Combination effects of alogliptin and pioglitazone on steatosis and hepatic fibrosis formation in a mouse model of non-alcoholic steatohepatitis.

This study aimed to evaluate the effects of combination therapy with a dipeptidyl peptidase-4 inhibitor, alogliptin, and a peroxisome proliferator-activated receptor-γ agonist, pioglitazone, in a preclinical model of nonalcoholic steatohepatitis using low-density lipoprotein receptor-knockout mice fed a modified choline-deficient l-amino acid-defined diet. Monotherapy with either alogliptin (10-200 mg/kg) or pioglitazone (6-20 mg/kg) significantly decreased hepatic triglyceride content and fibrosis. The concomitant treatment of alogliptin (30 mg/kg), pioglitazone (20 mg/kg) also decreased hepatic triglyceride and hepatic collagen-I mRNA at greater extent compared to monotherapy. Hepatic expression of CD11b mRNA and monocyte chemoattractant protein-1 were also reduced by the concomitant treatment. These results suggest that via an anti-inflammatory potential in addition to anti-metabolic effects, the combination therapy of alogliptin and pioglitazone may provide therapeutic benefits to type 2 diabetes patients with nonalcoholic steatohepatitis, which will be proven in controlled clinical trials.

Non-alcoholic steatohepatitis-associated hepatic fibrosis and hepatocellular carcinoma in a combined mouse model of genetic modification and dietary challenge.

AIM: Experimental models of non-alcoholic steatohepatitis (NASH) are still required for understanding the pathophysiology of this disease. This study aimed to examine whether disease progression is accelerated by combining dyslipidemic genetic modification and dietary challenges and develop NASH-associated hepatic fibrosis, cirrhosis, and carcinoma in a short period. METHODS: Low-density lipoprotein receptor knockout mice were fed a modified choline-deficient amino acid-defined diet, including 1 w/w% cholesterol and 41 kcal% fat, and was comprehensively profiled over 1 year. RESULTS: Microvesicular and macrovesicular steatosis in the liver was observed from the first week after starting the modified choline-deficient amino acid-defined diet. Macrovesicular steatosis was exacerbated with time and was observed in almost all hepatocytes at week 8, but slightly decreased at week 16. Infiltration of macrophages and neutrophils, and upregulation of hepatic inflammatory cytokines such as tumor necrosis factor-α and interleukin-1ß were also observed from week 1. Plasma hepatic transaminase activities were increased at week 1, reached a peak at week 4, and gradually decreased thereafter. In parallel with increases in hepatic gene expression of collagen-I, the hepatic fibrosis area expanded after week 4 and massively spread all over the liver by week 8. Hepatocellular hyperplasia was observed from week 24. Hepatocellular adenoma and carcinoma were observed from week 31 and 39, respectively. CONCLUSION: These results suggest that, in a rodent NASH model with the combination of genetic modification and dietary challenges, hepatic steatosis, inflammatory cell infiltration and hepatic injury, hepatic fibrosis, hepatocellular hyperplasia, adenoma, and carcinoma can be developed in a relatively short period.

Pharmacological evaluation of pioglitazone and candesartan cilexetil in a novel mouse model of non-alcoholic steatohepatitis, modified choline-deficient, amino acid-defined diet fed low-density lipoprotein receptor knockout mice.

AIM: Low-density lipoprotein receptor knockout (LDLR-KO) mice fed a modified choline-deficient and amino acid-defined (mCDAA) diet show non-alcoholic steatohepatitis (NASH)-like pathophysiology. In order to pharmacologically benchmark this model, effects of pioglitazone (a thiazolidinedione) and candesartan cilexetil (an angiotensin II type 1 receptor blocker) on steatosis and liver fibrosis were examined. METHODS: Pioglitazone (10 mg/kg) and candesartan cilexetil (3 mg/kg) were given orally once daily to LDLR-KO mice under mCDAA diet for 7 weeks. Blood biochemistry and hepatic histology were assessed, and hepatic gene expression levels and triglyceride content were measured. RESULTS: Pioglitazone suppressed hepatic COL1A1 gene expression by 43% and attenuated hepatic fibrosis areas by 49%. Pioglitazone also decreased plasma alanine aminotransferase levels, liver weight, hepatic triglyceride content, and hepatic expression of other fibrosis-related genes such as TGFB1, SPP1, TIMP1, and IL6. Candesartan cilexetil suppressed hepatic COL1A1 gene expression by 33%, whereas the other end-points including hepatic fibrosis areas were not affected. CONCLUSIONS: Pioglitazone showed anti-fibrotic effects accompanied by improving hepatic transaminase activity and hepatic lipid accumulation, but the effect of candesartan cilexetil was only limited, unlike previous reports for angiotensin II type 1 receptor blockers. As the pharmacological effects of pioglitazone in the current animal model are similar to those reported in patients with NASH, this model may represent some aspects of the pathophysiology of NASH. Further profiling using other agents or mechanisms that have been tested in the clinic will better clarify the utility of the animal model.

Antitumor activity of the selective pan-RAF inhibitor TAK-632 in BRAF inhibitor-resistant melanoma.

The mitogen-activated protein kinase (MAPK) pathway is particularly important for the survival and proliferation of melanoma cells. Somatic mutations in BRAF and NRAS are frequently observed in melanoma. Recently, the BRAF inhibitors vemurafenib and dabrafenib have emerged as promising agents for the treatment of melanoma patients with BRAF-activating mutations. However, as BRAF inhibitors induce RAF paradoxical activation via RAF dimerization in BRAF wild-type cells, rapid emergence of acquired resistance and secondary skin tumors as well as presence of few effective treatment options for melanoma bearing wild-type BRAF (including NRAS-mutant melanoma) are clinical concerns. Here, we demonstrate that the selective pan-RAF inhibitor TAK-632 suppresses RAF activity in BRAF wild-type cells with minimal RAF paradoxical activation. Our analysis using RNAi and TAK-632 in preclinical models reveals that the MAPK pathway of NRAS-mutated melanoma cells is highly dependent on RAF. We also show that TAK-632 induces RAF dimerization but inhibits the kinase activity of the RAF dimer, probably because of its slow dissociation from RAF. As a result, TAK-632 demonstrates potent antiproliferative effects both on NRAS-mutated melanoma cells and BRAF-mutated melanoma cells with acquired resistance to BRAF inhibitors through NRAS mutation or BRAF truncation. Furthermore, we demonstrate that the combination of TAK-632 and the MAPK kinase (MEK) inhibitor TAK-733 exhibits synergistic antiproliferative effects on these cells. Our findings characterize the unique features of TAK-632 as a pan-RAF inhibitor and provide rationale for its further investigation in NRAS-mutated melanoma and a subset of BRAF-mutated melanomas refractory to BRAF inhibitors.

Anti-angiogenic and anti-tumor effects of TAK-593, a potent and selective inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinase.

We recently reported that TAK-593, a novel imidazo[1,2-b]pyridazine derivative, is a highly potent and selective inhibitor of the vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor tyrosine kinase families. Moreover, TAK-593 exhibits a uniquely long-acting inhibitory profile towards VEGF receptor 2 (VEGFR2) and PDGF receptor ß (PDGFRß). In this study, we demonstrated that TAK-593 potently inhibits VEGF- and PDGF-stimulated cellular phosphorylation and proliferation of human umbilical vein endothelial cells and human coronary artery smooth muscle cells. TAK-593 also potently inhibits VEGF-induced tube formation of endothelial cells co-cultured with fibroblasts. Oral administration of TAK-593 exhibited strong anti-tumor effects against various human cancer xenografts along with good tolerability despite a low level of plasma exposure. Even after the blood and tissue concentrations of TAK-593 decreased below the detectable limit, a pharmacodynamic marker (phospho VEGFR2) was almost completely suppressed, indicating that its long duration of enzyme inhibition might contribute to the potent activity of TAK-593. Immunohistochemical staining indicated that TAK-593 showed anti-proliferative and pro-apoptotic effects on tumors along with a decrease of vessel density and inhibition of pericyte recruitment to microvessels in vivo. Furthermore, dynamic contrast-enhanced magnetic resonance imaging revealed that TAK-593 reduced tumor vessel permeability prior to the onset of anti-tumor activity. In conclusion, TAK-593 is an extremely potent VEGFR/PDGFR kinase inhibitor whose potent anti-angiogenic activity suggests therapeutic potential for the treatment of solid tumors.