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SR 142801, The first potent non-peptide antagonist of the tachykinin NK3 receptor
Life Sciences, Vol. 56, No. 1 pp. PL 27-32, 1995 Copyright 0 1994 Elsevier Science Ltd Printed in the USA. All rights resewed ww3205/95 $9.50 + .oo
Pergamon 0024-3205(94)00413-7 Ph?ARMACOLOGY LETTERS Accelerated Communication
SR 142801, THE FIRST POTENT NON-PEPTIDE ANTAGONIST OF THE TACHYKININ NK3 RECEPTOR X. Emonds-Altl, V. Proiettol,
D. Bichonl, J.P. Ducouxl, M. Heaulmel, B. Milouxz, M. Ponceletl, D. Van Broeckl, P. Vilainl, G. Neliat3, P. SoubriC1, G. Le Furl and J.C. Brelibel
Sanofi Recherche,
134184 Montpellier and 23 1676 Toulouse-Labtge, 3CEREP, 86600 Celle L’Evescault, France
France
(Submitted September 6, 1994; accepted September 27, 1994; received in final form October 13, 1994)
Abstract. SR 142801 is the first potent and selective non-peptide antagonist of the tachykinin NK3 receptor. It inhibited [MePhe7]NKB binding to its receptor from various species, including humans. SR 142801 was a competitive antagonist of [MePhe7]NKB_mediated contractions of guinea-pig ileum and inhibited the acetylcholine release following the activation of the guinea-pig ileum tachykinin NK3 receptor. In vivo, SR 142801 potently inhibited the turning behaviour induced by intrastriatal injection of senktide in gerbils, and appears as a powefil tool for investigation of the physiological and pathological role of NKB and its NK3 receptor. f@
Words: neurokinin
B, tachykinin
NK, receptor,
SR 142801
Introduction Neurokinin B (NKB) belongs to a group of neuropeptides named tachykinins, that includes substance P (SP) and neurokinin A (NKA). NKB preferentially interacts with the tachykinin NK3 receptor while SP and NKA are the preferred endogenous agonists of the tachykinin NKl and NK2 receptors, respectively (1,2). Progress in the understanding of the physiological and pathological role of tachykinins are highly dependent on the use of potent, specific and metabolically stable non-peptide antagonists of their receptors. Although several potent and specific non-peptide antagonists of the tachykinin NKl (3-5) and NK2 (6,7) receptors have been recently discovered, only peptide-type compounds showing specific pharmacological activity at the tachykinin NK3 receptor have been described (1,8). On the other hand, some non-peptide angiotensin receptor antagonists (DUP 753, L-158,809, EXP 3174) and the non-peptide tachykinin NK2 receptor antagonist, SR 48968, have been reported to interact in vitro at high concentrations with the tachykinin NK3 receptor (1,9-l 1). We now describe the first highly potent non-peptide antagonist of the tachykinin NK3 receptor, SR 142801 (Fig. I). Methods Drugs. All peptides were from Bachem (Switzerland) and were solubilized in organic solvents (ethanol or dimethylsulfoxyde). Radioactive ligands were purchased from Amersham (U.K.) and Du Pont NEN (USA). SR 142801 and its (R)-enantiomer (SR 142806) were synthesized at Sanofi Corresponding author : X. Emonds-Alt, Sanofi Recherche, 371 Rue du Professeur J. Blayac, F34184 Montpellier Cedex 04, France. Telephone : (33) 67-10-6710; Fax (33) 67-10-6767.
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SR 142801: NK3 Receptor Antagonist
Vol. 56, No. 1, 1995
Fig. 1 Structure of SR 142801 (S)-CN)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl) -4-phenylpiperidin-4-yl)-N-methylacetamide Recherche and were used as hydrochlorides. They were solubilized in organic solvents (ethanol or dimethylsulfoxyde) or suspended in distilled water by using 0.01 % of Tween 80. Binding assays. The affinity of SR 142801 for tachykinin receptors was evaluated in several receptor radioligand binding assays : (i) binding of [I25I]Bolton-Hunter labelled SP to NKI receptors of rat brain cortex, human lymphoblast cells (IM9) and human astrocytoma cells (U373MG, STTGl); (ii) binding of [I25I]iodohistidylNKA (or [I25I]neuropeptide r) to NK2 receptors of rat or hamster urinary bladder and guinea-pig ileum; (iii) binding of [ 125I]iodohistidyl-[MePhe7]NKB (or [ I25IlEledoisin) to tachykinin NK3 receptors of rat, guineapig and gerbil brain cortex. The effect of SR 142801 was also evaluated on the binding of [ 125I]iodohistidyl-[MePhe7]NKB to the human NK3 receptor, cloned and expressed in CHO cells according to Buell et al (12). All binding assays were conducted and analysed as previously described in details (3,6). In vitro assays. The in vitro pharmacological profile of SR 142801 was investigated by using several in vitro functional bioassays for tachykinin receptors (1,3,6) : (i) [MePhe7]NKB_induced contractions of rat portal vein and guinea-pig ileum; (ii) [ Sar9,Met(02) I I]SP-induced endothelium-dependent relaxation of rabbit pulmonary artery, previously contracted by 100 nM noradrenaline; (iii) [PAla8]NKA-(4-1O)-induced contractions of endothelium-denuded rabbit pulmonary artery. Experimental protocols were previously described in details (3,6). As already reported and discussed for some non-peptide antagonists of the tachykinin NKI and NK2 receptors (3,6,13), preliminary experiments have indicated that fill activity of SR 142801 was only observed after a long time of contact with the tissue and thus, in all experiments SR 142801 was added 140 min before the agonist. Antagonism was analysed according to Arunlakshana and Schild (14). On the other hand, antagonist activity of SR 142801 for tachykinin NK3 receptors was also evaluated by monitoring acetylcholine release following activation of these receptors in guinea-pig ileum. Longitudinal strips of guinea-pig ileum were incubated in oxygenated (95 % 02 - 5 % CO2) and thermostated (37 “C) Krebs-Henseleit physiological salt solution (20 ml), pH 7.4, containing 0.25 @i/ml [3H]choline. After 30 min incubation, strips were washed for 90 min in the Krebs-Henseleit physiological salt solution, containing 20 uM hemicholinium-3. Each strip was then incubated in 6 successive Krebs-Henseleit physiological salt solution (3 ml) at 6 min intervals. The agonist (senktide or [MePhe7]NKB) was introduced during the fourth incubation. SR 142801 was added to the incubation medium 10 min before the agonist and was then present until the end of the experiment. [3H]radioactivity was evaluated in the 6 incubation media and after solubilization, in the tissue.
Vol. 56, No. 1, 1995
SR 142801:
NK3 Receptor
PL-29
Antagonist
In vivo assays. SR 142801 activity at NK3 receptors was investigated on the turning behaviour induced by intrastriatal injection of senktide in gerbils. The general method has been described elsewhere (15,16) and was modified as follows. Free-hand microinjection (1 c(l) of se&tide or vehicle (water) was made slowly (2-3 s) in the right striatum of conscious, non-restrained gerbils (8 or more animals per group) by means of a 5 ul Hamilton microsyringe and a 10 mm calibrated needle (final length below the skin : 4.5 mm). The descending point of the needle was internal (2 mm) and caudal (2 mm) to the right orbitus. The animals did not show any behavioural sign of distress or pain during injection. Immediately after injection, they were placed individually in a cylindrical glass container. The number of complete contralateral rotations (i.e. away from the injected side) was visually recorded and cumulated over 3 periods of 2 min (2-4, 5-7, 8-10 min) post-intrastriatal injection. Statistical analysis was performed using ANOVA followed by the Dunnett’s t-test. SR 142801 activity at tachykinin NKl and NK2 receptors was investigated on [Sar9,Met(O2)I I]SP-induced hypotension in anaesthetized dogs and on [NlelO]NKA-(4-10)induced bronchoconstriction in anaesthetized guinea-pigs as previously described (3,6). Results Binding studies. Inhibition constants (Ki) for SR 142801 and SR 142806 obtained in the different radioligand binding assays for tachykinin receptors are given in table I. SR 142801 potently inhibited the binding of [125I]iodohistidyl-[MePhe7]NKB to tachykinin NK3 receptors from various species including humans. However, it was less active on tachykinin NK3 receptors from rat. Its (R)-enantiomer was less active, indicating a stereoselective action of SR 142801. On the other hand, SR142801 only moderately or slightly inhibited the binding of [125I]Bolton-Hunter labelled SP and [125I]iodohistidyl-NKA (or [125I]neuropeptide y) to tachykinin NKl and NK2 receptors, respectively. Finally, the selectivity of SR 142801 for tachykinin NK3 receptors was also demonstrated by its lack of activity at concentrations up to 1 uM in several binding assays for various neurotransmitter and neuropeptide receptors (3) except for calcium (verapamil site) and TABLE I Inhibition Constants (Ki) of SR 142801 and its (R)-enantiomer (SR 142806) in Tachykinin Receptor Radioligand Binding Assays Ki (nM)
Rat brain cortex IM9 U373MG STTGl Rat urinary bladder Guinea-pig ileum Hamster urinary bladder Rat brain cortex Guinea-pig brain cortex Gerbil brain cortex Human, expressed in CHO cells
Receptors
SR 142801
SR 142806
Ml ml =1 ml m2 m2 I=2 m3 m3 m3 m3
>2000 60t4 1374 + 130 428 + 24 10029 73 +9 >2000 1553 0.11 +0.01 0.42 + 0.04 0.21 + 0.03
>2000 220 + 36 3272 + 344 1146+49 248 + 76 350 2 48 >2000 406 + 60 26 t 3 30 5 9 3225
Values are means + S.E.M. obtained from at least 3 independent experiments performed in triplicate. Radioligands : [125I]Bolton-Hunter labelled SP for NKI receptors; [125I]iodohistidyl-NKA for NK2 receptors except of guinea-pig ileum where [ 125Ilneuropeptide y was used; [ 125IliodohistidyL [MePheT]NKB for NK3 receptors except of rat brain cortex where [ 125IlEledoisin was used.
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SR 142801: NK3 Receptor Antagonist
Vol. 56, No. 1, 1995
sodium (site 2) channels and opiate (binding of nalaxone) receptors where IC50 were in the range 0.1 to 1 l.tM (data not shown). In vitro studies. SR 142801 competitively antagonized [MePhe7]NKB-induced contractions of guinea-pig ileum. As shown in the figure 2, SR 142801 produced a concentration-dependent rightward shift of the concentration-response curves of [MePhe7]NKB. Assuming competitive antagonism, an apparent pA2 of 9.15 (8.93-9.37, 95 % confidence limits) was calculated by the method of Arunlakshana and Schild (14). On the other hand, SR 142801 did not antagonize [MePhe7]NKB-induced contractions of rat portal vein up to a concentration of 0.1 PM. A significant effect was only observed at 1 @4. Similarly, except at 1 pM, SR 142801 did not significantly antagonize [Sar9,Met(O2)1 l]SP-induced endothelium-dependent relaxation of rabbit pulmonary artery, previously contracted by 100 nM noradrenaline (a tachykinin NRl receptor functional assay) and [PAla8]m(4-1O)-induced contractions of endothelium-denuded rabbit pulmonary artery (a tachykinin NR2 receptor functional assay) (data not shown). Finally, antagonist activity of SR 142801 for tachykinin NK3 receptors was further studied on senktide- or [MePhe7]NKE3-induced release of [3H]acetylcholine from guinea-pig ileum. EC50 of senktide and [MePhe7]NKB were 0.24 + 0.12 nM and 4.3 + 1.6 nM (n=4), respectively. SR 142801 potently inhibited the acetvlcholine release with an ICso of 6.1 + 1.8 nM and 13.0 -+ 0.1 nM (n=4), respectively when-using 1 nM senktide and 10 g [MePhe$KEJ. Control0 SR
-11
SR
1428010 Control
142801 1nM
.
3nM
0
0.1 /AM
v
10nM
.
1pM
.
-10
-9
Log [Me Phe']-NKE
80
-8
-7
-6
-11
(M)
0.01 pM
-10
.
-9
Log [Me Phe7]-NKB
T/4
-8
-7 (M)
Fig. 2
Concentration-response curves for [MePhe7]NKB_induced contractions of guinea-pig ileum (left panel) and rat portal vein (right panel) in the absence (control) and in the presence of SR 142801. Values are means + S.E.M. (n=6-8). In vivo studies, Senktide injected at 0.1, 0.3, 1 and 3 pg dose-dependently (inverted U-shaped relationship) produced vigorous contralateral rotations in gerbils. When administered either intraperitoneally (i.p.) 30 min or orally (p.0.) 60 min before intrastriatal injection of 0.3 pg se&tide, SR 142801 dose-dependently inhibited this turning behaviour (Fig. 3). ID50 was 0.58 mg/kg i.p. (0.43-0.79, 95 % confidence limits) or 0.72 mgikg p.o. (0.5-1.0, 95 % confidence limits). When studied at 3 mg/kg p.o., SR 142801 showed a long acting effect that was significant at least until 8 h after se&tide injection. Its (R)-enantiomer (SR 142806) was inactive at least up to 1 mg/kg i.p. The in vivo pharmacological activity of SR 142801 was further evaluated on two animal models where the role of tachykinin NKl or NK2 receptor has been well characterized (3,7) : [Sar9,Met(02)1 l]SP-induced hypotension in anaesthetized dogs and l?JlelO]NKA-(4-10)-
SR 142801: NK3 Receptor Antagonist
Vol. 56, No. 1, 1995
PL-31
induced bronchoconstriction in anaesthetized guinea-pigs. When intravenously or intraperitoneally administered at 10 mg/kg, SR 142801 was completely inactive (data not shown).
25 -
20 P 5 t
15-
0 5 :2
lo-
S-
c
1
2
4
6
’ 6
‘I 24
TIME (h)
Fig. 3 Inhibition by SR 142801 of the turning behaviour induced by intrastriatal injection of 0.3 pg senktide in gerbils. Dose-effect relationships of SR 142801 when administered either intraperitoneally 30 min or orally 60 min before senktide injection (insert) and time-course study of the SR 142801 effect administered orally at 3 mg/kg. Values are means + S.E.M. (Dunnett’s t-test : * P< 0.05). C is the mean value in the control group. Discussion Our studies have demonstrated that SR 142801 is a competitive and highly potent non-peptide antagonist of the tachykinin NK3 receptor. However, its pharmacological activity was highly species dependent. In binding experiments, SR 142801 had a high affinity for the human, gerbil and guinea-pig tachykinin NK3 receptors. Its high affinity was confirmed in two in vitro functional assays for the guinea-pig NK3 receptors. It has been also confirmed in inhibiting different biochemical events ([Ca2+]i mobilization, inositol phosphate production, arachidonic acid release) following activation of the human NK3 receptor expressed in CHO cells (Oury-Donat et al, submitted for publication). Conversely, SR 142801 had a much lower affinity for the rat tachykinin NK3 receptors in binding and in vitro functional assays. These results clearly confirm previous studies (l,lO,l 1) suggesting that, as for tachykinin NKl and NK2 receptors (1,2), receptor differences among species also exist for the tachykinin NK3 receptor. On the other hand, the selectivity of SR 142801 for the tachykinin NK3 receptor has been shown by its lack of activity in two in vivo models where the role of tachykinin NKl or NK2 receptors has been well hypotension in dogs and mlelO]NKA-(4-lO)-induced established : [Sar9,Met(02)ll]SP-induced bronchoconstriction in guinea-pigs (3,6). Moreover, SR 142801 was only active at high concentrations in in vitro functional assays for tachykinin NKl and NK2 receptors and it was about lOOO-fold less active than specific non-peptide antagonists (SR 140333, SR 48968) of these receptors (3,6). Similarly, in binding assays, SR 142801 moderately interacted with some NKl and NK2 receptors, depending on the origin of these receptors. But compared to results reported for SR 140333 and SR 48968 (3,6), it had always 200- to lOOO-fold lower affinity than the specific antagonists (3,6). Finally, the potent antagonistic action of SR 142801 on the tachykinin NK3
PL-32
SR 142801:
NK3 Receptor
Antagonist
Vol. 56, No. 1, 1995
receptor has been also evidenced in vivo at the level of the central nervous system by inhibition of the turning behaviour induced by intrastriatal injection of senktide in gerbils. The compound can cross the blood brain barrier and is active by oral route with a long acting effect. In conclusion, SR 142801 may be useful to investigate the physiological and pathological role of NKB and its NK3 receptor. Acknowledgments We are grateful to C. Amal, N. Baudry, , N. Chabert, M. Fournier, I. Grossriether, C. Gueudet, P. Gueule, F. Guevara, 0. Hamelin, F. Lozano, P. Marquie and F. Ruffel-Seguier for their excellent technical assistance. References 1. D. REGOLI, Q.T. NGUYEN and D. JUKIC, Life Sci. 54 2035-2047 (1994). 2. C.A. MAGGI, R. PATACCHINI, P. ROVER0 and A. GIACHETTI, J. Autonom. Pharmacol. 13 23-93 (1993). 3. X. EMONDS-ALT, J.D. DOUTREMEPUICH, M. HEAULME, G. NELIAT, V. SANTUCCI, R. STEINBERG, P. VILAIN, D. BICHON, J.P. DUCOUX, V. PROIETTO, D. VAN BROCK, P. SOUBRIE, G. LE FUR and J.C. BRELIERE, Eur. J. Pharmacol. 250 403-413 (1993). 4. R.M. SNIDER, S.J.W. CONSTANTINE, J.A. LOWE, K.P. LONGO, W.S. LEBEL, H.A. WOODY, S.E. DROZDA, M.C. DESAI, F.J. VINICK, R.W. SPENCER and H.J. HESS, Science 25l_ 435-437 (1991). 5. C. GARRET, A. CARRUETTE, V. FARDIN, S. MOUSSAOUI, J.F. PEYRONEL, J.C. BLANCHARD and P.M. LADURON, Proc. Natl. Acad. Sci. USA @ 10208-10212 (1991). 6. X. EMONDS-ALT, P. VILAIN, P. GOULAOUIC, V. PROIETTO, D. VAN BROCK, C. ADVENIER, E. NALINE, G. NELIAT, G. LE FUR and J.C. BRELIERE, Life Sci. 50 PLlOl-PL106 (1992). 7. D.I. BALL, I.J.M. BERESFORD, G.P.A. WREN, Y.D. PENDRY, R.L.G. SHELDRICK, R.L.G. WALSH, M.P. TURPIN, R.M. HAGAN and R.A. COLEMAN, Br. J. Pharmacol. 112 48P (1994). 8. P. BODEN, J.M. EDEN, J. HODGSON, D.C. HORWELL, W. HOWSON, J. HUGHES, A.T. McKNlGHT, K. MEECHAM, M.C. PRITCHARD, J. RAPHY, G.S. RATCLIFFE, N. SUMAN-CHAUHAN and G.N. WOODRUFF, Bioorg. Med. Chem. Lett. 4 1679-1684 (1994). 9. L. CHRETIEN, G. GUILLEMETTE and D. REGOLI, Eur. J. Pharmacol. 256 73-78 (1994). 10. F. PETITET, J.C. BEAUJOUAN, M. SAFFROY, Y. TORRENS, and J. GLOWINSKI, Biochem. Biophys. Res. Commun. &l lSO-187 (1993). 11. F.Z. CHUNG, L.H. WU, M.A. VARTANIAN, K.J. WATLING, S. GUARD, G.N. WOODRUFF and D.L. OXENDER, Biochem. Biophys. Res. Commun. 198 967-972 (1994). 12. G. BUELL, M.F. SCHULZ, S.J. ARKINSTALL, K. MAURY, M. MISSOTTEN, N. ADAMI, F. TALABOT and E. KAWASHIMA, FEBS Lett. 299 90-95 (1992). 13. C.A. MAGGI, R. PATACCHINI, S. GIULIANI and A. GIACHETTI, Eur. J. Pharmacol. 234 83-90 (1993). 14. 0. ARUNLAKSHANA and H.O. SCHILD, Br. J. Pharmacol. Chemother. 14 48-58 (1959). 15. M. PONCELET, C. GUEUDET, X. EMONDS-ALT, J.C. BRELIERE, G. LE FUR and P. SOUBRIE, Neurosci. Lett. 149 40-42 (1993). 16. M. JUNG, R. CALASSI, J. MARUANI, M.C. BARNOUIN, J. SOUILHAC, M. PONCELET, C. GUEUDET, X. EMONDS-ALT, P. SOUBRIE, J.C. BRELIERE and G. LE FUR, Neuropharmacology 21167-179 (1994).
Pergamon 0024-3205(94)00413-7 Ph?ARMACOLOGY LETTERS Accelerated Communication
SR 142801, THE FIRST POTENT NON-PEPTIDE ANTAGONIST OF THE TACHYKININ NK3 RECEPTOR X. Emonds-Altl, V. Proiettol,
D. Bichonl, J.P. Ducouxl, M. Heaulmel, B. Milouxz, M. Ponceletl, D. Van Broeckl, P. Vilainl, G. Neliat3, P. SoubriC1, G. Le Furl and J.C. Brelibel
Sanofi Recherche,
134184 Montpellier and 23 1676 Toulouse-Labtge, 3CEREP, 86600 Celle L’Evescault, France
France
(Submitted September 6, 1994; accepted September 27, 1994; received in final form October 13, 1994)
Abstract. SR 142801 is the first potent and selective non-peptide antagonist of the tachykinin NK3 receptor. It inhibited [MePhe7]NKB binding to its receptor from various species, including humans. SR 142801 was a competitive antagonist of [MePhe7]NKB_mediated contractions of guinea-pig ileum and inhibited the acetylcholine release following the activation of the guinea-pig ileum tachykinin NK3 receptor. In vivo, SR 142801 potently inhibited the turning behaviour induced by intrastriatal injection of senktide in gerbils, and appears as a powefil tool for investigation of the physiological and pathological role of NKB and its NK3 receptor. f@
Words: neurokinin
B, tachykinin
NK, receptor,
SR 142801
Introduction Neurokinin B (NKB) belongs to a group of neuropeptides named tachykinins, that includes substance P (SP) and neurokinin A (NKA). NKB preferentially interacts with the tachykinin NK3 receptor while SP and NKA are the preferred endogenous agonists of the tachykinin NKl and NK2 receptors, respectively (1,2). Progress in the understanding of the physiological and pathological role of tachykinins are highly dependent on the use of potent, specific and metabolically stable non-peptide antagonists of their receptors. Although several potent and specific non-peptide antagonists of the tachykinin NKl (3-5) and NK2 (6,7) receptors have been recently discovered, only peptide-type compounds showing specific pharmacological activity at the tachykinin NK3 receptor have been described (1,8). On the other hand, some non-peptide angiotensin receptor antagonists (DUP 753, L-158,809, EXP 3174) and the non-peptide tachykinin NK2 receptor antagonist, SR 48968, have been reported to interact in vitro at high concentrations with the tachykinin NK3 receptor (1,9-l 1). We now describe the first highly potent non-peptide antagonist of the tachykinin NK3 receptor, SR 142801 (Fig. I). Methods Drugs. All peptides were from Bachem (Switzerland) and were solubilized in organic solvents (ethanol or dimethylsulfoxyde). Radioactive ligands were purchased from Amersham (U.K.) and Du Pont NEN (USA). SR 142801 and its (R)-enantiomer (SR 142806) were synthesized at Sanofi Corresponding author : X. Emonds-Alt, Sanofi Recherche, 371 Rue du Professeur J. Blayac, F34184 Montpellier Cedex 04, France. Telephone : (33) 67-10-6710; Fax (33) 67-10-6767.
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SR 142801: NK3 Receptor Antagonist
Vol. 56, No. 1, 1995
Fig. 1 Structure of SR 142801 (S)-CN)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl) -4-phenylpiperidin-4-yl)-N-methylacetamide Recherche and were used as hydrochlorides. They were solubilized in organic solvents (ethanol or dimethylsulfoxyde) or suspended in distilled water by using 0.01 % of Tween 80. Binding assays. The affinity of SR 142801 for tachykinin receptors was evaluated in several receptor radioligand binding assays : (i) binding of [I25I]Bolton-Hunter labelled SP to NKI receptors of rat brain cortex, human lymphoblast cells (IM9) and human astrocytoma cells (U373MG, STTGl); (ii) binding of [I25I]iodohistidylNKA (or [I25I]neuropeptide r) to NK2 receptors of rat or hamster urinary bladder and guinea-pig ileum; (iii) binding of [ 125I]iodohistidyl-[MePhe7]NKB (or [ I25IlEledoisin) to tachykinin NK3 receptors of rat, guineapig and gerbil brain cortex. The effect of SR 142801 was also evaluated on the binding of [ 125I]iodohistidyl-[MePhe7]NKB to the human NK3 receptor, cloned and expressed in CHO cells according to Buell et al (12). All binding assays were conducted and analysed as previously described in details (3,6). In vitro assays. The in vitro pharmacological profile of SR 142801 was investigated by using several in vitro functional bioassays for tachykinin receptors (1,3,6) : (i) [MePhe7]NKB_induced contractions of rat portal vein and guinea-pig ileum; (ii) [ Sar9,Met(02) I I]SP-induced endothelium-dependent relaxation of rabbit pulmonary artery, previously contracted by 100 nM noradrenaline; (iii) [PAla8]NKA-(4-1O)-induced contractions of endothelium-denuded rabbit pulmonary artery. Experimental protocols were previously described in details (3,6). As already reported and discussed for some non-peptide antagonists of the tachykinin NKI and NK2 receptors (3,6,13), preliminary experiments have indicated that fill activity of SR 142801 was only observed after a long time of contact with the tissue and thus, in all experiments SR 142801 was added 140 min before the agonist. Antagonism was analysed according to Arunlakshana and Schild (14). On the other hand, antagonist activity of SR 142801 for tachykinin NK3 receptors was also evaluated by monitoring acetylcholine release following activation of these receptors in guinea-pig ileum. Longitudinal strips of guinea-pig ileum were incubated in oxygenated (95 % 02 - 5 % CO2) and thermostated (37 “C) Krebs-Henseleit physiological salt solution (20 ml), pH 7.4, containing 0.25 @i/ml [3H]choline. After 30 min incubation, strips were washed for 90 min in the Krebs-Henseleit physiological salt solution, containing 20 uM hemicholinium-3. Each strip was then incubated in 6 successive Krebs-Henseleit physiological salt solution (3 ml) at 6 min intervals. The agonist (senktide or [MePhe7]NKB) was introduced during the fourth incubation. SR 142801 was added to the incubation medium 10 min before the agonist and was then present until the end of the experiment. [3H]radioactivity was evaluated in the 6 incubation media and after solubilization, in the tissue.
Vol. 56, No. 1, 1995
SR 142801:
NK3 Receptor
PL-29
Antagonist
In vivo assays. SR 142801 activity at NK3 receptors was investigated on the turning behaviour induced by intrastriatal injection of senktide in gerbils. The general method has been described elsewhere (15,16) and was modified as follows. Free-hand microinjection (1 c(l) of se&tide or vehicle (water) was made slowly (2-3 s) in the right striatum of conscious, non-restrained gerbils (8 or more animals per group) by means of a 5 ul Hamilton microsyringe and a 10 mm calibrated needle (final length below the skin : 4.5 mm). The descending point of the needle was internal (2 mm) and caudal (2 mm) to the right orbitus. The animals did not show any behavioural sign of distress or pain during injection. Immediately after injection, they were placed individually in a cylindrical glass container. The number of complete contralateral rotations (i.e. away from the injected side) was visually recorded and cumulated over 3 periods of 2 min (2-4, 5-7, 8-10 min) post-intrastriatal injection. Statistical analysis was performed using ANOVA followed by the Dunnett’s t-test. SR 142801 activity at tachykinin NKl and NK2 receptors was investigated on [Sar9,Met(O2)I I]SP-induced hypotension in anaesthetized dogs and on [NlelO]NKA-(4-10)induced bronchoconstriction in anaesthetized guinea-pigs as previously described (3,6). Results Binding studies. Inhibition constants (Ki) for SR 142801 and SR 142806 obtained in the different radioligand binding assays for tachykinin receptors are given in table I. SR 142801 potently inhibited the binding of [125I]iodohistidyl-[MePhe7]NKB to tachykinin NK3 receptors from various species including humans. However, it was less active on tachykinin NK3 receptors from rat. Its (R)-enantiomer was less active, indicating a stereoselective action of SR 142801. On the other hand, SR142801 only moderately or slightly inhibited the binding of [125I]Bolton-Hunter labelled SP and [125I]iodohistidyl-NKA (or [125I]neuropeptide y) to tachykinin NKl and NK2 receptors, respectively. Finally, the selectivity of SR 142801 for tachykinin NK3 receptors was also demonstrated by its lack of activity at concentrations up to 1 uM in several binding assays for various neurotransmitter and neuropeptide receptors (3) except for calcium (verapamil site) and TABLE I Inhibition Constants (Ki) of SR 142801 and its (R)-enantiomer (SR 142806) in Tachykinin Receptor Radioligand Binding Assays Ki (nM)
Rat brain cortex IM9 U373MG STTGl Rat urinary bladder Guinea-pig ileum Hamster urinary bladder Rat brain cortex Guinea-pig brain cortex Gerbil brain cortex Human, expressed in CHO cells
Receptors
SR 142801
SR 142806
Ml ml =1 ml m2 m2 I=2 m3 m3 m3 m3
>2000 60t4 1374 + 130 428 + 24 10029 73 +9 >2000 1553 0.11 +0.01 0.42 + 0.04 0.21 + 0.03
>2000 220 + 36 3272 + 344 1146+49 248 + 76 350 2 48 >2000 406 + 60 26 t 3 30 5 9 3225
Values are means + S.E.M. obtained from at least 3 independent experiments performed in triplicate. Radioligands : [125I]Bolton-Hunter labelled SP for NKI receptors; [125I]iodohistidyl-NKA for NK2 receptors except of guinea-pig ileum where [ 125Ilneuropeptide y was used; [ 125IliodohistidyL [MePheT]NKB for NK3 receptors except of rat brain cortex where [ 125IlEledoisin was used.
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SR 142801: NK3 Receptor Antagonist
Vol. 56, No. 1, 1995
sodium (site 2) channels and opiate (binding of nalaxone) receptors where IC50 were in the range 0.1 to 1 l.tM (data not shown). In vitro studies. SR 142801 competitively antagonized [MePhe7]NKB-induced contractions of guinea-pig ileum. As shown in the figure 2, SR 142801 produced a concentration-dependent rightward shift of the concentration-response curves of [MePhe7]NKB. Assuming competitive antagonism, an apparent pA2 of 9.15 (8.93-9.37, 95 % confidence limits) was calculated by the method of Arunlakshana and Schild (14). On the other hand, SR 142801 did not antagonize [MePhe7]NKB-induced contractions of rat portal vein up to a concentration of 0.1 PM. A significant effect was only observed at 1 @4. Similarly, except at 1 pM, SR 142801 did not significantly antagonize [Sar9,Met(O2)1 l]SP-induced endothelium-dependent relaxation of rabbit pulmonary artery, previously contracted by 100 nM noradrenaline (a tachykinin NRl receptor functional assay) and [PAla8]m(4-1O)-induced contractions of endothelium-denuded rabbit pulmonary artery (a tachykinin NR2 receptor functional assay) (data not shown). Finally, antagonist activity of SR 142801 for tachykinin NK3 receptors was further studied on senktide- or [MePhe7]NKE3-induced release of [3H]acetylcholine from guinea-pig ileum. EC50 of senktide and [MePhe7]NKB were 0.24 + 0.12 nM and 4.3 + 1.6 nM (n=4), respectively. SR 142801 potently inhibited the acetvlcholine release with an ICso of 6.1 + 1.8 nM and 13.0 -+ 0.1 nM (n=4), respectively when-using 1 nM senktide and 10 g [MePhe$KEJ. Control0 SR
-11
SR
1428010 Control
142801 1nM
.
3nM
0
0.1 /AM
v
10nM
.
1pM
.
-10
-9
Log [Me Phe']-NKE
80
-8
-7
-6
-11
(M)
0.01 pM
-10
.
-9
Log [Me Phe7]-NKB
T/4
-8
-7 (M)
Fig. 2
Concentration-response curves for [MePhe7]NKB_induced contractions of guinea-pig ileum (left panel) and rat portal vein (right panel) in the absence (control) and in the presence of SR 142801. Values are means + S.E.M. (n=6-8). In vivo studies, Senktide injected at 0.1, 0.3, 1 and 3 pg dose-dependently (inverted U-shaped relationship) produced vigorous contralateral rotations in gerbils. When administered either intraperitoneally (i.p.) 30 min or orally (p.0.) 60 min before intrastriatal injection of 0.3 pg se&tide, SR 142801 dose-dependently inhibited this turning behaviour (Fig. 3). ID50 was 0.58 mg/kg i.p. (0.43-0.79, 95 % confidence limits) or 0.72 mgikg p.o. (0.5-1.0, 95 % confidence limits). When studied at 3 mg/kg p.o., SR 142801 showed a long acting effect that was significant at least until 8 h after se&tide injection. Its (R)-enantiomer (SR 142806) was inactive at least up to 1 mg/kg i.p. The in vivo pharmacological activity of SR 142801 was further evaluated on two animal models where the role of tachykinin NKl or NK2 receptor has been well characterized (3,7) : [Sar9,Met(02)1 l]SP-induced hypotension in anaesthetized dogs and l?JlelO]NKA-(4-10)-
SR 142801: NK3 Receptor Antagonist
Vol. 56, No. 1, 1995
PL-31
induced bronchoconstriction in anaesthetized guinea-pigs. When intravenously or intraperitoneally administered at 10 mg/kg, SR 142801 was completely inactive (data not shown).
25 -
20 P 5 t
15-
0 5 :2
lo-
S-
c
1
2
4
6
’ 6
‘I 24
TIME (h)
Fig. 3 Inhibition by SR 142801 of the turning behaviour induced by intrastriatal injection of 0.3 pg senktide in gerbils. Dose-effect relationships of SR 142801 when administered either intraperitoneally 30 min or orally 60 min before senktide injection (insert) and time-course study of the SR 142801 effect administered orally at 3 mg/kg. Values are means + S.E.M. (Dunnett’s t-test : * P< 0.05). C is the mean value in the control group. Discussion Our studies have demonstrated that SR 142801 is a competitive and highly potent non-peptide antagonist of the tachykinin NK3 receptor. However, its pharmacological activity was highly species dependent. In binding experiments, SR 142801 had a high affinity for the human, gerbil and guinea-pig tachykinin NK3 receptors. Its high affinity was confirmed in two in vitro functional assays for the guinea-pig NK3 receptors. It has been also confirmed in inhibiting different biochemical events ([Ca2+]i mobilization, inositol phosphate production, arachidonic acid release) following activation of the human NK3 receptor expressed in CHO cells (Oury-Donat et al, submitted for publication). Conversely, SR 142801 had a much lower affinity for the rat tachykinin NK3 receptors in binding and in vitro functional assays. These results clearly confirm previous studies (l,lO,l 1) suggesting that, as for tachykinin NKl and NK2 receptors (1,2), receptor differences among species also exist for the tachykinin NK3 receptor. On the other hand, the selectivity of SR 142801 for the tachykinin NK3 receptor has been shown by its lack of activity in two in vivo models where the role of tachykinin NKl or NK2 receptors has been well hypotension in dogs and mlelO]NKA-(4-lO)-induced established : [Sar9,Met(02)ll]SP-induced bronchoconstriction in guinea-pigs (3,6). Moreover, SR 142801 was only active at high concentrations in in vitro functional assays for tachykinin NKl and NK2 receptors and it was about lOOO-fold less active than specific non-peptide antagonists (SR 140333, SR 48968) of these receptors (3,6). Similarly, in binding assays, SR 142801 moderately interacted with some NKl and NK2 receptors, depending on the origin of these receptors. But compared to results reported for SR 140333 and SR 48968 (3,6), it had always 200- to lOOO-fold lower affinity than the specific antagonists (3,6). Finally, the potent antagonistic action of SR 142801 on the tachykinin NK3
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SR 142801:
NK3 Receptor
Antagonist
Vol. 56, No. 1, 1995
receptor has been also evidenced in vivo at the level of the central nervous system by inhibition of the turning behaviour induced by intrastriatal injection of senktide in gerbils. The compound can cross the blood brain barrier and is active by oral route with a long acting effect. In conclusion, SR 142801 may be useful to investigate the physiological and pathological role of NKB and its NK3 receptor. Acknowledgments We are grateful to C. Amal, N. Baudry, , N. Chabert, M. Fournier, I. Grossriether, C. Gueudet, P. Gueule, F. Guevara, 0. Hamelin, F. Lozano, P. Marquie and F. Ruffel-Seguier for their excellent technical assistance. References 1. D. REGOLI, Q.T. NGUYEN and D. JUKIC, Life Sci. 54 2035-2047 (1994). 2. C.A. MAGGI, R. PATACCHINI, P. ROVER0 and A. GIACHETTI, J. Autonom. Pharmacol. 13 23-93 (1993). 3. X. EMONDS-ALT, J.D. DOUTREMEPUICH, M. HEAULME, G. NELIAT, V. SANTUCCI, R. STEINBERG, P. VILAIN, D. BICHON, J.P. DUCOUX, V. PROIETTO, D. VAN BROCK, P. SOUBRIE, G. LE FUR and J.C. BRELIERE, Eur. J. Pharmacol. 250 403-413 (1993). 4. R.M. SNIDER, S.J.W. CONSTANTINE, J.A. LOWE, K.P. LONGO, W.S. LEBEL, H.A. WOODY, S.E. DROZDA, M.C. DESAI, F.J. VINICK, R.W. SPENCER and H.J. HESS, Science 25l_ 435-437 (1991). 5. C. GARRET, A. CARRUETTE, V. FARDIN, S. MOUSSAOUI, J.F. PEYRONEL, J.C. BLANCHARD and P.M. LADURON, Proc. Natl. Acad. Sci. USA @ 10208-10212 (1991). 6. X. EMONDS-ALT, P. VILAIN, P. GOULAOUIC, V. PROIETTO, D. VAN BROCK, C. ADVENIER, E. NALINE, G. NELIAT, G. LE FUR and J.C. BRELIERE, Life Sci. 50 PLlOl-PL106 (1992). 7. D.I. BALL, I.J.M. BERESFORD, G.P.A. WREN, Y.D. PENDRY, R.L.G. SHELDRICK, R.L.G. WALSH, M.P. TURPIN, R.M. HAGAN and R.A. COLEMAN, Br. J. Pharmacol. 112 48P (1994). 8. P. BODEN, J.M. EDEN, J. HODGSON, D.C. HORWELL, W. HOWSON, J. HUGHES, A.T. McKNlGHT, K. MEECHAM, M.C. PRITCHARD, J. RAPHY, G.S. RATCLIFFE, N. SUMAN-CHAUHAN and G.N. WOODRUFF, Bioorg. Med. Chem. Lett. 4 1679-1684 (1994). 9. L. CHRETIEN, G. GUILLEMETTE and D. REGOLI, Eur. J. Pharmacol. 256 73-78 (1994). 10. F. PETITET, J.C. BEAUJOUAN, M. SAFFROY, Y. TORRENS, and J. GLOWINSKI, Biochem. Biophys. Res. Commun. &l lSO-187 (1993). 11. F.Z. CHUNG, L.H. WU, M.A. VARTANIAN, K.J. WATLING, S. GUARD, G.N. WOODRUFF and D.L. OXENDER, Biochem. Biophys. Res. Commun. 198 967-972 (1994). 12. G. BUELL, M.F. SCHULZ, S.J. ARKINSTALL, K. MAURY, M. MISSOTTEN, N. ADAMI, F. TALABOT and E. KAWASHIMA, FEBS Lett. 299 90-95 (1992). 13. C.A. MAGGI, R. PATACCHINI, S. GIULIANI and A. GIACHETTI, Eur. J. Pharmacol. 234 83-90 (1993). 14. 0. ARUNLAKSHANA and H.O. SCHILD, Br. J. Pharmacol. Chemother. 14 48-58 (1959). 15. M. PONCELET, C. GUEUDET, X. EMONDS-ALT, J.C. BRELIERE, G. LE FUR and P. SOUBRIE, Neurosci. Lett. 149 40-42 (1993). 16. M. JUNG, R. CALASSI, J. MARUANI, M.C. BARNOUIN, J. SOUILHAC, M. PONCELET, C. GUEUDET, X. EMONDS-ALT, P. SOUBRIE, J.C. BRELIERE and G. LE FUR, Neuropharmacology 21167-179 (1994).