RESUMO
NaV1.7 is an actively pursued, genetically validated, target for pain. Recently reported quinolinone sulfonamide inhibitors displayed promising selectivity profiles as well as efficacy in preclinical pain models; however, concerns about off-target liabilities associated with this series resulted in an effort to reduce the lipophilicity of these compounds. Successful prosecution of this strategy was challenging due to the opposing requirement for lipophilic inhibitors for NaV1.7 potency and in vivo clearance (CL). Deconstruction of the heterocyclic core of the quinolinone series and utilization of an intramolecular hydrogen bond to mimic the requisite pharmacophore enabled the introduction of polarity without adversely impacting CL. Ultimately, this strategy led to the identification of compound 29, which demonstrated favorable ADME and was efficacious in pre-clinical models of pain.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7 , Quinolonas , Humanos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor/tratamento farmacológico , Relação Estrutura-Atividade , Sulfanilamida , Sulfonamidas/química , Sulfonamidas/farmacologia , Ureia/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/químicaRESUMO
There is an unmet medical need for nonopioid pain therapies in human populations; several pathways are under investigation for possible therapeutic intervention. Tetrahydrobiopterin (BH4) has received attention recently as a mediator of neuropathic pain. Recent reports have implicated sepiapterin reductase (SPR) in this pain pathway as a regulator of BH4 production. To evaluate the role of SPR inhibition on BH4 reduction, we developed analytical methods to monitor the relationship between the plasma concentration of test article and endogenous pterins and applied these in the rat spinal nerve ligation pain model. Sepiapterin is an endogenous substrate, which accumulates upon inhibition of SPR. In response to a potent inhibitor of SPR, plasma concentrations of sepiapterin increased proportionally with exposure. An indirect-effect pharmacokinetic/pharmacodynamic model was developed to describe the relationship between the plasma pharmacokinetics of test article and plasma sepiapterin levels in the rat, which was used to determine an in vivo SPR IC50 value. SPR inhibition and mechanical allodynia were assessed coordinately with pterin biomarkers in plasma and at the site of neuronal injury (i.e., dorsal root ganglion). Upon daily oral administration for 3 consecutive days, unbound plasma concentrations of test article exceeded the unbound in vivo rat SPR IC90 throughout the dose intervals, leading to a 60% reduction in BH4 in the dorsal root ganglion. Despite evidence for pharmacological modulation of the BH4 pathway, there was no significant effect on the tactile paw withdrawal threshold relative to vehicle-treated controls.
Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/metabolismo , Hiperalgesia/metabolismo , Neuralgia/metabolismo , Medição da Dor/métodos , Animais , Biopterinas/análogos & derivados , Biopterinas/antagonistas & inibidores , Biopterinas/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Humanos , Hiperalgesia/tratamento farmacológico , Masculino , Neuralgia/tratamento farmacológico , Medição da Dor/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Tato/efeitos dos fármacos , Tato/fisiologiaRESUMO
Potent and selective antagonists of the voltage-gated sodium channel NaV1.7 represent a promising avenue for the development of new chronic pain therapies. We generated a small molecule atropisomer quinolone sulfonamide antagonist AMG8379 and a less active enantiomer AMG8380. Here we show that AMG8379 potently blocks human NaV1.7 channels with an IC50 of 8.5 nM and endogenous tetrodotoxin (TTX)-sensitive sodium channels in dorsal root ganglion (DRG) neurons with an IC50 of 3.1 nM in whole-cell patch clamp electrophysiology assays using a voltage protocol that interrogates channels in a partially inactivated state. AMG8379 was 100- to 1000-fold selective over other NaV family members, including NaV1.4 expressed in muscle and NaV1.5 expressed in the heart, as well as TTX-resistant NaV channels in DRG neurons. Using an ex vivo mouse skin-nerve preparation, AMG8379 blocked mechanically induced action potential firing in C-fibers in both a time-dependent and dose-dependent manner. AMG8379 similarly reduced the frequency of thermally induced C-fiber spiking, whereas AMG8380 affected neither mechanical nor thermal responses. In vivo target engagement of AMG8379 in mice was evaluated in multiple NaV1.7-dependent behavioral endpoints. AMG8379 dose-dependently inhibited intradermal histamine-induced scratching and intraplantar capsaicin-induced licking, and reversed UVB radiation skin burn-induced thermal hyperalgesia; notably, behavioral effects were not observed with AMG8380 at similar plasma exposure levels. AMG8379 is a potent and selective NaV1.7 inhibitor that blocks sodium current in heterologous cells as well as DRG neurons, inhibits action potential firing in peripheral nerve fibers, and exhibits pharmacodynamic effects in translatable models of both itch and pain.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Neurônios/efeitos dos fármacos , Dor/prevenção & controle , Dor/psicologia , Técnicas de Patch-Clamp , Prurido/prevenção & controle , Prurido/psicologia , Quinolonas/farmacologia , Bibliotecas de Moléculas Pequenas , Estereoisomerismo , Sulfonamidas/farmacologiaRESUMO
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
Cannabinoid receptors (CBRs) play an important role in a variety of physiological functions and have been considered drug targets for obesity and psychiatric disorders. In particular, the CB1R is highly expressed in brain regions crucial to learning and memory processes, and several lines of evidence indicate that pharmacological blockade of this receptor could have therapeutic applications in the treatment of cognitive disorders. In this study, we investigated whether MK-7128 (0.1, 0.3, and 1 mg/kg, orally), a novel and selective CB1R inverse agonist, could improve learning and memory deficits induced by scopolamine (1 mg/kg, subcutaneously) in mice. The investigators also assessed CB1R occupancy in the brain to ensure target engagement of MK-7128, and showed that MK-7128 significantly improved both Y-maze spontaneous alternation and object habituation performance in scopolamine-treated mice and inhibits the binding of radioiodinated AM251 in murine cortex and hippocampus. These data indicate that MK-7128 improves cognitive performance in a model of cholinergic hypofunction and suggest that efficacy is achieved at relatively low levels of CB1R occupancy in the brain. Our results extend earlier findings suggesting a role of CB1Rs in the modulation of memory processes and a potential therapeutic application for CB1R inverse agonists in cognitive disorders.
Assuntos
Azetidinas/farmacologia , Agonismo Inverso de Drogas , Transtornos da Memória/tratamento farmacológico , Oxidiazóis/farmacologia , Receptor CB1 de Canabinoide/agonistas , Animais , Azetidinas/administração & dosagem , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Oxidiazóis/administração & dosagem , Piperidinas/metabolismo , Ligação Proteica , Pirazóis/metabolismo , EscopolaminaRESUMO
Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.
Assuntos
Imunoconjugados , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/química , Venenos de Aranha/química , Bloqueadores do Canal de Sódio Disparado por Voltagem , Animais , Anticorpos/química , Descoberta de Drogas , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Masculino , Camundongos , Terapia de Alvo Molecular , Canal de Sódio Disparado por Voltagem NAV1.7/imunologia , Peptídeos/farmacocinética , Venenos de Aranha/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinéticaRESUMO
Inhibitors of the voltage-gated sodium channel NaV1.7 are being investigated as pain therapeutics due to compelling human genetics. We previously identified NaV1.7-inhibitory peptides GpTx-1 and JzTx-V from tarantula venom screens. Potency and selectivity were modulated through attribute-based positional scans of native residues via chemical synthesis. Herein, we report JzTx-V lead optimization to identify a pharmacodynamically active peptide variant. Molecular docking of peptide ensembles from NMR into a homology model-derived NaV1.7 structure supported prioritization of key residues clustered on a hydrophobic face of the disulfide-rich folded peptide for derivatization. Replacing Trp24 with 5-Br-Trp24 identified lead peptides with activity in electrophysiology assays in engineered and neuronal cells. 5-Br-Trp24 containing peptide AM-6120 was characterized in X-ray crystallography and pharmacokinetic studies and blocked histamine-induced pruritis in mice after subcutaneous administration, demonstrating systemic NaV1.7-dependent pharmacodynamics. Our data suggests a need for high target coverage based on plasma exposure for impacting in vivo end points with selectivity-optimized peptidic NaV1.7 inhibitors.
Assuntos
Descoberta de Drogas , Histamina/efeitos adversos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Prurido/tratamento farmacológico , Venenos de Aranha/química , Animais , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Canal de Sódio Disparado por Voltagem NAV1.7/química , Peptídeos/farmacocinética , Peptídeos/uso terapêutico , Conformação Proteica , Dobramento de Proteína , Prurido/induzido quimicamente , Relação Estrutura-Atividade , Distribuição Tecidual , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
Several reports have recently emerged regarding the identification of heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. The optimization of a series of internal NaV1.7 leads that address a number of metabolic liabilities including bioactivation, PXR activation, as well as CYP3A4 induction and inhibition led to the identification of potent and selective inhibitors that demonstrated favorable pharmacokinetic profiles and were devoid of the aforementioned liabilities. The key to achieving this within a series prone to transporter-mediated clearance was the identification of a small range of optimal cLogD values and the discovery of subtle PXR SAR that was not lipophilicity dependent. This enabled the identification of compound 20, which was advanced into a target engagement pharmacodynamic model where it exhibited robust reversal of histamine-induced scratching bouts in mice.
Assuntos
Isoquinolinas/química , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Sulfonamidas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Animais , Linhagem Celular , Citocromo P-450 CYP3A/biossíntese , Inibidores do Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/farmacocinética , Inibidores do Citocromo P-450 CYP3A/farmacologia , Cães , Indução Enzimática , Histamina , Humanos , Isoquinolinas/administração & dosagem , Isoquinolinas/farmacocinética , Masculino , Camundongos Endogâmicos C57BL , Receptor de Pregnano X , Prurido/induzido quimicamente , Prurido/prevenção & controle , Ratos , Receptores de Esteroides/agonistas , Relação Estrutura-Atividade , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
Because of its strong genetic validation, NaV1.7 has attracted significant interest as a target for the treatment of pain. We have previously reported on a number of structurally distinct bicyclic heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. Herein, we report the discovery and optimization of a series of atropisomeric quinolinone sulfonamide inhibitors [ Bicyclic sulfonamide compounds as sodium channel inhibitors and their preparation . WO 2014201206, 2014 ] of NaV1.7, which demonstrate nanomolar inhibition of NaV1.7 and exhibit high levels of selectivity over other sodium channel isoforms. After optimization of metabolic and pharmacokinetic properties, including PXR activation, CYP2C9 inhibition, and CYP3A4 TDI, several compounds were advanced into in vivo target engagement and efficacy models. When tested in mice, compound 39 (AM-0466) demonstrated robust pharmacodynamic activity in a NaV1.7-dependent model of histamine-induced pruritus (itch) and additionally in a capsaicin-induced nociception model of pain without any confounding effect in open-field activity.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Quinolonas/química , Sulfonamidas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Analgésicos/química , Analgésicos/farmacocinética , Analgésicos/farmacologia , Animais , Capsaicina , Linhagem Celular , Cães , Histamina , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Dor/induzido quimicamente , Dor/prevenção & controle , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Prurido/induzido quimicamente , Prurido/prevenção & controle , Quinolonas/administração & dosagem , Quinolonas/síntese química , Quinolonas/farmacocinética , Quinolonas/farmacologia , Ratos , Relação Estrutura-Atividade , Sulfonamidas/administração & dosagem , Sulfonamidas/síntese química , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
[This corrects the article DOI: 10.1021/acsmedchemlett.6b00243.].
RESUMO
The majority of potent and selective hNaV1.7 inhibitors possess common pharmacophoric features that include a heteroaryl sulfonamide headgroup and a lipophilic aromatic tail group. Recently, reports of similar aromatic tail groups in combination with an acyl sulfonamide headgroup have emerged, with the acyl sulfonamide bestowing levels of selectivity over hNaV1.5 comparable to the heteroaryl sulfonamide. Beginning with commercially available carboxylic acids that met selected pharmacophoric requirements in the lipophilic tail, a parallel synthetic approach was applied to rapidly generate the derived acyl sulfonamides. A biaryl acyl sulfonamide hit from this library was elaborated, optimizing for potency and selectivity with attention to physicochemical properties. The resulting novel leads are potent, ligand and lipophilic efficient, and selective over hNaV1.5. Representative lead 36 demonstrates selectivity over other human NaV isoforms and good pharmacokinetics in rodents. The biaryl acyl sulfonamides reported herein may also offer ADME advantages over known heteroaryl sulfonamide inhibitors.
Assuntos
Benzamidas/química , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Sulfonamidas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Animais , Benzamidas/síntese química , Benzamidas/farmacocinética , Benzamidas/farmacologia , Linhagem Celular , Feminino , Histamina , Humanos , Masculino , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Prurido/induzido quimicamente , Prurido/tratamento farmacológico , Ensaio Radioligante , Ratos , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/síntese química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
Human genetic evidence has identified the voltage-gated sodium channel NaV1.7 as an attractive target for the treatment of pain. We initially identified naphthalene sulfonamide 3 as a potent and selective inhibitor of NaV1.7. Optimization to reduce biliary clearance by balancing hydrophilicity and hydrophobicity (Log D) while maintaining NaV1.7 potency led to the identification of quinazoline 16 (AM-2099). Compound 16 demonstrated a favorable pharmacokinetic profile in rat and dog and demonstrated dose-dependent reduction of histamine-induced scratching bouts in a mouse behavioral model following oral dosing.
RESUMO
We report the identification of a PDE10A clinical candidate by optimizing potency and in vivo efficacy of promising keto-benzimidazole leads 1 and 2. Significant increase in biochemical potency was observed when the saturated rings on morpholine 1 and N-acetyl piperazine 2 were changed by a single atom to tetrahydropyran 3 and N-acetyl piperidine 5. A second single atom modification from pyrazines 3 and 5 to pyridines 4 and 6 improved the inhibitory activity of 4 but not 6. In the in vivo LC-MS/MS target occupancy (TO) study at 10 mg/kg, 3, 5, and 6 achieved 86-91% occupancy of PDE10A in the brain. Furthermore, both CNS TO and efficacy in PCP-LMA behavioral model were observed in a dose dependent manner. With superior in vivo TO, in vivo efficacy and in vivo PK profiles in multiple preclinical species, compound 5 (AMG 579) was advanced as our PDE10A clinical candidate.
Assuntos
Antipsicóticos/química , Benzimidazóis/química , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/metabolismo , Pirazinas/química , Administração Oral , Animais , Antipsicóticos/síntese química , Antipsicóticos/farmacologia , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Disponibilidade Biológica , Encéfalo/metabolismo , Cães , Humanos , Masculino , Atividade Motora/efeitos dos fármacos , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/química , Primatas , Conformação Proteica , Pirazinas/síntese química , Pirazinas/farmacologia , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Sustained attention is defined as the ability or capacity to remain focused on the occurrence of rare events over long periods of time. We describe here the development of a novel, operant-based attention task that can be learned by mice in 8-10 days. Mice were trained on a 2-choice visual discrimination task in an operant chamber, wherein the correct response on any given trial was a lever-press cued by a stimulus light. Upon reaching a criterion of greater than 80% correct responses, all subjects were tested in a mixed-trial attention paradigm combining four different stimulus durations within a single session (0.5, 1, 2, or 10 s). During attention testing, the percentage of correct responses decreased as a function of stimulus duration, indicating a performance decrement which parallels increasing attentional demand within the task. Pretreatment with the muscarinic-receptor antagonist scopolamine yielded a reliable, dose-dependent performance deficit whereas nicotine treatment improved the percentage of correct responses during trials with the greatest attentional demand. Moreover, medial prefrontal cortex lesions impaired attention performance without affecting acquisition or retention of the discrimination rule. These results underscore the utility of this task as a novel means of assessing attentional processes in mice in a relatively high-throughput manner.