RESUMO
The therapeutic potential of transient receptor potential vanilloid type 1 (TRPV1) antagonists for chronic pain has been recognized for more than a decade. However, preclinical and clinical data revealed that acute pharmacological blockade of TRPV1 perturbs thermoregulation, resulting in hyperthermia, which is a major hurdle for the clinical development of these drugs. Here, we describe the properties of 7-tert-butyl-6-(4-chloro-phenyl)-2-thioxo-2,3-dihydro-1H-pyrido[2,3-d]pyrimidin-4-one (BCTP), a TRPV1 antagonist with excellent analgesic properties that does not induce significant hyperthermia in rodents at doses providing maximal analgesia. BCTP is a classic polymodal inhibitor of TRPV1, blocking activation of the human channel by capsaicin and low pH with IC(50) values of 65.4 and 26.4 nM, respectively. Similar activity was observed with rat TRPV1, and the inhibition by BCTP was competitive and reversible. BCTP also blocked heat-induced activation of TRPV1. In rats, the inhibition of capsaicin-induced mechanical hyperalgesia was observed with a D(50) value of 2 mg/kg p.o. BCTP also reversed visceral hypersensitivity and somatic inflammatory pain, and using a model of neuropathic pain in TRPV1 null mice we confirmed that its analgesic properties were solely through the inhibition of TRPV1. We were surprised to find that BCTP administered orally induced only a maximal 0.6°C increase in core body temperature at the highest tested doses (30 and 100 mg/kg), contrasting markedly with N-[4-({6-[4-(trifluoromethyl)phenyl]pyrimidin-4-yl}oxy)-1,3-benzothiazol-2-yl]acetamide (AMG517), a clinically tested TRPV1 antagonist, which induced marked hyperthermia (>1°C) at doses eliciting submaximal reversal of capsaicin-induced hyperalgesia. The combined data indicate that TRPV1 antagonists with a classic polymodal inhibition profile can be identified where the analgesic action is separated from the effects on body temperature.
Assuntos
Analgésicos/farmacologia , Febre/tratamento farmacológico , Hipersensibilidade/tratamento farmacológico , Pirimidinonas/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Tionas/farmacologia , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Animais , Benzotiazóis/efeitos adversos , Temperatura Corporal/efeitos dos fármacos , Regulação da Temperatura Corporal/efeitos dos fármacos , Células CHO , Capsaicina/farmacologia , Cricetinae , Febre/metabolismo , Humanos , Hiperalgesia/induzido quimicamente , Hipersensibilidade/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pirimidinas/efeitos adversos , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Canais de Cátion TRPV/metabolismo , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Inflammatory responses are crucial for regeneration following peripheral nerve injury (PNI). PNI triggers inflammatory responses at the site of injury. The DNA-sensing receptor cyclic GMP-AMP synthase (cGAS) and its downstream effector stimulator of interferon genes (STING) sense foreign and self-DNA and trigger type I interferon (IFN) immune responses. We demonstrate here that following PNI, the cGAS/STING pathway is upregulated in the sciatic nerve of naive rats and dysregulated in old rats. In a nerve crush mouse model where STING is knocked out, myelin content in sciatic nerve is increased resulting in accelerated functional axon recovery. STING KO mice have lower macrophage number in sciatic nerve and decreased microglia activation in spinal cord 1 week post injury. STING activation regulated processing of colony stimulating factor 1 receptor (CSF1R) and microglia survival in vitro. Taking together, these data highlight a previously unrecognized role of STING in the regulation of nerve regeneration.
RESUMO
Assessment of myelin integrity in peripheral nerve injuries and pathologies has largely been limited to post-mortem analysis owing to the difficulty in obtaining biopsies without affecting nerve function. This is further encumbered by the small size of the tissue and its location. Therefore, the development of robust, non-invasive methods is highly attractive. In this study, we used magnetic resonance imaging (MRI) techniques, including magnetization transfer ratio (MTR), to longitudinally and non-invasively characterize both the sciatic nerve crush and lysolecithin (LCP) demyelination models of peripheral nerve injury in rodents. Electrophysiological, gene expression and histological assessments complemented the extensive MRI analyses in young and aged animals. In the nerve crush model, MTR analysis indicated a slower recovery in regions distal to the site of injury in aged animals, as well as incomplete recovery at six weeks post-crush when analyzing across the entire nerve surface. Similar regional impairments were also found in the LCP demyelination model. This research underlines the power of MTR for the study of peripheral nerve injury in small tissues such as the sciatic nerve of rodents and contributes new knowledge to the effect of aging on recovery after injury. A particular advantage of the approach is the translational potential to human neuropathies.
Assuntos
Fatores Etários , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/diagnóstico por imagem , Traumatismos dos Nervos Periféricos/fisiopatologia , Animais , Axônios/patologia , Biomarcadores/metabolismo , Modelos Animais de Doenças , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Regeneração Nervosa/efeitos dos fármacos , Ratos , Recuperação de Função Fisiológica/efeitos dos fármacos , Nervo Isquiático/lesões , Neuropatia Ciática/metabolismoRESUMO
Age-related loss of skeletal muscle innervation by motor neurons leads to impaired neuromuscular function and is a well-established clinical phenomenon. However, the underlying pathogenesis remains unclear. Studying mice, we find that the number of motor units (MUs) can be maintained by counteracting neurotoxic microglia in the aged spinal cord. We observe that marked innervation changes, detected by motor unit number estimation (MUNE), occur prior to loss of muscle function in aged mice. This coincides with gene expression changes indicative of neuronal remodeling and microglial activation in aged spinal cord. Voluntary exercise prevents loss of MUs and reverses microglia activation. Depleting microglia by CSF1R inhibition also prevents the age-related decline in MUNE and neuromuscular junction disruption, implying a causal link. Our results suggest that age-related changes in spinal cord microglia contribute to neuromuscular decline in aged mice and demonstrate that removal of aged neurotoxic microglia can prevent or reverse MU loss.
Assuntos
Envelhecimento/metabolismo , Microglia/metabolismo , Neurônios Motores/metabolismo , Condicionamento Físico Animal/fisiologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Envelhecimento/patologia , Animais , Linhagem Celular , Bases de Dados Genéticas , Humanos , Células-Tronco Pluripotentes Induzidas , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/enzimologia , Microglia/fisiologia , Neurônios Motores/citologia , Neurônios Motores/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Junção Neuromuscular/metabolismo , Plasticidade Neuronal/genética , RNA-Seq , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Medula Espinal/enzimologia , Medula Espinal/metabolismo , Medula Espinal/fisiopatologiaRESUMO
Vanilloid receptor 1 (VR1, TRPV1) is a cation-selective ion channel that is expressed on primary afferent neurons and is upregulated following inflammation and nerve damage. Blockers of this channel may have utility in the treatment of chronic nociceptive and neuropathic pain. Here, we describe the optimization from a high throughput screening hit, of a series of 6-aryl-7-isopropylquinazolinones that are TRPV1 antagonists in vitro. We also demonstrate that one compound is active in vivo against capsaicin-induced hyperalgesia and in models of neuropathic and nociceptive pain in the rat.
Assuntos
Dor/tratamento farmacológico , Quinazolinas/síntese química , Canais de Cátion TRPV/antagonistas & inibidores , Animais , Barreira Hematoencefálica/metabolismo , Células CHO , Células CACO-2 , Permeabilidade da Membrana Celular , Doença Crônica , Cricetinae , Cricetulus , Modelos Animais de Doenças , Humanos , Técnicas In Vitro , Camundongos , Testes para Micronúcleos , Microssomos Hepáticos/metabolismo , Quinazolinas/farmacocinética , Quinazolinas/farmacologia , Ratos , Solubilidade , Relação Estrutura-Atividade , Canais de Cátion TRPV/genéticaRESUMO
We describe the properties of a novel nonpeptide kinin B1 receptor antagonist, NVP-SAA164, and demonstrate its in vivo activity in models of inflammatory pain in transgenic mice expressing the human B1 receptor. NVP-SAA164 showed high affinity for the human B1 receptor expressed in HEK293 cells (K(i) 8 nM), and inhibited increases in intracellular calcium induced by desArg10kallidin (desArg10KD) (IC50 33 nM). While a similar high affinity was observed in monkey fibroblasts (K(i) 7.7 nM), NVP-SAA164 showed no affinity for the rat B1 receptor expressed in Cos-7 cells. In transgenic mice in which the native B1 receptor was deleted and the gene encoding the human B1 receptor was inserted (hB1 knockin, hB1-KI), hB1 receptor mRNA was induced in tissues following LPS treatment. No mRNA encoding the mouse or human B1 receptor was detected in mouse B1 receptor knockout (mB1-KO) mice following LPS treatment. Freund's complete adjuvant-induced mechanical hyperalgesia was similar in wild-type and hB(1)-KI mice, but was significantly reduced in mB1-KO animals. Mechanical hyperalgesia induced by injection of the B1 agonist desArg10KD into the contralateral paw 24 h following FCA injection was similar in wild-type and hB1-KI mice, but was absent in mB1-KO animals. Oral administration of NVP-SAA164 produced a dose-related reversal of FCA-induced mechanical hyperalgesia and desArg10KD-induced hyperalgesia in hB1-KI mice, but was inactive against inflammatory pain in wild-type mice. These data demonstrate the use of transgenic technology to investigate the in vivo efficacy of species selective agents and show that NVP-SAA164 is a novel orally active B1 receptor antagonist, providing further support for the utility of B1 receptor antagonists in inflammatory pain conditions in man.
Assuntos
Analgésicos/uso terapêutico , Benzamidas/uso terapêutico , Antagonistas de Receptor B1 da Bradicinina , Hiperalgesia/tratamento farmacológico , Receptor B1 da Bradicinina/metabolismo , Sulfonamidas/uso terapêutico , Administração Oral , Analgésicos/química , Analgésicos/farmacologia , Animais , Benzamidas/química , Benzamidas/farmacologia , Células COS , Linhagem Celular , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Hiperalgesia/genética , Hiperalgesia/metabolismo , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ratos , Receptor B1 da Bradicinina/genética , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Capsaicin and analogues are valuable analgesic agents when administered to mammals, including humans. However, their pungency and the effects on the cardiovascular and respiratory systems through their general activation of small calibre (nociceptive) primary afferents severely limit their use. Recently, structure activity analysis revealed that the initial pungent and general excitatory effects can be prevented by structural modifications in such a way that the analgesic activity is retained. In this paper we present SDZ 249-665, a capsaicin analogue which produced analgesia in the mouse and anti-hyperalgesic effects in the rat and guinea pig. SDZ 249-665 was administered p.o., s.c. and i.v. in models of nociceptive pain, such as tail flick latency in response to a noxious thermal stimulus and acetic acid-induced writhing in mice, and in models of inflammatory mechanical hyperalgesia induced by turpentine or carrageenan in the rat and guinea pig, respectively. SDZ 249-665 was effective in the tail flick and the writhing assays and produced significant anti-hyperalgesic effects in the inflammatory models. The efficacy of SDZ 245-665 was similar to that of capsaicin, however, it was significantly more potent. SDZ 249-665 did not produce any irritancy in a nose wipe assay in guinea pigs or an eye irritancy assay in rats, while capsaicin was clearly irritant in both cases. Furthermore, unlike capsaicin, SDZ 249-665 did not produce unwanted side effects such as bronchoconstriction and blood pressure changes in the analgesic/anti-hyperalgesic dose range. Thus, a clear analgesic therapeutic window exists for SDZ 249-665. In summary, SDZ 249-665 is a potent orally active, analgesic/anti-hyperalgesic agent in mouse, rat and guinea pig. It lacks the excitatory effects associated with capsaicin and other close analogues, and therefore provides a clear therapeutic window for use in painful conditions. In addition to this favourable profile, no sign of tolerance was detected after a 5 day repeated dose treatment.
Assuntos
Analgésicos/farmacologia , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Hiperalgesia/tratamento farmacológico , Dor/tratamento farmacológico , Analgésicos/química , Animais , Comportamento Animal , Piscadela/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Broncoconstrição/efeitos dos fármacos , Capsaicina/química , Carragenina , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Tolerância a Medicamentos , Olho , Feminino , Gânglios Espinais/citologia , Cobaias , Membro Posterior , Hiperalgesia/induzido quimicamente , Irritantes , Masculino , Camundongos , Camundongos Endogâmicos , Nociceptores/efeitos dos fármacos , Nariz , Odorantes , Dor/induzido quimicamente , Medição da Dor/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Terebintina , Ureia/análogos & derivadosRESUMO
A high throughput screening campaign identified 5-(2-chlorophenyl)indazole compound 4 as an antagonist of the transient receptor potential A1 (TRPA1) ion channel with IC50 = 1.23 µM. Hit to lead medicinal chemistry optimization established the SAR around the indazole ring system, demonstrating that a trifluoromethyl group at the 2-position of the phenyl ring in combination with various substituents at the 6-position of the indazole ring greatly contributed to improvements in vitro activity. Further lead optimization resulted in the identification of compound 31, a potent and selective antagonist of TRPA1 in vitro (IC50 = 0.015 µM), which has moderate oral bioavailability in rodents and demonstrates robust activity in vivo in several rodent models of inflammatory pain.
Assuntos
Indazóis/química , Proteínas do Tecido Nervoso/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Administração Oral , Analgésicos/química , Analgésicos/farmacocinética , Analgésicos/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Disponibilidade Biológica , Células CHO , Canais de Cálcio , Cricetulus , Adjuvante de Freund , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Indazóis/farmacocinética , Indazóis/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Mostardeira , Óleos de Plantas , Ratos Wistar , Especificidade da Espécie , Relação Estrutura-Atividade , Canal de Cátion TRPA1 , Canais de Cátion TRPC/antagonistas & inibidoresRESUMO
BACKGROUND: Prokineticin 2 (PROK2) is an inflammatory cytokine-like molecule expressed predominantly by macrophages and neutrophils infiltrating sites of tissue damage. Given the established role of prokineticin signaling on gastrointestinal function, we have explored Prok2 gene expression in inflammatory conditions of the gastrointestinal tract and assessed the possible consequences on gut physiology. METHODS: Prokineticin expression was examined in normal and colitic tissues using qPCR and immunohistochemistry. Functional responses to PROK2 were studied using calcium imaging and a novel antagonist, Compound 3, used to determine the role of PROK2 and prokineticin receptors in inflammatory visceral pain and ion transport. KEY RESULTS: Prok2 gene expression was up-regulated in biopsy samples from ulcerative colitis patients, and similar elevations were observed in rodent models of inflammatory colitis. Prokineticin receptor 1 (PKR1) was localized to the enteric neurons and extrinsic sensory neurons, whereas Pkr2 expression was restricted to sensory ganglia. In rats, PROK2-increased intracellular calcium levels in cultured enteric and dorsal root ganglia neurons, which was blocked by Compound 3. Moreover, PROK2 acting at prokineticin receptors stimulated intrinsic neuronally mediated ion transport in rat ileal mucosa. In vivo, Compound 3 reversed intracolonic mustard oil-induced referred allodynia and TNBS-induced visceral hypersensitivity, but not non-inflammatory, stress-induced visceral pain. CONCLUSIONS & INFERENCES: Elevated Prok2 levels, as a consequence of gastrointestinal tract inflammation, induce visceral pain via prokineticin receptors. This observation, together with the finding that PROK2 can modulate intestinal ion transport, raises the possibility that inhibitors of PROK2 signaling may have clinical utility in gastrointestinal disorders, such as irritable bowel syndrome and inflammatory bowel disease.