In vitro profile of some opioid pentapeptide analogues

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European Journal of Pharmacology, 49 (1978) 313--317 © Elsevier/North-Holland Biomedical Press

Short communication IN V I T R O P R O F I L E OF SOME OPIOID PENTAPEPTIDE A N A L O G U E S JOHN S. SHAW and MICHAEL J. TURNBULL

Department of Biology, I.C.I. Limited, Alderley Park, Macclesfield, Cheshire, U.K. Received 20 March 1978, accepted 7 April 1978

J.S. SHAW and M.J. TURNBULL, In vitro profile of some opioid pentapeptide analogues, European J. Pharmacol. 49 (1978) 313--317. The opiate agonist potency of thirteen synthetic enkephalin pentapeptides has been examined on the electrically stimulated guinea pig ileum and mouse vas deferens preparations in comparison with methionine and leucine enkephalins, ~-endorphin and normorphine. Their antagonism by naloxone (Ke) was also assessed on each preparation. Our findings are compatible with, and are discussed in the context of, the hypothesis that these preparations possess at least two populations of receptor. Enkephalin analogue

Mouse vas deferens

Naloxone

1. Introduction

Since the isolation and identification b y Hughes et al. (1975a) of the t w o pentapeptides leucine and methionine enkephalin, several workers have demonstrated that these compounds produce similar effects to the classical opiate agonists in a variety of test situations. The enkephalins have thus been demonstrated to inhibit the contractions of the field stimulated guinea pig ileum (Lord et al., 1976) and mouse vas deferens (Hughes et al., 1975a) preparations. They also bind to opiate receptors in guinea pig brain homogenates {Lord et al., 1976). However, it has recently b e c o m e apparent that differences do exist between the enkephalins and the classical opiate agonists. Thus, whilst the classical c o m p o u n d s are more potent in the guinea pig ileum assay than on ~he mouse vas deferens, the reverse is true of t h e enkephalins. Furthermore, whilst similar Concentrations of naloxone are required to antagonise the classical opiates on the vas and ileum, and to reverse the enkephalins on the

Guinea pig ileum

Opiate receptor

ileum, considerably more naloxone is required to antagonise the enkephalins on the mouse vas deferens (Lord et al., 1976). These findings were interpreted as evidence for more than one t y p e of opiate receptor. Subsequently, Lord et al. (1977) demonstrated that although relative resistance to the action of naloxone on the vas was c o m m o n to all the opioid peptides which t h e y examined, some analogues of enkephalin did not exhibit the expected increase in p o t e n c y on the mouse vas deferens compared to their ileum activity. The larger opioid peptide ~-endorphin (Bradbury et al., 1976) also appeared to fall into this latter category. Thus, the opioid peptides can be distinguished from the classical opiate agonists on the grounds of their resistance to naloxone antagonism on the mouse vas deferens, and may be further sub
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phalin analogues. The susceptibility of these agonists to the narcotic antagonist naloxone has also been determined.

2. Materials and methods

J.S. SHAW, M.J. TURNBULL

enkephalin determined immediately before and after the test compound. Antagonist potency was expressed as Ke (Kosterlitz and Watt, 1968} and was calculated from complete dose--response curves in the presence and absence of naloxone.

2.1. Drugs 3. Results Normorphine was a gift from Burroughs Wellcome Ltd., and naloxone was donated b y Endo Laboratories Inc. The enkephalin analogues and the human ~ n d o r p h i n were synthesised b y Dr. J.S. Morley's Group, Chemistry Department, ICI Pharmaceuticals Division; the purity of analogues was established as > 9 5 % by the criteria given previously (Dutta et al., 1977). All drugs were prepared as solutions in distilled water. 2.2. Guinea pig ileum assay The coaxially stimulated guinea pig ileum preparation was similar to that described b y Kosterlitz and Watt (1968), although it was not found necessary to add mepyramine or hexamethonium to the Krebs solution. The stimuli (0.5 msec, 0.1 Hz at 1.5 X maximal voltage) were generated b y a Grass S88 stimulator. The contractions were recorded isometrically using an Ether UF1 d y n a m o m e t e r and a Devices MX4 chart recorder. 2.3. Mouse vas deferens assay The method employed was a modification of that described b y Hughes et al. (1975b). In our preparation a larger (9 ml) organ bath, filled with Mg2÷-free Krebs solution was employed. Short trains of stimuli (1 msec pulses at 50 Hz for 100 msec) at a voltage of 1.25 X maximal were repeated at 10 sec intervals. The contractions were recorded using a Palmer 4 1 1 / 1 3 2 6 isotonic transducer with 100 mg tension and a Servoscribe chart recorder. In both preparations, agonist p o t e n c y (ICs0) was calculated from dose--response plots and expressed as a ratio to the ICs0 of methionine

The potencies of the compounds relative to methionine enkephalin in both the guinea pig ileum and mouse vas deferens preparations are listed in table 1, as are the Ke values for naloxone against each agonist. Relative potencies are used in preference to ICs0 values since the former were subject to less variation. The ICs0 values for methionine enkephalin were 149 + 15.8 nM in the guinea pig ileum and 97.1 + 11.0 nM in the mouse vas deferens. In each tissue the Ke values for naloxone covered approximately a two-fold range. However, in the ileum only the two extremes of the range differed significantly from each other (P < 0.05, Student's t-test) whereas in the vas deferens all the c o m p o u n d s tested were significantly different (P < 0.05) from either the higher or lower extreme of the range and the difference between the extremes was highly significant (P < 0.001). Table 2 lists, in ascending order, the ratio between p o t e n c y on the guinea pig ileum and the mouse vas deferens assays for each comp o u n d and serves to demonstrate the large range of ratios obtained. In fact, there was no significant correlation between p o t e n c y on the ileum and the vas, nor b e t w e e n the values on the two preparations.

4. D i s c u s s i o n

The results presented in table 1 clearly demonstrate several points. First, the concentration o f naloxone required to antagonise the opioid peptides on the mouse vas deferens is considerably greater than that required to antagonise either the same corn-

ENKEPHALIN ANALOGUES IN VITRO

315

TABLE 1 The agonist potencies, and reversal by naloxone, of the enkephalins, normorphine and ~-endorphin in the guinea pig ileum and the mouse vas deferens. Mouse vas deferens

Guinea pig ileum

R 1

K e 2 (nM)

R I

Met-enkephalin Leu-enkephatin Tyr-D-Ala-Gly-Phe-Met -NH2 Tyr -D-Ser-Gly-Phe-Met -OMe Tyr-D-Met -Gly-Phe-Leu-OMe Tyr -D-Met-Gly-Phe-Leu-NH2 Tyr -D-Ala-Gly-Phe-Pro-NH2 Tyr -D-Ser-Gly-Phe-Pro-NH2 Tyr-D-Met ~:]ly-Phe-Pro-NH2 Tyr-D-Ala~:~ly-Phe-Pro-NHEt Tyr-D-Ser~:~ly-Phe-Pro-NHEt Me Tyr -Gly-Gly-Phe-Leu-NH2 Me Tyr-D-Ala-Gly-Phe-Leu-NH2 Me Tyr -D-Ser-Gly-Phe-Pro-NHEt OH

1 3.63 (1.02) 3.81 (0.81) 29.5 (3.95) 2.99 (0.24) 3.68 (0.39) 0.49 (0.07) 0.19 (0.09) 0.97(0.10) 0.53 (0.09) 0.31 (o.o6) 0.85 (0.16) 6.4 (0.65) 0.07 (0.013)

28.3 (4.3) 3 21.4 (4.04)4 36.6 (2.8) 3 29.2(4.57)3 25.4 (2.59) 3,4 23.9 (3.26) 3,4 22.4 (3.71) 4 20.8 (2.76) 4 21.8 (3.8) 4 23.4 (4.11)4 22.1 (4.23)4 16.4 (1.61)4 33.2 (2.81) 3 15.3 (1.73) 4

H 2 N ~ CO-D-Ala-Gly-Phe-Leu-OMe Normorphine ~-Endorphin

1.14 (0.21) 0.25 (0.06) 0.50 (0.02)

19.8 (1.25) 4 4.75 (0.59) 3,4 30.5 (2.11)3

1 0.48 7.75 14.3 63 27.5 5.49 2.20 29.2 2.72 2.11 4.85 10.78 2.18

K e 2 (nM)

(0.02) (0.64) (2.17) (12.3) (4.7) (0.89) (0.18) (2.94) (0.32) (0.42) (0.84) (2.7) (0.37)

7.67 (1.41) 2.17 (0.27) 4.52(1.23)

1.94 1.74 2.91 2.45 1.86 1.31 2.43 1.92 2.13 1.85 1.91 2.44 1.73 1.76

(0.21) (0.21) (0.68) (0.37)6 (0.37) (0.15)s (0.51) (0.27) (0.22) (0.39) (0.29) (0.53) (0.16) (0.36)

1.69 (0.12) s 1.83 (0.18) 2.53 (0.27)6

I Agonist potency relative to methionine enkephalin measured in the same experiment. Values are means with S.E.M. in parentheses. 2 The Ke values were determined from complete dose--response curves in the presence and absence of naloxone. 3 Significantly different from Me Tyr-D-Ser-Gly-Phe-Pro-NHEt (P < 0.05, Student's t-test). 4 Significantly different from Tyr-D-Ala-Gly-Phe-Met-NH 2 (P < 0.05). s Significantly different from ~-endorphin (P < 0.05). 6 Significantly different from Tyr-D-Met~31y-Phe-Leu-NH2 (P < 0.05).

pounds on the guinea pig ileum, or normorphine on both the vas and the ileum. This observation confirms and extends the findings of Waterfield et al. (1977). A second point to emerge from this study is that the Ke value for naloxone against the peptides is not a constant on the mouse vas deferens, and probably also varies on the guinea pig ileum. This is consistent with the recent finding of Ronai et al. (1977) who demonstrated t h a t the Ke for naltrexone against another group of enkephalin analogues was subject to a four-fold variation. However, on the guinea pig ileum the variation was

much less marked. In an a t t e m p t to explain these findings Ronai et al. suggested that the interaction between a peptide and the receptor could induce a change in the receptor, thus altering the kinetics of the agonistantagonist interaction. Whilst we have no evidence to either support or refute this hypothesis, an alternative explanation could be that two populations of receptor exist in the mouse vas deferens, one o f which is relatively resistant to naloxone. Thus, the Ke value for naloxone against a particular agonist would depend on that compound's relative affinity for the two receptor types. If this

316

J.S. SHAW, M.J. TURNBULL

TABLE 2 Enkephalin analogues ranked in order of the ratio of their potency on the guinea pig ileum and mouse vas deferens preparations. Enkephalin analogue Tyr-Gly-Gly-Phe-Leu-OH Tyr -D-Ser -Gly-Phe-Met-OMe Tyr-Gly-Gly-Phe-Met-OH Me Tyr-D-Aia-Gly-Phe-Leu-NH2 Me Tyr -D-Ala-Gly-Phe-Met-NH2 Me Tyr -D-Ala-Gly-Phe-Pro-NHEt Me Tyr-Gly-Gly-Phe-Leu-NH2 OH

tt2N~CO-D-Ala-Gly-Phe-Leu-OMe 'ryr-D-Ser-Gly-Phe-Pro-NHEt Tyr -D-Met-Gly-Phe-Leu-NH2 Tyr-D-Ala-Gly-Phe-Pro-NH2 Tyr-D-Ser-Gly-Phe-Pro-NH2 Tyr-D-Met-Gly-Phe-Leu-OMe Tyr-D-Met-Gly-Phe-Pro-NH2 Me Tyr-D-Ser-Gly-Phe-Pro-NHEt

Potency ratio ileum/vas 0.13 0.48 1~ 1.68 2.03 5.13 5.7

6.7 6.8 7.5 11.2 11.6 21.1 30.1 31.1

were the case, a c o m p o u n d such as normorphine with a low Ke would act predominantly at a naloxone-sensitive receptor, whereas a peptide with a high Ke (e.g./~-endorphin) would act at a receptor which is relatively insensitive to naloxone. Compounds with intermediate values (e.g. leucine-enkephalin) would act at both receptor types. This hypothesis thus offers an explanation for both the range of Ke values found amongst the peptides, and the large difference between the Ke values against the peptides and the classical opiate agonists. Another finding which emerged from our study was that the agonist potency of the peptides on the mouse vas deferens appeared to bear little resemblence to the potency in the guinea pig assay (table 2). Similar results were obtained b y Lord et al. (1977) with a different group of enkephalin analogues, although they also tested ~-endorphin and ob-

tained results similar to those reported in this communication. As an explanation, Lord et al. suggested that the effects of the peptides on the guinea pig ileum may be mediated via a single type of receptor (p-receptor) whilst on the mouse vas deferens b o t h p-receptors and a second class of opiate receptor (the 8receptor) is involved. However, it is probably an oversimplification to explain all differences between the vas deferens and the ileum in terms of different receptor populations since other factors, such as enzymic stability of the peptides are almost certain to be of importance. Nevertheless, the findings of our study lend support to the hypothesis that the receptor populations of the guinea pig ileum and mouse vas deferens are different. Furthermore, if, as suggested b y Lord et al. (1977), the ileum receptor and the naloxone sensitive receptor of the vas deferens are identical (i.e. p-receptors), c o m p o u n d s with a high ileum/vas potency ratio would be acting predominantly at p-receptors and would thus be expected to be relatively easily reversed b y naloxone on the vas. Conversely, c o m p o u n d s with low potency ratios, which would be acting predominantly at 6-receptors, would be more difficult to antagonise. Thus if the ileum/vas ratios were to be unaffected b y such factors as enzymic degradation, the ratios would be expected to vary inversely with the Ke on the mouse vas deferens. Such a correlation does, in fact, hold if only analogues with a D-amino acid in position 2 are considered. Thus the peptides with the three highest Ke values also have the lowest potency ratios. Similarly, the c o m p o u n d with the lowest Ke has the highest potency ratio. Whilst we have no data concerning the stability of these peptides in our test situation, we and others (Pert et al., 1976) have shown that D-Ala2-enkephalin amides are relatively stable in the presence of tissue homogenates. Thus, whilst far from being conclusive, these findings lend support to the hypothesis that/~-receptors occur in b o t h the guinea pig ileum and the mouse vas deferens, whereas 5-receptors occur only in the vas. It therefore follows that enkephalins act at both

ENKEPHALIN ANALOGUES IN VITRO

p- and 8-receptors since they are both potent agonist on the guinea pig ileum (p-receptors) and relatively resistant to the antagonist action of naloxone on the mouse vas deferens. However, the physiological role of these receptors remains to be elucidated.

References Bradbury, A.F., D.G. Smyth, C.R. Snell, N.J.M. Birdsall and E.C. Hulme, 1976, C-fragment of lipotropin has a high affinity for brain opiate receptom, Nature 260, 793. Dutta, A.S., J.J. Gormley, C.F. Hayward, J.S. Morley, J.S. Shaw, G.J. Stacey and M.J. Turnbull, 1977, Analgesia following intravenous administration of enkephalin analogues, Life Sci. 21,559. Hughes, J., H.W. Kosterlitz and F.M. Leslie, 1975b, Effect of morphine on adrenergic transmission in the mouse vas deferens. Assessment of agonist and antagonist potencies of narcotic analgesics, Brit. J. Pharmacol. 53, 371. Hughes, J., T.W. Smith, H.W. Kosterlitz, L.A. Fother-

317 gill, B.A. Morgan and H.R. Morris, 1975a, Identification of two related pentapeptides from the brain with potent opiate agonist activity, Nature 258, 577. Kosterlitz, H.W. and A.J. Watt, 1968, Kinetic parameters of narcotic agonists and antagonists, with particular reference to N-allylnoroxymorphone (Naloxone), Brit. J. Pharmacol. 33,266. Lord, J.A.H., A.A. Waterfield, J. Hughes and H.W. Kosterlitz, 1976, Multiple opiate receptors, in: Opiates and Endogenous Opioid Peptides, ed. H.W. Kosterlitz (North-Holland Publishing Co., Amsterdam) p. 275. Lord, J.A.H., A.A. Waterfieid, J. Hughes and H.W. Kosterlitz, 1977, Endogenous opioid peptides: multiple agonists and receptors, Nature 267,495. Pert, C.B., A. Pert, J.K. Chang and B.T.W. Fong, 1976, (D-AIa2)-Met-enkephalinamide: A potent, long-lasting synthetic pentapeptide analgesic, Science 194,330. R6nai, A.S., I. Berz6tei and S. Bajusz, 1977, Differentiation between opioid peptides by naltrexone, European J. Pharmacol. 45,393. Waterfield, A.A., R.W.J. Smokcum, J. Hughes, H.W. Kosterlitz and G. Henderson, 1977, In vitro pharmacology of the opioid peptides, enkephalins and endorphins, European J. Pharmacol. 43, 107.