Influence of dipeptides on precipitated morphine withdrawal in the mouse

Peptides. Vol. 4. pp. 417-419, 1983. ' Ankho International Inc. Printed in the U.S.A.

Influence of Dipeptides on Precipitated Morphine Withdrawal in the Mouse G , ~ B O R L. K O V , ~ C S , * L U C I A S Z O N T A G H ? * L A J O S B A L A S P I R I + AND GYULA TELEGDY*

*Institute o f Pathophysiolo,~,y and +lnstitttte ~[" Medical Chemistry University Medical School, Szeged, Httngal3' R e c e i v e d 7 F e b r u a r y 1983 K o v A c s , G. L.. L. SZONTAGH, L. BALASPIRI AN D G. TELEGDY. htlhu'nce ql'dipeptides on prucipitaled mot7~him' withdrawal in the motoe. PEPTIDES 4(4) 417-419. 1983.--Effects of various dipeptides on naloxone-precipitated morphine withdrawal were studied in the mouse. Mice were rendered dependent on morphine by implantation of morphine pellets and the withdrawal syndrome was measured by the httency of the onset of stereotyped jumpings. In accordance with previous data, subcutaneous injection of Z-prolyI-D-leucine significantly delayed the onset of morphine withdrawal. The alI-L enantiomer of the dipeptide (Z-L-prolyI-L-leucine) did not affect morphine withdrawal in the dose studied. Replacement of L-proline by L-glutamate or L-pyroglutamate IZ-L-glutamyI-L-leucine and L-pyroglutamyI-L-leucine) resulted in dipeptides which were more potent towards morphine withdrawal than Z-prolyI-D-leucine. Z-L-glycyI-L-proline attenuated the morphine withdrawal syndrome more effectively than Z-L-prolyI-D-leucine, but Z-L-leucyI-L-glycine was ineffective in this respect. The data reveal that certain dipeptides--which in their nonprotected forms are normal sequences of endogenous peptides--affect morphine withdrawal more potently than Z-prolyI-D-leucine, a synthetic dipeptide known to attenuate morphine dependence. Dipeptides

Morphine withdrawal

Naloxone

Mouse

R E C E N T studies indicate that neurohypophyseal hormones (oxytocin and vasopressin) affect drug addiction [6,8]. The active core of oxytocin in this respect resides in the C-terminal part of the molecule. Accordingly, the C-terminal tripeptide (H-L-Pro-L-Leu-L-Gly-NHe)----which has been shown to exert some naloxone-like actions [2,3]--facilitates the development of tolerance to and dependence on narcotic analgesics 18]. According to others, however, the tripeptide as well as the cyclic form of the C-terminal dipeptide Icyclo-L-Leu-L-Gly-NH_,) inhibit the development of physical dependence on chronic morphine challenge [ 1, 7, 9]. The synthetic dipeptide Z-Pro-D-Leu, which contains an N-terminal protective Z-group (N-benzyloxycarbonyl) and the enzymatically more stable D enantiomer of leucine, is a compound highly active in attenuating morphine dependence [4, 5, 7, 101. Because of the obvious theoretical and potential clinical importance of non-narcotic compounds which attenuate physical dependence on opiates, a number of analogs and derivatives of the C-terminal portion of oxytocin have been analysed for their effects on morphine withdrawal [7,9]. In these studies the main emphasis was on replacing the leucine moiety by other amino acids, and some derivatives appeared to be potent. In the present experiment the effects of various dipeptides were studied on naloxone-induced precipitated morphine withdrawal in the mouse. In most of the dipeptides examined, the L enantiomer of leucine was retained in the

molecule and L-proline was replaced by L-glutamate or L-pryoglutamate. The influence of L-Gly-L-Pro and L-Leu-L-Gly on morphine withdrawal was also studied. The dipeptides were protected with the N-benzyloxycarbonyl group. METHOD

A nimal,s Male CPLP mice of an inbred strain, weighing 25-30 g, were used. The mice were maintained on a standard illumination schedule of 12 hr (lights on at 6.00 a.m.) with water and food available ad lib. The animals were housed 10 per cage.

Naloxo/te-Precipitated Withdrau'al Swtdrome The naloxone-precipitated withdrawal syndrome in morphine-dependent mice was investigated according to previously published techniques [4]. Briefly, the animals were lightly anesthetized with ether vapor and a morphine pellet which consisted of morphine-HCI (37.5 rag) polyvinyl chloride (PVC, 75 mg) and Ca-stearate I1.5 mg) was implanted under the skin on the neck. The diameter of the pellet was 6 mm and the hardness was 14+1 Strong-Cobb units. Four days after pellet implantation, naloxone (1.0 mg/kg, Endo, Lab., Du Pont de Nemours, Gmbh) was injected subcutaneously and the precipitated abstinence syndrome was measured by the latency of the appearance of

'Present address: Blood Transfusion Center, University Medical School, Szeged.

417

418

KovAcs TABLE

E7 AL.

1

INFLUENCE OF DIPEPT1DES ON THE LATENCY OF ONSET OF NALOXONE-PRECIPITATED MORPHINE WITHDRAWAL IN MOUSE Significance Versus Treatments Control Z-L-Pro-D-Leu

Z-L-Pro-L-Leu Z-L-Pglu-L-Leu

L-Pglu-L-Leu

Z-L-GIn-L-Leu

Z-L-Gly-L-Pro

Z-L-Leu-L-Gly

Dose

No.

-0.5 #g 5.0 keg 50.0 keg 50.0 keg 0.5 keg 5.0 keg 50.0 keg 0.5 keg 5.0 keg 50.0 keg 0.5 keg 5.0 keg 50.0 ~g 0.5 keg 5.0 keg 50.0/*g 5t).0 keg

80 7 5 13 18 8 9 9 7 7 19 6 8 14 7 7 16 15

Latency of Onset 170 167 168 217 153 208 251 433 174 162 274 189 220 365 177 243 330 177

_+ 6* + 12 ± 37 + 31 + 11 _+ 17 + 15 + 80 _+ 22 + 23 + 37 ± 25 + 12 + 61 16 + "~'~ + 34 ± 23 +

Latency in Percent 100e~ 98% 99% 128% 90c7~,

122% 148% 255% 102% 95% 161% I I 1% 129c~ 215% 1(14% 143% 194% 104%

Control

Z-L-Pro-D-Leu' (50 keg)

-NS NS 0.05 NS NS 0.05 0.01 NS NS 0.05 NS 0.05 0.02 NS 0.05 I1.1)1 NS

-----NS 0.05 -NS -NS 0.05 - -

NS (I.(/5 --

*Mean ± S.E.M. in seconds. -Calculated for groups in which the mean was higher than in the group treated with 50 keg of Z-L-Pro-D-Leu.

s t e r e o t y p e d j u m p i n g from a c i r c u l a r p l a t f o r m 35 c m in diamet e r a n d 70 c m high. A c u t - o f f time of 900 sec was used. ~lr e a t m e n t s

P e p t i d e s w e r e s y n t h e t i z e d by o n e o f the a u t h o r s (L.B.). T h e r e f e r e n c e s u b s t a n c e ( L - P r o - D - L e u ) was a c q u i r e d from t w o different s o u r c e s ( P e n i n s u l a L a b o r a t o r i e s and s y n t h e tized by L.B.). Since the two p r e p a r a t i o n s gave identical results, data with this peptide are illustrated in a single group. All p e p t i d e s were d i s s o l v e d in physiological saline c o n t a i n i n g 6 vol% e t h a n o l . C o n t r o l mice r e c e i v e d the vehicle. A v o l u m e o f 0.2 ml was injected s u b c u t a n e o u s l y . Treatm e n t s w e r e g i v e n 2 h o u r s prior to the i m p l a n t a t i o n o f the m o r p h i n e pellet a n d r e p e a t e d on the t w o s u b s e q u e n t days. P e p t i d e t r e a t m e n t w a s not given on the f o u r t h day, w h e n m o r p h i n e w i t h d r a w a l was precipitated. S t a t i . s t i c a l A nal3"si,s

Data were a n a l y s e d b y A N O V A , followed by the Scheffe test for multiple c o m p a r i s o n s . A p r o b a b i l i t y level o f 0.05 was a c c e p t e d as a significant difference. RESULTS D a t a are s u m m a r i z e d in T a b l e 1. In the c o n t r o l group, s t e r e o t y p e d w i t h d r a w a l j u m p i n g a p p e a r e d 170_+6 sec after injection o f n a l o x o n e . Z - L - P r o - D - L e u r e s u l t e d in a signific a n t and d o s e - d e p e n d e n t delay in the o n s e t o f w i t h d r a w a l j u m p i n g s with 50 /,g b e i n g the lowest effective dose. Rep l a c e m e n t o f D - l e u c i n e by L-leucine ( Z - L - P r o - L - L e u ) resulted in a d i p e p t i d e w h i c h did not affect m o r p h i n e withdrawal. C o m p o u n d s in w h i c h L-proline w a s r e p l a c e d by L - p y r o g l u t a m a t e ( Z - L - P g l u - L - L e u ) or by L - g l u t a m a t e (Z-

L - G I n - L - L e u ) a t t e n u a t e d m o r p h i n e w i t h d r a w a l significantly m o r e (about ten times) p o t e n t l y t h a n the r e f e r e n c e substance. T h e n o n p r o t e c t e d form o f L - P g l u - L - L e u a t t e n u a t e d m o r p h i n e w i t h d r a w a l c o n s i d e r a b l y , but this effect was not significantly different f i o m that i n d u c e d by the r e f e r e n c e dipeptide. Z - L - P g l u - L - H i s and Z - L - L e u - L - G l y did not influe n c e the w i t h d r a w a l j u m p i n g , w h e r e a s Z - L - G I y - L - P r o att e n u a t e d m o r p h i n e w i t h d r a w a l m o r e p o t e n t l y t h a n the refere n c e s u b s t a n c e (Table 1). DISCUSSION T h e p r e s e n t data c o n f i r m p r e v i o u s results of different l a b o r a t o r i e s [4, 5, 10] that the s y n t h e t i c dipeptide ZL - P r o - D - L e u d e l a y s the o n s e t of n a l o x o n e - p r e c i p i t a t e d stere o t y p e d w i t h d r a w a l j u m p i n g in the m o r p h i n e - d e p e n d e n t m o u s e . This effect was s h o w n to be related to a t t e n u a t i o n of m o r p h i n e d e p e n d e n c e by the dipeptide, of w h i c h the all-L a n a l o g is k n o w n to be p r e s e n t in the m o l e c u l e of o x y t o c i n (in positions 7 a n d 8 o f the n o n a p e p t i d e ) . The all-L analog o f the dipeptide ( Z - L - P r o - L - L e u ) , however, was ineffective t o w a r d s m o r p h i n e w i t h d r a w a l in the a m o u n t studied in this e x p e r i m e n t ( 5 0 / , g ) . This finding might be e x p l a i n e d by the fact that the D e n a n t i o m e r o f leucine is m o r e stable against e n z y m a t i c d e g r a d a t i o n t h a n the naturally o c c u r r i n g L e n a n t i o m e r . As c o n c e r n s o t h e r p a r a m e t e r s of m o r p h i n e w i t h d r a w a l (e.g., decline in b o d y t e m p e r a t u r e d u r i n g precipit a t e d withdrawal), R i t z m a n n e t a / . 17] f o u n d Z - L - P r o - L - L e u as active as the d i p e p t i d e c o n t a i n i n g the D e n a n t i o m e r o f leucine, suggesting the a l t e r n a t i v e e x p l a n a t i o n t h a t different p a r a m e t e r s o f m o r p h i n e w i t h d r a w a l (e.g., s t e r e o t y p e d j u m p ing r e s p o n s e and regulation o f b o d y t e m p e r a t u r e ) exhibit diff e r e n t rates o f sensitivity t o w a r d s peptide t r e a t m e n t s . O n e aim o f lhe p r e s e n t e x p e r i m e n t was to i n v e s t i g a t e the

DIPEPTIDES AND MORPHINE WITHDRAWAL

419

effects of dipeptides which contain the naturally occurring L-leucine and in which L-proline was replaced by other amino acids. Replacement by L-glutamate or L-pyroglutamate, resulted in dipeptides which were about ten times more effective than the reference substance. The analog of L-Pglu-L-Leu in which the N-terminal was not protected by benzyloxycarbonyl was less effective than the same dipeptide with N-terminal protection. One possible explanation for this finding is that L-Pglu-L-Leu undergoes a relatively fast enzymatic inactivation and the N-terminal protection prevents this degradation. It is of interest to compare our findings with those of Ritzmann et al. [7]: they also replaced the proline residue by pyroglutamate, but in the tripeptide L - P r o - L - L e u - L - G I y and not in the dipeptide L-Pro-L-Leu as in our case. In their experiment the substituted tripeptide appeared to be inactive. Whether this difference is related to the presence of the C-terminal glycinamide in the tripeptide or to other factors is not clear as yet. In the present study the dipeptide in which L-proline was substituted by L-glutamate has been found active whereas Ritzmann et ,/. 17] described an active dipeptide in which L-leucine was replaced by L-glutamate. The latter authors, however, did not investigate the effect of L-Glu-L-Leu. Interestingly, L-pGlu-L-His did not influence morphine withdrawal in the mouse, suggesting that the presence of leucine together with pyroglutamate might be important for an effect on morphine withdrawal.

Finally, the effects of two dipeptides in which the L-glycine had been introduced were studied on morphine withdrawal. While Z-L-Gly-L-Pro attenuated morphine withdrawal more effectively than Z-L-Pro-D-Leu, ZL-Leu-L-GIy was ineffective. In accordance with these findings, Ritzmann et al. I7] found the dipeptide ZL-Leu-L-Pro-NH., ineffective towards morphine withdrawal. Cyclo (L-Leu-L-Gly) on the other hand, was found to be very active in attenuating morphine withdrawal 17]. In conclusion, the data suggest that the naloxoneprecipitated stereotyped withdrawal jumping in morphinedependent mouse can be attenuated by the synthetic dipeptide Z-L-Pro-D-Leu. Results of interchange of the amino acids indicate that Z-L-pGlu-L-Leu, Z-L-Gln-L-Leu and Z-L-GIy-L-Pro exert more potent effects on morphine withdrawal. Nonprotected forms of the latter peptides contain amino acid sequences which normally occur in endogenous peptides (L-Gln-L-Leu: porcine intestinal peptide, insulin, parathyroid hormone, corticotropin releasing factor; L-pGlu-L-Leu: neurotensin, locust adipokinetic hormone; L-Gly-L-Pro: pancreatic polypeptide, sauvagine, /3-MSH, etc.). Whether sufficient amounts of these dipeptides might be generated from endogenous peptides to affect the adaptive response of the organism to a chronic challenge with narcotic analgesics is a matter of speculation as yet.

REFERENCES

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6. Krivoy, W. A., E. Zimmermann and S. Lande. Facilitation of development of resistance to morphine analgesia by desglycinamide-lysine-vasopressin. Pro(' Nail Acad Sci USA 71: 1852-1856, 1974. 7. Ritzmann, R. F., R. Walter, H. N. Bhargaw~ and W. A. Krivoy. The inhibition of the development of tolerance to and physical dependence on morphine by peptides. In: N~,tlropeptide.s and Net,'al ]ransmi,s,sion, edited by C. Ajmone Marsan and W. Z. Traczyk. New York: Raven Press, 1980, pp. 237-244. 8. Van Ree, J. M. and D. De Wied. ProlyI-LeucyI-Glycinamide (PLG) facilitates morphine dependence. Li/~' S¢i 19: 1331-1340, 1976. 9. Walter, R., R. F. Ritzmann, H. N. Bhargava and L. B. Flexner. Prolyl-leucyl-glycinamide, cyclo (leucylglycine), and derivatives block development of physical dependence on morphine in mice. Proc Nail A('ad Sci USA 76: 518-520, 1979. 10. Walter, R., R. F. Ritzmann, H. N. Bhargawl, T. C. Rainbow, L. B. Flexner and W. A. Krivoy. Inhibition by Z-Pro-D-Leu of development of tolerance to and physical dependence on morphine in mice. Pro~ Natl Acad Sci USA 75: 4573-4576, 1978.