Selective Estrogen Receptor Modulators Enhance CNS Remyelination Independent of Estrogen Receptors.

A significant unmet need for patients with multiple sclerosis (MS) is the lack of U.S. Food and Drug Administration (FDA)-approved remyelinating therapies. We have identified a compelling remyelinating agent, bazedoxifene (BZA), a European Medicines Agency (EMA)-approved (and FDA-approved in combination with conjugated estrogens) selective estrogen receptor (ER) modulator (SERM) that could move quickly from bench to bedside. This therapy stands out as a tolerable alternative to previously identified remyelinating agents and other candidates within this family. Using an unbiased high-throughput screen, with subsequent validation in both murine and human oligodendrocyte precursor cells (OPCs) and coculture systems, we find that BZA enhances differentiation of OPCs into functional oligodendrocytes. Using an in vivo murine model of focal demyelination, we find that BZA enhances OPC differentiation and remyelination. Of critical importance, we find that BZA acts independently of its presumed target, the ER, in both in vitro and in vivo systems. Using a massive computational data integration approach, we independently identify six possible candidate targets through which SERMs may mediate their effect on remyelination. Of particular interest, we identify EBP (encoding 3ß-hydroxysteroid-Δ8,Δ7-isomerase), a key enzyme in the cholesterol biosynthesis pathway, which was previously implicated as a target for remyelination. These findings provide valuable insights into the implications for SERMs in remyelination for MS and hormonal research at large.SIGNIFICANCE STATEMENT Therapeutics targeted at remyelination failure, which results in axonal degeneration and ultimately disease progression, represent a large unmet need in the multiple sclerosis (MS) population. Here, we have validated a tolerable European Medicines Agency-approved (U.S. Food and Drug Administration-approved in combination with conjugated estrogens) selective estrogen receptor (ER) modulator (SERM), bazedoxifene (BZA), as a potent agent of oligodendrocyte precursor cell (OPC) differentiation and remyelination. SERMs, which were developed as nuclear ER-α and ER-ß agonists/antagonists, have previously been implicated in remyelination and neuroprotection, following a heavy focus on estrogens with underwhelming and conflicting results. We show that nuclear ERs are not required for SERMs to mediate their potent effects on OPC differentiation and remyelination in vivo and highlight EBP, an enzyme in the cholesterol biosynthesis pathway that could potentially act as a target for SERMs.

Discovery of potent and selective PPARα/δ dual antagonists and initial biological studies.

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.

In vitro and in vivo pharmacology of NXT629, a novel and selective PPARα antagonist.

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.

Identification of the first potent, selective and bioavailable PPARα antagonist.

The discovery and SAR of a novel series of potent and selective PPARα antagonists are herein described. Exploration of replacements for the labile acyl sulfonamide linker led to a biaryl sulfonamide series of which compound 33 proved to be suitable for further profiling in vivo. Compound 33 demonstrated excellent potency, selectivity against other nuclear hormone receptors, and good pharmacokinetics in mouse.

The antinociceptive effect of the asthma drug ibudilast in rat models of peripheral and central neuropathic pain.

Ibudilast, an asthma drug, has demonstrated antinociceptive effects in several rat models of peripheral neuropathic pain, and a proposed mechanism of action is the inhibition of release of the cytokine tumor necrosis factor-α (TNF-α) from activated spinal microglia. Spinal glial activation has also been demonstrated in rat models of central neuropathic pain following spinal cord injury (SCI). The current study evaluated the effect of a short course of treatment with ibudilast on SCI-induced pain, and for comparison, following a chronic constriction injury (CCI; the Bennett model) of the sciatic nerve in rats. The effects of ibudilast treatment on spinal (SCI and CCI rats), and nerve tissue (CCI only) TNF-α content were also evaluated. Following an acute midthoracic SCI with a microvascular clip (20-g force), hindpaw withdrawal thresholds were significantly decreased, indicating below-level cutaneous tactile hypersensitivity. Likewise, unilateral loose ligation of the sciatic nerve led to a robust ipsilateral tactile hypersensitivity. Rats were treated with either ibudilast (10 mg/kg IP) or vehicle (2 mL/kg) during the period of robust and steady hindpaw hypersensitivity for each model--CCI rats were treated 14-16 days post-surgery, and SCI rats were treated 30-32 days post-surgery--and tested daily. Ibudilast ameliorated hindpaw hypersensitivity in both SCI and CCI rats, whereas vehicle treatment had no effect. Interestingly, repeated treatment led to increased baseline thresholds, beyond the duration of the drug half-life, suggesting persistent changes in neuropathic pain processing. In SCI rats, an increase in TNF-α content in spinal tissue rostral to the SCI was observed. Ibudilast treatment did not significantly alter this increase. In rats with a CCI, TNF-α content was markedly increased in the ipsilateral sciatic nerve and was partially reduced following ibudilast, but not vehicle, treatment. Ibudilast could be useful for the treatment of neuropathic pain of central as well as peripheral origin.

Estrogen receptor (ER) subtype agonists alter monoamine levels in the female rat brain.

We assessed the effects of subtype-selective ER agonists on monoamine levels in discrete regions of the female rat brain. Ovariectomized (ovx) rats were treated for 4 days with vehicle, 17ß-estradiol (E; 0.05mg/kg), an ERß agonist (C19; 3mg/kg) or an ERα agonist (PPT; 3mg/kg) and samples from brain regions were assessed for monoamines and metabolites. We also assessed effects of ERß modulation on baseline and fenfluramine-induced release of monoamines in hippocampus using microdialysis. In the first study, E and the ERα agonist increased norepinephrine in cortex and all three ER ligands increased it in the ventral hippocampus. Changes in levels of the noradrenergic metabolite, MHPG and the dopaminergic metabolite, DOPAC were noted in brain areas of ER ligand-treated animals. E also increased levels of 5HIAA in three brain areas. In the microdialysis study, there were no differences among groups in baseline levels of monoamines. However, E and the ERß agonist increased levels of the dopaminergic metabolite, HVA following fenfluramine. In summary, activation of the two nuclear ERs with selective agonists affects monoamine and metabolite levels in discrete brain areas, a number of which are known to play key roles in cognitive and affective function.

Pharmacology of AM803, a novel selective five-lipoxygenase-activating protein (FLAP) inhibitor in rodent models of acute inflammation.

We evaluated the in vivo pharmacological properties of AM803 3-[3-tert-butylsulfanyl-1-[4-(6-ethoxy-pyridin-3-yl)-benzyl]-5-(5-methyl-pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic acid, a selective five-lipoxygenase-activating protein (FLAP) inhibitor, using rat and mouse models of acute inflammation. Oral administration of AM803 (1 mg/kg) resulted in sustained inhibition of ex vivo ionophore-challenged whole blood LTB4 biosynthesis with >90% inhibition for up to 12 h and an EC50 of approximately 7 nM. When rat lungs were challenged in vivo with calcium-ionophore, AM803 inhibited LTB4 and cysteinyl leukotriene (CysLT) production with ED50s of 0.12 mg/kg and 0.37 mg/kg, respectively. The inhibition measured 16 h following a single oral dose of 3 mg/kg was 86% and 41% for LTB4 and CysLTs, respectively. In an acute inflammation setting, AM803 dose-dependently reduced LTB4, CysLTs, plasma protein extravasation and neutrophil influx induced by peritoneal zymosan injection. Finally, AM803 increased survival time in mice exposed to a lethal intravenous injection of platelet activating factor (PAF). The magnitude of effect was similar to that of an inhibitor of five-lipoxygenase (5-LO) and LTA4 hydrolase but superior to a leukotriene CysLT1 receptor antagonist. In summary, AM803 is a novel, potent and selective FLAP inhibitor that has excellent pharmacodynamic properties in vivo and is effective in animal models of acute inflammation and in a model of lethal shock.

Therapeutic efficacy of AM156, a novel prostanoid DP2 receptor antagonist, in murine models of allergic rhinitis and house dust mite-induced pulmonary inflammation.

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.

Pharmacological blockade of the DP2 receptor inhibits cigarette smoke-induced inflammation, mucus cell metaplasia, and epithelial hyperplasia in the mouse lung.

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.

A novel 5-lipoxygenase-activating protein inhibitor, AM679, reduces inflammation in the respiratory syncytial virus-infected mouse eye.

Respiratory syncytial virus (RSV) is an important cause of viral respiratory disease in children, and RSV bronchiolitis has been associated with the development of asthma in childhood. RSV spreads from the eye and nose to the human respiratory tract. Correlative studies of humans and direct infection studies of BALB/c mice have established the eye as a significant pathway of entry of RSV to the lung. At the same time, RSV infection of the eye produces symptoms resembling allergic conjunctivitis. Cysteinyl leukotrienes (CysLTs) are known promoters of allergy and inflammation, and the first step in their biogenesis from arachidonic acid is catalyzed by 5-lipoxygenase (5-LO) in concert with the 5-LO-activating protein (FLAP). We have recently developed a novel compound, AM679, which is a topically applied and potent inhibitor of FLAP. Here we show with the BALB/c mouse eye RSV infection model that AM679 markedly reduced the RSV-driven ocular pathology as well as the synthesis of CysLTs in the eye. In addition, AM679 decreased the production of the Th2 cell cytokine interleukin-4 but did not increase the viral load in the eye or the lung. These results suggest that FLAP inhibitors may be therapeutic for RSV-driven eye disease and possibly other inflammatory eye indications.

Pharmacological characterization of 3-[3-tert-butylsulfanyl-1-[4-(6-methoxy-pyridin-3-yl)-benzyl]-5-(pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic acid (AM103), a novel selective 5-lipoxygenase-activating protein inhibitor that reduces acute and chronic inflammation.

Leukotrienes (LTs) are proinflammatory lipid mediators synthesized by the conversion of arachidonic acid (AA) to LTA(4) by the enzyme 5-lipoxygenase (5-LO) in the presence of 5-LO-activating protein (FLAP). 3-[3-tert-Butylsulfanyl-1-[4-(6-methoxy-pyridin-3-yl)-benzyl]-5-(pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic acid (AM103) is a novel selective FLAP inhibitor in development for the treatment of respiratory conditions such as asthma. In a rat ex vivo whole-blood calcium ionophore-induced LTB(4) assay, AM103 (administered orally at 1 mg/kg) displayed >50% inhibition for up to 6 h with a calculated EC(50) of approximately 60 nM. When rat lung was challenged in vivo with calcium ionophore, AM103 inhibited LTB(4) and cysteinyl leukotriene (CysLT) production with ED(50) values of 0.8 and 1 mg/kg, respectively. In this model, the EC(50) derived from plasma AM103 was approximately 330 nM for inhibition of both LTB(4) and CysLT. In an acute inflammation setting, AM103 displayed dose-dependent inhibition of LTB(4), CysLT, and plasma protein extravasation induced by peritoneal zymosan injection. In a model of chronic lung inflammation using ovalbumin-primed and challenged BALB/c mice, AM103 reduced the concentrations of eosinophil peroxidase, CysLTs, and interleukin-5 in the bronchoalveolar lavage fluid. Finally, AM103 increased survival time in mice exposed to a lethal intravenous injection of platelet-activating factor. In summary, AM103 is a novel, potent and selective FLAP inhibitor that has excellent pharmacodynamic properties in vivo and is effective in animal models of acute and chronic inflammation and in a model of lethal shock.