RESUMO
The voltage-gated sodium channel NaV1.7 has received much attention from the scientific community due to compelling human genetic data linking gain- and loss-of-function mutations to pain phenotypes. Despite this genetic validation of NaV1.7 as a target for pain, high quality pharmacological tools facilitate further understanding of target biology, establishment of target coverage requirements and subsequent progression into the clinic. Within the sulfonamide class of inhibitors, reduced potency on rat NaV1.7 versus human NaV1.7 was observed, rendering in vivo rat pharmacology studies challenging. Herein, we report the discovery and optimization of novel benzoxazine sulfonamide inhibitors of human, rat and mouse NaV1.7 which enabled pharmacological assessment in traditional behavioral rodent models of pain and in turn, established a connection between formalin-induced pain and histamine-induced pruritus in mice. The latter represents a simple and efficient means of measuring target engagement.
Assuntos
Benzoxazinas/química , Benzoxazinas/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Analgésicos/química , Analgésicos/farmacocinética , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Benzoxazinas/farmacocinética , Benzoxazinas/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Dor/tratamento farmacológico , Dor/metabolismo , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapêutico , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
Clinical genetic data have shown that the product of the SCN9A gene, voltage-gated sodium ion channel Nav1.7, is a key control point for pain perception and a possible target for a next generation of analgesics. Sodium channels, however, historically have been difficult drug targets, and many of the existing structure-activity relationships (SAR) have been defined on pharmacologically modified channels with indirect reporter assays. Herein we describe the discovery, optimization, and SAR of potent aminopyrimidinone Nav1.7 antagonists using electrophysiology-based assays that measure the ligand-receptor interaction directly. Within this series, rapid functionalization at the polysubstituted aminopyrimidinone head group enabled exploration of SAR and of pharmacokinetic properties. Lead optimized N-Me-aminopyrimidinone 9 exhibited improved Nav1.7 potency, minimal off-target hERG liability, and improved rat PK properties.
Assuntos
Pirimidinonas/farmacologia , Canais de Sódio/metabolismo , Animais , Relação Dose-Resposta a Droga , Humanos , Ligantes , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Canal de Sódio Disparado por Voltagem NAV1.7 , Pirimidinonas/síntese química , Pirimidinonas/química , Ratos , Relação Estrutura-AtividadeRESUMO
Recent evidence suggests that the P2X(7) receptor may play a role in the pathophysiology of preclinical models of pain and inflammation. Therefore, pharmacological agents that target this receptor may potentially have clinical utility as anti-inflammatory and analgesic therapy. We investigated and characterized the previously reported P2X(7) antagonist N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, hydrochloride salt (AACBA; GSK314181A). In vitro, AACBA was a relatively potent inhibitor of both human P2X(7)-mediated calcium flux and quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triemethylammonio)propyl]-diiodide (YO-PRO-1) uptake assays, with IC(50) values of approximately 18 and 85 nM, respectively. Compared with the human receptor, AACBA was less potent at the rat P2X(7) receptor, with IC(50) values of 29 and 980 nM in the calcium flux and YO-PRO-1 assays, respectively. In acute in vivo models of pain and inflammation, AACBA dose-dependently reduced lipopolysaccharide-induced plasma interleukin-6 release and prevented or reversed carrageenan-induced paw edema and mechanical hypersensitivity. In chronic in vivo models of pain and inflammation, AACBA produced a prophylactic, but not therapeutic-like, prevention of the clinical signs and histopathological damage of collagen-induced arthritis. Finally, AACBA could not reverse L(5) spinal nerve ligation-induced tactile allodynia when given therapeutically. Consistent with previous literature, these results suggest that P2X(7) receptors do play a role in animal models of pain and inflammation. Further study of P2X(7) antagonists both in preclinical and clinical studies will help elucidate the role of the P2X(7) receptor in pain and inflammatory mechanisms and may help identify potential clinical benefits of such molecules.
Assuntos
Adamantano/análogos & derivados , Anti-Inflamatórios não Esteroides/farmacologia , Benzamidas/farmacologia , Inflamação/tratamento farmacológico , Dor/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2 , Adamantano/farmacologia , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Benzoxazóis , Cálcio/metabolismo , Modelos Animais de Doenças , Humanos , Concentração Inibidora 50 , Compostos de Quinolínio , Ratos , Receptores Purinérgicos P2X7RESUMO
Current therapies to treat persistent pain and neuropathic pain are limited by poor efficacy, side effects and risk of addiction. Here, we present a novel class of potent selective, central nervous system (CNS)-penetrant potentiators of glycine receptors (GlyRs), ligand-gated ion channels expressed in the CNS. AM-1488 increased the response to exogenous glycine in mouse spinal cord and significantly reversed mechanical allodynia induced by nerve injury in a mouse model of neuropathic pain. We obtained an X-ray crystal structure of human homopentameric GlyRα3 in complex with AM-3607, a potentiator of the same class with increased potency, and the agonist glycine, at 2.6-Å resolution. AM-3607 binds a novel allosteric site between subunits, which is adjacent to the orthosteric site where glycine binds. Our results provide new insights into the potentiation of cysteine-loop receptors by positive allosteric modulators and hold promise in structure-based design of GlyR modulators for the treatment of neuropathic pain.
Assuntos
Receptores de Glicina/química , Regulação Alostérica , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Glicina/química , Células HEK293 , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Subunidades Proteicas/químicaRESUMO
While genetic evidence shows that the Nav1.7 voltage-gated sodium ion channel is a key regulator of pain, it is unclear exactly how Nav1.7 governs neuronal firing and what biophysical, physiological, and distribution properties of a pharmacological Nav1.7 inhibitor are required to produce analgesia. Here we characterize a series of aminotriazine inhibitors of Nav1.7 in vitro and in rodent models of pain and test the effects of the previously reported "compound 52" aminotriazine inhibitor on the spiking properties of nociceptors in vivo. Multiple aminotriazines, including some with low terminal brain to plasma concentration ratios, showed analgesic efficacy in the formalin model of pain. Effective concentrations were consistent with the in vitro potency as measured on partially-inactivated Nav1.7 but were far below concentrations required to inhibit non-inactivated Nav1.7. Compound 52 also reversed thermal hyperalgesia in the complete Freund's adjuvant (CFA) model of pain. To study neuronal mechanisms, electrophysiological recordings were made in vivo from single nociceptive fibers from the rat tibial nerve one day after CFA injection. Compound 52 reduced the spontaneous firing of C-fiber nociceptors from approximately 0.7 Hz to 0.2 Hz and decreased the number of action potentials evoked by suprathreshold tactile and heat stimuli. It did not, however, appreciably alter the C-fiber thresholds for response to tactile or thermal stimuli. Surprisingly, compound 52 did not affect spontaneous activity or evoked responses of Aδ-fiber nociceptors. Results suggest that inhibition of inactivated states of TTX-S channels, mostly likely Nav1.7, in the peripheral nervous system produces analgesia by regulating the spontaneous discharge of C-fiber nociceptors.
Assuntos
Analgésicos/uso terapêutico , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Fibras Nervosas Amielínicas/metabolismo , Nociceptores/metabolismo , Dor/tratamento farmacológico , Tetrodotoxina/farmacologia , Potenciais de Ação/fisiologia , Analgesia/métodos , Animais , Formaldeído/farmacologia , Adjuvante de Freund/farmacologia , Masculino , Dor/induzido quimicamente , Manejo da Dor , Medição da Dor/métodos , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
Clinical genetic studies have shown that loss of Nav1.7 function leads to the complete loss of acute pain perception. The global deletion is reported lethal in mice, however, and studies of mice with promoter-specific deletions of Nav1.7 have suggested that the role of Nav1.7 in pain transduction depends on the precise form of pain. We developed genetic and animal husbandry strategies that overcame the neonatal-lethal phenotype and enabled construction of a global Nav1.7 knockout mouse. Knockouts were anatomically normal, reached adulthood, and had phenotype wholly analogous to human congenital indifference to pain (CIP): compared to littermates, knockouts showed no defects in mechanical sensitivity or overall movement yet were completely insensitive to painful tactile, thermal, and chemical stimuli and were anosmic. Knockouts also showed no painful behaviors resulting from peripheral injection of nonselective sodium channel activators, did not develop complete Freund's adjuvant-induced thermal hyperalgesia, and were insensitive to intra-dermal histamine injection. Tetrodotoxin-sensitive sodium current recorded from cell bodies of isolated sensory neurons and the mechanically-evoked spiking of C-fibers in a skin-nerve preparation each were reduced but not eliminated in tissue from knockouts compared to littermates. Results support a role for Nav1.7 that is conserved between rodents and humans and suggest several possibly translatable biomarkers for the study of Nav1.7-targeted therapeutics. Results further suggest that Nav1.7 may retain its key role in persistent as well as acute forms of pain.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/deficiência , Insensibilidade Congênita à Dor/etiologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/fisiologia , Fibras Nervosas Amielínicas/fisiologia , Sistema Nervoso/patologia , Sistema Nervoso/fisiopatologia , Transtornos do Olfato/genética , Transtornos do Olfato/fisiopatologia , Insensibilidade Congênita à Dor/genética , Insensibilidade Congênita à Dor/fisiopatologia , Limiar da Dor/fisiologia , Fenótipo , Células Receptoras Sensoriais/fisiologiaRESUMO
Creating a robust and unbiased assay for the study of current and novel analgesics has been a daunting task. Traditional rodent models of pain and inflammation typically rely on a negative reaction to various forms of evoked stimuli to elicit a pain response and are subject to rater interpretation. Recently, models such as weight bearing and gait analysis have been developed to address these drawbacks while detecting a drug's analgesic properties. We have recently developed the Reduction of Spontaneous Activity by Adjuvant (RSAA) model as a quick, unbiased method for the testing of potential analgesics. Rats, following prior administration of an activity-decreasing inflammatory insult, will positively increase spontaneous locomotor exploration when given single doses of known analgesics. The RSAA model capitalizes on a rat's spontaneous exploratory behavior in a novel environment with the aid of computer tracking software to quantify movement and eliminate rater bias.
Assuntos
Comportamento Exploratório/fisiologia , Inflamação/fisiopatologia , Atividade Motora/fisiologia , Dor/fisiopatologia , Anfetamina/farmacologia , Anfetamina/uso terapêutico , Analgésicos Opioides/farmacologia , Analgésicos Opioides/uso terapêutico , Animais , Artrite Experimental/fisiopatologia , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Estimulantes do Sistema Nervoso Central/farmacologia , Estimulantes do Sistema Nervoso Central/uso terapêutico , Modelos Animais de Doenças , Meio Ambiente , Comportamento Exploratório/efeitos dos fármacos , Masculino , Morfina/farmacologia , Morfina/uso terapêutico , Atividade Motora/efeitos dos fármacos , Dor/tratamento farmacológico , Medição da Dor/instrumentação , Medição da Dor/métodos , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Translating promising analgesic compounds into reliable pain therapeutics in humans is made particularly challenging by the difficulty in measuring the pain quantitatively. This problem is manifest not only in clinical settings in which patient pain assessments involve mostly subjective measures but also in preclinical settings wherein laboratory animals, most commonly rodents, are typically evaluated in stimulus-evoked response tests. OBJECTIVE: Given the limitations of traditional pain tests, we sought out new approaches to measure pain, and analgesia, in laboratory animals. METHODS: We reviewed the peer reviewed literature to identify pain tests that could be utilized in preclinical settings to understand the effects of new and established analgesics. RESULTS/CONCLUSIONS: The tests identified include weight bearing differential, suppression of feeding, reduction in locomotor activity, gait analysis, conditioning models and functional MRI. Although the pharmacology of known and new analgesics has not been broadly established in these models, they hold the promise of better predictive utility for the discovery of pain relievers.
RESUMO
The majority of rodent models used to evaluate analgesic drug effects rely on evoked measures of nociceptive thresholds as primary outcomes. These approaches are often time-consuming, requiring extensive habituation sessions and repeated presentations of eliciting stimuli, and are prone to false-positive outcomes due to sedation or tester subjectivity. Here, we describe the reduction of spontaneous activity by adjuvant (RSAA) model as an objective and quantifiable behavioral model of inflammatory pain that can predict the analgesic activity of a variety of agents following single-dose administration. In the RSAA model, activity was measured in nonhabituated rats using standard, photocell-based monitors. Bilateral inflammation of the knee joints by complete Freund's adjuvant (CFA) reduced the normal level of activity (horizontal locomotion and vertical rearing) by approximately 60% in a novel environment. This reduction in activity was dose-dependently reversed by ibuprofen, rofecoxib, celecoxib, piroxicam, and dexamethasone, whereas gabapentin and amitriptyline were inactive. Morphine significantly reversed the activity-suppressing effects of CFA, at 1 mg/kg s.c., but at higher doses locomotor activity progressively declined, coincident with the induction of sedation. In contrast to morphine and anti-inflammatory therapies, amphetamine did not affect vertical rearing, even though it increased horizontal locomotion. Thus, unlike standard measures of analgesia such as alteration in thermal or mechanical sensitivity, the RSAA model operationally defines analgesia as a drug-induced increase in spontaneous behavior (vertical rearing in a novel environment). We conclude that the RSAA model is valuable as an objective measure of analgesic efficacy that is not dependent on an evoked stimulus response.