European JournalofPharmacology, 217 (1992) 191-195

191

© 1992 Elsevier Science Publishers B.V. AI1 rights reserved 0014-2999/92/$05.00

EJP 52525

Antiinflammatory and analgesic activity of a non-peptide substance P receptor antagonist A t s u s h i N a g a h i s a a, Y o s h i h i t o K a n a i a, O s a m u Suga a, K a n a T a n i g u c h i a, M e g u m i Tsuchiya ~, J o h n A. L o w e III b a n d H a n s - J i i r g e n Hess b Department of Medicinal Biology, Central Research Division, Pfizer Inc., 5-2 Taketoyo, Aichi 470-23, Japan and b Department of Medicinal Chemistry, Central Research Division, Pfizer Inc., Groton, CT06340, USA Received 21 November 1991, revised MS received 26 February 1992, accepted 14 April 1992

CP-96,345, a potent non-peptide antagonist of the substance P (SP) receptor, inhibited SP-, neurokinin A (NKA)- and neurokinin B-induced plasma extravasation in guinea pig dorsal skin. The inhibition was specific for the three tachykinins; CP-96,345 was not active against plasma leakage caused by histamine, bradykinin, platelet-activating factor or leukotriene D4. CP-96,345 inhibited capsaicin-induced plasma extravasation in the ureter, an inflammatory response caused by neuropeptides released from afferent C-fibers. Thus, the NK 1 receptor appears to play a major role in vascular permeability increases induced by exogenous and endogenous tachykinins. In contrast, CP-96,345 was inactive against SP- and NKA-induced contraction of guinea pig ureter, suggesting that the smooth muscle contraction is not NKl-mediated. CP-96,345 exhibited analgesic activity in acetic acid-induced abdominal stretching in mice, indicating for the first time that SP plays a critical role in this model. The results of these studies support a pathophysiological role of SP and the NK~ receptor under acute neurogenic inflammatory conditions and in pain. Neurogenic inflammation; Plasma extravasation; Substance P antagonists; Tachykinins; Capsaicin; Pain

I. Introduction

Primary afferent neurons contain neuropeptides that are released following nerve stimulation at the central and peripheral terminals of C-fibers (Lembeck, 1987). Substance P (SP) is a member of a family of neuropeptides called tachykinins, which includes neurokinin A (NKA) and neurokinin B (NKB) (Pernow, 1983). The three tachykinin receptors, designated as NK~, NK 2 and NK3, show preferred affinity for SP, NKA and NKB, respectively (Lavielle et al., 1988; Regoli et al., 1988). In peripheral tissues, SP has been shown to cause vasodilatation and plasma extravasation, effects implicated in neurogenic inflammatory responses (Lembeck and Holzer, 1979; Payan, 1989; Barnes et al., 1990). Literature reports also suggest that SP plays a role in chronic disease states such as arthritis (Levine et al., 1986), asthma (Barnes, 1986), rhinitis (Stj~irne et al., 1989), inflammatory bowel disease (Mantyh et al.,

Correspondence to: A. Nagahisa, Department of Medicinal Biology, Central Research Division, Pfizer Inc., 5-2 Taketoyo, Aichi 470-23, Japan. Tel. 81.569.72 6805, fax 81.569.72 6840.

1988; Golden et al., 1989) and fibromyalgia (Littlejohn et al., 1987; Vaer0y et al., 1988, 1989) as well as in pain transmission (Otsuka and Yanagisawa, 1987). In an attempt to better define the role of SP in inflammation, much work has focused on the use of capsaicin which depletes SP from primary afferent neurons. Capsaicin, however, causes sensory neurons to release multiple pro-inflammatory peptides including SP, NKA and calcitonin gene-related peptide (CGRP) in peripheral tissues (Bevan and Szolcs~nyi, 1990; Maggi and Pierau, 1991). Each peptide can bind to its own receptor and exert effects alone or synergistically. Thus, it has been difficult to define the role of a single ligand-receptor interaction, e.g. SP at the NK 1 receptor (Payan, 1989; Barnes et al., 1990). While peptide antagonists have been developed (Regoli et al., 1988), the lack of potency and selectivity, as well as the peptidic nature of these molecules has limited their use in in vivo studies. The discovery of CP-96,345 ((2S, 3 S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)methyl)-l-azabicyclo[2,2,2]octan-3-amine), a potent and specific nonpeptide NK l receptor antagonist (Snider et al., 1991; McLean et al., 1991; Constantine et al., 1991), offers an opportunity to study the role of SP in a

192 variety of pathophysiologic conditions. Here we describe the in vivo pharmacological characterization of CP-96,345 in models of acute inflammation and pain.

2. Materials and methods

2.1. Plasma extravasation in the skin Plasma extravasation was induced by intradermal administration of SP or other vasoactive mediators at submaximal doses (50 /zl of 0.1% bovine serum albumin (BSA)-saline solution) in dorsal skin of pentobarbital (25 mg/kg i.p.)-anesthetized non-fasted male Hartley guinea pigs weighing 450-500 g. CP-96,345 and CP-96,344 (dihydrochloride salts) were dissolved in 0.1% methyl cellulose-water (MC) and given p.o. 1 h before SP challenge (3 pmol/site). Evans blue dye (30 mg/kg) was administered i.v. 5 min before challenge. Ten minutes after the SP injection, the animals were killed, the dorsal skin was removed, and the blue spots were punched out using a cork borer (11.5 mm outer diameter). Tissue dye content was quantitated after overnight formamide extraction at 600 nm absorbance.

2.2. Capsaicin-induced plasma extravasation Plasma extravasation was induced by i.p. injection of capsaicin (30 mM stock solution in 70% ethanol diluted 1000-fold to a final concentration of 30 /zM in 0.1% BSA-saline, 10 ml injection) into male Hartley guinea pigs pentobarbital-anesthetized (25 mg/kg i.p.) and fasted overnight. CP-96,345 and CP-96,344 (dihydrochloride salts) were dissolved in 0.1% MC and given p.o. 1 h before capsaicin challenge. Evans blue dye (30 mg/kg) was administered i.v. 5 min before challenge. The animals were killed 10 min after capsaicin injection and both right and left ureter were removed. Tissue dye content was quantitated as before.

2.3. Contractions of guinea pig isolated ureter The ureter preparations (2.5-3 cm) were mounted in 20-ml tissue baths containing a physiological salt solution (composition in mM: NaC1 137.9, KCI 2.7, CaC1 z 1.8, MgC1z 0.5, NaHCO 3 11.9, NaH2PO 4 1.1, glucose 5.6) bubbled with 95% 02-5% CO z and maintained at 37°C. Contractions were recorded isometrically using Kent model TRN001/002 force transducers which supplied analog signals continuously to a Macintosh II personal computer equipped with a MacLab analog to digital interface hardware. The resting tension was adjusted to 0.2 g during an equilibration period of 1 h.

2. 4. Acetic acid-induced abdominal stretching Male ddY mice, weighing 14-18 g, were fasted overnight. CP-96,345 and CP-96,344 (methanesulfonic acid salts) were dissolved in 0.1% MC and given p.o. 0.5 h before acetic acid injection (0.7%, 0.16 ml/10 g body weight). The animals were placed in clear beakers (one per beaker) and the stretching response was counted 10 to 20 min after the acetic acid injection (10-min interval). Piroxicam (3 mg/kg p.o.), used as a positive control, gave 64% inhibition (P NKB >I NKA. CP96,345, at 10 mg/kg p.o., inhibited 69, 87 and 75% of the SP- (3 pmol/site), NKA- (10 pmol/site) and NKBinduced (10 pmol/site) plasma extravasation, respec-

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Fig. 1. CP-96,345 inhibition of SP-induced plasma protein extravasation. Plasma extravasation was induced by intradermal administration of SP (50/xl) in dorsal skin of pentobarbital-anesthetized guinea pigs. Each column represents the m e a n and S.E.M. for five animals (N = 6 for control and basal group). CP-96,345 and CP-96,344 (dihydrochloride salts) were given p.o. 1 h before SP challenge (3 pmol/site). * P < 0.05 compared to control (1-way A N O V A , Dunnett's test).

tively. At 10 mg/kg p.o., CP-96,345 was inactive against extravasation induced by histamine, bradykinin, PAF or leukotriene D4 (table 1).

3.2. Capsaicin-induced plasma extrauasation Capsaicin (30 /~M in BSA-saline) injected i.p. into anesthetized guinea pigs induced plasma extravasation in several tissues, including ureter, bile duct and umbilical ligaments. Oral administration of CP-96,345, 1 h prior to the capsaicin injection, inhibited the plasma leakage in all three tissues. The ureter was the most convenient tissue in which to conduct quantitative exTABLE l Effect of CP-96,345 (10 m g / k g p.o.) on plasma extravasation induced by vasoactive pro-inflammatory mediators in guinea pig dorsal skin. Plasma extravasation was induced by intradermal administration (50 pA) of appropriate concentrations of mediators dissolved in 0.1% BSA-saline. CP-96,345 (methanesulfonic acid salts) was given 1 h before the challenge. T h e values are the m e a n s and S.E.M., N = 4 - 6 for each group. T h e % inhibition was calculated after subtracting the basal leakage (data not shown) from each of the CP-96,345-treated and the control values. Agonist

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Dose (mg/kg) Fig. 2. CP-96,345 inhibition of capsaicin-induced plasma extravasation in guinea pig ureter. Plasma extravasation was induced by intraperitoneal injection of capsaicin (10 ml of 30 /.tM) into anesthetized guinea pigs. Each column represents the m e a n and S.E.M. for four to six guinea pigs. CP-96,345 and CP-96,344 (dihydrochloride salts) were given p.o. 1 h before capsaicin challenge. * P < 0.05, • * P < 0.01 compared to control (l-way A N O V A , D u n n e t t ' s test).

periments and CP-96,345 showed dose-related inhibition, with an EDs0 and (c.1.) of 0.96 (0.43-1.5) mg/kg p.o. (fig. 2). Essentially complete inhibition (92%) was observed at a dose of 6 mg/kg p.o., while the inactive enantiomer CP-96,344 (6 mg/kg p.o.) showed no significant reduction.

3.3. Contractions of guinea pig isolated ureter In tissue bath experiments, concentrations >~ 10 -~ M of tachykinins caused transient and repetitive contractions of the ureter, and the rank order of potency was NKA > NKB >> SP (data not shown). CP-96,345 (10 7 M) was inactive against the tachykinin-induced effect (n = 3). Typical tracings showing the contractile

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i'0 100 0 Control 5 10 Dose (mglkg) Capsalcin (mglkg) Fig. 4. (A) CP-96,345 inhibition of acetic acid-induced abdominal stretching in mice. CP-96,345 and CP-96,344 (methanesulfonic acid salts) were given p.o. 0.5 h before acetic acid injection. Twelve mice were used per group and the values represent the means and S.E.M. (B) Effect of systemic capsaicin pretreatment on acetic acid-induced abdominal stretching. Eight to twelve mice were used per group and values represent the mean and S.E.M. The number above each column indicates % inhibition. For both figures, * P < 0.01 compared to the appropriate control (1-way ANOVA, Dunnen's test).

response of the guinea pig ureter to SP and NKA in the absence and presence of CP-96,345 are shown in fig. 3.

3.4. Acetic acid-induced abdominal stretching CP-96,345 inhibited acetic acid-induced abdominal stretching in a dose-dependent manner, with an EDs0 and (c.l.) of 10.7 (5.2-16.3) mg/kg p.o. (fig. 4A). CP96,344 at 30 mg/kg p.o. was inactive, suggesting that SP antagonism is required for activity. The abdominal stretching response was completely inhibited after systemic capsaicin pretreatment (fig. 4B).

4. Discussion

'CP-96,345 is a specific non-peptide NK~ receptor antagonist which does not possess activity at NK 2 and NK3, the other two tachykinin receptors (Snider et al., 1991). Plasma extravasation studies with guinea pig dorsal skin show that CP-96,345 antagonism is specific for tachykinins and NK 1 is the receptor responsible for the neurogenic inflammatory response caused by the exogenously applied peptides. Capsaicin has been shown to increase vascular permeability and contract smooth muscle in the guinea pig ureter. These effects have been attributed to neuropeptides released from primary afferent C-fibers (Saria et al., 1983; Lundberg et al., 1984; Hua et al., 1986). The ability of CP-96,345 to inhibit the ureter plasma leakage suggests strongly that the NK] receptor alone is responsible for the extravasation caused by endogenous neuropeptides released by capsaicin. In contrast, the NK t receptor appears not to be involved in guinea pig ureter smooth muscle contrac-

tion. CP-96,345 was completely inactive to prevent the SP- and NKA-induced ureter contraction at a concentration (10 -7 M) 50- to 200-fold higher than previously shown to be effective in binding and in tissue bath studies (Snider et al., 1991). Moreover, NKA was approximately two orders of magnitude more potent than SP to elicit the response. Thus, NK 2 (and/or NK 3) receptor stimulation apparently mediates the contractile response to tachykinins. The results of our studies show that there is a clear functional separation of the tachykinin receptors in the guinea pig ureter. Acetic acid-induced abdominal stretching in mice is a model that has been used to predict the analgesic potential of non-steroidal anti-inflammatory drugs. Prostaglandins play a major role in the response, as cyclooxygenase inhibitors exhibit excellent activity in this model. In the present study, systemic capsaicin pretreatment completely inhibited the stretching response, presumably due to depletion of peptide neurotransmitters. The stereospecific inhibition by CP-96,345 indicates for the first time that neuropeptides, SP in particular, play a critical role in this model. It has been shown recently that CP-96,345 also inhibits mustard oil-induced inflammation in the rat foot (Lembeck et al., 1992). The NKa receptor may therefore be solely responsible for the vascular permeability changes caused by tachykinins in other species including man. We conclude that NK] receptor antagonism represents a new approach to the treatment of inflammatory diseases and pain.

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