RESUMEN
Lysophosphatidic acid (LPA) is a bioactive phospholipid that signals through a family of at least six G protein-coupled receptors designated LPA1â6. LPA type 1 receptor (LPA1) exhibits widespread tissue distribution and regulates a variety of physiological and pathological cellular functions. Here, we evaluated the in vitro pharmacology, pharmacokinetic, and pharmacodynamic properties of the LPA1-selective antagonist AM095 (sodium, {4'-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetate) and assessed the effects of AM095 in rodent models of lung and kidney fibrosis and dermal wound healing. In vitro, AM095 was a potent LPA1 receptor antagonist because it inhibited GTPγS binding to Chinese hamster ovary (CHO) cell membranes overexpressing recombinant human or mouse LPA1 with IC50 values of 0.98 and 0.73 µM, respectively, and exhibited no LPA1 agonism. In functional assays, AM095 inhibited LPA-driven chemotaxis of CHO cells overexpressing mouse LPA1 (IC50= 778 nM) and human A2058 melanoma cells (IC50 = 233 nM). In vivo, we demonstrated that AM095: 1) had high oral bioavailability and a moderate half-life and was well tolerated at the doses tested in rats and dogs after oral and intravenous dosing, 2) dose-dependently reduced LPA-stimulated histamine release, 3) attenuated bleomycin-induced increases in collagen, protein, and inflammatory cell infiltration in bronchalveolar lavage fluid, and 4) decreased kidney fibrosis in a mouse unilateral ureteral obstruction model. Despite its antifibrotic activity, AM095 had no effect on normal wound healing after incisional and excisional wounding in rats. These data demonstrate that AM095 is an LPA1 receptor antagonist with good oral exposure and antifibrotic activity in rodent models.
Asunto(s)
Antifibrinolíticos/administración & dosificación , Antifibrinolíticos/farmacocinética , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Administración Oral , Animales , Antifibrinolíticos/química , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Perros , Humanos , Masculino , Ratones , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Receptores del Ácido Lisofosfatídico/metabolismoRESUMEN
BACKGROUND AND PURPOSE: The aim of this study was to assess the potential of an antagonist selective for the lysophosphatidic acid receptor, LPA(1), in treating lung fibrosis We evaluated the in vitro and in vivo pharmacological properties of the high affinity, selective, oral LPA(1)-antagonist (4'-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (AM966). EXPERIMENTAL APPROACH: The potency and selectivity of AM966 for LPA(1) receptors was determined in vitro by calcium flux and cell chemotaxis assays using recombinant and native cell cultures. The in vivo efficacy of AM966 to reduce tissue injury, vascular leakage, inflammation and fibrosis was assessed at several time points in the mouse bleomycin model. KEY RESULTS: AM966 was a potent antagonist of LPA(1) receptors, with selectivity for this receptor over the other LPA receptors. In vitro, AM966 inhibited LPA-stimulated intracellular calcium release (IC(50)= 17 nM) from Chinese hamster ovary cells stably expressing human LPA(1) receptors and inhibited LPA-induced chemotaxis (IC(50)= 181 nM) of human IMR-90 lung fibroblasts expressing LPA(1) receptors. AM966 demonstrated a good pharmacokinetic profile following oral dosing in mice. In the mouse, AM966 reduced lung injury, vascular leakage, inflammation and fibrosis at multiple time points following intratracheal bleomycin instillation. AM966 also decreased lactate dehydrogenase activity and tissue inhibitor of metalloproteinase-1, transforming growth factor beta1, hyaluronan and matrix metalloproteinase-7, in bronchoalveolar lavage fluid. CONCLUSIONS AND IMPLICATIONS: These findings demonstrate that AM966 is a potent, selective, orally bioavailable LPA(1) receptor antagonist that may be beneficial in treating lung injury and fibrosis, as well as other diseases that are characterized by pathological inflammation, oedema and fibrosis.
Asunto(s)
Carbamatos/uso terapéutico , Pulmón/efectos de los fármacos , Fenilacetatos/uso terapéutico , Fibrosis Pulmonar/tratamiento farmacológico , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Administración Oral , Animales , Bleomicina/farmacología , Líquido del Lavado Bronquioalveolar/química , Células CHO , Calcio/metabolismo , Carbamatos/administración & dosificación , Carbamatos/farmacocinética , Carbamatos/farmacología , Línea Celular Tumoral , Quimiotaxis/efectos de los fármacos , Colágeno/metabolismo , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Fenilacetatos/administración & dosificación , Fenilacetatos/farmacocinética , Fenilacetatos/farmacología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Ratas , Receptores del Ácido Lisofosfatídico/genética , TransfecciónRESUMEN
Biochemical, behavioral and electrophysiological evidence suggests interactions between pathways containing neuronal nicotinic acetylcholine receptors (NAChRs) and excitatory amino acid receptors. Recently, protective effects of nicotine against N-methyl-D-aspartate (NMDA)-induced toxicity in primary cortical cultures were reported. To address possible interactions between NAChR and NMDA receptor containing pathways, several NAChR agonists were evaluated for their effects on NMDA-evoked [3H]acetylcholine ([3H]ACh) release from slices of rat striatum. Nicotine, cytisine and epibatidine had no effect on NMDA-evoked release or basal release of [3H]ACh over a wide range of concentrations. Lobeline and dimethylphenylpiperazinium iodide (DMPP), however, decreased basal [3H]ACh release and attenuated NMDA-evoked [3H]ACh release with EC50 values of 35 and 155 microM, respectively. The NAChR antagonists, dihydro-beta-erythroidine (DH beta E) and d-tubocurarine had no effect on NMDA-evoked [3H]ACh release, whereas mecamylamine attenuated the NMDA-evoked [3H]ACh evoked release with an EC50 value of 144 microM. Methyllycaconitine (MLA), a highly selective and potent antagonist of the alpha-bungarotoxin-sensitive alpha 7 NAChR subtype, also had no effect on NMDA-evoked [3H]ACh release at concentrations upto 10 microM. The inhibitory effects of DMPP and lobeline on NMDA-evoked [3H[ACh release were relatively insensitive to mecamylamine, d-tubocurarine, MLA and DH beta E. In addition, DMPP or lobeline-induced attenuation of basal [3H]ACh release was insensitive to blockade by sulpiride, a dopamine (D2) receptor antagonist. In contrast to their effects on NMDA-evoked striatal [3H]ACh release, both DMPP and lobeline increased basal release of striatal [3H]DA and hippocampal [3H]norepinephrine ([3H]NE) and did not attenuate NMDA-evoked release of these two transmitters. Instead, DMPP and lobeline appeared to have an additive effect on both NMDA-evoked hippocampal [3H]NE release and striatal [3H]DA release. These pharmacological results suggest that the inhibitory effects on lobeline and DMPP on striatal [3H]ACh release are independent of their interactions with classical NAChRs or the NMDA receptor complex itself.
Asunto(s)
Acetilcolina/metabolismo , Yoduro de Dimetilfenilpiperazina/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Lobelina/farmacología , N-Metilaspartato/farmacología , Neostriado/efectos de los fármacos , Agonistas Nicotínicos/farmacología , Animales , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Neostriado/metabolismo , Ratas , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/fisiologíaRESUMEN
Nicotine, the prototypical agonist for neuronal nicotinic acetylcholine receptors (NAChR), nonselectively activates NAChR limiting its use in elucidating the function of NAChR subtypes. SIB-1765F is a subtype selective NAChR agonist that displaces [3H]-nicotine binding with an IC50 of 4.6 nM and [3H]-cytisine binding with an IC50 of 12.2 nM which is 2000- to 6000-fold lower than its displacement of [3H]-QNB or [125I]-alpha-bungarotoxin. SIB-1765F did not inhibit human or rat cholinesterases or the uptake of [3H]-DA in synaptosomal preparations. SIB-1765F mimicked (-)-nicotine in stimulating [3H]-DA release from rat striatal and olfactory tubercle slices, with EC50 values of 99.6 and 39.6 microM, respectively. Such stimulation was sensitive to mecamylamine and DH beta E. SIB-1765F also released endogenous DA in the striatum and the nucleus accumbens as measured by in vivo microdialysis. SIB-1765F was less efficacious than (-)-nicotine at stimulating [3H]-NE release from rat hippocampal slices; in contrast, SIB-1765F increased [3H]-NE release from rat thalamic and cortical slices with efficacies approaching those of (-)-nicotine. Similar to (-)-nicotine and (+/-)-epibatidine, subcutaneous administration of SIB-1765F increased the turnover rate of dopamine ex vivo both in the striatum and olfactory tubercles in a mecamylamine-sensitive manner. Because the release of striatal DA and hippocampal NE appears to be regulated by distinct NAChR, differential effects of SIB-1765F on striatal DA and hippocampal NE release supports the NAChR subtype selectivity of SIB-1765F compared to (-)-nicotine. This is further demonstrated by observations showing that SIB-1765F has a higher affinity for h alpha 4 beta 2 NAChR relative to h alpha 4 beta 4 NAChRs in displacing [3H]-epibatidine binding and increasing cytosolic CA+2 concentration in cell lines stably expressing h alpha 4 beta 2 or h alpha 4 beta 4.
Asunto(s)
Canales Iónicos/agonistas , Agonistas Nicotínicos/farmacología , Piridinas/farmacología , Pirrolidinas/farmacología , Animales , Calcio/metabolismo , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Masculino , Microdiálisis , Norepinefrina/metabolismo , Vías Olfatorias/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/metabolismoAsunto(s)
Antiparkinsonianos/síntesis química , Antiparkinsonianos/farmacología , Neuronas/fisiología , Piridinas/síntesis química , Piridinas/farmacología , Pirrolidinas/síntesis química , Pirrolidinas/farmacología , Receptores Nicotínicos/fisiología , Animales , Antiparkinsonianos/química , Línea Celular , Femenino , Humanos , Cinética , Estructura Molecular , Oocitos/fisiología , Técnicas de Placa-Clamp , Piridinas/química , Pirrolidinas/química , Receptores Nicotínicos/efectos de los fármacos , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Estereoisomerismo , XenopusRESUMEN
In the present investigation, anti-nociceptive effects of neuronal nicotinic acetylcholine receptor (NAChR) ligands, (+)- and (-)-nicotine, cytisine, methylcarbamylcholine (MCC), dimethylphenylpiperazinium iodide (DMPP), and (+/-)-epibatidine were evaluated in the rat tail-flick assay both after subcutaneous (s.c.) and intracerebroventricular (i.c.v.) administration. The pharmacology of the tail-flick response to NAChR ligands after s.c. and i.c.v. routes was similar. Epibatidine was the most potent ligand examined with a longer duration of action than any other agonist. (-)-Nicotine was more active than (+)-nicotine indicating stereospecificity. ICV administration studies indicated an apparent partial agonist activity for (+)-nicotine in the tail-flick response. Tail-flick responses to NAChR agonists are independent of opioid and muscarinic pathways and appear to be mediated both by central and peripheral NAChR recognition sites. Central administration of MCC activates both NAChR and muscarinic anti-nociceptive mechanisms. Studies employing the alpha-adrenergic receptor alkylating agent, phenoxybenzamine or the noradrenergic neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), suggested that the NAChR-noradrenergic and NAChR-serotoninergic interactions play an important role in the tail-flick response. Studies employing a selective alpha-bungarotoxin-sensitive NAChR receptor antagonist, methyllycaconitine (MLA), suggested a minimal role for these receptors in the tail-flick response. The biochemical studies also indicated that a sub-population of NAChR receptors are located pre-synaptically on noradrenergic and/or serotoninergic pathways in the hippocampus.
Asunto(s)
Aconitina/análogos & derivados , Neuronas/efectos de los fármacos , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Dimensión del Dolor/métodos , Receptores Nicotínicos/efectos de los fármacos , Aconitina/farmacología , Animales , Cromatografía Líquida de Alta Presión , Electroquímica , Estudios de Evaluación como Asunto , Inyecciones Intraventriculares , Inyecciones Subcutáneas , Ligandos , Masculino , Norepinefrina/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The effect of the neuronal acetylcholine-gated ion channel receptor agonist (+/-)-epibatidine was studied on neurotransmitter release in vitro and motor behavior in vivo. (+/-)-Epibatidine (3-300 nM) caused a concentration- and calcium-dependent release of [3H]-dopamine from striatal slices and [3H]-norepinephrine release from hippocampal and thalamic slices. (+/-)-Epibatidine-induced neurotransmitter release was inhibited in all three regions by mecamylamine (3 microM). In contrast, D-tubocurarine (10-100 microM) inhibited only (+/-)-epibatidine-induced [3H]-norepinephrine release from the hippocampus and the thalamus. Conversely, dihydro beta-erythroidine (3-100 microM) inhibited (+/-)-epibatidine-induced [3H]-dopamine release in the striatum without significantly altering [3H]-norepinephrine release from either the hippocampus or the thalamus. This is consistent with the observation that different nAChRs modulate dopamine release as compared with norepinephrine release. The effect of (+/-)-epibatidine on both [3H]-dopamine and [3H]-norepinephrine release was tetrodotoxin-sensitive, suggesting the involvement of sodium channels. (+/-)-Epibatidine (1-3 micrograms/kg s.c.) produced ipsilateral turning in the unilaterally [6(OH)-DA]-lesioned rat. This effect was mimicked by (-)-nicotine (0.35 mg/kg s.c.). Both (+/-)-epibatidine- and (-)-nicotine-induced turning were significantly inhibited by mecamylamine (3 mg/kg s.c.), indicating that the turning response was mediated by nAChRs. (+/-)-Epibatidine also increased locomotor activity in a dose-dependent manner. (+/-)-Epibatidine-induced hyperactivity was blocked by D1 and D2 receptor antagonists, SCH 23390 and eticlopride, respectively, suggesting that both dopamine receptor subtypes might be required for the locomotor effect of (+/-)-epibatidine. These results demonstrate that (+/-)-epibatidine displays nAChR agonist activity in the rat CNS and that certain effects are mediated via nAChR-stimulated catecholamine release and subsequent activation of corresponding receptors.