Application of convolutional neural network for analyzing hepatic fibrosis in mice.

Recently, with the development of computer vision using artificial intelligence (AI), clinical research on diagnosis and prediction using medical image data has increased. In this study, we applied AI methods to analyze hepatic fibrosis in mice to determine whether an AI algorithm can be used to analyze lesions. Whole slide image (WSI) Sirius Red staining was used to examine hepatic fibrosis. The Xception network, an AI algorithm, was used to train normal and fibrotic lesion identification. We compared the results from two analyses, that is, pathologists' grades and researchers' annotations, to observe whether the automated algorithm can support toxicological pathologists efficiently as a new apparatus. The accuracies of the trained model computed from the training and validation datasets were greater than 99%, and that obtained by testing the model was 100%. In the comparison between analyses, all analyses showed significant differences in the results for each group. Furthermore, both normalized fibrosis grades inferred from the trained model annotated the fibrosis area, and the grades assigned by the pathologists showed significant correlations. Notably, the deep learning algorithm derived the highest correlation with the pathologists' average grade. Owing to the correlation outcomes, we conclude that the trained model might produce results comparable to those of the pathologists' grading of the Sirius Red-stained WSI fibrosis. This study illustrates that the deep learning algorithm can potentially be used for analyzing fibrotic lesions in combination with Sirius Red-stained WSIs as a second opinion tool in non-clinical research.

A Comparative Systematic Analysis of The Influence of Microplastics on Colon Cells, Mouse and Colon Organoids.

BACKGROUND: Microplastics (MPs) are small fragments from any type of plastic formed from various sources, including plastic waste and microfibers from clothing. MPs degrades slowly, resulting in a high probability of human inhalation, ingestion and accumulation in bodies and tissues. As its impact on humans is a prolonged event, the evaluation of its toxicity and influence on human health are critical. In particular, MPs can enter the human digestive system through food and beverage consumption, and its effect on the human colon needs to be carefully examined. METHODS: We monitored the influence of small MPs (50 and 100 nm) on human colon cells, human colon organoids and also examined their toxicity and changes in gene expression in vivo in a mouse model. RESULTS: The data suggested that 5 mg/mL concentrations of 50 and 100 nm MPs induced a > 20% decrease in colon organoid viability and an increase in the expression of inflammatory-, apoptosis- and immunity-related genes. In addition, in vivo data suggested that 50 nm MPs accumulate in various mouse organs, including the colon, liver, pancreas and testicles after 7 d of exposure. CONCLUSION: Taken together, our data suggest that smaller MPs can induce more toxic effects in the human colon and that human colon organoids have the potential to be used as a predictive tool for colon toxicity.

Development of in vitro three-dimensional drug screening system for obesity-related metabolic syndrome.

Metabolic syndrome is increasingly common, and closely related with overweight or obesity. In the obese state, macrophages infiltrate to the adipose tissue (AT), resulting in chronic inflammation and insulin resistance in the AT cells. Recently, attention has been paid to the role of AT macrophages in metabolic disorders should be applied to the initial drug screening step, but it was difficult to mimic the inflammatory adipocytes using the traditional 2-dimensional (2D) culture. In this study, we developed the 3-dimensional (3D) culture system to overcome this limitation. After adipogenic differentiation, lipid droplets were highly accumulated in cells, and differentiation of preadipocytes was not declined by macrophage co-culture. However, only co-cultured cells expressed the insulin resistance features. Compare to mono-cultured adipocytes, co-cultured adipocytes showed reduced glucose uptake and GLUT4 did not translocated to cell membrane even though treatment of high concentration of insulin. Using 3D co-culture model, we develop a microwell-scale drug screening protocol to test anti-obesity effect. 3D cultured cells reacted more sensitive to drugs, and PPARγ antagonist GW9662 (10, 20 µM) repressed adipogenic differentiation in a concentration-dependent manner in 3D co-cultured cells.

Toxicity Assessment of SiO2 and TiO2 in Normal Colon Cells, In Vivo and in Human Colon Organoids.

We conducted systemic assessments on the toxicity of silicon dioxide (SiO2) and titanium dioxide (TiO2) nanoparticles using different forms of normal colon cells (CCD-18Co), in vivo and in human colon organoids. The in vivo acute oral toxicity data showed that the LD50 values are greater than 2000 mg/kg for both the SiO2 and TiO2 nanoparticles; however, the SiO2 and TiO2 nanoparticles induced cytotoxicity in two-dimensional CCD-18Co cells and three-dimensional CCD-18Co spheroids and human colon organoids, with IC50 values of 0.6, 0.8 and 0.3 mM for SiO2 and 2.5, 1.1 and 12.5 mM for TiO2 nanoparticles, respectively. The data suggest that, when SiO2 and TiO2 are in nanoparticle form, cytotoxicity is induced; thus, care should be taken with these materials.

A Novel Selective 11ß-HSD1 Inhibitor, (E)-4-(2-(6-(2,6-Dichloro-4-(Trifluoromethyl)Phenyl)-4-Methyl-1,1-Dioxido-1,2,6-Thiadiazinan-2-yl)Acetamido)Adamantan-1-Carboxamide (KR-67607), Prevents BAC-Induced Dry Eye Syndrome.

Dry eye syndrome is the most common eye disease and it is caused by various reasons. As the balance of the tear film that protects the eyes is broken due to various causes, it becomes impossible to properly protect the eyes. In this study, the protective effects and underlying mechanisms of topical (E)-4-(2-(6-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-methyl-1,1-dioxido-1,2,6-thiadiazinan-2-yl)acetamido)adamantan-1-carboxamide (KR-67607), a novel selective 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) inhibitor, were investigated in benzalkonium chloride (BAC)-induced dry eye syndrome. BAC-treated rat eyes induced significant increases in ocular surface damage, decreased corneal thickness, corneal basement membrane destruction in the conjunctival epithelium, and expression of pro-inflammatory cytokines tumor necrosis factor-α and 11ß-HSD1. These effects of BAC were reversed by topical KR-67607 treatment. Furthermore, KR-67607 decreased 4-hydroxynonenal expression and increased antioxidant and mucus secretion in BAC-treated rat eyes. Taken together, a novel selective 11ß-HSD1 inhibitor can prevent BAC-induced dry eye syndrome by inhibiting pro-inflammatory cytokine and reactive oxygen species expression via the inhibition of both 11ß-HSD1 activity and expression.

Development of a three-dimensional in vitro co-culture model to increase drug selectivity for humans.

AIM: Insulin resistance is a metabolic state where insulin sensitivity is lower than normal condition and strongly related to type 2 diabetes. However, an in vitro model mimicking insulin resistance is rare and thus screening drugs for insulin resistance severely depends on an in vivo model. Here, to increase anti-diabetic drug selectivity for humans, 3D ADMSCs and macrophages were co-cultured with in-house fabricated co-culture plates. MATERIAL AND METHODS: 3D co-culture plates were designed to load ADMSCs and RAW264.7 cells containing hydrogels in separate wells while allowing cell-cell interaction with co-culturing media. Hydrogels were constructed using a 3D cell-printing system containing 20 mg/ml alginate, 0.5 mg/ml gelatin and 0.5 mg/ml type I collagen. Cells containing hydrogels in 3D co-culture plates were incubated for 10 min to allow stabilization before the experiment. 3D co-culture plates were incubated with the CaCl2 solution for 5 min to complete the cross linking of alginate hydrogel. Cells in 3D co-culture plates were cultured for up to 12 days depending on the experiment and wells containing adipocytes and macrophages were separated and used for assays. RESULTS: KR-1, KR-2 and KR-3 compounds were applied during differentiation (12 days) in 3D co-cultured mouse 3T3-L1 adipocytes and 3D co-cultured human ADMSCs. Glucose uptake assay using 2-DG6P and 2-NBDG and western blot analysis were performed to investigate changes of insulin resistance in the 3D co-cultured model for interspecies selectivity of drug screening. KR-1 (mouse potent enantiomer) and KR-3 (racemic mixture) showed improvement of 2-DG and 2-NBDG uptake compared with KR-2 (human potent enantiomer) in 3D co-cultured 3T3-L1 adipocytes. In connection with insulin resistance in a 3D 3T3-L1 co-cultured model, KR-1 and KR-3 showed improvement of insulin sensitivity compared to KR-2 by markedly increasing GLUT4 expression. In contrast to the result of 3D co-cultured 3T3-L1 adipocytes, KR-1 failed to significantly improve 2-DG and 2-NBDG uptake in 3D co-cultured ADMSC adipocytes. Results of 2-NBDG accumulation and western blot analysis also showed that KR-2 and KR-3 improved insulin sensitivity relatively better than KR-1. CONCLUSIONS: Our 3D co-culture model with/without 3D co-culture plates can successfully mimic insulin resistance while allowing investigation of the effects of anti-obesity or anti-diabetic drugs on human or mouse co-culturing cell type. This 3D co-culture system may accelerate screening of drugs for insulin resistance depending on species.

Optimization of cyclic sulfamide derivatives as 11ß-hydroxysteroid dehydrogenase 1 inhibitors for the potential treatment of ischemic brain injury.

The 11ß-hydroxysteroiddehydrogenase type 1(11ß-HSD1), acortisolregenerating enzyme that amplifies tissue glucocorticoidlevels, plays an important role in diabetes, obesity, and glaucoma and is recognized as a potential therapeutic target for various disease conditions. Moreover, a recent study demonstrated that selective 11ß-HSD1 inhibitor can attenuate ischemic brain injury. This prompted us to optimize cyclic sulfamide derivative for aiming to treat ischemic brain injury. Among the synthesized compounds, 6e has an excellent in vitro activivity with an IC50 value of 1 nM toward human and mouse 11ß-HSD1 and showed good 11ß-HSD1 inhibition in ex vivo study using brain tissue isolated from mice. Furthermore, in the transient middle cerebral artery occlusion model in mice, 6e treatment significantly attenuated infarct volume and neurological deficit following cerebral ischemia/reperfusion injury. Additionally, binding modes of 6e for human and mouse 11ß-HSD1 were suggested.

Anticancer effect of XAV939 is observed by inhibiting lactose dehydrogenase A in a 3-dimensional culture of colorectal cancer cells.

XAV939, a tankyrase inhibitor, exerts an anticancer effect in 3-dimensional (3D) cultured SW480 cells, however this is not exhibited in 2-dimensional (2D) cultured SW480 cells. In the current study, XAV939 induced a 3.7-fold increase in cellular apoptosis in 3D culture but not in the 2D culture. However, no significant changes were indicated in cell cycle distribution in the 2D or 3D culture. Based on the observation that protein expression, which was associated with the glycolytic pathway, was increased in the 3D culture, the effect of XAV939 on the patterns of glycolytic protein expression was assessed. XAV939 was revealed to decrease lactose dehydrogenase A (LDHA) expression in 3D cultured SW480 cells, but only exerted a small effect in the 2D culture. The coadministration of XAV939 with the LDHA inhibitor FX11 decreased proliferation in 3D cultured SW480 cells compared with the single administration of FX11, while there was no additive effect in the 2D culture. The lactate assay also indicated that XAV939 decreased lactate secretion in the 3D cell culture but not in the 2D culture. These results suggest that XAV939 exerts an anticancer effect through inhibition of LDHA in the 3D culture.

Protective effect of a novel selective 11ß-HSD1 inhibitor on eye ischemia-reperfusion induced glaucoma.

Glaucoma is one of the leading causes of preventable blindness, affecting > 2 million people in the United States. Recently, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitors were found to exert preventive effects against glaucoma. However, there is no evidence for the role of 11ß-HSD1 inhibitors against glaucoma. Here, we developed a novel 11ß-HSD1 inhibitor, (1R,2S,3S,5R,6S,7S)-6-(2-(6-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-methyl-1,1-dioxido-1,2,6-thiadiazinan-2-yl)acetamido)-adamantane-2-carboxamide (KR-67607) and showed its protective effects against ischemia-reperfusion-induced eye injury. We demonstrate that KR-67607 effectively reduced cortisol levels in mouse eyes and maintained the trabecular meshwork (TM) structure in the presence of transient ischemic stress. Furthermore, KR-67607 reversed the elevation of intra-ocular pressure (IOP), suggesting that the TM structure maintained by KR-67607 prevented the excessive rise in IOP that exacerbates glaucoma. KR-67607 was shown to have a higher specificity for 11ß-HSD1 than carbenoxolone (CBX) in vitro. Moreover, KR-67607 reduced apoptosis and the structural disruption of TM cells. Antioxidation was the major protective pathway of KR-67607 against chemically-induced ischemia-reperfusion in TM cells and the glucocorticoid receptor (GR) was closely associated with this pathway. When TM cells undergo ischemic stress, GR is activated and then translocates to the cell nucleus where it interferes with Nrf-2-mediated antioxidant gene expression. However, when KR-67607 inhibited GR translocation, Nrf-2 was able to induce antioxidant gene transcription, which consequently, enhanced the antioxidant capacity of the cells. In conclusion, our current work describes a novel selective 11ß-HSD1 inhibitor for glaucoma treatment and provides evidence of its physiological role in anti-oxidative pathways in the TM.

Cyp7a1 is continuously increased with disrupted Fxr-mediated feedback inhibition in hypercholesterolemic TALLYHO/Jng mice.

TALLYHO/Jng (TH) mice reveal hypercholesterolemia at an early age before their plasma glucose levels have increased. The increased plasma cholesterol should be related to bile acids (BAs) metabolism, because cholesterol is the precursor of BAs and BAs change cholesterol metabolism in a feedback manner. We analyzed the BAs pool size, BAs composition, and expression levels of several proteins that have key roles in BAs synthesis, excretion, and reabsorption and compared them to those of C57BL/6 (B6) mice to study BAs metabolism in TH mice. TH mice exhibited an increased total BAs pool size, increased BAs content in the cecum feces, and an increased ratio of muricholic acid (MCA)/cholic acid (CA). The mRNA and protein levels of cholesterol 7 alpha-hydroxylase (Cyp7a1) and the ATP-binding cassette sub-family G member 5 (Abcg5) were elevated in the liver but not in the apical sodium bile acid transporter (Asbt) and organic solute transporters (Osts) in the ileum. These results indicate that synthesis and the excretion of BAs from the liver to the gallbladder might be elevated, but the reabsorption rate of BAs in the ileum might be reduced. The declined expression of fibroblast growth factor 15 (Fgf15) and fibroblast growth factor receptor 4 (Fgfr4) was respectively identified in the ileum and the liver, indicating the disrupted feedback inhibition of Cyp7a1. Consequently, hypercholesterolemia in TH mice might increase the BAs amounts via the interrupted Fxr/Fgf15/Fgfr4-mediated feedback regulation of Cyp7a1.

Synthesis and biological evaluation of thiazole derivatives as GPR119 agonists.

A series of 4-(phenoxymethyl)thiazole derivatives was synthesized and evaluated for their GPR119 agonistic effect. Several 4-(phenoxymethyl)thiazoles with pyrrolidine-2,5-dione moieties showed potent GPR119 agonistic activities. Among them, compound 27 and 32d showed good in vitro activity with an EC50 value of 49 nM and 18 nM, respectively with improved human and rat liver microsomal stability compare with MBX-2982. Compound 27 &32d did not exhibit significant CYP inhibition, hERG binding, and cytotoxicity. Moreover, these compounds lowered the glucose excursion in mice in an oral glucose-tolerance test.

Protective effects of carbenoxolone, an 11ß-HSD1 inhibitor, against chemical induced dry eye syndrome.

Dry eye syndrome (DES) is a disorder of the eye due to tear deficiency or excessive evaporation that causes damage to the eye and is associated with discomfort and dryness. 11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) is an enzyme that converts inactive cortisone to active cortisol. Recently, 11ß-HSD1 has been expressed in human and rodent eyes and has been recognized as a target of glaucoma. In this study, the therapeutic effects and underlying mechanisms of topical carbenoxolone, an 11ß-HSD1 inhibitor, were investigated in benzalkonium chloride (BAC)-treated human conjunctival epithelial cells and a rat DES model. In the in vitro study, carbenoxolone dose-dependently inhibited cell death and 11ß-HSD1 activity in BAC-treated human conjunctival epithelial cells. For the in vivo study, carbenoxolone or a solvent was administered to the BAC-induced DES model twice daily. BAC-treated rat eyes showed significant increases in ocular surface damage, a reduction of tears, decrease corneal thickness, corneal basement membrane destruction, apoptosis in the conjunctival epithelium, and expression of pro-inflammatory cytokines (TNF-α and IL-6) and 11ß-HSD1. These effects of BAC were reversed by topical carbenoxolone treatment. These results demonstrate that carbenoxolone can prevent DES by inhibiting pro-inflammatory cytokine expression and cell death of the corneal and conjunctival epithelium via inhibition of both 11ß-HSD1 activity and expression in the eyes of BAC-treated rats. It is suggested that topical 11ß-HSD1 inhibitors may provide a new therapeutic window in the prevention and/or treatment of DES.

Carbenoxolone prevents chemical eye ischemia-reperfusion-induced cell death via 11ß-hydroxysteroid dehydrogenase type 1 inhibition.

Glaucoma is one of the leading causes of preventable blindness diseases, affecting more than 2 million people in the United States. Recently, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitors were found to exert preventive effects against glaucoma. Therefore, we investigated whether carbenoxolone (CBX), an 11ß-HSD1 inhibitor, prevents chemical ischemia-reperfusion-induced cell death in human trabecular meshwork (HTM) cells. The present study demonstrated that CBX inhibited cell death caused by iodoacetic acid (IAA)-induced ischemia-reperfusion, and its effect was associated with the inhibition of 11ß-HSD1 expression and activity. Furthermore, CBX reversed the IAA-induced structural damage on filamentous actin in HTM cells. In IAA-treated cells, the levels of 11ß-HSD1 and the apoptosis-related factors Bax and FASL were increased throughout the reperfusion period, and CBX was able to attenuate the expression of 11ß-HSD1 and the apoptosis-related factors. CBX also effectively suppressed IAA-induced intracellular ROS formation and cytochrome c release, which are involved in the mitochondrial apoptosis pathway. In addition, IAA-induced chemical ischemia-reperfusion stimulated TNF-α expression and NF-κB p65 phosphorylation, and these effects were attenuated by CBX. 11ß-HSD1 RNAi also suppressed IAA-induced cell apoptosis via reduction of oxidative stress and inhibition of the pro-inflammatory pathway. Taken together, the present study demonstrated that the inhibition of 11ß-HSD1 protected the TM against chemical ischemia-reperfusion injury, suggesting that the use of 11ß-HSD1 inhibitors could be a useful strategy for glaucoma therapy.

Anti-inflammatory effect of a selective 11ß-hydroxysteroid dehydrogenase type 1 inhibitor via the stimulation of heme oxygenase-1 in LPS-activated mice and J774.1 murine macrophages.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) converts inactive cortisone to the active cortisol. 11ß-HSD1 may be involved in the resolution of inflammation. In the present study, we investigate the anti-inflammatory effects of 2-(3-benzoyl)-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344), a selective 11ß-HSD1 inhibitor, in lipopolysaccharide (LPS)-activated C57BL/6J mice and macrophages. LPS increased 11ß-HSD1 activity and expression in macrophages, which was inhibited by KR-66344. In addition, KR-66344 increased survival rate in LPS treated C57BL/6J mice. HO-1 mRNA expression level was increased by KR-66344, and this effect was reversed by the HO competitive inhibitor, ZnPP, in macrophages. Moreover, ZnPP reversed the suppression of ROS formation and cell death induced by KR-66344. ZnPP also suppressed animal survival rate in LPS plus KR-66344 treated C57BL/6J mice. In the spleen of LPS-treated mice, KR-66344 prevented cell death via suppression of inflammation, followed by inhibition of ROS, iNOS and COX-2 expression. Furthermore, LPS increased NFκB-p65 and MAPK phosphorylation, and these effects were abolished by pretreatment with KR-66344. Taken together, KR-66344 protects against LPS-induced animal death and spleen injury by inhibition of inflammation via induction of HO-1 and inhibition of 11ß-HSD1 activity. Thus, we concluded that the selective 11ß-HSD1 inhibitor may provide a novel strategy in the prevention/treatment of inflammatory disorders in patients.

Identification of a novel 11ß-HSD1 inhibitor from a high-throughput screen of natural product extracts.

Selective inhibitors of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) have considerable potential as a treatment for metabolic syndrome including type 2 diabetes mellitus and obesity. To identify 11ß-HSD1 inhibitors, we conducted high-throughput screening (HTS) of active natural product extracts from the Korea Chemical Bank, including Tanshinone I, Tanshinone IIA, and flavanone derivatives, and 2- and 3-phenyl-4H-chromen-4-one. Then Tanshinone IIA and its derivatives were targeted for the development of a lead compound according to the HTS results. However, the mechanism for anti-adipogenic effect through 11ß-HSD1 enzyme inhibition by Tanshinone IIA is not clear. Tanshinone IIA (2a) concentration-dependently inhibited 11ß-HSD1 activity in human and mouse 11ß-HSD1 overexpressed cells and 3T3-L1 adipocytes. Tanshinone IIA (2a) also inhibited 11ß-HSD1 enzyme activities in murine liver and fats. Furthermore, Tanshinone IIA (2a)-suppressed adipocyte differentiation of cortisone-induced adipogenesis in 3T3-L1 cells was associated with the suppression of the cortisone-induced adipogenesis-specific markers mRNA and protein expression. In 3T3-L1 preadipocytes, Tanshinone IIA (2a)-inhibited cortisone induced reactive oxygen species formation in a concentration-dependent manner. Thus, these results support the therapeutic potential of Tanshinone IIA (2a) as a 11ß-HSD1 inhibitor in metabolic syndrome patients.

Synthesis and biological evaluation of thienopyrimidine derivatives as GPR119 agonists.

A series of thienopyrimidine derivatives was synthesized and evaluated for their GPR119 agonistic ability. Several thienopyrimidine derivatives containing R(1) and R(2) substituents displayed potent GPR119 agonistic activity. Among them, compound 5d, which is a prototype, showed good in vitro activity with an EC50 value of 3 nM and human and rat liver microsomal stability. Compound 5d exhibited no CYP inhibition and induction, Herg binding, or mutagenic potential. Compound 5d showed increase insulin secretion in beta TC-6 cell and lowered the glucose excursion in mice in an oral glucose-tolerance test.

A novel 11ß-HSD1 inhibitor improves diabesity and osteoblast differentiation.

Selective inhibitors of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) have considerable potential as treatment for osteoporosis as well as metabolic syndrome including type 2 diabetes mellitus. Here, we investigated the anti-diabetic, anti-adipogenic, and anti-osteoporotic activity of KR-67500, as a novel selective 11ß-HSD1 inhibitor. Cellular 11ß-HSD1 activity was tested based on a homogeneous time-resolved fluorescence method. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) levels were measured in diet-induced obese (DIO)-C57BL/6 mice administered KR-67500 (50  mg/kg per day, p.o.) for 28 days and, additionally, its anti-diabetic effect was evaluated by OGTT and ITT. The in vitro anti-adipogenic effect of KR-67500 was determined by Oil Red O Staining. The in vitro anti-osteoporotic activity of KR-67500 was evaluated using bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation model systems. KR-67500 improved the in vivo glucose tolerance and insulin sensitivity in DIO-C57BL/6 mice. KR-67500 suppressed cortisone-induced differentiation of 3T3-L1 cells into adipocytes. KR-67500 enhanced BMP2-induced osteoblastogenesis in C2C12 cells and inhibited RANKL-induced osteoclastogenesis in mouse bone marrow-derived macrophages. KR-67500, a new selective 11ß-HSD1 inhibitor, may provide a new therapeutic window in the prevention and/or treatment of type 2 diabetes, obesity, and/or osteoporosis.

Anti-diabetic and anti-inflammatory effect of a novel selective 11ß-HSD1 inhibitor in the diet-induced obese mice.

It has been reported that the selective inhibitors of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) have considerable potential for treating type 2 diabetes mellitus, metabolic syndrome and inflammation. In the present study, we investigated the anti-diabetic and anti-inflammatory effects of N-(5-carbamoyladamantan-2-yl)-3-((2-fluorophenyl) sulfonyl)thiazolidine-2-carboxamide (KR-67105), a novel 11ß-HSD1 inhibitor, in diabetic mice model and preadipocyte model. KR-67105 concentration dependently inhibited 11ß-HSD1 activity in human and mouse 11ß-HSD1 overexpressing cells and mouse 3T3-L1 adipocytes. Furthermore, KR-67105 concentration-dependently inhibited 11ß-HSD1 activity in the ex vivo assay of C57BL/6 mice. In the study with diet-induced obese (DIO) mice, the administration of KR-67105 (100mg/kg/day, orally for 28 days) improved the glucose tolerance and insulin sensitivity as determined by the oral glucose tolerance test and the insulin tolerance test. Anti-diabetic effect by KR-67105 was associated with the suppression of diabetic related genes expression in liver and fat. Furthermore, KR-67105 suppressed 11ß-HSD1 activity in liver and fat of diabetic mice, but showed no effect on adrenal grand weight/body weight ratio and plasma corticosterone concentration in diabetic mice. In 3T3-L1 preadipocytes, cortisone induced the mRNA of inflammatory cytokines and 11ß-HSD1 and reactive oxygen species formation. This effect was abolished by co-incubation with KR-67105 in a concentration-dependent manner. Moreover, KR-67105 attenuated cortisone induced iNOS expression and phosphorylation of NF-κB p65, p38 MAPK, and ERK1/2 in preadipocytes. Taken together, it is concluded that a selective 11ß-HSD1 inhibitor, KR-67105, may provide a new therapeutic window in the prevention and treatment of type 2 diabetes with chronic inflammation without toxicity.

Synthesis and biological evaluation of aminobenzimidazole derivatives with a phenylcyclohexyl acetic acid group as anti-obesity and anti-diabetic agents.

A series of benzimidazole derivatives with a phenylcyclohexyl acetic acid group as DGAT-1 inhibitors was developed. Among the benzimidazole series, compound 5k showed submicromolar in vitro activity toward human and mouse DGAT-1, good selectivity toward DGAT-2, human liver metabolic stability, and pharmacokinetic (PK) and safety profiles such as hERG, CYP and acute toxicity. Additionally, 5k showed good in vivo efficacy in 4weeks study with DIO mouse model.

Anti-diabetic and anti-adipogenic effects of a novel selective 11ß-hydroxysteroid dehydrogenase type 1 inhibitor in the diet-induced obese mice.

Glucocorticoid excess (Cushing's syndrome) causes metabolic syndrome such as visceral obesity, insulin resistance, diabetes mellitus, dyslipidaemia and hypertension. The selective inhibitors of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) have considerable potential for treating type 2 diabetes mellitus and metabolic syndrome. In the present study, we investigated the anti-diabetic and anti-adipogenic effects of 4-(2-(1,1-dioxido-6-(2,4,6-trichlorophenyl)-1,2,6-thiadiazinan-2-yl)acetamido)adamantane-1-carboxamide (KR-67183), a novel selective 11ß-HSD1 inhibitor; we also investigated the underlying molecular mechanisms in the cortisone-induced 3T3-L1 adipogenesis model system and diet-induced obese (DIO) mice. KR-67183 concentration-dependently inhibited 11ß-HSD1 activity in human and mouse 11ß-HSD1 over-expressed cells and in the ex vivo assay of C57BL/6 mice. In the study with DIO mice, the administration of KR-67183 (20 and 50mg/kg/day, orally for 28 days) improved the glucose tolerance and insulin sensitivity with suppressed 11ß-HSD1 activity in the liver and fat. However, KR-67183 showed no change in the adrenal gland weight/body weight ratio and plasma corticosterone concentration in DIO mice. Further, KR-67183 suppressed adipocyte differentiation on cortisone-induced adipogenesis in 3T3-L1 cells is associated with the suppression of the cortisone-induced mRNA levels of FABP4, PPARγ2 and GLUT4, and 11ß-HSD1 activity. Taken together, it is suggested that a selective 11ß-HSD1 inhibitor, KR-67183, may provide a new therapeutic window in the prevention and treatment without toxicity in type 2 diabetes with obesity.