Xavier Emonds-Alt

Role of tachykinins in castor oil diarrhoea in rats

We set out to ascertain the role of tachykinins, neurokinin A and substance P, in castor oil‐induced diarrhoea in rats as disclosed by the inhibitory effect of the non‐peptide NK1‐ and NK2‐receptor antagonists, SR 140333 and SR 48968, respectively. SR 48968 (0.02 to 20 μg kg−1, s.c. or p.o.), and the opioid receptor agonist loperamide (1–10 mg kg−1, p.o.), dose‐dependently prevented castor oil effects: % inhibition vs castor oil, diarrhoea 0 to 100, increase in faecal mass 7 to 90 and water content 16 to 90. SR 140333 (0.02 to 20 μg kg−1, s.c.) and the platelet activating factor antagonist SR 27417 (5 to 500 μg kg−1, p.o.) did not prevent the increase in faecal water content, but reduced faecal mass (35 to 66%) and diarrhoea (0 to 57%). The R‐enantiomers of tachykinin NK1 and NK2 receptor antagonists, SR 140603 and SR 48605 (both at 2 or 20 μg kg−1, s.c.) had no effect other than reducing faecal mass at the highest dose tested. SR 48968 (20 μg kg−1, p.o.) but not loperamide (10 mg kg−1, p.o.) given 24 h before castor oil, still slightly but significantly reduced by 30% the increase of faecal mass output; both treatments significantly reduced (30 to 70%) the effect of castor oil on faecal water content, although the incidence of diarrhoea was only slightly less than in controls. In castor oil‐treated rats, naloxone (2 mg kg−1, s.c.) completely blocked the antidiarrhoeal action of loperamide (10 mg kg−1, p.o.) but not of SR 48968 (20 μg kg−1, p.o.); a similar result was obtained on faecal mass and water content. Castor oil strongly increased the occurrence of manometrically recorded propulsive giant contractions (500 to 1000% over control values) of transverse and distal colon, this effect being significantly prevented (80 to 100%) by SR 48968 and loperamide and partially by SR 140333 (35% distal colon, 70% transverse colon). In castor oil free rats, loperamide but not SR 48968 or SR 140333 significantly reduced by 50% the gastrointestinal transit of a charcoal test meal, as well as 24 h faecal mass output. Consistently, loperamide, unlike the tachykinin receptor antagonists, had a dramatic effect on manometric recordings of intestinal motility, reducing all kinds of colonic contractions. Our findings suggest that castor oil diarrhoea in rats entails activation of NK1 and NK2 receptors by endogenous tachykinins, whose antagonists may have a potential as antidiarrhoeal agents free from the constipating action of opioids.

In vitro characterizat of the non-peptide tachykinin NK1 and NK2-receptor antagonists, SR140333 and SR48968 in different rat and guinea-pig intestinal segments

We investigated the potent non-peptide tachykinin receptor antagonists SR140333 and SR48968 for their ability to prevent the contraction of isolated intestinal tissues elicited by the non-selective agonists substance P (SP) and neurokinin A (NKA), or by [Sar9,Met(O2)11]SP and [beta-Ala8]NKA-(4-10) that are selective agonists for NK1 and NK2 receptors, respectively. In guinea-pig ileum, containing mainly NK1-receptors: SR140333 caused a pseudo-irreversible blockade of contractions induced by either SP (KB, 0.01 nM) or [Sar9,Met(O2)11]SP (KB, 0.03 nM); SR140333 but not SR48968, dose-dependently (IC50, 0.06 nM) antagonized the contractions elicited by capsaicin. In rat duodenum, containing mainly NK2 receptors, SR48968 caused a parallel rightward shift of the concentration-response curves of [beta-Ala8]NKA-(4-10) (pA2, 9.5), but not of NKA. In rat esophageal tunica muscularis mucosae, SR48968 non-competitively antagonized [beta-Ala8]NKA-(4-10) and NKA. SR48968 and SR140333 thus appear to be potent tachykinin receptor antagonists, selective for intestinal receptors respectively of the NK2 and NK1 type. The results also suggest that rat esophagus might contain a NK2-receptor subtype different from that of rat duodenum.

Influence of (+/-)-CP-96,345 and SR 48968 on electrical field stimulation of the isolated guinea-pig main bronchus.

The aim of this study was to investigate the effects of (+/-)-CP-96,345 and SR 48968, two new nonpeptide antagonists of neurokinin NK1 and NK2 receptors, respectively, on the response of isolated guinea pig main bronchi to electrical field stimulation (EFS). Bronchi were stimulated transmurally with biphasic pulses (16 Hz, 1 ms, 320 mA for 10 s) in the presence of indomethacin (10(-6) M) and propranolol (10(-6) M). Two successive contractile responses were observed. Both responses were abolished by tetrodotoxin (10(-6) M) whereas only the first rapid phase was abolished by atropine (10(-6) M). The late and prolonged second phase was strongly reduced by the neurokinin A (NK2) receptor antagonist SR 48968 (10(-11) to 10(-8) M) with an EC50 of 0.056 nM and a maximal inhibition of 83.3 +/- 10.8% (10(-8) M, n = 4). This second response was partially inhibited by the substance P (NK1) receptors antagonist (+/-)-CP-96,345 (10(-8) to 10(-6) M). An incubation of 2 h was necessary for SR 48968 to inhibit the EFS-evoked noncholinergic contraction. These results confirm that EFS of guinea-pig bronchi involves stimulation of cholinergic and noncholinergic excitatory nerves and demonstrate that the new developed tachykinin receptors nonpeptide antagonists (+/-)-CP-96,345 and especially SR 48968 are potent inhibitors of the noncholinergic contraction induced by EFS of the isolated guinea-pig main bronchus.

Comparative effects of nonpeptide tachykinin receptor antagonists on experimental gut inflammation in rats and guinea-pigs

Previous studies have shown tachykinins implicated in gut inflammation. The aim of this work was to evaluate the effect of treatments with tachykinin NK1, NK2, and NK3 selective receptor antagonists on the development of gut inflammation induced by trinitrobenzenesulfonic acid (TNBS) in rats and guinea-pigs. On day 0, rats and guinea-pigs received an intraluminal instillation of TNBS/ethanol (40 mg/kg). Each group was daily treated with intraperitoneally injected NK1 (SR 140333; 0.3 mg/kg/day), NK2 (SR 48968; 5 mg/kg/day), or NK3 (SR 142801; 1, 5, or 10 mg/kg/day) receptor antagonists or their vehicle. On day 4, inflammatory levels were evaluated by measuring gut permeability, myeloperoxidase activity, macro- and microscopic damage scores. In TNBS treated rats, daily administration of SR 140333 (0.3 mg/kg/day) and SR 48968 (5 mg/kg/day) reduced colonic inflammation. In TNBS treated guinea-pigs, daily administration of SR 48968 (5 mg/kg/day) and SR 142801 (at 5 and 10 mg/kg/day) attenuated significantly ileal injury. These results suggest that non-peptide tachykinin receptor antagonists are potent anti-inflammatory agents on gut inflammation in rats and guinea-pigs. However, their activity depends upon the animal species and type of receptor considered.

Antitussive effect of SR 48968, a non-peptide tachykinin NK2, receptor antagonist

The antitussive effects of SR 48968, a non-peptide tachykinin NK2 receptor antagonist, were investigated on citric acid-induced cough in the unanesthetized guinea-pig and compared with the effects of codeine. SR 48968 (0.01-0.3 mg/kg i.p.) inhibited in a dose-dependent manner the number of coughs induced by inhalation of an aqueous solution of citric acid with an ED50 of 0.1 mg/kg (0.17 mumol/kg). Under similar conditions, the codeine ED50 was 8 mg/kg (27 mumol/kg). Naloxone, an opioid receptor antagonist, abolished the effects of codeine but did not modify the effects of SR 48968. These data suggest that NK2 receptor stimulation might play an important role in the regulation of the cough reflex and that SR 48968 could be a potential antitussive agent.

In vitro characterization of tachykinin NK2-receptors modulating motor responses of human colonic muscle strips

1 Human in vitro preparations of transverse or distal colonic circular smooth muscle were potently and dose‐dependently contracted by neurokinin A (EC50, 4.9 nm), the tachykinin NK2‐receptor selective agonist [β‐Ala8]neurokinin A (4–10) ([β‐Ala8]NKA (4–10)) (EC50, 5.0 nm), neurokinin B (EC50, 5.3 nm) and substance P (EC50, 160 nm), but not by the tachykinin NK1‐receptor selective agonist [Sar9Met(O2)11] substance P, or the NK3‐receptor selective agonists, senktide and [MePhe7] neurokinin B. No regional differences between transverse and distal colon were observed in response to [β‐Ala8]NKA (4–10). 2 Atropine (1 μm) and tetrodotoxin (1 μm) did not significantly inhibit responses to [β‐Ala8]NKA (4–10), neurokinin A, substance P or neurokinin B. 3 The newly developed non‐peptide antagonists for tachykinin NK2‐receptors SR 48968, SR 144190 and its N‐demethyl (SR 144743) and N,N‐demethyl (SR 144782) metabolites, were used to challenge agonist responses, as appropriate. SR 144190 and the metabolites all potently and competitively antagonized the response to [β‐Ala8]NKA (4–10), with similar potency (Schild plot pA2 values 9.4, 9.4 and 9.3, slope = 1). SR 48968 antagonism was not competitive: the Schild plot slope was biphasic with a high (X intercept∼9.3) and a low (X intercept 8.4, slope 1.6) affinity site. Co‐incubation of SR 48968 (10, 100 nm) and SR 144782 (10 nm) produced additive effects; in this experimental condition, SR 48968 apparent affinity (pKB) was 8.2. In addition, SR 144782 (0.1 μm) antagonized responses to neurokinin A, substance P and neurokinin B, with pKB consistent with its affinity for tachykinin NK2‐receptors. The potent and selective NK1 and NK3‐receptor antagonists, SR 140333 and SR 142801 (both 0.1 μm), failed to inhibit contractions induced by SP or NKB. 4 In conclusion, the in vitro mechanical responses of circular smooth muscle preparations from human colon are strongly consistent with the presence of non‐neuronal tachykinin NK2‐receptors, but not tachykinin NK1‐ or NK3‐receptors. Our findings with SR 48968 suggest the existence of two tachykinin NK2‐receptor subtypes, that it seems to distinguish, unlike SR 144190 and its metabolites. However, the precise nature of SR 48968 allotopic antagonism remains to be elucidated, since allosteric effects at the tachykinin NK2‐receptor might well account for the complexity of the observed interaction.

Pharmacological profile and chemical synthesis of SR 48968, a non-peptide antagonist of the neurokinin A (NK2) receptor

Abstract SR 48968 is a potent, competitive and selective non-peptide antagonist of the neurokinin A (NK2)_receptor. The synthesis of SR 48968 is described. Structure activity relationship is shown using binding and pharmacological results.

Effect of substance P on cytokine production by human astrocytic cells and blood mononuclear cells: characterization of novel tachykinin receptor antagonists

Substance P (SP) has been reported to induce inflammatory cytokine production in human neuroglial cells and peripheral lymphoid cells as well. In order to evaluate the potency of novel non‐peptide antagonists of the tachykinin receptors as inhibitors of SP‐induced cytokines, we used the astrocytoma cell line U373MG and blood mononuclear cells as models of central and peripheral SP‐target cells, respectively. In the first part of this study, we showed that SR 140333, an NK1 tachykinin receptor antagonist, was able to inhibit strongly the SP‐induced production of interleukin (IL)‐6 and IL‐8 in the astrocytoma cell line. The antagonistic activity of SR 140333 toward SP‐induced cytokine production was specific and could not be attributed to a general anti‐cytokine effect, since cytokine release induced by another inflammatory protein such as IL‐1β was not blocked by this compound. In addition, NK2 and NK3 agonist neuropeptides were at least 1000‐fold less effective than SP, while SR 48968 and SR 142801 which are selective NK2 and NK3 receptor antagonists, respectively, displayed a 2.5–3 orders of magnitude lower inhibitory potency than SR 140333. All these data indicated that SR 140333 blocked SP‐induced cytokine production in U373MG astrocytic cells via a specific NK1 receptor‐mediated process. Since SP has also been described to trigger peripheral blood mononuclear cells (PBMNC) or monocytes to release inflammatory cytokines, we attempted, in the second part of this study, to evaluate the potential antagonistic effect of our compounds on these cells. Experiments on human PBMNC from different donors were carried out to determine first their pattern of cytokine production upon SP stimulation. Surprisingly, we noticed that SP at concentrations ranging from 0.1 to 1000 nM was unable to stimulate the release of any inflammatory cytokine tested. This raises the question of the specificity of the reported in vitro effects of SP on cytokine production by human peripheral immune cells.

Neurokinin B- and specific tachykinin NK3 receptor agonists-induced airway hyperresponsiveness in the guinea-pig

The aim of this study was to determine whether neurokinin B (NKB) or specific agonists of tachykinin NK3 receptors, [MePhe7]NKB and senktide, were able to induce airway hyperresponsiveness in guinea‐pigs. The effects of these compounds were compared to those of substance P (SP), neurokinin A (NKA) and the preferential tachykinin NK1 ([Sar9, Met(02)11]SP) or NK2 ([βAla8]NKA (4‐10)) receptor agonists. In guinea‐pigs pretreated with phosphoramidon (10−4 M aerosol for 10 min) and salbutamol (8.7×10−3 M for 10 min), all tachykinins administrated by aerosol (3×10−7 to 10−4 M) induced airway hyperresponsiveness 24 h later, displayed by an exaggerated response to the bronchoconstrictor effect of acetylcholine (i.v.). The rank order of potency was: [βAla8]NKA (4‐10)>NKA=NKB=senktide=[MePhe7]NKB=[Sar9,Met(02)11]SP>SP. Airway hyperresponsiveness induced by [MePhe7]NKB was prevented by the tachykinin NK3 (SR 142801) and NK2 (SR 48968) receptor antagonists. Bronchoconstriction induced by tachykinins administered by aerosol was also determined. SP, NKA, NKB and the tachykinin NK1 and NK2 receptor agonist induced bronchoconstriction. The rank order of potency was: NKA=[βAla8]NKA (4‐10)>NKB=SP=[Sar9,Met(02)11]SP. Under similar conditions, and for concentrations which induce airway hyperresponsiveness, senktide and [MePhe7]NKB failed to induce bronchoconstriction. It is concluded that tachykinin NK3‐receptor stimulation can induce airway hyperresponsiveness and that this effect is not related to the ability of tachykinins to induce bronchoconstriction.

SSR240600 [(R)-2-(1-{2-[4-{2-[3,5-Bis(trifluoromethyl)phenyl]acetyl}-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl}-4-piperidinyl)-2-methylpropanamide], a Centrally Active Nonpeptide Antagonist of the Tachykinin Neurokinin 1 Receptor: II. Neurochemical and Behavioral Characterization

SSR240600 [(R)-2-(1-{2-[4-{2-[3,5-bis(trifluoromethyl)phenyl]acetyl}-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl}-4-piperidinyl)-2-methylpropanamide], a new nonpeptide tachykinin neurokinin 1 (NK1) receptor antagonist, was evaluated against the neurochemical, electrophysiological, and behavioral effects provoked by direct activation of brain tachykinin NK1 receptors or by stress in guinea pigs. SSR240600 (0.1–10 mg/kg i.p. or p.o.) antagonized the excitatory effect of i.c.v. infusion of [Sar9,Met(O2)11]substance P (SP) on the release of acetylcholine in the striatum of anesthetized and awake guinea pigs. This antagonistic action was still observed after repeated administration of SSR240600 (5 days, 10 mg/kg p.o., once a day). SSR240600 (10 mg/kg i.p.) inhibited the phosphorylation of the cAMP response element-binding protein in various brain regions induced by i.c.v. administration of [Sar9,Met(O2)11]SP. In slice preparations, neuronal firing of the locus coeruleus (LC) neurons elicited by the application of [Sar9,Met(O2)11]SP was suppressed by SSR240600 at 100 nM. Norepinephrine release in the prefrontal cortex, elicited either by an intra-LC application of [Sar9,Met(O2)11]SP or by an i.c.v administration of corticotropin-releasing factor, was reduced by SSR240600 (0.3–1 mg/kg and 1–10 mg/kg i.p., respectively). SSR240600 (1–10 mg/kg i.p.) inhibited vocalizations induced in adult guinea pigs by an i.c.v. administration of the NK1 receptor agonist, GR73632 [d-Ala-[l-Pro9,Me-Leu8]substance P(7-11)]. Furthermore, SSR240600 (1–10 mg/kg i.p.) inhibited distress vocalizations produced in guinea pig pups by maternal separation. SSR240600 also reduced maternal separation-induced increase in the number of neurons displaying NK1 receptor internalization in the amygdala. Finally, SSR240600 counteracted the increase in body temperature induced by isolation stress. In conclusion, SSR240600 is able to antagonize various NK1receptor-mediated as well as stress-mediated effects in the guinea pig.