European Journal of Pharmacoh>gy, 211 (1992) 75-8(1

75

© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 52265

Different effects of peptidase inhibitors on dermophin- and on [D-Arg~]dermorphin-induced antinociceptive activity S h i n o b u S a k u r a d a , H i r o m i W a t a n a b e , T s u k a s a S a k u r a d a , K e n s u k e Kisara, Y u s u k e Sasaki i and Kenji Suzuki Departments of Pharmacology and t Biochemistry, Tohoku College of t'harmacy, 4-4-I Komatsushima, Aoba-ku, Sendai 981. Japan Received 9 July 1991. revised MS received 30 October 1991, accepted 12 November 1991

The antinociceptive effects produced by the intracerebroventricular (i.c.v.) injection of dermorphin and [D-Arg2]dermorphin were compared in conscious mice, using the combined administration of peptidase inhibitors. Nociception was assessed using a tail pressure assay. Dermorphin-induced antinociception was not potentiated by simultaneous administration of amastatin or captopril as judged from the EDso values. Co-administration of dermorphin and amastatin gave a longer duration than with dermorphin alone, whereas there was no significant effect on duration with captopril. The antinociceptive activity of dermorphin was significantly enhanced when the heptapeptide was injected simultaneously with both peptidase inhibitors. This result indicates that the heptapeptide sequence is required for the full expression of intrinsic opioid activity of dermorphin. In contrast, co-administration of amastatin brought about a significant enhancement of the antinociceptive activity induced by i.c.v. administration of [D-ArgZ]dermorphin, whereas the effect of [D-Arg2]dermorphin was markedly decreased by the concurrent administration of captopril or thiorphan. The potency of captopril was much greater than that of thiorphan. The present results suggest that [D-Arg2]dermorphin may be transformed metabolically to a peptide which has potent antinociceptive activity. Dermorphin; [D-Arg2]dermorphin; Captopril; Amastatin; Antinociceptive activity

1. Introduction

The amphibian heptapeptide, dermorphin (H-TyrD-Ala-Phe-Gly-Tyr-Pro-Ser-NHz), presents the unique feature of having a D-amino acid residue in its sequence (Erspamer and Melchiorri, 1980) and possesses potent and long-lasting peripheral and central opioidlike activity (Erspamer et al., 1981). When administered i.c.v, to mice, this opioid peptide is much more potent than morphine to produce an antinociceptive effect. Studies on the structure-activity relationship have shown that the N-terminal 1-4 sequence of dermorphin is the minimal fragment required for full opioid activity (Broccardo et al., 1981). An endogenous enkephalin-releasing dipeptide, kyotorphin, that does not bind to opioid receptors was isolated from bovine brain (Takagi et al., 1979). The antinociceptive effect produced by central administration of L-Tyr-D-Arg, a kyotorphin analogue, was more potent and long-lasting than that of kyotorphin or

Correspondence to: S. Sakurada, Department of Pharmacology, Tohoku College of Pharmacy, 4-4-1 Komatsushima, Aoba-ku, Sendai 981, Japan.

[MetS]enkephalin (Sakurada et al., 1982). We have recently reported that [D-Arg2]dermorphin and its shorter homologues were more potent and had a more long-lasting effect than morphine, but less so than dermorphin (Kisara et al., 1986). Regarding thc structure-activity relationship, the presence of the N-terminal tripeptide of [D-ArgZ]dcrmorphin is of crucial importance for the manifestation of full intrinsic opioidlike antinociccptive activity of [D-Arg2]dermorphin (Kisara et al., 1986). The main enzymatically degraded product of dermorphin (Ncgri and lmprota, 1982) or [D-Arg2]dermorphin (Sasaki et al., 1985) in the brain is the corresponding N-terminal 1-4 fragment. Little information is available concerning the enzymes degrading dermorphins in the central nervous system. The present studies aimed to clarify the role of peptidase activity in modulating the antinociception induced by dermorphin and its analogue, using the combined administration of peptidase inhibitors. To this end, the aminopeptidase inhibitor, amastatin, the angiotensin-converting enzyme inhibitor, captopril, and thiorphan (an inhibitor of EC 24.11) were tested for their ability to modulate the antinociceptive activity induced by i.c.v, administration of dermorphin or [D-ArgZ]dermorphin.

76 2. Materials

and

TABLE 1

methods

M a l e mice ( S t d - d d y ) weighing 2 2 - 2 5 g were held in g r o u p s o f 20 in a t e m p e r a t u r e - and l i g h t - c o n t r o l l e d room (23_+ I ° C , 12 h light starting at 09:00 h). All mice had free access to l a b o r a t o r y chow a n d t a p water, except d u r i n g e x p e r i m e n t s , which were p e r f o r m e d between 10:00 and 18:00 h. A n t i n o c i c e p t i v e activity was m e a s u r e d by the tail p r e s s u r e m e t h o d as previously d e s c r i b e d ( S a k u r a d a et al., 1982). Briefly, increasing m e c h a n i c a l p r e s s u r e was a p p l i e d to the base o f the tail as i n c r e m e n t s o f 20 m m H g / s , and biting o r struggling b e h a v i o u r o f mice to which p r e s s u r e was a p p l i e d m e c h a n i c a l l y was an indication of r e s p o n s e threshold. T h e trials were t e r m i n a t e d at the level o f 200 m m Hg to p r e v e n t tail tissue d a m a g e . F o r the tail p r e s s u r e assay, the m e a n _+ S.E. of the p r e s s u r e level was p l o t t e d . To o b t a i n the d o s e - r e sponse curve, the dose was p l o t t e d against % m a x i m u m possible effect ( % M P E ) c a l c u l a t e d using the following e q u a t i o n : % M P E = ( P 2 - P 1 / 2 0 0 - P1) × 100 w h e r e PI is the r e s p o n s e p r e s s u r e b e f o r e p e p t i d e injection (ram Hg) a n d P2 is the r e s p o n s e p r e s s u r e (mm Hg) after p e p t i d e injection (mm Hg). G r o u p s o f I0 u n a n a e s t h e t i z e d mice were injected with a H a m i l t o n syringe into the right-side lateral c e r e b r o v e n t r i c l e , using a v o l u m e of 1 0 / z l at a rate of 10-15 s. T h e EDso values and t h e i r 95% c o n f i d e n c e limits were d e t e r m i n e d by the m e t h o d of Litchfield a n d Wilcoxon (1949). Statistical significance of the d a t a was e s t i m a t e d by a mixed two-factor analysis of variance ( A N O V A ) with D u n n e t t ' s test ( D u n n e t t , 1964). T h e c o m p o u n d s used were a m a s t a t i n ( P e p t i d e Institute Inc., O s a k a , J a p a n ) , c a p t o p r i l (Sankyo Co. Ltd.,

80

..

o

.".

Effccts of i.c.v, administration of captopril and/or amastalin on dermorphin- and [D-Arg2]dermorphin-induced antinociceptive activity in mice. EDs0 values were calculated from the value obtained at the time of peak effect; 95% confidence limits are in parentheses. Drugs

ED.~o (pmol/mouse)

Dermorphin Dermorphin+thiorphan (50 p.g) Derrnorphin + captopril (50 p.g) Dermorphin + amastatin (10 P-B) Derrnorphin+captopril+amastatin [D-Arg']Dcrmorphin [D-Arg2]Dcrmorphin + thiorphan [D-Arg2JDermorphin + captopril (50 p.g) [D-ArgZ]Dermorphin +amastatin (10 p.g) [D-Arg2]Dermorphin-(1-4)

6.9 (4.5- 10.6) 6.6 (4.2- 10.4) 6.1 (3.8- 9.7) 6.6 (4.4- 10.0) 2.0 (1.2- 2.3) 20.0 (12.6- 31.7) 50.0 (20.6-121.4) 350.0(197.5-620.2) 15.(I (8.8- 25.5) 8.2 (5.6- 11.8)

Tokyo, J a p a n ) and t h i o r p h a n ( M e i j i s e i k a Co. Ltd., Tokyo, J a p a n ) . D e r m o r p h i n a n d [ D - A r g 2 ] d e r m o r p h i n were s y n t h e t i z e d by c o n v e n t i o n a l liquid p h a s e m e t h o d s in o u r laboratory. All c o m p o u n d s were dissolved in R i n g e r solution a n d a d m i n i s t e r e d i.c.v.

3, Results

3.1. Effects of arnastatin on dermorphin- and [DArg 2]dermorphin-induced antinociception W h e n a d m i n i s t e r e d i.c.v, to mice, [ D - A r g 2 ] d e r m o r p h i n and d e r m o r p h i n e x h i b i t e d p o t e n t a n t i n o c i c e p tive activity, the effect reaching a m a x i m u m at 10-15 min. [ D - A r g 2 ] d e r m o r p h i n had a l o n g e r d u r a t i o n o f

• ..

o" I ;

..

!

3o

:]den.onm.

'9o 12o "rime after inioclion (rain)

Fig. I. Comparison of lime course of the effect of i.c.v, administered [D-ArgZ]derrnorphin alone and in combination with amastalin in the mouse fail pressure test. The doses of [D-Arg 2]dermorphin used were 68.4 (i), 40.5 (o), 23.9 ( • ), 14.2 ( z~) and 8.4 ( v ) pmol. Amastatin (I0 ~,g) was co-administered i.c.v, with each dose of [D-Arg2]dermorphin. * P < 0.05, * * P < (1.01 when compared to Ringer solution-treated controls (square symbols in the right panel) or controls treated with Ringer solution and arnastatin (square symbols in the left panel). "'P < (1.01 when compared to [D-Arg2]dermorphin akrne.

77 a c t i o n t h a n d e r m o r p h i n , t h o u g h d e r m o r p h i n was m o r e p o t e n t t h a n its a n a l o g u e , as s e e n f r o m t h e v a l u e s o f E D s 0 d e t e r m i n e d f r o m t h e p e a k t i m e e f f e c t ( t a b l e 1, figs. 1 a n d 4). A single i n j e c t i o n o f t h e a m i n o p e p t i d a s e i n h i b i t o r , a m a s t a t i n 10 t , g , w a s w i t h o u t e f f e c t in p r o d u c i n g a n t i n o c i c e p t i v e activity in t h e assay u s e d h e r e . T h e a n t i n o c i c e p t i v e e f f e c t p r o d u c e d by i.c.v, a d m i n i s tration of [D-Arg2]dermorphin together with amastatin was m o r e p o t e n t a n d l o n g - l a s t i n g t h a n t h a t o f [DA r g 2 ] d e r m o r p h i n a l o n e , t h e d i f f e r e n c e b e i n g statistically s i g n i f i c a n t (fig. 1). A m a s t a t i n i n c r e a s e d t h e a n t i n o c i c e p t i v e e f f e c t s o f [ D - A r g 2 ] d e r m o r p h i n (14.2 a n d 23.9 p m o l ) , a l t h o u g h t h e i n c r e a s e was not statistically significant. A t 30, 45, 60, 90 a n d 120 min, c o - a d m i n i s t r a t i o n o f [ D - A r g 2 ] d e r m o r p h i n (40.5 p m o l ) a n d amastatin significantly increased the antinociceptive e f f e c t as c o m p a r e d to [ D - A r g 2 ] d e r m o r p h i n a l o n e . T h e e f f e c t o f [ D - A r g 2 ] d e r m o r p h i n 40.5 p m o l p e a k e d at 30 m i n p o s t - i n j e c t i o n a n d h a d d i s a p p e a r e d at 240 min. [ D - A r g 2 ] d e r m o r p h i n (40.5 p m o l ) in c o m b i n a t i o n w i t h a m a s t a t i n w a s as p o t e n t as 68.4 p m o l [ D - A r g 2 ] d e r m o r p h i n a l o n e (fig. 1). C o - a d m i n i s t r a t i o n o f d e r m o r phin and amastatin did not potentiate the dermorphin-induced a n t i n o c i c e p t i o n w i t h r e s p e c t to

t h e i r E D s 0 v a l u e s ( t a b l e 11. H o w e v e r , a m a s t a t i n p r o d u c e d a s i g n i f i c a n t i n c r e a s e in d u r a t i o n o f t h e d e r m o r p h i n e f f e c t ( t a b l e 2).

3.2. l~ffects of captopril or thiorphan on dermorphinand [ D-Arg 2]dermorphin-induced antinociception The [D-Arg2]dermorphin-induccd antinociception was m a r k e d l y r e d u c e d by c o - a d m i n i s t r a t i o n o f t h e ang i o t e n s i n - c o n v e r t i n g e n z y m e i n h i b i t o r , c a p t o p r i l (fig. 2). W i t h this i n h i b i t o r (5(I g g ) t h e d e g r e e o f a n t i n o c i c e p t i v e activity was 2 0 % less, e v e n at a d o s e o f 136.8 p m o l o f [ D - A r g 2 ] d e r m o r p h i n , w h i c h i n d u c e s 100% o f M P E at the p e a k t i m e . In t h e p r e s e n c e o f 511 p,g c a p t o p r i l , t h e ED.s0 v a l u e o f [ D - A r g 2 ] d e r m o r p h i n was 350 p m o l ( t a b l e 1). T h i o r p h a n , an i n h i b i t o r o f E C 24.11, p r o d u c e d a d o s e - d e p e n d e n t i n h i b i t i o n o f [DA r g 2 ] d e r m o r p h i n - i n d u c e d a n t i n o c i c e p t i o n , w h i c h was s i m i l a r to t h e i n h i b i t i o n w i t h c a p t o p r i l (fig. 3). T h e t i m e o f p e a k a n t i n o c i c e p t i v e e f f e c t was d e l a y e d f r o m 30 to 90 m i n a f t e r c o - a d m i n i s t r a t i o n o f [ D - A r g 2 ] d e r morphin and thiorphan. The dose-response curve of [ D - A r g 2 ] d e r m o r p h i n was s h i f t e d to the right in t h e p r e s e n c e o f c a p t o p r i l o r t h i o r p h a n (fig. 3). T h c in-

TABLE 2 Time course of the effects of dermorphin co-administered with captopril or amastatin in mice. Each value was expressed as % of MPE. A dose of 50 p,I/mouse of captopril or 10 p,g/mouse of amastatin was co-administered i.c.v, with dermorphin. Treatment

Dermorphin (2.6 pmol) Dermorphin + captopril Dermorphin + amastatin Dermorphin (4.5 pmol) Dermorphin + captopril Dermorphin + amastatin Dermorphin (7.6 pmol) Dermorphin + captopril Dermorphin + amastatin Dermorphin (12.9 pmol) Dermorphin + captopril Dermorphin + amastatin

Time after injection (min) 5

10

15

30

45

611

27.0 + 5.5 31.5 t 7.3 12.2 + 4.6

17.4 + 4.4 26.9 ± 3.2 14.2 + 6.1

23.2 _+ 6.3 27.0 +_ 6.4 27.7 + 7.1

11.7 _+ 4.3 31.3 + 3.9 26.6 + 8.1

10.6 + 6.2 17.8 +_ 4.8 20.1 + 4.8

6.2 +_ 4.3 14.5 ± 4.2 211.3_+ 7.(1

30.0 + 5.7 30.0 + 3.4 16.5 + 3.1

33.3 ± 4.7 39.11_+ 7.6 18.6 + 5.7

37.4 +_ 4.9 45.0 ± 5.8 30.8 _+ 6.5

31 .(1± 9.5 37.7 _+ 6.7 29.1 + 6.2

31.9 _+ 3.9 34.8 _+ 8.7 22.0 +_ 5.7

29.11 + 6.0 51.1 + 9.6 37.9 + 8.4

48.8 + 8.2 63.9 + 7.2 36.7 + 5.4

40.0 _+ 4.5 52.6 +_ 6.4 45.2 _+ 6.7

32.4 + 5.4 46.9 +_ 11.9 57.0 + 6.7

60.3 + 8.3 55.4 + 7.7 32.1 + 9.3

76.2 ± 7.1 72.6 ± 9.7 64.2 + 10.7

69.9 + 8.6 68.6 + 12.9 68.4 _+ 9.3

59.3 +_ 12.1 48.8 + 12.3 72.2 + 9.9

a p < 0.05 and b p < 0.01 when compared with dermorphin alone.

911

1211

16.1 _+ 4.1 24.3 ± 5.6 9.7 +_ 6.6

11.8 + 4.9 12.6 + 4.9 11.2 _+ 3.2

10.1 +_ 4.2 3.5 +_ 2.5 9.7 _+ 2.2

21.5 + 5.9 38.2 +_ 8.9 47.9 + 5.4

19.0 _+ 4.11 23.0 _+ 5.3 29.4 +_ 6.8

18.3 _+ 4.8 18.8 + 3.8 26.5 _+ 6.5 ~'

5.7 _+ 2.9 5.9 -2.6 18.2 ± 4.4 "

411.7 _+ 7.6 46.6 +_ 2.0 63,8 ± 11.1

29.4 _+ 11.3 26.9 _+ 9.8 48.4 ± 12.7 ~

9.7 +_ 3.6 28.4 + 5.0 44.6 _+ 9.1 h

7.9 + 5.9 19.7 +_ 5.2 37.8 + 10.6 ~'

'.

78

100

Captopril + [D-Ar gTJdermorl~n

em

80

~

80 =*

o

8o 50

50

i 20 u

20

~. 5

I.~6 0 **

5 i

,De

i

i

0.3

1

~40

•

i

i

¢

i

#

3

10

30

1O0

300

1000

Dole (ncnol ' ~ U l O )

Fig. 3. Effects of captopril and thiorphan on the antinociccptivc activity produced by i.c.v. [l)-ArgZ]dermorphin. Captopril (circle symbols) or thiorphan (square symbols) was co-administered i.c.v. with [D-Arg2]dermorphin (136.8 pmol). The horizontal axis represents the dose of captopril and thiorphan.

2O

-2(

o

is

45

ao

Time

after injection

(mm)

Fig. 2. Time course of the effect of i.e.,,', administered [D-ArgZJder morphin with captopril in the mouse tail pressure lest. The doses of [D-Arg2]dermorphin used were 681.7 (e), 390 (,.~) and 136.8 (zx) pmol. Captopril (50 p.g) was co-administered i.c.v, with each dose of [D-Arg2]dermorphin. * P < (1.05, * * < (1.01 when compared to controis treated with Ringer solution and captopril (square symbols).

hibitory potency of thiorphan o n the antinociceptive activity induced by [D-Arg2]dermorphin was 100 times less than that of captopril. However, the antinociccplive effect of the parent heptapetide, dermorphin, was not significantly affected by captopril or thiorphan (table 2).

3.3. Potentiation of dermorphin-induced antinociception by captopril and amastatin Simultaneous injection of dermorphin with two peptidase inhibitors, captopril and amastatin, brought about a marked potentiation and increased duration of dermorphin-induced antinociception in mice (fig. 4). Even a small dose of dermorphin (1.3 pmol) in combination with both peptidase inhibitors had a morc potent and long-lasting effect than 2.7 pmol of dermorphin alone. The EDs. value of dermorphin was reduced by one-third when it was combined with both peptidase inhibitors and the difference was statistically significant (table 1).

100

80

o°

~ 60

**

i

I

;0

,:5

"6 240

20

-20

.

0

.

.

15

.

3"o

.

.:=.~-v,~--/~,~

4"s

6o 9 0 1 2 0 1 8 0 0

....

* .;, , .:,-=

is

Time

after

60 9 0 1 2 0 injection (rain)

Fig. 4. Comparison of time course of the effect of i.c.v, dermorphin alone and in combination with amastatin and captopril in the mouse tail pressure test. The doses of dcrmorphin used were 12.9 (e), 7.6 (o), 4.5 ( • ) , 2.7 (zx) and 1.3 ( • ) pmol. Amastatin (1(1 p,g) and captopril (50 ,u,g) were co-administered i.c.v, with each dose of dermorphin. * P < 0.05, ** P < 0.01 when compared to Ringer solution treated-controls (square symbols in the right panel) and controls treated with amatatin and captopril (square symbols in the left panel). "P < 0.05, "'P < 0.01 when compared to dcmorphin alone.

79

4. Discussion

Dcrmorphin, a heptapcptide with exceedingly potent and long-lasting opioid activity is the first example of a new class of opioid peptides occurring in amphibian skin (Erspamcr and Mclchiorri, 1980). Thc chemical structure of dcrmorphin presents the unique feature of having a D-AIa residuc incorporated in its N-terminal sequence. The L-Ala dermorphin is virtually inactive. Likewise, in cnkcphalin analogues, the substitution of Gly 2 with D-AIa resulted in pcptides with considerably increased activity, as rcprescnted by [D-Ala2,MctS]cnkephalinamidc (Pcrt et al., 1976) or FK-33824 (Rocmer ct al., 1977). The rcleasc of Tyr from these derivatives having the Tyr ~-D-Ala2 unit was slower than from cnkephalin upon incubation with mousc brain extract (Huguenin and Maurer, 1980). The 1-4 fragment of dermorphin, Tyr-D-Ala-Phc-Gly, was more slowly cleaved by aminopcptidase M than was the tetrapeptidc having thc L-amino acid in the second position (Sasaki ct al., 1985). Inferring from evidencc that aminopeptidasc is important in the tcrmination of the response produced by enkcphalin analogues and the N-tcrminal 1-4 fragment of dcrmorphin, the release of Tyr might be the determinant of dcrmorphin catabolism. This concept is supported by data suggesting that Dcs-Tyr~-dermorphin is biologically inactive (Suzuki et al., 1985). In thc present study, concurrent administration of amastatin did not affect antinociception produccd by 2.6 and 4.5 pmol of dermorphin. Howevcr, larger doses (7.6 and 12.9 pmol) of dermorphin in combination with amastatin, produced antinociceptivc effects which lasted much longer than that of dcrmorphin alone (table 2). These data suggest that enzymatic resistance of the Tyr~-D-Ala 2 bond in the molecule of dermorphin or thc 1-4 fragment (TyrD-Ala-Phc-Giy) may bc intensified by brain aminopeptidase inhibition with amastatin. Dcrmorphin is rapidly cleaved at Gly4-Tyr5 in rat kidney to release thc N-terminal 1-4 fragment (Scalia et al., 1986) as one of the main degradation products, while this fragment is only formed slowly by brain pcptidascs (Negri and Improta, 1982). In the previous study (Sato et al., 1987), wc havc found that the antinociceptive effect of the tctrapeptide ('Fyr-D-AlaPhe-Gly, EDs0 valuc = 510.0 pmol/mousc determined by the tail pressure method) was approximately 84 times less potent than that of the parcnt peptide when administered i.c.v. This indicates that cleavage of thc Glya-Tyr 5 bond may be one of the inactivating mechanisms for dermorphin. The duration of antinociception was not significantly changed when dermorphin was co-administered with captopril (table 2). When a combination of amastatin and captopril was included, substantial inhibition of the cleavage of three bonds, Tyr ~D-AIa 2, Gly4-Tyr s and Pro6-Scr 7 was observed on in-

cubation with rat brain enzyme (Sasaki ct al., 1985). Together with the above finding, the marked cnhancement of dcrmorphin-induccd antinociception by captopril and amastatin (fig. 4) clearly indicates that a full sequence of dermorphin is required to express the most powerful intrinsic opioid activity. In fact, even the removal of one C-terminal amino acid from the dermorphin molecule was found to diminish the activity rclativc to that of the parent hcptapcptidc in the structure-activity relationship study (Broccardo et al., 1981). Our present findings suggest that the predominant catabolic pathway modulating dermorphin-induced antinociception by neuropcptidascs may bc cleavage of the Tyr t-D-Ala 2 bond, and the other inactivating process may produce the N-terminal 1-4 fragmcnt which is less potcnt than its parent heptapeptide. Captopril (Stincet al., 1980) and thiorphan (Roques ct al., 1980) were found to facilitate the antinociccptivc cffcct of cnkephalins. However, enkcphalinase and angiotcnsin-convcrting enzyme, in spite of their similarities, are not identical. Captopril, an inhibitor of angiotensin converting enzyme, displays a specificity pattern, being much morc potent on this cnzymc than on cnkephalinase. This enzyme inhibitor is 20 timcs more potent than thiorphan as an inhibitor of angiotensinconverting enzyme (Roques ct al., 1980). The antinociceptive activity of [D-Arg2]dermorphin was markedly decreased by captopril or thiorphan. Furthermore, infcrring from the fact that the inhibitory potency of captopril was 100 times greater than that of thiorphan, angiotcnsin-convcrting enzyme but not enkcphalinase may be a key enzyme to cleave the Gly4-Tyr 5 bond of [D-Arg2]dermorphin. Thc prescnt in vivo experimental rcsults indicate that captopril can show a remarkably different degree of potency in reducing thc antinociception of [D-Arg2]dermorphin. In fact, thc antinociccptive cffect of thc D-Arg2-substituted dermorphin 1-4 fragment was greater than that of [D-Arg2]der morphin. The present results suggest that [D-Arg2]der morphin, after systemic administration, may be rapidly metabolized to Tyr-D-Arg-Phe-Gly, which was found to have not only resistance to cnzymatic attack but also high affinity in competing for the binding of [3H]dihydromorphine (unpublished data). In contrast, the corresponding dermorphin tetrapeptide may only bc an intermediary fragment in an inactivation process.

References Brcx:cardo, M., V. Erspamer, G.F. Erspamer. G. Imix~rta, G. Linaria, P. Melchiorri and P.C. Montecucchi, 1981, Pharmacological data on dermorphins, new class of potent opioid peptides from amphibian skin, Br. J. Pharmacol. 73, 625. Dunnett. C.W.. 1964, New tables for muhiplc comparisons with a control, Biometrics 20, 482.

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