Identification and characterisation of SB-366791, a potent and selective vanilloid receptor (VR1/TRPV1) antagonist.

Vanilloid receptor-1 (TRPV1) is a non-selective cation channel, predominantly expressed by peripheral sensory neurones, which is known to play a key role in the detection of noxious painful stimuli, such as capsaicin, acid and heat. To date, a number of antagonists have been used to study the physiological role of TRPV1; however, antagonists such as capsazepine are somewhat compromised by non-selective actions at other receptors and apparent modality-specific properties. SB-366791 is a novel, potent, and selective, cinnamide TRPV1 antagonist isolated via high-throughput screening of a large chemical library. In a FLIPR-based Ca(2+)-assay, SB-366791 produced a concentration-dependent inhibition of the response to capsaicin with an apparent pK(b) of 7.74 +/- 0.08. Schild analysis indicated a competitive mechanism of action with a pA2 of 7.71. In electrophysiological experiments, SB-366791 was demonstrated to be an effective antagonist of hTRPV1 when activated by different modalities, such as capsaicin, acid or noxious heat (50 degrees C). Unlike capsazepine, SB-366791 was also an effective antagonist vs. the acid-mediated activation of rTRPV1. With the aim of defining a useful tool compound, we also profiled SB-366791 in a wide range of selectivity assays. SB-366791 had a good selectivity profile exhibiting little or no effect in a panel of 47 binding assays (containing a wide range of G-protein-coupled receptors and ion channels) and a number of electrophysiological assays including hippocampal synaptic transmission and action potential firing of locus coeruleus or dorsal raphe neurones. Furthermore, unlike capsazepine, SB-366791 had no effect on either the hyperpolarisation-activated current (I(h)) or Voltage-gated Ca(2+)-channels (VGCC) in cultured rodent sensory neurones. In summary, SB-366791 is a new TRPV1 antagonist with high potency and an improved selectivity profile with respect to other commonly used TRPV1 antagonists. SB-366791 may therefore prove to be a useful tool to further study the biology of TRPV1.

Orexinergic neurons and barbiturate anesthesia.

Orexins (OXs) regulate sleep with possible interactions with brain noradrenergic neurons. In addition, noradrenergic activity affects barbiturate anesthesia. As we have also recently reported that OXs selectively evoke norepinephrine release from rat cerebrocortical slices we hypothesized that barbiturate anesthesia may result from of an interaction with central orexinergic systems. To test this hypothesis, we performed a series of in vivo and in vitro studies in rats. In vivo, the effects of i.c.v. OX A, B and SB-334867-A (OX1 receptor antagonist) on pentobarbital, thiopental or phenobarbital-induced anesthesia times (loss of righting reflex) was assessed. In vitro effects of barbiturates and SB-334867-A on OX-evoked norepinephrine release from cerebrocortical slice was examined. In Chinese hamster ovary cells expressing human OX1/OX2 receptors OX A- and B-evoked increases in intracellular Ca2+ were measured with and without barbiturates. OX A and B significantly decreased pentobarbital, thiopental and phenobarbital anesthesia times by 15-40%. SB-334867-A increased thiopental-induced anesthesia time by approximately by 40%, and reversed the decrease produced by OX A. In vitro, all anesthetic barbiturates inhibited OX-evoked norepinephrine release with clinically relevant IC50 values. A GABAA antagonist, bicuculline, did not modify the inhibitory effects of thiopental and the GABAA agonist, muscimol, did not inhibit norepinephrine release. In addition there was no interaction of barbiturates with either OX1 or OX2 receptors. Collectively our data suggest that orexinergic neurons may be an important target for barbiturates, and GABAA, OX1 and OX2 receptors may not be involved in this interaction.

Comparison of effects of anandamide at recombinant and endogenous rat vanilloid receptors.

BACKGROUND: Anandamide, an endogenous lipid, activates both cannabinoid (CB(1)) and vanilloid (VR1) receptors, both of which are co-expressed in rat dorsal root ganglion (DRG) cells. Activation of either receptor results in analgesia but the relative contribution of CB(1) and VR1 in anandamide-induced analgesia remains controversial. Here we compare the in vitro pharmacology of recombinant and endogenous VR1 receptors using calcium imaging, in clonal and DRG cells, respectively. We also consider the contribution of CB(1) and VR1 receptors to anandamide-induced analgesia. METHODS: Using a Flurometric Imaging Plate Reader (FLIPR), calcium imaging has been used to study the effects of several vanilloid and cannabinoid ligands in rat VR1-transfected HEK293 (rVR1-HEK) cells and in DRG cells. The effect of pre-exposure of several vanilloid and cannabinoids has also been compared in DRG cells. RESULTS: The VR1 agonists capsaicin, olvanil, (N-(4-hydroxyphenyl-arachinoylamide) (AM404) and anandamide caused a concentration-dependent increase in intracellular calcium concentration ([Ca(2+)](i)), with similar temporal profiles in both rVR1-HEK and DRG cells, and potency (pEC(50)) values of 8.25 (SEM 0.11), 8.37 (0.04), 6.96 (0.06), 5.85 (0.01) and 7.45 (0.10), 7.55 (0.07), 6.10 (0.13), approximately 5.5, respectively. These responses were inhibited by the VR1 antagonist capsazepine (1 micro M). In contrast, application of synthetic cannabinoid antagonists failed to inhibit the anandamide-induced increase in [Ca(2+)](i). Reapplication of VR1 agonists significantly inhibited a subsequent challenge to either capsaicin or anandamide in either cell type, whilst pre-exposure to cannabinoid agonists were without effect. CONCLUSION: Here we provide evidence that the pharmacology of recombinant rVR1 receptors is similar to those endogenously expressed in DRG cells. Moreover, we have shown that VR1, but not CB(1), receptors are involved in anandamide-induced responses in dorsal root primary neurones in vitro. Therefore, the analgesic properties of anandamide are likely to be mediated, at least in part, by VR1 activation in DRG cells in vivo.

TRPV3 is a temperature-sensitive vanilloid receptor-like protein.

Vanilloid receptor-1 (VR1, also known as TRPV1) is a thermosensitive, nonselective cation channel that is expressed by capsaicin-sensitive sensory afferents and is activated by noxious heat, acidic pH and the alkaloid irritant capsaicin. Although VR1 gene disruption results in a loss of capsaicin responses, it has minimal effects on thermal nociception. This and other experiments--such as those showing the existence of capsaicin-insensitive heat sensors in sensory neurons--suggest the existence of thermosensitive receptors distinct from VR1. Here we identify a member of the vanilloid receptor/TRP gene family, vanilloid receptor-like protein 3 (VRL3, also known as TRPV3), which is heat-sensitive but capsaicin-insensitive. VRL3 is coded for by a 2,370-base-pair open reading frame, transcribed from a gene adjacent to VR1, and is structurally homologous to VR1. VRL3 responds to noxious heat with a threshold of about 39 degrees C and is co-expressed in dorsal root ganglion neurons with VR1. Furthermore, when heterologously expressed, VRL3 is able to associate with VR1 and may modulate its responses. Hence, not only is VRL3 a thermosensitive ion channel but it may represent an additional vanilloid receptor subunit involved in the formation of heteromeric vanilloid receptor channels.

Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1.

The vanilloid receptor-1 (VR1) is a heat-gated ion channel that is responsible for the burning sensation elicited by capsaicin. A similar sensation is reported by patients with esophagitis when they consume alcoholic beverages or are administered alcohol by injection as a medical treatment. We report here that ethanol activates primary sensory neurons, resulting in neuropeptide release or plasma extravasation in the esophagus, spinal cord or skin. Sensory neurons from trigeminal or dorsal root ganglia as well as VR1-expressing HEK293 cells responded to ethanol in a concentration-dependent and capsazepine-sensitive fashion. Ethanol potentiated the response of VR1 to capsaicin, protons and heat and lowered the threshold for heat activation of VR1 from approximately 42 degrees C to approximately 34 degrees C. This provides a likely mechanistic explanation for the ethanol-induced sensory responses that occur at body temperature and for the sensitivity of inflamed tissues to ethanol, such as might be found in esophagitis, neuralgia or wounds.

Cannabinoid activation of recombinant and endogenous vanilloid receptors.

The effects of three structurally related cannabinoids on human and rat recombinant vanilloid VR1 receptors expressed in human embryonic kidney (HEK293) cells and at endogenous vanilloid receptors in the rat isolated mesenteric arterial bed were studied. In the recombinant cells, all three were full agonists, causing concentration-dependent increases in [Ca(2+)](i) (FLIPR), with a rank order of potency relative to the vanilloids capsaicin and olvanil, of olvanil> or =capsaicin>AM404 ((allZ)-N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide)>anandamide>methanandamide. These responses were inhibited by the vanilloid VR1 receptor antagonist, capsazepine. In the mesenteric arterial bed, vasorelaxation was evoked by these ligands with a similar order of potency. The AM404-induced vasorelaxation was virtually abolished by capsaicin pretreatment. AM404 inhibition of capsaicin-sensitive sensory neurotransmission was blocked by ruthenium red, but not by cannabinoid CB(1) and CB(2) receptor antagonists. AM404 had no effect on relaxations to calcitonin gene-related peptide. These data demonstrate that the vasorelaxant and sensory neuromodulator properties of AM404 in the rat isolated mesenteric arterial bed are mediated by vanilloid VR1 receptors.

1,3-Biarylureas as selective non-peptide antagonists of the orexin-1 receptor.

This communication reports SARs for the first orexin-1 receptor antagonist series of 1-aryl-3-quinolin-4-yl and 1-aryl-3-naphthyridin-4-yl ureas. One of these compounds, 31 (SB-334867), has excellent selectivity for the orexin-1 receptor, blood-brain barrier permeability and shows in vivo activity following ip dosing.

Low pH modulation of recombinant vanilloid receptors and perivascular capsaicin-sensitive sensory neurotransmission.

The effect of low pH on capsaicin-sensitive sensory neurotransmission in the rat isolated mesenteric arterial bed and at recombinant (rVR1) vanilloid receptors was investigated. Mesenteric sensory neurogenic vasorelaxation elicited by electrical field stimulation was reversibly inhibited by lowering pH from 7.4 to 6.9 and 6.3. Capsaicin-induced vasorelaxation was not different at pH 6.9, but was attenuated at pH 6.3. Vasorelaxation to calcitonin gene-related peptide, the principal sensory motor neurotransmitter in rat mesenteric arteries, was not different at pH 6.9 or pH 6.3. In rVR1-transfected HEK293 cells, acidic conditions enhanced the affinities of capsaicin and capsazepine at rVR1, but did not affect the potency of carbachol at endogenous muscarinic receptors. Following inactivation of endogenous acid-sensitive ion channels, lowering pH (6.0-4.5) directly increased [Ca2+]i in rVR1-HEK293 cells (EC50 5.5). This response was abolished by 1 microM capsazepine. In conclusion, a decrease in pH (to 6.9 and 6.3) enhances the affinity of capsaicin at rVR1, but inhibits sensory neurotransmission in the rat mesenteric arterial bed. This likely explains why there is no evidence of an enhancement of sensitivity to capsaicin at endogenous vanilloid receptors, as observed with rVR1. When pH is reduced still further (6.0-5.5) there is direct activation of rVR1.

Characterisation using FLIPR of human vanilloid VR1 receptor pharmacology.

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.

SB-334867-A: the first selective orexin-1 receptor antagonist.

The pharmacology of various peptide and non-peptide ligands was studied in Chinese hamster ovary (CHO) cells stably expressing human orexin-1 (OX(1)) or orexin-2 (OX(2)) receptors by measuring intracellular calcium ([Ca(2+)](i)) using Fluo-3AM. Orexin-A and orexin-B increased [Ca(2+)](i) in CHO-OX(1) (pEC(50)=8.38+/-0.04 and 7.26+/-0.05 respectively, n=12) and CHO-OX(2) (pEC(50)=8.20+/-0.03 and 8.26+/-0.04 respectively, n=8) cells. However, neuropeptide Y and secretin (10 pM - 10 microM) displayed neither agonist nor antagonist properties in either cell-line. SB-334867-A (1-(2-Methyylbenzoxanzol-6-yl)-3-[1,5]naphthyridin-4-yl-urea hydrochloride) inhibited the orexin-A (10 nM) and orexin-B (100 nM)-induced calcium responses (pK(B)=7.27+/-0.04 and 7.23+/-0.03 respectively, n=8), but had no effect on the UTP (3 microM)-induced calcium response in CHO-OX(1) cells. SB-334867-A (10 microM) also inhibited OX(2) mediated calcium responses (32.7+/-1.9% versus orexin-A). SB-334867-A was devoid of agonist properties in either cell-line. In conclusion, SB-334867-A is a non-peptide OX(1) selective receptor antagonist.

Structure-activity analysis of truncated orexin-A analogues at the orexin-1 receptor.

Truncated peptide analogues of orexin-A were prepared and their biological activity assesed at the orexin-1 receptor. Progressive N-terminal deletions identified the minimum C-terminal sequence required for maintaining a significant agonist effect, whilst an alanine scan and other pertinent substitutions identified key side-chain and stereochemical requirements for receptor activation.

Pharmacological characterisation of human 5-HT2 receptor subtypes.

Prompted by conflicting literature, this study compared the pharmacology of human 5-hydroxytryptamine2 (5-HT2) receptors expressed in SH-SY5Y cells using a fluorometric imaging plate reader (FLIPR) based Ca2+ assay. 5-Hydroxytryptamine (5-HT) increased intracellular calcium concentration ([Ca2+]i) at 5-HT2A, 5-HT2B and 5-HT2C receptors (pEC(50)=7.73+/-0.03, 8.86+/-0.04 and 7.99+/-0.04, respectively) and these responses were inhibited by mesulergine (pKB=7.42+/-0.06, 8.77+/-0.10 and 9.52+/-0.11). A range of selective agonists and antagonists displayed the expected pharmacology at each receptor subtype. Sodium butyrate pretreatment increased receptor expression in SH-SY5Y/5-HT2B (15-fold) and SH-SY5Y/5-HT2C cells (7-fold) and increased agonist potencies and relative efficacies. In contrast, sodium butyrate pretreatment of SH-SY5Y/5-HT(2A) cells did not affect receptor expression. The present study provides a direct comparison of agonist and antagonist pharmacology at 5-HT(2) receptor subtypes in a homogenous system and confirms that agonist potency and efficacy varies with the level of receptor expression.

Characterisation of an endogenous bombesin receptor in CHO/DG44 cells.

Bombesin and its receptors have been shown to have a role regulating circadian rhythms in the hamster suprachiasmatic and dorsal raphe nuclei and have been implicated in the regulation of sleep. We have identified and characterised a bombesin receptor endogenously expressed in a Chinese hamster ovary cell line (CHO/DG44). Using a range of bombesin-like peptides, we demonstrate that this receptor displays bombesin BB2 receptor-like pharmacology. We also show that this receptor signals through inositol-[1,4,5]-trisphosphate and protein kinase C and thus provides a useful model system to aid in the interpretation of hamster suprachiasmatic nucleus studies of mammalian circadian rhythm.

Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology.

The vanilloid receptor (VR1) is a ligand-gated ion channel, which plays an important role in nociceptive processing. Therefore, a pharmacological characterization of the recently cloned rat VR1 (rVR1) was undertaken. HEK293 cells stable expressing rVR1 (rVR1-HEK293) were loaded with Fluo-3AM and then incubated at 25 degrees C for 30 min with or without various antagonists or signal transduction modifying agents. Then intracellular calcium concentrations ([Ca(2+)](i)) were monitored using FLIPR, before and after the addition of various agonists. The rank order of potency of agonists (resiniferatoxin (RTX)>capsaicin>olvanil>PPAHV) was as expected, and all were full agonists. The potencies of capsaicin and olvanil, but not RTX or PPAHV, were enhanced at pH 6.4 (pEC(50) values of 7.47+/-0.06, 7.16+/-0.06, 8.19+/-0.06 and 6.02+/-0.03 respectively at pH 7.4 vs 7.71+/-0.05, 7.58+/-0.14, 8.10+/-0.05 and 6.04+/-0.08 at pH 6.4). Capsazepine, isovelleral and ruthenium red all inhibited the capsaicin (100 nM)-induced Ca(2+) response in rVR1-HEK293 cells, with pK(B) values of 7.52+/-0.08, 6.92+/-0.11 and 8.09+/-0.12 respectively (n=6 each). The response to RTX and olvanil were also inhibited by these compounds. None displayed any agonist-like activity. The removal of extracellular Ca(2+) abolished, whilst inhibition of protein kinase C with chelerythrine chloride (10 microM) partially (approximately 20%) inhibited, the capsaicin (10 microM)-induced Ca(2+) response. However, tetrodotoxin (3 microM), nimodipine (10 microM), omega-GVIA conotoxin (1 microM), thapsigargin (1 microM), U73122 (3 microM) or H-89 (3 microM) had no effect on the capsaicin (100 nM)-induced response. In conclusion, the recombinant rVR1 stably expressed in HEK293 cells acts as a ligand-gated Ca(2+) channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor.

The hypocretins are weak agonists at recombinant human orexin-1 and orexin-2 receptors.

The pharmacology of the orexin-like peptides, hypocretin-1 and hypocretin-2, was studied in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX(1)) or orexin-2 (OX(2)) receptors by measuring intracellular calcium ([Ca(2+)](i)) using Fluo-3AM. Orexin-A and orexin-B increased [Ca(2+)](i) in CHO-OX(1) (pEC(50)=7. 99+/-0.05 and 7.00+/-0.10 respectively, n=8) and CHO-OX(2) (pEC(50)=8.30+/-0.05 and 8.21+/-0.07 respectively, n=5). However, hypocretin-1 and hypocretin-2 were markedly less potent, with pEC(50) values of 5.31+/-0.04 and 5.41+/-0.04 respectively in CHO-OX(2) cells (n=5). In CHO-OX(1) cells 10 microM hypocretin-1 only elicited a 37.5+/-3.4% response whilst 10 microM hypocretin-2 elicited a 18.0+/-2.1% response (n=8). Desensitisation of OX(1) or OX(2) with orexin-A (100 nM) abolished the response to orexin-A (10 nM) and the hypocretins (10 microM), but not to UTP (3 microM). In conclusion, the hypocretins are only weak agonists at the orexin receptors.

The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1).

The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 microM) and capsaicin (1 microM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 microM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC(50) values of 5. 94+/-0.06 (n=5) and 7.13+/-0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK(B) of 7.40+/-0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1.

Molecular and pharmacological characterization of a functional tachykinin NK3 receptor cloned from the rabbit iris sphincter muscle.

1. A functional tachykinin NK3 receptor was cloned from the rabbit iris sphincter muscle and its distribution investigated in ocular tissues. 2. Standard polymerase chain reaction (PCR) techniques were used to clone a full length rabbit NK3 receptor cDNA consisting of 1404 nucleotides. This cDNA encoded a protein of 467 amino acids with 91 and 87% homology to the human and rat NK3 receptors respectively. 3. In CHO-K1 cells transiently expressing the recombinant rabbit NK3 receptor, the relative order of potency of NKB>>NKA>/=SP to displace [125I]-[MePhe7]-NKB binding and to increase intracellular calcium, together with the high affinity of NK3 selective agonists (e.g. senktide, [MePhe7]-NKB) and antagonists (e.g. SR 142801, SB 223412) in both assays was consistent with NK3 receptor pharmacology. In binding and functional experiments, agonist concentration response curves were shallow (0.7 - 0.8), suggesting the possibility of multiple affinity states of the receptor. 4. Quantitative real time PCR analysis revealed highest expression of rabbit NK3 receptor mRNA in iris sphincter muscle, lower expression in retina and iris dilator muscle, and no expression in lens and cornea. In situ hybridization histochemistry revealed discrete specific localization of NK3 receptor mRNA in the iris muscle and associated ciliary processes. Discrete specific labelling of NK3 receptors with the selective NK3 receptor agonist [125I]-[MePhe7]-NKB was also observed in the ciliary processes using autoradiography. 5. Our study reveals a high molecular similarity between rabbit and human NK3 receptor mRNAs, as predicted from previous pharmacological studies, and provide the first evidence that NK3 receptors are precisely located on ciliary processes in the rabbit eye. In addition, there could be two affinity states of the receptor which may correspond to the typical and 'atypical' NK3 receptor subtypes previously reported.

Characterization of recombinant human orexin receptor pharmacology in a Chinese hamster ovary cell-line using FLIPR.

The cellular mechanisms underlying the physiological effects of the orexins are poorly understood. Therefore, the pharmacology of the recombinant human orexin receptors was studied using FLIPR. Intracellular calcium ([Ca2+]i) was monitored in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX1) or orexin-2 (OX2) receptors using Fluo-3AM. Orexin-A and orexin-B increased [Ca2+]i in a concentration dependent manner in CHO-OX1 (pEC50=8.03+/-0.08 and 7. 30+/-0.08 respectively, n=5) and CHO-OX2 (pEC50=8.18+/-0.10 and 8. 43+/-0.09 respectively, n=5) cells. This response was typified as a rapid peak in [Ca2+]i (maximal at 6 - 8 s), followed by a gradually declining secondary phase. Thapsigargin (3 microM) or U73122 (3 microM) abolished the response. In calcium-free conditions the peak response was unaffected but the secondary phase was shortened, returning to basal values within 90 s. Calcium (1.5 mM) replacement restored the secondary phase. In conclusion, orexins cause a phospholipase C-mediated release of calcium from intracellular stores, with subsequent calcium influx.

Identification of (-)-cis-6-acetyl-4S-(3-chloro-4-fluoro-benzoylamino)- 3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3S-ol as a potential antimigraine agent.

Optimisation of novel cis- and trans-4-(substituted-amido)benzopyran-3-ol derivatives has led to the identification of SB-220453 20 with an in vivo pre-clinical CNS profile predictive of potential antimigraine activity.