Naunyn-Schmiedeberg's Arch Pharmacol DOI 10.1007/s00210-015-1112-7
ORIGINAL ARTICLE
Reno-protection of G004, a novel anti-diabetic sulfonylurea in db/db mice Xiaohui Tong & Haijian Ma & Sarah Wambui Amadi & Lingman Ma & Guanzhong Wu
Received: 2 October 2014 / Accepted: 24 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract 1-[4-[2-(4-Bromobenzene-sulfonamino)ethyl] phenylsulfonyl]-3-(trans-4-methylcyclohexyl) urea (G004, CAS865483-06-3) is a synthetic sulfonylurea, incorporating the hypoglycemic active structure of glimepiride (CAS 93479-97-1) and anti-TXA2 receptor (TP) active structure of BM-531(CAS 284464-46-6). In this study, we evaluated the effect of G004 on hyperglycemia and dyslipidemia as well as diabetic nephropathy (DN) in db/db mice by gavage over 90 consecutive days of treatment. The fasting blood glucose (FBG), glucose, and insulin tolerance as well as dyslipidemia were effectively ameliorated in db/db mice treated with G004. Interestingly, renal histological results of db/db mice revealed that G004 markedly reversed the expansion of mesangial extracellular matrix (ECM), the early hallmark of DN. Indeed, G004 treatment downregulated the renal expressions of type 4 collagen (Col IV) and transforming growth factor-β1 (TGF-β1) in db/db mice. In addition, imbalance in expressions of matrix metalloproteinase-9 (MMP-9) and its tissue inhibitor-1 (TIMP-1) in db/db mice kidneys was observed. However, G004 increased and decreased the expressions of MMP-9 and TIMP-1, respectively. It is well known that TGF-β pathway signaling plays an essential role in hyperglycemia-induced cell protein synthesis. On the other hand, MMP/TIMP system is responsible for the breakdown and turnover of ECM. Thus, we speculate that G004 possibly attenuated ECM accumulation via remodeling the synthesis and degradation of ECM component Col IV through modulation in TGF-β1 and MMP-9/TIMP-1 expressions in kidneys of db/db mice. Results from this study provide a strong rationale for G004 to be an efficient glucose-controlling agent with significant reno-protective properties.
X. Tong : H. Ma : S. W. Amadi : L. Ma : G. Wu (*) China Pharmaceutical University, Nanjing 210009, China e-mail: [email protected]
Keywords G004 . Glimepiride . db/db mice
Introduction Type 2 diabetes mellitus (T2DM), affecting more than 90 % of diabetic patients, is a heterogeneous and polygenic metabolic disease, characterized by hyperglycemia and insulin resistance (Giorgino et al. 2005). The pathological mechanisms of this disease are mainly attributed to impaired insulin secretion by pancreatic β cells and insulin resistance in target tissues, leading to hyperglycemia. Diabetic nephropathy (DN), one of the major long-term microvascular complications, is the major cause of morbidity and premature mortality in individuals with T2DM. End-stage renal disease in patients with T2DM has increased dramatically worldwide during the last few decades, and diabetes is associated with worse survival among patients undergoing dialysis. However, there is currently no definitive treatment for DN. T2DM patients are associated with platelet abnormalities, alterations in coagulation factors, and disturbances in endothelial cells that lead to a hypercoagulable state (Giona et al. 2006). Recently, reports suggest that platelet hyper-activation might contribute to the increased risk of cardiovascular events of DN (Tarnow et al. 2009). Moreover, blood clotting initiates platelets to release various bioactive substances, including the latent TGF-β (Grainger et al. 1995; Wakefield et al. 1988). And activation of this plateletderived latent TGF-β by shear (Miyazono 2008) contributes to production of extracellular matrix (ECM) proteins. G004 is chemically described as 1-[4-[2-(4-bromobenzenesulfonamino)ethyl]phenylsulfonyl]-3-(trans-4methylcyclohexyl) urea, a combination of glimepiride, the third generation sulfonylurea, as core nucleus with antiplatelet aggregation group. It has been reported that G004 could induce a non-insulin-dependent glucose consumption in HepG2 cells. Additionally, G004 exhibited an excellent
Naunyn-Schmiedeberg's Arch Pharmacol
ability to prevent collagen- and epinephrine-induced mice mortality (Wu et al. 2009). G004 also significantly inhibited platelet aggregation induced by U-46619, a stable analog of TXA2, in both human and rat whole blood. Lately, suppression and relaxation of G004 on atherosclerotic plaque formation in apoE−/− mice and on thoracic aorta of SD rat in vitro were also reported, respectively (Lu et al. 2012). Therefore, these results lead us to hypothesize that G004 might be useful in the prevention and treatment of DN. Db/db mice are a widely used spontaneous model of T2DM and develop diabetic complications similar to those in human condition. Db/db mice develop renal hypertrophy, glomerular enlargement, albuminuria, and mesangial matrix expansion, which are key common features in human with DN (Sharma et al. 2003; Adhikary et al. 2004). In the present study, the C57BLKS db/db mice were used to evaluate the effects of G004 on diabetes and DN, and the efficacy was compared with glimepiride and pioglitazone which are widely used anti-diabetic drugs.
Research Center of Nanjing University (Nanjing, China) and were housed at 20±2 °C and 55 % humidity with a 12:12 h light-dark cycle. The mice were allowed free access to standard chow diet (Qinglongshan Animal Inc., Nanjing, China) comprising 24.0 % protein, 3.5 % lipids, and 60.5 % carbohydrate and water. Group composition was as follows: (1) db/ m mice (n=10), (2) db/db mice (vehicle, n=10), (3) pioglitazone (10 mg/kg, n=10), (4) glimepiride (10 mg/kg, n=10), (5) G004 (20 mg/kg, n=10), (6) G004 (10 mg/kg, n=10), and (7) G004 (5 mg/kg, n=10). Drugs were administrated orally to db/db mice daily for 12 weeks. Body weight and food intake were measured every 2 weeks. After 12 weeks of administration, blood samples were collected, mixed with heparin, and centrifuged at 1000 rpm for 15 min at 4 °C and plasma was isolated. Kidneys were harvested, divided in two, and either snap-frozen or stored in 10 % formalin. Animals were handled and cared for in accordance with the guidelines of Science and Technology Department of Jiangsu Province (SYXK 2012– 0035), and all procedures were approved by the Committee for the Supervision of Animal Experiments of China Pharmaceutical University.
Materials and methods Measurements of glycemia controlling parameters Materials G004 was synthesized and provided by the Department of Medicinal Chemistry, China Pharmaceutical University (purity 98.5 %, Nanjing, China), glimepiride and pioglitazone were purchased from Tianjin Medicine Research Institute Co. Ltd (Tianjin, China) (Fig. 1). All other chemicals were of reagent grade quality and obtained from commercial sources.
Fasting blood glucose (FBG) was determined on days 0, 7, 45 and 90 of the treatment. On day 45, an oral glucose tolerance test (OGTT) was performed after 12-h fasting. Glucose solution (2 g/kg) or corresponding drugs dissolved in glucose solution were gavaged. Blood glucose concentrations were determined by glucometer measurement before glucose challenge and at 0.5, 1, 2 and 4 h thereafter. Plasma HbA1c was determined in the end of the experiment.
Animals and treatment Insulin sensitivity assessment Eight-week-old male C57BLKS/J db/db mice and their lean db/m littermates were purchased from Model Animal
Insulin sensitivity in db/db mice was measured by the insulin tolerance test (ITT) which is a simple and accurate method for screening insulin resistance. ITT was performed after 12-h fasting. Vehicle or corresponding drugs were coadministrated orally with glucose (2 g/kg) by gavage after which insulin (0.5 U/kg) was subcutaneously injected. Blood glucose levels were determined by glucometer at 0, 0.5, 1, 2, and 4 h after glucose administration, respectively. Measurement of plasma and hepatic lipid parameters
Fig. 1 Chemical structure of G004 (a) and glimepiride (b)
Plasma levels of triglyceride (TG) and total cholesterol (TC) were measured using enzymatic kits (Wako, Osaka, Japan). Plasma free fatty acid (FFA) concentrations were measured by spectrophotometric commercial kits (Sigma Chemical Co.). Liver samples (100 mg) of each mouse were homogenized in 0.9 mL mixture of chloroform and methanol (2:1, v/v), total lipids of the liver homogenates were extracted 12 h later, and
Naunyn-Schmiedeberg's Arch Pharmacol
the amounts of TG and TC were determined. Content of FFAs in the liver (100 mg) of each mouse was determined according to kits instruction. Histopathological study Kidney samples fixed in 10 % formalin were embedded in paraffin, and 4-μm-thick sections were cut for morphological study. The histology was assessed following hematoxylineosin (H&E) staining. Then, the severity of renal pathological development was scored on an arbitrary scale from 0 to 3 in a blinded manner using light microscopy (Olympus BX-50, Olympus Optical, Tokyo, Japan).
Positive staining (dark brown) for Col IV, TGF-β, MMP-9, and TIMP-1 in each glomerulus was quantified by two investigators in a double-blind manner at ×400 magnification using Image Pro Plus and expressed as the ratio of the mean of normal mice. Two glomeruli were examined in each animal (a total of 20 glomeruli for each group). Statistical analysis The data are expressed as the mean±standard error of the mean (SEM). Difference between the groups was examined for statistical significance using ANOVA followed by a Dunnett’s post hoc test. A value of p
ORIGINAL ARTICLE
Reno-protection of G004, a novel anti-diabetic sulfonylurea in db/db mice Xiaohui Tong & Haijian Ma & Sarah Wambui Amadi & Lingman Ma & Guanzhong Wu
Received: 2 October 2014 / Accepted: 24 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract 1-[4-[2-(4-Bromobenzene-sulfonamino)ethyl] phenylsulfonyl]-3-(trans-4-methylcyclohexyl) urea (G004, CAS865483-06-3) is a synthetic sulfonylurea, incorporating the hypoglycemic active structure of glimepiride (CAS 93479-97-1) and anti-TXA2 receptor (TP) active structure of BM-531(CAS 284464-46-6). In this study, we evaluated the effect of G004 on hyperglycemia and dyslipidemia as well as diabetic nephropathy (DN) in db/db mice by gavage over 90 consecutive days of treatment. The fasting blood glucose (FBG), glucose, and insulin tolerance as well as dyslipidemia were effectively ameliorated in db/db mice treated with G004. Interestingly, renal histological results of db/db mice revealed that G004 markedly reversed the expansion of mesangial extracellular matrix (ECM), the early hallmark of DN. Indeed, G004 treatment downregulated the renal expressions of type 4 collagen (Col IV) and transforming growth factor-β1 (TGF-β1) in db/db mice. In addition, imbalance in expressions of matrix metalloproteinase-9 (MMP-9) and its tissue inhibitor-1 (TIMP-1) in db/db mice kidneys was observed. However, G004 increased and decreased the expressions of MMP-9 and TIMP-1, respectively. It is well known that TGF-β pathway signaling plays an essential role in hyperglycemia-induced cell protein synthesis. On the other hand, MMP/TIMP system is responsible for the breakdown and turnover of ECM. Thus, we speculate that G004 possibly attenuated ECM accumulation via remodeling the synthesis and degradation of ECM component Col IV through modulation in TGF-β1 and MMP-9/TIMP-1 expressions in kidneys of db/db mice. Results from this study provide a strong rationale for G004 to be an efficient glucose-controlling agent with significant reno-protective properties.
X. Tong : H. Ma : S. W. Amadi : L. Ma : G. Wu (*) China Pharmaceutical University, Nanjing 210009, China e-mail: [email protected]
Keywords G004 . Glimepiride . db/db mice
Introduction Type 2 diabetes mellitus (T2DM), affecting more than 90 % of diabetic patients, is a heterogeneous and polygenic metabolic disease, characterized by hyperglycemia and insulin resistance (Giorgino et al. 2005). The pathological mechanisms of this disease are mainly attributed to impaired insulin secretion by pancreatic β cells and insulin resistance in target tissues, leading to hyperglycemia. Diabetic nephropathy (DN), one of the major long-term microvascular complications, is the major cause of morbidity and premature mortality in individuals with T2DM. End-stage renal disease in patients with T2DM has increased dramatically worldwide during the last few decades, and diabetes is associated with worse survival among patients undergoing dialysis. However, there is currently no definitive treatment for DN. T2DM patients are associated with platelet abnormalities, alterations in coagulation factors, and disturbances in endothelial cells that lead to a hypercoagulable state (Giona et al. 2006). Recently, reports suggest that platelet hyper-activation might contribute to the increased risk of cardiovascular events of DN (Tarnow et al. 2009). Moreover, blood clotting initiates platelets to release various bioactive substances, including the latent TGF-β (Grainger et al. 1995; Wakefield et al. 1988). And activation of this plateletderived latent TGF-β by shear (Miyazono 2008) contributes to production of extracellular matrix (ECM) proteins. G004 is chemically described as 1-[4-[2-(4-bromobenzenesulfonamino)ethyl]phenylsulfonyl]-3-(trans-4methylcyclohexyl) urea, a combination of glimepiride, the third generation sulfonylurea, as core nucleus with antiplatelet aggregation group. It has been reported that G004 could induce a non-insulin-dependent glucose consumption in HepG2 cells. Additionally, G004 exhibited an excellent
Naunyn-Schmiedeberg's Arch Pharmacol
ability to prevent collagen- and epinephrine-induced mice mortality (Wu et al. 2009). G004 also significantly inhibited platelet aggregation induced by U-46619, a stable analog of TXA2, in both human and rat whole blood. Lately, suppression and relaxation of G004 on atherosclerotic plaque formation in apoE−/− mice and on thoracic aorta of SD rat in vitro were also reported, respectively (Lu et al. 2012). Therefore, these results lead us to hypothesize that G004 might be useful in the prevention and treatment of DN. Db/db mice are a widely used spontaneous model of T2DM and develop diabetic complications similar to those in human condition. Db/db mice develop renal hypertrophy, glomerular enlargement, albuminuria, and mesangial matrix expansion, which are key common features in human with DN (Sharma et al. 2003; Adhikary et al. 2004). In the present study, the C57BLKS db/db mice were used to evaluate the effects of G004 on diabetes and DN, and the efficacy was compared with glimepiride and pioglitazone which are widely used anti-diabetic drugs.
Research Center of Nanjing University (Nanjing, China) and were housed at 20±2 °C and 55 % humidity with a 12:12 h light-dark cycle. The mice were allowed free access to standard chow diet (Qinglongshan Animal Inc., Nanjing, China) comprising 24.0 % protein, 3.5 % lipids, and 60.5 % carbohydrate and water. Group composition was as follows: (1) db/ m mice (n=10), (2) db/db mice (vehicle, n=10), (3) pioglitazone (10 mg/kg, n=10), (4) glimepiride (10 mg/kg, n=10), (5) G004 (20 mg/kg, n=10), (6) G004 (10 mg/kg, n=10), and (7) G004 (5 mg/kg, n=10). Drugs were administrated orally to db/db mice daily for 12 weeks. Body weight and food intake were measured every 2 weeks. After 12 weeks of administration, blood samples were collected, mixed with heparin, and centrifuged at 1000 rpm for 15 min at 4 °C and plasma was isolated. Kidneys were harvested, divided in two, and either snap-frozen or stored in 10 % formalin. Animals were handled and cared for in accordance with the guidelines of Science and Technology Department of Jiangsu Province (SYXK 2012– 0035), and all procedures were approved by the Committee for the Supervision of Animal Experiments of China Pharmaceutical University.
Materials and methods Measurements of glycemia controlling parameters Materials G004 was synthesized and provided by the Department of Medicinal Chemistry, China Pharmaceutical University (purity 98.5 %, Nanjing, China), glimepiride and pioglitazone were purchased from Tianjin Medicine Research Institute Co. Ltd (Tianjin, China) (Fig. 1). All other chemicals were of reagent grade quality and obtained from commercial sources.
Fasting blood glucose (FBG) was determined on days 0, 7, 45 and 90 of the treatment. On day 45, an oral glucose tolerance test (OGTT) was performed after 12-h fasting. Glucose solution (2 g/kg) or corresponding drugs dissolved in glucose solution were gavaged. Blood glucose concentrations were determined by glucometer measurement before glucose challenge and at 0.5, 1, 2 and 4 h thereafter. Plasma HbA1c was determined in the end of the experiment.
Animals and treatment Insulin sensitivity assessment Eight-week-old male C57BLKS/J db/db mice and their lean db/m littermates were purchased from Model Animal
Insulin sensitivity in db/db mice was measured by the insulin tolerance test (ITT) which is a simple and accurate method for screening insulin resistance. ITT was performed after 12-h fasting. Vehicle or corresponding drugs were coadministrated orally with glucose (2 g/kg) by gavage after which insulin (0.5 U/kg) was subcutaneously injected. Blood glucose levels were determined by glucometer at 0, 0.5, 1, 2, and 4 h after glucose administration, respectively. Measurement of plasma and hepatic lipid parameters
Fig. 1 Chemical structure of G004 (a) and glimepiride (b)
Plasma levels of triglyceride (TG) and total cholesterol (TC) were measured using enzymatic kits (Wako, Osaka, Japan). Plasma free fatty acid (FFA) concentrations were measured by spectrophotometric commercial kits (Sigma Chemical Co.). Liver samples (100 mg) of each mouse were homogenized in 0.9 mL mixture of chloroform and methanol (2:1, v/v), total lipids of the liver homogenates were extracted 12 h later, and
Naunyn-Schmiedeberg's Arch Pharmacol
the amounts of TG and TC were determined. Content of FFAs in the liver (100 mg) of each mouse was determined according to kits instruction. Histopathological study Kidney samples fixed in 10 % formalin were embedded in paraffin, and 4-μm-thick sections were cut for morphological study. The histology was assessed following hematoxylineosin (H&E) staining. Then, the severity of renal pathological development was scored on an arbitrary scale from 0 to 3 in a blinded manner using light microscopy (Olympus BX-50, Olympus Optical, Tokyo, Japan).
Positive staining (dark brown) for Col IV, TGF-β, MMP-9, and TIMP-1 in each glomerulus was quantified by two investigators in a double-blind manner at ×400 magnification using Image Pro Plus and expressed as the ratio of the mean of normal mice. Two glomeruli were examined in each animal (a total of 20 glomeruli for each group). Statistical analysis The data are expressed as the mean±standard error of the mean (SEM). Difference between the groups was examined for statistical significance using ANOVA followed by a Dunnett’s post hoc test. A value of p
