Serotonin pharmacology in the gastrointestinal tract: a review.

Serotonin (5-hydroxytryptamine or 5-HT) plays a critical physiological role in the regulation of gastrointestinal (GI) function. 5-HT dysfunction may also be involved in the pathophysiology of a number of functional GI disorders, such as chronic constipation, irritable bowel syndrome and functional dyspepsia. This article describes the role of 5-HT in the enteric nervous system (ENS) of the mammalian GI tract and the receptors with which it interacts. Existing serotonergic therapies that have proven effective in the treatment of GI functional disorders and the potential of drugs currently in development are also highlighted. Advances in our understanding of the physiological and pathophysiological roles of 5-HT in the ENS and the identification of selective receptor ligands bodes well for the future development of more efficacious therapies for patients with functional GI disorders.

The in vitro pharmacological profile of TD-5108, a selective 5-HT(4) receptor agonist with high intrinsic activity.

The in vitro pharmacological profile of TD-5108, a novel, selective 5-HT(4) receptor agonist, was compared to that of clinically efficacious gastroprokinetic 5-HT(4) receptor agonists. TD-5108 produced an elevation of cyclic adenosine monophosphate in human embryonic kidney 293 cells expressing the human recombinant 5-HT(4(c)) (h5-HT(4(c))) receptor (pEC(50) = 8.3) and 5-HT(4) receptor-mediated relaxation of the rat esophagus (pEC(50) = 7.9) and contraction of the guinea pig colon (pEC(50) = 7.9). In all in vitro assays, TD-5108 was a high intrinsic activity agonist, unlike tegaserod, mosapride, and cisapride which, in the majority of test systems, had lower intrinsic activity. TD-5108 had high affinity (pK (i) = 7.7) and selectivity (> or =25-fold) for h5-HT(4(c)) receptors over other biogenic amine receptors. TD-5108 was >500-fold selective over other 5-HT receptors (including h5-HT(2B) and h5-HT(3A)) and, at 3 microM, had no effect on human ether-à-go-go-related gene K+ channels. In conclusion, TD-5108 is a selective 5-HT(4) receptor agonist in vitro. The high intrinsic activity and preferential binding of TD-5108 to 5-HT4 over other 5-HT receptors may result in an improved clinical profile for the treatment of gastrointestinal disorders of reduced motility.

The in vivo gastrointestinal activity of TD-5108, a selective 5-HT(4) receptor agonist with high intrinsic activity.

The in vivo preclinical pharmacodynamic profile of TD-5108, a selective 5-HT(4) receptor agonist with high intrinsic activity, was compared to that of the clinically studied gastrointestinal pro-kinetic agents, tegaserod, cisapride and mosapride. The activity of TD-5108 was evaluated in guinea pig colonic transit, rat oesophageal relaxation and dog gastrointestinal smooth muscle contractility models. Subcutaneous administration of TD-5108, tegaserod, cisapride and mosapride increased guinea pig colonic transit (rank order of potencies: TD-5108 > tegaserod > cisapride > mosapride). Following intravenous and intraduodenal dosing, TD-5108, tegaserod, cisapride and mosapride produced dose-dependent relaxation of the rat oesophagus. On a molar basis, TD-5108 was approximately twofold less potent than tegaserod following intravenous dosing but 6- or 86-fold more potent than cisapride or mosapride, respectively, and 9- or 18-fold more potent than tegaserod or cisapride, respectively, after intraduodenal administration. Orally dosed TD-5108 increased the contractility of the canine antrum, duodenum and jejunum with higher potency than tegaserod. The selective 5-HT(4) receptor agonist, TD-5108, demonstrates robust in vivo activity in the guinea pig, rat and dog gastrointestinal tracts.

A comparison of the pharmacological properties of guinea-pig and human recombinant 5-HT4 receptors.

BACKGROUND AND PURPOSE: 5-HT(4) receptor agonists are used therapeutically to treat disorders of reduced gastrointestinal motility. Since such compounds are evaluated in guinea-pigs, we cloned, expressed and pharmacologically characterized the guinea-pig 5-HT(4) and human 5-HT(4(b)) splice variant, which share 95% homology. The functional properties of guinea-pig 5-HT(4(b)) receptors were compared with native receptors in guinea-pig colon. EXPERIMENTAL APPROACH: Membrane radioligand binding and whole cell cAMP accumulation assays were used to determine the affinities, potencies and intrinsic activities (IA). Contraction of the guinea-pig distal colon longitudinal muscle myenteric plexus preparation (LMMP) was monitored to evaluate functional activity. KEY RESULTS: pK(i) values for guinea-pig and human recombinant receptors, and guinea-pig striatum 5-HT(4) receptors, were in agreement, as were the potency and IA values for guinea-pig and human 5-HT(4) receptors expressed at a similar density ( approximately 0.2 pmol mg(-1) protein). Tegaserod was a potent (pEC(50)=8.4 and 8.7, respectively), full agonist at both guinea-pig and human 5-HT(4) receptors. In contrast, in the LMMP preparation, tegaserod was a potent, partial agonist (pEC(50)=8.2; IA=66%). CONCLUSIONS AND IMPLICATIONS: Close agreement between the pharmacological properties of guinea-pig and human 5-HT(4) receptors support the use of guinea-pig model systems for the identification of 5-HT(4) receptor therapeutics. However, the mechanisms underlying the different agonist properties of tegaserod in recombinant and isolated tissue preparations, and the extent to which these impact the clinical efficacy of tegaserod as a prokinetic agent, remain to be determined.

The in vitro pharmacology of the peripherally restricted opioid receptor antagonists, alvimopan, ADL 08-0011 and methylnaltrexone.

This study characterized the pharmacology of the peripherally restricted opioid receptor antagonists, alvimopan, its metabolite, ADL 08-0011, and methylnaltrexone. The activities of the compounds were investigated with respect to human or guinea pig opioid receptor binding and function in recombinant cell lines and mechanical responsiveness of the guinea pig ileum. Alvimopan and ADL 08-0011 had higher binding affinity than methylnaltrexone at human mu opioid receptors (pK (i) values of 9.6, 9.6, and 8.0, respectively). The compounds had different selectivities for the mu receptor over human delta and guinea pig kappa opioid receptors. ADL 08-0011 had the highest mu receptor selectivity. With respect to their mu opioid receptor functional activity ([(35)S]GTPgammaS incorporation), methylnaltrexone had a positive intrinsic activity, consistent with partial agonism, unlike alvimopan and ADL 08-0011, which had negative intrinsic activities. Alvimopan, ADL 08-0011, and methylnaltrexone antagonized inhibitory responses mediated by the mu opioid agonist, endomorphin-1 (pA (2) values of 9.6, 9.4, and 7.6, respectively) and by U69593, a kappa opioid agonist (pA (2) values of 8.4, 7.2, and 6.7, respectively). In morphine-naive guinea pig ileum, methylnaltrexone reduced, while alvimopan and ADL 08-0011 increased, the amplitude of electrically evoked contractions and spontaneous mechanical activity. In tissue from morphine-dependent animals, alvimopan and ADL 08-0011 increased spontaneous activity to a greater degree than methylnaltrexone. The data suggested that alvimopan-induced contractions resulted predominantly from an interaction with kappa opioid receptors. It is concluded that alvimopan, ADL 08-0011, and methylnaltrexone differ in their in vitro pharmacological properties, particularly with respect to opioid receptor subtype selectivity and intrinsic activity. The clinical significance of the data from this study remains to be determined.

Vaccine-induced antibodies inhibit CETP activity in vivo and reduce aortic lesions in a rabbit model of atherosclerosis.

Using a vaccine approach, we immunized New Zealand White rabbits with a peptide containing a region of cholesteryl ester transfer protein (CETP) known to be required for neutral lipid transfer function. These rabbits had significantly reduced plasma CETP activity and an altered lipoprotein profile. In a cholesterol-fed rabbit model of atherosclerosis, the fraction of plasma cholesterol in HDL was 42% higher and the fraction of plasma cholesterol in LDL was 24% lower in the CETP-vaccinated group than in the control-vaccinated group. Moreover, the percentage of the aorta surface exhibiting atherosclerotic lesion was 39.6% smaller in the CETP-vaccinated rabbits than in controls. The data reported here demonstrate that CETP activity can be reduced in vivo by vaccination with a peptide derived from CETP and support the concept that inhibition of CETP activity in vivo can be antiatherogenic. In addition, these studies suggest that vaccination against a self-antigen is a viable therapeutic strategy for disease management.

Novel class of amino acid antagonists at non-N-methyl-D-aspartic acid excitatory amino acid receptors. Synthesis, in vitro and in vivo pharmacology, and neuroprotection.

The isoxazole amino acid 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA) (1), which is a highly selective agonist at the AMPA subtype of excitatory amino acid (EAA) receptors, has been used as a lead for the development of two novel EAA receptor antagonists. One of the compounds, 2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionic acid (AMOA, 7), was synthesized via O-alkylation by ethyl chloroacetate of the amino acid protected AMPA derivative 4. The other compound, 2-amino-3-[2-(3-hydroxy-5-methylisoxazol-4-yl)-methyl-5-methyl-3-+ ++oxoisoxazolin -4-yl]propionic acid (AMNH, 14) was synthesized with use of 4-(chloromethyl)-3-methoxy-5-methylisoxazole (8) as the starting material. The intermediate 4-(chloromethyl)-2-(3-methoxy-5-methylisoxazol-4-yl)methyl-5-me thylisoxazolin- 3-one (11) was converted into the acetamidomalonate (12), which was stepwise deprotected to give 14. Compounds 7 and 14 were stable in aqueous solution at pH values close to physiological pH. Neither 7 nor 14 showed detectable affinities for the receptor, ion channel, or modulatory sites of the N-methyl-D-aspartic acid (NMDA) receptor complex. Quantitative receptor autoradiographic and conventional binding techniques were used to study the affinities of 7 and 14 for non-NMDA receptor sites. Both compounds were inhibitors of the binding of [3H]AMPA (IC50 = 90 and 29 microM, respectively). Compounds 14 and 7 were both very weak inhibitors of the high-affinity binding of radioactive kainic acid [( 3H]KAIN). Compound 14, but not 7, was, however, shown to be an inhibitor of low-affinity [3H]KAIN binding (IC50 = 40 microM) as determined in the presence of 100 mM calcium chloride. In the rat cortical slice preparation, 7 was shown to antagonize excitation induced by 1 with some selectivity, whereas 14 proved to be a rather selective antagonist of KAIN-induced excitation. Both antagonists showed very weak effects on the excitatory effects of NMDA. Compound 7 was a poor antagonist of excitation by quisqualic acid (2), whereas 14 did not affect excitation by this nonselective AMPA receptor agonist. On cat spinal neurones, both 7 and 14 reduced excitations by 1 and KAIN, but, again, the excitatory effects of 2 were much less sensitive. Compound 14 and, in particular, 7 effectively protected rat striatal neurones against the neurotoxic effects of KAIN, whereas the toxic effects of 1 were reduced only by 7. Neither antagonist showed protection against the cell damage caused by intrastriatal injection of the NMDA agonist quinolinic acid.(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of the trigeminal ganglion on carotid blood flow in anaesthetized guinea-pigs.

1. The influence of the trigeminal ganglion on the carotid circulation has been investigated by measuring electrical stimulation-induced alterations in carotid arterial blood flow and resistance in anaesthetized guinea-pigs. The effects of several receptor antagonists were assessed to determine which neurotransmitters are involved in regulating carotid blood flow. 2. Arterial blood pressure and carotid vascular resistance were reduced by electrical stimulation (0.5 mA, 1 ms, 5 Hz, 60 s) of the trigeminal ganglion ipsilateral to the carotid artery from which flow was measured. No consistent effect of electrical stimulation on carotid blood flow was observed. However, when guinea-pigs were pretreated with guanethidine (30 mg kg-1, s.c., 24 h prior to experiments), stimulation produced little change in blood pressure, while carotid blood flow was increased and vascular resistance decreased, consistent with vasodilatation in the cranial circulation. Stimulation of the trigeminal ganglion contralateral to the carotid artery from which blood flow was measured, had little effect on either carotid blood flow or vascular resistance. 3. In animals pretreated with guanethidine, intravenous administration of the vasoactive intestinal polypeptide (VIP) receptor antagonist, [p-Cl-D-Phe6,Leu17]-VIP (50 micrograms kg-1) significantly attenuated the increase in carotid blood flow and decrease in carotid vascular resistance evoked by trigeminal ganglion stimulation. Responses evoked by trigeminal ganglion stimulation were, however, unaffected by intravenous injection of the tachykinin NK1 receptor antagonists, GR82334 (0.3 mg kg-1) and CP-99,994 (0.4 mg kg-1), calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP8-37 (0.9 mg kg-1) and the ganglion blocking agent, hexamethonium (10 mg kg-1). 4. It is concluded that in the guanethidine-pretreated guinea-pig, electrical stimulation of the trigeminal ganglion increases carotid blood flow and produces an accompanying decrease in carotid vascular resistance, consistent with the dilatation of carotid blood vessels. The transmitter mediating this effect is most likely to be VIP.

Neurokinin-induced changes in pial artery diameter in the anaesthetized guinea-pig.

1. The effects of selective neurokinin agents on pial artery diameter, measured with an on-line image analyser, have been studied in anaesthetized guinea-pigs in order to characterize the neurokinin receptors present on pial arteries. 2. Perivascular injection of either substance P (0.01-1 microM) or the selective NK1 receptor agonists, substance P methyl ester (SPOMe, 0.01-1 microM) and GR73632 (0.1 microM), increased pial artery diameter. 3. In contrast, the selective NK2 receptor agonist, GR64349 (1 microM), produced a small vasoconstriction while the NK3 receptor-selective agonist, senktide (1 microM) was inactive. 4. Co-administration of GR82334 (1 microM), a selective NK1 receptor antagonist, inhibited the vasodilatation produced by SPOMe (0.1 microM) but not that caused by calcitonin gene-related peptide (CGRP, 0.01 microM). 5. The results are consistent with an involvement of NK1 receptors in the neurokinin-induced increase in guinea-pig pial artery diameter.

Effects of calcium channel antagonists on action potential conduction and transmitter release in the guinea-pig vas deferens.

The effects of the Ca2+ channel antagonists amlodipine, cobalt, diltiazem, nifedipine and verapamil and the local anaesthetic lignocaine were investigated on action potential conduction in and on evoked transmitter release from sympathetic nerves in the guinea-pig isolated vas deferens. Transmitter release was investigated by measurement of evoked (trains of pulses at 1 and 2 Hz, 0.1-0.5 ms supramaximal voltage) excitatory junction potentials (e.j.ps) using microelectrodes; tension was recorded simultaneously; tritium [3H] overflow from vasa preincubated (37 degrees C, 30 min) in Krebs solution containing either [3H]-noradrenaline (NA, 25 microCi ml-1, 2 X 10(-6) M NA) or [3H]-adenosine (50 microCi ml-1, 1 X 10(-6) M adenosine). Amlodipine (0.5-2 X 10(-4) M), verapamil (0.5-2 X 10(-4) M), diltiazem (1-8 X 10(-4) M), lignocaine (0.1-2 X 10(-3) M) and cobalt (2-6 X 10(-2) M) in descending order of potency, but not nifedipine (1-5 X 10(-3) M), increased the latency and inhibited, then abolished, the amplitude and number of action potentials in a concentration-dependent manner. Amlodipine (0.5-1 X 10(-4) M), verapamil (1-2 X 10(-4) M), diltiazem (1-5 X 10(-4) M) and cobalt (1 X 10(-3) M), in descending order of potency, but not nifedipine (5 X 10(-4) M), inhibited then abolished evoked e.j.ps in a concentration-dependent manner. Cobalt inhibited e.j.ps at a lower concentration than that (2-6 X 10(-2) M) required to block action potential conduction. In unstimulated tissues, the resting [3H] overflow following preincubation with [3H]-NA consisted largely of 4-hydroxy 3-methoxymandelic acid (VMA), 4-hydroxy 3-methoxy phenylglycol (MOPEG), 3,4 dihydroxyphenylglycol (DOPEG) and NA; stimulated tissues (300 pulses at 20 Hz, 0.5 ms supramaximal voltage) released mainly NA. Verapamil (0.1-1 X 10(-4) M), amlodipine (0.05-1 X 10(-4) M) and nifedipine (1-5 X 10(-4) M), but not cobalt (2 X 10(-3) M), increased, significantly, the resting overflow of 3H comprising mainly DOPEG. Cobalt (2 X 10(-3) M) inhibited, significantly, the stimulation-evoked overflow of 3H. Verapamil (1 X 10(-4) M) had little effect on the resting overflow of 3H following preincubation with [3H]-adenosine. Diltiazem (5 X 10(-4) M) and cobalt (2 X 10(-3) M) both inhibited evoked 3H overflow. Nifedipine (5 X 10(-4) M) was ineffective. 6 The effectiveness of Ca2+ channel antagonists at pre- and postjunctional sites differ; the results are discussed in terms of the selectivity of these drugs for each site and their differential effects on transmitter release.

The pharmacology of GR203040, a novel, potent and selective non-peptide tachykinin NK1 receptor antagonist.

1. The in vitro and in vivo pharmacology of GR203040 ((2S, 3S)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-y l)-amine), a novel, highly potent and selective non-peptide tachykinin NK1 receptor antagonist, was investigated in the present study. 2. GR203040 potently inhibited [3H]-substance P binding to human NK1 receptors expressed in Chinese hamster ovary (CHO) and U373 MG astrocytoma cells, and NK1 receptors in ferret and gerbil cortex (pKi values of 10.3, 10.5, 10.1 and 10.1 respectively). GR203040 had lower affinity at rat NK1 receptors (pKi = 8.6) and little affinity for human NK2 receptors (pKi < 5.0) in CHO cells and NK3 receptors in guinea-pig cortex (pKi < 6.0). With the exception of the histamine H1 receptor (pIC50 = 7.5). GR203040 had little affinity (pIC50 < 6.0) at all non-NK1 receptors and ion channels examined. Furthermore, GR203040 produced only weak inhibition of Na+ currents in SH-SY5Y neuroblastoma and superior cervical ganglion cells (pIC50 values < 4.0). GR203040 produced only weak antagonism of Ca(2+)-evoked contractions of rat isolated portal vein (pKn = 4.1). The enantiomer of GR203040, GR205608 (2R, 3R)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-y l)-amine), had 10,000 fold lower affinity at the human NK1 receptor expressed in CHO cells (pKi = 6.3). 3. In gerbil ex vivo binding experiments, GR203040 produced a dose-dependent inhibition of the binding of [3H]-substance P to cerebral cortical membranes (ED50 = 15 micrograms kg-1 s.c. and 0.42 mg kg-1 p.o.). At 10 micrograms kg-1 s.c., the inhibition of [3H]-substance P binding was maintained for > 6 h. In the rat, GR203040 was less potent (ED50 = 15.4 mg kg-1 s.c.) probably reflecting, at least in part, its lower affinity at the rat NK1 receptor. 4. In guinea-pig isolated ileum and dog isolated middle cerebral and basilar arteries, GR203040 produced a rightward displacement of the concentration-effect curves to substance P methyl ester (SPOMe) with suppression of the maximum agonist response (apparent pKB values of 11.9, 11.2 and 11.1 respectively). 5. In anaesthetized rabbits, GR203040 antagonized reductions in carotid arterial vascular resistance evoked by SPOMe, injected via the lingual artery (DR10 (i.e. the dose producing a dose-ratio of 10) = 1.1 micrograms kg-1, i.v.). At a dose 20 fold greater than its DR10 value (i.e. 22 micrograms kg-1, i.v.), significant antagonism was evident more than 2 h after GR203040 administration. 6. In anaesthetized rats, GR203040 (3 and 10 mg kg-1, i.v.) produced a dose-dependent inhibition of plasma protein extravasation in dura mater, conjunctiva, eyelid and lip in response to electrical stimulation of the trigeminal ganglion. 7. It is concluded that GR203040 is one of the most potent and selective NK1 receptor antagonists yet described, and as such, has considerable potential as a pharmacological tool to characterize the physiological and pathological roles of substance P and NK1 receptors. GR203040 may also have potential as a novel therapeutic agent for the treatment of conditions such as migraine, emesis and pain.

The 5-HT4 receptor agonist, tegaserod, is a potent 5-HT2B receptor antagonist in vitro and in vivo.

1 Tegaserod (Zelnorm) is a potent 5-hydroxytryptamine4 (5-HT4) receptor agonist with clinical efficacy in disorders associated with reduced gastrointestinal motility and transit. The present study investigated the interaction of tegaserod with 5-HT2 receptors, and compared its potency in this respect to its 5-HT4 receptor agonist activity. 2 Tegaserod had significant binding affinity for human recombinant 5-HT2A, 5-HT2B and 5-HT2C receptors (pKi=7.5, 8.4 and 7.0, respectively). The 5-HT2B receptor-binding affinity of tegaserod was identical to that at human recombinant 5-HT4(c) receptors (mean pKi=8.4) in human embryonic kidney-293 (HEK-293) cells stably transfected with the human 5-HT4(c) receptor. 3 Tegaserod (0.1-3 microm) inhibited 5-HT-mediated contraction of the rat isolated stomach fundus potently (pA2=8.3), consistent with 5-HT(2B) receptor antagonist activity. Tegaserod produced, with similar potency, an elevation of adenosine 3',5' cyclic monophosphate in HEK-293 cells stably transfected with the human 5-HT4(c) receptor (mean pEC50=8.6), as well as 5-HT4) receptor-mediated relaxation of the rat isolated oesophagus (mean pEC50=8.2) and contraction of the guinea-pig isolated colon (mean pEC50=8.3). 4 Following subcutaneous administration, tegaserod (0.3 or 1 mg kg(-1)) inhibited contractions of the stomach fundus in anaesthetized rats in response to intravenous dosing of alpha-methyl 5-HT (0.03 mg kg(-1)) and BW 723C86 (0.3 mg kg(-1)), selective 5-HT2B receptor agonists. At similar doses, tegaserod (1 and 3 mg kg(-1) subcutaneously) evoked a 5-HT4 receptor-mediated increase in colonic transit in conscious guinea-pigs. 5 The data from this study indicate that tegaserod antagonizes 5-HT2B receptors at concentrations similar to those that activate 5-HT4 receptors. It remains to be determined whether this 5-HT2B receptor antagonist activity of tegaserod contributes to its clinical profile.

The activity of GR205171, a potent non-peptide tachykinin NK1 receptor antagonist, in the trigeminovascular system.

The in vivo activity of GR205171, a novel, highly potent non-peptide tachykinin NK1 receptor antagonist, has been investigated in the trigeminovascular system in order to assess its potential as an acute therapy for migraine headache. In anaesthetised rabbits, GR205171 attenuated reductions in carotid arterial vascular resistance evoked by the tachykinin NK1 receptor agonist, substance P methyl ester (SPOMe), injected via the lingual artery (DR30 (i.e., the dose producing a dose-ratio of 30) = 0.4 microgram/kg, i.v.). In anaesthetised rats, GR205171 (0.1 and 1 mg/kg, i.v.) produced a dose-dependent inhibition of plasma protein extravasation (PPE) in dura mater, conjunctiva, eyelid and lip in response to electrical stimulation of the trigeminal ganglion. In anaesthetised guinea-pigs, GR205171 (1.10 and 100 micrograms/kg, i.v.) inhibited, by up to approximately 60%, expression of c-fos in the trigeminal nucleus caudalis in response to electrical stimulation of the trigeminal ganglion. It is concluded that GR205171 is a potent antagonist of NK1 receptor-mediated cranial vasodilatation, dural PPE and expression of c-fos in the trigeminal nucleus caudalis. Such a profile of action suggests that GR205171 may have potential as a novel therapeutic agent in the treatment of migraine headache.

GR205171: a novel antagonist with high affinity for the tachykinin NK1 receptor, and potent broad-spectrum anti-emetic activity.

It has been demonstrated recently that antagonists of the tachykinin NK1 receptor, specifically CP-99,994 and GR203040, possess anti-emetic activity in a range of species. To optimise this activity, a series of analogues based around the structure of GR203040 have been synthesised and their affinity at the human tachykinin NK1 receptor determined. In addition, the potency of these analogues to inhibit emesis induced in the ferret by whole-body X-irradiation has been examined. A range of substitution at the C-1 position of the tetrazole moiety in GR203040 were explored in vitro and in vivo. The trifluoromethyl compound, GR205171, was the most potent antagonist with regard to the ability to inhibit emesis induced by X-irradiation. This compound was demonstrated to have a broad spectrum of anti-emetic activity, inhibiting emesis in the ferret induced by cisplatin, cyclophosphamide, morphine, ipecacuanha and copper sulphate. Furthermore, emesis was also inhibited in the house-musk shrew, Suncus murinus, when induced by either motion or cisplatin, and in the dog when induced by ipecacuanha. GR205171 has the most potent anti-emetic activity of any tachykinin NK1 receptor antagonist described to date. The compound is orally active in the ferret and dog, long-lasting, and warrants further investigation as a potential broad-spectrum anti-emetic agent.

The influence of neurokinins and calcitonin gene-related peptide on cerebral blood flow in anaesthetized guinea-pigs.

The effects of systemically-administered human alpha calcitonin gene-related peptide (h.alpha CGRP), substance P and the selective neurokinin receptor agonists, GR73632 (NK1) and GR64349 (NK2) on cerebral blood flow (CBF) were studied in anaesthetized guinea-pigs using a laser-Doppler flowmeter. h.alpha CGRP (0.1 and 0.3 nmol/kg), substance P (0.03-1.0 nmol/kg), GR73632 (0.03-0.3 nmol/kg) and GR64349 (0.3 nmol/kg) each, following intra-carotid artery injection, reduced transiently (< 5 min) blood pressure and CBF. GR73632 (0.1 and 0.3 nmol/kg) and GR64349 (0.3 nmol/kg), but not h.alpha CGRP (0.01-0.3 nmol/kg) or substance P(0.01-1.0 nmol/kg), then produced a more prolonged increase in CBF, the peak effect occurring 10-15 min after injection. It is likely that this increase in CBF was due to their bronchoconstrictor activity, rather than a direct effect on the cerebrovasculature; arterial PaCO2 levels were increased and PaO2 decreased by both compounds. Following pretreatment with urea (5 M) to disrupt the blood brain barrier, h.alpha CGRP (0.1 nmol/kg) produced a significant increase in CBF (13 +/- 4%), implying that access to its receptors on the cerebrovascular smooth muscle had been achieved. Substance P (0.1 nmol/kg) remained inactive. The study has demonstrated that compounds acting on neuropeptide receptors have little direct influence on CBF following systemic administration. CGRP requires access to its receptors on the cerebrovascular smooth muscle, while selective NK1 and NK2 receptor agonists increase CBF, probably indirectly via their bronchoconstrictor activity. The lack of effect of substance P may be due to its rapid breakdown by peptidases, a property not shared by the selective neurokinin agonists.

NK1 and CGRP receptor-mediated dilatation of the carotid arterial bed of the anaesthetized rabbit.

The present study has investigated the effects of alpha and beta calcitonin gene-related peptide (CGRP), and the tachykinin neurokinin1 (NK1) receptor agonist, substance P methyl ester (SPOMe), on carotid vascular resistance, following their injection into the carotid artery bed of the anaesthetized rabbit. The involvement of CGRP and NK1 receptors in nicotine-induced alterations in carotid vascular resistance has also been characterized. alpha-or beta CGRP (1 and 10 pmolkg-1 i.a.) and SPOMe (0.01 and 0.1 pmolkg-1 i.a.) caused dose-related increases in carotid arterial blood flow associated with decreases in carotid arterial vascular resistance with little effect on arterial blood pressure. The selective CGRP receptor antagonist, CGRP8-37 (0.34 mumolkg-1 i.v.), caused a rightward displacement of the dose-response curves to both alpha- and beta CGRP; mean dose-ratios, 5 min after antagonist administration, were 14 and 24 respectively. The selective NK1 receptor antagonist, CP99 994 (0.23 mumolkg-1 i.v.), caused a rightward shift in the dose-response curve to SPOMe; mean dose-ratios, 15 and 75 min after antagonist administration, were 42 and 16 respectively. CGRP8-37 (0.34 mumolkg-1) had no effect on decreases in carotid arterial vascular resistance produced by SPOMe, and CP99 994 (0.23 mumolkg-1 i.v.) had no effect on vasodilator responses produced by either alpha- or beta CGRP. Intracarotid injection of nicotine (0.002-2 mumolkg-1) caused dose-dependent transient, followed by a more prolonged, increase in carotid blood flow and reduction in arterial vascular resistance. The prolonged carotid vasodilator response produced by nicotine (0.2 mumolkg-1) was markedly attenuated by CGRP8-37 (0.34 mumolkg-1 i.v.) but unaffected by CP99 994 (1.15 mumolkg-1 i.v.) suggesting a role for CGRP, and not substance P, in this vasodilation. Neither receptor antagonist affected the transient response produced by nicotine. This study has demonstrated that intracarotid injection of NK1 and CGRP receptor agonists to the anaesthetized rabbit results in an increase in carotid blood flow and a reduction in vascular resistance, indicative of vasodilatation of this artery bed. CGRP mediates the nicotine-induced dilatation of the carotid vascular bed, consistent with its release from sensory nerves. This model should prove useful for the in vivo characterization of NK1 or CGRP receptor agonist and antagonist activities, and in the study of neurogenically induced vasodilation.

The involvement of calcitonin gene-related peptide (CGRP) and substance P in feline pial artery diameter responses evoked by capsaicin.

The effects of capsaicin and selective neuropeptide antagonists on pial artery diameter were measured using an on-line image analyser in anaesthetised cats, in order to monitor the effects of mediators released in response to activation of trigeminal nerves. Perivascular injection of CGRP (10(-8) M) and the neurokinin-1 (NK1) receptor agonist substance P methyl ester, SPOMe (10(-6) M) produced an increase in pial artery diameter. The vasodilatory action of these agonists was significantly and selectively inhibited using the CGRP receptor antagonist, CGRP8-37 (10(-6) M), and the NK1 receptor antagonist, CP99994 (10(-6) M) respectively. Capsaicin (10(-8)-10(-5) M) produced a biphasic response upon perivascular injection that was concentration dependent. At 10(-6) M capsaicin an initial transient vasoconstriction was observed followed by a longer-lasting vasodilatation. The vasodilator component was significantly reduced by CGRP8-37 (10(-6) M) or CP99994 (10(-6) M). These results show that chemical (capsaicin) activation of trigeminal nerves leads to vasodilatation of feline arteries in situ. This vasodilatation is mediated via the activation of CGRP and NK1 receptors probably via the efferent release of CGRP and a substance P-like peptide.

GR127935: a potent and selective 5-HT1D receptor antagonist.

GR127935 is the most potent 5-HT1D receptor antagonist yet described, possessing nanomolar affinity at human 5-HT1D receptors. Sumatriptan-induced contractions of the dog isolated basilar artery and saphenous vein are antagonised by GR127935 in an insurmountable manner indicative of its slow dissociation from the 5-HT1D receptor. 5-HT1D receptor-mediated hypothermia and rotational behaviour in guinea-pigs are antagonised potently, and with long duration, by GR127935, administered by a variety of routes. GR127935 also blocks central 5-HT1D autoreceptors in vitro and in vivo. GR127935 has much lower affinity at other 5-HT, and non-5-HT, receptors. In functional studies, GR127935 fails to affect 5-HT2 receptor-mediated 'wet dog shakes' in guinea-pigs and 5-HT1A receptor-mediated inhibition of 5-HT release in rat dorsal raphé nucleus. The compound has a good safety profile in all species tested. It is concluded that GR127935 is a useful pharmacological tool to characterise 5-HT1D receptor function.

Attenuation of Fos-like immunoreactivity in the trigeminal nucleus caudalis following trigeminovascular activation in the anaesthetised guinea-pig.

The present study has examined the involvement of sensory neurotransmitters in activating neurones in the trigeminal nucleus caudalis following stimulation of the trigeminovascular system in anaesthetised guinea-pigs. Electrical stimulation of the right trigeminal ganglion produced a unilateral expression of Fos-like immunoreactivity (Fos-LI) in the trigeminal nucleus caudalis. The tachykinin NK1 receptor antagonist, GR205171 (100 micrograms/kg i.v.) and the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (1 mg/kg i.v.) each inhibited expression of Fos-LI following electrical stimulation. The calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP8-37 (1.3 mg/kg i.v.), administered following rostral intracarotid infusion of mannitol to disrupt the blood-brain barrier, tended to reduce Fos-LI evoked by electrical stimulation, although this failed to reach statistical significance. Capsaicin (10 nmol in 0.1 ml), administered intracisternally, produced a bilateral expression of Fos-LI in the trigeminal nucleus caudalis. This expression was unaffected by the peripherally acting NK1 receptor antagonist, GR82334 (0.2 mg/kg i.v.) or CGRP8-37 (1.3 mg/kg i.v.). The centrally penetrant NK1 receptor antagonist, GR205171 (100 micrograms/kg i.v.), inhibited significantly Fos-LI evoked by intracisternal capsaicin administration. It is concluded that the sensory neurotransmitters, substance P and glutamate are released centrally following activation of the trigeminovascular system and that each may be involved in activation of cells in the trigeminal nucleus caudalis.

Trigeminal ganglion stimulation increases facial skin blood flow in the rat: a major role for calcitonin gene-related peptide.

Activation of the trigeminovascular system leads to neurogenic inflammation within the dura mater and cerebral vasodilatation. These processes have been implicated in the pathogenesis of migraine headache. Neurogenic vasodilator responses to trigeminal ganglion stimulation were investigated in rat facial skin, an area innervated by the trigeminal nerve. Microvascular blood flow changes in the facial skin were measured in anaesthetised rats, using laser Doppler flowmetry. Electrical stimulation of the trigeminal ganglion caused an ipsilateral increase in facial skin blood flow which was found to be frequency dependent (0.5-10 Hz). The role of several neuropeptides in these blood flow responses was studied using selective receptor antagonists. The calcitonin gene-related peptide antagonist, CGRP8-37 (400 nmol.kg-1, i.v.) had no effect on resting levels of facial skin blood flow, but markedly inhibited responses induced by trigeminal ganglion stimulation (5 Hz, 10 V, 1 ms for 30 s). However, neither the neurokinin-1 (NK1) receptor antagonist, RP67580 (0.23 or 2.3 mumol.kg-1, i.v.) nor the vasoactive intestinal peptide (VIP) antagonist, [p-Cl-D-Phe6,Leu17]-VIP (15 or 30 nmol.kg-1, i.v.) had any effect on these responses. These results suggest that CGRP is the major neuropeptide involved in the vasodilator response to trigeminal ganglion stimulation in rat facial skin. Clarification of the mechanisms involved in this neurogenic vasodilator response may aid the development of drugs that target the trigeminovascular system during migraine headache.