The peripheral NK-1/NK-2 receptor antagonist MDL 105,172A inhibits tachykinin-mediated respiratory effects in guinea-pigs.

1. Stimulation of sensory nerves causes release of tachykinins, including substance P (SP) and neurokinin A (NKA), which produce a variety of respiratory effects via NK-1 and NK-2 receptors, respectively. Hence, development of a compound which could potently and equivalently antagonize both receptors was pursued. 2. MDL 105,172A ((R)-1-[3-(3,4-dicholorophenyl)-1-(3,4,5-trimethoxybenzoyl)- 3-pyrrolidinyl]-4- phenyl-piperidine-4-morpholinecarboxamide) exhibited high affinity for NK-1 (4.34 nM) and NK-2 (2.05 nM) receptors. In vitro, the compound antagonized SP (pA2 = 8.36) or NKA (pA2 = 8.61)-induced inositol phosphate accumulation in tachykinin monoreceptor cell lines. 3. In anaesthetized guinea-pigs, MDL 105,172A inhibited SP-induced plasma protein extravasation (ED50 = 1 mg kg-1, i.v.) and [beta-Ala8]NKA 4-10-induced bronchoconstriction (ED50 = 0.5 mg kg-1, i.v.) indicating NK-1 and NK-2 antagonism, respectively. 4. Capsaicin was used to elicit respiratory effects in anaesthetized and conscious guinea-pigs; the latter were inhibited by MDL 105,172A following i.v. (ED50 = 1 mg kg-1) or oral (ED50 = 20 mg kg-1) administration. Hence, MDL 105,172A can inhibit pulmonary responses to tachykinins released endogenously in the airways. 5. At doses up to 200 mg kg-1, p.o., MDL 105,172A failed to inhibit repetitive hind paw tapping induced by i.c.v GR 73632, and NK-1 selective agonist, in gerbils, whereas CP-99,994 (0.87 mg kg-1, s.c.) completely ablated the effect. These data suggest that MDL 105,172A does not penetrate the central nervous system (CNS) and its tachykinin antagonism is restricted to the periphery. 6. MDL 105,172A is a non-peptide, potent, equivalent antagonist of NK-1 and NK-2 receptors. Its ability to inhibit both exogenously administered as well as endogenously released tachykinins support its use in examining the role of sensory neuropeptides in diseases associated with neurogenic inflammation including asthma.

In vitro and in vivo characterization of MDL 105,212A, a nonpeptide NK-1/NK-2 tachykinin receptor antagonist.

We have identified and characterized a novel, potent, nonselective tachykinin receptor antagonist, MDL 105,212A [(R)-1-[2-[3-(3,4- dichlorophenyl)-1-(3,4,5-trimethoxybenzoyl)-pyrrolidin-3-yl] -ethyl]- 4-phenylpiperidine-4-carboxamide, hydrochloride]. The compound binds with low nanomolar affinity and species specificity to human NK-1 and NK-2 receptors as well as to guinea pig NK-3 receptors. In vitro functional assays are consistent with potent competitive antagonism of substance P-(SP) or neurokinin A-(NKA) induced [3H]-inositol phosphate accumulation in NK-1 or NK-2 monoreceptor cell lines with pA2 values of 8.19 and 8.67, respectively. Its ability to inhibit SP, NKA and capsaicin-mediated respiratory effects was examined in guinea pigs in vivo. MDL 105,212A attenuated SP-induced airway plasma protein extravasation (ED50 = 0.20 mg/kg, i.v.), NKA-induced respiratory collapse (ED50 = 5 mg/kg, i.v) and inhibited capsaicin-induced increases in pulmonary insufflation pressure (ED50 = 0.5 mg/kg, i.v.). Conscious guinea pigs responded to capsaicin aerosol exposure with dyspnea, coughs and gasps (significant respiratory events) and plasma protein extravasation. MDL 105,212A inhibited these responses in a dose-dependent manner after i.v. (ED50 = 5 mg/kg) or oral (ED50 = 50 mg/kg) administration. These data suggest that MDL 105,212A is a potent NK-1 and NK-2 receptor antagonist based on in vitro activity and its ability to inhibit SP and NKA mediated respiratory effects in vivo after exogenous administration or endogenous release and hence may be a useful therapeutic agent in neuroinflammatory disorders such as asthma in which a role for both tachykinins in the pathogenesis of the disease has been postulated.

Parainfluenza virus type 3 induced alterations in tachykinin NK1 receptors, substance P levels and respiratory functions in guinea pig airways.

We have investigated the effects of parainfluenza virus type 3 (PI-3) on sensory neuropeptide levels, tachykinin receptors and their functions in guinea pig airways during the course of respiratory viral infection. PI-3 infected guinea pigs were hyperresponsive to methacholine and substance P aerosols as determined by earlier onset of dyspnea in these animals as compared with control on post-inoculation day (PID) 7 but not 19. In addition, plasma protein extravasation produced in response to the tachykinin was increased in infected airways during the first week post inoculation. Infected guinea pig trachea did not respond any differently to methacholine when smooth muscle contraction and [3H]inositol phosphate accumulation were measured although the magnitude of substance P effects using in vitro tests was significantly greater than control on post-inoculation day 7 but not 19. Trachea from PI-3 infected animals were characterized by reductions in substance P-like immunoreactivity, tachykinin NK1 receptor number and agonist affinity during the first post-inoculation week. Substance P levels or tachykinin NK1 receptor numbers or affinity were not altered in trachea of guinea pigs 4 days after treatment with lipopolysaccharide. These data suggest substance P release occurs during critical periods of respiratory viral infection which are temporally correlated with airway hyperresponsiveness. Despite apparent down-regulation of tachykinin NK1 receptors, substance P-mediated functions remained enhanced suggesting some alterations in post-receptor mechanisms.

Tachykinin-mediated respiratory effects in conscious guinea pigs: modulation by NK1 and NK2 receptor antagonists.

Tachykinins, in particular neurokinin A and substance P, produce a number of airway effects which may contribute to respiratory diseases such as asthma. We examined the ability of aerosolized substance P, neurokinin A or capsaicin to produce respiratory alterations in conscious guinea pigs using modified whole body plethysmography. Substance P-mediated dyspnea and significant respiratory events were inhibited by the NK1 receptor antagonist, CP-96,345. Neurokinin A-mediated respiratory effects were ablated by the NK2 receptor antagonists: MEN 10207, MDL 29,913 and SR 48,968, the latter being the most potent. The peptide-based antagonist, MEN 10207, produced respiratory effects itself suggesting partial agonist activity. The cyclic hexapeptide, MDL 29,913, relaxed airway smooth muscle via mechanisms other than tachykinin antagonism. NK2 but not NK1 receptor antagonists were able to delay the onset of capsaicin-induced dyspnea, although alone they did not usually (in approximately 10% of the animals) eliminate the response. However, when NK2 receptor antagonists were combined with CP-96,345, the incidence of dyspnea induced by capsaicin decreased significantly (40%) suggesting that both tachykinins contribute to dyspnea in this system.

A new class of high affinity ligands for the neurokinin A NK2 receptor: psi (CH2NR) reduced peptide bond analogues of neurokinin A4-10.

Analogues of [Leu10]NKA4-10 were synthesized in which each of the amide bonds was sequentially replaced with the reduced amide psi (CH2NH) bond to determine the effect of this structural modification on the antagonism of NKA binding to the HUB NK2 receptor. [psi (CH2-NH)9,Leu10]NKA4-10 (6) retained significant affinity for the NK2 receptor (IC50 = 115 nM) and showed weak partial stimulation of PI turnover (approximately 10-15% of NKA maximum). 6 behaves as a competitive antagonist of NKA-stimulated PI turnover with a pA2 = 6.7. The secondary amine of the psi (CH2NH) moiety of 6 was converted to a tertiary amine by alkylation. This modification was found to have a small effect upon receptor affinity but did result in attenuation of partial agonist activity. A combination of amino acid substitutions and psi (CH2NH) alkylation yielded [beta Ala8,psi (CH2N(CH2)2CH3)9,Phe10]NKA4-10 (21) which has very high affinity for the HUB NK2 receptor. This compound inhibited [125I]NKA binding with an IC50 = 1 nM which is equal to the receptor affinity of NKA. Compound 21 also shows very weak partial agonism of PI turnover (< or = 5% of NKA maximum) which makes this the most potent member of a new class of NKA ligands: psi(CH2NR)9-NKA4-10 analogues which potently antagonize NKA binding and possess minimal partial agonist activity.

Comparison of the effects of neuropeptide K and neuropeptide gamma with neurokinin A at NK2 receptors in the hamster urinary bladder.

Neuropeptide K (NPK) and neuropeptide gamma (NP gamma) are two endogenous N-terminally extended forms of neurokinin A (NKA). Here, we compared their effects with those of NKA on 125I-NKA binding, phosphatidylinositol (PI) turnover and smooth muscle contraction in the hamster urinary bladder. NPK, NP gamma and NKA were equipotent in competing 125I-NKA from NK2 receptors in crude hamster bladder membranes. All three peptides stimulated PI turnover by approximately 750% with similar potency. In a third series of experiments, these peptides had similar efficacy in inducing a dose-dependent contraction of bladder smooth muscle. The NK2 receptor selective antagonist L-659,877 (cyclo[Leu-Met-Gln-Trp-Phe-Gly]) inhibited the stimulation of PI turnover and bladder contractions induced by all three tachykinins. The present results show that NKA, NPK and NP gamma display a similar biological profile. The N-terminal extensions of NPK and NP gamma appear not to influence binding of these peptides to NK2 receptors, NK2 receptor mediated stimulation of PI turnover, or smooth muscle contraction in hamster urinary bladder.

Characterization of a tachykinin peptide NK2 receptor transfected into murine fibroblast B82 cells.

Membranes isolated from a murine fibroblast B82 cell line (SKLKB82#3) transfected with the bovine stomach cDNA pSKR56S exhibited binding of [His(125I)1]neurokinin A (125I-NKA) to a single population of sites with a Bmax of 147 fmol/mg of protein and a Kd of 0.59 nM. Control cell lines had little or no specific binding. The ligand binding in SKLKB82#3 cells was reversible and was inhibited by peptides in the potency rank of neuropeptide gamma greater than neuropeptide K greater than neurokinin A greater than [10-norleucine]neurokinin A-(4-10) greater than substance P much greater than senktide (succinyl-Asp-Phe-MePhe-Gly-Leu-Met-NH2). Specific binding was enhanced by Mn2+, Mg2+, and Ca2+ and was inhibited by guanine nucleotide analogues. Thus, SKLKB82#3 cells have been transfected with NK2 receptors that have become associated with an endogenous guanine nucleotide-binding protein. In comparison with membranes from the hamster urinary bladder, a tissue enriched in NK2 receptors, NK2 receptor antagonists displayed markedly different potencies, either more or less potent, in inhibiting specific binding in membranes of the transfected cells. Furthermore, inhibition of 125I-NKA binding by nucleotide analogues was markedly different in SKLKB82#3 cells compared with hamster bladder tissue. The different binding profile in the cells is not due to an artefact introduced during cDNA transfection because a similar profile was also observed in bovine stomach membranes. These results may indicate the existence of two distinct NK2 receptors.

C-terminal modifications of neuropeptide Y and its analogs leading to selectivity for the mouse brain receptor over the porcine spleen receptor.

Neuropeptide Y (NPY) is known to bind to at least two types of receptors (Y1 & Y2). One type (Y2) is able to bind and undergo activation by both NPY and its C-terminal fragments with good potency while the other (Y1) requires the full length of NPY for good potency. For most NPY analogs that have been examined, potency for the Y2 system (porcine spleen) is greater than or equal to that for the Y1 system (mouse brain), since the Y2 system is generally less selective. However, modifications of NPY and its analogs at position 34 can lead to materials with some Y1 selectivity. For example, [Pro34]-pNPY binds to mouse brain with an affinity of 0.14 nM. Its affinity for porcine spleen is 140 nM. [His34]-pNPY was also found to be Y1 selective (19-fold), but not to the degree of the [Pro34] analog (1000-fold). The Pro34 modification in the Y2 selective C-terminal fragment NPY (20-36) converted it into an essentially non-selective analog. The selectivity from the Pro34 substitution results from a loss of Y2 binding potency along with little effect on the Y1-receptor binding. Therefore, Y1 and Y2 receptors have differing requirements for the C-terminal region of NPY in addition to their different requirements for NPY's N-terminus.

[Leu9 psi(CH2NH)Leu10]-neurokinin A (4-10) (MDL 28,564) distinguishes tissue tachykinin peptide NK2 receptors.

The neurokinin A analogue, MDL 28,564 (Asp-Ser-Phe-Val-Gly-Leu-CH2NH-Leu-NH2), inhibited 125I-NKA binding to hamster urinary bladder NK2 receptors with a KI of 130 nM. For rat submaxillary gland NK1 receptors and cerebral cortical NK3 receptors, the KI's for MDL 28,564 were greater than 250 microM and greater than 500 microM, respectively. MDL 28,564 did not relax dog carotid artery (NK1 tissue) or contract rat portal vein (NK3 tissue). In guinea-pig trachea tissues, MDL 28,564 stimulated phosphatidylinositol turnover and induced contraction with maximum effects similar to those of neurokinin A. In hamster urinary bladder tissue, MDL 28,564 stimulated phosphatidylinositol turnover with maximum effect only 10% of that of neurokinin A, did not produce sustained contraction itself and antagonized NKA-induced contraction. MDL 28,564 also produced full contraction in rabbit pulmonary artery (NK2 tissue) but was inactive in rat vas deferens (NK2 tissue). These data with MDL 28,564 are consistent with the NK2 receptors in guinea-pig trachea and rabbit pulmonary artery being different from those in hamster urinary bladder and rat vas deferens.

Agonist and antagonist binding to tachykinin peptide NK-2 receptors.

The binding of tachykinin peptides and fragments to NK-2 receptor sites in hamster urinary bladder membranes was examined and compared to binding to NK-1 receptor sites in rat submandibular gland. Neurokinin A (NKA) and its C-terminal fragments bound with highest NK-2 affinity and selectivity. N-terminal fragments of NKA did not bind to either type of receptor. Kassinin and eledoisin were NK-2 selective while physalaemin, phyllomedusin, and uperolein were NK-1 selective. Of fifteen tachykinin antagonists examined, none exhibited appreciable affinity or selectivity (relative to agonists) for NK-1, NK-2, or rat cerebral cortical NK-3 receptor sites. NKA binding to NK-2 sites was stimulated by Mn++ greater than Mg++ greater than Ca++. At the optimal concentration, the Mn++ stimulation was due to both an increased Bmax and increased affinity. The nonhydrolyzable guanine nucleotide, GppNHp, reduced agonist binding but not antagonist binding to NK-2 receptor sites. The nucleotide effect was due to a reduction in both Bmax and affinity and was potentiated by Mn++. The results indicate that tachykinin NK-2 receptor sites possess distinct structural requirements for agonists and are linked to a G-protein coupling system.