Pharmacology of MEN 11467: a potent new selective and orally- effective peptidomimetic tachykinin NK1 receptor antagonist

Neuropeptides (2001) 35(3&4), 137±147 ß 2001 Harcourt Publishers Ltd doi: 10.1054/npep.2001.0855, available online at http://www.idealibrary.com on

Pharmacology of MEN 11467: a potent new selective and orally-effective peptidomimetic tachykinin NK1 receptor antagonist R. Cirillo,1 M. Astolfi,1 B. Conte,1 G. Lopez,1 M. Parlani,1 G. Sacco,1 R. Terracciano,2 C. I. Fincham,2 A. Sisto,2 S. Evangelista,1 C. A. Maggi,1 S. Manzini1 Departments of Pharmacology1 and Departments of Chemistry2 Menarini Ricerche SpA, Pomezia ± Roma, Italy

Summary We have investigated the pharmacological properties of MEN 11467, a novel partially retro-inverse peptidomimetic antagonist of tachykinin NK1 receptors. MEN 11467 potently inhibits the binding of [3H] substance P (SP) to tachykinin NK1 receptors in the IM9 limphoblastoid cell line (p Ki ˆ 9.4 + 0.1). MEN 11467 is highly specific for the human tachykinin NK1 receptors, since it has negligible effects (p Ki < 6) on the binding of specific ligands to tachykinin NK2 or NK3 receptors and to a panel of 30 receptors ion channels unrelated to tachykinin receptors. The antagonism exerted by MEN 11467 at tachykinin NK1 receptors is insurmountable in saturation binding experiments, both KD and Bmax of SP were significantly reduced by MEN 11467 (0.3±10 nM). In the guinea-pig isolated ileum, MEN 11467 (0.03±1 nM) produced a nonparallel rightward shift of the concentration±response curve to SP methylester with a concomitant reduction of the Emax to the agonist (p KB ˆ 10.7 + 0.1). Moreover the antagonist activity of MEN 11467 was hardly reversible despite prolonged washout. In vivo, MEN 11467 produced a long lasting ( > 2±3 h) dosedependent antagonism of bronchoconstriction induced by the selective tachykinin NK1 receptor agonist, [Sar9, Met(O2)11]SP in anaesthetized guinea-pigs (ID50s0 ˆ 29 + 5, 31 + 12 and 670 + 270 mg/kg, after intravenous, intranasal and intraduodenal administration, respectively), without affecting bronchoconstriction induced by methacholine. After oral administration MEN 11467 produced a dose-dependent inhibition of plasma protein extravasation induced in guinea-pig bronchi by [Sar9, Met(O2)11] (ID50 ˆ 6.7 + 2 mg/kg) or by antigen challenge in sensitized animals (ID50 ˆ 1.3 mg/kg). After i.v. administration MEN 11467 weakly inhibited the GR 73632-induced foot tapping behaviour in gerbil (ED50 ˆ 2.96 + 2 mg/kg), indicating a poor ability to block central tachykinin NK1 receptors. These results demonstrate that MEN 11467 is a potent, highly selective and orally effective insurmountable pseudopeptide antagonist of peripheral tachykinin NK1 receptors with a long duration of action. ß 2001 Harcourt Publishers Ltd INTRODUCTION The mammalian tachykinins (substance P, neurokinin A and neurokinin B) are widely distributed throughout the central and peripheral nervous systems, where they act as neurotransmitters or neuromodulators (Maggi et al., 1993; Otsuka and Yoshioka, 1993). Substance P (SP) and Received 4 June 2000 Accepted 15 March 2001 Dr Stefano Manzini, Preclinical Development Department, Menarini Ricerche SpA, Via Tito Speri 10, I-00040 Pomezia ± Roma, Italy. Tel.: ‡ 39 06 911841; Fax: ‡ 39 06 9100220.

neurokinin A (NKA), albeit with different af®nities, are now considered as the physiological ligands for tachykinins NK1 receptors (Maggi and Schwartz, 1997) which is abundantly expressed in both the central and peripheral nervous systems of various mammalian species, including humans (Quartara and Maggi, 1998). At central level tachykinin NK1 receptors are involved in regulating various behavioural, endocrine and autonomic functions: centrally active tachykinin NK1 receptor antagonists are thought to be a novel class of therapeutic agents endowed with antidepressant, anxiolytic, antiemetic and analgesic properties as indicated by the results 137

138 Cirillo et al.

of preclinical studies (Quartara and Maggi, 1998) and by clinical studies with tachykinin NK1 receptor antagonists in humans (Dionne et al., 1998; Kramer et al., 1998; Diemunsch et al., 1999; Hesketh et al., 1999). In the peripheral nervous system the activation of tachykinin NK1 receptors determines several biological effects (vasodilation, increase in vascular permeability to plasma proteins, recruitment/activation of in¯ammatory cells, stimulation of secretions, smooth muscle contraction) collectively known as `neurogenic in¯ammation' ( Jancso' et al., 1968; Maggi et al., 1993) which is thought to play a role in the pathogenesis of chronic in¯ammatory diseases such as asthma, chronic in¯ammatory bowel diseases (Crohn's disease and ulcerative colitis), rheumatoid arthritis or psoriasis. Thus a number of therapeutic opportunities could arise from blockade of peripheral tachykinin NK1 receptors as well. Our understanding of the biological actions of SP and the involvement of tachykinin NK1 receptors has progressed signi®cantly following the identi®cation of several peptide and non-peptide tachykinin NK1 receptor antagonists with high af®nity and selectivity (Quartara and Maggi, 1998). Among them, peptide antagonists of tachykinin NK1 receptor are generally characterized by a short duration of action in vivo and by a low oral bioavailability (Hagan et al., 1991; Fujii et al., 1992; Hagiwara et al., 1992; Hashimoto et al., 1992). We now report on the discovery of MEN 11467 (Fig. 1) which is the result of the stepwise modi®cation of the peptide backbone of the peptidomimetic analogues of FK 888, MEN 10725 and MEN 10930 (Sisto et al., 1994; Astol® et al., 1997). An intermediate result of progressive

site-modi®cation of the peptide structure with a single retro-inverse amide bond at the C-terminal, led to the discovery of MEN 11149 which, despite further improvements in potency and selectivity, is hampered by a short duration of action after oral administration (Cirillo et al., 1998). MEN 11467 is characterized by the presence of a 1,2 (1R,2S) cis- diaminocyclohexane moiety and of a D-(2-naphthyl)alanine residue, this inserted in the peptide chain in a reversed direction, resulting in two consecutive retro-inverso modi®cations of the peptide amide bonds starting from the C-terminus. The introduction of these modi®cations preserves the analogue from enzymatic processing, being the reversed amide bonds are no longer recognisable by hydrolytic enzymes, but still maintains the proper three-dimensional assembly of the aromatic rings which is crucial for high af®nity interaction with tachykinin NK1 receptors (Pispisa et al., 1996).

MATERIALS AND METHODS Tachykinin NK1 receptor binding in human lymphoblastoma IM9 cells Tachykinin NK1 receptor binding was assessed using the human lymphoblastoma IM9 cell line. The IM9 cell line was obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured as previously described (Goso et al., 1994). Binding to IM9 cells was determined by incubating 4  106 cells/tube with 0.3 nM [3H]SP in the presence of increasing concentrations of MEN 11467 for 60 min at room temperature. The reaction was terminated

O

O

NH

CH3 N

O

NH

NH

CH3

Fig. 1 Chemical structure of MEN 11467. Neuropeptides (2001) 35(3&4), 137±147

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MEN 11467: a tachykinin NK1 antagonist 139

by centrifugation in a Beckman 12 microfuge (12,000  g for 6 min). Non-speci®c binding was determined in the presence of 10 mM SP. To evaluate the type of the antagonism of MEN 11467 on [3H]SP binding to IM9 cells, families of homologous competition curves for SP were obtained in the absence or presence of increasing concentrations of MEN 11467 (0.3±10 nM). Tachykinin NK2 and NK3 receptor binding In order to assess selectivity, the interactions of MEN 11467 with tachykinin NK2 receptors were investigated using both CHO cells transfected with human NK2 receptor (Catalioto et al., 1998) and hamster urinary bladder (Goso et al., 1995). The interaction with tachykinin NK3 receptors was investigated using guinea-pig cerebral cortex membranes (Renzetti et al., 1991). CHO cells transfected with human tachykinin NK2 receptor were purchased from Dr. J.K. Krause (Washington University, St. Louis, USA) and cultured as described elsewhere (Catalioto et al., 1998). hCHO cell membranes (60±63 mg of protein) were incubated for 30 min at 208C with 120±145 pM [125I]NKA in the presence of MEN 11467 (0.01 nM±10 mM). Non-speci®c binding was determined in the presence of 1 mM cold NKA. The binding of [3H] [b-Ala8]NKA(4±10) to hamster urinary bladder membranes (tachykinin NK2 receptors) was evaluated by incubating in triplicate aliquots of membrane preparation (100 mg) in 0.5 ml buffer (50 mM TrisHCl, pH 7.4 containing 2 mM MnCl2, 0.1% bovine serum albumin, 4 mM chymostatin, 40 mM bacitracin, 4 mM leupeptin and 1 mM thiorphan) for 90 min at room temperature in the presence of 0.5 mM [3H][b-Ala8] NKA-(4±10). Non-speci®c binding was determined in the presence of 1 mM non-radioactive [b-Ala8] NKA-(4±10). The af®nity of MEN 11467 for tachykinin NK3 receptors was investigated by measuring the displacement of [3H]senktide to guinea-pig cerebral cortex membranes. Membranes (ca. 0.8 mg/tube) were incubated at 208C for 1 h in the presence of 1 nM [3H]senktide in a ®nal volume of 0.5 ml Krebs-HEPES buffer, pH 7.4, composed of (mM): NaCl (120), KCl (4.8), CaCl2 (1), MgSO4 (1) and HEPES (20) plus MnCl2 (2), bovine serum albumin 0.1% and peptidase inhibitors (concentrations in brackets expressed as mg/ml): bacitracin (40), chymostatin (2), trypsin inhibitor (4), phosphoramidon (1.1). Non-speci®c binding was determined in the presence of 1 mM non radioactive senktide. Binding to receptors/ion channels unrelated to tachykinin receptors The af®nity of MEN 11467 for a panel of 30 binding sites (receptors ion channels) unrelated to tachykinin receptors ß 2001 Harcourt Publishers Ltd

was investigated at Cerep (Celle l'Evescault, France) according to their established proprietary protocols. Radioligands, tissue preparations and incubation conditions are summarized in Table 2 Contraction of guinea-pig ileum induced by SP methyl ester Male Dunkin-Hartley guinea-pigs (Charles River, Calco, Italy; 400±450 g) were killed by cervical dislocation. A segment of ileum (about 10 cm) was removed. A 1 ml glass pipette was placed in the ileal lumen and the longitudinal muscle layer was carefully removed and divided in 4 equal segments (1.5±2 cm long). Each segment was mounted under an initial tension of 0.5 g in a 5-ml organ bath containing Krebs solution (in the presence of atropine 1 mM, ( + )-chlorpheniramine 1 mM and indomethacin 3 mM) gassed with 5% CO2 and 95% O2 at 378C. Responses were recorded with an isometric transducer connected to a 7050 Unirecord polygraph (U. Basile, Comerio, Italy). The preparation was left to stabilize for 60 min, and three responses to 100 nM substance P methyl ester at 15 min intervals were then obtained to achieve stabilization in the preparation. After 15 min, a ®rst cumulative concentration±response curve to SP methyl ester (1±300 nM) was obtained. After 15 min, MEN 11467 (0.03±1 nM) was incubated for 60 or 120 min and then a second curve for the agonist was determined. The data were expressed as percentages of the maximum response to the ®rst agonist cumulative concentration±response curve. A kinetic study was also performed in order to investigate the rate of drug dissociation from receptors. Tissue were stimulated every 20 min by a submaximally effective concentration of SP methyl ester (30 nM). After obtaining three reproducible responses in the absence of the antagonist, MEN 11467 at 0.5 nM or FK888 at 5 nM or the vehicle were incubated for 60 min and then responses to SP methyl ester were produced every 20 min. After washout of the antagonist, the responses to SP methyl ester were challenged every 20 min for a further 4 h period. In each group the responses to SP methylester were expressed as % of the respective control before application of the antagonist. Bronchoconstriction in guinea-pigs Bronchoconstriction was induced with [Sar9,Met(O2)11]SP in male Dunkin Hartley guinea-pigs (300±400 g). The animals were anaesthetized with intraperitoneal urethane (1.2 g/kg) and arti®cially ventilated (tidal volume ˆ 10 ml/kg; 60 strokes/min; U. Basile 7025 rodent ventilator). d-Tubucurarine (3.9 mmol/kg i.v.) was administered to prevent spontaneous respiratory movements. The jugular Neuropeptides (2001) 35(3&4), 137±147

140 Cirillo et al.

vein was cannulated and body temperature was maintained at 378C with a heating pad. A side-arm from the tracheal cannula was attached to a Statham pressure transducer connected to a 8805D (Hewlett Packard, Andover, MA, USA) preampli®er to measure pulmonary in¯ation pressure. [Sar9,Met(O2)11]SP was intravenously administered at the dose of 1 nmol/kg 15, 30 and 45 min before and up to 180 min after the intravenous administration of vehicle, or of MEN 11467 by the intravenous (10±300 nmol/kg), intranasal (30±100 nmol/ kg) and intraduodenal (0.5±5 mmol/kg) routes. Bronchoconstriction was quanti®ed as an increase in pulmonary in¯ation pressure and recorded on a Hewlett Packard 7754A polygraph. To investigate the speci®city of NK1 receptor antagonism, a dose±response curve for methacholine (3±50 mg/kg i.v.)-induced bronchoconstriction was performed in anaesthetized guinea-pigs in animals that had received vehicle or MEN 11467 at 300 nmol/kg i.p. administered 30 min before the test. [Sar9,Met(O2)11]SP-induced plasma protein extravasation in guinea-pig bronchi Male albino Dunkin-Hartley guinea-pigs (Charles River, Calco, Italy) weighing 300±500 g were anaesthetized with urethane (1.2 g/kg i.p.) and the jugular vein was cannulated for drug administration. Evans blue dye (1.5% in saline containing 105 IU heparin/ml) was administered over 10 sec at the dose of 20 mg/kg/2 ml. After 5 min, [Sar9,Met(O2)11]SP (1 nmol/ml/kg) was injected to produce the leakage of the dye bound to albumins. After a further 5 min, the chest was opened and a needle was inserted into the left ventricle and kept in place. At this stage, the right atrium was cut to allow the blood to drain and the perfusion (saline at room temperature) started at the rate of 10 ml/min during 10 min. At the end, the caudal segment of the trachea and the main stem bronchi were removed for the subsequent assay. Tissues were blotted, weighed and incubated in 2 ml formamide for 24 h at 508C. The extravasation of Evans blue-labeled albumins from the circulation in the tissues was evaluated by measuring the optical density of the dye extracted by formamide at the wavelength of 620 nm with a spectrophotometer (Beckman DU-7). The amount of Evans blue extravasated in the tissue was expressed in ng per mg of tissue (wet weight). The value of dye extravasated in the absence of the NK1 stimulation (20.8 + 3 ng of Evans blue/mg tissue, n ˆ 6) was subtracted for all the data reported in Results or in Figures. The oral effect of MEN 11467 (0.3±10 mg/kg) was investigated with a 180-min pretreatment period before the challenge with the agonist in conscious animals which were anaesthetized 2 h later for the surgical preparation. Neuropeptides (2001) 35(3&4), 137±147

Antigen-induced plasma protein extravasation in bronchi of sensitized guinea-pigs Experiments were performed in male Dunkin Hartley albino guinea-pigs (350±550 g; Charles River, Calco, Italy). The antigen sensibilization was performed by administering to each animal 100 mg of ovoalbumin i.p. followed by a second injection (100 mg i.p.) one week later. Experiments were carried out 2±3 weeks after the last administration. Three hours later, plasma protein extravasation was determined in pentobarbital-anaesthetized animals (30±50 mg/ kg i.p.) by an i.v. challenge with ovoalbumin. MEN 11467 or FK 888 (3 mg/kg) was orally administered to conscious animals. Approximately 2 h later, under pentobarbitalinduced anaesthesia (30±50 mg/kg i.p.), the jugular vein was cannulated for drug injection. DL-thiorphan (1.3 mg/kg) and diphenhydramine (5 mg/kg) were slowly injected over 10 min. At the end, Evans blue (30 mg/kg in saline containing 105 IU heparin/ml) was administered over 10 sec. Five min later, 1 mg/kg i.v. ovoalbumin was injected to produce the leakage of the dye bound to albumins. The procedure to collect bronchi and to determine Evans blue content is similar to that above described. The basal value of dye extravasated without antigen stimulation (22.1 + 4 ng/mg tissue, n ˆ 6) was subtracted for all the data reported in Results or in Figures. GR 73632-induced foot tapping in gerbils The ability of MEN 11467 to block central tachykinin NK1 receptors was evaluated by measuring its activity toward the food tapping behaviour induced by central administration of a selective tachykinin NK1 receptor agonist GR73632 as described by Bristow and Young (1994) and by Rupniak and Williams (1994). Brie¯y, male and female mongolian gerbils (Charles River, Calco, Italy; 40±80 g) were anaesthetized by inhalation of an iso¯uorane/oxygen mixture. After exposing the skull, GR 73632 (3 pmol) was administered by i.c.v. into the lateral ventricles in animals that just 2 min before had received by i.v. route the vehicle (2% tween 80) or MEN 11467 (1±10 mg/kg). The CNS-penetrant tachykinin NK1 receptor antagonist CP 122721 was used as positive control (0.003±0.1 mg/kg; McLean et al., 1996). Following a brief period of recovery, the duration of repetitive hind foot tapping was recorded over a 5-min period. In order to evaluate the duration of action, in separate experiments MEN 11467 (1±10 mg/kg) was administered intravenously at 5±120 min before administration of GR 73632. Data analysis All the data in the text, tables and ®gures are means + S.E.M. Binding data were analyzed by means of ß 2001 Harcourt Publishers Ltd

MEN 11467: a tachykinin NK1 antagonist 141

LIGAND, a non-linear curve-®tting program (Munson and Rodbard, 1980). Families of homologous competition curves for [3H]substance P in the absence and in the presence of increasing concentrations of the antagonist MEN 11467 were simultaneously analyzed with the LIGAND program. An apparent pKB was derived from a doublereciprocal regression plot from at least 3 or 4 couples of curves (control and in the presence of the antagonist) for each concentration of antagonist (Kenakin, 1993). IC50 and ID50 values were calculated by means of the least squares method, considering the curves linear between 20 and 80% of the maximal effect. ID50 values for the antibronchospastic effect were determined considering the inhibition at peak effect (30, 90 and 90 min after the intravenous, intranasal and oral administration of the antagonist, respectively). Signi®cance was assessed by oneway analysis of variance followed by Bonferroni's test. Chemicals MEN 11467 (1R,2S)-2-N[1 (H)indol-3-yl-carbonyl] -1-N{N ÿ ( p -tolylacetyl)-N-(methyl)-D-3(2-naphthyl)alanyl} diaminolohexane) was synthesized at the Chemistry Department of Menarini Richerche, (Pomezia, Italy). FK 888 (N2-[(4R)-4-hydroxy-hydroxy-1-(1-methyl-1H-indol3-yl) carbonyl-L-propyl]-N-methyl-N-phenylmethyl-3(2-napthyl)-L-alaninamide) and CP 122,721, [( ‡ )-(2S,3S)3-(2-methoxy-5-tri¯uoromethoxybenzyl) amino-2-phenylpiperidine], were synthesized at Tocris Cookson Ltd., (Langford, Bristol, England). Senktide and [Sar9, Met(O2)11]-SP were from Peninsula Laboratories Inc, (Belmont, CA, USA). GR 73632 was from Neosystem, Strasbourg, France. SP-methyl ester was from Bachem, (Bubendorf, Switzerland). SP was from Novabiochem, (Switzerland). [3H]SP (speci®c activity ˆ 46 Ci/mmol) was purchased from Amersham International, (Buckinghamshire, UK). [3H]Senktide (speci®c activity ˆ 46.8 Ci/mmol) and [3H][bAla8] -NKA-(4±10) (speci®c activity ˆ 87.5 Ci/ mmol) were obtained from New England Nuclear, Boston,

USA.[125I]NKA(speci®cactivity ˆ 2,000Ci/mmol)wasfrom Amersham (Amersham, UK). If not otherwise stated all other materials were from Sigma Co. (St. Louis, MA, USA). MEN 11467 was dissolved in dimethylsulphoxide or ethanol (maximal ®nal concentration ˆ 0.3%) in all the in vitro experiments. For the in vivo studies, MEN 11467 was solubilized in DMSO (bronchoconstriction, intravenous administration) or in saline containing 2±6% Tween 80 (bronchoconstriction, intranasal and intraduodenal administration; plasma protein extravasation; GR 73632induced foot tapping in gerbils).

RESULTS Binding affinity for tachykinin receptors The af®nity of MEN 11467 for the tachykinin NK1 receptor was assessed by measuring the displacement of [3H]SP binding to human IM9 cells. MEN 11467 inhibited [3H]SP binding to IM9 cells in a concentration-dependent manner yielding a pKi value of 9.4 + 0.1 (n ˆ 7). The nature of MEN 11467 antagonism was studied by Scatchard analysis of speci®c [3H] SP binding to IM9 cells in the absence and presence of increasing concentrations (1±30 nM) of MEN 11467 or of 10 nM FK 888. MEN 11467 (n ˆ 4 for each concentration of the antagonist) concentration-dependently increased KD values with a concentration±dependent signi®cant decrease of Bmax values. On the other hand FK 888 produced a signi®cant increase of KD leaving unaffected the Bmax value (Table 1). The af®nity of MEN 11467 for human tachykinin NK2 receptor expressed in transfected CHO cells ( p Ki ˆ 6.4 + 0.1, n ˆ 4) was about three orders of magnitude lower than that measured for tachykinin NK1 receptor. Likewise MEN 11467 displayed poor af®nity for the binding of [3H][bAla8]NKA (4±10) to hamster urinary bladder membranes (tachykinin NK2 receptors, ( p Ki ˆ 5.9 ‡ 0.1, n ˆ 6) or that of [3H]senktide to guinea-pig cerebral cortex membranes (tachykinin NK3 receptors, pKi < 5, n ˆ 6).

Table 1 Effects of MEN 11467 and FK 888 on the binding parameters of [3H]substance P to tachykinin NK1 receptors expressed on IM9 cells.

Control MEN 11467 0.3 nM MEN 11467 1 nM MEN 11467 3 nM MEN 11467 10 nM FK 888 10 nM

KD (nM)

Bmax (sites/cell)

0.16 + 0.02 0.30 + 0.04** 0.55 + 0.08** 0.86 + 0.15** 1.3 + 0.3** 1.9 + 0.3**

5092 + 325 4789 + 335 4199 + 377* 3328 + 399** 1966 + 373** 5876 + 290

Data are the means + S.E.M. of at least 4 separate experiments performed in triplicate. Statistical analysis was performed with the LIGAND program. *P < 0.05; **P < 0.01 vs control value

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142 Cirillo et al.

Binding to receptors/ion channels unrelated to tachykinin receptors MEN 11467 lacked appreciable af®nity for L-type voltagesensitive calcium channel, displacing [3H]desmethoxyverapamil binding with a pKi < 5 + 0.1 (n ˆ 3). MEN 11467 was essentially inactive in an extensive receptogram for a number of receptors and ion channels. In all assays, MEN 11467 had Ki values greater than 1 mM (Table 2). Antagonism at tachykinin NK1 receptors in the guinea-pig ileum MEN 11467 produced a concentration±dependent, nonparallel rightward shift of the concentration±response curve of SP methyl ester in the guinea-pig isolated and a concomitant concentration-dependent inhibition of the Emax to the agonist (Fig. 2A). A pKB value of 10.7 + 0.1 for noncompetitive antagonism was calculated with the double-reciprocal regression plot from at least 3 or 4 couples of curves (control and in the presence of the antagonist) for each concentration of antagonist (Kenakin, 1993).

On the other hand, FK 888 (10, 30 and 100 nM) produced a rightward parallel shift of the concentration±response curve without depression of the maximal response, (slope of schild plot ÿ 0.94 + 0.13), yielding an apparent pA2 value of 8.8 + 0.2 (n ˆ 6; data not shown). Owing to the very different type of antagonism exerted by MEN 11467 and FK 888 in the guinea-pig isolated ileum assay it appeared of interest to explore whether the compounds also differ in terms of reversibility of the antagonism by washout. MEN 11467 was further examined in a kinetic study aimed to evaluate the rate of dissociation from tachykinin NK1 receptor in comparison to FK 888. MEN 11467 (0.5 nM) and FK 888 (5 nM) time-dependently inhibited the response to tachykinin NK1 receptor agonist: at 60 from beginning of superfusion with the antagonists the residual responses to SP methylester averaged 23 + 4 and 39 + 6% of control in the presence of MEN 11467 (0.5 nM) and FK 888 (5 nM, respectively, n ˆ 5 for each drug). The reversibility of the antagonistic effect by washout was markedly different for the two drugs: at 4 h from the beginning of washout of MEN 11467, the response to SP methylester was still markedly depressed (38 + 5% of

Table 2 Affinity profile of MEN 11467 in different receptor assays. Binding site

Radioligand, tissue, incubation conditions

Ki (mM)

a1 (non sel.) a2 (non sel.) b1 b2 BDZc BDZp B2 D1 D2 ETB GABAA GABAB H1 H2 Muscarinic Nicotinic NK2(h) NK3(h) NMDA  k m 5-HT1A 5-HT2A 5-HT3 Somatostatin Ca2 ‡ channel (L, DHP site) Ca2 ‡ channel (N) Na ‡ chann. (site 1) Na ‡ chann. (site 2)

[3H]parazoson (0.25 nM), rat cerebral cortex, 60 min/258C [3H]RX821002 (0.5 nM), rat cerebral cortex, 30 min/228C [3H]( ÿ )CGP 12177 (0.5 nM), rat heart, 20 min/258C [3H]( ÿ )CGP 12177 (0.4 nM), guinea-pig lung, 20 min/258C [3H]flunitrazepam (0.4 nM), rat cerebral cortex, 60 min/248C [3H]PK 11195 (0.2 nM), rat heart, 15 min/258C [3H]NPC 17731 (30 pM), guinea-pig ileum, 90 min/228C [3H]SCH 23390 (0.3 nM), rat straitum, 45 min/258C [3H]YM-09151-2 (0.1 nM), rat straitum, 60 min/258C [125I]endothelin-1 (10 pM), rat cerebellum, 60 min/378C [3H]muscimol (2.5 nM), rat cerebral cortex, 10 min/48C [3H]GABA (10 nM), rat cerebral cortex, 10 min/228C [3H]pyrilamine (1 nM), guinea-pig lung, 15 min/258C [125I]APT (0.1 nM), guinea-pig straitum, 150 min/228C [3H]QNB (50 nM), rat cerebral cortex, 120 min/258C [3H]cytisine (1.5 nM), rat cerebral cortex, 75 min/48C [125I]NKA (0.2 nM), hCHO cells, 60 min/228C [3H]senktide (5 nM), hCHO cells, 60 min/228C [3H]CGP 39653 (2 nM), rat cerebral cortex, 60 min/48C [3H]DPDPE (1.5 nM), guinea-pig cerebral cortex, 120 min/258C [3H]U 69593 (0.7 nM), guinea-pig cerebellum, 80 min/258C [3H]DAMGO (1 nM), rat cerebral cortex, 60 min/258C [3H]8-OH-DPAT (0.5 nM), rat cerebral cortex, 30 min/258C [3H]ketanserin (0.5 nM), rat cerebral cortex, 15 min/378C [3H]BRL 43694 (1 nM), N1E-115 cells, 180 min/48C [125I]Tyr11-somatostatin (50 pM), AtT-20 cells, 60 min/378C [3H]PN 200-110 (40 pM), rat cerebral cortex, 90 min/258C [125I]w-conotoxin GVIA (1 pM), rat cerebr. cortex, 30 min/258C [3H]saxitoxin (2 nM), rat cerebral cortex, 30 min/228C [3H]batrachotoxinin (10 nM), rat cerebral cortex, 60 min/258C

>1 >1 >1 >1 >1 >1 >1 >1 >1 >1 >1 >1 >1 >1 >1 >1 1 >1 >1 >1 >1 >1 >1 >1 >1 >1 >1 >1 >1 >1

Abbreviations: a1, a2 ˆ adrenoceptor; b1, b2 ˆ adrenoceptor; BDZc and BDZp ˆ central and peripheral benzodiazepine; B2 ˆ bradykinin; D1 and D2 ˆ dopamine; ETB ˆ endothelin; GABAA and GABAB ˆ g-amino-n-butyric acid; h ˆ human; H1 and H2 ˆ histamine; , k and m ˆ opioid; 5-HT1A, 5-HT2A and 5-HT3 ˆ serotonin. Neuropeptides (2001) 35(3&4), 137±147

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MEN 11467: a tachykinin NK1 antagonist 143

100

A

% maximal effect

80

60

40

20

0 10-9

10-8

10-7

10-6

10-5

10-4

[M]

% second response to SPOMe

120

Controls FK888 (5nM) MEN 11467 (0.5nM)

B

100

Administration of MEN 11467 by the intravenous, intranasal or intraduodenal administration resulted in dosedependent inhibition of the bronchoconstriction induced by [Sar9,Met(02)11]SP. ID50 values calculated at their corresponding time-to-peak of the inhibitory effect were as follows: 29 + 5, 31 + 12 and 670 + 270 mg/kg for the intravenous, intranasal and intraduodenal route, respectively (Fig. 3A, B, C). The duration of action after the intravenous administration exceeded the observation time of the experiment ( >3 h). After intranasal or intraduodenal administration a slower onset of action was observed, peaking at about 1 h from administration and the antagonist activity was thereafter persistent throughout the observation period ( >2h) (Fig. 3B, C). The methacholine-induced dose±response curve for bronchoconstriction was unaffected by MEN 11467: the ED50 values were 8.5 + 1.3 (n ˆ 7) and 9.1 + 2 (n ˆ 4) mg/kg in vehicle- and MEN 11467-treated animals, respectively (data not shown).

80

Effect on plasma protein extravasation induced by [Sar9,Met(02)11]SP

60 40 20 0 0

40

80

160

200

220

240

280

320

Orally-administered MEN 11467, (3 h before) produced a dose-dependent inhibition of plasma protein extravasation induced [Sar9,Met(02)11]±SP in guinea-pig bronchi with an ID50 ˆ 6.7 + 2 mg/kg (Fig. 4). FK 888 at 10 mg/ kg was ineffective (Fig. 4).

Time (min) Fig. 2 N K1 antagonism of MEN 11467 in isolated guinea-pig ileum. A) Concentration±response curves for substance P methyl ester-induced contractions of guinea-pig ileum in the presence of vehicle ( u , n ˆ 20) or 0.03 ( j , n ˆ 6), 0.1 (n, n ˆ 6), 0.3 (s, n ˆ 4) and 1 nM (d, n ˆ 4) MEN 11467. Values are expressed as percentages relative to the maximum response obtained in the first cumulative agonist-induced contraction curve. B) Timedependent variations of contractile responses of guinea-pig ileum stimulated every 20 min by substance P methyl ester (30 nM) in the presence of vehicle (n ˆ 11) and after addition of 0.5 nM MEN 11467 (n ˆ 5) or 5 nM FK 888 (n ˆ 7). The antagonists were left to incubate for 60 min before removal. Results are expressed as percentages of the response to substance P methyl ester obtained at time 0.

control, n ˆ 5) whereas a partial recovery was evident after washout of FK 888 (68 + 9% of control, Fig. 2B). Effect of MEN 11467 on bronchoconstriction induced by [Sar9,Met(02)11]SP in anesthetized guinea-pigs In each experimental group the inhibitory effect of MEN 11467 on the bronchoconstriction produced by the tachykinin NK1 receptor agonist was compared to the response to the agonist in vehicle-treated groups which showed non-statistically signi®cant time-dependent variations. ß 2001 Harcourt Publishers Ltd

Antigen-induced plasma protein extravasation in bronchi of sensitized guinea-pigs In guinea-pigs sensitized to ovoalbumin, the i.v. administration of the antigen produced a plasma protein extravasation (evaluated as the amount of Evans blue leakage) in main bronchi averaging 99 + 22 ng/mg tissue (n ˆ 8; Fig. 5). Pretreatment with MEN 11467 (1±3 mg/kg p.o.; 3 before antigen challenge) produced a dose-dependent inhibition of plasma protein extravasation with an ID50 of 1.3 mg/kg, whereas FK 888 was ineffective (Fig. 5).

Inhibition of GR 73632-induced foot tapping in gerbils MEN 11467, administered intravenously 5 min before, only weakly inhibited the foot tapping induced by the central administration of GR 73632 (ED50 ˆ 3 + 2 mg/kg; Fig. 6A). Conversely, CP 122721 potently antagonized the NK1-mediated behavioural effect (ED50 ˆ 6.4 + 2 mg/kg i.v.; Fig. 6A). In the same experimental conditions, FK 888 up to 3 mg/kg i.v. inhibited only marginally ( ÿ 28 + 11%, n ˆ 5) the foot tapping induced by GR 73632. Neuropeptides (2001) 35(3&4), 137±147

144 Cirillo et al.

p<0.05

A 20 ng Evans blue/mg tissue

80

% inhibition

0 −20 −40 −60 −80

70 60 50 40 30 20 10 0 Vehicle

−100 30

60

90

120

150

180

Time (min)

B 40 20

10

10

mg/kg p.o.

FK 888

Fig. 4 Plasma protein extravasation induced by [Sar9,Met(02)11] substance P (1 nmol/kg i.v.) in guinea-pig bronchi. Effect of the oral treatment with vehicle (n ˆ 17), MEN 11467 at 0.3 (n ˆ 5), 3 (n ˆ 5) and 10 (n ˆ 4) or FK 888 at 10 mg/kg (n ˆ 4). Each antagonist or its vehicle was administered 180 min before the agonist challenge.

p<0.01

0 140 −20 −40 −60 −80 −100 5

30

60

90

120

150

180

Time (min)

C

ng Evans blue/mg tissue

% inhibition

3 MEN 11467

5

120 100 80 60 40 20 0 Vehicle

1

3

MEN 11467

60

1

mg/kg p.o.

FK 888

Fig. 5 Effect of oral administration of vehicle (n ˆ 8), MEN 11467 at 1 (n ˆ 3) and 3 (n ˆ 7) or FK 888 at 10 mg/kg (n ˆ 3) on plasma protein extravasation induced by ovalbumin in bronchi of sensitized guinea-pig. Each antagonist or its vehicle was administered orally 180 min before the agonist challenge.

40 % inhibition

0.3

20 0 −20 −40 −60 −80 −100 5

30

60

90

120 150 180

The question was raised as to whether MEN 11467 could have a slow rate of penetration into the CNS: however as shown in Fig. 6B when administered up to 120 min before the administration of GR 73632, MEN 11467 signi®cantly inhibited the foot-tapping behaviour only at doses of 3±10 mg/kg i.v.

Time (min) Fig. 3 Inhibitory effect of MEN 11467, administered through different routes, on bronchoconstriction induced by [Sar9, Met(02)11]substance P (1 nmol/kg i.v.) in anaesthetized guinea-pigs. After 2 or more reproducible control responses to the agonist had been established at 30 min interval, the antagonists or the vehicle were administered either intravenously (A), intranasally (B) or intraduodenally (C). Responses are expressed as % variation of the control response to the agonist. Each point represents the mean + s.e.m. of at least 5±8 determinations. Neuropeptides (2001) 35(3&4), 137±147

DISCUSSION MEN 11467 is the prototype of a new class of partially retro-inverse peptidomimetic antagonists of the tachykinin NK1 receptor in which the presence of a 1,2 (1R, 2S) cisdiaminocyclohexane moiety and of a D-(2-naphthyl)alanine residue, both inserted in the peptide chain in a reversed direction, determined the presence of two ß 2001 Harcourt Publishers Ltd

MEN 11467: a tachykinin NK1 antagonist 145

A 100

% inhibition

80 60 40 CP 122721

MEN 11467

20 0 0.003

0.03

0.1

1

3

10 mg/kg i.v.

B 100 10 mg/kg i.v.

% inhibition

80 60 40 20

3 mg/kg i.v. 1 mg/kg i.v.

0 5

15

60

120

180

(min)

Fig. 6 Effect of MEN 11467 administered intravenously on GR 73632-induced foot tapping in gerbils. A) Comparison with the CNS-penetrant NK1 antagonist CP 122721 administered intravenously. B) Duration of the inhibitory effect of different i.v. doses of MEN 11467.

consecutive retro-inverse-peptide bonds. The modi®cations to the backbone structure led to a more favourable pharmacological pro®le of MEN 11467 if compared to the structurally related compounds FK 888 and MEN 11149 (Palma et al., 1999). The af®nity of MEN 11467 for human tachykinin NK1 receptors, as evaluated in binding studies, is in the subnanomolar range ( pKi ˆ 9.4 + 0.1) which is approximately 4 orders of magnitude higher than that found on murine tachykinin NK1 receptor. Such high discriminatory power between human and murine tachykinin NK1 receptor has been reported also for other tachykinin NK1 antagonists like CP 96345, FK 888, LY 303870 and MEN 10930 (Beresford et al., 1991; Fujii et al., 1992; Gitter et al., 1995; Astol® et al., 1997). The af®nity of MEN 11467 for the human NK1 receptor is approximately one order of magnitude higher than that of FK 888 ( pKi ˆ 8.9; Cirillo et al., 1998) and the structurally related MEN 11149 ( pKi ˆ 8.5; Cirillo et al., 1998). ß 2001 Harcourt Publishers Ltd

The af®nity of MEN 11467 for tackykinin NK1 receptors, measured in functional experiments in longitudinal smooth muscle preparations from guinea-pig ileum, appears to be greater ( pKB ˆ 10.7 + 0.1) than that reported for FK 888 ( pA2 ˆ 8.8; Cirillo et al., 1998) or MEN 11149 ( pKB ˆ 9.6; Cirillo et al., 1998). This high value of apparent af®nity is likely linked to the unsurmountable type of antagonism documented in both radioligand binding experiments (concomitant decrease of both KD and Bmax values of the tritiated SP) and in functional experiments in the guinea-pig ileum: a similar noncompetitive behaviour in displacing SP binding from human tachykinin NK1 receptor has been reported previously for some nonpeptide antagonists such as CP 122721 (McLean et al., 1996) and SDZ NKT 343 (Walpole et al., 1998) as well as for MEN 11149 (Cirillo et al., 1998), a close analog of MEN 11467. The unsourmontable type of antagonism at tachykinin NK1 receptor likely correlates with poor reversibility of the compound from tachykinin NK1 receptors in guinea-pig ileum: these experiments demonstrated that, even after prolonged washout, the exposure to a very low concentration of MEN 11467 (0.5 nM) determines an almost irreversible type of antagonism toward contractions induced by SP methylester. This characteristic is likely to account, at least partially, for the long duration of action of the compound at peripheral tachykinin NK1 receptors. MEN 11467 also shows a high (at least 3 orders of magnitude) degree of selectivity for tachykinin NK1 versus NK2 and NK3 receptors, whereas its af®nity for other non-tachykinin receptors is negligible ( >1 mM). Moreover, the compound exhibits a negligible af®nity for a number of ion channels, a drawback encountered for some nonpeptide tachykinin NK1 receptor antagonists (Maggi et al., 1993). The high potency and selectivity of MEN 11467 are paralleled by a high potency and long duration of action in blocking peripheral tachykinin NK1 receptors: at this level the compound is also active after intraduoneal and oral administration indicating its suitability for further pharmaceutical development by these routes of administration. Interestingly, MEN 11467 is poorly effective in blocking the GR73632 foot-tapping behaviour in gerbils. Since the pharmacology of gerbil tachykinin NK1 receptor, in terms of af®nity for nonpeptide receptor antagonists is identical to that of the human tachykinin NK1 receptor (Beresford et al., 1991; Gitter et al., 1991; Rupniak et al., 1997), we interpret these observations as indication that MEN 11467 has a poor ability to penetrate the CNS. As mentioned in the Introduction, there are a number of possible therapeutic indications to be exploited for centrally-active tachykinin NK1 receptor antagonists. However, the abundant expression of both tachykinins and tachykinin NK1 receptors in the mammalian CNS Neuropeptides (2001) 35(3&4), 137±147

146 Cirillo et al.

make it quite possible that blockade of central NK1 receptors may determine undesired effects, especially during repeated administration of the drug. Although this point remains a matter of speculation, it appears conceivable that, if targeted for blocking peripheral tachykinin NK1 receptors, a peripherally acting compound may be better tolerated than CNS-penetrating drugs for treatment of diseases in the peripheral nervous system such as asthma or in¯ammatory bowel diseases. In conclusion, the present data indicate that MEN 11467 is a potent and selective antagonist suitable for producing long lasting blockade of peripheral tachykinin NK1 receptors after systemic administration. The noncompetitive and hardly reversible interaction with tachykinin NK1 receptors make this compound an interesting drug candidate for treatment of diseases in which blockade of peripheral tachykinin NK1 receptor can be devised to be of therapeutic relevance. ACKNOWLEDGEMENTS This work was supported in part by Istituto Mobiliare Italiano (contract n.56665). We would like to thank Miss C. Azzurrini for her secretarial assistance and Dr. Patacchini R. for helpful advice and suggestions. REFERENCES Astolfi M, Patacchini R, Maggi M, Manzini S (1997) Improved discriminatory properties between human and murine tachykinin NK-1 receptors of MEN 10930: a new potent and competitive antagonist. Neuropeptides 31: 373±379. Beresford IJM, Birch PJ, Hagan RM, Ireland SJ (1991) Investigation into species variants in tachykinin NK-1 receptors by use of the non-peptide antagonist, CP 96,345. Br J Pharmacol 104: 292±293. Bristow LJ, Young L (1994) Chromodacryorrhea and repetitive hind paw tapping: models of peripheral and central tachykinin NK1 receptor activation in gerbils. Eur J Pharmacol 253: 245±252. Catalioto RM, Criscuoli M, Cucchi P, et al. (1998) MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist. Br J Pharmacol 123: 81±91. Cirillo R, Astolfi M, Conte B, et al. (1998) Pharmacology of the peptidomimetic, MEN 11149, a new potent, selective and orally active tachykinin NK1 receptor antagonist. Eur J Pharmacol 341: 201±209. Diemunsch P, Schoeffler P, Bryssine B, et al. (1999) Antiemetic activity of the NK1 receptor antagonist GR205171 in the treatment of established postoperative nausea and vomiting after major gynaecological surgery. Br J Ananesthesia 82: 274±276. Dionne RA, Max MB, Gordon SM, et al. (1998) The substance P receptor antagonist CP99994 reduces acute postoperative pain. Clin Pharmacol Ther 64: 562±568. Fujii T, Murai M, Morimoto H, et al. (1992) Pharmacological profile of a high affinity dipeptide NK1 receptor antagonist. FK 888. Br J Pharmacol 107: 785±789. Gitter BD, Bruns RD, Howbert JJ, et al. (1995) Pharmacological characterization of LY303870: a novel, potent and selective

Neuropeptides (2001) 35(3&4), 137±147

nonpeptide substance P (neurokinin-1) receptor antagonist. J Pharmacol Exp Ther 275: 737±744. Gitter BD, Waters DC, Bruns RF, Mason NR, Nixon J, Howbert JJ (1991) Species differentes in affinities of non-peptide antagonists for substance P receptors. Eur J Pharmacol 197: 237±238. Goso C, Astolfi M, Parlani M, Manzini S (1995) [3H] [b-Ala8] neurokinin A-(4±10): a novel, selective radioligand for the tachykinin NK2 receptor. Eur J Pharmacol 294: 239±245. Goso C, Potier E, Manzini S, Szallasi A (1994) Comparison of tachykinin NK1 receptors in human IM9 and U373 MG cells, using antagonist (FK888, ( ‡ )-CP-96, 345, and RP 67580) binding. Eur J Pharmacol 254: 221±227. Hagan RM, Ireland SJ, Bailey F, McBride C, Jordan CC, Ward P (1991) A spirolactam conformationally-constrained analogue of physalaemin which is a peptidase-resistant, selective neurokinin NK1 receptor antagonist. Br J Pharmacol 102: 168P. Hagiwara D, Miyake H, Morimoto H, Murai M, Fujii T, Matsuo M (1992) Studies of neurokinin antagonists. The design of novel tripeptides possessing the glutaminyl-D-tryptophylphenylalanine sequence as substance P antagonists. J Med Chem 35: 2015±2025. Hashimoto M, Hayashi K, Murai M, et al. (1992) WS9326A, a novel tachykinin antagonist isolated from streptomyces violaceusinger no. 9326. II Biological and pharmacological properties of WS9326A and tetrahydro-WS9326A (FK224). J Antibiotics 45: 1064. Hesketh PJ, Gralla RJ, Webb RT, et al. (1999) Randomized phase II study of the NK1 receptor antagonist CJ-11, 974 in the control of cisplatin-induced emesis. J Clin Oncology 17: 338±343. Jancso N,JancsoÂ-Gabor A, Szolcsanyi J (1968) The role of sensory nerve endings in neurogenic inflammation induced in human skin and in the eye and paw of the rat. Br J Pharmacol 32: 32±41. Kenakin TP (1993) In: Pharmacological analysis of drug-receptor interaction, 2nd ed. New York: Raven Press. 27±32. Kramer MS, Cutler N, Feighner J, et al. (1998) Distinct mechanism for antidepressant activity by blockade of central SP receptors. Science 281: 1640±1645. Maggi CA, Patacchini R, Rovero P, Giachetti A (1993) Tachykinin receptors and tachykinin receptor antagonists. J Autonom Pharmacol 13: 958±962. Maggi CA, Schwartz TW (1997) The dual nature of the tachykinin NK-1 receptor. Trends Pharmacol Sciences 18: 351±355. McLean S, Ganong A, Seymour PA, et al. (1996) Characterization of CP-122, 721; a nonpeptide antagonist of the neurokinin NK1 receptor. J Pharmacol Exp Ther 277: 900±908. Munson PJ, Rodbard D (1980) LIGAND: a versatile computerized approach for the characterization of ligand-binding systems. Anal Biochem 107: 220±239. Otsuka M, Yoshioka K. (1993) Neurotransmitter functions of mammalian tachykinins. Physiol Rev 73: 229±307. Palma C, Nardelli F, Manzini S (1999) Correlation between binding characteristics and functional antagonism in human glioma cells by tachykinin NK1 receptor antagonist. Eur J Pharmacol 374: 435±443. Pispisa B, Cavalieri F, Venanzi M, Sisto A (1996) Absorption, emission, and chiroptical spectra of neurokinin 1 tachykinin receptor antagonists: the role of charge-transfer states on the biological activity. Biopolymers 40: 529±542. Quartara L, Maggi CA (1998) The tachykinin NK1 receptor. Part I: ligands and mechanisms of cellular activation. Neuropeptides 31: 537±563. Renzetti AR, Barsacchi P, Criscuoli M, Lucacchini A (1991) Characterization of NK3 binding sites in rat and guinea pig cortical

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membranes by the selective ligand [3H]senktide. Neuropeptides 18: 107±114. Rupniak NMJ, Tattershall FD, Williams AR, et al. (1997) In vitro and in vivo predictors of the anti-emetic activity of tachykinin NK1 receptor antagonists. Eur J Pharmacol 326: 201±209. Rupniak NMJ, Williams A (1994) Differential inhibition of foot tapping and chromodacryorrhoea in gerbils by CNS peentrant and non-penetrant tachykinin NK1 receptor antagonists. Eur J Pharmacol 265: 179±183.

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Sisto A, Bonelli F, Fincham CI, et al. (1994) Synthesis and biological evaluation of novel NK1 tachykinin receptor antagonists: the use of cycloalkyl amino acids as a template. Biopolymers 36: 511±524. Walpole CSJ, Brown MCS, James IF, et al. (1998) Comparative general pharmacology of SDZ NKT 343, a novel, selective NK1 receptor antagonist. Br J Pharmacol 124: 83±92.

Neuropeptides (2001) 35(3&4), 137±147