N-4-methansulfonamidobenzyl-N'-2-substituted-4-tert-butyl-benzyl thioureas as potent vanilloid receptor antagonistic ligands

A series of N-4-methansulfonamidobenzyl-N'-2-substituted-4-tert-butylbenzyl thioureas were prepared for the study of their agonistic/antagonistic activities to the vanilloid receptor in rat DRG neurons. Their structure-activity relationship reveals that there is a space for another hydrophobic binding interaction around 2-position in 4-tert-butylbenzyl region. Among the prepared derivatives, 6n show the highest antagonistic activity against the vanilloid receptor (IC(50)=15 nM).     

Biarylcarboxybenzamide derivatives as potent vanilloid receptor (VR1) antagonistic ligands

Seventeen biarylcarboxybenzamide derivatives were prepared for the study of their agonistic/antagonistic activities to the vanilloid receptor (VR1) in rat DRG neurons. The replacement of the piperazine moiety of the lead compound 1 with phenyl ring showed quite enhanced antagonistic activity. Among the prepared derivatives, N-(4-tert-butylphenyl)-4-pyridine-2-yl-benzamide (2, IC(50)=31 nM) and N-(4-tert-butylphenyl)-4-(3-methylpyridine-2-yl)benzamide (3g, IC(50)=31 nM), showed 5-fold higher antagonistic activity than 1 in (45)Ca(2+)-influx assay.     

N-[4-(methylsulfonylamino)benzyl]thiourea analogues as vanilloid receptor antagonists: analysis of structure-activity relationships for the "C-Region"

We recently reported that N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl] thiourea (2) was a high affinity antagonist of the vanilloid receptor with a binding affinity of K(i)=63 nM and an antagonism of K(i)=53.9 nM in rat VR1 heterologously expressed in Chinese hamster ovary (CHO) cells (Mol. Pharmacol. 2002, 62, 947-956). In an effort to further improve binding affinity and antagonistic potency, we have modified the C-region of the lead 4-t-butylbenzyl group with diverse surrogates, such as araalkyl, alkyl, 4-alkynylbenzyl, indanyl, 3,3-diarylpropyl, 4-alkoxybenzyl, 4-substituted piperazine and piperidine. The lipophilic surrogates, arylalkyl and alkyl, conferred modest decreases in binding affinities and antagonistic potencies; the groups having heteroatoms resulted in dramatic decreases. Our findings indicate that 4-t-butylbenzyl is one of the most favorable groups for high receptor binding and potent antagonism to VR1 in this structural series.     

Synthesis of N,N',N"-trisubstituted thiourea derivatives and their antagonist effect on the vanilloid receptor

Twenty-seven N,N',N"-trisubstituted thiourea derivatives were prepared. Among them, 1-[3-(4'-hydroxy-3'-methoxy-phenyl)-propyl]-1,3-diphenethyl-thiourea (8l, IC(50)=0.32 microM), showed 2-fold higher antagonistic activity than that of capsazepine (3, IC(50)=0.65 microM) against the vanilloid receptor in a (45)Ca(2+)-influx assay.     

Novel non-vanilloid VR1 antagonist of high analgesic effects and its structural requirement for VR1 antagonistic effects

A novel non-vanilloid VR1 antagonist consisting of a new vanilloid equivalent exhibits excellent analgesic effects as well as highly potent antagonistic activities in both capsaicin single channel and calcium uptake assays. In addition, the structural requirement for the vanilloid equivalent of the potent VR1 antagonist has also been elucidated.     

Analysis of structure-activity relationships for the 'B-region' of N-(3-acyloxy-2-benzylpropyl)-N(')-[4-(methylsulfonylamino)benzyl]thiourea analogues as vanilloid receptor antagonists: discovery of an N-hydroxythiourea analogue with potent analgesic activity

The structural modifications on the B-region of the potent and high affinity vanilloid receptor (VR1) lead ligand N-(3-acyloxy-2-benzylpropyl)-N(')-[4-(methylsulfonylamino)benzyl]thiourea were investigated by the replacement of the thiourea with diverse isosteric functional groups. Structure-activity analysis indicated that the A-region in this series was the primary factor in determining the agonistic/antagonistic activities regardless of the B-region. The N(C)-hydroxy thiourea analogues (12, 13) showed excellent analgesic activities in the acetic acid writhing assay compared to the parent thiourea analogues.     

Novel benzodiazepines derivatives as analgesic modulating for Transient receptor potential vanilloid 1

A new series of derivatives of 3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)propanoic acid were designed and synthesized as analgesic modulating for Transient receptor potential vanilloid 1. They were investigated for TRPV1 antagonistic activity in vitro, analgesic activity and sedative activity in vivo and aqueous solubility. Preliminary studies identified 3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-N,N-dimethylpropanamide(Compound 11), as a potent analgesic modulating for TRPV1 with potent activity and good aqueous solubility.     

3-Acyloxy-2-phenalkylpropyl amides and esters of homovanillic acid as novel vanilloid receptor agonists

A series of 3-acyloxy-2-phenalkylpropyl amides and esters of homovanillic acid were designed and synthesized as vanilloid receptor agonists containing the three principal pharmacophores of resiniferatoxin. Amide analogues 23, 5 and 11 were found to be potent agonists in vanilloid receptor assay both for ligand binding and for activation.     

Cardiovascular pharmacology of anandamide

The fatty acid amide anandamide produces hypotension and a decrease in systemic vascular resistance in vivo. A drop in blood pressure is also seen with synthetic cannabinoid (CB) receptor agonists. The hypotensive responses to anandamide and synthetic cannabinoids are absent in CB1 receptor gene knockout mice. In isolated arteries and perfused vascular beds, anandamide induces vasodilator responses, which cannot be mimicked by synthetic cannabinoids. Instead, vanilloid receptors on perivascular sensory nerves play a key role in these effects of anandamide. Activation of vanilloid receptors by anandamide triggers the release of sensory neuropeptides such as the vasodilator calcitonin gene-related peptide (CGRP). Anandamide is detected in blood and in many cells of the cardiovascular system, and macrophage-derived anandamide may be involved in several hypotensive clinical conditions. Interestingly, cannabinoid and vanilloid receptors display an overlap in ligand recognition properties, and the frequently used CB1 receptor antagonist SR141716A also inhibits vanilloid receptor-mediated responses. The presence of anandamide in endothelial cells, neurones and activated macrophages (monocytes), and its ability to activate CB and vanilloid receptors make this lipid a potential bioregulator in the cardiovascular system.     

Role of Vanilloid Receptors in the Capsaicin-Mediated Induction of iNOS in PC12 Cells

The vanilloid receptor 1(VR1) is a nonselective cation channel that is activated by pungent vanilloid compound, extracellular protons, or noxious heat. mRNA of VR1 and vanilloid receptor 1-like receptor (VRL1) were expressed in PC12 cells, and only VRI mRNA was detected in glioma and A10 cell lines. VRI protein was demonstrated in PC12 cells by immunocytochemistry and Western blotting. Capsaicin (CPS), the VRI receptor agonist, led to an increase in intracellular calcium ion, and this effect was blocked by pretreatment with VR1 receptor antagonist capsazepin (CPZ). Treatment of PC12 cells with low concentration of CPS (5-50 microM) increased reactive oxygen species (ROS) production, and inducible nitric oxide synthase (iNOS) was expressed after CPS treatment for 24 h. These CPS-induced changes are inhibited by pretreatment of CPZ. These findings suggest that CPS-induced iNOS expression through the VR1 and/or VRL1-mediated pathway, and this may explain the CPS-mediated physiological and pathological effects in neuron system.     

Molecular basis for species-specific sensitivity to "hot" chili peppers

Chili peppers produce the pungent vanilloid compound capsaicin, which offers protection from predatory mammals. Birds are indifferent to the pain-producing effects of capsaicin and therefore serve as vectors for seed dispersal. Here, we determine the molecular basis for this species-specific behavioral response by identifying a domain of the rat vanilloid receptor that confers sensitivity to capsaicin to the normally insensitive chicken ortholog. Like its mammalian counterpart, the chicken receptor is activated by heat or protons, consistent with the fact that both mammals and birds detect noxious heat and experience thermal hypersensitivity. Our findings provide a molecular basis for the ecological phenomenon of directed deterence and suggest that the capacity to detect capsaicin-like inflammatory substances is a recent acquisition of mammalian vanilloid receptors.     

Vanilloid receptor 1 regulates multiple calcium compartments and contributes to Ca2+-induced Ca2+ release in sensory neurons

Vanilloid receptor 1 belongs to the transient receptor potential ion channel family and transduces sensations of noxious heat and inflammatory hyperalgesia in nociceptive neurons. These neurons contain two vanilloid receptor pools, one in the plasma membrane and the other in the endoplasmic reticulum. The present experiments characterize these two pools and their functional significance using calcium imaging and 45Ca uptake in stably transfected cells or dorsal root ganglion neurons. The plasma membrane localized receptor is directly activated by vanilloids. The endoplasmic reticulum pool was demonstrated to be independently activated with 20 microm capsaicin or 1.6 microm resiniferatoxin using a bathing solution containing 10 microm Ruthenium Red (to selectively block plasma membrane-localized receptors) and 100 microm EGTA. We also demonstrate an overlap between the endoplasmic reticulum-localized vanilloid receptor regulated stores and thapsigargin-sensitive stores. Direct depletion of calcium via activation of endoplasmic reticulum-localized vanilloid receptor 1 triggered store operated calcium entry. Furthermore, we found that, in the presence of low extracellular calcium (10(-5) m), either 2 microm capsaicin or 0.1 nm-1.6 microm resiniferatoxin caused a pronounced calcium-induced calcium release in either vanilloid receptor-expressing neurons or heterologous expression systems. This phenomenon may allow new insight into how nociceptive neuron function in response to a variety of nociceptive stimuli both acutely and during prolonged nociceptive signaling.     

Molecular cloning of an N-terminal splice variant of the capsaicin receptor. Loss of N-terminal domain suggests functional divergence among capsaicin receptor subtypes

Recently a cDNA clone, vanilloid receptor subtype-1 (VR1), was isolated and found to encode an ion channel that is activated by both capsaicin, the pain producing compound in chili peppers, and by noxious thermal stimuli. Subsequently, two related cDNAs have been isolated, a stretch inactivating channel with mechanosensitive properties and a vanilloid receptor-like protein that is responsive to high temperatures (52-53 degrees C). Here, we report the isolation of a vanilloid receptor 5'-splice variant (VR.5'sv) which differs from VR1 by elimination of the majority of the intracellular N-terminal domain and ankyrin repeat elements. Both VR.5'sv and VR1 mRNA were shown to be expressed in tissues reportedly responsive to capsaicin including dorsal root ganglion, brain, and peripheral blood mononuclear cells. Functional expression of VR.5'sv in Xenopus oocytes and mammalian cells showed no sensitivity to capsaicin, the potent vanilloid resiniferatoxin, hydrogen ions (pH 6.2), or noxious thermal stimuli (50 degrees C). Since VR.5'sv is otherwise identical to VR1 throughout its transmembrane spanning domains and C-terminal region, these results support the hypothesis that the N-terminal intracellular domain is essential for the formation of functional receptors activated by vanilloid compounds and noxious thermal stimuli.     

Naphthol derivatives as TRPV1 inhibitors for the treatment of urinary incontinence

We have identified naphthol derivatives as inhibitors of the vanilloid receptor TRPV1 by high throughput screening. The initial lead showed high clearance in rats and has been optimized by enhancing the acidity of the phenol group. Compound 6b has reduced clearance, improved potency and is active in rat cystometry models of urinary incontinence after intravenous administration.     

Ruthenium red as a capsaicin antagonist.

Definition of the physiological and pharmacological properties of primary afferent neurons by the use of capsaicin and its analogues (e.g. resiniferatoxin) has represented one of the most active areas of research of the last decade (1-4 for reviews). In the past 3 years many important advancements have been made in this field, dealing with: a) discovery of the capsaicin (or 'vanilloid' receptor (5); b) discovery of capsazepine as a competitive receptor antagonist at the vanilloid receptor (6); c) definition of the cation channel coupled with the vanilloid receptor and the ionic basis for excitation and "desensitization" of primary afferents by capsaicin and related substances (7,8) and d) discovery of ruthenium red as a functional capsaicin antagonist. The aim of the present article is to briefly review the pharmacology of ruthenium red as a capsaicin antagonist and attempting to define the usefulness and the limits of this substance as a tool in sensory neuron research.     

Involvement of peripheral TRPV1 in TMJ hyperalgesia induced by ethanol withdrawal

Ethanol withdrawal increases nociception after the injection of formalin into the rat's temporomandibular joint (TMJ). Little is known about the neurological basis for hyperalgesia induced by ethanol withdrawal, but it has been reported that ethanol can potentiate the response of transient receptor potential vanilloid receptor-1 (TRPV1) in superficial tissues. The present study was designed to test the hypothesis that peripheral TRPV1 could be involved on nociceptive behavioral responses induced by the injection of formalin into the TMJ region of rats exposed to chronic ethanol administration and ethanol withdrawal. Behavioral hyperalgesia was verified 12 h after ethanol withdrawal in rats that drank an ethanol solution (6.5%) for 10 days. In another group submitted to the same ethanol regimen, the selective vanilloid receptor antagonist capsazepine (300, 600 or 1200 microg/25 microl) or an equal volume of vehicle were injected into the TMJ regions 30 min before the TMJ formalin test. The local injections of capsazepine reduced the increased nociceptive responses induced by ethanol withdrawal. The effect of capsazepine on rats that did not drink ethanol was not significant. These results indicate that the peripheral TRPV1 can contribute to the hyperalgesia induced by ethanol withdrawal on deep pain conditions.     

Role of TRPV4-P2X7 Pathway in Neuropathic Pain in Rats with Chronic Compression of the Dorsal Root Ganglion

Neurochemical Research - Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable non-selective cation channel that is involved in the development of neuropathic pain. P2X7 receptor...     

Effect of bradykinin analogues on the B1 receptor of rat ileum

The longitudinal muscle of isolated rat ileum is a sensitive bioassay suitable for testing compounds with antagonistic effects on the B(1) receptor. Bradykinin analogues with replacement of proline by alkyl-substituted phenylalanine at position 7 are effective on this receptor as entire molecules and have a stronger antagonistic effect than on the B(2) receptor. A corresponding desArg(9)-compound has a specific effect on the B(1) receptor and a very high antagonistic potency. [LNMPhe(2)]bradykinin as a compound without any replacement at position 7 or 8 shows antagonistic activity as well.     

Synthesis and structure-activity relationship studies of tyrosine-based antagonists at the human P2X7 receptor

Analogues of the P2X(7) receptor antagonist KN-62, modified at the piperazine and arylsulfonyl groups, were synthesized and assayed at the human P2X(7) receptor for inhibition of BzATP-induced effects, that is, uptake of a fluorescent dye (ethidium bromide) in stably transfected HEK293 cells and IL-1beta release in differentiated THP-1 cells. Substitution of the arylsulfonyl moiety with a nitro group increased antagonistic potency relative to methyl substitution, such that compound 21 was slightly more potent than KN-62. Substitution with D-tyrosine in 36 and sterically bulky tyrosyl 2,6-dimethyl groups [corrected] in 9 enhanced antagonistic potency.     

Transient receptor potential channel polymorphisms are associated with the somatosensory function in neuropathic pain patients

Transient receptor potential channels are important mediators of thermal and mechanical stimuli and play an important role in neuropathic pain. The contribution of hereditary variants in the genes of transient receptor potential channels to neuropathic pain is unknown. We investigated the frequency of transient receptor potential ankyrin 1, transient receptor potential melastin 8 and transient receptor potential vanilloid 1 single nucleotide polymorphisms and their impact on somatosensory abnormalities in neuropathic pain patients. Within the German Research Network on Neuropathic Pain (Deutscher Forscbungsverbund Neuropathischer Schmerz) 371 neuropathic pain patients were phenotypically characterized using standardized quantitative sensory testing. Pyrosequencing was employed to determine a total of eleven single nucleotide polymorphisms in transient receptor potential channel genes of the neuropathic pain patients and a cohort of 253 German healthy volunteers. Associations of quantitative sensory testing parameters and single nucleotide polymorphisms between and within groups and subgroups, based on sensory phenotypes, were analyzed. Single nucleotide polymorphisms frequencies did not differ between both the cohorts. However, in neuropathic pain patients transient receptor potential ankyrin 1 710G>A (rs920829, E179K) was associated with the presence of paradoxical heat sensation (p = 0.03), and transient receptor potential vanilloid 1 1911A>G (rs8065080, I585V) with cold hypoalgesia (p = 0.0035). Two main subgroups characterized by preserved (1) and impaired (2) sensory function were identified. In subgroup 1 transient receptor potential vanilloid 1 1911A>G led to significantly less heat hyperalgesia, pinprick hyperalgesia and mechanical hypaesthesia (p = 0.006, p = 0.005 and p<0.001) and transient receptor potential vanilloid 1 1103C>G (rs222747, M315I) to cold hypaesthesia (p = 0.002), but there was absence of associations in subgroup 2. In this study we found no evidence that genetic variants of transient receptor potential channels are involved in the expression of neuropathic pain, but transient receptor potential channel polymorphisms contributed significantly to the somatosensory abnormalities of neuropathic pain patients.