RESUMEN
The time courses of levels of multiple plasma and cerebrospinal fluid (CSF) cytokines in patients with Alzheimer disease (AD) and in age-matched control subjects were compared. Interleukin (IL)-1ß, IL-2, IL-6, IL-8, IL-10, IL-12p70, granulocyte-macrophage colony-stimulating factor, interferon-γ, and tumor necrosis factor alpha levels were measured 7 times over a 24-hour period in plasma and CSF using a lumbar catheter. Baseline plasma and CSF cytokine levels were found to be similar in AD and control subjects. However, the CSF levels of all measured cytokines, except IL-6 and IL-8, diverged over time between AD and control subjects, such that CSF cytokine levels in AD subjects were higher than in controls. This difference was greatest at 24 hours after the insertion of the lumbar catheter. In contrast, no differences in cytokine trajectories were seen in plasma. These data suggest that the neuroinflammatory response to lumbar catheter placement differs between AD and control subjects.
Asunto(s)
Enfermedad de Alzheimer/líquido cefalorraquídeo , Biomarcadores/líquido cefalorraquídeo , Citocinas/líquido cefalorraquídeo , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Three novel series of histamine H(4) receptor (H(4)R) antagonists containing the 2-aminopyrimidine motif are reported. The best of these compounds display good in vitro potency in both functional and binding assays. In addition, representative compounds are able to completely block itch responses when dosed ip in a mouse model of H(4)-agonist induced scratching, thus demonstrating their activities as H(4)R antagonists.
Asunto(s)
Aminopiridinas/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Humanos , Ratones , Receptores Histamínicos , Receptores Histamínicos H4RESUMEN
Guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays were established and utilized as a reliable and high-capacity functional assay for determining antagonist and inverse agonist pharmacological parameters of novel histamine H(3) ligands, at the recombinant human H(3) receptor. [(35)S]GTPgammaS binding assays were performed with membranes prepared from human embryonic kidney 293 cells stably expressing the full-length (445 amino acids) human H(3) receptor isoform, at approximately 1 pmol/mg of protein. Utilizing robotic liquid handling, assay filtration, and scintillation counting in a 96-well format, concentration-response curves were determined for up to 40 compounds per assay. The imidazole-containing H(3) receptor antagonist ciproxifan and the non-imidazole antagonist ABT-239 inhibited (R)-alpha-methylhistamine (RAMH)-stimulated [(35)S]GTPgammaS binding in a competitive manner, and negative logarithm of the dissociation equilibrium constant (pK(b)) values determined for nearly 200 structurally diverse H(3) antagonists were very similar to the respective negative logarithm of the equilibrium inhibition constant values from N-alpha-[(3)H]methylhistamine competition binding assays. H(3) antagonists also concentration-dependently decreased basal [(35)S]GTPgammaS binding, thereby displaying inverse agonism at the constitutively active H(3) receptor. At maximally effective concentrations, non-imidazole H(3) antagonists inhibited basal [(35)S]GTPgammaS binding by approximately 20%. For over 100 of these antagonists, negative logarithm of the 50% effective concentration values for inverse agonism were very similar to the respective pK(b) values. Both H(3) receptor agonist-dependent and -independent (constitutive) [(35)S]GTPgammaS binding were sensitive to changes in assay concentrations of sodium, magnesium, and the guanine nucleotide GDP; however, the potency of ABT-239 for inhibition of RAMH-stimulated [(35)S]GTPgammaS binding was not significantly affected. These robust and reliable [(35)S]GTPgammaS binding assays have become one of the important tools in our pharmacological analysis and development of novel histamine H(3) receptor antagonists/inverse agonists.
Asunto(s)
Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Agonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos H3/farmacología , Receptores Histamínicos H3/efectos de los fármacos , Radioisótopos de Azufre , Benzofuranos/farmacología , Línea Celular , Agonismo Inverso de Drogas , Humanos , Ligandos , Metilhistaminas/farmacología , Pirrolidinas/farmacologíaRESUMEN
In this article, we pharmacologically characterized two naturally occurring human histamine H3 receptor (hH3R) isoforms, hH3R(445) and hH3R(365). These abundantly expressed splice variants differ by a deletion of 80 amino acids in the intracellular loop 3. In this report, we show that the hH3R(365) is differentially expressed compared with the hH3R(445) and has a higher affinity and potency for H3R agonists and conversely a lower potency and affinity for H3R inverse agonists. Furthermore, we show a higher constitutive signaling of the hH3R(365) compared with the hH3R(445) in both guanosine-5'-O-(3-[35S]thio) triphosphate binding and cAMP assays, likely explaining the observed differences in hH3R pharmacology of the two isoforms. Because H3R ligands are beneficial in animal models of obesity, epilepsy, and cognitive diseases such as Alzheimer's disease and attention deficit hyperactivity disorder and currently entered clinical trails, these differences in H3R pharmacology of these two isoforms are of great importance for a detailed understanding of the action of H3R ligands.
Asunto(s)
Empalme Alternativo , Aminoácidos , Receptores Histamínicos H3 , Eliminación de Secuencia , Secuencia de Aminoácidos , Aminoácidos/genética , Animales , Unión Competitiva , Encéfalo/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Clonación Molecular , AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Ligandos , Datos de Secuencia Molecular , Unión Proteica , Isoformas de Proteínas , Ensayo de Unión Radioligante , Ratas , Receptores Histamínicos H3/química , Receptores Histamínicos H3/genética , Receptores Histamínicos H3/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Three novel heterocyclic benzofurans A-688057 (1), A-687136 (2), and A-698418 (3) were profiled for their in vitro and in vivo properties as a new series of histamine H(3) receptor antagonists. The compounds were all found to have nanomolar potency in vitro at histamine H(3) receptors, and when profiled in vivo for CNS activity, all were found active in an animal behavioral model of attention. The compound with the most benign profile versus CNS side effects was selected for greater scrutiny of its in vitro properties and overall drug-likeness. This compound, A-688057, in addition to its potent and robust efficacy in two rodent behavioral models at blood levels ranging 0.2-19 nM, possessed other favorable features, including high selectivity for H(3) receptors (H(3), K(i)=1.5 nM) versus off-target receptors and channels (including the hERG K(+) channel, K(i)>9000 nM), low molecular weight (295), high solubility, moderate lipophilicity (logD(pH7.4)=2.05), and good CNS penetration (blood/brain 3.4x). In vitro toxicological tests indicated low potential for phospholipidosis, genotoxicity, and CYP(450) inhibition. Even though pharmacokinetic testing uncovered only moderate to poor oral bioavailability in rat (26%), dog (30%), and monkey (8%), and only moderate blood half-lives after i.v. administration (t(1/2) in rat of 2.9h, 1.7h in dog, 1.8h in monkey), suggesting poor human pharmacokinetics, the data overall indicated that A-688057 has an excellent profile for use as a pharmacological tool compound.
Asunto(s)
Conducta Animal/efectos de los fármacos , Antagonistas de los Receptores Histamínicos/farmacología , Receptores Histamínicos H3/fisiología , Animales , Conducta Animal/fisiología , Benzofuranos/química , Benzofuranos/farmacología , Perros , Haplorrinos , Antagonistas de los Receptores Histamínicos/sangre , Humanos , Ratas , Receptores Histamínicos H3/efectos de los fármacosRESUMEN
1. A-349821 is a selective histamine H3 receptor antagonist/inverse agonist. Herein, binding of the novel non-imidazole H3 receptor radioligand [3H]A-349821 to membranes expressing native or recombinant H3 receptors from rat or human sources was characterized and compared with the binding of the agonist [3H]N--methylhistamine ([3H]NMH). 2. [3H]A-349821 bound with high affinity and specificity to an apparent single class of saturable sites and recognized human H3 receptors with 10-fold higher affinity compared to rat H3 receptors. [3H]A-349821 detected larger populations of receptors compared to [3H]NMH. 3. Displacement of [3H]A-349821 binding by H3 receptor antagonists/inverse agonists was monophasic, suggesting recognition of a single binding site, while that of H3 receptor agonists was biphasic, suggesting recognition of both high- and low-affinity H3 receptor sites. 4. pKi values of high-affinity binding sites for H3 receptor competitors utilizing [3H]A-349821 were highly correlated with pKi values obtained with [3H]NalphaMH, consistent with labelling of H3 receptors by [3H]A-349821. 5. Unlike assays utilizing [3H]NMH, addition of GDP had no effect on saturation parameters measured with [3H]A-349821, while displacement of [3H]A-349821 binding by the H3 receptor agonist histamine was sensitive to GDP. 6. In conclusion, [3H]A-349821 labels interconvertible high- and low-affinity states of the H3 receptor, and displays improved selectivity over imidazole-containing H3 receptor antagonist radioligands. [3H]A-349821 competition studies showed significant differences in the proportions and potencies of high- and low-affinity sites across species, providing new information about the fundamental pharmacological nature of H3 receptors.
Asunto(s)
Compuestos de Bifenilo/farmacocinética , Antagonistas de los Receptores Histamínicos/farmacología , Ensayo de Unión Radioligante/métodos , Receptores Histamínicos H3/química , Receptores Histamínicos H3/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Células Cultivadas , Guanosina Difosfato/farmacología , Histamina/farmacología , Agonistas de los Receptores Histamínicos/farmacología , Humanos , Imidazoles/farmacología , Metilhistaminas/farmacología , Modelos Biológicos , Unión Proteica/efectos de los fármacos , Ratas , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
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.
Asunto(s)
Bioensayo/métodos , Calcio/análisis , Capsaicina/análogos & derivados , Receptores de Droga/efectos de los fármacos , Receptores de Droga/metabolismo , Secuencia de Aminoácidos , Animales , Bioensayo/instrumentación , Calcio/metabolismo , Capsaicina/farmacología , Células Cultivadas , Diterpenos/metabolismo , Diterpenos/farmacología , Fluorescencia , Humanos , Cinética , Ligandos , Datos de Secuencia Molecular , Ratas , Receptores de Droga/genética , Rojo de Rutenio/farmacología , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Histamine H3 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 H3 receptor ligand, displaying high affinity for recombinant rat and human H3 receptors, with pKi values of 9.4 and 8.8, respectively, and high selectivity for the H3 receptor versus H1, H2, and H4 histamine receptors. A-349821 is a potent H3 receptor antagonist in a variety of models using recombinant human and rat receptors, reversing agonist induced changes in cyclic AMP formation (pKb= 8.2 and pKb= 8.1, respectively), [35S]-GTPgammaS binding (pKb= 9.3 and pKb= 8.6, respectively) and calcium levels (human pKb= 8.3). In native systems, A-349821 competitively reversed agonist induced inhibition of electric field stimulated guinea-pig ileum (pA2= 9.5) and histamine-mediated inhibition of [3H]-histamine release from rat brain cortical synaptosomes (pKb= 9.2). Additionally, A-349821 inhibited constitutive GTPgammaS binding at both rat and human H3 receptors with respective pEC50 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)-alpha-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 1-10mg/kg, with the 1mg/kg dose showing comparable efficacy to a fully efficacious dose of ciproxifan (3mg/kg). These doses of A-349821 were without effect on spontaneous locomotor activity. Thus, A-349821 is a novel, selective non-imidazole H3 antagonist/inverse agonist with balanced high potency across species and favorable cognition enhancing effects in rats.
Asunto(s)
Compuestos de Bifenilo/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Histamina/metabolismo , Receptores Histamínicos H3/metabolismo , Animales , Reacción de Prevención/efectos de los fármacos , Perros , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cobayas , Humanos , Masculino , Actividad Motora/efectos de los fármacos , Neurotransmisores/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Receptores Histamínicos H3/genética , Proteínas Recombinantes/antagonistas & inhibidores , Radioisótopos de AzufreRESUMEN
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.
Asunto(s)
Fármacos Antiobesidad/farmacología , Compuestos de Bifenilo/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Nitrilos/farmacología , Pirrolidinas/farmacología , Receptores Histamínicos H3/efectos de los fármacos , Tejido Adiposo/efectos de los fármacos , Animales , Depresores del Apetito/farmacología , Conducta Animal/efectos de los fármacos , Composición Corporal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Calcio/metabolismo , Clonación Molecular , Diagnóstico por Imagen , Dieta , Grasas de la Dieta/farmacología , Fenfluramina/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Ensayo de Unión Radioligante , Ratas , Pérdida de Peso/efectos de los fármacosRESUMEN
Cerebrospinal fluid (CSF) is commonly used for assessing biomarkers of drug efficacy or disease progression in the central nervous system. Studies of CSF from pre-clinical species can characterize biomarkers for use in clinical trials. However, obtaining CSF from pre-clinical species, particularly rodents, can be challenging due to small body sizes, and consequently, low volumes of CSF. Surgical cannulation of rats is commonly used to allow for CSF withdrawal from the cisterna magna. However, cannulae do not remain patent over multiple days, making chronic studies on the same rats difficult. Moreover, CSF biomarkers may be affected by cannulation. Thus cannulation may contribute confounding factors to the understanding of CSF biomarkers. To determine the potential impact on biomarkers, CSF was analyzed from cannulated rats, surgically implanted with catheters as well as from non-cannulated rats. Brain protein biomarkers (αII-spectrin SBDP150 and total tau) and albumin, were measured in the CSF using ELISA assays. Overall, cannulated rat CSF had elevated levels of the biomarkers examined compared to non-cannulated rat CSF. Additionally, the variation in biomarker levels observed among CSF from cannulated rats was greater than that observed for non-cannulated rat CSF. These results demonstrate that in some cases, biomarker assessment using CSF from cannulated rats may differ from that of non-cannulated animals and may contribute confounding factors to biomarker measurements and assay development for clinical use.
Asunto(s)
Biomarcadores/líquido cefalorraquídeo , Catéteres de Permanencia , Cisterna Magna/metabolismo , Albúminas/líquido cefalorraquídeo , Animales , Barrera Hematoencefálica , Cisterna Magna/cirugía , Ensayo de Inmunoadsorción Enzimática , Masculino , Ratas , Ratas Sprague-Dawley , Espectrina/líquido cefalorraquídeo , Proteínas tau/líquido cefalorraquídeoRESUMEN
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.
Asunto(s)
Analgésicos/farmacología , Modelos Animales de Enfermedad , Inflamación/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Analgésicos/uso terapéutico , Animales , Masculino , Ratones , Ratones Endogámicos BALB C , Ensayo de Unión Radioligante , Ratas , Receptores Histamínicos , Receptores Histamínicos H4RESUMEN
A new histamine H3 receptor (H3R) antagonist chemotype 1 was designed by combining key pharmacophoric elements from two different precursor structural series and then simplifying and optimizing the resulting combined structural features. First, analogues were made based on a previously identified conessine-based H3R antagonist series. While the first analogues 11 and 15 showed no antagonistic activity to H3R, the mere addition of a key moiety found in the reference compound 7 (ABT-239) elevated the series to high potency at H3R. The hybrid structure (16b) was judged too synthetically demanding to enable an extensive SAR study, thus forcing a strategy to simplify the chemical structure. The resulting (3aR,6aR)-5-alkyl-1-aryl-octahydropyrrolo[3,4-b]pyrrole series proved to be highly potent, as exemplified by 17a having a human H3 K(i) of 0.54 nM, rat H3 K(i) of 4.57 nM, and excellent pharmacokinetics (PK) profile in rats (oral bioavailability of 39% and t(1/2) of 2.4 h).
Asunto(s)
Antagonistas de los Receptores Histamínicos H3/síntesis química , Pirroles/síntesis química , Animales , Diseño de Fármacos , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/metabolismo , Antagonistas de los Receptores Histamínicos H3/farmacocinética , Antagonistas de los Receptores Histamínicos H3/farmacología , Humanos , Pirroles/farmacocinética , Pirroles/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
We have recently identified three splice isoforms of the histamine H(3) receptor in multiple brain regions of cynomolgus monkey (Macaca fascicularis). Two of the novel isoforms displayed a deletion in the third intracellular loop (H(3)(413) and H(3)(410)), the third isoform H(3)(335) displayed a deletion in the i3 intracellular loop and a complete deletion of the putative fifth transmembrane domain TM5. We have confirmed by RT-PCR the expression of full-length H(3)(445) mRNA as well as H(3)(413), H(3)(410), and H(3)(335) splice isoform mRNA in multiple monkey brain regions including the frontal, parietal and occipital cortex, parahippocampal gyrus, hippocampus, amygdala, caudate nucleus, putamen, thalamus, hypothalamus, and cerebellum. The full-length isoform H(3)(445) was predominant in all of the regions tested, followed by H(3)(335), with the H(3)(413) and H(3)(410) being of low abundance. When expressed in C6 cells, H(3)(445), H(3)(413), and H(3)(410) exhibit high affinity binding to the agonist ligand [(3)H]-(N)-alpha-methylhistamine with respective pK(D) values of 9.7, 9.7, and 9.6. As expected, the H(3)(335) isoform did not display any saturable binding with [(3)H]-(N)-alpha-methylhistamine. The histamine H(3) receptor agonists histamine, (R)-alpha-methylhistamine, imetit and proxyfan were able to activate calcium mobilization responses through H(3)(445), H(3)(413) and H(3)(410) receptors when they were co-expressed with the chimeric G alpha(qi5)-protein in HEK293 cells, while no response was elicited in cells expressing the H(3)(335) isoform. The existence of multiple H(3) receptor splice isoforms across species raises the possibility that isoform specific properties including ligand affinity, signal transduction coupling, and brain localization may differentially contribute to observed in vivo effects of histamine H(3) receptor antagonists.
Asunto(s)
Expresión Génica , ARN Mensajero/metabolismo , Receptores Histamínicos H3/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Clonación Molecular , Agonistas de los Receptores Histamínicos/farmacología , Humanos , Ligandos , Macaca fascicularis , Masculino , Unión Proteica , Isoformas de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Especificidad de la EspecieRESUMEN
The naturally occurring alkaloid, conessine (6), was discovered to bind to histamine H3 receptors in a radioligand-based high-throughput screen. Conessine displayed high affinity at both rat and human H3 receptors (pKi = 7.61 and 8.27) and generally high selectivity against other sites, including histamine receptors H1, H2, and H4. Conessine was found to efficiently penetrate the CNS and reach very high brain concentrations. Although the very slow CNS clearance and strong binding to adrenergic receptors discouraged focus on conessine itself for further development, its potency and novel steroid-based skeleton motivated further chemical investigation. Modification based on introducing diversity at the 3-nitrogen position generated a new series of H3 antagonists with higher in vitro potency, improved target selectivity, and more favorable drug-like properties. One optimized analogue (13c) was examined in detail and was found to be efficacious in animal behavioral model of cognition.
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Alcaloides/farmacocinética , Antagonistas de los Receptores Histamínicos/farmacocinética , Receptores Histamínicos H3/efectos de los fármacos , Alcaloides/química , Alcaloides/farmacología , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Química Encefálica , Cognición/efectos de los fármacos , Antagonistas de los Receptores Histamínicos/química , Antagonistas de los Receptores Histamínicos/farmacología , Humanos , Ensayo de Unión Radioligante , RatasRESUMEN
A new structural class of histamine H 4 receptor antagonists (6-14) was designed based on rotationally restricted 2,4-diaminopyrimidines. Series compounds showed potent and selective in vitro H 4 antagonism across multiple species, good CNS penetration, improved PK properties compared to reference H 4 antagonists, functional H 4 antagonism in cellular and in vivo pharmacological assays, and in vivo anti-inflammatory and antinociceptive efficacy. One compound, 10 (A-943931), combined the best features of the series in a single molecule and is an excellent tool compound to probe H 4 pharmacology. It is a potent H 4 antagonist in functional assays across species (FLIPR Ca (2+) flux, K b < 5.7 nM), has high (>190x) selectivity for H 4, and combines good PK in rats and mice (t 1/2 of 2.6 and 1.6 h, oral bioavailability of 37% and 90%) with anti-inflammatory activity (ED 50 = 37 micromol/kg, mouse) and efficacy in pain models (thermal hyperalgesia, ED 50 = 72 micromol/kg, rat).
Asunto(s)
Aminas/química , Antiinflamatorios/síntesis química , Antagonistas de los Receptores Histamínicos/síntesis química , Antagonistas de los Receptores Histamínicos/uso terapéutico , Dolor/tratamiento farmacológico , Pirimidinas/síntesis química , Receptores Histamínicos/metabolismo , Animales , Antiinflamatorios/química , Antiinflamatorios/clasificación , Antiinflamatorios/uso terapéutico , Modelos Animales de Enfermedad , Antagonistas de los Receptores Histamínicos/química , Antagonistas de los Receptores Histamínicos/clasificación , Ligandos , Ratones , Estructura Molecular , Pirimidinas/química , Pirimidinas/clasificación , Pirimidinas/uso terapéutico , RatasRESUMEN
A series of 2-aminopyrimidines was synthesized as ligands of the histamine H4 receptor (H4R). Working in part from a pyrimidine hit that was identified in an HTS campaign, SAR studies were carried out to optimize the potency, which led to compound 3, 4- tert-butyl-6-(4-methylpiperazin-1-yl)pyrimidin-2-ylamine. We further studied this compound by systematically modifying the core pyrimidine moiety, the methylpiperazine at position 4, the NH2 at position 2, and positions 5 and 6 of the pyrimidine ring. The pyrimidine 6 position benefited the most from this optimization, especially in analogs in which the 6- tert-butyl was replaced with aromatic and secondary amine moieties. The highlight of the optimization campaign was compound 4, 4-[2-amino-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl]benzonitrile, which was potent in vitro and was active as an anti-inflammatory agent in an animal model and had antinociceptive activity in a pain model, which supports the potential of H 4R antagonists in pain.
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Antagonistas de los Receptores Histamínicos/síntesis química , Antagonistas de los Receptores Histamínicos/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacología , Receptores Histamínicos/metabolismo , Animales , Biomarcadores , Antagonistas de los Receptores Histamínicos/química , Humanos , Hiperplasia/inducido químicamente , Hiperplasia/prevención & control , Ligandos , Locomoción/efectos de los fármacos , Ratones , Estructura Molecular , Pirimidinas/química , Ratas , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
cis-4-(Piperazin-1-yl)-5,6,7a,8,9,10,11,11a-octahydrobenzofuro[2,3-h]quinazolin-2-amine, 4 (A-987306) is a new histamine H(4) antagonist. The compound is potent in H(4) receptor binding assays (rat H(4), K(i) = 3.4 nM, human H(4) K(i) = 5.8 nM) and demonstrated potent functional antagonism in vitro at human, rat, and mouse H(4) receptors in cell-based FLIPR assays. Compound 4 also demonstrated H(4) antagonism in vivo in mice, blocking H(4)-agonist induced scratch responses, and showed anti-inflammatory activity in mice in a peritonitis model. Most interesting was the high potency and efficacy of this compound in blocking pain responses, where it showed an ED(50) of 42 mumol/kg (ip) in a rat post-carrageenan thermal hyperalgesia model of inflammatory pain.
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Antiinflamatorios no Esteroideos/farmacología , Benzofuranos/farmacología , Hiperalgesia/tratamiento farmacológico , Dolor/prevención & control , Quinazolinas/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Benzofuranos/síntesis química , Benzofuranos/química , Carragenina , Modelos Animales de Enfermedad , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Hiperalgesia/inducido químicamente , Ligandos , Ratones , Estructura Molecular , Dolor/fisiopatología , Peritonitis/tratamiento farmacológico , Quinazolinas/síntesis química , Quinazolinas/química , Ratas , Receptores Histamínicos , Receptores Histamínicos H4 , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Previously reported pharmacological studies using the imidazole-containing histamine H3 receptor ligands GT-2331 (Cipralisant) and proxyfan resulted in a range of classifications (antagonist, agonist, and protean) for these compounds. We examined the role that the signaling system, with particular emphasis on the type of G protein, had on the pharmacology observed for H3 ligands. Ligands were assessed using assays measuring neurotransmitter release, cAMP, and guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding. Whereas clobenpropit and ciproxifan were consistently antagonists, GT-2331, proxyfan, and imetit exhibited differential activity. Although GT-2331 and proxyfan exhibited little agonist activity in neurotransmitter release assays, both demonstrated full agonism relative to (R)-alpha-methylhistamine in cAMP assays. In [35S]GTPgammaS binding assays, GT-2331 and proxyfan demonstrated partial agonism. Imetit showed full agonism in most assays, but it was slightly less efficacious in a neurotransmitter release assay and in [35S]GTPgammaS binding at the human H3 receptor. To further examine these ligands, we coexpressed G alpha16 or chimeric G alpha q/i5 in human embryonic kidney cells expressing the human H3 receptor and assayed intracellular calcium and cAMP levels. GT-2331, proxyfan, and imetit demonstrated full agonism in all assays of cAMP activity. However, in cells expressing G alpha16, they exhibited minimal agonism in calcium mobilization assays, whereas imetit showed partial agonism. When G alpha q/i5 was used, the activity of both GT-2331 and proxyfan increased, whereas imetit became a full agonist. These results demonstrate that GT-2331 and proxyfan's differential pharmacology at the H3 receptor depends on the type of G protein used and provide indirect evidence for differential ligand-bound active states that mediate signaling by the H3 receptor.
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Proteínas de Unión al GTP/fisiología , Receptores Histamínicos H3/efectos de los fármacos , Animales , AMP Cíclico/metabolismo , Estimulación Eléctrica , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cobayas , Agonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Liberación de Histamina/efectos de los fármacos , Íleon/efectos de los fármacos , Íleon/metabolismo , Imidazoles/farmacología , Ligandos , Masculino , Membranas/metabolismo , Neurotransmisores/metabolismo , Conformación Proteica , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
Histamine H3 receptor antagonists are being developed to treat a variety of neurological and cognitive disorders that may be ameliorated by enhancement of central neurotransmitter release. Here, we present the in vitro pharmacological and in vivo pharmacokinetic profiles for the nonimidazole, benzofuran ligand ABT-239 [4-(2-{2-[(2R)-2-methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzonitrile] and compare it with several previously described imidazole and nonimidazole H3 receptor antagonists. ABT-239 binds to recombinant human and rat H3 receptors with high affinity, with pK(i) values of 9.4 and 8.9, respectively, and is over 1000-fold selective versus human H1, H2, and H4 histamine receptors. ABT-239 is a potent H3 receptor antagonist at recombinant human and rat receptors, reversing agonist-induced changes in cAMP formation (pK(b) = 7.9 and 7.6, respectively), guanosine 5'-O-(3-[35S]thio) triphosphate ([35S]GTPgammaS) binding (pK(b) = 9.0 and 8.3, respectively), and calcium mobilization (human pK(b) = 7.9). ABT-239 also competitively reversed histamine-mediated inhibition of [3H]histamine release from rat brain cortical synaptosomes (pK(b) = 7.7) and agonist-induced inhibition of contractile responses in electric field stimulated guinea pig ileal segments (pA2 = 8.7). Additionally, ABT-239 is a potent inverse agonist, inhibiting constitutive [35S]GTPgammaS binding at both rat and human H3 receptors with respective pEC50 values of 8.9 and 8.2. ABT-239 demonstrates good pharmacokinetic characteristics in rat, dog, and monkey with t1/2 values ranging from 4 to 29 h, corresponding with clearance values and metabolic turnover in liver microsomes from these species, and good oral bioavailability ranging from 52 to 89%. Thus, ABT-239 is a selective, nonimidazole H3 receptor antagonist/inverse agonist with similar high potency in both human and rat and favorable drug-like properties.
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Benzofuranos/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Pirrolidinas/farmacología , Receptores Histamínicos H3/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , Benzofuranos/farmacocinética , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Clonación Molecular , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cobayas , Antagonistas de los Receptores Histamínicos/farmacocinética , Liberación de Histamina/efectos de los fármacos , Íleon/efectos de los fármacos , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Neurotransmisores/metabolismo , Pirrolidinas/farmacocinética , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-DawleyRESUMEN
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.