Beneficial Effects of a Curcumin Derivative and Transforming Growth Factor-ß Receptor I Inhibitor Combination on Nonalcoholic Steatohepatitis.

BACKGRUOUND: Curcumin 2005-8 (Cur5-8), a derivative of curcumin, improves fatty liver disease via AMP-activated protein kinase activation and autophagy regulation. EW-7197 (vactosertib) is a small molecule inhibitor of transforming growth factor ß (TGF-ß) receptor I and may scavenge reactive oxygen species and ameliorate fibrosis through the SMAD2/3 canonical pathway. This study aimed to determine whether co-administering these two drugs having different mechanisms is beneficial. METHODS: Hepatocellular fibrosis was induced in mouse hepatocytes (alpha mouse liver 12 [AML12]) and human hepatic stellate cells (LX-2) using TGF-ß (2 ng/mL). The cells were then treated with Cur5-8 (1 µM), EW-7197 (0.5 µM), or both. In animal experiments were also conducted during which, methionine-choline deficient diet, Cur5-8 (100 mg/kg), and EW-7197 (20 mg/kg) were administered orally to 8-week-old C57BL/6J mice for 6 weeks. RESULTS: TGF-ß-induced cell morphological changes were improved by EW-7197, and lipid accumulation was restored on the administration of EW-7197 in combination with Cur5-8. In a nonalcoholic steatohepatitis (NASH)-induced mouse model, 6 weeks of EW-7197 and Cur5-8 co-administration alleviated liver fibrosis and improved the nonalcoholic fatty liver disease (NAFLD) activity score. CONCLUSION: Co-administering Cur5-8 and EW-7197 to NASH-induced mice and fibrotic hepatocytes reduced liver fibrosis and steatohepatitis while maintaining the advantages of both drugs. This is the first study to show the effect of the drug combination against NASH and NAFLD. Similar effects in other animal models will confirm its potential as a new therapeutic agent.

Vactosertib, TGF-ß receptor I inhibitor, augments the sensitization of the anti-cancer activity of gemcitabine in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) exhibits a pronounced extracellular matrix (ECM)-rich response, which is produced by an excessive amount of transforming growth factor ß (TGF-ß), resulting in tumor progression and metastasis. In addition, TGF-ß signaling contributes to rapidly acquired resistance and incomplete response to gemcitabine. Recently, selective inhibitors of the TGF-ß signaling pathway have shown promise in PDAC treatment, particularly as an option for augmenting responses to chemotherapy. Here, we investigated the synergistic anticancer effects of a small-molecule TGF-ß receptor I kinase inhibitor (vactosertib/EW-7197) in the presence of gemcitabine, and its mechanism of action in pancreatic cancer. Vactosertib sensitized pancreatic cancer cells to gemcitabine by synergistically inhibiting their viability. Importantly, the combination of vactosertib and gemcitabine significantly attenuated the expression of major ECM components, including collagens, fibronectin, and α-SMA, in pancreatic cancer compared with gemcitabine alone. This resulted in potent induction of mitochondrial-mediated apoptosis, gemcitabine-mediated cytotoxicity, and inhibition of tumor ECM by vactosertib. Additionally, the combination decreased metastasis through inhibition of migration and invasion, and exhibited synergistic anti-cancer activity by inhibiting the TGF-ß/Smad2 pathway in pancreatic cancer cells. Furthermore, co-treatment significantly suppressed tumor growth in orthotopic models. Therefore, our findings demonstrate that vactosertib synergistically increased the antitumor activity of gemcitabine via inhibition of ECM component production by inhibiting the TGF-ß/Smad2 signaling pathway. This suggests that the combination of vactosertib and gemcitabine may be a potential treatment option for patients with pancreatic cancer.

Different routes of administering EW-7197 versus EW-7197⋠HBr for preventing peritoneal adhesion in a rat model.

BACKGROUND: The relatively low aqueous solubility of EW-7197 that was administered orally may have affected the desired concentration in the systemic circulation for treating peritoneal adhesion. This experimental study aimed to compare the efficacy of different routes of administering EW-7197 (2-fluoro-N-[(5-[6-methylpyridin-2-yl]-4-[(1,2,4)triazolo(1,5-a)pyridin-6-yl]-1H-imidazol-2-yl)methyl]aniline) and EW-7197·hydrobromide (HBr), with improved aqueous solubility, for inhibiting peritoneal adhesion in a rat model. METHODS: After peritoneal adhesion induction, 30 male Sprague-Dawley rats were randomly divided into 5 groups with 6 rats in each: group A, sham control; group B, orally administered 25 mg/kg of EW-7197·HBr for 7 days; group C, locally administered 25 mg/kg of EW-7197·HBr; group D, orally administered 20 mg/kg of EW-7197 for 7 days; and group E, locally administered 20 mg/kg of EW-7197. Gross examination, histologic staining (hematoxylin and eosin and Masson's trichrome), and immunohistochemical analyses (Ki-67 and α-smooth muscle actin marker [α-SMA]) were performed to evaluate the efficacy of both drugs. RESULTS: All procedures were technically successful. All treatment groups, except for group C, showed significantly reduced incidence, quality, tenacity, fibrosis, and collagen deposition scores and lowered expressions of Ki-67- and α-SMA-positive cells compared with group A. When comparing between groups, all scores were significantly lower in group B than in group C (all P < .001), whereas no significant difference was noted in any of the scores between groups D and E and groups B and E (all P > .05). CONCLUSION: Orally administering EW-7197·HBr and both orally and locally administering EW-7197 significantly prevented peritoneal adhesion formation, and orally administering EW-7197·HBr was the most effective overall.

Discovery of (E)-3-(3-((2-Cyano-4'-dimethylaminobiphenyl-4-ylmethyl)cyclohexanecarbonylamino)-5-fluorophenyl)acrylic Acid Methyl Ester, an Intestine-Specific, FXR Partial Agonist for the Treatment of Nonalcoholic Steatohepatitis.

A series of fexaramine analogs were synthesized and evaluated to develop an intestine-selective/specific FXR partial agonist. Introduction of both a CN substituent at the C-2 in the biphenyl ring and a fluorine at the C-5 in the aniline ring in fexaramine markedly increased FXR agonistic activity. 27c showed 53 ± 3% maximum efficacy relative to GW4064 in an FXR agonist assay. A substantial amount of 27c was absorbed in the intestine after oral administration in rats, and then it was rapidly metabolized to inactive carboxylic acid 44 by serum esterases. In CDAHFD-fed mice, oral administration of 27c strongly induced multiple intestinal FXR target genes, FGF15, SHP, IBABP, and OST-α, but failed to activate SHP in the liver. 27c significantly reduced the liver fibrogenesis area, hepatic fibrosis markers, and serum level of AST. Rational optimization of fexaramine has led to the identification of an intestine-specific FXR partial agonist 27c.

EW-7197 Attenuates the Progression of Diabetic Nephropathy in db/db Mice through Suppression of Fibrogenesis and Inflammation.

BACKGROUND: Diabetic nephropathy (DN) is characterized by albuminuria and accumulation of extracellular matrix (ECM) in kidney. Transforming growth factor-ß (TGF-ß) plays a central role in promoting ECM accumulation. We aimed to examine the effects of EW-7197, an inhibitor of TGF-ß type 1 receptor kinase (ALK5), in retarding the progression of DN, both in vivo, using a diabetic mouse model (db/db mice), and in vitro, in podocytes and mesangial cells. METHODS: In vivo study: 8-week-old db/db mice were orally administered EW-7197 at a dose of 5 or 20 mg/kg/day for 10 weeks. Metabolic parameters and renal function were monitored. Glomerular histomorphology and renal protein expression were evaluated by histochemical staining and Western blot analyses, respectively. In vitro study: DN was induced by high glucose (30 mM) in podocytes and TGF-ß (2 ng/mL) in mesangial cells. Cells were treated with EW-7197 (500 nM) for 24 hours and the mechanism associated with the attenuation of DN was investigated. RESULTS: Enhanced albuminuria and glomerular morphohistological changes were observed in db/db compared to that of the nondiabetic (db/m) mice. These alterations were associated with the activation of the TGF-ß signaling pathway. Treatment with EW-7197 significantly inhibited TGF-ß signaling, inflammation, apoptosis, reactive oxygen species, and endoplasmic reticulum stress in diabetic mice and renal cells. CONCLUSION: EW-7197 exhibits renoprotective effect in DN. EW-7197 alleviates renal fibrosis and inflammation in diabetes by inhibiting downstream TGF-ß signaling, thereby retarding the progression of DN. Our study supports EW-7197 as a therapeutically beneficial compound to treat DN.

Synthesis, biological evaluation and molecular modelling of 2,4-disubstituted-5-(6-alkylpyridin-2-yl)-1H-imidazoles as ALK5 inhibitors.

A series of 2,4-disubstituted-5-(6-alkylpyridin-2-yl)-1H-imidazoles, 7a-c, 11a-h, and 16a-h has been synthesised and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. Incorporation of a quinoxalin-6-yl moiety and a methylene linker at the 4- and 2-position of the imidazole ring, respectively, and a m-CONH2 substituent in the phenyl ring generated a highly potent and selective ALK5 inhibitor 11e. Docking model of ALK5 in complex with 11e showed that it fitted well in the ATP-binding pocket with favourable interactions.

Vactosertib, a Novel, Orally Bioavailable Activin Receptor-Like Kinase 5 Inhibitor, Promotes Regression of Fibrotic Plaques in a Rat Model of Peyronie's Disease.

PURPOSE: To examine the therapeutic effect of Vactosertib, a small molecule inhibitor of transforming growth factor-ß (TGF-ß) type I receptor (activin receptor-like kinase-5, ALK5), in an experimental model of Peyronie's disease (PD) and determining anti-fibrotic mechanisms of Vactosertib in primary fibroblasts derived from human PD plaques. MATERIALS AND METHODS: Male rats were randomly divided into three groups (n=6 per group); control rats without treatment; PD rats receiving vehicle; and PD rats receiving Vactosertib (10 mg/kg). PD-like plaques were induced by administering 100 µL of each of human fibrin and thrombin solutions into the tunica albuginea on days 0 and 5. Vactosertib was given orally five times a week for 2 weeks. On day 30, we performed electrical stimulation of the cavernous nerve to measure erectile function, and the penis was obtained for histological examination. Fibroblasts isolated from human PD plaques were used to determine the anti-fibrotic effects of Vactosertib in vitro. RESULTS: Vactosertib induced significant regression of fibrotic plaques in PD rats in vivo through reduced infiltration of inflammatory cells and reduced expression of phospho-Smad2, which recovered erectile function. Vactosertib also abrogated TGF-ß1-induced enhancement of extracellular matrix protein production and hydroxyproline content in PD fibroblasts in vitro by hindering the TGF-ß1-induced Smad2/3 phosphorylation and nuclear translocation, and fibroblast-to-myofibroblast transdifferentiation. CONCLUSIONS: In view of the critical role of TGF-ß and the Smad pathway in the pathogenesis of PD, inhibition of this pathway with an ALK5 inhibitor may represent a novel, targeted therapy for PD.

EW-7197, an oral transforming growth factor ß type I receptor kinase inhibitor, for preventing peritoneal adhesion formation in a rat model.

BACKGROUND: EW-7197 is an oral transforming growth factor ß type I receptor kinase inhibitor currently undergoing phase I clinical trials for cancer treatment in the United States. This study evaluates whether EW-7197 prevents peritoneal adhesion formation in a rat model. METHODS: Forty-eight female Wistar rats underwent peritoneal adhesion induction by the creation of peritoneal ischemic buttons and were randomly divided into 4 groups of 12 each. The control group received 0.3 mL vehicle by oral gavage once daily for 7 days after adhesion induction. The 10 mg and 20 mg groups received 10 or 20 mg/kg EW-7197 phosphate dissolved in 0.3 mL vehicle by oral gavage once daily for 7 days after adhesion induction. The rebound group received 20 mg/kg EW-7197 phosphate dissolved in 0.3 mL vehicle by oral gavage once daily for 7 days after adhesion induction followed by 0.3 mL vehicle only by gavage once daily for an additional 21 days. After the respective treatments were completed, the animals were euthanized. RESULTS: All rats survived until the end of the study without complications. EW-7197 reduced the incidence, quality, and tenacity of peritoneal adhesions in a dose-dependent manner. Fibrosis and collagen production were reduced in EW-7197-treated peritoneal ischemic buttons. Transforming growth factor ß/Smad2/3 signaling and mesothelial-to-mesenchymal transition were inhibited in EW-7197-treated peritoneal ischemic buttons. Discontinuation of EW-7197 was not associated with rebound effects. CONCLUSION: EW-7197 prevented peritoneal adhesion formation potentially via inhibition of transforming growth factor ß1/Smad2/3-induced mesothelial-to-mesenchymal transition in a rat model.

EW-7197 eluting nano-fiber covered self-expandable metallic stent to prevent granulation tissue formation in a canine urethral model.

PURPOSE: To evaluate an EW-7197-eluting nanofiber-covered stent (NFCS) for suppressing granulation tissue formation after stent placement in a canine urethral model. MATERIALS AND METHODS: All experiments were approved by the committee of animal research. A total of 12 NFCSs were placed in the proximal and distal urethras of six dogs. Dogs were divided into two groups with 3 dogs each. The control stent (CS) group received NFCSs and the drug stent (DS) group received EW-7197 (1000 µg)-eluting NFCSs. All dogs were sacrificed 8 weeks after stent placement Histologic findings of the stented urethra were compared using the Mann-Whitney U test. RESULTS: Stent placement was technically successful in all dogs without procedure-related complications. On urethrographic analysis, the mean luminal diameter was significantly larger in the DS group than in the CS group at 4 and 8 weeks after stent placement (all p < 0.001). On histological examination, mean thicknesses of the papillary projection, thickness of submucosal fibrosis, number of epithelial layers, and degree of collagen deposition were significantly lower in the DS group than in the CS group (all p < 0.001), whereas the mean degree of inflammatory cell infiltration was not significantly different (p > 0.05). CONCLUSION: The EW-7197-eluting NFCS is effective and safe for suppressing granulation tissue formation after stent placement in a canine urethral model.

EW-7197, an activin-like kinase 5 inhibitor, suppresses granulation tissue after stent placement in rat esophagus.

BACKGROUND AND AIMS: Self-expanding metallic stent (SEMS) placement is a well-established method for treating malignant esophageal strictures; however, this procedure has not gained widespread acceptance for treating benign esophageal strictures because of granulation tissue formation. The aim of the present study was to investigate whether EW-7197, a novel per-oral transforming growth factor-ß type I receptor kinase inhibitor, suppressed granulation tissue formation after SEMS placement in the rat esophagus. METHODS: Sixty rats underwent SEMS placement and were randomly divided into 4 groups. Group A (n = 20) received vehicle-treated control for 4 weeks. Group B (n = 20) received 20 mg/kg/day EW-7197 for 4 weeks. Group C (n = 10) received 20 mg/kg/day EW-7197 for 4 weeks followed by vehicle-treated control for 4 weeks. Group D (n = 10) received 20 mg/kg/day EW-7197 for 8 weeks. RESULTS: SEMS placement was technically successful in all rats. Eleven rats, however, were excluded because of stent migration (n = 9) and procedure-related death (n = 2). The luminal diameter in group A was significantly smaller than those in groups B, C, and D (all P < .001). The percentage of granulation tissue area, number of epithelial layers, thickness of submucosal fibrosis, percentage of connective tissue area, and degree of collagen deposition were significantly higher in group A than in groups B, C, and D (all P < .001); however, there were no significant differences among groups B, C, and D. EW-7197 decreased the expression levels of phospho-Smad 3, N-cadherin, fibronectin, α-smooth muscle actin, and transforming growth factor-ß1 and increased the expression level of E-cadherin (all P < .01). CONCLUSIONS: EW-7197 suppressed granulation tissue formation after SEMS placement in the rat esophagus.

Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-ß signaling.

Cancer upregulated gene 2 (CUG2) enhances cell migration and invasion, but the underlying mechanism has not been revealed. Herein, CUG2 decreased the expression of E-cadherin and increased the expression of N-cadherin and vimentin, characteristics of the epithelial-mesenchymal transition (EMT). A CUG2 deletion mutant, lacking interaction with nucleophosmin 1 (NPM1), or suppression of NPM1 reduced wound healing and cell invasion, indicating that CUG2-mediated EMT requires NPM1. CUG2 enhanced activation of Smad2/3 and expression of Snail and Twist, while the CUG2 silence decreased these TGF-ß signaling pathways, leading to suppression of EMT. NPM silence also inhibited the CUG2-induced TGF-ß signaling. These results suggest that TGF-ß signaling is involved in CUG2-induced EMT. Treatment with EW-7197, a novel inhibitor of TGF-ß signaling, diminished CUG2-mediated EMT and inhibition of Akt, ERK, JNK, and p38 MAPK, non-canonical TGF-ß signaling molecules, also decreased expression of Smad2/3, Snail and Twist, leading to inhibition of EMT. The results confirm that TGF-ß signaling is essential for CUG2-mediated EMT. Interestingly, TGF-ß enhanced CUG2 expression. We further found that both CUG2-induced TGF-ß production and TGF-ß-induced CUG2 up-regulation required a physical interaction between Sp1 and Smad2/3 in the CUG2 and TGF-ß promoter, as demonstrated by a promoter reporter assay, immunoprecipitation, and ChIP assay. These results indicated close crosstalk between CUG2 and TGF-ß. Conversely, suppression of CUG2 or NPM1 did not completely inhibit TGF-ß-induced EMT, indicating that the effect of TGF-ß on EMT is dominant over the effect of CUG2 on EMT. Collectively, our findings suggest that CUG2 induces the EMT via TGF-ß signaling.

TGF-ß Type I Receptor Kinase Inhibitor EW-7197 Suppresses Cholestatic Liver Fibrosis by Inhibiting HIF1α-Induced Epithelial Mesenchymal Transition.

BACKGROUND/AIMS: Hypoxia is an environmental factor that aggravates liver fibrosis. HIF1α activates hepatic stellate cells (HSCs) and increases transforming growth factor-ß (TGF-ß) signaling and the epithelial mesenchymal transition (EMT), accelerating the progression of fibrosis. We evaluated the anti-fibrotic therapeutic potential of a small-molecule inhibitor of TGF-ß type I receptor kinase, EW-7197, on HIF1α-derived TGF-ß signaling in cholestatic liver fibrosis. METHODS: We used a bile duct ligation (BDL)-operated rat model to characterize the role of HIF1α-derived TGF-ß signaling in liver fibrosis. Cellular assays were performed in LX-2 cells (human immortalized HSCs). The anti-fibrotic effects of EW-7197 in liver tissues and HSCs were investigated via biochemical assays, immunohistochemistry (IHC), immunofluorescence (IF), chromatin immunoprecipitation (ChIP) assays, real-time PCR, and western blotting. RESULTS: In our BDL rat model, orally administered EW-7197 inhibited fibrosis and attenuated HIF1α-induced activation of HSCs and EMT in vivo. In addition, EW-7197 inhibited HIF1α-derived HSC activation and expression of EMT markers in LX-2 cells in vitro. CONCLUSION: This study suggests that EW-7197 exhibits potential as a treatment for liver fibrosis because it inhibits HIF1α-induced TGF-ß signaling.

Novel oral transforming growth factor-ß signaling inhibitor EW-7197 eradicates CML-initiating cells.

Recent strategies for treating CML patients have focused on investigating new combinations of tyrosine kinase inhibitors (TKIs) as well as identifying novel translational research agents that can eradicate CML leukemia-initiating cells (CML-LICs). However, little is known about the therapeutic benefits such CML-LIC targeting therapies might bring to CML patients. In this study, we investigated the therapeutic potential of EW-7197, an orally bioavailable transforming growth factor-ß signaling inhibitor which has recently been approved as an Investigational New Drug (NIH, USA), to suppress CML-LICs in vivo. Compared to TKI treatment alone, administration of TKI plus EW-7197 to CML-affected mice significantly delayed disease relapse and prolonged survival. Notably, combined treatment with EW-7197 plus TKI was effective in eliminating CML-LICs even if they expressed the TKI-resistant T315I mutant BCR-ABL1 oncogene. Collectively, these results indicate that EW-7197 may be a promising candidate for a new therapeutic that can greatly benefit CML patients by working in combination with TKIs to eradicate CML-LICs.

Synthesis and biological evaluation of 5-(fluoro-substituted-6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)imidazoles as inhibitors of transforming growth factor-ß type I receptor kinase.

To further optimize a clinical candidate 5 (EW-7197), a series of 5-(3-, 4-, or 5-fluoro-substituted-6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)imidazoles 19a-l have been synthesized and evaluated for their TGF-ß type I receptor kinase (ALK5) and p38α MAP kinase inhibitory activity in an enzyme assay. The 5-(5-fluoro-substituted-6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)imidazoles 19h-l displayed the similar level of potency to that of 5 against both ALK5 (IC50=7.68-13.70 nM) and p38α MAP kinase (IC50=1240-3370 nM). Among them, 19j inhibited ALK5 with IC50 value of 7.68 nM in a kinase assay and displayed 82% inhibition at 100 nM in a luciferase reporter assay.

Combinatorial TGF-ß attenuation with paclitaxel inhibits the epithelial-to-mesenchymal transition and breast cancer stem-like cells.

Distant relapse after chemotherapy is an important clinical issue for treating breast cancer patients and results from the development of cancer stem-like cells (CSCs) during chemotherapy. Here we report that blocking epithelial-to-mesenchymal transition (EMT) suppresses paclitaxel-induced CSCs properties by using a MDA-MB-231-xenografted mice model (in vivo), and breast cancer cell lines (in vitro). Paclitaxel, one of the cytotoxic taxane-drugs such as docetaxel, increases mesenchymal markers (Vimentin and Fibronectin) and decreases an epithelial marker (Zo-1). Blocking TGF-ß signaling with the TGF-ß type I receptor kinase (ALK5) inhibitor, EW-7197, suppresses paclitaxel-induced EMT and CSC properties such as mammosphere-forming efficiency (MSFE), aldehyde dehydrogenase (ALDH) activity, CD44+/CD24- ratio, and pluripotency regulators (Oct4, Nanog, Klf4, Myc, and Sox2). The combinatorial treatment of EW-7197 improves the therapeutic effect of paclitaxel by decreasing the lung metastasis and increasing the survival time in vivo. We confirmed that Snail is increased by paclitaxel-induced intracellular reactive oxygen species (ROS) and EW-7197 suppresses the paclitaxel-induced Snail and EMT by attenuating paclitaxel-induced intracellular ROS. Knock-down of SNAI1 suppresses paclitaxel-induced EMT and CSC properties. These data together suggest that blocking the Snail-induced EMT with the ALK5 inhibitor attenuates metastasis after paclitaxel-therapy and that this combinatorial approach could prove useful in treating breast cancer.

Phosphorylation status determines the opposing functions of Smad2/Smad3 as STAT3 cofactors in TH17 differentiation.

Transforming growth factor-ß (TGF-ß) and interleukin-6 (IL-6) are the pivotal cytokines to induce IL-17-producing CD4(+) T helper cells (TH17); yet their signalling network remains largely unknown. Here we show that the highly homologous TGF-ß receptor-regulated Smads (R-Smads): Smad2 and Smad3 oppositely modify STAT3-induced transcription of IL-17A and retinoic acid receptor-related orphan nuclear receptor, RORγt encoded by Rorc, by acting as a co-activator and co-repressor of STAT3, respectively. Smad2 linker phosphorylated by extracellular signal-regulated kinase (ERK) at the serine 255 residue interacts with STAT3 and p300 to transactivate, whereas carboxy-terminal unphosphorylated Smad3 interacts with STAT3 and protein inhibitor of activated STAT3 (PIAS3) to repress the Rorc and Il17a genes. Our work uncovers carboxy-terminal phosphorylation-independent noncanonical R-Smad-STAT3 signalling network in TH17 differentiation.

EW-7197 inhibits hepatic, renal, and pulmonary fibrosis by blocking TGF-ß/Smad and ROS signaling.

Fibrosis is an inherent response to chronic damage upon immense apoptosis or necrosis. Transforming growth factor-beta1 (TGF-ß1) signaling plays a key role in the fibrotic response to chronic liver injury. To develop anti-fibrotic therapeutics, we synthesized a novel small-molecule inhibitor of the TGF-ß type I receptor kinase (ALK5), EW-7197, and evaluated its therapeutic potential in carbon tetrachloride (CCl4) mouse, bile duct ligation (BDL) rat, bleomycin (BLM) mouse, and unilateral ureteral obstruction (UUO) mouse models. Western blot, immunofluorescence, siRNA, and ChIP analysis were carried out to characterize EW-7197 as a TGF-ß/Smad signaling inhibitor in LX-2, Hepa1c1c7, NRK52E, and MRC5 cells. In vivo anti-fibrotic activities of EW-7197 were examined by microarray, immunohistochemistry, western blotting, and a survival study in the animal models. EW-7197 decreased the expression of collagen, α-smooth muscle actin (α-SMA), fibronectin, 4-hydroxy-2, 3-nonenal, and integrins in the livers of CCl4 mice and BDL rats, in the lungs of BLM mice, and in the kidneys of UUO mice. Furthermore, EW-7197 extended the lifespan of CCl4 mice, BDL rats, and BLM mice. EW-7197 blocked the TGF-ß1-stimulated production of reactive oxygen species (ROS), collagen, and α-SMA in LX-2 cells and hepatic stellate cells (HSCs) isolated from mice. Moreover, EW-7197 attenuated TGF-ß- and ROS-induced HSCs activation to myofibroblasts as well as extracellular matrix accumulation. The mechanism of EW-7197 appeared to be blockade of both TGF-ß1/Smad2/3 and ROS signaling to exert an anti-fibrotic activity. This study shows that EW-7197 has a strong potential as an anti-fibrosis therapeutic agent via inhibition of TGF-ß-/Smad2/3 and ROS signaling.

An novel inhibitor of TGF-ß type I receptor, IN-1130, blocks breast cancer lung metastasis through inhibition of epithelial-mesenchymal transition.

TGF-ß signaling plays an important role in breast cancer progression and metastasis. Epithelial-mesenchymal transition (EMT) is an important step in the progression of solid tumors to metastatic disease. We previously reported that IN-1130, a novel transforming growth factor-ß type I receptor kinase (ALK5) inhibitor, suppressed renal fibrosis in obstructive nephropathy (Moon et al., 2006). Here, we show that IN-1130 suppressed EMT and the lung metastasis of mammary tumors in mouse models. Treating human and mouse cell lines with IN-1130 inhibited TGF-ß-mediated transcriptional activation, the phosphorylation and nuclear translocation of Smad2, and TGF-ß-induced-EMT, which induces morphological changes in epithelial cells. Additionally, we demonstrated that IN-1130 blocked TGF-ß-induced 4T1 mammary cancer cell migration and invasion. The TGF-ß-mediated increase in matrix metalloproteinase (MMP)-2 and MMP-9 expression was restored by IN-1130 co-treatment with TGF-ß in human epithelial cells and in 4T1 cells. Furthermore, we found that lung metastasis from primary breast cancer was inhibited by IN-1130 in both 4T1-xenografted BALB/c mice and MMTV/c-Neu transgenic mice without any change in primary tumor volume. IN-1130 prolonged the life span of tumor-bearing mice. In summary, this study indicated that IN-1130 has therapeutic potential for preventing breast cancer metastasis to the lung.

Discovery of N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline (EW-7197): a highly potent, selective, and orally bioavailable inhibitor of TGF-ß type I receptor kinase as cancer immunotherapeutic/antifibrotic agent.

A series of 2-substituted-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)imidazoles was synthesized and evaluated to optimize a prototype inhibitor of TGF-ß type I receptor kinase (ALK5), 6. Combination of replacement of a quinoxalin-6-yl moiety of 6 with a [1,2,4]triazolo[1,5-a]pyridin-6-yl moiety, insertion of a methyleneamino linker, and a o-F substituent in the phenyl ring markedly increased ALK5 inhibitory activity, kinase selectivity, and oral bioavailability. The 12b (EW-7197) inhibited ALK5 with IC50 value of 0.013 µM in a kinase assay and with IC50 values of 0.0165 and 0.0121 µM in HaCaT (3TP-luc) stable cells and 4T1 (3TP-luc) stable cells, respectively, in a luciferase assay. Selectivity profiling of 12b using a panel of 320 protein kinases revealed that it is a highly selective ALK5/ALK4 inhibitor. Pharmacokinetic study with 12b·HCl in rats showed an oral bioavailability of 51% with high systemic exposure (AUC) of 1426 ng × h/mL and maximum plasma concentration (Cmax) of 1620 ng/mL. Rational optimization of 6 has led to the identification of a highly potent, selective, and orally bioavailable ALK5 inhibitor 12b.

EW-7197, a novel ALK-5 kinase inhibitor, potently inhibits breast to lung metastasis.

Advanced tumors produce an excessive amount of transforming growth factor ß (TGFß), which promotes tumor progression at late stages of malignancy. The purpose of this study was to develop anti-TGFß therapeutics for cancer. We synthesized a novel small-molecule TGFß receptor I kinase (activin receptor-like kinase 5) inhibitor termed N-[[4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl]methyl]-2-fluoroaniline (EW-7197), and we investigated its potential antimetastatic efficacy in mouse mammary tumor virus (MMTV)/c-Neu mice and 4T1 orthotopic-grafted mice. EW-7197 inhibited Smad/TGFß signaling, cell migration, invasion, and lung metastasis in MMTV/c-Neu mice and 4T1 orthotopic-grafted mice. EW-7197 also inhibited the epithelial-to-mesenchymal transition (EMT) in both TGFß-treated breast cancer cells and 4T1 orthotopic-grafted mice. Furthermore, EW-7197 enhanced cytotoxic T lymphocyte activity in 4T1 orthotopic-grafted mice and increased the survival time of 4T1-Luc and 4T1 breast tumor-bearing mice. In summary, EW-7197 showed potent in vivo antimetastatic activity, indicating its potential for use as an anticancer therapy.