Improvement of physiochemical properties of the tetrahydroazepinoindole series of farnesoid X receptor (FXR) agonists: beneficial modulation of lipids in primates.

In an effort to develop orally active farnesoid X receptor (FXR) agonists, a series of tetrahydroazepinoindoles with appended solubilizing amine functionalities were synthesized. The crystal structure of the previously disclosed FXR agonist, 1 (FXR-450), aided in the design of compounds with tethered solubilizing functionalities designed to reach the solvent cavity around the hFXR receptor. These compounds were soluble in 0.5% methylcellulose/2% Tween-80 in water (MC/T) for oral administration. In vitro and in vivo optimization led to the identification of 14dd and 14cc, which in a dose-dependent fashion regulated low density lipoprotein cholesterol (LDLc) in low density lipoprotein receptor knockout (LDLR(-/-)) mice. Compound 14cc was dosed in female rhesus monkeys for 4 weeks at 60 mg/kg daily in MC/T vehicle. After 7 days, triglyceride (TG) levels and very low density lipoprotein cholesterol (VLDLc) levels were significantly decreased and LDLc was decreased 63%. These data are the first to demonstrate the dramatic lowering of serum LDLc levels by a FXR agonist in primates and supports the potential utility of 14cc in treating dyslipidemia in humans beyond just TG lowering.

Oligomerization is required for the activity of recombinant soluble LOX-1.

LOX-1 is a scavenger receptor that functions as the primary receptor for oxidized low-density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX-1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX-1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand-binding function of cell surface LOX-1, we generated a series of recombinant human LOX-1-crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand-binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX-1 is required for high-avidity binding of ligands, we found that LOX-1-Fc fusion protein containing four ligand-binding domains per Fc dimer, but not the one containing two ligand-binding domains, exhibited ligand-binding activity. Optimal ligand-binding activity could be achieved via crosslinking of LOX-1-Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX-1-Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX-1. These findings demonstrate that functional oligomerization is required for recombinant LOX-1-Fc to function as an effective antagonist.

Pyrrole[2,3-d]azepino compounds as agonists of the farnesoid X receptor (FXR).

Pyrrole[2,3-d]azepines have been identified as potent agonists of the farnesoid X receptor (FXR). Based on the planar X-ray crystal structure of WAY-362450 1 in the ligand binding domain and molecular modeling studies, non-planar reduced compounds were designed which led to agonists that exhibit high aqueous solubility and retain moderate in vitro potency.

Farnesoid X receptor agonist WAY-362450 attenuates liver inflammation and fibrosis in murine model of non-alcoholic steatohepatitis.

BACKGROUND/AIMS: The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily, which plays an essential role in the regulation of enterohepatic circulation and lipid homeostasis. Here we investigated whether WAY-362450, a synthetic potent FXR agonist, could protect against non-alcoholic steatohepatitis (NASH) in mice fed a methionine and choline-deficient (MCD) diet. METHODS: Male C57BL/6 mice on the MCD diet were treated with or without WAY-362450 (30 mg/kg) for 4 weeks. RESULTS: The elevations of serum ALT and AST activities induced by the MCD diet were decreased with WAY-362450 treatment. In terms of liver histology, while WAY-362450 treatment showed no impact on hepatic triglyceride accumulation, it significantly reduced inflammatory cell infiltration and hepatic fibrosis. The reduction in inflammatory cell infiltration correlated with deceased serum levels of keratinocyte derived chemokine (mKC) and MCP 1 and decreased hepatic gene expression of MCP-1 and VCAM-1. The reduction of hepatic fibrosis by WAY-362450 treatment corresponded to a reduction in hepatic gene expression of fibrosis markers. The positive effects of WAY-362450 were FXR-dependent since no protection was observed in MCD diet-fed FXR deficient mice. CONCLUSIONS: These findings demonstrate that FXR agonists may be useful for the treatment of non-alcoholic steatohepatitis.

CRP is a novel ligand for the oxidized LDL receptor LOX-1.

C-reactive protein (CRP) is a risk factor for cardiovascular events and functions to amplify vascular inflammation through promoting endothelial dysfunction. Lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 (LOX-1) is the primary endothelial receptor for oxLDL, and both its expression and function are associated with vascular inflammation. As a scavenger receptor, LOX-1 is capable of binding to a variety of structurally unrelated ligands. Evidence is provided that demonstrates that CRP can act as a novel ligand for LOX-1. The direct interaction between these two proteins was demonstrated with purified protein in both ELISA and AlphaScreen assays. This interaction could be disrupted with known LOX-1 ligands, such as oxLDL and carrageenan. Moreover, the CRP interaction with cell surface-expressed LOX-1 was confirmed in cell-based immunofluorescent-binding studies. Mutagenesis studies demonstrated that the arginine residues forming the basic spine structure on the LOX-1 ligand-binding interface were dispensable for CRP binding, suggesting a novel ligand-binding mechanism for LOX-1, distinct from that used for oxLDL binding. The treatment of human endothelial cells with CRP led to the activation of proinflammatory genes including IL-8, ICAM-1, and VCAM-1. The inductions of these genes by CRP were LOX-1 dependent, as demonstrated by their attenuation in cells transfected with LOX-1 small-interfering RNA. Our study identifies and characterizes the direct interaction between LOX-1 and CRP and suggests that this interaction may mediate CRP-induced endothelial dysfunction.

A synthetic farnesoid X receptor (FXR) agonist promotes cholesterol lowering in models of dyslipidemia.

The nuclear hormone receptor farnesoid X receptor (FXR) plays a critical role in the regulation of bile acid, triglyceride (TG), and cholesterol homeostasis. WAY-362450 (FXR-450/XL335) is a potent synthetic FXR agonist as characterized in luciferase reporter assays and in mediating FXR target gene regulation in primary human and immortalized mouse hepatocytes. In vivo, WAY-362450 dose dependently decreased serum TG levels after 7 days of oral dosing in western diet-fed low-density lipoprotein receptor-/- mice and in the diabetic mouse strains KK-Ay and db/db comparable to that achieved with the peroxisome proliferator activated receptor-alpha agonist, fenofibrate. WAY-362450 treatment also reduced serum cholesterol levels via reductions in LDLc, VLDLc, and HDLc lipoprotein fractions that were not accompanied by hepatic cholesterol accumulation. This cholesterol lowering was dependent on FXR as demonstrated in a hypothyroid-induced hypercholesterolemia setting in FXR-/- mice. In fructose-fed models, WAY-362450 also decreased TG and VLDLc levels in rats and hamsters but significantly increased HDLc levels in rats while reducing HDLc levels in hamsters. The differential effect of WAY-362450 on HDLc is likely due to a murine-specific induction of endothelial lipase and scavenger receptor-BI that does not occur in rats. These studies demonstrate a consistent ability of WAY-362450 to lower both serum TG and cholesterol levels and suggest that synthetic FXR agonists may have clinical utility in the treatment of mixed dyslipidemia.

Suppression of interleukin-6-induced C-reactive protein expression by FXR agonists.

C-reactive protein (CRP), a human acute-phase protein, is a risk factor for future cardiovascular events and exerts direct pro-inflammatory and pro-atherogenic properties. The farnesoid X receptor (FXR), a member of the nuclear hormone receptor superfamily, plays an essential role in the regulation of enterohepatic circulation and lipid homeostasis. In this study, we report that two synthetic FXR agonists, WAY-362450 and GW4064, suppressed interleukin-6-induced CRP expression in human Hep3B hepatoma cells. Knockdown of FXR by short interfering RNA attenuated the inhibitory effect of the FXR agonists and also increased the ability of interleukin-6 to induce CRP production. Furthermore, treatment of wild type C57BL/6 mice with the FXR agonist, WAY-362450, attenuated lipopolysaccharide-induced serum amyloid P component and serum amyloid A3 mRNA levels in the liver, whereas no effect was observed in FXR knockout mice. These data provide new evidence for direct anti-inflammatory properties of FXR.

Estrogen receptor dependent inhibitors of NF-kappaB transcriptional activation-1 synthesis and biological evaluation of substituted 2-cyanopropanoic acid derivatives: pathway selective inhibitors of NF-kappaB, a potential treatment for rheumatoid arthritis.

Pathway selective ligands of the estrogen receptor inhibit transcriptional activation of proinflammatory genes mediated by NF-kappaB. Substituted 2-cyanopropanoic acid derivatives were developed leading to the discovery of WAY-204688, an orally active, pathway selective, estrogen receptor dependent anti-inflammatory agent. This propanamide was shown to be orally active in preclinical models of inflammatory diseases, such as rheumatoid arthritis, without the proliferative effect associated with traditional estrogens.

Estrogen receptor ligands in the control of pathogenic inflammation.

Inflammation is recognized as a key component in a number of diseases, including rheumatoid arthritis, inflammatory bowel disease and atherosclerosis. Although well known for their classic effects on the reproductive tract and action by means of estrogen response elements in gene promoters, estrogens are also known to possess anti-inflammatory activity. This was originally highlighted with the observation that pregnancy ameliorates symptoms of rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. Furthermore, the antagonistic cross talk between nuclear factor kappaB and estrogen receptor signaling pathways has been well documented. Recently, novel estrogen receptor ligands, pathway-selective ligands and estrogen receptor beta-selective ligands have been identified which demonstrate potent anti-inflammatory activity; these ligands are being analyzed for their therapeutic potential in pathogenic inflammation.

The pathway-selective estrogen receptor ligand WAY-169916 displays differential activity in ischemia-reperfusion injury models.

We previously reported on the development of a pathway-selective estrogen receptor (ER) ligand, WAY-169916, that has ER-dependent antiinflammatory activity and is devoid of classic ER transcriptional activity. In the current study, WAY-169916 and 17beta-estradiol (17beta-E2) were evaluated for protective activity in models of cardiac ischemia-reperfusion injury. In rats subjected to cardiac ischemia-reperfusion injury by occlusion of the left coronary artery, infarct size relative to the area at risk in the left ventricle was significantly attenuated by a single dose of 17beta-E2 (20 microg/kg, SC), and WAY-169916 administered SC (10 mg/kg) or IV (1 mg/kg) during the ischemia phase. In isolated hearts perfused on a Langendorff apparatus and subjected to global ischemia and reperfusion, 17beta-E2 and WAY-169916 both had direct cardioprotective activity when perfused at 1 microM but their effects varied between different end points. Perfusion with 17beta-E2 only improved recovery of left ventricle-developed pressure. Perfusion with WAY-169916 attenuated the elevation in perfusion pressure, diastolic pressure, and release of creatine kinase after ischemia. In contrast to 17alpha-ethinylestradiol, WAY-169916 had no classic estrogen effects on uterine weight or total serum cholesterol in rats treated for 4 days. The data demonstrate that the pathway-selective ER ligand WAY-169916 displays differential activity in vivo on different cardiovascular end points.

Bile acids induce adhesion molecule expression in endothelial cells through activation of reactive oxygen species, NF-kappaB, and p38.

Bile acids are synthesized in the liver, stored in gallbladder, and secreted into the intestine to aid in the absorption of lipid-soluble nutrients. In addition, bile acids also actively participate in regulation of gene expression through their ability to act as ligands for the nuclear receptor farnesoid X receptor or by activating kinase signaling pathways. Under cholestatic conditions, elevated levels of bile acids in the liver induce hepatic inflammation, and because bile acid levels are also elevated in the circulation, they might also induce vascular inflammation. To test this hypothesis, primary human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells were treated with bile acids, and the expression of ICAM-1, VCAM-1, and E-selectin were monitored. The three major bile acids found in the circulation, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid, all strongly induced both the mRNA and protein expression of ICAM-1 and VCAM-1. To delineate the mechanism, the experiments were conducted in the presence of various kinase inhibitors. The results demonstrate that the bile acid-mediated induction of adhesion molecule expression occurs by stimulation of NF-kappaB and p38 MAPK signaling pathways through the elevation in reactive oxygen species. The bile acid-induced cell surface expression of ICAM-1 and VCAM-1 was sufficient to result in the increased adhesion of THP-1 monocytes to the HUVEC, suggesting that elevated levels of bile acids in the circulation may cause endothelium dysfunction and contribute to the initiation of early events associated with vascular lesion formation.

Control of chronic inflammation with pathway selective estrogen receptor ligands.

The discovery of novel intervention points in the inflammatory pathway has been a focus of drug development in recent years. We have identified pathway selective ligands for the estrogen receptor (ER) that inhibit NF-kappaB mediated inflammatory gene expression causing a reduction of cytokines, chemokines, adhesion molecules and inflammatory enzymes. SAR development of a series of 4-(Indazol-3-yl)-phenols has led to the identification of WAY-169916 an orally active non-steroidal ligand with the potential use in the treatment of inflammatory diseases without the classical proliferative effects associated with non-selective estrogens.

Synthesis and activity of a new class of pathway-selective estrogen receptor ligands: hydroxybenzoyl-3,4-dihydroquinoxalin-2(1H)-ones.

The anti-inflammatory activity of non-selective estrogens has been attributed to their ability to antagonize the activity of nuclear factor kappaB (NF-kappaB), a known mediator of inflammatory responses. Here we report the identification of a potent new class of pathway-selective ER ligands that selectively antagonize NF-kappaB functional activity, while exhibiting a lack of classical estrogenic effect.

Substituted 4-hydroxyphenyl sulfonamides as pathway-selective estrogen receptor ligands.

The transcription factor nuclear factor-kappaB (NF-kappaB) is a key component in the onset of inflammation. We describe here a series of 4-hydroxyphenyl sulfonamide estrogen receptor (ER) ligands that selectively inhibit NK-kappaB transcriptional activity but are devoid of conventional estrogenic activity.

The activity of pathway-selective estrogen receptor ligands in experimental septic shock.

Estrogen receptors (ER) are widely expressed in multiple genital and nongenital tissues. Upon engagement of these receptors, multiple genes are affected in target tissues via estrogen response elements. Nonsteroidal pathway-selective ER ligands have recently been identified that inhibit NF-kappaB transcriptional activity and are devoid of conventional estrogenic activities on genital tissues. These pathway-selective ligands are potent anti-inflammatory agents in vivo and may prove to be of therapeutic utility in systemic inflammatory states. These pathway-selective ER ligands were tested in the murine listeriosis model, the neutropenic rat model, and the mouse cecal ligation and puncture model. WAY-204688 did not have any significant activity after systemic infection by Listeria monocytogenes. In the neutropenic rat model, WAY-204688 provided a significant survival benefit against an otherwise lethal challenge of Pseudomonas aeruginosa 12.4.4 compared with the control group (88% versus 25% survival; P < 0.05). Preservation of mucosal weight and prevention of histopathologic changes were observed with the administration of WAY-204688. Similar findings were observed in a cecal ligation and puncture model with WAY-204688 and a related compound WAY-169916. These results indicate that oral administration of these pathway-selective ER ligands preserved gastrointestinal barrier function and improve outcome in experimental models of systemic infection and inflammation. These agents may prove to be useful clinically as a novel treatment strategy for severe sepsis.

The utility of pathway selective estrogen receptor ligands that inhibit nuclear factor-kappa B transcriptional activity in models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that produces synovial proliferation and joint erosions. The pathologic lesions of RA are driven through the production of inflammatory mediators in the synovium mediated, in part, by the transcription factor NF-kappaB. We have identified a non-steroidal estrogen receptor ligand, WAY-169916, that selectively inhibits NF-kappaB transcriptional activity but is devoid of conventional estrogenic activity. The activity of WAY-169916 was monitored in two models of arthritis, the HLA-B27 transgenic rat and the Lewis rat adjuvant-induced model, after daily oral administration. In both models, a near complete reversal in hindpaw scores was observed as well as marked improvements in the histological scores. In the Lewis rat adjuvant model, WAY-169916 markedly suppresses the adjuvant induction of three serum acute phase proteins: haptoglobin, alpha1-acid glycoprotein (alpha1-AGP), and C-reactive protein (CRP). Gene expression experiments also demonstrate a global suppression of adjuvant-induced gene expression in the spleen, liver, and popliteal lymph nodes. Finally, WAY-169916 was effective in suppressing tumor necrosis factor-alpha-mediated inflammatory gene expression in fibroblast-like synoviocytes isolated from patients with RA. Together, these data suggest the utility of WAY-169916, and other compounds in its class, in treating RA through global suppression of inflammation via selective blockade of NF-kappaB transcriptional activity.

Bile acid signaling through FXR induces intracellular adhesion molecule-1 expression in mouse liver and human hepatocytes.

Previous studies have demonstrated a dramatic induction of inflammatory gene expression in livers from mice fed a high-fat, high-cholesterol diet containing cholate after 3-5 wk. To determine the contribution of cholate in mediating these inductions, C57BL/6 mice were fed a chow diet supplemented with increasing concentrations of cholic acid (CA) for 5 days. A dose-dependent induction in the hepatic levels of TNF-alpha, VCAM-1, ICAM-1, and SAA-2 mRNA were observed. As positive controls, a dose-dependent repression of cholesterol 7alpha-hydroxylase and a dose-dependent induction of small heterodimer partner (SHP) expression were also observed, suggesting that farnesoid X receptor (FXR) was activated. In addition, ICAM-1 and SHP mRNA levels were also induced in primary human hepatocytes when treated with chenodeoxycholic acid or GW4064, a FXR-selective agonist. The involvement of FXR in CA-induced inflammatory gene expression was further investigated in the human hepatic cell line HepG2. Both ICAM-1 and SHP expression were induced in a dose- and time-dependent manner by treatment with the FXR-selective agonist GW4064. Moreover, the induction of ICAM-1 by GW4064 was inhibited by the FXR antagonist guggulsterone or with transfection of FXR siRNA. Finally, the activity of FXR was mapped to a retinoic acid response element (RARE) site containing an imbedded farnesoid X response element (FXRE) on the human ICAM-1 promoter and FXR and retinoid X receptor were demonstrated to bind to this site. Finally, FXR-mediated activation of ICAM-1 could be further enhanced by TNF-alpha cotreatment in hepatocytes, suggesting a potential cooperation between cytokine and bile acid-signaling pathways during hepatic inflammatory events.

Identification of pathway-selective estrogen receptor ligands that inhibit NF-kappaB transcriptional activity.

Inflammation is now recognized as a key component in a number of diseases such as atherosclerosis, rheumatoid arthritis, and inflammatory bowel disease. The transcription factor NF-kappaB has been shown to be involved in both the early and late stages of the inflammatory-proliferative process. In this report, we describe the identification of the pathway-selective estrogen receptor (ER) ligand, WAY-169916, that inhibits NF-kappaB transcriptional activity but is devoid of conventional estrogenic activity. This pathway-selective ligand does not promote the classic actions of estrogens such as stimulation of uterine proliferation or ER-mediated gene expression, but is a potent antiinflammatory agent, as demonstrated in the HLA-B27 transgenic rat model of inflammatory bowel disease. Our results indicate the potential utility of pathway-selective ER ligands such as WAY-169916 in the treatment of chronic inflammatory diseases.

Synthesis and activity of substituted 4-(indazol-3-yl)phenols as pathway-selective estrogen receptor ligands useful in the treatment of rheumatoid arthritis.

Pathway-selective ligands for the estrogen receptor (ER) inhibit NF-kappaB-mediated inflammatory gene expression causing a reduction of cytokines, chemokines, adhesion molecules, and inflammatory enzymes. SAR development of a series of 4-(indazol-3-yl)phenols has led to the identification of WAY-169916 an orally active nonsteroidal ligand with the potential use in the treatment of rheumatoid arthritis without the classical proliferative effects associated with estrogens.

Beneficial effects of estrogen treatment in the HLA-B27 transgenic rat model of inflammatory bowel disease.

A well-established model of bowel inflammation is the HLA-B27 transgenic rat that exhibits a spontaneous disease phenotype resulting in chronic diarrhea caused by immune cell activation. Estrogens have previously been shown to modulate the immune system, and both estrogen receptors (ERalpha and ERbeta) are present in the intestine and cells of the immune system. Therefore, the ability of estrogen to ameliorate disease progression in the HLA-B27 transgenic rat was determined. HLA-B27 transgenic rats with chronic diarrhea were treated with 17alpha-ethynyl-17beta-estradiol (EE) for 5 days. EE treatment dramatically improved stool scores after only 3 days. Histological scores of the degree of ulceration, inflammatory cell infiltration, fibrosis, and lesion depth of the colon were also improved by EE treatment. Because neutrophil infiltration into the colon is involved in the development and propagation of disease, myeloperoxidase (MPO) activity was measured. MPO levels were reduced by 80% by EE treatment. Cotreatment with the pure ER antagonist ICI-182780 (ICI) blocked the effects of EE on stool character, MPO activity, and histology scores, strongly suggesting that the activity of EE is mediated through ER. Mast cell proteases can promote neutrophil infiltration, and gene expression analysis demonstrated that mast cell protease 1, 3, and 4 mRNA were all decreased in colons from estrogen-treated rats. In addition, a direct effect of estrogen on bone marrow-derived mast cell activity was demonstrated, suggesting that ER-mediated inactivation of mast cells may contribute to the improvement in the clinical sign and histological scores in this model.