Correlation between pharmacological and opiate receptor binding activities of tetrapeptide acylhydrazide analogs of enkephalin

European Journal o f Pharmacology, 72 (1981) 297--304

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Elsevier/North-Holland Biomedical Press

CORRELATION BETWEEN PHARMACOLOGICAL AND OPIATE RECEPTOR BINDING ACTIVITIES OF TETRAPEPTIDE ACYLHYDRAZIDE ANALOGS OF ENKEPHALIN KIYOHISA KAWAI *, HARUMITSU ISHII, T A K A Y U K I DOI, SHIGEO TAMURA, ** SUSUMU SHINAGAWA and ** MASAHIKO FUJINO

Medicinal Research Laboratories, ** Chemical Research Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., 1 7-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532, Japan Received 13 January 1981, revised MS received 24 March 1981, accepted 7 April 1981

K. KAWAI, H. ISHII, T. DOI, S. TAMURA, S. SHINAGAWA and M. FUJINO, Correlation between pharmacological and opiate receptor binding activities o f tetrapeptide acylhydrazide analogs o f enkephalin, European J. Pharmacol. 72 (1981) 297--304. The pharmacological and opiate receptor binding activities of four synthetic tetrapeptide acylhydrazide analogs of enkephalin (EK compound) were compared with those of reference compounds. EK-159 administered subcutaneously was less analgesic, while EK-209, EK-259 and EK-272 were more potent than morphine in the hot plate, Haffner's and phenylquinone writhing tests in mice. EK-272 being the most active was comparable to what was found with FK-33824. ICs0 values of EK compounds in a sodium-free medium in the opiate receptor binding assay were lower than the values seen with morphine. The binding activities of EK compounds in 100 mM NaCi medium showed a clearer correlation with their analgesic activities than was seen in the absence of sodium ion. The binding affinity of EK-272 was the highest and the sodium response ratio (ICs0 + NaC1/ICs0 -- NaC1) was slightly lower than that of pentazocine. The analgesic action in rodents, respiratory inhibition in rabbits, inhibition of intestinal movement in mice, and hyperthermic action in rats, were all qualitatively, but not quantitatively similar to the effects seen with morphine. The analgesic action of EK compounds was relatively resistant to the antagonizing effect of naloxone and the EK compounds modified the analgesic action of morphine. These properties were inversely correlated with the sodium response ratios. Enkephalin analog

Analgesia

Opiate receptor binding

1. Introduction With the establishment of enkephalin as an endogenous morphine-like peptide in the mammalian brain as well as in the other tissues (Hughes et al., 1975), investigations have centered on possible clinically available analgesic derivatives of the peptide. FK33824 (Sandoz), one of the derivatives, is several times more analgesic than morphine in experimental animals (Roemer et al., 1977; Pless et al., 1979) and is reportedly also effective for the clinical t r e a t m e n t of cancer-asso-

* To w h o m correspondence should be addressed: 1785, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532, Japan.

ciated pain (Cardinaux, 1979). When given subcutaneously, several derivatives among a series of synthetic tetrapeptide acylhydrazide analogs of enkephalin from the Takeda laboratory proved to be considerably more active than morphine when analgesia was evaluated with the hot plate technique in mice (Fujino et al., 1979). Since the mixed agonist-antagonists have low physical dependence liability, some compounds such as pentazocine, butorphanol and buprenorphine have been introduced for clinical application (Pircio et al., 1976; Cowan et al., 1977). At present, it is unclear whether the sodium response ratio obtained in the opiate receptor binding assay, as a predictor of narcotic antagonistic property of opiates (Pert and Snyder, 1974), is an effective

0014-2999/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press

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K. K A W A I E T AL.

predictor of antagonistic properties of opioid peptides. The present study was an a t t e m p t to elucidate the correlation between the pharmacological properties of 4 representative tetrapeptides and their receptor binding activity. 2. Materials and methods

Male Ta : CF1/b and Slc: ICR" mice, male Jcl : SD rats and male albino rabbits bred by Seiwa Experimental Animals {Fukuoka-ken) were used. Chemical structures of enkephalins and 4 tetrapeptide acylhydrazides synthesized by Fujino et al. {1979) are shown in fig. 1. Morphine HC1 (Takeda), FK-33824 (Sandoz), levorphanol tartrate (Hoffman La Roche), pentazocine (Grelan), naloxone HC1 (Endo Lab.), levallorphan tartrate (Hoffman La Roche), dextrallorphan HBr (Hoffman La Roche) and [3H]naloxone HC1 (New England Nucl. Co., 15.2 Ci/mmol) were used. 2.1. Analgesic assays Test c o m p o u n d s were dissolved in physiological saline, and administered subcutaneously (s.c.). All solutions were coded and tests were carried o u t under blind conditions. H o t plate test: groups of 10 CFI/b mice weighing 18--23 g were used for the analgesic evaluation, following the method of Woolfe and Macdonald {1944). The animals were placed on a h o t plate the temperature of which was 55 + 0.5°C. Mice which licked their hind feet or exhibited jumping behavior within 20 sec were selected and given the test compounds s.c. The lag time for such Met-Enkephalin H-Tyr-Gly-Gly- Phe-Met-OH Leu-Enkephalin H-Tyr-Gly-Gly-Phe-Leu-OH EK-159 EK-209 EK-259 EK-272

H-Tyr-D-Ala-Gly-Phe-NHNHCOCH2CH2CH3. AcOH H-Tyr-D-Ala-Gly-MePhe-NHNHCOCH2CH3.AcOH H-Tyr- D-Met(O)-Gly-Phe-NHNHCOCH2CH3.AcOH H-Tyr- D-Met(O)-Gly-MePhe-NHNHCOCH2CH3.AcOH

Fig. 1. Chemical structures of enkephalins and E K compounds.

responses was measured 10, 30 and 60 min later. The lack of the nociceptive response for more than 60 sec was regarded as positive. The values for % analgesia {number of positive animals/number of tested animals ×100) in the respective dosage groups obtained at the time of the maximal analgesic response were used for calculation of the EDs0 values of test c o m p o u n d s following the probit method. The analgesic potencies of test c o m p o u n d s relative to that of morphine were calculated following the parallel line assay (Finney, 1964). Haffner's m e t h o d (1929): groups of 10 Sic : ICR mice weighing 18--22 g were used. After turning back, biting and squeaking of the animal had been seen in response to pinching the tail r o o t by a forceps, the same mechanical stimulation was repeated 15, 30, 45 and 60 min after the s.c. administration of the test compounds. The lack of nociceptive responses was regarded as an indication of analgesia. Phenylquinone writhing (Siegmund et al., 1957): groups of 10 Slc: ICR mice weighing 18--22 g were given i.p. 0.02% phenylquinone in 5% ethanol saline in a dose of 0.1 ml/10 g b o d y weight 30 min after administration of the compounds. The frequency of abdominal writhing and stretching was then counted for 20 min. A decrease of over 50% from the mean frequency of the control animals was regarded as an indication of analgesia. Modification of analgesic activity of test compounds b y combined use of naloxone: following the h o t plate method, the values of % analgesia in the respective dosage groups were obtained 10, 30 and 60 min after the administration of test compounds. The EDs0 of test c o m p o u n d s alone or in combination with 0.05 mg/kg s.c. of naloxone was calculated. Modification of analgesic activity of morphine by combined use of test compounds: following Haffner's method, the modification of analgesic activity of 12.5 mg/kg s.c. of morphine, previously confirmed to produce 100% analgesia with the combined subcutaneous administration of the test compounds, was observed after 30, 60 and 120 min.

TETRAPEPTIDE ACYLHYDRAZIDE ANALOGS OF ENKEPHALIN

2.2. Opiate receptor binding assay Rats weighing 180--200 g were decapitated and the brains rapidly removed. The brains minus cerebella were homogenized in 30 vol of 50 mM Tris-HC1 buffer (pH 7.5 at 25°C) in a Polytron homogenizer (PT-10) with a setting at 6 for 20 sec and centrifuged for 10 min at 40 000 × g. The pellets were suspended in 30 vol of Tris buffer and incubated for 30 min at 37°C. The homogenate was recentrifuged for 10 min at 4 0 0 0 0 × g and the pellets were resuspended in an a m o u n t of ice-cold Tris buffer equal to the original volume. A mixture in a final volume of 2.0 ml, consisting of 2 nM [3H]naloxone, the test compound and 50 pg/ml of bacitracin in the presence of 1 pM levallorphan or l p M dextrallorphan in Tris buffer, was incubated for 3 h at 0 °C. Bacitracin was used to exclude the possibility of enzymatic degradation of enkephalin analogs (Simantov and Snyder, 1976). The reaction was terminated by filtration on a glass fiber filter (Whatman GF/B). The filters were washed under vacuum with two 5 ml portions of ice-cold Tris-buffer, and left overnight in a 5 ml scintillation cocktail (1 vol water, 3 vol Triton X-100, 6 vol toluene, 0.7 weight/vol % PPO and 0.05 weight/vol % POPOP). Radioactivity was counted by liquid scintillation spectrometry (Atoka LSC-653) at 30% efficiency. Specific opiate receptor binding was defined as the difference in binding in the presence of levallorphan and in the presence of dextrallorphan. All data are means of 4-6 experiments. In each experiment, a compound was tested in triplicate at 4--8 different concentrations between 10-9--10 -7 M. ICs0, the concentration which inhibited [aH]naloxone binding by 50%, was obtained from a log-probit graph. The protein c o n t e n t was determined by the m e t h o d of Lowry et al. (1951). Mean specific [3H]naloxone bindings in the absence and presence of 100 mM sodium ion were 65% and 84% of the total bindings, respectively. Mean concentration of

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protein in the reaction mixture was 0.6 mg/ ml.

2.3. Respiratory depression The expiratory pressure changes of the respiratory tract in unanesthetized rabbits were recorded via an Ugo Basile recording microdynamometer, following the m e t h o d of Gaddum (1941). Depression of the respiratory frequency and pressure by injecting test compounds into the marginal ear vein were compared to the maximal effects.

2.4. Inhibition of intestinal charcoal transfer Groups of 10 Slc: ICR mice weighing 18-22 g were fasted for 6 h and then treated s.c. with test compounds. Thirty min later, 0.2 ml of a slime mixture consisting of 1 g of charcoal powder, 0.5 g of gum acacia and 7 ml of water was administered p.o. The animals were killed by cervical dislocation 15 min later, and the intestine from the pylorus to the cecum was removed to determine the transfer of the charcoal.

2.5. Febrile response in rats Groups of 6 rats weighing 190--210 g were fasted for 16 h, and then deprived of drinking water. Rectal temperature was measured 3 times (basal value). The effect of the s.c. administration of test compounds was evaluated hourly for 4 h. The deviations of the mean temperature in the treated group from the ones of the control group were summed within 4 h to obtain a measure of the total hyperthermic effect.

3. Results

3.1. Analgesic activity Hot plate test: EK compounds showed a dose-dependent analgesia in mice. Parallelism was observed among the dose-response lines

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TABLE 1 Comparative analgesic activities of EK compounds in the hot plate, Haffner's and phenylquinone writhing tests in mice. Compound

Hot plate test

Haffner's test

EDs0 mg/kg s.c.

Relative potency

P.T. a

1 0.67 (0.47--0.92) 1.60 (1.13--2.23)

30 10 30

3.73 (2.11--5.34) 8.15 (5.45--11.3) 2.14 (1.26--3.20)

EDs0 mg/kg s.c.

Morphine EK-159 EK-209

1.76 (1.44--2.40) 2.57 (2.00--4.35) 1.19 (0.90--2.21)

EK-259

0.41 (0.33--0.57)

4.43 (3.24--6.05)

60

0.37 (0.21--0.61)

EK-272 FK-33 824

0.31 (0.17--0.49) 0.36 (0.21--0.69)

5.66 (3.54--10.0) 4.99 (3.05--8.60)

60 30

0.31 (0.18--0.54) 0.39 (0.27--0.60)

Levorphanol

0.36 (0.18--0.47)

5.60 (3.50--10.0)

30

0.625 ca: max.

Pentazoeine

9.90 (2.80--25.8)

0.18 (0.11--0.33)

10

a Peak time (min).

b

b

60 ca: max.

95% confidence limits in parentheses.

of EK compounds, morphine, FK-33824, levorphanol and pentazocine. The EDs0 values and relative potencies are shown in table 1. The time of the peak analgesia after the s.c. administration of the test c o m p o u n d s varied considerably; 10 min for EK-159 and pentazocine, 30 min for EK-209, FK-33824, morphine and levorphanol, and 60 min for EK-259 and EK-272. EK-159 was less analgesic than morphine, whereas the other EK c o m p o u n d s were more active. Haffner's method: the analgesic activity of EK-159 was less than that of morphine,

whereas the activity of the other EK compounds was superior to that of the latter. EKTABLE 3 Inhibitory effects of EK compounds and reference compounds on morphine analgesia (12.5 mg/kg) in Haffner's test. Compound

Dose (mg/kg s.c.)

% inhibition

EK-159

0.625 1.25 2.5

20 33.3 30

EK-209

0.313 0.625 1.25

30 43 20

EK-259

0.04 0.08 0.16

25 53.3 40

TABLE 2 EDs0 ratios of EK compounds, alone and in combination with naloxone (0.05 mg/kg) in the hot plate test. Compound

EDs0 ratio (with naloxone/alone)

EK-272

0.02 0.04 0.08

30 60 30

Morphine EK-159 EK-209 EK-259 EK-272 FK-33 824 Levorphanol

3.55 2.63 2.32 1.86 1.61 2.66 5.30

FK-33 824

0.02 0.04 0.08

10 26.7 20

(2.43--5.78) a (1.94--3.68) (1.70--3.16) (1.31--3.00) (1.05--2.36) (2.01--3.61) (3.91--7.44)

a 95% confidence limits in parentheses.

ADs0 mg/kg Pentazocine Naloxone

8.39 (3.98--13.5) 0.063 (0.005--0.24)

TETRAPEPTIDE ACYLHYDRAZIDE ANALOGS OF ENKEPHALIN

301

Phenylquinone writhing test Relative potency

P.T. a

EDs0 mg/kg s.c.

Relative potency

1 0.47 (0.28-0.75) 1.72 (0.99-2.90) 9.34 (4.72-17.5) 10.9 (4.78-22.3) 9.29 (5.16-16.6

60 60 30 60 60 45

0.39 (0.10-0.56) 0.39 (0.19--0.88) 0.11 (0.04-0.24) 0.06 (0.01-0.11) 0.015 (0.006-0.026) 0.013 (0.006-0.045)

0.91 (0.33-1.81) 3.50 (1.45-6.83) 6.16 (3.02-11.6) 23.0 (10.6-47.2) 19.0 (2.14--69.5)

60% analgesia

45

0.10 (0.05-0.13)

4.11 (2.56-7.11)

40% analgesia

30

1.07 (0.20-4.54)

0.35 (0.12--0.68)

259 and EK-272 in particular showed a p o t e n t activity which was comparable t o t hat seen with FK-33824. The peak analgesic times o f 5 o f 8 c o m p o u n d s were longer than those observed in the h o t plate test. Since the efficacy o f levorphanol and pentazocine was low, the EDs0 could n o t be estimated. P h e n y l q u i n o n e writhing: phenylquinoneinduced writhing in mice was markedly sensit i v e to the s.c. t r e a t m e n t with each of the

TABLE 4 Potencies of opiates and EK compounds in competing for the [3H]naloxone binding sites. Compound

Morphine EK-159 EK-209 EK-259 EK-272 FK-33 824 Met-enkephalin ~-Endorphin Levorphanol Pentazoeine Levallorphan Naloxone

ICs0 (nM)

Ratio +Na/--Na

--NaCI

+100 mM NaC1

11 7.6 5.0 6.0 4.5 3.9 19 6.5 6.0 31 1.4 5.8

710 710 200 88 39 230 500 58o 72 310 2.5 2.8

65 93 40 15 8.7 59 26 89 12 10 1.8 0.48

1

c o m p o u n d s . EDs0 values of EK-159, EK-209, EK-259, EK-272, FK-33824, m orphi ne, levorphanol and pentazocine were about 1/7, 1/11, 1/7, 1/21, 1/28, 1/5, 1/4 and 1/9 of those obtained with the h o t plate test. EK272 which had the greatest anti-writhing effect was 23 times as active as morphine. EK159 was less p o t e n t than morphine. Modification of the analgesic activity o f test c o m p o u n d s by c o m b i n e d use with naloxone: as shown in table 2, the analgesic activity of each of the c o m p o u n d s in the h o t plate test was antagonized by nal oxone at 0.05 mg/kg s.c. However, the antagonizing effect was most evident with levorphanol, followed in decreasing order by morphine, FK33824, EK-159, EK-209, EK-259 and EK272. Modification of the analgesic activity of m o r p h i n e by c o m b i n e d use of test compounds: the analgesic activity of m orphi ne in Haffner's m e t h o d was modified by the small doses of each of the peptide c o m p o u n d s {table 3). T he effect of EK-272 was clearer than the effects o f the other peptides, including FK-33824. The dose ranges were narrow and bell-shaped dose-response relationships were observed. In contrast, b o t h pentazocine and n a l o x o n e dose-dependently antagonized the analgesic action of morphine, giving clear-cut ADs0 values.

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K. KAWAI ET AL.

3.2. Opiate receptor binding activity The ICs0 of morphine, in competing for [3H]naloxone binding to the rat brain homogenates in the absence of sodium ion was 11 nM (table 4). ICs0 values of EK compounds ranged from 4.5 to 7.6 nM. Pentazocine was the least active of the compounds tested. The ICs0 values, e)~cept that of

3.3. Respiratory depression Administered i.v. each of the EK compounds decreased dose-dependently the respiratory rate (fig. 2) and the expiratory pressure (data not shown) in unanesthetized rabbits. EK-159 and EK-209 were less inhibitory than morphine, while EK-259 and EK-272 were inhibitory to a greater extent.

A

°

naloxone, were increased to different extents in the presence of 100 mM NaC1; the sodium response ratios of morphine, EK-159, EK-209 and FK-33824 were higher than 40, the ratio of EK-259 was 15, and that of EK-272 was 8.7. This value was slightly lower than that of pentazocine.

50-

3.4. Inhibition of intestinal charcoal transfer

0 log

mg/kg I.Y.

B

EK-259~

m

E

K

-

~

8

"~ 5O

Intestinal charcoal transfer in mice was inhibited dose-dependently by morphine and EK compounds. EK-159, EK-209 and EK-259 were more potent than morphine. The inhibitory activity of EK-272 was marked and was comparable to that of FK-33824.

3.5. Febrile response in rats

.~_ .;_-

Om61~

0:5

0 .'125

log

i

J 2;5

mg/kg S.C.

°C

C

Morphine and EK compounds increased rectal temperature. The hyperthermic effect of EK-272 was the most potent, followed in decreasing order by EK-259, morphine, EK209 and EK-159. EK-159 was weak in this respect.

Morphine EK-209

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

/

. EK-15g

~

o

0.I~5

01~5

Om'~

t i'0

~0

4~0

log mglkgs.c. Fig. 2. (A) Inhibitory effects of enkephalin analogs and morphine on the respiratory rate in unanesthetized rabbits. (B) Inhibitory effects on charcoal transfer in the small intestine of mice. (C) Hyperthermic effects on normal body temperature in rats.

The characteristics of 4 EK compounds regarding pharmacological effects and receptor binding activity were compared with those of reference compounds. EK-159 was less analgesic than morphine in the h o t plate, Haffner's and phenylquinone w r i t h i n g t e s t s , while the other EK compounds were more active than morphine. In particular, EK-272 was 5.66--23 times as active as morphine in

TETRAPEPTIDE ACYLHYDRAZIDE ANALOGS OF ENKEPHALIN these tests. The replacement D-Ala 2-~ DMet(O) 2 and N-methylation of Phe 4 resulted in a further increase in analgesic action and a decrease in the sodium response ratio. EK159 and EK-209, of the D-Ala 2 type, showed a similar analgesic pattern to that of morphine in so far as they were less effective in Haffner's test ~han in the h o t plate test. EK259 and EK-272, of the D-Met(O) 2 type, were similar to FK-33824 in that they were equieffective in both tests and were most active in the phenylquinone writhing test. ICs0 values of EK compounds ranged from 4.5 to 7.6 nM in a sodium-free medium, thereby indicating binding activities higher than that of morphine. However, the binding activities did not correlate so clearly with their analgesic effects. The binding activities in 100 mM NaC1 medium showed a significant correlation. The latter observations suggest binding activities of these compounds at the sites of action in vivo. The sodium response ratios of pure agonists, pure antagonists and the mixed agonistantagonists are over 10, 1 and 1--10, respectively (Pert and Snyder, 1974). Therefore, EK-159, EK-209, EK-259, FK-33824, morphine and levorphanol could be classified as pure agonists, though the ratios of EK-259 and levorphanol were near 10. The ratios of EK-272, pentazocine and levallorphan were between 1.8 and 10. It has been reported that pentazocine is less sensitive to the antagonizing effect of naloxone than is morphine (Tallarida et al., 1979). Similarly, the analgesic actions of EK compounds, particularly EK-272, proved to be resistant to the naloxone antagonism. Numerous receptor binding and bioassay studies clearly demonstrated the existence of multiple types of opiate receptors. Thus, the analgesic actions of e.g. EK272 may be partly mediated by the non-p type receptor. The validity of the sodium response ratio as predictor of a narcotic antagonistic property of the synthetic opioid peptides remains unknown. It has n o t been proven that opioid peptides have narcotic antagonistic properties.

303

In the case of Haffner's test, we observed an inhibitory effect on the analgesic action of morphine by EK compounds, particularly EK-259 and EK-272. However, the maximal inhibition seen with either c o m p o u n d did not exceed 60%. EK compounds seem to act as agonists in the higher dose ranges. In the case of the h o t plate test, there was little inhibitory effect on the morphine analgesia by these peptides (data n o t shown). All the EK compounds induced respiratory depression in rabbits, inhibition of the intestinal movement in mice and elevation of the body temperature in rats. Morley (1980) has observed that H-Tyr-D-Ala-Gly-Phe (6H)Leu-OH was relatively free from respiratory depressant activity, at least in doses which induced analgesia. In the case of EK compounds, some deviation was found with the respiratory depression by EK-209 (relatively weak), the intestinal movement inhibition by EK-259 and EK-272 (relatively potent), and the hyperthermia by EK-159 (relatively weak). The pharmacological features of the EK compounds we tested differed clearly from those of morphine, thereby indicating the physiological significance of the sodium response ratios of opioid peptides. The search for peptides with lower sodium response ratios is the subject of ongoing studies. Acknowledgements We thank Endo Laboratories for the generous supply of naloxone, Hoffmann La Roche Co. for levallorphan tartrate and dextrallorphan HBr, and M. Ohara, Kyushu University for critical reading of the manuscript.

References

Cardinaux, F., 1979, Details of Sandoz' enkephalin FK-33824, SCRIP No. 382, p. 13. Cowan, A., J.W. Lewis and L.R. Macfarlane, 1977, Agonist and antagonist properties of buprenorphine, a new antinociceptive agent, Br. J. Pharmacol. 60,537.

304 Finney, D.J., 1964, Statistical methods in biological assay, 2nd ed., (Charles Griffin, Inc., Ltd., London). Fujino, M., S. Shinagawa, K. Kawai and H. Ishii, 1979, Tetrapeptide acyl-hydrazide analogs o f enkephalin, Naturwissenschaften 6 6 , 6 2 5 . Gaddum, J.H., 1941, A method of recording the respiration, J. Physiol. 9 9 , 2 5 7 . Haffner, F., 1929, Experimentelle priifung schmerzstillender mittel, Dt. Med. Wsch. 5 5 , 7 3 1 . Hughes, J., T.W. Smith, H.W. Kosterlitz, L.A. Fothergill, B.A. Morgan and H.R. Morris, 1975, Identification of two related pentapeptides from the brain with potent opiate agonist activity, Nature 258, 577. Lowry, D.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193,265. Morley, J.S., 1980, Structure-activity relationships of enkephalin-like peptides, Ann. Rev. Pharmacol. Toxicol. 20, 81. Pert, C.B. and S.H. Snyder, 1974, Opiate receptor binding of agonists and antagonists affected differentially by sodium, Mol. Pharmacol. 10, 868. Pircio, A.W., J.A. Glylys, R.L. Cavanagh, J.P. Buyniski and M.E. Bierwagen, 1976, The pharmacology of butorphanol, a 3,14-dihydroxymorphinan narcotic

K. KAWAI ET AL. antagonist analgesic, Arch. int. Pharmacodyn. 220, 231. Pless, J., W. Bauer, F. Cardinaux, A. Closse, D. Hauser, R. Huguenin, D. Roemer, H. Beuscher and R.C. Hill, 1979, Synthesis, opiate receptor binding and analgesic activity of enkephalin analogues, Helv. Chim. Acta 62, 398. Roemer, D., H.H. Buescher, R.C. Hill, J. Pless, W. Bauer, F. Cardinaux, A. Closse, D. Hauser and R. Huguenin, 1977, A synthetic enkephalin analogue with prolonged parenteral and oral analgesic activity, Nature 268, 547. Siegmund, E., R. Cadmus and G. Lu, 1957, A method for evaluating both non-narcotic and narcotic analgesics, Proc. Soc. Exp. Biol. Med. 95, 729. Simantov, R. and S.H. Snyder, 1976, Morphine-like peptides, leucine enkephalin and methionine enkephalin: Interactions with the opiate receptor, Mol. Pharmacol. 12, 987. Tallarida, R.J., A Cowan and M.W. Adler, 1979, pA2 and receptor differentiation: A statistical analysis of competitive antagonism, Life Sci. 25, 637. Woolfe, G. and A.A. Macdonald, 1944, Evaluation of the analgesic action of pethidine hydrochloride (demerol), J. Pharmacol. Exp. Ther. 80, 300.