RESUMO
Multiple sclerosis (MS) is a chronic and debilitating neurological disease that results in inflammatory demyelination. While endogenous remyelination helps to recover function, this restorative process tends to become less efficient over time. Currently, intense efforts aimed at the mechanisms that promote remyelination are being considered promising therapeutic approaches. The M1 muscarinic acetylcholine receptor (M1R) was previously identified as a negative regulator of oligodendrocyte differentiation and myelination. Here, we validate M1R as a target for remyelination by characterizing expression in human and rodent oligodendroglial cells (including those in human MS tissue) using a highly selective M1R probe. As a breakthrough to conventional methodology, we conjugated a fluorophore to a highly M1R selective peptide (MT7) which targets the M1R in the subnanomolar range. This allows for exceptional detection of M1R protein expression in the human CNS. More importantly, we introduce PIPE-307, a brain-penetrant, small-molecule antagonist with favorable drug-like properties that selectively targets M1R. We evaluate PIPE-307 in a series of in vitro and in vivo studies to characterize potency and selectivity for M1R over M2-5R and confirm the sufficiency of blocking this receptor to promote differentiation and remyelination. Further, PIPE-307 displays significant efficacy in the mouse experimental autoimmune encephalomyelitis model of MS as evaluated by quantifying disability, histology, electron microscopy, and visual evoked potentials. Together, these findings support targeting M1R for remyelination and support further development of PIPE-307 for clinical studies.
Assuntos
Esclerose Múltipla , Oligodendroglia , Receptor Muscarínico M1 , Remielinização , Animais , Humanos , Camundongos , Ratos , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Camundongos Endogâmicos C57BL , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Antagonistas Muscarínicos/farmacologia , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Oligodendroglia/efeitos dos fármacos , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M1/antagonistas & inibidores , Remielinização/efeitos dos fármacosRESUMO
Multiple sclerosis (MS) is a chronic neurological disease characterized by inflammatory demyelination that disrupts neuronal transmission resulting in neurodegeneration progressive disability. While current treatments focus on immunosuppression to limit inflammation and further myelin loss, no approved therapies effectively promote remyelination to mitigate the progressive disability associated with chronic demyelination. Lysophosphatidic acid (LPA) is a pro-inflammatory lipid that is upregulated in MS patient plasma and cerebrospinal fluid (CSF). LPA activates the LPA1 receptor, resulting in elevated CNS cytokine and chemokine levels, infiltration of immune cells, and microglial/astrocyte activation. This results in a neuroinflammatory response leading to demyelination and suppressed remyelination. A medicinal chemistry effort identified PIPE-791, an oral, brain-penetrant, LPA1 antagonist. PIPE-791 was characterized in vitro and in vivo and was found to be a potent, selective LPA1 antagonist with slow receptor off-rate kinetics. In vitro, PIPE-791 induced OPC differentiation and promoted remyelination following a demyelinating insult. PIPE-791 further mitigated the macrophage-mediated inhibition of OPC differentiation and inhibited microglial and fibroblast activation. In vivo, the compound readily crossed the blood-brain barrier and blocked LPA1 in the CNS after oral dosing. Direct dosing of PIPE-791 in vivo increased oligodendrocyte number, and in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS, we observed that PIPE-791 promoted myelination, reduced neuroinflammation, and restored visual evoked potential latencies (VEP). These findings support targeting LPA1 for remyelination and encourage development of PIPE-791 for treating MS patients with advantages not seen with current immunosuppressive disease modifying therapies.
Assuntos
Esclerose Múltipla , Receptores de Ácidos Lisofosfatídicos , Remielinização , Animais , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/metabolismo , Remielinização/efeitos dos fármacos , Humanos , Camundongos , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/metabolismo , Oligodendroglia/metabolismo , Oligodendroglia/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Diferenciação Celular/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Lisofosfolipídeos/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacosRESUMO
Structure-activity relationship studies led to the discovery of PIPE-3297, a fully efficacious and selective kappa opioid receptor (KOR) agonist. PIPE-3297, a potent activator of G-protein signaling (GTPγS EC50 = 1.1 nM, 91% Emax), did not elicit a ß-arrestin-2 recruitment functional response (Emax < 10%). Receptor occupancy experiments performed with the novel KOR radiotracer [3H]-PIPE-3113 revealed that subcutaneous (s.c.) administration of PIPE-3297 at 30 mg/kg in mice achieved 90% occupancy of the KOR in the CNS 1 h post dose. A single subcutaneous dose of PIPE-3297 in healthy mice produced a statistically significant increase of mature oligodendrocytes (P < 0.0001) in the KOR-enriched striatum, an effect that was not observed in animals predosed with the selective KOR antagonist norbinaltorphimine. An equivalent dose given to mice in an open-field activity-monitoring system revealed a small KOR-independent decrease in total locomotor activity versus vehicle measured between 60 and 75 min post dose. Daily doses of PIPE-3297 at both 3 and 30 mg/kg s.c. reduced the disease score in the mouse experimental autoimmune encephalomyelitis (EAE) model. Visually evoked potential (VEP) N1 latencies were also significantly improved versus vehicle in both dose groups, and latencies matched those of untreated animals. Taken together, these findings highlight the potential therapeutic value of functionally selective G-protein KOR agonists in demyelinating disease, which may avoid the sedating side effects typically associated with classical nonbiased KOR agonists.
Assuntos
Receptores Opioides kappa , Transdução de Sinais , Camundongos , Animais , beta-Arrestina 2/farmacologia , Receptores Opioides kappa/agonistas , Proteínas de Ligação ao GTP/metabolismo , Antagonistas de Entorpecentes/farmacologia , Analgésicos Opioides/farmacologiaRESUMO
The discovery of PIPE-359, a brain-penetrant and selective antagonist of the muscarinic acetylcholine receptor subtype 1 is described. Starting from a literature-reported M1 antagonist, linker replacement and structure-activity relationship investigations of the eastern 1-(pyridinyl)piperazine led to the identification of a novel, potent, and selective antagonist with good MDCKII-MDR1 permeability. Continued semi-iterative positional scanning facilitated improvements in the metabolic and hERG profiles, which ultimately delivered PIPE-359. This advanced drug candidate exhibited robust efficacy in mouse myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalitis (EAE), a preclinical model for multiple sclerosis.
RESUMO
We previously published on the design and synthesis of novel, potent and selective PPARα antagonists suitable for either i.p. or oral in vivo administration for the potential treatment of cancer. Described herein is SAR for a subsequent program, where we set out to identify selective and potent PPARα/δ dual antagonist molecules. Emerging literature indicates that both PPARα and PPARδ antagonism may be helpful in curbing the proliferation of certain types of cancer. This dual antagonism could also be used to study PPARs in other settings. After testing for selective and dual potency, off-target counter screening, metabolic stability, oral bioavailability and associated toxicity, compound 11, the first reported PPARα/δ dual antagonist was chosen for more advanced preclinical evaluation.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Neoplasias Ovarianas/tratamento farmacológico , PPAR alfa/antagonistas & inibidores , PPAR delta/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , PPAR alfa/metabolismo , PPAR delta/metabolismo , Ratos , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/químicaRESUMO
Peroxisome-proliferator activated receptors (PPAR) are members of the nuclear hormone receptor superfamily which regulate gene transcription. PPARα is a key regulator of lipid homeostasis and a negative regulator of inflammation. Under conditions of metabolic stress such as fasting or glucose deprivation, PPARα is upregulated in order to control gene expression necessary for processing alternate fuel sources (e.g. fatty acid oxidation) and thereby promote maintenance of cell viability. Clinically, PPARα expression is upregulated in diseased tissues such as melanoma, chronic lymphocytic leukemia, ovarian and prostate cancer. This may allow for cellular proliferation and metastasis. Importantly, genetic knockouts of PPARα have been shown to be protected against tumor growth in a variety of syngeneic tumors models. We hypothesized that a potent and selective PPARα antagonist could represent a novel cancer therapy. Early in our discovery research, we identified NXT629 (Bravo et al., 2014). Herein we describe the pharmacology of NXT629 and demonstrate that it is a potent and selective PPARα antagonist. We identify NXT629 as a valuable tool for use in in vivo assessment of PPARα due to its good systemic exposure following intraperitoneal injection. We explore the in vivo pharmacology of NXT629 and demonstrate that it is efficacious in pharmacodynamic models that are driven by PPARα. Finally, we probe the efficacy of NXT629 in disease models where PPARα knockouts have shown to be protected. We believe that PPARα antagonists will be beneficial in diseases such as ovarian cancer and melanoma where PPARα and fatty acid oxidation may be involved.
Assuntos
Aminopiridinas/farmacologia , PPAR alfa/antagonistas & inibidores , Sulfonamidas/farmacologia , Aminopiridinas/farmacocinética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/sangue , Humanos , Camundongos , Metástase Neoplásica , Neovascularização Fisiológica/efeitos dos fármacos , Ratos , Sulfonamidas/farmacocinéticaRESUMO
Demyelination in MS disrupts nerve signals and contributes to axon degeneration. While remyelination promises to restore lost function, it remains unclear whether remyelination will prevent axonal loss. Inflammatory demyelination is accompanied by significant neuronal loss in the experimental autoimmune encephalomyelitis (EAE) mouse model and evidence for remyelination in this model is complicated by ongoing inflammation, degeneration and possible remyelination. Demonstrating the functional significance of remyelination necessitates selectively altering the timing of remyelination relative to inflammation and degeneration. We demonstrate accelerated remyelination after EAE induction by direct lineage analysis and hypothesize that newly formed myelin remains stable at the height of inflammation due in part to the absence of MOG expression in immature myelin. Oligodendroglial-specific genetic ablation of the M1 muscarinic receptor, a potent negative regulator of oligodendrocyte differentiation and myelination, results in accelerated remyelination, preventing axonal loss and improving functional recovery. Together our findings demonstrate that accelerated remyelination supports axonal integrity and neuronal function after inflammatory demyelination.
RESUMO
BACKGROUND: Allergic conjunctivitis is characterized by itchy, watery and swollen eyes which occur in response to exposure to seasonal or environmental allergens. The early phase reaction of allergic conjunctivitis is primarily mediated by mast cell degranulation while the late phase reaction is driven by Th2 cells and eosinophils. Prostaglandin D(2) (PGD(2)), released from mast cells, is present in allergic conjunctival tears and may elicit classical allergic responses via interaction with the high-affinity DP2 receptor (chemoattractant receptor-homologous molecule expressed on Th2 cells, CRTh2). Furthermore, antagonism of this receptor is well known to inhibit eosinophil chemotaxis, basophil activation and Th2 cytokine production. PGD(2), therefore, may be involved in both early and late phase reactions in response to allergen challenge. METHODS: Thus, we explored whether our novel and selective DP2 antagonist AM156 would be efficacious in animal models of allergic conjunctivitis. Furthermore, as respiratory syncytial virus (RSV) has been implicated in the pathogenesis of allergic conjunctivitis, we examined the effects of DP2 antagonism in a murine model of RSV ocular infection. RESULTS: Utilizing a guinea pig ovalbumin model and a murine ragweed model we demonstrated that AM156 reduces redness, discharge and swelling in response to allergen challenge. These effects were equal to or greater than those of current clinical treatment options for allergic conjunctivitis including topical corticosteroids and a dual-mechanism antihistamine and decongestant. AM156 significantly reduced RSV-induced ocular inflammation and IL-4 production. CONCLUSION: These results suggest that a topical DP2 antagonist such as AM156 may represent a novel therapeutic for allergic conjunctivitis.
Assuntos
Antialérgicos/uso terapêutico , Benzilaminas/uso terapêutico , Conjuntivite Alérgica/tratamento farmacológico , Conjuntivite Viral/tratamento farmacológico , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Administração Tópica , Alérgenos/imunologia , Ambrosia/imunologia , Animais , Conjuntivite Alérgica/imunologia , Conjuntivite Alérgica/metabolismo , Conjuntivite Viral/imunologia , Conjuntivite Viral/metabolismo , Modelos Animais de Doenças , Feminino , Cobaias , Interleucina-4/imunologia , Interleucina-4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/metabolismoRESUMO
The prostaglandin D(2) (PGD(2)) receptor type 2 (DP2) is a G protein-coupled receptor that has been shown to be involved in a variety of allergic diseases, including allergic rhinitis, asthma, and atopic dermatitis. In this study, we describe the preclinical pharmacological and pharmacokinetic properties of the small-molecule DP2 antagonist [2'-(3-benzyl-1-ethyl-ureidomethyl)-6-methoxy-4'-trifluoromethyl-biphenyl-3-yl]-acetic acid (AM211). We determine that AM211 has high affinity for human, mouse, rat, and guinea pig DP2 and it shows selectivity over other prostanoid receptors and enzymes. Antagonist activity of AM211 at the DP2 receptor was confirmed by inhibition of PGD(2)-stimulated guanosine 5'-O-[γ-thio]triphosphate binding to membranes expressing human DP2. A basophil activation assay and a whole-blood assay of eosinophil shape change were used to demonstrate the ability of AM211 to potently antagonize PGD(2)-stimulated functional responses in relevant human cells and in the context of a physiologically relevant environment. AM211 exhibits good oral bioavailability in rats and dogs and dose-dependently inhibits 13,14-dihydro-15-keto-PGD(2)-induced leukocytosis in a guinea pig pharmacodynamic assay. AM211 demonstrates efficacy in two animal models of allergic inflammation, including an ovalbumin-induced lung inflammation model in guinea pigs and an ovalbumin-induced mouse model of allergic rhinitis. AM211 represents a potent and selective antagonist of DP2 that may be used clinically to evaluate the role of DP2 in T helper 2-driven allergic inflammatory diseases.
Assuntos
Modelos Animais de Doenças , Compostos de Metilureia/uso terapêutico , Fenilacetatos/uso terapêutico , Antagonistas de Prostaglandina/uso terapêutico , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Rinite Alérgica Perene/tratamento farmacológico , Adulto , Animais , Cães , Feminino , Cobaias , Células HEK293 , Humanos , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Masculino , Compostos de Metilureia/química , Compostos de Metilureia/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Fenilacetatos/química , Fenilacetatos/farmacologia , Pneumonia/tratamento farmacológico , Pneumonia/imunologia , Pneumonia/metabolismo , Antagonistas de Prostaglandina/química , Antagonistas de Prostaglandina/farmacologia , Ligação Proteica/fisiologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/imunologia , Receptores de Prostaglandina/metabolismo , Rinite Alérgica Perene/imunologia , Rinite Alérgica Perene/metabolismoRESUMO
Prostaglandin D(2) (PGD(2)) is derived from arachidonic acid and binds with high affinity to the G protein coupled receptors prostanoid DP(1) and DP(2). Interaction with DP(2) results in cell chemotaxis, eosinophil degranulation, eosinophil shape change, adhesion molecule upregulation and Th2 cytokine production. In allergic rhinitis and allergic asthma PGD(2) is released from mast cells in response to allergen challenge and may trigger symptoms such as sneezing, rhinorrhea, pruritus, mucus hypersecretion and pulmonary inflammation. In Japan, ramatroban, a dual prostanoid DP(2)/prostanoid TP receptor antagonist, is marketed for allergic rhinitis while selective DP(2) antagonists are currently under investigation as therapeutics for asthma and allergic rhinitis. In the studies described herein, we investigated the efficacy of AM156, a novel selective prostanoid DP(2) receptor antagonist, in murine models of allergic rhinitis and asthma. AM156 inhibited sneezing and nasal rubs in a model of allergic rhinitis. AM156 inhibited pulmonary inflammation and mucus hypersecretion induced by chronic inhalation of house dust mite. These results suggest that selective prostanoid DP(2) receptor antagonists such as AM156 may provide beneficial effects for the clinical treatment of diseases such as allergic rhinitis and asthma.
Assuntos
Antialérgicos/uso terapêutico , Asma/tratamento farmacológico , Benzilaminas/uso terapêutico , Pulmão/patologia , Pneumonia/tratamento farmacológico , Pyroglyphidae/imunologia , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Rinite Alérgica Perene/tratamento farmacológico , Animais , Antialérgicos/farmacologia , Benzilaminas/farmacologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Imunoglobulina E/sangue , Pulmão/imunologia , Metaplasia/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mucinas/metabolismo , Pneumonia/imunologia , Pneumonia/metabolismo , Rinite Alérgica Perene/metabolismoRESUMO
Prostaglandin D(2) (PGD(2)) is one of a family of biologically active lipids derived from arachidonic acid via the action of COX-1 and COX-2. PGD(2) is released from mast cells and binds primarily to two G protein-coupled receptors, namely DP1 and DP2, the latter also known as chemoattractant receptor-homologous molecule expressed on Th2 cells. DP2 is predominantly expressed on eosinophils, Th2 cells, and basophils, but it is also expressed to a lesser extent on monocytes, mast cells, and epithelial cells. Interaction of PGD(2) and its active metabolites with DP2 results in cellular chemotaxis, degranulation, up-regulation of adhesion molecules, and cytokine production. Chronic obstructive pulmonary disease (COPD) is a chronic progressive inflammatory disease characterized by elevated lung neutrophils, macrophages, and CD8+ T lymphocytes and mucus hypersecretion. Cigarette smoke contributes to the etiology of COPD and was used here as a provoking agent in a murine model of COPD. In an acute model, {2'-[(cyclopropanecarbonyl-ethyl-amino)-methyl]-6-methoxy-4'-trifluoro-methyl-biphenyl-3-yl}-acetic acid, sodium salt (AM156) and (5-{2-[(benzoyloxycarbonyl-ethyl-amino)-methyl]-4-trifluoromethyl-phenyl}-pyridin-3-yl)-acetic acid, sodium salt) (AM206), potent DP2 receptor antagonists, dose-dependently inhibited influx of neutrophils and lymphocytes to smoke-exposed airways. In a subchronic model, AM156 and AM206 inhibited neutrophil and lymphocyte trafficking to the airways. Furthermore, AM156 and AM206 treatment inhibited mucus cell metaplasia and prevented the thickening of the airway epithelial layer induced by cigarette smoke. These data suggest that DP2 receptor antagonism may represent a novel therapy for COPD or other conditions characterized by neutrophil influx, mucus hypersecretion, and airway remodeling.
Assuntos
Pulmão/efeitos dos fármacos , Muco/metabolismo , Doença Pulmonar Obstrutiva Crônica/prevenção & controle , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Mucosa Respiratória/efeitos dos fármacos , Fumar/efeitos adversos , Animais , Benzilaminas/farmacocinética , Benzilaminas/farmacologia , Linhagem Celular , Movimento Celular , Feminino , Cobaias , Humanos , Técnicas In Vitro , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Pulmão/imunologia , Pulmão/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Masculino , Metaplasia , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Niacina/análogos & derivados , Niacina/farmacocinética , Niacina/farmacologia , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/patologiaRESUMO
The aim of the present study was to examine the ability of the nonpungent vanilloid VR1 receptor agonist, olvanil, to activate the afferent and efferent function of capsaicin-sensitive C-fibers in guinea pig airways. We found that while capsaicin (10 nM-10 microM) and resiniferatoxin (0.1 nM-1.0 microM) evoked a robust contraction of the guinea pig trachea in vitro, olvanil (10 nM-10 microM) was a weak spasmogen. In addition, pretreatment with olvanil caused only a minor reduction of subsequent responses to capsaicin or resiniferatoxin. Using single fiber recording from guinea pig airway C-fibers, we found that olvanil (10 microM) did not evoke action potential discharge although these fibers responded vigorously to capsaicin after prolonged treatment with olvanil (10 microM). These findings are indicative of significant differences in the relative sensitivity of vanilloid VR1 receptor-transfected cells and the peripheral terminals of airway C-fibers to pungent and nonpungent vanilloid VR1 receptor agonists.