TRPV4 channels promote vascular permeability in retinal vascular disease.

This study aimed to determine the role of transient receptor potential vanilloid 4 (TRPV4), a calcium (Ca2+)-permeable cation channel, in the pathophysiology of retinal vascular disease. The retinal vein occlusion (RVO) murine model was created by irradiating retinal veins using lasers. TRPV4 expression and localization were evaluated in RVO mice retinas. In addition, we examined the effects of TRPV4 antagonists (RQ-00317310, HC-067047, GSK2193874, and GSK2798745) on retinal edema, blood flow, and ischemic areas in RVO mice. Furthermore, changes in the retinal expression of tumor necrosis factor (TNF)-α and aquaporin4 (AQP4) by RQ-00317310 were analyzed using Western blot. We also assessed the barrier integrity of epithelial cell monolayers using trans-endothelial electrical resistance (TEER) in Human Retinal Microvascular Endothelial Cells (HRMECs). The expression of TRPV4 was significantly increased and co-localized with glutamine synthetase (GS), a Müller glial marker, in the ganglion cell layer (GCL) of the RVO mice. Moreover, RQ-00317310 administration ameliorated the development of retinal edema and ischemia in RVO mice. In addition, the up regulation of TNF-α and down-regulation of AQP4 were lessened by the treatment with RQ-00317310. Treatment with GSK1016790A, a TRPV4 agonist, increased vascular permeability, while RQ-00317310 treatment decreased vascular endothelial growth factor (VEGF)- or TRPV4-induced retinal vascular hyperpermeability in HRMECs. These findings suggest that TRPV4 plays a role in the development of retinal edema and ischemia. Thus, TRPV4 could be a new therapeutic target against the pathological symptoms of retinal vascular diseases.

Intravenous administration of the selective toll-like receptor 7 agonist DSR-29133 leads to anti-tumor efficacy in murine solid tumor models which can be potentiated by combination with fractionated radiotherapy.

Strategies to augment anti-cancer immune responses have recently demonstrated therapeutic utility. To date clinical success has been achieved through targeting co-inhibitory checkpoints such as CTLA-4, PD-1, and PD-L1. However, approaches that target co-activatory pathways are also being actively being developed. Here we report that the novel TLR7-selective agonist DSR-29133 is well tolerated in mice and leads to acute immune activation. Administration of DSR-29133 leads to the induction of IFNα/γ, IP-10, TNFα, IL-1Ra and IL-12p70, and to a reduction in tumor burden in syngeneic models of renal cancer (Renca), metastatic osteosarcoma (LM8) and colorectal cancer (CT26). Moreover, we show that the efficacy of DSR-29133 was significantly improved when administered in combination with low-dose fractionated radiotherapy (RT). Effective combination therapy required weekly administration of DSR-29133 commencing on day 1 of a fractionated RT treatment cycle, whereas no enhancement of radiation response was observed when DSR-29133 was administered at the end of the fractionated RT cycle. Combined therapy resulted in curative responses in a high proportion of mice bearing established CT26 tumors which was dependent on the activity of CD8+ T-cells but independent of CD4+ T-cells and NK/NKT cells. Moreover, long-term surviving mice originally treated with DSR-29133 and RT were protected by a tumor-specific memory immune response which could prevent tumor growth upon rechallenge. These results demonstrate that DSR-29133 is a potent selective TLR7 agonist that when administered intravenously can induce anti-tumor immune responses that can be further enhanced through combination with low-dose fractionated RT.

TLR7 tolerance is independent of the type I IFN pathway and leads to loss of anti-tumor efficacy in mice.

Systemic administration of small molecule toll-like receptor (TLR)-7 agonists leads to potent activation of innate immunity and to the generation of anti-tumor immune responses. However, activation of TLRs with small molecule agonists may lead to the induction of TLR tolerance, defined as a state of hyporesponsiveness to subsequent agonism, which may limit immune activation, the generation of anti-tumor responses and clinical response. Our data reveal that dose scheduling impacts on the efficacy of systemic therapy with the selective TLR7 agonist, 6-amino-2-(butylamino)-9-((6-(2-(dimethylamino)ethoxy)pyridin-3-yl)methyl)-7,9-dihydro-8H-purin-8-one (DSR-6434). In a preclinical model of renal cell cancer, systemic administration of DSR-6434 dosed once weekly resulted in a significant anti-tumor response. However, twice weekly dosing of DSR-6434 led to the induction of TLR tolerance, and no anti-tumor response was observed. We show that TLR7 tolerance was independent of type I interferon (IFN) negative feedback because induction of TLR7 tolerance was also observed in IFN-α/ß receptor knockout mice treated with DSR-6434. Moreover, our data demonstrate that treatment of bone marrow-derived plasmacytoid dendritic cells (BM-pDC) with DSR-6434 led to downregulation of TLR7 expression. From our data, dose scheduling of systemically administered TLR7 agonists can impact on anti-tumor activity through the induction of TLR tolerance. Furthermore, TLR7 expression on pDC may be a useful biomarker of TLR7 tolerance and aid in the optimization of dosing schedules involving systemically administered TLR7 agonists.

Discovery of 3H-imidazo[4,5-c]quinolin-4(5H)-ones as potent and selective dipeptidyl peptidase IV (DPP-4) inhibitors: use of a carboxylate prodrug to improve bioavailability.

We have previously reported a novel series of 3H-imidazo[4,5-c]quinolin-4(5H)-ones with potent dipeptidyl peptidase IV (DPP-4) inhibitory activity. However, these compounds showed poor oral absorption. We attempted in this study esterification of the carboxylic acid moiety to improve the compounds 1-4 plasma concentrations. Our efforts yielded 10h with a 5-methyl-2-oxo-1,3-dioxol-4-yl methyl ester as an S9/plasma-cleavable functionality. Compound 10h showed significantly high oral absorption and potent DPP-4 inhibition in vivo and decreased Zucker fatty rats glucose levels in the oral glucose tolerance test. Optimization of the ester moiety revealed that rapid conversion to the carboxyl form in both liver S9 fractions and serum was important for prodrugs not to be detected in the plasma after oral administration. In particular, lability in the serum was found to be an important characteristic. Through our investigation, we were able to develop a novel efficient synthetic method for construction of 3H-imidazo[4,5-c]quinolin-4(5H)-ones using intramolecular radical cyclization.

2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727): a potent, orally active dipeptidyl peptidase IV inhibitor without mechanism-based inactivation of CYP3A.

We report on the identification of 2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727) (7a) as a potent and orally active DPP-4 inhibitor without mechanism-based inactivation of CYP3A. Compound 7a showed good DPP-4 inhibitory activity (IC(50)=1.1 nM), excellent selectivity against related peptidases and other off-targets, good pharmacokinetic and pharmacodynamic profile, great in vivo efficacy in Zucker-fatty rat, and no safety concerns both in vitro and in vivo.

Discovery of new chemotype dipeptidyl peptidase IV inhibitors having (R)-3-amino-3-methyl piperidine as a pharmacophore.

Structures containing the (R)-3-amino-3-methyl piperidine unit as a new pharmacophore moiety have been shown to possess moderate inhibitory activity for DPP-4 with good pharmacokinetics profile. One of these compounds was found to have good oral bioavailability and PK/PD profile in ZF-rat.

Weight loss and lipolysis promote a dynamic immune response in murine adipose tissue.

Obesity elicits an immune response characterized by myeloid cell recruitment to key metabolic organs, including adipose tissue. However, the response of immune cells to nonpathologic metabolic stimuli has been less well studied, and the factors that regulate the metabolic-dependent accumulation of immune cells are incompletely understood. Here we characterized the response of adipose tissue macrophages (ATMs) to weight loss and fasting in mice and identified a role for lipolysis in ATM recruitment and accumulation. We found that the immune response to weight loss was dynamic; caloric restriction of high-fat diet-fed mice led to an initial increase in ATM recruitment, whereas ATM content decreased following an extended period of weight loss. The peak in ATM number coincided with the peak in the circulating concentrations of FFA and adipose tissue lipolysis, suggesting that lipolysis drives ATM accumulation. Indeed, fasting or pharmacologically induced lipolysis rapidly increased ATM accumulation, adipose tissue chemoattractant activity, and lipid uptake by ATMs. Conversely, dietary and genetic manipulations that reduced lipolysis decreased ATM accumulation. Depletion of macrophages in adipose tissue cultures increased expression of adipose triglyceride lipase and genes regulated by FFA, and increased lipolysis. These data suggest that local lipid fluxes are central regulators of ATM recruitment and that once recruited, ATMs form lipid-laden macrophages that can buffer local increases in lipid concentration.

C-C chemokine receptor 2 (CCR2) regulates the hepatic recruitment of myeloid cells that promote obesity-induced hepatic steatosis.

OBJECTIVE: Obesity induces a program of systemic inflammation that is implicated in the development of many of its clinical sequelae. Hepatic inflammation is a feature of obesity-induced liver disease, and our previous studies demonstrated reduced hepatic steatosis in obese mice deficient in the C-C chemokine receptor 2 (CCR2) that regulates myeloid cell recruitment. This suggests that a myeloid cell population is recruited to the liver in obesity and contributes to nonalcoholic fatty liver disease. RESEARCH DESIGN AND METHODS: We used fluorescence-activated cell sorting to measure hepatic leukocyte populations in genetic and diet forms of murine obesity. We characterized in vivo models that increase and decrease an obesity-regulated CCR2-expressing population of hepatic leukocytes. Finally, using an in vitro co-culture system, we measured the ability of these cells to modulate a hepatocyte program of lipid metabolism. RESULTS: We demonstrate that obesity activates hepatocyte expression of C-C chemokine ligand 2 (CCL2/MCP-1) leading to hepatic recruitment of CCR2(+) myeloid cells that promote hepatosteatosis. The quantity of these cells correlates with body mass and in obese mice represents the second largest immune cell population in the liver. Hepatic expression of CCL2 increases their recruitment and in the presence of dietary fat induces hepatosteatosis. These cells activate hepatic transcription of genes responsible for fatty acid esterification and steatosis. CONCLUSIONS: Obesity induces hepatic recruitment of a myeloid cell population that promotes hepatocyte lipid storage. These findings demonstrate that recruitment of myeloid cells to metabolic tissues is a common feature of obesity, not limited to adipose tissue.

Combination therapy with SMP-534 and an angiotensin-converting enzyme inhibitor provides additional renoprotection in 5/6 nephrectomized rats.

The number of patients with chronic kidney disease (CKD) has continuously grown worldwide. Treatment with antihypertensive agents reduces the rate of progression of CKD, however, there is still a large unmet need to develop strategies for the treatment of CKD. Although we have previously reported that the antifibrotic agent, SMP-534 inhibits the progression of CKD, it is unknown whether combination therapy with SMP-534 and antihypertensive agent shows additive effects on CKD. In present study, we examined whether combination therapy with SMP-534 and the antihypertensive agent, lisinopril is more effective than single therapy with SMP-534 or lisinopril on five-sixths nephrectomized (5/6Nx) rat model. Combination therapy with SMP-534 (50 mg/kg) and lisinopril (5 mg/kg) significantly decreased urinary albumin excretion, blood urea nitrogen (BUN) and serum creatinine and increased creatinine clearance in 5/6Nx rats. On the other hands, single treatment with SMP-534 or lisinopril did not improve renal function at this dose. In addition, combination therapy with SMP-534 and lisinopril significantly decreased extracellular matrix (ECM) accumulation in renal glomeruli and tubulointerstitial injury. These data suggest that combination therapy with an antifibrotic agent and an antihypertensive agent may offer a new therapeutic option for suppressing the progression of CKD.

The anti-fibrotic agent SMP-534 attenuates bleomycin-induced pulmonary fibrosis in hamsters.

Pulmonary fibrosis is a progressive and lethal lung disease characterized by accumulation of ECM and loss of pulmonary function. However, no cure exists for this disease, and current treatments often fail to slow its progression or relieve its symptoms. We have previously reported that the anti-fibrotic agent SMP-534 has beneficial effects on renal fibrosis in animal model of nephropathy. In this study, we examined whether SMP-534 has beneficial effects on pulmonary fibrosis in bleomycin-treated hamsters. Treatment with SMP-534 [low dose (70 mg/kg) or high dose (110 mg/kg)] counteracted inhibition of body weight increase induced by bleomycin. In addition, SMP-534 significantly inhibited bleomycin-induced increase in lung hydroxyproline level, an index of collagen formation. Moreover, SMP-534 significantly ameliorated histological pulmonary fibrotic changes induced by bleomycin. The results of this study indicate that the anti-fibrotic agent SMP-534 may offer a new therapeutic option for the treatment of pulmonary fibrosis.

Chronic administration of SMP-534 ameliorates renal dysfunction in 5/6 nephrectomized rats.

BACKGROUND/AIMS: Chronic kidney disease (CKD) is the common cause of end-stage renal disease. Antihypertensive agents are clinically used to inhibit the progression of CKD. However, these agents cannot completely prevent progression to renal failure. We have previously reported that 5-chloro-2-{(1E)-3-[2-(4-methoxybenzoyl)-4-methyl-1H- pyrrol-1-yl]prop-1en-1-yl}-N-(methylsulfonyl)benzamide (SMP-534) improves renal disease and prevents the production of extracellular matrix in vitro. Additionally, SMP-534 inhibits glomerular fibrosis and provides renoprotection in vivo. In the present study, we investigated the effect of SMP-534 on renal dysfunction in a 5/6 nephrectomized (5/6Nx) rat model. METHOD: Five groups of rats were studied: sham operated, 5/6Nx + vehicle, 5/6Nx + SMP-534 30 mg/kg, 5/6Nx + SMP-534 60 mg/kg and 5/6Nx + SMP-534 90 mg/kg. Treatment with SMP-534 began 13 weeks after surgery, when hypertension and renal insufficiency had developed. Serum creatinine, blood urea nitrogen levels, creatinine clearance and urinary albumin were measured at specific time points. RESULTS: Serum creatinine and blood urea nitrogen levels were significantly reduced in SMP-534-treated groups. In addition, SMP-534 dose-dependently suppressed the increase in urinary albumin excretion observed in 5/6Nx rats. Moreover, survival rates were improved in SMP-534-treated groups. CONCLUSION: We have shown in this study that chronic oral administration of SMP-534 improves renal dysfunction in 5/6Nx rats. These findings indicate that SMP-534 may be a new therapeutic agent for the treatment of CKD.

Amelioration of established diabetic nephropathy by combined treatment with SMP-534 (antifibrotic agent) and losartan in db/db mice.

BACKGROUND/AIMS: Diabetic nephropathy is the main cause of end-stage renal disease. Previously we have demonstrated that SMP-534 (an antifibrotic agent) prevents the development of diabetic nephropathy in db/db mouse and that combined treatment with SMP-534 and losartan (antihypertensive agents) markedly prevents the development of diabetic nephropathy compared with single treatment. SMP-534 or losartan was prophylactically administered to db/db mice before the onset of diabetic nephropathy. In the present study, we evaluated the efficacy of combined treatment when administration was started after the onset of diabetic nephropathy. METHODS: db/db mice were raised untreated until 17 weeks of age, by which time increase of urinary albumin was noted, and then treated with SMP-534 and/or losartan for another 8 weeks. Biochemical and histological analyses were performed at 25 weeks of age. RESULTS: Combined treatment with SMP-534 and losartan markedly prevented the increase of urinary albumin and ameliorated the progression of mesangial matrix expansion, even when administration was started long after the increase of urinary albumin. CONCLUSION: The study results indicate that a combination of SMP-534 and losartan might be a valuable therapeutic approach for the treatment of diabetic nephropathy even when administration is started after the onset of diabetic nephropathy.

Enhanced effect of combined treatment with SMP-534 (antifibrotic agent) and losartan in diabetic nephropathy.

BACKGROUND/AIMS: Diabetic nephropathy is now the most common cause of end-stage renal disease. It is also clear that the current therapy, angiotensin II blockage, cannot prevent the progression of diabetic nephropathy. We had previously demonstrated that an antifibrotic agent, SMP-534, reduced extracellular matrix production induced by transforming growth factor-beta in vitro, and that SMP-534 prevented renal fibrosis and urinary albumin in diabetic db/db mice via a nonantihypertensive mechanism. We expected that combined use of SMP-534 and losartan would produce a more highly renoprotective action. METHODS: We examined the effects of combined treatment with SMP-534 and losartan on urinary albumin and glomerular fibrosis in db/db mice. Diet containing these agents was provided from age 9 to 25 weeks. Blood and urine analyses were performed at 8, 17, and 25 weeks. At the end of the study, kidney tissues were histologically analyzed. RESULTS: SMP-534 significantly suppressed an increase in urinary albumin excretion and ameliorated the progression of glomerular fibrosis in db/db mice, whereas losartan did not. Combined treatment with SMP-534 and losartan markedly prevented the increase of urinary albumin excretion compared with treatment with either SMP-534 or losartan alone. In contrast, renal histological analysis revealed that combined treatment did not significantly prevent an increase of mesangial expansion in the kidney compared with treatment with SMP-534 alone. CONCLUSION: A combination of the two agents, SMP-534 and losartan, might be a valuable therapeutic approach for the treatment of diabetic nephropathy.

SMP-534 ameliorates progression of glomerular fibrosis and urinary albumin in diabetic db/db mice.

Diabetic nephropathy is currently the most common cause of end-stage renal disease. Diabetic nephropathy patients, whether insulin dependent or not, develop fibrotic changes in glomeruli that manifest as overt nephropathy. Previously, we demonstrated that 5-chloro-2-{(1E)-3-[2-(4-methoxybenzoyl)-4-methyl-1H-pyrrol-1-yl]prop-1-en-1-yl}-N-(methylsulfonyl)benzamide (SMP-534) reduces extracellular matrix (ECM) production induced by transforming growth factor-beta (TGF-beta) in vitro and prevents the accumulation of ECM in glomeruli in rat Thy-1 nephritis models. In this study, we examined the long-term effects of SMP-534 on renal insufficiency and glomerulosclerosis in db/db mice, which are models of type 2 diabetes. A diet containing SMP-534 was given to the mice from the age of 9 to 25 wk, and blood and urine analysis were performed at 8, 17, and 25 wk. At the end of study, kidney tissues were analyzed histologically. Treatment with SMP-534 dose dependently suppressed the increase of urinary albumin and type IV collagen excretion in db/db mice. The renal histological analysis showed that SMP-534 dose dependently suppressed the increase of mesangial expansion in the kidney. In the immunohistological analysis, fibronectin and type IV collagen expression were lower in SMP-534-treated db/db mice compared with vehicle-treated db/db mice. This study suggested that SMP-534 ameliorated the increase of ECM production in kidney of db/db mice, possibly through the inhibition of TGF-beta action. Hence, antifibrotic agents such as SMP-534 might be a new therapeutic option for the treatment of diabetic nephropathy.

SMP-534 inhibits TGF-beta-induced ECM production in fibroblast cells and reduces mesangial matrix accumulation in experimental glomerulonephritis.

Transforming growth factor-beta (TGF-beta) is a potent fibrotic factor responsible for the synthesis of extracellular matrix (ECM) and is implicated as the major determinant in pathogenesis of chronic fibroses, including kidney. The novel small compound SMP-534 reduced ECM production induced by TGF-beta in fibroblast cells. SMP-534 inhibited TGF-beta-induced p38 mitogen-activated protein kinase (p38) activation but did not inhibit epidermal growth factor (EGF)-induced extracellular signal-related kinase (ERK) activation. We also found that oral administration of SMP-534 dose dependently lowered hydroxyproline contents in the cortical region of the kidney in rat anti-Thy-1 nephritis models. In periodic acid-Schiff staining of kidney sections, ECM accumulation was reduced by SMP-534 treatment. These data indicate that SMP-534 has potential in therapy for fibrotic diseases, including nephropathy.

Brain-derived neurotrophic factor (BDNF) regulates glucose and energy metabolism in diabetic mice.

Neurotrophins are important regulators in the embryogenesis, development and functioning of nervous systems. In addition to the efficacy of brain-derived neurotrophic factor (BDNF) in neurological disorders, we have found that BDNF demonstrates endocrinological functions and reduces food intake and blood glucose concentration in rodent obese diabetic models, such as C57BL/KsJ-db/db mice. The hypoglycemic effect of BDNF was found to be stronger in younger db/db mice with hyperinsulinemia than in older mice. While BDNF itself did not alter blood glucose in normal mice and streptozotocin (STZ)-treated mice, BDNF enhanced the hypoglycemic effect of insulin in STZ-treated mice. These data indicate that BDNF needs endogenous or exogenous insulin to show hypoglycemic action. In addition, BDNF treatment enhanced energy expenditure in db/db mice. The efficacy of BDNF in regulating glucose and energy metabolism was reproduced through intracerebroventricular administration, suggesting that BDNF acted directly on the hypothalamus, the autonomic center of the brain.