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.

Pharmacology of AM211, a potent and selective prostaglandin D2 receptor type 2 antagonist that is active in animal models of allergic inflammation.

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.

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.

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.