Non-peptide Substance Research Articles

96-3 - Nonpeptide substance P and other tachykinin receptor, antagonists as a novel pharmacologic and therapeutic class

96-3 - Nonpeptide substance P and other tachykinin receptor, antagonists as a novel pharmacologic and therapeutic class

Molecular characterization and functional expression of a substance P receptor from the sympathetic ganglion of Rana catesbeiana

Substance P is an important neuropeptide neurotransmitter in the central, autonomic and enteric nervous systems. In sympathetic ganglia, substance P is thought to play a role in modulating synaptic transmission. Release of substance P by neuronal stimulation or direct application of substance P to ganglionic neurons increases neuronal excitability. An amphibian substance P receptor complementary DNA has been cloned and characterized from bullfrog, Rana catesbeiana, sympathetic ganglion complementary DNA libraries. The deduced primary structure contains features indicative of a seven transmembrane domain G-protein-coupled receptor. The deduced protein sequence shows 69% identity to previously cloned mammalian substance P receptors. In situ hybridization analysis performed on bullfrog sympathetic ganglia using digoxigenin-labelled complementary RNA probe demonstrated that approximately 75% of the principal neurons displayed reaction product above background levels. Radioligand binding studies were performed on stably transfected cells with [ 125I]Tyr-1-substance P as the ligand. Substance P had an IC 50 of 16 nM and the agonist potency profile was substance P>neurokinin A>>neurokinin B. The order of potency for three tachykinins to increase intracellular calcium when applied to a stably transfected clonal cell line was substance P>neurokinin A>>neurokinin B. This order of agonist potency also held for inhibition of the M-type potassium current in intact bullfrog sympathetic neurons. The non-peptide substance P antagonists CP-96345 and RP-67580 at concentrations that block mammalian substance P receptors had little or no effect on the responses to substance P at the bullfrog receptor. Overall, these results demonstrate that the cloned sequence has the features consistent with and characteristic of a substance P receptor. The results are discussed with reference to the established pharmacology of the bullfrog substance P receptor and known structure–activity relationships of mammalian tachykinin receptors.

γδ T cells: Their immunobiology and role in malaria infections

The status of research on γδ T cells is reviewed. Recent research shows that γδ T cells may see antigens in an immunoglobulin-like manner and that non-peptidic substance can be antigens for these cells. Considerable advances have been made in defining the immunobiology of γδ T cells, with evidence for sentinel, protective and immunoregulatory roles. Research on γδ T cells in malaria infections suggests that γδ T cells are mediators of protective immunity, most probably through the production of Th1 cytokines such as TNFα, TNFδ and IFNγ and that excessive production of such cytokines may contribute to pathology. Our data on the features of the peripheral blood γδ T cell response in humans infected with Plasmodium falciparum show that there is considerable variation between individuals in the relative expension of γδ T lymphocytes fellewing primary or secondary infection. They confirm that activation of γδ T cells occurs during P. falciparum infection and that activated cells can persist for many weeks after treatment. The possibility that γδ T cells have an immunoregulatory function in malaria infections is proposed.

Ether extract of fetal calf serum protects cultured rat cortical neurons against glutamate cytotoxicity.

The effects of an ether extract of fetal calf serum (EE-FCS) on glutamate-induced cytotoxicity were examined using primary cultures of rat cortical neurons. The simultaneous addition of EE-FCS and glutamate reduced glutamate cytotoxicity in a concentration-dependent manner. EE-FCS also reduced the cytotoxicity induced by S-nitrosocysteine, a nitric oxide (NO) donor. The findings indicate that EE-FCS contains lipid-soluble, non-peptide substances with neuroprotective actions against NO-mediated glutamate neurotoxicity.

Ether Extract of Fetal Calf Serum Protects Cultured Rat Cortical Neurons against Glutamate Cytotoxicity

The effects of an ether extract of fetal calf serum (EE-FCS) on glutamate-induced cytotoxicity were examined using primary cultures of rat cortical neurons. The simultaneous addition of EE-FCS and glutamate reduced glutamate cytotoxicity in a concentration-dependent manner. EE-FCS also reduced the cytotoxicity induced by S-nitrosocysteine, a nitric oxide (NO) donor. The findings indicate that EE-FCS contains lipid-soluble, non-peptide substances with neuroprotective actions against NO-mediated glutamate neurotoxicity.

Practical enantiospecific synthesis of RPR 111905: A novel non-peptide substance P antagonist

The synthesis of enantiomerically pure RPR 111905 was achieved in twelve steps with an overall yield of 6.9%. The synthetic strategy was based on a Diels-Alder reaction to generate the bicyclic framework and the asymmetry was introduced by resolution.

Synthesis and biological evaluation of novel NK‐1 tachykinin receptor antagonists: The use of cycloalkyl amino acids as a template

In the course of a program aimed at synthesizing novel, potent NK-1 tachykinin receptor antagonists, we developed upon a bioactive model by comparing the low energy structures of a series of peptide and nonpeptide Substance P antagonists. The comparison was based on the superimposition of the aromatic rings, assuming that the rest of the molecule behaves predominantly as a template to arrange the key aromatic groups in the right spatial position. A series of 2-aminocyclohexane carboxylic acid analogues were then selected as the best templates for reproducing the postulated bioactive structure, leading to several pseudo-peptides with interesting biological activity. According to the molecular modeling, these compounds exhibit a neat parallel facing of the indolyl and naphthyl groups at about 3 A distance. Ultraviolet absorption and steady state fluorescence measurements support this conclusion, showing a linear correlation between the spectral properties and the binding affinity of these analogues. Stacking of the indole ring with naphthalene gives rise to a complex characterized by a well-defined molar extinction coefficient. Consistently, steady state and lifetime fluorescence measurements suggest that the quenching process is ascribable to ground-state interactions between the chromophores. Implications of the pi stacking propensity of aromatic groups in the biological activity of the compounds examined are briefly discussed.

Involvement of Substance P in Ultraviolet Irradiation‐induced Inflammation in Rat Skin

The possible involvement of substance P released from primary afferents in rat skin was investigated in cutaneous inflammation following ultraviolet (UV) irradiation. Recordings from c-fibres innervating the UV-exposed hindpaw skin showed long-lasting low-frequency (0.8-1.25 Hz) spontaneous activity. Spontaneously active c-fibres increased to constitute 35.3% of the total population 72 h after UV exposure. Immunohistochemical analysis of substance P-containing nerve fibres in hindpaw skin revealed a significant increase in substance P immunoreactivity 24 h after UV irradiation. Average length of substance P-immunolabelled nerve fibres was about two times higher in UV-exposed compared to control skin. UV-induced oedema was investigated in rat ears using an ear-swelling test. Intradermal injection of either peptide (Spantide) or nonpeptide (CP-96,345) substance P antagonists and epicutaneous application of CP-96,345 reduced UV-induced oedema significantly in the late phase of sunburn (> 12 h after UV exposure). The UV-induced increase in skin blood flow was investigated in hindpaw skin up to 72 h by the laser Doppler technique. Epicutaneous application of CP-96,345 reduced erythema significantly between 12 and 72 h after UV exposure. Thus, our findings suggest the involvement of neurogenic substance P as a proinflammatory mediator in the late phase of UV-induced cutaneous inflammation in rats.

Substance P increases microvascular permeability via nitric oxide-mediated convective pathways.

The goal of these studies was to examine the effects of substance P, a tachykinin neuropeptide, on pathways of microvascular permeability. Individual frog mesenteric venular capillaries were cannulated, and albumin apparent permeability coefficients (Ps) were determined by quantitative fluorescence microscopy. Ps of albumin (PsAlb) rose from 6.8 +/- 1.8 (SE) cm.s-1.10(7) at control to 22.3 +/- 2.3 cm.s-1.10(7) when substance P (10(-11) M) was perfused. The effect of increased microvessel permeability induced by substance P (10(-11) M) was blocked with the nonpeptide substance P receptor antagonist CP-96,345 and NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase. PsAlb increased 0.99 cm.s-1.10(7) for every cmH2O increase in microvessel pressure after treatment of the vessel with substance P, demonstrating coupling of albumin flux to transvascular water flow. In conclusion, the mechanism of increased microvessel permeability in response to substance P appears to be the result of receptor-mediated increase in nitric oxide production and formation of water-filled convective pathways presumably located between adjacent endothelial cells.

Noxious peripheral stimulation produces antinociception mediated via substance P and opioid mechanisms in the rat tail-flick test

Physiological experiments were run to examine the effects of noxious thermal stimulation of one hindpaw on the tail-flick reflex in the lightly anesthetized rat. Male Sprague-Dawley rats were anesthetized with an i.p. injection of a mixture of Na-pentobarbital (20 mg/kg) and chloral hydrate (120 mg/kg). After baseline readings were taken in the tail-flick test, either a non-noxious or a noxious stimulus was applied which consisted of immersion of one hindpaw in water at 40, 45, 50 or 55°C for 1.5 min. After immersion, tail-flick readings were taken at 3-min intervals for at least 16 min. Paw immersion in water at 55°C induced an antinociceptive response, consisting of an increase in the reaction time, at 0.5 min after immersion. Recovery to baseline levels occurred over the next 3–6 min. Immersion at lower temperatures provoked smaller antinociceptive responses, except at 40°C, where readings remained around the baseline values. The increase in reaction time in response to immersion at 55°C was attenuated or blocked by the novel, nonpeptide substance P (NK-1) receptor antagonist, CP-96,345, administered s.c. 30 or 60 min, respectively, prior to paw immersion. Similar injection of CP-96,344, the inactive stereoisomer, had no effect on the response, while another NK-1 receptor antagonist, CP-99,994, also attenuated the antinociceptive effect of the immersion. The increase in reaction time induced by immersion at 55°C was absent in animals treated neonatally with capsaicin. This nociceptive response was blocked by the broad spectrum opiate antagonist, naloxone (10 mg/kg i.p.) given 20 min before paw immersion. The data provide evidence that a noxious stimulus produces an intensity-related heterosegmental antinociceptive effect and that C-fibers, and NK-1 and opiate receptors are involved in mediating this effect.

Substance P binding in normal neonatal foreskin.

The distribution of binding sites for NTE-biotinyl-[Arg3]-substance P (SPB) was demonstrated in neonatal foreskin using a conjugate of streptavidin with horseradish peroxidase. The observed binding is reversible, and may be abrogated by either the non-peptide substance P receptor antagonist, CP-96,345, or by unlabelled substance P. The generalized epidermal distribution and focal dermal localization of SPB binding suggest that although NK-1 receptors are abundant in human neonatal foreskin, neuromodulatory mechanisms may play a significant role in epidermal physiology.

Specific residues at the top of transmembrane segment V and VI of the neurokinin-1 receptor involved in binding of the nonpeptide antagonist CP 96,345 [corrected

Previously we have found that binding of the nonpeptide substance P antagonist, CP 96,345, to the neurokinin-1 (NK-1) receptor was critically dependent on two short segments adjacent to the top of transmembrane segments (TM) V and VI, called segments A (residues 183-195) and D (residues 271-276), respectively. In the present study we have systematically performed substitutions of nonconserved residues within these two segments with residues from the homologous NK-3 and/or NK-2 receptor. In segment A, deletion of residues Glu193 and Lys194, which are not present in the NK-3 receptor, or substituting them with leucines as in the NK-2 receptor, decreased the affinity of CP 96,345 10- and 22-fold, respectively. Surprisingly, switching the position of Glu193 and Lys194 did not affect the affinity of CP 96,345, suggesting that, rather than interacting directly with CP 96,345, an interaction of these residues with one another is important for CP 96,345 binding. In segment D substitution of Tyr272 with threonine as in the NK-2 receptor and with alanine as in the NK-3 receptor decreased the affinity of CP 96,345 7- and 24-fold, respectively. Mutation of the preceding Pro271 to glycine alone did not affect CP 96,345 binding, but, combined with the mutation of Tyr272 to threonine, the affinity decreased 28-fold. A series of CP 96,345 analogues with modifications of the major chemical moieties exhibited equally reduced affinity as that of CP 96,345 for the Tyr272- and Lys193-Glu194-substituted constructs, except CP 95,555, which lacks one of the phenyl rings in the benzhydryl group and which was almost unaffected by these mutations. In conclusion, our data indicate a direct interaction between CP 96,345 and Tyr272, which are located at the top of TM VI likely in close spatial proximity to the previously identified interaction point, His197, at the top of the adjacent TM V. Furthermore, the data demonstrated a critical involvement in CP 96,345 binding of Lys193 and Glu194 located one alpha-helical turn above His197.

Mediation by bradykinin of rat paw oedema induced by collagenase from Clostridium histolyticum.

1. Collagenases are thought to play a major role in the pathology of gas gangrene caused by Clostridium histolyticum, because they can destroy the connective tissue barriers. We investigated possible mediators involved in the oedema formation and plasma protein extravasation which follow the injection of a collagenase (EC 3.4.24.3) from Clostridium histolyticum into one hind paw of anaesthetized rats. 2. The magnitude of the oedema following a subplantar injection was dependent on the dose of collagenase (30, 100 and 300 micrograms) injected. It reached its maximum within 30 min and remained unchanged for at least 5 h. Plasma protein extravasation into the paw was most pronounced within 20 min of the injection. Heat-inactivated collagenase was ineffective. 3. The B2 bradykinin (BK) antagonist icatibant (D-Arg-[Hyp3-Thi5-D-Tic7- Oic8] bradykinin, formerly named Hoe-140) reduced oedema formation in a dose-dependent manner with a maximal reduction of around 65% at a dose of 100 nmol kg-1 (s.c.). A significant effect could already be observed at a dose of 10 nmol kg-1. The duration of the effect of icatibant (100 nmol kg-1) was found to be at least 3 h. These results demonstrate the high potency and long duration of action of icatibant. Pretreatment of rats with the bradykinin B1 antagonist, des-Arg9-[Leu8]-BK did not affect collagenase-induced paw oedema. Thus, the observed collagenase-induced effects are mainly mediated by BK through activation of B2 receptors. 4. Pretreatment of adult rats with capsaicin (125 mg kg-1, s.c.) three weeks before the collagenase injection caused a significant attenuation of the paw oedema and of plasma extravasation but was significantly less effective than icatibant (100 nmol kg-1, s.c.). The non-peptide substance P antagonist,CP-96,345 (l0 micromol kg-1, i.v.) significantly reduced collagenase-induced oedema formation to a degree comparable with that seen after capsaicin pretreatment. The inhibition by the substance P antagonist was significantly smaller than that seen after icatibant. The inhibitory effect of icatibant in capsaicin pretreated rats, or of icatibant together with CP-96,345 in untreated rats, was not greater than that oficatibant alone in rats treated with the vehicle for either capsaicin or CP-96,345. CP-96,344(10 micromol kg-1, i.v.), the inactive enantiomer of CP-96,345, did not affect collagenase-induced paw oedema. In capsaicin-pretreated rats, CP-96,345 (10 micromol kg-1, i.v.) did not reduce collagenase-induced paw oedema.The subplantar injection of bradykinin (30 nmol) induced a paw oedema comparable with that induced by collagenase (100 microg). CP-96,345 (10 micromol kg-1, i.v.), but not CP-96,344 (1O micromol kg-1, i.v.),significantly reduced the bradykinin-induced paw oedema. These findings indicate that collagenase leads to the release of bradykinin; bradykinin then stimulates afferent C-fibre terminals and causes the release of substance P and probably also neurokinin A, which augment the oedema-inducing effect of bradykinin.5. Indomethacin or mepyramine plus cimetidine failed to inhibit collagenase-induced paw oedema.Thus, prostaglandins and histamine do not seem to be involved in collagenase-induced paw oedema.6. After subplantar injection of collagenase, the sensitivity scores in a modified formalin-test rapidly increased during the first 10 min. This increase was abolished by pretreatment with icatibant(100 nmol kg-1, s.c.) indicating that the stimulation of nociceptive afferent neurones following injection of collagenase is due to the action of released kinins.7. In conclusion, bradykinin appears to be the main mediator of inflammation induced by a collagenase from Clostridium histolyticum. As well as having direct relevance to a known pathological condition,collagenase-induced paw oedema could prove to be a useful model in inflammation research and in the investigation of bradykinin antagonists. The present results might provide an experimental basis for clinical investigations of the effects of icatibant in infectious diseases where the release of collagenases from bacteria causes rapid spreading of inflammation.

N-alkyl quinuclidinium substance P antagonists

The synthesis and SAR of bridgehead N-alkylated analogues of the nonpeptide substance P antagonist CP-96,345 are described. The results indicate that the bridgehead nitrogen may provide more of an anchoring function rather than being in intimate contact with the receptor.

2-Aryl-1-azabicyclo[2.2.2]octanes as novel nonpeptide substance P antagonists

The synthesis and SAR of a series of 2-aryl-1-azabicyclo[2.2.2]octanes structurally related to the nonpeptide substance P antagonists CP-96,345 and CP-99,994 are described. The novel SAR observed at the 2-position in these derivatives represents a hybrid between that seen in the two previous series.

Topography of a binding site for small amnestic peptides deduced from structure-activity studies: relation to amnestic effect of amyloid beta protein.

Four peptides homologous to amyloid beta protein containing the Val-Phe-Phe (VFF) sequence administered intracerebroventricularly after training caused amnesia for footshock active avoidance training in mice. Results with VFF and other peptides containing VFF or portions thereof were used to generate a topographic map for a hypothetical binding surface for amnestic peptides, termed Z. Effects on retention of footshock active avoidance training were rationalized in terms of fit to Z, making possible design of potential memory-modulating peptidic and nonpeptidic substances. Three peptides that neither improved nor impaired retention blocked the amnestic effects of beta-(12-28), a peptide homologous to amyloid beta protein, opening the way to development of substances that can antagonize the neurotoxic effects of amyloid beta protein on neural structures and thus attenuate symptoms and progression of Alzheimer disease.

Synthesis, in vitro binding profile, and autoradiographic analysis of [3H]-cis-3-[(2-methoxybenzyl)amino]-2-phenylpiperidine, a highly potent and selective nonpeptide substance P receptor antagonist radioligand.

The synthesis of a highly potent and selective NK1 receptor antagonist radioligand, [3H]-cis-3-[(2-methoxybenzyl)amino]-2-phenylpiperidine (6a) is described. The in vitro binding pharmacology and autoradiographic distribution of 6a in guinea pig brain following peripheral administration are also reported.

Variations in affinities for the NK 1 receptor: differences between the non-peptide substance P antagonists RP 67580 and CP-96,345 and the agonist septide

Variations in affinities for the NK 1 receptor: differences between the non-peptide substance P antagonists RP 67580 and CP-96,345 and the agonist septide

Effect of CP-96,345 a non-peptide substance P antagonist, capsaicin, resiniferatoxin and ruthenium red on nociception

Effect of CP-96,345 a non-peptide substance P antagonist, capsaicin, resiniferatoxin and ruthenium red on nociception

Antiinflammatory and analgesic activity of CP-96,345: on orally active non-peptide substance P receptor antagonist

Antiinflammatory and analgesic activity of CP-96,345: on orally active non-peptide substance P receptor antagonist