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The substance P (neurokinin-1) receptor belongs to the family of seven putative transmembrane domain receptors that are coupled via G proteins to phospholipase C activation. Homologous desensitization of substance P-stimulated responses has been described in various systems. The rat neurokinin-1 receptor and a truncated mutant lacking the carboxyl-terminal region were expressed in Chinese hamster ovary cells to examine the mechanisms of substance P-induced desensitization. Wild-type and truncated receptor-bearing cells were indistinguishable in agonist binding affinity and EC50 of substance P-induced accumulation of 3H-inositol phosphates. Substance P-induced responses continued for 30-45 min in cells expressing wild-type and truncated receptors as well as in rat LRM-55 and human U373 cells, which express endogenous neurokinin-1 receptors. In transfected cells expressing the wild-type receptor, CP-96,345 added 15 min after substance P blocked further responses, demonstrating the continuing presence of responsive receptors. The rates of accumulation of 3H-inositol phosphates were four times greater in the initial 15 s of stimulation than for the next 20 min for both wild-type and truncated receptor types. This decrease in rate of substance P-stimulated phosphatidylinositol hydrolysis is therefore not dependent on the carboxyl-terminal region of the rat neurokinin-1 receptor, which contains 26 serine and threonine residues. These results are discussed in relation to current ideas regarding neurokinin-1 receptor desensitization.

The synthesis and structure-activity relationships of a series of aza-tricyclic analogs of the quinuclidine substance P (SP) antagonist 1 are described. The SP receptor affinity of these compounds was found to vary according to the size of the new ring fused to the quinuclidine and the mode of fusion. Correlations between receptor affinity and (1) the steric bulk of the newly introduced ring fusion and (2) the dihedral angle between the benzhydryl and benzylamino substituents of these aza-tricyclic compounds were explored.

Polymorphonuclear neutrophils (PMNs) accumulate within the airways during acute and chronic bronchitis and can adhere to bronchial epithelium. Substance P (SP), a neuropeptide released from the primary afferent nerve endings, has been shown to have a proinflammatory effect on PMNs. To test the hypothesis that SP could modulate PMN bronchial epithelial cell adherence, bovine bronchial epithelial cells (BBECs) were isolated and cultured, and the capacity of SP to modulate PMN-BBEC adherence was evaluated. SP interacted with BBECs to induce an increase in PMN adhesion (14.7 +/- 1.2% vs 5.3 +/- 0.7% adherence, p < 0.01). The effect of SP was both time- and dose-dependent with maximal responses at 6 h and 10(-10) M. The effect was reproduced by the carboxyl-terminal sequence of the molecule (SP 6-11). Importantly, pretreatment of the BBECs with the tachykinin SP receptor (NK1) antagonist, CP-96,345, significantly reduced the increase in adhesion induced by SP (p < 0.01). Furthermore, treatment of the BBECs with antibodies against CD11a (LFA-1), CD11b (MAC-1), or ICAM-1 significantly decreased SP-induced adherence (p < 0.01 all comparisons). Conversely, SP stimulation of PMN induced a dose-dependent, rapid (within 5 min) increase in adherence. This effect was also mediated by the carboxy end of the molecule and was decreased by CP-96,345, again suggesting that this effect was NK1 mediated. These data demonstrate the SP has the capacity for modulating PMN-BBEC interactions and suggest an important role for SP in modulating airway inflammation.

Toxin A from Clostridium difficile mediates acute inflammatory enterocolitis in experimental animals, while cholera toxin causes noninflammatory secretory diarrhea. The purpose of this study was to investigate whether an antagonist to the peptide substance P, a constituent of primary sensory neurons known to participate in inflammatory responses, would inhibit toxin A-mediated enteritis in the rat ileum. Pretreatment of rats with CP-96,345 (2.5 mg per kg of body weight), a substance P antagonist, dramatically inhibited fluid secretion (P < 0.01) and mannitol permeability (P < 0.01) in ileal loops exposed to toxin A. The protective effects, which were dose dependent, caused a significant reduction of inflammation in the lamina propria, reduction of the necrosis of intestinal epithelial cells, and complete inhibition of toxin A-mediated release of rat mast cell protease II, a specific product of rat mucosal mast cells. An inactive enantiomer of the substance P antagonist, CP-96,344, had no effect. In contrast, pretreatment with CP-96,345 had no inhibitory effect on the intestinal effects caused by administration of cholera toxin into the ileal loops. From these data, we conclude that the peptide substance P is involved in the secretory and inflammatory effects of toxin A but not of cholera toxin.

Studies with CP-96,345, a potent, selective, orally active, nonpeptide NK1 receptor antagonist, have provided considerable insight into SP pharmacology. Rather than being a primary neurotransmitter, SP prolongs the nociception produced by other neurotransmitters. By controlling endothelial permeability, SP plays a major role in inflammation and inflammatory aspects of asthma, possibly by regulating the access of neutrophils to an inflammatory site. These results indicate potential therapeutic applications for SP antagonists in the treatment of chronic pain, inflammation, and inflammatory aspects of asthma, and signal a new era in the clinical management of these important diseases.

The molecular mechanism of action for two chemically distinct and highly selective, nonpeptide antagonists, CP-96,345 and SR-48,968, was studied by development of a series of chimeric constructs between their respective target receptors, the NK1 (substance P) and NK2 (neurokinin A) receptors. The binding affinities of the natural peptide ligands, substance P and neurokinin A, were not affected by exchanging almost the entire C-terminal half of the NK1 receptor with the corresponding segment of the NK2 receptor. In contrast, it was found that transfer from the NK2 to the NK1 receptor of a segment corresponding to transmembrane segment VI, the amino-terminal half of transmembrane segment VII, and the connecting extracellular loop 3 completely switched the susceptibility for the nonpeptide antagonists. This chimeric exchange, corresponding to 17 nonconserved residues, conveyed full susceptibility for the NK2-specific compound SR-48,968 to the previously unresponsive NK1 receptor--i.e., the Ki value for inhibition of binding of 125I-labeled substance P decreased from > 10,000 to 0.97 nM. At the same time the affinity for the NK1-selective compound CP-96,345 decreased > 30-fold. The actual binding site for SR-48,968 was localized to this region of the NK2 receptor by use of [3H]SR-48,968, which did not bind to the NK1 receptor but bound with similar high affinities to the wild-type NK2 receptor and to the chimeric NK1 receptor with the NK2 receptor segment incorporated around transmembrane segments VI and VII, Kd = 1.5 nM and 1.0 nM, respectively. Our data indicate that two chemically very different nonpeptide antagonists, CP-96,345 and SR-48,968, act through epitopes located around transmembrane segment VI on their respective target receptors and that at least the nonconserved residues in these epitopes are not important for the binding of the natural peptide ligands, substance P and neurokinin A.

CP 96,345 is a nonpeptide high affinity antagonist of the substance P (NK1) receptor. The radiosynthesis of [11C]CP 96,345 suitable for Positron Emission Tomography (PET) applications is described. [11C]CP 96,345 was prepared by O-methylation of a desmethyl precursor via in situ generation of its phenolate salt. The in vivo tissue distribution of [11C]CP 96,345 in guinea pigs (n = 2) at 5 and 30 min was determined. Uptake was low in brain (approximately 0.04% dose/g) and highest (approximately 1-2% dose/g) in the spleen and lungs. The present findings indicate that the use of [11C]CP 96,345 in PET might be more applicable to the study of substance P receptors in peripheral tissues involved with inflammatory disease and arthritis.

The non-peptide substance P (SP) antagonist CP-96,345 has a 90-fold selectivity for the human neurokinin-1 (NK-1) or SP receptor over the rat NK-1 receptor, while the agonist SP shows no such selectivity. The cloned NK-1 receptors from these two species have primary protein structures that differ in only 22 of 407 residues. Wild type, chimeric, and point-mutated NK-1 receptors have been created and functionally expressed to understand the structural basis of this species selectivity. Residue 290 in the seventh putative membrane-spanning region is responsible for 20-fold of the affinity difference seen for CP-96,345 between the rat and human NK-1 receptors. Residues in the second extracellular loop and in membrane-spanning region six contribute the additional 3-5-fold of the species difference. The direct or indirect action of these residues with this nonpeptidic antagonist is discussed. This study serves to emphasize the need for species-appropriate in vitro models for the screening of antagonists in the search for therapeutics, and provides useful information to model nonpeptidic antagonist interactions with peptide hormone receptors.

We describe the structure-activity relationship development of a series of quinuclidines which culminated in the first potent, selective, nonpeptide substance P (SP) antagonist, (2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxy-phenyl)methyl]-1- azabicyclo[2.2.2]octan-3-amine, 3 (CP-96,345). Compound 3 is a potent displacer of [3H]SP binding in human IM-9 cells and blocks SP-induced and capsaicin-induced plasma extravasation, as well as SP-induced salivation in the rat in vivo. This compound may both help to further our understanding of the interactions of small molecules with peptide receptors and serve to evaluate the therapeutic potential of a SP antagonist.

CP-96,345 [(2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl) methyl]-1-azabicyclo[2.2.2]octan-3-amine) antagonism of substance P-stimulated salivation was investigated in pentobarbital-anesthetized rats. Administered either intraperitoneally or orally, CP-96,345 produced dose-dependent inhibition of the sialogogic response elicited by substance P, with a median effective dose of 12-24 mumol/kg (5-10 mg/kg) of body weight, but had no effect on acetylcholine-stimulated salivation. CP-96,345 produced concentration-dependent inhibition of [3H]substance P binding to rat submaxillary gland membranes, with a median effective concentration of 34 +/- 3.6 nM. These biological activities were confined to CP-96,345 in that the 2R,3R enantiomer (CP-96,344) was without effect.