The cloned vanilloid receptor 1 (VR1) is a ligand-gated calcium channel that is believed to be the capsaicin-activated vanilloid receptor found in native tissues, based on similarities regarding ...molecular mass, tissue distribution, and electrophysiological properties. Using a Fluorescent Imaging Plate Reader (FLIPR), along with Fluo-3 to signal intracellular calcium levels (Ca(++)(i)), rat VR1 (rVR1) and a human orthologue (hVR1) were pharmacologically characterized with various VR1 ligands. HEK-293 cells, stably expressing rVR1 or hVR1, exhibited dose-dependent increases in Ca(++)(i) when challenged with capsaicin (EC(50)s congruent with 10 nM). Responses to capsaicin were blocked by the VR1 antagonist capsazepine and were dependent on VR1 expression. Potencies for 10 structurally diverse VR1 agonists revealed rVR1 potencies highly correlated to that of hVR1 (R(2) = 0.973). However, a subset of agonists (tinyatoxin, gingerol, and zingerone) was approximately 10-fold more potent for rVR1 compared to hVR1. Schild analysis for blockade of capsaicin-induced responses by capsazepine was consistent with competitive antagonism, whereas ruthenium red displayed noncompetitive antagonism. Compared to rVR1, hVR1 was more sensitive to blockade by both antagonists. For both rVR1 and hVR1, time-response waveforms elicited by resiniferatoxin increased more gradually compared to other agonists. Tinyatoxin also displayed slow responses with hVR1 but showed rapid responses with rVR1. Thus, FLIPR technology can be used to readily reveal differences between rVR1 and hVR1 pharmacology with respect to potencies, efficacies, and kinetics for several VR1 ligands.
The histamine H(4) receptor (H(4)R) is expressed primarily on cells involved in inflammation and immune responses. To determine the potential role of H(4)R in pain transmission, the effects of ...JNJ7777120, a potent and selective H(4) antagonist, were characterized in preclinical pain models. Administration of JNJ7777120 fully blocked neutrophil influx observed in a mouse zymosan-induced peritonitis model (ED(50)=17 mg/kg s.c., 95% CI=8.5-26) in a mast cell-dependent manner. JNJ7777120 potently reversed thermal hyperalgesia observed following intraplantar carrageenan injection of acute inflammatory pain (ED(50)=22 mg/kg i.p., 95% CI=10-35) in rats and significantly decreased the myeloperoxide activity in the carrageenan-injected paw. In contrast, no effects were produced by either H(1)R antagonist diphenhydramine, H(2)R antagonists ranitidine, or H(3)R antagonist ABT-239. JNJ7777120 also exhibited robust anti-nociceptive activity in persistent inflammatory (CFA) pain with an ED(50) of 29 mg/kg i.p. (95% CI=19-40) and effectively reversed monoiodoacetate (MIA)-induced osteoarthritic joint pain. This compound also produced dose-dependent anti-allodynic effects in the spinal nerve ligation (ED(50)=60 mg/kg) and sciatic nerve constriction injury (ED(50)=88 mg/kg) models of chronic neuropathic pain, as well as in a skin-incision model of acute post-operative pain (ED(50)=68 mg/kg). In addition, the analgesic effects of JNJ7777120 were maintained following repeated administration and were evident at the doses that did not cause neurologic deficits in rotarod test. Our results demonstrate that selective blockade of H(4) receptors in vivo produces significant anti-nociception in animal models of inflammatory and neuropathic pain.
Background and purpose:
The histamine H
4
receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In ...this report, we describe the
in vitro
and
in vivo
anti‐inflammatory properties of a potent histamine H
4
receptor antagonist, A‐940894 (4‐piperazin‐1‐yl‐6,7‐dihydro‐5H‐benzo6,7cyclohepta1,2‐dpyrimidin‐2‐ylamine).
Experimental approach:
We have analysed the pharmacological profile of A‐940894 at mouse native, rat recombinant and human recombinant and native, histamine H
4
receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan‐induced peritonitis.
Key results:
A‐940894 potently binds to both human and rat histamine H
4
receptors and exhibits considerably lower affinity for the human histamine H
1
, H
2
or H
3
receptors. It potently blocked histamine‐evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine‐induced shape change of mouse bone marrow‐derived mast cells and chemotaxis of human eosinophils
in vitro
. In a mouse mast cell‐dependent model of zymosan‐induced peritonitis, A‐940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D
2
levels. Finally, A‐940894 has good pharmacokinetic properties, including half‐life and oral bioavailability in rats and mice.
Conclusions and Implications:
These data suggest that A‐940894 is a potent and selective histamine H
4
receptor antagonist with pharmacokinetic properties suitable for long‐term
in vivo
testing and could serve as a useful tool for the further characterization of histamine H
4
receptor pharmacology.
Histamine affects homeostatic mechanisms, including food and water consumption, by acting on central nervous system (CNS) receptors. Presynaptic histamine H
3 receptors regulate release of histamine ...and other neurotransmitters, and histamine H
3 receptor antagonists enhance neurotransmitter release. A-331440 {4′-3-(3(
R)-(dimethylamino)-pyrrolidin-1-yl)-propoxy-biphenyl-4-carbonitrile} is a histamine H
3 receptor antagonist which binds potently and selectively to both human and rat histamine H
3 receptors (
K
i≤25 nM). Mice were stabilized on a high-fat diet (45 kcal % lard) prior to 28-day oral b.i.d. dosing for measurement of obesity-related parameters. A-331440 administered at 0.5 mg/kg had no significant effect on weight, whereas 5 mg/kg decreased weight comparably to dexfenfluramine (10 mg/kg). A-331440 administered at 15 mg/kg reduced weight to a level comparable to mice on the low-fat diet. The two higher doses reduced body fat and the highest dose also normalized an insulin tolerance test. These data show that the histamine H
3 receptor antagonist, A-331440, has potential as an antiobesity agent.
Histamine H
3 receptors regulate the release of a variety of central neurotransmitters involved in cognitive processes. A-349821 ((4′-(3-((
...R,R)2,5-dimethyl-pyrrolidin-1-yl)-propoxy)-biphenyl-4-yl)-morpholin-4-yl-methanone) is a novel, non-imidazole H
3 receptor ligand, displaying high affinity for recombinant rat and human H
3 receptors, with p
K
i values of 9.4 and 8.8, respectively, and high selectivity for the H
3 receptor versus H
1, H
2, and H
4 histamine receptors. A-349821 is a potent H
3 receptor antagonist in a variety of models using recombinant human and rat receptors, reversing agonist induced changes in cyclic AMP formation (p
K
b = 8.2 and p
K
b = 8.1, respectively),
35
S
-GTPγS binding (p
K
b = 9.3 and p
K
b = 8.6, respectively) and calcium levels (human p
K
b = 8.3). In native systems, A-349821 competitively reversed agonist induced inhibition of electric field stimulated guinea-pig ileum (p
A
2 = 9.5) and histamine-mediated inhibition of
3
H
-histamine release from rat brain cortical synaptosomes (p
K
b = 9.2). Additionally, A-349821 inhibited constitutive GTPγS binding at both rat and human H
3 receptors with respective pEC
50 values of 9.1 and 8.6, demonstrating potent inverse agonist properties. In behavioral studies, A-349821 (0.4
mg/kg—4
mg/kg) potently blocked (
R)-α-methylhistamine-induced dipsogenia in mice. The compound also enhanced cognitive activity in a five-trial inhibitory avoidance model in spontaneously hypertensive rat (SHR) pups at doses of 1–10
mg/kg, with the 1
mg/kg dose showing comparable efficacy to a fully efficacious dose of ciproxifan (3
mg/kg). These doses of A-349821 were without effect on spontaneous locomotor activity. Thus, A-349821 is a novel, selective non-imidazole H
3 antagonist/inverse agonist with balanced high potency across species and favorable cognition enhancing effects in rats.
Background and purpose: The histamine H4 receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In ...this report, we describe the in vitro and in vivo anti‐inflammatory properties of a potent histamine H4 receptor antagonist, A‐940894 (4‐piperazin‐1‐yl‐6,7‐dihydro‐5H‐benzo6,7cyclohepta1,2‐dpyrimidin‐2‐ylamine).
Experimental approach: We have analysed the pharmacological profile of A‐940894 at mouse native, rat recombinant and human recombinant and native, histamine H4 receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan‐induced peritonitis.
Key results: A‐940894 potently binds to both human and rat histamine H4 receptors and exhibits considerably lower affinity for the human histamine H1, H2 or H3 receptors. It potently blocked histamine‐evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine‐induced shape change of mouse bone marrow‐derived mast cells and chemotaxis of human eosinophils in vitro. In a mouse mast cell‐dependent model of zymosan‐induced peritonitis, A‐940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D2 levels. Finally, A‐940894 has good pharmacokinetic properties, including half‐life and oral bioavailability in rats and mice.
Conclusions and Implications: These data suggest that A‐940894 is a potent and selective histamine H4 receptor antagonist with pharmacokinetic properties suitable for long‐term in vivo testing and could serve as a useful tool for the further characterization of histamine H4 receptor pharmacology.
Selective radioligands for histamine H
3 receptors have been used to characterize H
3 receptor pharmacology by radioligand binding assays and to determine H
3 receptor distribution by tissue ...autoradiography. Here we report the synthesis and receptor binding characterization of
3HA-317920 (furan-2-carboxylic acid(2-{4-3-(3,5-
3H4-cyclopropanecarbonyl-phenoxy)-propyl-piperazin-1-yl}-1-methyl-2-oxo-ethyl)-amide), a high affinity inverse agonist radioligand for the rat H
3 receptor. The binding of
3HA-317920 to rat cortical and cloned H
3 receptors revealed fast on- and slower off-rate kinetics with calculated
K
d values in agreement with those determined in saturation binding assays (0.2
nM for both receptors). Further, we compared
3HA-317920 with the agonist
3H(
N)-α-methylhistamine (
3HNαMH) as radioligand tools to study receptor pharmacology. Agonists and antagonists displaced
3HNαMH with one-site binding characteristics and Hill slopes approached unity. In contrast, although antagonists exhibited one-site binding,
3HA-317920 displacement by agonists was best fit by two-site binding models, and the potencies of the high affinity, GDP-sensitive sites correlated with the potencies defined in
3HNαMH binding. Unlike
125Iiodoproxyfan,
3HA-317920 exhibits potent and selective binding to rat H
3 receptors with low binding to non-H
3 sites, including cytochrome P450. These findings show that
3HA-317920 is a potent rat H
3 receptor antagonist radioligand and has utility for studying H
3 receptor pharmacology.
Fiduxosin is a new alpha(1)-adrenoceptor antagonist targeted for the treatment of symptomatic benign prostatic hyperplasia. The purpose of this study was to determine and compare the potencies of the ...alpha(1)-adrenoceptor antagonists terazosin, doxazosin, tamsulosin, and fiduxosin, based on relationships between plasma drug concentrations and blockade of phenylephrine (PE)-induced intraurethral (IUP) and mean arterial pressure (MAP) responses after single oral dosing in conscious male beagle dogs. Magnitude of blockade and plasma concentrations were evaluated at selected time points over 24 h. All drugs produced dose-dependent antagonism of PE-induced IUP and MAP responses. When IUP and MAP blockade effects were plotted against drug plasma concentrations, direct relationships were observed that were well described by the sigmoidal maximal effect model. IUP IC(50) values for terazosin, doxazosin, tamsulosin, and fiduxosin were 48.6, 48.7, 0.42, and 261 ng/ml, respectively. MAP IC(50) values were 12.2, 13.8, 1.07, and 1904 ng/ml, respectively. Uroselectivity index values, defined as MAP IC(50)/IUP IC(50), were 0.25, 0.28, 2.6, and 7.3, respectively. These results extend previous observations with terazosin in this model, showing that doxazosin exhibits a uroselectivity index comparable to terazosin, consistent with the lack of alpha(1)-adrenoceptor subtype selectivity or uroselectivity of these drugs. Tamsulosin, an alpha(1a)-/alpha(1d)-subtype selective agent, had an index value approximately 10-fold greater than the nonselective drugs. Based on its pharmacokinetic profile and a relative uroselectivity 29-fold greater than the nonselective drugs, fiduxosin is expected to exhibit greater selectivity for urethral compared with vascular alpha(1)-adrenoceptors in human and should be a novel, long-acting, uroselective alpha(1)-adrenoceptor antagonist.
Fiduxosin is an alpha(1)-adrenoceptor antagonist with higher affinity for alpha(1A)-adrenoceptors and for alpha(1D)-adrenoceptors than for alpha(1B)-adrenoceptors. Our hypothesis is that such a ...compound with higher affinity for subtypes implicated in the control of lower urinary tract function and lower affinity for a subtype implicated in the control of arterial pressure could result in a superior clinical profile for the treatment of lower urinary tract symptoms suggestive of benign prostatic obstruction. The purpose of this study was to evaluate the potency and selectivity of fiduxosin for effects on prostatic intraurethral pressure (IUP) versus mean arterial pressure (MAP) relative to current clinical standards, terazosin and tamsulosin, in conscious dogs. Phenylephrine (PE)-induced increases in IUP and MAP were determined before and at various time points after an oral dose of each antagonist. Hypotensive potency was also determined. All three antagonists caused dose- and time-dependent blockade of the IUP and MAP pressor effects of PE. The IUP ED(50) values of fiduxosin, tamsulosin, and terazosin were 0.24, 0.004, and 0.23 mg/kg p.o., respectively. The corresponding MAP ED(50) values were 1.79, 0.006, and 0.09 mg/kg p.o. The rank order of IUP selectivity (ratio) was fiduxosin (7.5-fold), tamsulosin (1.5-fold), and terazosin (0.4 = 2.5-fold MAP-selective). Tamsulosin and terazosin caused dose-dependent hypotension, whereas no change in arterial pressure was seen after fiduxosin. These data, illustrating a superior selectivity profile of fiduxosin, are consistent with our hypothesis.
Recent evidence suggests that the level of interleukin-6 (IL-6) is elevated in Alzheimer's disease (AD) brains. IL-6 is produced by reactive glial cells and could potentially affect neuronal ...survival. Understanding the biochemical mechanism that regulates the production and release of IL-6 by astrocytic cells may help to identify potential targets for therapeutic intervention in AD. In the present study, glial fibrillary acidic protein-positive human U373MG astrocytoma cells were used as a model of reactive astrocytes. Production of IL-6 in response to drug treatment was monitored with an ELISA assay. Histamine (1-100 microM), substance P (SP; 1-100 nM), and human interleukin-1 beta (IL-1 beta; 1-30 pM) stimulated the release of IL-6 in a time- and concentration-dependent manner, with EC50 values of 4.5 microM, 8 nM, and 4.5 pM, respectively. The respective effects of histamine, SP, and IL-1 beta were effectively blocked by the histamine H1, SP, and IL-1 receptor antagonists, supporting a receptor-mediated event for these agents. Both histamine and SP enhanced the formation of inositol phosphates and increase intracellular calcium levels, suggesting that the phosphatidylinositol bisphosphate/protein kinase C pathway may be involved in the IL-6 release process. Indeed, phorbol 12-myristate 13-acetate, a protein kinase C activator, also evoked IL-6 release from the U373MG cells. On the other hand, IL-1 beta, which produces a much more robust release of IL-6 than histamine or SP, has no effect on inositol phosphate formation or intracellular calcium levels.
PDF
