. Both peptides suppressed completely signs of 12-hr morphine deprivation, as does the prototype mu-receptor agonist morphine. The peptides were 100 and 2000 times more potent, respectively, after i.c.v. than s.c. injection. Thus, although peptidaseresistant, these compounds have restricted entrance into the central nervous system after systemic administration. The i.c.v, administration of compounds in rhesus monkeys should prove to be a valuable tool in the study of peptide ligands for opiate receptors. Centrally acting compounds which are large molecular weight and poorly soluble in lipids have difficulty penetrating the blood-brain barrier (BBB). Some compounds (e.g., peptides) are in addition rapidly metabolized in peripheral tissues. Thus, these compounds may be more readily and economically studied by injecting them directly into the cerebroventricular system. This technique is particularly useful when investigating activities of endogenous opioid peptides and their analogs. The single-dose suppression (SDS) test in morphinedependent rhesus monkeys is a well-established method for determining various activities of opioids in non-human primates. In this procedure, dependent monkeys (3 mg/kg/6 hr) are deprived of two consecutive maintenance doses of morphine. Single doses of test agents are then administered to the withdrawn animals and their behavior is monitored. Compounds which act at mureceptors suppress signs of abstinence in morphine-deprived dependent monkeys ( i ) . Very few peptides have been studied, in part, perhaps, due to the presumed rapid systemic degradation and poor central nervous system penetration. We have now established a procedure for intracerebroventricular (i.c.v.) administration of opioids in rhesus monkeys. In preparation for 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
362
Intraventricular
Peptides in Monkeys
Vol. 33, Sup. I, 1983
studies w i t h rapidly d e g r a d e d (systemically inactive) compounds, we have characterized the effects of two enzyme-resistant peptides, FK 33-824 and m e t k e p h a m i d , in the SDS test after systemic and i.c.v, administration. The r e l a t i v e systemic and i.c.v, potencies for suppressing morphine-abstinence were compared. MATERIALS
AND M E T H O D S
Cannulation Mature m o r p h i n e - d e p e n d e n t rhesus m o n k e y s w e i g h i n g 4-6 kg w e r e a n e s t h e t i z e d w i t h k e t a m i n e and p e n t o b a r b i t a l . They were i m p l a n t e d w i t h stainless steel c a n n u l a (Plastic Products) d i r e c t e d towards the lateral cerebral v e n t r i c l e a c c o r d i n g to the atlas of Snider and Lee (2). S t a n d a r d aseptic and stereotaxic techniques were used. Cannula placements were verified by r a d i o g r a p h y i m m e d i a t e l y after surgery and every 6 weeks thereafter. Anesthetized monkeys were x-rayed before and after i n j e c t i o n of m e t r i z a m i d e (15 mg; Winthrop). Only those animals w i t h e v i d e n c e of dye in the c e r e b r o v e n t r i c u l a r space w e r e used. SDS P r o c e d u r e Rhesus m o n k e y s m a i n t a i n e d on 3 mg/kg m o r p h i n e sulfate (s.c.) every 6 hr for at least 90 days w e r e d e p r i v e d of m o r p h i n e for 12-16 hr. Single doses of test agents w e r e then a d m i n i s t e r e d by s.c. or i.c.v, routes. The severity of withdrawal was rated at regular intervals by two e x p e r i e n c e d observers, a c c o r d i n g to the p r o c e d u r e s o u t l i n e d by D e n e a u and Seevers (3) and V i l l a r r e a l (I). Three to five animals were observed simultaneously. Monkeys were tested at w e e k l y intervals. Each test agent was given to at least 3 m o n k e y s per dose. The w i t h d r a w a l severity scores at each time point were averaged. C o m p o u n d s and Injections FK 33-824 (Sandoz), m e t k e p h a m i d (LY 127623, Eli Lilly) and m o r p h i n e sulfate (Mallinckrodt) were d i s s o l v e d in sterile water. Doses refer to the p a r t i c u l a r salt. The m o n k e y s are t r a i n e d to come out of their cages for s.c. injections (0.i ml/kg). They are p l a c e d in r e s t r a i n i n g chairs for i.c.v, injections. Sterile or aseptic solutions are injected slowly i.c.v, in v o l u m e s of 100-500 ul. RESULTS
and D I S C U S S I O N
M e t k e p h a m i d and FK 33-824 s u p p r e s s e d m o r p h i n e - a b s t i n e n c e in a dose-related m a n n e r following b o t h routes of a d m i n i s t r a t i o n (FIG 1 and 2). This is in a g r e e m e n t w i t h p r e v i o u s w o r k indicating that m e t k e p h a m i d and FK 33-824 have m o r p h i n e - l i k e effects (4,5,6,7,8,9). The m o n k e y s w e i g h an a v e r a g e of 5 kg. Thus, FK 33-824 is a p p r o x i m a t e l y 100 times more potent [(5x5.6)/0.3] in c o m p l e t e l y s u p p r e s s i n g a b s t i n e n c e w h e n given by central c o m p a r e d to systemic routes. Metkephamid is a p p r o x i m a t e l y 2000 times [(5xi00)/0.3] m o r e p o t e n t after i.c.v, i n j e c t i o n (assuming that a systemic dose of 100 m g / k g w o u l d c o m p l e t e l y suppress abstinence). This is in c o m p a r i s o n to morphine, w h i c h is only 5 times more potent after i.c.v, administration (unpublished observations). Unlike morphine, the h i g h e s t i.c.v, doses of FK 33-824 and m e t k e p h a m i d caused severe ataxia and apnea, such that n a l o x o n e had to be a d m i n i s t e r e d as an antidote. The e f f e c t of
Vol. 33, Sup. I, 1 9 8 3
Intraventricular Peptidesin Monkeys
SUBCUTANEOUS
363
INTRACEREBROVENTRICULAR
iI
control~ 1.7 mg/kg
>,
0.3 mg 3 0.
~
o
mg/kg
,~js.6 mg/kg
\
~ I 2 ,3 time (hours)
P.I.
4
5
P.I.
2
3
4
5
time
6
7
I~
22
24
28
(hour:~)
FIG. 1 Effect of FK 33-824 on 12-hr morphine abstinence.
INTRACEREBROVENTRICULAR
SUBCUTANEOUS
controls
1
0 mg/kg :30 m g / k g
>.
"c_~,
~
~770.i
mcj
/
,), 0.3 mcj
mg/kg £
P,I,
~
2
{.ime (hours)
3
4
S
P,I.
1
2 time
FIG. 2 Effect of metkephamid on 12-hr morphine abstinence.
'~ (heur~)
4
5
364
Intraventricular
Peptides in Monkeys
Vol. 33, Sup. I, 1983
the p e p t i d e s lasted longer than naloxone, however. W h e r e a s an i.c.v, dose of m o r p h i n e that suppresses abstinence completely lasts for 5 hours (unpublished observations), FK 33-824 comp l e t e l y s u p p r e s s e d w i t h d r a w a l signs for more than 13 hours after i.c.v, injection. The d r a m a t i c increase in p o t e n c y and d u r a t i o n of action of FK 33-824 and m e t k e p h a m i d after i.c.v, i n j e c t i o n i l l u s t r a t e s the e f f e c t i v e n e s s of the BBB. Therefore, e n z y m a t i c s t a b i l i z a t i o n is not always s u f f i c i e n t for a c c u r a t e a s s e s s m e n t of p e p t i d e efficacy. We p r e v i o u s l y found that w h e r e a s FK 33-824 and metk e p h a m i d share d i s c r i m i n a t i v e stimulus p r o p e r t i e s w i t h m o r p h i n e like compounds, they were r e l a t i v e l y i n e f f e c t i v e as r e i n f o r c e r s after systemic a d m i n i s t r a t i o n in m o n k e y s (5,7). At that time it was s u g g e s t e d that the BBB was r e s p o n s i b l e for the d e l a y e d onset of the d i s c r i m i n a t i v e stimulus effect. It is now a p p a r e n t that the BBB does inhibit entry of FK 33-824 and m e t k e p h a m i d into the central n e r v o u s system, w h i c h is r e q u i r e d for drug d i s c r i m i n a tion (i0). This p r o b a b l y also a c c o u n t s for the limited reinforcing effects of systemically administered morphine-like peptides. Assessment of o p i o i d p e p t i d e e f f i c a c y may be best d e t e r m i n e d by central a d m i n i s t r a t i o n . The i.c.v, a d m i n i s t r a t i o n of c o m p o u n d s in rhesus m o n k e y s should prove to be a v a l u a b l e tool in the study of p e p t i d e ligands for opiate receptors. ACKNOWLEDGEMENTS This study was s u p p o r t e d by USPHS grant DA-00254. Metk e p h a m i d and FK 33-824 w e r e g e n e r o u s l y supplied by Drs. R.C.A. F r e d r i c k s o n and E. S m i t h w i c k (Eli Lilly, Co.) and Dr. D. Roemer (Sandoz, Ltd.), respectively. We also thank J. G o o d r i c h and F. Adams for their t e c h n i c a l assistance. REFERENCES I.
J.E. VILLARREAL, A g o n i s t and A n t a g o n i s t A c t i o n s of N a r c o t i c A n a l g e s i c Drugs, H.W. Kosterlitz, H.O.J. C o l l i e r and J.E. Villarreal, eds. p. 73-93, U n i v e r s i t y Park Press, B a l t i m o r e (1973). 2. R.S. SNIDER and J.C. LEE, A S t e r e o t a x i c Atlas of the M o n k e y Brain, U n i v e r s i t y of Chicago Press, Chicago (1961). 3. G.A. D E N E A U and M.H. SEEVERS, Proc. 25th Ann Meet, CPDD, NAS (1963) A d d e n d u m 25. 4. ROEMER, D., H.H. BUESCHER, R.C. HILL, J. PLESS, W. BAUER, F. CARDINAUX, A. CLOSSE, D. H A U S E R and R. HUGUENIN, Nature 268 547-549 (1977). 5. J.H. WOODS, D.W. HEIN, S. HERLING, A.M. YOUNG and R.J. VALENTINO, E n d o g e n o u s and E x o g e n o u s Opiate A g o n i s t s and Antagonists, E.L. Way, ed. p. 443-445, P e r g a m o n Press, New York (1979). 6. HERLING, S., E.H. COALE, JR, R.J. VALENTINO, D.W. HEIN and J.H. WOODS, J. Pharmacol. Exp. Ther. 214 139-146 (1980). 7. J.H. WOODS, S. H E R L I N G and A.M. YOUNG, N e u r o p e p t i d e s 1 409-419 (1981). 8. COWAN, A., F.C. T O R T E L L A and M.W. ADLER, Eur. J. Pharmacol. 71 117-121 (1981). 9. LEANDER, J.D. and C.R. WOOD, Peptides 3 771-773 (1982). I0. R.J. VALENTINO, S. HERLING, J.H. WOODS, F. M E D Z I H R A D S K Y and H. MERZ, J. Pharmacol. Exp. Ther. 217 652-659 (1981).