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1.
Nat Med ; 30(2): 443-454, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38321220

RESUMO

Compromised vascular endothelial barrier function is a salient feature of diabetic complications such as sight-threatening diabetic macular edema (DME). Current standards of care for DME manage aspects of the disease, but require frequent intravitreal administration and are poorly effective in large subsets of patients. Here we provide evidence that an elevated burden of senescent cells in the retina triggers cardinal features of DME pathology and conduct an initial test of senolytic therapy in patients with DME. In cell culture models, sustained hyperglycemia provoked cellular senescence in subsets of vascular endothelial cells displaying perturbed transendothelial junctions associated with poor barrier function and leading to micro-inflammation. Pharmacological elimination of senescent cells in a mouse model of DME reduces diabetes-induced retinal vascular leakage and preserves retinal function. We then conducted a phase 1 single ascending dose safety study of UBX1325 (foselutoclax), a senolytic small-molecule inhibitor of BCL-xL, in patients with advanced DME for whom anti-vascular endothelial growth factor therapy was no longer considered beneficial. The primary objective of assessment of safety and tolerability of UBX1325 was achieved. Collectively, our data suggest that therapeutic targeting of senescent cells in the diabetic retina with a BCL-xL inhibitor may provide a long-lasting, disease-modifying intervention for DME. This hypothesis will need to be verified in larger clinical trials. ClinicalTrials.gov identifier: NCT04537884 .


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Edema Macular , Animais , Camundongos , Humanos , Edema Macular/tratamento farmacológico , Edema Macular/etiologia , Retinopatia Diabética/tratamento farmacológico , Inibidores da Angiogênese/uso terapêutico , Células Endoteliais , Senoterapia , Senescência Celular
2.
Nature ; 448(7150): 204-8, 2007 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-17538622

RESUMO

Sensory nerve fibres can detect changes in temperature over a remarkably wide range, a process that has been proposed to involve direct activation of thermosensitive excitatory transient receptor potential (TRP) ion channels. One such channel--TRP melastatin 8 (TRPM8) or cold and menthol receptor 1 (CMR1)--is activated by chemical cooling agents (such as menthol) or when ambient temperatures drop below approximately 26 degrees C, suggesting that it mediates the detection of cold thermal stimuli by primary afferent sensory neurons. However, some studies have questioned the contribution of TRPM8 to cold detection or proposed that other excitatory or inhibitory channels are more critical to this sensory modality in vivo. Here we show that cultured sensory neurons and intact sensory nerve fibres from TRPM8-deficient mice exhibit profoundly diminished responses to cold. These animals also show clear behavioural deficits in their ability to discriminate between cold and warm surfaces, or to respond to evaporative cooling. At the same time, TRPM8 mutant mice are not completely insensitive to cold as they avoid contact with surfaces below 10 degrees C, albeit with reduced efficiency. Thus, our findings demonstrate an essential and predominant role for TRPM8 in thermosensation over a wide range of cold temperatures, validating the hypothesis that TRP channels are the principal sensors of thermal stimuli in the peripheral nervous system.


Assuntos
Temperatura Baixa , Canais de Cátion TRPM/fisiologia , Sensação Térmica , Animais , Feminino , Gânglios Sensitivos/fisiologia , Marcação de Genes , Masculino , Mentol/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Canais de Cátion TRPM/genética
3.
J Pharmacol Exp Ther ; 341(1): 137-45, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22235148

RESUMO

Translation of central nervous system occupancy and clinical effect from animal models to humans has remained elusive for many pharmacological targets. The current studies evaluate the ability of a rodent pharmacokinetic/pharmacodynamic (PK/PD) modeling approach to translate ex vivo occupancy determined in rats to that observed after positron emission tomography (PET) imaging in humans for the dual serotonin transporter (SERT) and norepinephrine transporter (NET) inhibitor duloxetine. Ex vivo transporter occupancy in rat spinal cord was evaluated after single oral doses of 0.3 to 60 mg/kg. A novel methodology, based on the initial rates of association of transporter selective radioligands to tissue homogenates, was developed and validated for the assessment of ex vivo transporter occupancy. Duloxetine exhibited selectivity for occupancy of SERT over NET in rat spinal cord with ED(50) values of 1 and 9 mg/kg, respectively. Corresponding EC(50) values for the inhibition of SERT and NET based on unbound duloxetine spinal cord concentrations were 0.5 and 8 nM. An effect compartment PK/PD modeling approach was used to analyze the relationship between the time course of duloxetine plasma concentration and SERT and NET occupancy. Duloxetine inhibited SERT and NET in rat spinal cord with a plasma EC(50) of 2.95 and 59.0 ng/ml, respectively. Similar plasma EC(50) values for the inhibition of SERT (2.29-3.7 ng/ml) have been reported from human PET studies. This study illustrates the value of translational PK/PD modeling approaches and suggests that the preclinical modeling approach used in the current study is capable of predicting plasma concentrations associated with 50% occupancy of SERT in the human central nervous system.


Assuntos
Modelos Neurológicos , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Tiofenos/farmacocinética , Animais , Relação Dose-Resposta a Droga , Cloridrato de Duloxetina , Humanos , Masculino , Valor Preditivo dos Testes , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Tiofenos/metabolismo
4.
Nat Neurosci ; 11(7): 772-9, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18568022

RESUMO

In traditional folk medicine, Xanthoxylum plants are referred to as 'toothache trees' because their anesthetic or counter-irritant properties render them useful in the treatment of pain. Psychophysical studies have identified hydroxy-alpha-sanshool as the compound most responsible for the unique tingling and buzzing sensations produced by Szechuan peppercorns or other Xanthoxylum preparations. Although it is generally agreed that sanshool elicits its effects by activating somatosensory neurons, the underlying cellular and molecular mechanisms remain a matter of debate. Here we show that hydroxy-alpha-sanshool excites two types of sensory neurons, including small-diameter unmyelinated cells that respond to capsaicin (but not mustard oil) as well as large-diameter myelinated neurons that express the neurotrophin receptor TrkC. We found that hydroxy-alpha-sanshool excites neurons through a unique mechanism involving inhibition of pH- and anesthetic-sensitive two-pore potassium channels (KCNK3, KCNK9 and KCNK18), providing a framework for understanding the unique and complex psychophysical sensations associated with the Szechuan pepper experience.


Assuntos
Amidas/farmacologia , Neurônios Aferentes/efeitos dos fármacos , Piper nigrum/química , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Capsaicina/farmacologia , Células Cultivadas , Estimulação Elétrica/métodos , Gânglios Sensitivos/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , Proteínas de Neurofilamentos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Receptor trkC/metabolismo , Canal de Cátion TRPA1 , Canais de Cátion TRPV/deficiência , Canais de Potencial de Receptor Transitório/deficiência
5.
Cell Metab ; 33(4): 818-832.e7, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33548171

RESUMO

Attenuating pathological angiogenesis in diseases characterized by neovascularization such as diabetic retinopathy has transformed standards of care. Yet little is known about the molecular signatures discriminating physiological blood vessels from their diseased counterparts, leading to off-target effects of therapy. We demonstrate that in contrast to healthy blood vessels, pathological vessels engage pathways of cellular senescence. Senescent (p16INK4A-expressing) cells accumulate in retinas of patients with diabetic retinopathy and during peak destructive neovascularization in a mouse model of retinopathy. Using either genetic approaches that clear p16INK4A-expressing cells or small molecule inhibitors of the anti-apoptotic protein BCL-xL, we show that senolysis suppresses pathological angiogenesis. Single-cell analysis revealed that subsets of endothelial cells with senescence signatures and expressing Col1a1 are no longer detected in BCL-xL-inhibitor-treated retinas, yielding a retina conducive to physiological vascular repair. These findings provide mechanistic evidence supporting the development of BCL-xL inhibitors as potential treatments for neovascular retinal disease.


Assuntos
Senescência Celular , Doenças Retinianas/patologia , Proteína bcl-X/metabolismo , Animais , Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Cadeia alfa 1 do Colágeno Tipo I/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Flavonóis/química , Flavonóis/farmacologia , Flavonóis/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Patológica , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/metabolismo , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Proteína bcl-X/antagonistas & inibidores
6.
Neuron ; 51(2): 201-12, 2006 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-16846855

RESUMO

Transient receptor potential (TRP) channels mediate numerous sensory transduction processes and are thought to function as tetramers. TRP channel physiology is well studied; however, comparatively little is understood regarding TRP channel assembly. Here, we identify an autonomously folded assembly domain from the cold- and menthol-gated channel TRPM8. We show that the TRPM8 cytoplasmic C-terminal domain contains a coiled coil that is necessary for channel assembly and sufficient for tetramer formation. Cell biological experiments indicate that coiled-coil formation is required for proper channel maturation and trafficking and that the coiled-coil domain alone can act as a dominant-negative inhibitor of functional channel expression. Our data define an authentic TRP modular assembly domain, establish a clear role for coiled coils in ion channel assembly, demonstrate that coiled-coil assembly domains are a general feature of TRPM channels, and delineate a new tool that should be of general use in dissecting TRPM channel function.


Assuntos
Temperatura Baixa , Canais de Cátion TRPM/química , Canais de Cátion TRPM/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Sequência Conservada , Feminino , Humanos , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Xenopus
7.
ACS Med Chem Lett ; 10(12): 1641-1647, 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31857840

RESUMO

The effects of opioids in the central nervous system (CNS) provide significant benefit in the treatment of pain but can also lead to physical dependence and addiction, which has contributed to a growing opioid epidemic in the United States. Gastrointestinal dysfunction is an additional serious consequence of opioid use, and this can be treated with a localized drug distribution of a non-CNS penetrant, peripherally restricted opioid receptor antagonist. Herein, we describe the application of Theravance's multivalent approach to drug discovery coupled with a physicochemical property design strategy by which the N-substituted-endo-3-(8-aza-bicyclo[3.2.1]oct-3-yl)-phenyl carboxamide series of µ-opioid receptor antagonists was optimized to afford the orally absorbed, non-CNS penetrant, Phase 3 ready clinical compound axelopran (TD-1211) 19i as a potential treatment for opioid-induced constipation.

8.
Naunyn Schmiedebergs Arch Pharmacol ; 386(6): 479-91, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23549670

RESUMO

The clinical efficacy of opioid receptor antagonists for the treatment of opioid-induced constipation (OIC) is established. Peripherally selective antagonists are intended to provide OIC symptom relief without compromising the analgesic effects of centrally penetrant opioid agonists. We describe the in vitro profile of a novel opioid receptor antagonist, TD-1211, at recombinant (human µ and δ, and guinea pig κ) and rodent native opioid receptors. TD-1211 bound with high affinity to human recombinant µ and δ, and guinea pig κ receptors expressed in CHO-K1 cells (pK d = 9.7, 8.6, and 9.9, respectively). The in vitro receptor selectivity of TD-1211 (µ ≈ κ > δ) is similar to that for the peripherally-selective opioid receptor antagonist methylnaltrexone, but contrasts with the µ selectivity of alvimopan. Functionally, TD-1211 behaved as an antagonist at all three receptor types in both recombinant expression systems (pK b = 9.6, 8.8 and 9.5, at µ, δ, and κ, respectively) and rodent native tissue preparations (µ and κ pA2s = 10.1 and 8.8, respectively (guinea pig ileum), and δ pK b = 8.4 (hamster vas deferens)). TD-1211 displayed a high degree of selectivity for opioid receptors over a broad panel of cellular targets. These in vitro data justified investigation of the preclinical in vivo activity of TD-1211 (Armstrong et al., Naunyn-Schmiedeberg's Arch Pharm, 2013).


Assuntos
Benzamidas/farmacologia , Antagonistas de Entorpecentes/farmacologia , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides mu/antagonistas & inibidores , Tropanos/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Cobaias , Humanos , Técnicas In Vitro , Masculino , Mesocricetus , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Piperidinas/farmacologia , Compostos de Amônio Quaternário/farmacologia , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo
9.
PLoS One ; 8(9): e74891, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098676

RESUMO

Multimodal analgesia is designed to optimize pain relief by coadministering drugs with distinct mechanisms of action or by combining multiple pharmacologies within a single molecule. In clinical settings, combinations of monoamine reuptake inhibitors and opioid receptor agonists have been explored and one currently available analgesic, tapentadol, functions as both a µ-opioid receptor agonist and a norepinephrine transporter inhibitor. However, it is unclear whether the combination of selective norepinephrine reuptake inhibition and µ-receptor agonism achieves an optimal antinociceptive synergy. In this study, we assessed the pharmacodynamic interactions between morphine and monoamine reuptake inhibitors that possess different affinities and selectivities for norepinephrine and serotonin transporters. Using the rat formalin model, in conjunction with measurements of ex vivo transporter occupancy, we show that neither the norepinephrine-selective inhibitor, esreboxetine, nor the serotonin-selective reuptake inhibitor, fluoxetine, produce antinociceptive synergy with morphine. Atomoxetine, a monoamine reuptake inhibitor that achieves higher levels of norepinephrine than serotonin transporter occupancy, exhibited robust antinociceptive synergy with morphine. Similarly, a fixed-dose combination of esreboxetine and fluoxetine which achieves comparable levels of transporter occupancy potentiated the antinociceptive response to morphine. By contrast, duloxetine, a monoamine reuptake inhibitor that achieves higher serotonin than norepinephrine transporter occupancy, failed to potentiate the antinociceptive response to morphine. However, when duloxetine was coadministered with the 5-HT3 receptor antagonist, ondansetron, potentiation of the antinociceptive response to morphine was revealed. These results support the notion that inhibition of both serotonin and norepinephrine transporters is required for monoamine reuptake inhibitor and opioid-mediated antinociceptive synergy; yet, excess serotonin, acting via 5-HT3 receptors, may reduce the potential for synergistic interactions. Thus, in the rat formalin model, the balance between norepinephrine and serotonin transporter inhibition influences the degree of antinociceptive synergy observed between monoamine reuptake inhibitors and morphine.


Assuntos
Analgesia/métodos , Morfina/metabolismo , Inibidores da Captação de Neurotransmissores/metabolismo , Dor Nociceptiva/tratamento farmacológico , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Animais , Cloridrato de Atomoxetina , Monoaminas Biogênicas/metabolismo , Cromatografia Líquida , Sinergismo Farmacológico , Cloridrato de Duloxetina , Fluoxetina , Formaldeído , Morfolinas , Inibidores da Captação de Neurotransmissores/farmacocinética , Ondansetron , Propilaminas , Ratos , Teste de Desempenho do Rota-Rod , Espectrometria de Massas em Tandem , Tiofenos
10.
PLoS One ; 8(1): e55001, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23383028

RESUMO

Little is known about the molecular mechanisms underlying mammalian touch transduction. To identify novel candidate transducers, we examined the molecular and cellular basis of touch in one of the most sensitive tactile organs in the animal kingdom, the star of the star-nosed mole. Our findings demonstrate that the trigeminal ganglia innervating the star are enriched in tactile-sensitive neurons, resulting in a higher proportion of light touch fibers and lower proportion of nociceptors compared to the dorsal root ganglia innervating the rest of the body. We exploit this difference using transcriptome analysis of the star-nosed mole sensory ganglia to identify novel candidate mammalian touch and pain transducers. The most enriched candidates are also expressed in mouse somatosesensory ganglia, suggesting they may mediate transduction in diverse species and are not unique to moles. These findings highlight the utility of examining diverse and specialized species to address fundamental questions in mammalian biology.


Assuntos
Toupeiras/fisiologia , Percepção do Tato/fisiologia , Animais , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/patologia , Gânglios Espinais/fisiologia , Gânglios Espinais/fisiopatologia , Perfilação da Expressão Gênica , Mecanotransdução Celular , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Neurônios/patologia , Nociceptividade/fisiologia , Dor/genética , Dor/patologia , Dor/fisiopatologia , Gânglio Trigeminal/citologia , Gânglio Trigeminal/patologia , Gânglio Trigeminal/fisiologia , Gânglio Trigeminal/fisiopatologia
11.
Naunyn Schmiedebergs Arch Pharmacol ; 386(6): 471-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23512167

RESUMO

The in vivo preclinical pharmacodynamic profile of TD-1211, a selective opioid receptor antagonist currently under development for the treatment of opioid-induced constipation, was compared to that of the clinically studied opioid antagonists, naltrexone, alvimopan, and ADL 08-0011 (the primary active metabolite of alvimopan). The oral activity of TD-1211 was evaluated in models of gastrointestinal (GI) and central nervous system (CNS) function in the rat and dog. Oral administration of TD-1211, naltrexone, and ADL 08-0011 reversed loperamide-induced inhibition of gastric emptying and castor oil-induced diarrhea in rats and nonproductive GI circular smooth muscle contractility in dogs. Alvimopan was only efficacious in the castor oil model. Oral administration of naltrexone and ADL 08-0011, but not TD-1211 or alvimopan, was associated with a CNS withdrawal response in morphine-dependent mice, inhibition of morphine-induced anti-nociception in rat and dog hot plate tests, and hypothermia and sedation in dogs. It is concluded that TD-1211 has potent in vivo GI activity, consistent with opioid receptor antagonism, but has no significant CNS activity. The data from these studies support the clinical development of TD-1211 as a novel treatment for opioid-induced GI dysfunction.


Assuntos
Benzamidas/farmacologia , Sistema Nervoso Central/efeitos dos fármacos , Trato Gastrointestinal/efeitos dos fármacos , Antagonistas de Entorpecentes/farmacologia , Tropanos/farmacologia , Administração Oral , Analgésicos Opioides/antagonistas & inibidores , Analgésicos Opioides/farmacologia , Animais , Benzamidas/administração & dosagem , Sistema Nervoso Central/metabolismo , Cães , Feminino , Trato Gastrointestinal/metabolismo , Masculino , Camundongos , Morfina/antagonistas & inibidores , Morfina/farmacologia , Naltrexona/administração & dosagem , Naltrexona/farmacologia , Antagonistas de Entorpecentes/administração & dosagem , Dor/tratamento farmacológico , Piperidinas/administração & dosagem , Piperidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Síndrome de Abstinência a Substâncias/etiologia , Tropanos/administração & dosagem
12.
Neuropharmacology ; 61(1-2): 69-79, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21392515

RESUMO

There remains an urgent need for therapeutic agents that provide improved symptomatic treatment and attenuate disease progression in patients with Alzheimer's disease (AD). 5-HT(4) receptors are widely expressed in those CNS areas which receive substantial cholinergic input and are involved in cognition. The ability of 5-HT(4) receptor agonists to increase acetylcholine (ACh) release and reduce cognitive impairment in both animals and humans has been demonstrated. In addition, 5-HT(4) receptor agonist modulation of levels of the amyloid precursor protein (APP) derived peptides, soluble amyloid precursor protein (sAPPα) and amyloid beta protein (Aß) in the CNS has been reported. In this study, the preclinical properties of three structurally-distinct 5-HT(4) receptor selective agonists, PRX-03140, velusetrag and TD-8954, were studied to assess their potential for symptomatic and disease-modifying benefit in the treatment of AD. All three compounds exhibited high affinity for the rat 5-HT(4) receptor but could be discriminated on the basis of their agonist activity. In cAMP accumulation and sAPPα secretion assays using recombinant HEK293f-5-HT(4(d))-APP(695) cells, velusetrag and TD-8954 were potent, full agonists, relative to 5-HT, whereas PRX-03140 was a partial agonist (intrinsic activity 18%, relative to 5-HT). In a guinea pig colon isolated tissue preparation, TD-8954 exhibited lower intrinsic activity than velusetrag, and PRX-03140 had negligible agonist activity. In the rat Morris water maze (MWM) cognition test, velusetrag and TD-8954 (0.1 mg/kg), but not PRX-03140 (0.03-1 mg/kg), significantly reversed the scopolamine-induced spatial learning deficit via activation of 5-HT(4) receptors. Coadministration of subefficacious doses of the acetylcholinesterase inhibitor (AChEi), donepezil (0.1 mg/kg, i.p.), and either velusetrag or TD-8954 (0.01 mg/kg i.p.) resulted in reversal of the scopolamine-induced cognitive deficit. Pharmacokinetic data indicated that the CNS penetration for all three 5-HT(4) receptor agonists was relatively low. However, the pharmacodynamic-pharmacokinetic relationships in the MWM model for velusetrag and TD-8954 were consistent with their respective receptor pharmacology (binding affinity and intrinsic efficacy) and CNS penetration properties. Collectively, these findings support a potential role for potent and efficacious 5-HT(4) receptor agonists in the treatment of AD.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Cognição/fisiologia , Modificação Traducional de Proteínas/fisiologia , Receptores 5-HT4 de Serotonina/fisiologia , Agonistas do Receptor 5-HT4 de Serotonina/farmacologia , Animais , Cognição/efeitos dos fármacos , Relação Dose-Resposta a Droga , Cobaias , Células HEK293 , Humanos , Masculino , Ligação Proteica/fisiologia , Modificação Traducional de Proteínas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Agonistas do Receptor 5-HT4 de Serotonina/farmacocinética
13.
Front Pharmacol ; 2: 25, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21687517

RESUMO

This study evaluated the in vitro and in vivo pharmacological properties of TD-8954, a potent and selective 5-HT(4) receptor agonist. TD-8954 had high affinity (pK(i) = 9.4) for human recombinant 5-HT(4(c)) (h5-HT(4(c))) receptors, and selectivity (>2,000-fold) over all other 5-hydroxytryptamine (5-HT) receptors and non-5-HT receptors, ion channels, enzymes and transporters tested (n = 78). TD-8954 produced an elevation of cAMP in HEK-293 cells expressing the h5-HT(4(c)) receptor (pEC(50) = 9.3), and contracted the guinea pig colonic longitudinal muscle/myenteric plexus preparation (pEC(50) = 8.6). TD-8954 had moderate intrinsic activity in the in vitro assays. In conscious guinea pigs, subcutaneous administration of TD-8954 (0.03-3 mg/kg) increased the colonic transit of carmine red dye, reducing the time taken for its excretion. Following intraduodenal dosing to anesthetized rats, TD-8954 (0.03-10 mg/kg) evoked a dose-dependent relaxation of the esophagus. Following oral administration to conscious dogs, TD-8954 (10 and 30 µg/kg) produced an increase in contractility of the antrum, duodenum, and jejunum. In a single ascending oral dose study in healthy human subjects, TD-8954 (0.1-20 mg) increased bowel movement frequency and reduced the time to first stool. It is concluded that TD-8954 is a potent and selective 5-HT(4) receptor agonist in vitro, with robust in vivo stimulatory activity in the gastrointestinal (GI) tract of guinea pigs, rats, dogs, and humans. TD-8954 may have clinical utility in patients with disorders of reduced GI motility.

14.
J Pharmacol Toxicol Methods ; 61(2): 192-204, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20036748

RESUMO

INTRODUCTION: Monoamine reuptake inhibitors treat a wide range of CNS disorders, including depression, obesity, and pain. The in vitro pharmacological properties of these inhibitors are determined routinely using radioligand binding and/or neurotransmitter uptake assays. Measurements from such studies can be influenced by assay design and ligand-specific characteristics, both of which may contribute to discrepancies in literature reports. METHODS: We modified traditional methodologies to identify and account for factors that can confound in vitro potency determinations. Apparent equilibrium binding affinities (pK(i) values) were determined in either HEK293 cells stably-transfected with human recombinant serotonin (SERT) or norepinephrine (NET) transporters, or membranes prepared from these cell lines. Care was taken to ensure that apparent affinities were measured under conditions that minimized ligand depletion and established equilibrium for both the radioligand and the compound of interest. An unlabelled ligand kinetic method was used to approximate inhibitor binding kinetic constants and corresponding dissociation half lives. To measure inhibitory effects on substrate uptake, both radiolabeled neurotransmitter ([(3)H]-5-HT or [(3)H]-NE) and fluorescence-based assays were used. The time-dependent nature of functional inhibition was examined using a fluorescent substrate uptake assay which provided real-time measurements of NET and SERT function. RESULTS: SERT and NET inhibitors displayed a range of affinities, potencies, and inhibition modes by binding and functional uptake assays. Binding kinetic profiles for this panel of inhibitors were diverse, and affected in vitro measures using the former techniques. DISCUSSION: In the present study we describe key features of in vitro assay methodology that can influence the apparent pharmacological profiles of standard SERT and/or NET inhibitors. Such information can serve as a foundation for understanding the in vitro profiles of monoamine reuptake inhibitors in the context of their clinical efficacy and tolerability.


Assuntos
Inibidores da Captação Adrenérgica/farmacologia , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/antagonistas & inibidores , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Algoritmos , Sítios de Ligação , Linhagem Celular , Corantes Fluorescentes , Humanos , Cinética , Ligantes , Norepinefrina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/genética , Ligação Proteica , Ensaio Radioligante , Proteínas Recombinantes , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Temperatura
15.
Anat Rec (Hoboken) ; 290(5): 437-48, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17387732

RESUMO

Eimer's organ is a small, densely innervated sensory structure found on the glabrous rhinarium of most talpid moles. This structure consists of an epidermal papilla containing a central circular column of cells associated with intraepidermal free nerve endings, Merkel cell neurite complexes, and lamellated corpuscles. The free nerve endings within the central cell column form a ring invested in the margins of the column, surrounding 1-2 fibers that pass through the center of the column. A group of small-diameter nociceptive free nerve endings that are immunoreactive for substance P surrounds this central ring of larger-diameter free nerve endings. Transmission electron microscopy revealed a high concentration of tonofibrils in the epidermal cells of the central column, suggesting they are more rigid than the surrounding keratinocytes and may play a mechanical role in transducing stimuli to the different receptor terminals. The intraepidermal free nerve endings within the central column begin to degrade 15 microm from the base of the stratum corneum and do not appear to be active within the keratinized outer layer. The peripheral free nerve endings are structurally distinct from their counterparts in the central column and immunocytochemical double labeling with myelin basic protein and substance P indicates these afferents are unmyelinated. Merkel cell-neurite complexes and lamellated corpuscles are similar in morphology to those found in a range of other mammalian skin.


Assuntos
Epiderme/ultraestrutura , Células de Merkel/ultraestrutura , Toupeiras/anatomia & histologia , Neuritos/ultraestrutura , Animais , Epiderme/química , Epiderme/inervação , Imuno-Histoquímica , Mecanotransdução Celular , Células de Merkel/química , Microscopia Eletrônica de Transmissão , Toupeiras/fisiologia , Proteína Básica da Mielina/análise , Terminações Nervosas/ultraestrutura , Neuritos/química , Proteínas de Neurofilamentos/análise , Nociceptores/ultraestrutura , Nariz , Corpúsculos de Pacini/ultraestrutura , Substância P/análise
16.
Proc Natl Acad Sci U S A ; 103(24): 9339-44, 2006 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-16751268

RESUMO

Talpid moles are small insectivores that live in dark underground tunnels. They depend heavily on touch to navigate and find food. Most species have an array of complex epidermal sensory structures called Eimer's organs that cover the tip of the nose. In this study, the anatomy of Eimer's organ was examined in the coast mole and star-nosed mole by using the fluorescent styryl pyridinium dye AM1-43 and immunocytochemical staining for neurofilament 200 and substance P. In addition, DiI was used to label neural components of Eimer's organ. AM1-43 labeled all of the Eimer's organ receptors after systemic injection, suggesting a role in mechanotransduction. Immunostaining with neurofilament 200 and substance P labeled distinct subtypes of sensory fibers. Substance P labeled a group of free nerve endings along the outer edge of Eimer's organ, indicating a nociceptive role for these fibers. In contrast, neurofilament 200 labeled a more central set of nerve endings, suggesting that these fibers function as low-threshold mechanoreceptors. By labeling subsets of trigeminal afferents distant from the receptor array with DiI, we revealed innervation patterns indicating that one afferent supplies the outer, substance P-positive set of free nerve endings, whereas several afferents differentially innervate the central free nerve endings. Our results suggest that the free nerve endings innervating Eimer's organ are largely mechanosensitive and may play an important role in the rapid sensory discrimination observed in these species.


Assuntos
Vias Aferentes/anatomia & histologia , Toupeiras , Fibras Nervosas/ultraestrutura , Dor/metabolismo , Tato/fisiologia , Vias Aferentes/metabolismo , Animais , Carbocianinas/metabolismo , Corantes Fluorescentes/metabolismo , Imuno-Histoquímica , Células de Merkel/citologia , Células de Merkel/metabolismo , Toupeiras/anatomia & histologia , Toupeiras/fisiologia , Terminações Nervosas/citologia , Terminações Nervosas/metabolismo , Fibras Nervosas/metabolismo , Proteínas de Neurofilamentos/metabolismo , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Substância P/metabolismo
17.
Cell ; 124(6): 1269-82, 2006 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-16564016

RESUMO

TRPA1 is an excitatory ion channel targeted by pungent irritants from mustard and garlic. TRPA1 has been proposed to function in diverse sensory processes, including thermal (cold) nociception, hearing, and inflammatory pain. Using TRPA1-deficient mice, we now show that this channel is the sole target through which mustard oil and garlic activate primary afferent nociceptors to produce inflammatory pain. TRPA1 is also targeted by environmental irritants, such as acrolein, that account for toxic and inflammatory actions of tear gas, vehicle exhaust, and metabolic byproducts of chemotherapeutic agents. TRPA1-deficient mice display normal cold sensitivity and unimpaired auditory function, suggesting that this channel is not required for the initial detection of noxious cold or sound. However, TRPA1-deficient mice exhibit pronounced deficits in bradykinin-evoked nociceptor excitation and pain hypersensitivity. Thus, TRPA1 is an important component of the transduction machinery through which environmental irritants and endogenous proalgesic agents depolarize nociceptors to elicit inflammatory pain.


Assuntos
Alho , Inflamação , Nociceptores/imunologia , Dor , Canais de Potencial de Receptor Transitório/efeitos dos fármacos , Canais de Potencial de Receptor Transitório/genética , Acroleína/toxicidade , Animais , Temperatura Baixa , Potenciais Evocados Auditivos do Tronco Encefálico , Inflamação/imunologia , Exposição por Inalação , Camundongos , Camundongos Knockout , Estrutura Molecular , Nociceptores/efeitos dos fármacos , Canal de Cátion TRPA1 , Termorreceptores/fisiologia , Canais de Potencial de Receptor Transitório/metabolismo
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