Pharmacological assessment of spantide II analogues

ELSEVIER

European Journal of Pharmacology 260 (1994) 121-128

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Pharmacological assessment of spantide II analogues Zun-Yi Wang a, Dong-Mei Feng b, Ya-Li Wang h, Steven R. Tung c, Kin Wong c, Gary R. Strichartz c, Karl Folkers b, Roll H~kanson a,, a Department of Pharmacology, University ofLund, Lund, Sweden b Institute for Biomedical Research, University of Texas at Austin, Austin, TX,, USA cAnesthesia Research Laboratories, Harvard Medical School, Brigham and Women's Hospital, Boston, MA, USA Received 23 February 1994; revised MS received 22 April 1994; accepted 26 April 1994

Abstract

We have studied the structure-activity relationship of a series of tachykinin receptor antagonists based on spantide II. Fifteen novel peptides were tested for their ability to antagonize the electrically evoked tachykinin receptor-mediated response in the isolated rabbit iris sphincter muscle. Substitution or deletion of one to three amino acids in the spantide II sequence caused significant changes in biological activity. Eight of the novel analogues were found to be as potent as or more potent than spantide II and some were found to have better water solubility. We tested the selectivity for different tachykinin receptors of spantide II and two of the eight most potent analogues. They all interacted with tachykinin NK 1 (rabbit jugular vein) and tachykinin NK 2 (rabbit pulmonary artery) receptors with pA 2 values of about 6.5-7.5 at the NK 1 receptor and of 5.9-7.2 at the NK 2 receptor, while being inactive at the tachykinin NK 3 receptor (rat portal vein). Spantide II and the novel analogues were without effect on electrically evoked cholinergic responses of the isolated rabbit iris sphincter and on electrically evoked sympathetic responses of the guinea-pig vas deferens; moreover, they were without local anaesthetic-like effects on action potentials of the frog sciatic nerve, which suggests that they do not produce a general neurosuppressive effect. They were as effective as or slightly less effective than spantide II in causing histamine release from rat peritoneal mast cells.

Key words: Spantide II; Tachykinin receptor antagonist; Structure-activity relationship

1. Introduction

During the last decade numerous tachykinin receptor antagonists have been described; they are either peptides or non-peptides. Antagonists of the peptide category may be either full length substance P and neurokinin A analogues or truncated C-terminal analogues of six to eight residues (Leander et al., 1981; Folkers et al., 1984; Regoli et al., 1984; Hall and Morton, 1991; Maggi et al., 1993). Their usefulness has been restricted by low potency, instability in blood, and histamine-releasing properties (H/ikanson et al., 1983; Folkers et al., 1984; Devillier et al., 1985; Maggi et al., 1993). In addition, several of them had local anaesthetic-like properties (Post et al., 1985) and produced

* Corresponding author. Tel. 46 46 107587, fax 46 46 104429. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 2 4 8 - 6

neurotoxic effects when applied intrathecally (Post and Paulson, 1985). The full length substance P analogue, spantide (Folkers et al., 1984), has been widely used as a tachykinin antagonist in the past. A more recently developed analogue, spantide II, was found to have higher potency and to cause less histamine release than spantide and to be without neurotoxicity (H~kanson et al., 1990; 1991; Folkers et al., 1990; Wiesenfeld-Hallin et al., 1990; Maggi et al., 1991). It was relatively selective for tachykinin N K 1 receptors (Maggi et al., 1991). Recently developed non-peptide tachykinin antagonists, such as CP-96,345, {[(2S,3S)-cis-2-(diphenylmethyl)-N-(2-methoxyphenyl)-methyl]-l-azabicyclo[2.2.2]octan-3-amine} (Snider et al., 1991), R P 67580, {(3aR,7aR)-7,7-diphenyl-2-[1-imino-2-(2-methoxyphenyl)ethyl]perhydroisoindol-4-one} (Garret et al., 1991) and SR 48968, {(S)-N-methyl-N[4-(4-acetylamino-4phenyl piperidino)-2-(3,4-dichlorophenyl)butyl]benz-

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z.-Y. Wanget al. / European Journalof Pharmacology 260 (1994) 121-128

amide} (Emonds-Alt et al., 1992), have been found to induce effects not related to tachykinin receptor antagonism, including local anaesthesia and general neurosuppression (Boyle et al., 1991; Lecci et al., 1991; Schmidt et al., 1992; Wang and H~kanson, 1992a; Rupniak et al., 1993; Tamura et al., 1993; Wang et al., 1994). In the present study, the biological activities of spantide II and fifteen spantide II analogues were tested in vitro.

tagonist, the other being exposed to the vehicle (control). In each experiment the suppression of the electrically evoked contraction in the drug-exposed preparation was compared directly to the equivalent response in the control preparation (set as 100%) and expressed as percent of control (Wang et al., 1994). Concentration-response curves were obtained. In preliminary experiments, spantide II was tested with incubation times from 5 to 30 min and the maximal effect of the drug was obtained after 15 min. Thus, we chose an incubation time of 15 min for each concentration of antagonist.

2. Materials and methods

2.1. General Adult pigmented rabbits of either sex (1.5-3.0 kg), male Sprague-Dawley rats (200-300 g), male guinea pigs (200-250 g) and bullfrogs (Rana catesbeiana) of either sex (250-300 g) were used. The animals were killed by a blow on the neck and exsanguinated. Iris sphincter muscle from the rabbit (Wahlestedt et al., 1985), helicoidal strips of jugular vein (Nantel et al., 1990) and rings of pulmonary artery (endothelium-denuded) (D'Orl6ans-Juste et al., 1985) from the rabbit, helicoidal strips of the rat portal vein (Mastrangelo et al., 1986) and vas deferens from the guinea-pig (Stjernquist et al., 1983) were prepared and mounted vertically on a Perspex holder or mounted on two L-shaped stainless steel wires (rabbit pulmonary artery). The isometric tensions were measured by a Grass FT03 force-displacement transducer. The preparation was stretched with a force of 1.5 mN (iris sphincter) or 5-10 mN (jugular vein, pulmonary artery, portal vein and vas deferens). The modified Krebs solution (Wahlestedt et al., 1985) was bubbled with a gas mixture of 7% CO 2 in 0 2 giving a pH of 7.2-7.3 at 37°C. Before the start of each experiment the preparation was allowed to equilibrate for 60-90 min. Electrical stimulation with square wave pulses (25 V, voltage drop 14-17 V over the electrodes, 0.3-1 ms duration) was applied by means of platinum electrodes connected to a Grass $4C stimulator. The preparations were stimulated either with single pulses (iris sphincter muscle) or with trains of pulses (iris sphincter muscle and vas deferens) lasting 3-10 s, the pulse frequency varying from 1 to 20 Hz. All electrically evoked responses could be abolished by 10 - 6 M tetrodotoxin, a nerve conduction blocker (Kao, 1966).

2.2. Effects on electrically evoked, tachykinin-mediated contractions in the iris sphincter The two iris sphincter halves, mounted in separate baths, were stimulated in parallel, one being exposed to increasing concentrations of tachykinin receptor an-

2.3. Effects on contractile tachyMnin receptor agonists

responses

to selective

Concentration-response curves were made by adding the selective agonists in a cumulative manner. [Sar9,Met(O2)11]substance P induced contractions of the isolated jugular vein (tachykinin NK 1 receptor) starting from 10 -1° M with a maximal response at 10 -7 M. Similarly, [/3-AlaS]neurokinin A-(4-10) induced contractions of the d e n u d e d pulmonary artery (tachykinin NK 2 receptor) starting from 10-10 M with a maximal response at 10 -7 M. Each preparation could be used for two reproducible concentration-response curves. Concentration-response curves were obtained in the absence and presence of antagonist. Each concentration of antagonist was applied 15 min before the agonist. Each preparation of rat portal vein (tachykinin NK 3 receptor) was challenged repeatedly with [MePhe7] neurokinin B at 10 -8 M, a concentration producing half-maximal response (Mastrangelo et al., 1986). Applications were repeated with a 45-min interval (extensive washing) in order to avoid receptor desensitization. After reproducibility of the response to [MePhe7] neurokinin B had been attained (usually after 2-3 applications), antagonist was applied at a concentration of 10 -5 M 15 min before renewed application of the agonist.

2.4. Effects on electrically evoked, non-tachykininmediated contractions in the iris sphincter of rabbit and in the vas deferens of guinea-pig Two preparations from the same animal were mounted in separate baths. One was exposed to increasing concentrations of antagonist, the other being exposed to the vehicle. Repeated electrical stimulation caused reproducible contractile responses throughout the experiment (Leander and H~kanson, 1985; Wang et al., 1994). The effects of the antagonists on the electrically evoked contractile responses were recorded. Each concentration of antagonist was applied 15 rain before electrical stimulation.

z.-Y. Wanget al. / European Journal of Pharmacology 260 (1994) 121-128 2.5. Effects on action potentials in the sciatic nerves of frog Bullfrogs were killed by decapitation after hypothermic general anaesthesia (30 min at -20°C). The sciatic nerves were excised, desheathed and split longitudinally into two bundles. Each preparation was mounted in a sucrose-gap chamber as previously described (Strong et al., 1978; Hahin and Strichartz, 1981). One end of the nerve was stimulated in drug-free Ringer solution with A g / A g C I or platinum electrodes, using isolated square-wave supramaximal current pulses of 0.05 ms duration (Grass Instrument Co., Braintree, MA, Model $44). Similar Ag/AgCI or platinum electrodes were used to record potential changes between two regions of the nerve separated by a sucrose gap. These changes in potential were amplified and recorded as compound action potentials on an a.c.-coupled, dual-beam storage oscilloscope (Tektronix 5113). All sucrose-gap experiments were conducted at 21-24°C. The nerve was first stimulated to determine a supramaximal stimulus. Next, the flow of sucrose was begun, rapidly (20-30 ml/min) for 30 s to flush out the system, and then maintained at 2-3 ml/min for the duration of the experiment. The test chamber held 350 tzl solution and contained a 10-mm length of nerve. During the stabilization period, the solution in the test chamber was changed twice with normal frog Ringer solution to eliminate potential 'solution switching artifacts'. This baseline period usually lasted 20-30 min. Afterward, stimulation once every min showed steady compound action potentials, changing in amplitude by less than 5% over a 15-min period. Compound action potentials were measured in frog Ringer solution containing (mM): NaC1 110; KC1 2.5; CaC1z 2.0; morpholino-propanesulfonic acid 5.0; the pH was adjusted to 7.2 with NaOH.

2.6. Effects on histamine release from rat peritoneal cells The experiments were conducted as described earlier (H~kanson et al., 1983; 1990; Folkers et al., 1984). Briefly, a male Sprague-Dawley rat (250-300 g) was killed by decapitation under light diethyl ether anesthesia. Peritoneal saline washings were collected and the cells, consisting of 3-6% mast ceils (Kurose and Saeki, 1981), were spun down in a centrifuge and washed twice in a buffer salt solution containing 145 mM NaC1, 2.7 mM KC1 and 10% (v/v) S6rensen phosphate buffer (Na2HPO 4 + KH2PO4, pH 7.0). The cells were suspended in 10 ml of the buffer solution to which was added 0.1% bovine serum albumin and 1 mM CaC1 z at room temperature. Aliquots of 0.9 ml were transferred to polypropylene test tubes and preincubated for 5 min at 37°C before adding 0.1 ml of drug solution. Experiments were performed in duplicate.

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The mixture was incubated for a further 5 min and the reaction was interrupted by placing the tubes on ice. They were then centrifuged at 600 x g for 5 min at 4°C. Aliquots (0.1 ml) of the supernatant were taken for fluorometric assay of histamine (H[lkanson and R6nnberg, 1974). The sediment was suspended in 1 ml redistilled water (causing lysis of the cells) and centrifuged at 600 x g for 5 min. Aliquots of the supernatant were also taken for histamine determination. The histamine released was expressed as percent of the the total amount of histamine present in the peritoneal cells.

2. Z Drugs Spantide II and its analogues were synthesized by the solid-phase method as described elsewhere (Folkers et al., 1990). Their structures are given in Table 1. The purity of these peptides, analysed by high performance liquid chromatography, was greater than 98%. [Sar9,Met(O2)U]substance P, [/3-AlaS]neurokinin A (4-10), [MePheT]neurokinin B and neurokinin A were purchased from Peninsula Europe, St. Helens, Merseyside, UK. Analogues II, III, V, VI and VII could be dissolved in water while the other analogues had to be dissolved in 0.1-0.5 M acetic acid to give a concentration of 10 -3 M. Water was used to dilute further.

2.8. Analysis of results plCs0 values (the negative logarithm of the molar concentration of the antagonist producing 50% inhibition of the electrically evoked contraction) were calculated by linear regression analysis of the results in the 10-90% response interval. The pD 2 value is the negative logarithm of the molar concentration of agonist producing 50% of maximal response. The pA 2 values (the negative logarithm of the concentration of antagonist that reduces the effect of a double dose of an agonist to that of a single dose) and the slopes of the Schild plots were calculated as described (Tallarida et al., 1979).

3. Results

3.1. Effects on electrically evoked, tachykinin-mediated responses In the presence of 10 -6 M atropine and 5 x 10 -6 M guanethidine, the contractile response of the rabbit iris sphincter muscle to electrical stimulation (20 Hz, 25 V, 10 s) is mediated by tachykinins (Wahlestedt et al., 1985; Wang and H~kanson, 1992b). None of the drugs affected the basal tone of the sphincter muscle. All spantide II analogues tested inhibited the tachykinin-

Z.-Y. Wanget al. / European Journal of Pharmacology 260 (1994) 121-128

124

tested induced a reversible inhibition in that the contractile response of the iris sphincter muscle to electrical stimulation invariably recovered after extensive washing for 90-120 min.

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3.2. Effects on the contractile responses to selective tachykinin receptor agonists

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Spantide II and analogue IV and VII (because of their high potency and good water solubility) were selected for further examination with respect to their selectivity for the various tachykinin receptors. As shown in Figs. 2 and 3, a selective tachykinin NK 1 receptor agonist, [SarD,Met(o2)n]substance P, contracted the jugular vein with a pD 2 value of 8.5 while a selective tachykinin NK 2 receptor agonist, [/3Ala8]neurokinin A, contracted the pulmonary artery with a pD 2 value of 8.1. Spantide II and analogues IV and VII competitively and concentration dependently inhibited the responses induced by [Sar9,Met(O2)11] substance P in the jugular vein or by [/3-Ala 8]neurokinin A in the pulmonary artery (Figs. 2 and 3). The pA 2 values were 7.5 for both analogues IV and VII in the jugular vein (tachykinin NK 1 receptors) and 6.5 and 7.2 in the pulmonary artery (tachykinin NK 2 receptors), respectively, the pA 2 values for spantide II being 6.5 in the jugular vein and 5.9 in the pulmonary artery. Spantide II and analogues IV and VII at a concentration of 10 -5 M did not affect the response induced by the selective tachykinin NK 3 receptor agonist, [MePhe7]neurokinin B, in the rat portal vein (Fig. 4).

iI)

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Log M Fig. 1. The tachykinin-mediated contractile response of rabbit iris sphincter was inhibited by spantide II and its analogues concentration dependently; three of them are illustrated: spantide II (o), analogue IV (e) and analogue XI ( • ) . Drug-exposed preparations were stimulated in parallel with control preparations and the contraction of the drug-exposed preparation was expressed as percentage of that of the control.

mediated contraction in a concentration-dependent manner (Fig. 1). The amino acid sequences and plCs0 values are given in Table 1. The plCs0 values of analogues II, III, IV, V, VII and VIII were 0.1-0.5 log units higher than that of spantide II. All antagonists

Table 1 Amino acid sequences of spantide II (analogue I) and its analogues and their inhibitory effects on electrically evoked, tachykinin-mediated responses in the rabbit iris sphincter

I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI

1

2

3

4

5

6

7

8

9

10

D-Lys(Nic) ILys D-ILys o-Lys(Nic) ILys D-ILys o-Arg o-Arg o-Arg D-Pal o-Lys(Nic) D-Lys(Nic) D-Lys(Nic) D-Lys(Nic) D-Lys(Nic) D-Lys(Nic)

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro

Pal D-Pal D-Pal Pal p-Pal D-Pal Pal Nle Nle Pal Pal Pal Pal Pal Pal Pal

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro

D-Phe(CI2) D-Phe(Cl 2) D-Phe(Cl 2) D-Phe(Cl 2) D-Phe(CI 2) o-Phe(Cl 2) D-Phe(Cl 2) D-Phe(CI 2) D-PHe(CI 2) o-Phe(Cl 2) D-Nal D-Nal D-Phe(CI 2) D-Phe(C12) D-Phe(CI 2) D-Phe(CI 2)

Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn

D-TIP D-TIP o-Tip D-TIP D-TIP D-TIP D-TIP D-TIP D-TIP D-TIP D-TIP D-TIp D-TIp D-TIp D-TIp D-TIP

Phe Pbe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe

D-TIP D-TIP D-TIP D-Pal D-Pal D-Pal D-Pal D-TIP D-Pal D-TIP D-TIP D-TIP D-Pal D°Trp o-Tip p-Pal

Leu NIe-NH 2 spantide II Leu NIe-NH 2 Leu NIe-NH 2 Leu NIe-NH 2 Leu NIe-NH 2 Leu Nle-NH 2 Leu NIe-NH 2 Leu NIe-NH 2 Leu Nle-NH 2 Leu NIe-NH 2 Leu NIe-NH 2 Leu D-Leu-NH 2 Leu D-Ala-NH 2 Leu o-Leu-NH 2 Leu-NH 2 Leu-NH 2

11

pICs0 6.1 6.4 6.5 6.6 6.4 6.1 6.4 6.2 6.1 5.7 5.1 4.9 5.2 5.4 5.8

_+ 0.1 + 0.3 + 0.3 + 0.2 _+ 0.3 +_ 0.5 _+ 0.2 +_ 0.03 + 0.06 + 0.2 _ + + + +

0.1 0.1 0.1 0.1 0.1

- , a weak antagonist; a reliable plCs0 value could not be calculated. Means + S.E.M. n = 6-12. Lys(Nic) represents N-nicotinyllysine. Pal represents 3-(3-pyridyl)-alanine. ILys represents isopropyl-lysine. Phe(Cl 2) represents 3-(3,4-dichlorophenyl)-alanine. Nal represents 2-naphthylalanine.

Z. - Y. Wang et al. / European Journal of Pharmacology 260 (1994) 121-128

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Fig. 2. Rabbit jugular vein (tachykinin NK 1 receptor system). (A) Cumulative concentration-response curves illustrating the contractions induced by [Sar9,Met(O2)ll]substance P in the absence 0 and presence of analogue IV: 10 -7 M (e), 3 x 10 -7 ( • ) and 10 -6 ( • ) M. The responses are expressed as % of the maximum. Each value represents the mean of 6-18 experiments. Vertical bars give S.E.M. (B) Schild plot analysis reveals a slope of 0.996, suggesting competitive antagonism..

3.3. Effects on non-tachykinin-mediated neurotransmission The iris sphincter muscle responds to single-pulse stimulation (1 pulse/60 s, 25 V) with a twitch-like contraction. This contraction can be blocked by 10 -6 M atropine (Wahlestedt et al., 1985). The vas deferens responds to low-frequency stimulation (5 Hz, 25 V, 3 s) with a twitch-like contraction, which is unaffected by 10 -6 M atropine but abolished by 5 × 10 -6 M

guanethidine (Stjernquist et al., 1983). Thus, the electrically evoked contractions in these preparations are not mediated by tachykinins. Spantide II and analogues IV and VII were without effects on the electrically evoked contractions (not shown). Spantide II and analogues IV and VII at concentrations up to 10 - 4 M applied for 30 min were without effect on the compound action potentials of the frog sciatic nerve (not shown). Neither tonic inhibition (assayed by stimulation at 1 min -1) nor use-dependent,

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Fig. 3. Rabbit pulmonary artery (endothelium-denuded) (tachykinin NK 2 receptor system). (A) Cumulative concentration-response curves illustrating the contractions induced by [/3-AlaS]neurokinin A in the absence (<3) and presence of analogue IV: 3 × 10 -7 (o), 10 -6 ( • ) and 3 x 10 -6 M ( • ) . The responses are expressed as percentage of the response evoked by 137.7 mM KC1 buffer solutions (Wahlestedt et al., 1985) applied at the beginning of each experiment. Each value represents the mean of 6-18 experiments. Vertical bars give S.E.M. (B) Schild plot analysis reveals a slope of -1.2.

126

Z. -Y. Wang et al. / European Journal of Pharmacology 260 (1994) 121-128

(MePhe-) NKB 10.8 M

I 1 rain

(MePhe:) NKB A n a l o g u e IV 10 -5 M Fig. 4. Originaltracingsshowingthat analogueIV was withouteffect on the contractileresponsesinducedby 10-8 M [MePhe7]neurokinin B in the rat portal vein (tachykininNK3 receptor system).

phasic inhibition (assayed by short bursts of stimuli at 5-20 Hz) was detectable. 3.4. Effects on histamine release f r o m rat peritoneal mast cells

Spantide II and analogues IV and VII, at a concentration of 3 × 10 -5 M, caused histamine release, 56.0 + 12.9%, 64.9 + 7.8% and 31.6 + 4.0%, respectively, from rat peritoneal mast cells (n = 3).

4. Discussion

It is generally accepted that the biological activity of substance P resides in the C-terminal hexapeptide region with the N-terminus contributing to receptor recognition and binding (Maggio, 1988; Folkers et al., 1990; Wang et al., 1993). This view forms the basis for the design of peptide-type tachykinin receptor antagonists, and the results of studies of structure-activity relationships have provided further guidelines for preparing new generations of tachykinin receptor antagonists (Escher et al., 1985; Folkers et al., 1985; Liungquist et al., 1989; Maggi et al., 1993). The analogues tested in the present study were synthesized based on spantide II with one to three substitutions. All the novel analogues were found to inhibit tachykinin-mediated responses. It has long been recognized that D-Trp in position 7 and 9 is critical for antagonistic potency (Liungqvist et al., 1991). Interestingly, when D-Trp 9 was replaced by D-Pal, a weakly basic aromatic amino acid, the plCs0

was increased by 0.5 log unit (analogue IV) and its solubility in water was improved (the peptide could be dissolved in 0.1 M acetic acid unlike spantide II which had to be dissolved in 0.5 M acetic acid). D-Arg in position 1 yielded a virtually equipotent analogue VII, which could be dissolved in water at a concentration of 10 -3 M. Similarly, in analogues VIII and IX, D-mrg in position 1 led to improved water solubility without much reduction in the potency. Interestingly, introducing ILys or D-ILys into position 1 yielded potent analogues (II, III, V and VI), that could be dissolved in water at a concentration of 10 -3 M. From previous studies it has been suggested that either the size a n d / o r the hydrophobicity of the amino acid in position 5 may be crucial for the conversion of agonist to antagonist (Zacharia et al., 1991). Substitution of D-Phe(CI 2) by D-Nal in position 5 led to a considerable decrease in antagonistic potency (analogues XI and XII). Replacement of Nle with D-Ala or o-Leu or deletion of Nle in position 11 also caused a great loss of activity (analogues XV to XVI), suggesting that Nle in position 11 is important for maintaining antagonistic potency. Nle in position 3 (analogues VIII and IX) did not affect the potency compared to spantide II. Analogues IV and VII, which had high antagonistic potency and good water solubility, were selected together with spantide II for further study of their interaction with the various tachykinin receptor types. In a previous study, spantide II was found to be about 25 times more active at tachykinin NK 1 receptors (the jugular vein, pA 2 value of 6.8) than at tachykinin NK 2 receptors (the pulmonary artery, pA 2 value of 5.4) (Patacchini et al., 1992). In the present study, spantide II was found to have pA 2 values of 6.5 in the jugular vein and of 5.9 in the pulmonary artery, i.e. only an about 4-fold difference in affinity between NK 1 and N K 2 receptors. At present, we have no satisfactory explanation for this discrepancy. Analogues IV and VII had pA 2 values which were 1 log unit higher than that of spantide II at the tachykinin NK1 receptors. At the tachykinin NK 2 receptors the pA 2 values were about 0.6-1.2 log units higher than that of spantide II. Spantide II and, particularly, analogues IV and VII, which antagonized NK 1 and NK 2 receptors with fairly similar potency, may be regarded as non-selective tachykinin receptor antagonists (although they were inactive at NK 3 receptors). Since both tachykinin NK 1 and NK 2 receptors are involved jointly in numerous pathophysiological processes, non-selective tachykinin receptor antagonists may offer greater therapeutic promise for alleviating symptoms of diseases such as asthma, than more selective tachykinin receptor antagonists. Analogues IV and VII, like spantide II, induced mast-cell histamine release, suggesting that it may prove difficult to eliminate this side-effect, which may limit the use-

Z.-Y. Wang et al. / European Journal of Pharmacology 260 (1994) 121-128

fulness of peptide-type tachykinin receptor antagonists (Maggi et al., 1993). Recently, non-peptide tachykinin receptor antagonists, such as CP-96,345, RP 67580 (both favouring the tachykinin NK 1 receptor) and SR 48968 (favouring the tachykinin NK 2 receptor), have been introduced (Garret et al., 1991; Snider et al., 1991; Emonds-Alt et al., 1992). In the rabbit iris sphincter muscle, the pICs0 values of CP-96,345 and RP 67580 were 5.5 and 5.4 respectively (Wang et al., 1994). Thus, in this particular preparation, both spantide II and the two novel spantide II analogues were about 10 times more potent than the non-peptide antagonists tested (Wang et al., 1994). In addition, we found CP-96,345, RP 67580 and SR 48968 to have non-specific effects on neurotransmission (Wang and H~kanson, 1992a; Wang et al., 1994), in that they inhibited the electrically evoked cholinergic contractions of the iris sphincter muscle of rabbit, the sympathetic contractions of vas deferens and the action potentials of frog sciatic nerves in a concentration-dependent manner (Wang and H~kanson,1992a; Wang et al., 1994). Unlike the non-peptide antagonists, neither spantide II nor analogues IV and VII affected these non-tachykinin-mediated neural responses. In conclusion, our study suggests that D-Pal in position 9 and D-Arg, ILys or D-ILys in position 1 improve potency and water solubility of spantide-type tachykinin receptor antagonists. These antagonists are non-selective with high affinity for both tachykinin NK 1 and NK 2 receptors. Our results suggest that analogues IV and VII as well as spantide II have fewer side-effects than the non-peptide tachykinin receptor antagonists described to date.

Acknowledgements This study was supported by grants from the Swedish Medical Research Council (04X-1007) and from the Medical Faculty of Lund, Sweden and by a UPHS grant (GM 15904). Dr. S.R. Tung is a fellow of the Harvard Anesthesia Centre for Training and Research.

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