Toxic dinoflagellate Centrodinium punctatum (Cleve) F.J.R. Taylor: An examination on the responses in growth and toxin contents to drastic changes of temperature and salinity.

To understand environmental effects affecting paralytic shellfish toxin production of Centrodinium punctatum, this study examined the growth responses, and toxin contents and profiles of a C. punctatum culture exposed to drastic changes of temperature (5-30 °C) and salinity (15-40). C. punctatum grew over a temperature range of 15-25 °C, with an optimum of 20 °C., and over a salinity range of 25-40, with optimum salinities of 30-35. This suggests that C. punctatum prefers relatively warm waters and an oceanic habitat for its growth and can adapt to significant changes of salinity levels. When C. punctatum was cultivated at different temperature and salinity levels, the PST profile included four major analogs (STX, neoSTX, GTX1 and GTX4, constituted >80 % of the profile), while low amounts of doSTX and traces of dc-STX and dc-GTX2 were also observed. Interestingly, though overall toxin contents did not change significantly with temperature, increases in the proportion of STX, and decreases in proportions in GTX1 and GTX4 were observed with higher temperatures. Salinity did not affect either toxin contents or profile from 25 to 35. However, the total toxin content dropped to approximately half at salinity 40, suggesting this salinity may induce metabolic changes in C. punctatum.

Bloom development of toxic dinoflagellate Alexandrium catenella (Group I) in Jinhae-Masan Bay, Korea: Germination strategy of resting cysts in relation to temperature and salinity.

To better understand the role of resting cysts in the outbreak of paralytic shellfish poisoning and bloom dynamics in Jinhae-Masan Bay, Korea, this study investigated the germination features of ellipsoidal Alexandrium cysts isolated from sediments collected in winter and summer under different combinations of temperature and salinity. Morphology and phylogeny of germling cells revealed that the ellipsoidal Alexandrium cysts belong to Alexandrium catenella (Group I). The cysts could germinate across a wide range of temperature (5-25 °C) with germination success within 5 days, indicating that continuous seeding for the maintenance of vegetative cells in the water column may occur through the year without an endogenous clock to regulate germination timing. In addition, the cyst germination of A. catenella (Group I) was not controlled by seasonal salinity changes. Based on the results, this study provides a schematic scenario of the bloom development of A. catenella (Group I) in Jinhae-Masan Bay, Korea.

Alexandrium catenella (Group I) and A. pacificum (Group IV) cyst germination, distribution, and toxicity in Jinhae-Masan Bay, Korea.

To better understand the outbreaks of paralytic shellfish poisoning and bloom dynamics caused by Alexandrium species in Jinhae-Masan Bay, Korea, the germination and distributions of ellipsoidal Alexandrium cysts were investigated, and paralytic shellfish toxins (PSTs) profiles and contents were determined using strains established from germling cells. The phylogeny and morphological observations revealed that the germinated vegetative cells from ellipsoidal cysts collected from the surface sediments in Jinhae-Masan Bay belong to Alexandrium catenella (Group I) and A. pacificum (Group IV) nested within A. tamarense species complex. Cyst germinations of A. catenella (Group I) were observed at only 10 °C, whereas cysts of A. pacificum (Group IV) could germinate at temperature ranges of 10 to 25 °C. Maximum germination success (85%) for isolated cysts occurred at 15 °C, and the germling cells were A. pacificum (Group IV). The results indicate that the variation in water temperature in Jinhae-Masan Bay can control the seasonal variations in germination of cysts of A. catenella (Group I) and A. pacificum (Group IV). The germination rates of ellipsoidal Alexandrium cysts were different among sampling sites in Jinhae-Masan Bay, probably because of differences in distribution and abundance of A. catenella (Group I) and A. pacificum (Group IV) in the sediments. The ellipsoidal Alexandrium cyst concentrations were much higher in February than in August, however the distributions were similar. Gonyautoxins 3 and 4 (GTX-3 and GTX-4) contributed a large proportion (>90%) of the toxins produced by strains A. catenella (Group I) and A. pacificum (Group IV) established from germling cells, and the total cellular contents were higher in A. catenella (Group I) than in A. pacificum (Group IV).

A new potentially toxic dinoflagellate Fukuyoa koreansis sp. nov. (Gonyaulacales, Dinophyceae) from Korean coastal waters: Morphology, phylogeny, and effects of temperature and salinity on growth.

To clarify an unspecified toxic Gambierdiscus-like species isolated from seawaters off Jeju Island, Korea, its morphology and molecular phylogeny based on the small subunit (SSU) and partial large subunit (LSU) rRNA gene sequences were examined. Cells were narrow in ventral view and broad in lateral view with a smooth surface. The round thecal pores were evenly distributed, with an average diameter of 0.41 µm. Cell depth, width and height were 51.7 ± 4.5 µm, 43.0 ± 4.2 µm and 55.0 ± 4.7 µm, respectively, and depth-to-width (D/W) and height-to-width (H/W) ratios were 1.1 ± 0.2 µm and 1.3 ± 0.02 µm, respectively. The nucleus was located in the hypotheca. Scanning electron microscope observations revealed that the cells displayed a plate formula of Po, 4', 6'', 6c, 6s, 5''' and 2''', and transmission electron microscope observation demonstrated that the cells contained crystal-like particles. Morphological features indicated that the unspecified Korean isolate belonged to the genus Fukuyoa, and based on the H/W and D/W ratios, the apical pore H/W ratio and thecal pore size, it could be differentiated from other Fukuyoa species. The phylogenetic analyses based on the SSU and LSU rRNA sequences revealed that the Korean isolate was nested within the genus Fukuyoa with high support, and it grouped with F. cf. yasumotoi isolated from Japan. Based on the morpho-molecular data, a new species, Fukuyoa koreansis sp. nov. is proposed. The maximum growth rate (0.254 d-1) of F. koreansis was observed at 25°C and a salinity of 25. The required levels of temperature and salinity for growth distinguished Fukuyoa koreansis from Gambierdiscus species.

Prevention and treatment effect of evogliptin on hepatic steatosis in high-fat-fed animal models.

Dipeptidyl peptidase 4 (DPP4) is an adipokine that interrupts insulin signaling. The resulting insulin resistance exacerbates hepatic steatosis. We previously reported that the novel DPP4 inhibitor evogliptin improves insulin resistance. This study aimed to verify the therapeutic potential of evogliptin for fatty liver. Evogliptin treatment was initiated simultaneously with a high-fat diet (HFD) feeding in normal mice and in a post-24 week HFD-fed rats. In a prevention study, insulin sensitivity was preserved in evogliptin-treated mice after a 16-week treatment. Overall plasma lipid levels stayed lower and hepatic lipid accumulation was drastically suppressed by evogliptin treatment. Evogliptin reduced hepatic expression of Srebf1, a key transcriptional factor for lipogenesis. Additionally, DPP4 inhibitor-treated mice showed less weight gain. In a treatment study, after evogliptin treatment for 14 weeks in pre-established HFD-fed obese rats, weight loss was marginal, while hepatic lipid accumulation and liver damage assessed by measuring plasma aminotransferase levels were completely resolved, suggesting weight loss-independent beneficial effects on fatty liver. Moreover, reduction in plasma non-esterified fatty acids supported the improvement of insulin resistance by evogliptin treatment. Conclusively, our findings suggest that evogliptin treatment ameliorates fatty liver by increasing insulin sensitivity and suppressing lipogenesis.

In vivo gastric residence and gastroprotective effect of floating gastroretentive tablet of DA-9601, an extract of Artemisia asiatica, in beagle dogs.

OBJECTIVE: DA-9601, an extract of Artemisia asiatica containing eupatilin and jaceosidin as active compounds, has been prescribed to treat gastritis in Asia. In recent times, sustained-release, floating gastroretentive (GR) tablets of DA-9601 are available on the market. In the present study, the physical properties and in vitro drug release profile, in vivo gastric residence time, and gastroprotective effect of GR tablet were compared to those of immediate release (IR) tablets of DA-9601. METHOD: In vitro buoyancy behavior (floating lag time and duration) and release profile of eupatilin were assessed in acidic medium. The in vivo intragastric behaviors of the barium sulfate-loaded IR and GR tablets were evaluated in beagle dogs by radiographic studies. Local gastroprotective effect was compared in an experimentally induced gastric lesion in beagle dogs after oral administration of IR (three times per day) or GR (twice daily) tablets for 15 days. RESULTS: Upon contact with gastric juice, a low-density floating tablet (apparent density of 0.93 g/cm(3)) was buoyant on the medium and was upheld for 14 hours, providing sustained drug release profile, whereas the IR tablet disintegrated within 10 minutes, showing complete drug release within 2 hours. In vivo radiographic studies showed that the GR tablet was retained for >4 hours in the stomach. Both DA-9601 formulations remarkably alleviated gastric mucosal injury compared to placebo group, when observed by gastric endoscopy. CONCLUSION: Twice-daily GR tablets exhibited a prolonged gastric residence time and a remarkable mucosal restoration effect in animal models. Therefore, the GR system of DA-9601 could be a substitute dosage form for the treatment of gastritis, while reducing the dosing frequency and thus improving patient compliance.

Hepatic role in an early glucose-lowering effect by a novel dipeptidyl peptidase 4 inhibitor, evogliptin, in a rodent model of type 2 diabetes.

Although multiple dipeptidyl peptidase 4 (DPP4) inhibitors have shown glucose-lowering effects by preserving pancreatic cells in high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice, the hepatic role in regulation of glucose homeostasis by DPP4 inhibitors in HFD/STZ mice remains elusive. In herein study, parallel comparison of effects on the liver (expression of gluconeogenic genes and the linked signaling molecules) and pancreas (islet morphology and relative area of alpha or beta cells) in combination with glucose-lowering effects were made at the end of 2- and 10-week of evogliptin treatment in HFD/STZ mice. Significant control of hyperglycemia was observed from the second week and persisted during 10-week treatment of 0.3% evogliptin in HFD/STZ mice. This effect was accompanied by increased level of plasma glucagon-like peptide-1 and preserved pancreas islet structure. Furthermore, the hepatic increases in gluconeogenic gene expression in HFD/STZ mice was significantly reduced by evogliptin treatment, which was accompanied by the suppression of cAMP response element-binding protein (CREB) phosphorylation and expression of transducer of regulated CREB protein 2. This hepatic effect of evogliptin treatment was reproduced in 2-week study, however, pancreatic beta-cell area was not altered yet although the expression of pancreatic and duodenal homeobox protein 1 was increased. We conclude that the suppression of hepatic gluconeogenesis by evogliptin is followed by preservation of pancreatic islet, leading to remarkable and persistent glucose-lowering effect in HFD/STZ mice. Our findings provide further insight for the hepatic role in DPP4 inhibitor-mediated glucose control in diabetes.

Beneficial Effects of Evogliptin, a Novel Dipeptidyl Peptidase 4 Inhibitor, on Adiposity with Increased Ppargc1a in White Adipose Tissue in Obese Mice.

Although dipeptidyl peptidase 4 (DPP4) is an adipokine known to positively correlate with adiposity, the effects of pharmacological DPP4 inhibition on body composition have not been fully understood. This study was aimed to assess the effects of DPP4 inhibitors on adiposity for the first time in the established obese mice model. The weight loss effects of multiple DPP4 inhibitors were compared after a 4 week treatment in diet-induced obese mice. In addition, a 2 week study was performed to explore and compare the acute effects of evogliptin, a novel DPP4 inhibitor, and exenatide, a glucagon-like peptide-1 (GLP-1) analogue, on whole body composition, energy consumption, various plasma adipokines and gene expression in white adipose tissue (WAT). After the 4 week treatment, weight loss and blood glucose reductions were consistently observed with multiple DPP4 inhibitors. Moreover, after 2-week treatment, evogliptin dose-dependently reduced whole body fat mass while increasing the proportion of smaller adipocytes. However, insulin sensitivity or plasma lipid levels were not significantly altered. In addition to increased active GLP-1 levels by plasma DPP4 inhibition, evogliptin also enhanced basal metabolic rate without reduction in caloric intake, in contrast to exenatide; this finding suggested evogliptin's effects may be mediated by pathways other than via GLP-1. Evogliptin treatment also differentially increased Ppargc1a expression, a key metabolic regulator, in WAT, but not in skeletal muscle and brown adipose tissue. The increased expression of the downstream mitochondrial gene, Cox4i1, was also suggestive of the potential metabolic alteration in WAT by DPP4 inhibitors. We are the first to demonstrate that pharmacological DPP4 inhibition by evogliptin directly causes fat loss in established obese mice. In contradistinction to exenatide, the fat-loss effect of DPP4 inhibitor is partly attributed to enhanced energy expenditure along with metabolic changes in WAT. These results provide insight into the regulation of energy storage in WAT caused by DPP4 inhibition.

Proteomic analysis of INS-1 rat insulinoma cells: ER stress effects and the protective role of exenatide, a GLP-1 receptor agonist.

Beta cell death caused by endoplasmic reticulum (ER) stress is a key factor aggravating type 2 diabetes. Exenatide, a glucagon-like peptide (GLP)-1 receptor agonist, prevents beta cell death induced by thapsigargin, a selective inhibitor of ER calcium storage. Here, we report on our proteomic studies designed to elucidate the underlying mechanisms. We conducted comparative proteomic analyses of cellular protein profiles during thapsigargin-induced cell death in the absence and presence of exenatide in INS-1 rat insulinoma cells. Thapsigargin altered cellular proteins involved in metabolic processes and protein folding, whose alterations were variably modified by exenatide treatment. We categorized the proteins with thapsigargin initiated alterations into three groups: those whose alterations were 1) reversed by exenatide, 2) exaggerated by exenatide, and 3) unchanged by exenatide. The most significant effect of thapsigargin on INS-1 cells relevant to their apoptosis was the appearance of newly modified spots of heat shock proteins, thimet oligopeptidase and 14-3-3ß, ε, and θ, and the prevention of their appearance by exenatide, suggesting that these proteins play major roles. We also found that various modifications in 14-3-3 isoforms, which precede their appearance and promote INS-1 cell death. This study provides insights into the mechanisms in ER stress-caused INS-1 cell death and its prevention by exenatide.

Differential protective effects of exenatide, an agonist of GLP-1 receptor and Piragliatin, a glucokinase activator in beta cell response to streptozotocin-induced and endoplasmic reticulum stresses.

BACKGROUND: Agonists of glucagon-like peptide-1 receptor (GLP-1R) and glucokinase activators (GKA) act as antidiabetic agents by their ability protect beta cells, and stimulate insulin secretion. Oxidative and endoplasmic reticulum (ER) stresses aggravate type 2 diabetes by causing beta cell loss. It was shown that GLP-1R agonists protect beta cells from oxidative and ER stresses. On the other hand, little is known regarding how GKAs protect beta cells. We hypothesized that GKAs protect beta cells by mechanisms distinct from those underlying GLP-1R agonist and tested our hypothesis by comparing the molecular effects of exenatide, a GLP-1R agonist, and piragliatin, a GKA, on INS-1 cells under oxidative and ER-induced stresses. METHODS: BETA CELLS WERE TREATED WITH STREPTOZOTOCIN (STZ) TO INDUCE OXIDATIVE STRESS AND WITH PALMITATE OR THAPSIGARGIN (TG) TO INDUCE ER STRESS RESPECTIVELY, AND THE EFFECTS OF EXENATIDE AND PIRAGLIATIN ON THESE CELLS WERE INVESTIGATED BY: a) characterizing the kinases involved employing specific kinase inhibitors, and b) by identifying the differentially regulated proteins in response to stresses with proteomic analysis. RESULTS: Exenatide protected INS-1 cells from both ER and STZ-induced death. In contrast, piragliatin rescued the cells only from STZ-induced stress. Akt activation by exenatide appeared to contribute to its protective effects of beta cells while enhanced glucose utilization was the contributing factor in the case of piragliatin. Also, exenatide, not piragliatin, blocked changes in proteins 14-3-3ß, ε and θ, and preserved the 14-3-3θ levels under the ER stress. Isoform-specific modifications of 14-3-3, and the reduction of 14-3-3θ, commonly associated with beta cell death were assessed. CONCLUSIONS: Exenatide and piragliatin exert distinct effects on beta cell survival and thus on type 2 diabetes. This study which confirmed our hypothesis is also the first to observe specific modulation of 14-3-3 isoform in stress-induced beta cell death associated with progressive deterioration of type 2 diabetes.

Dipeptidyl peptidase-4 inhibitor with ß-amino amide scaffold: synthesis, SAR and biological evaluation.

Inhibitors of dipeptidyl peptidase-4 (DPP4) have been shown to be effective treatments for type 2 diabetes. Several series of ß-amino amide containing piperazine derivatives have been prepared and evaluated as a inhibitor of DPP4. Finally compound 5m was selected for further evaluation.

DA-1229, a novel and potent DPP4 inhibitor, improves insulin resistance and delays the onset of diabetes.

AIM: To characterize the pharmacodynamic profile of DA-1229, a novel dipeptidyl peptidase (DPP) 4 inhibitor. MAIN METHODS: Enzyme inhibition assays against DPP4, DPP8 and DPP9. Antidiabetic effects of DA-1229 in HF-DIO mice and young db/db mice. KEY FINDINGS: DA-1229 was shown to potently inhibit the DPP4 enzyme in human and murine soluble forms and the human membrane-bound form with IC(50) values of 0.98, 3.59 and 1.26 nM, respectively. As a reversible and competitive inhibitor, DA-1229 was more selective to human DPP4 (6000-fold) than to human DPP8 and DPP9. DA-1229 (0.1-3mg/kg) dose-dependently inhibited plasma DPP4 activity, leading to increased levels of plasma GLP-1 and insulin, and thereby lowering blood glucose levels in mice. In high fat diet-fed (HF) mice, a single oral dose of 100mg/kg of DA-1229 reduced plasma DPP4 activity by over 80% during a 24h period. Long-term treatment with DA-1229 for 8 weeks revealed significant improvements in glucose intolerance and insulin resistance, accompanied by significant body weight reduction. However, it remains unclear whether there is a direct causal relationship between DPP4 inhibition and body weight reduction. In young db/db mice, the DA-1229 treatment significantly reduced blood glucose excursions for the first 2 weeks, resulting in significantly lower levels of HbA1c at the end of the study. Furthermore, the pancreatic insulin content of the treatment group was significantly higher than that of the db/db control. SIGNIFICANCE: DA-1229 as a novel and selective DPP4 inhibitor improves the insulin sensitivity in HF mice and delays the onset of diabetes in young db/db mice.

Two small molecule agonists of glucagon-like peptide-1 receptor modulate the receptor activation response differently.

The glucagon-like peptide-1 receptor (GLP-1R) is a target for type 2 diabetes treatment. Due to the inconvenience of peptide therapeutics, small-molecule GLP-1R agonists have been studied. Compound 2 (6,7-dichloro-2-methylsulfonyl-2-N-tert-butylaminoquinoxaline) and compound B (4-(3-(benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine) have been described as small molecule, ago-allosteric modulators of GLP-1R. However, their modes of action at the GLP-1R have not been elucidated. Thus, in this study, we compared the mechanisms of action between these two compounds. When compound 2 was treated with endogenous or exogenous peptide agonists (GLP-1 and exenatide) or fragments of peptide agonists (GLP-1(9-36), Ex3, Ex4, and Ex5), the response curve of these peptide agonists shifted left without a change in maximum efficacy. In contrast, compound B potentiated the response and increased maximum efficacy. However, N-terminal truncated orthosteric antagonists including Ex7, Ex9, and Ex10, augmented the response of compound 2 at the GLP-1R but did not alter compound B activity. Intriguingly, when we co-treated compound 2 with compound B in CHO cells expressing full-length hGLP-1R or N-terminal extracellular domain-truncated GLP-1R, the activation of both types of receptors increased additively, implying that the N-terminus of the receptor is not involved in the modulation by compound agonists. We confirmed that these two compounds increased calcium influx by different patterns in CHO cells expressing GLP-1R. Taken together, our findings suggest that compounds 2 and B have different modes of action to activate GLP-1R. Further study to identify the putative binding sites will help in the discovery of orally available GLP-1R agonists.

Glucose exposure pattern determines glucagon-like peptide 1 receptor expression and signaling through endoplasmic reticulum stress in rat insulinoma cells.

Repeated fluctuation in plasma glucose levels, as well as chronic hyperglycemia, is an important phenomenon frequently observed in diabetic patients. Recently, several studies have reported that glucose fluctuation, compared to chronic hyperglycemia, mediates more adverse effects due to induced oxidative and/or endoplasmic reticulum (ER) stress. In type 2 diabetes, stimulation of insulin secretion by glucagon-like peptide-1 (GLP-1) has been found to be reduced, and the results of recent studies have shown that the expression of the GLP-1 receptor (GLP-1R) is reduced by chronic hyperglycemia. However, GLP-1R signaling in glucose fluctuation has not been elucidated clearly. In this study, we hypothesized that intermittent high glucose (IHG) conditions also reduced GLP-1-mediated cellular signaling via reduction in GLP-1R expression. To evaluate this hypothesis, rat insulinoma cells (INS-1) were exposed for 72 h to either sustained high glucose (SHG) conditions (30 mM glucose) or IHG conditions (11 and 30 mM glucose, alternating every 12h). In comparison to both the SHG and control groups, IHG conditions induced a more significant impairment of insulin release and calcium influx in response to 1nM GLP-1 treatment. In addition, the activity of caspase 3/7 as well as the gene expression of binding protein (Bip) and C/EBP homologous protein (CHOP), molecular markers of ER stress, was significantly higher in IHG-treated cells than in SHG-treated cells. Interestingly, the expression level of GLP-1R was significantly lower under IHG conditions than under SHG conditions. Collectively, these findings indicated that glucose fluctuation reduces GLP-1R expression through ER stress more profoundly than sustained hyperglycemia, which may contribute to the diminished response of GLP-1.

The development of non-peptide glucagon-like peptide-1 receptor agonist for the treatment of type 2 diabetes.

Glucagon-like peptide-1 (GLP-1) is the main member of the incretin family and stimulates insulin secretion by binding with its specific receptor on pancreatic ß-cells. In addition, GLP-1 exerts broad beneficial effects on the glucose regulation by suppressing food intake and delaying stomach emptying. Now, long acting GLP-1 analogs including exenatide and liraglutide have been approved for the treatment of diabetes mellitus type 2, however long-term injection can limit their use for these chronic patients. In this report, the authors provide a review on the development of non-peptide GLP-1 receptor agonists and introduce a novel agonist DA-15864.

Discovery of DA-1229: a potent, long acting dipeptidyl peptidase-4 inhibitor for the treatment of type 2 diabetes.

A series of ß-amino amide containing substituted piperazine-2-one derivatives was synthesized and evaluated as inhibitors of dipeptidyl pepdidase-4 (DPP-4) for the treatment of type 2 diabetes. As results of intensive SAR study of the series, (R)-4-[(R)-3-amino-4-(2,4,5-trifluorophenyl)-butanoyl]-3-(t-butoxymethyl)-piperazin-2-one (DA-1229) displayed potent DPP-4 inhibition pattern in several animal models, was selected for clinical development.

A novel dipeptidyl peptidase IV inhibitor DA-1229 ameliorates streptozotocin-induced diabetes by increasing ß-cell replication and neogenesis.

We studied the effect of a novel dipeptidyl peptidase IV (DPP IV) inhibitor, DA-1229, on blood glucose profile and pancreatic ß-cell mass in established diabetes after streptozotocin (STZ) treatment. Mice that developed diabetes after administration of STZ 100mg/kg were treated with DA-1229 for 13 weeks. DA-1229 significantly reduced plasma DPP IV activity, and enhanced glucagon-like peptide 1 (GLP-1) levels. In STZ-treated mice fed DA-1229 (STZ-DA), blood glucose levels were significantly lower than those in diabetic mice fed normal chow (STZ-NC). Basal and glucose-stimulated insulin secretion and glucose tolerance assessed by intraperitoneal glucose tolerance test were significantly improved by DA-1229 administration. Volume density of ß-cell was significantly increased in STZ-DA mice compared to STZ-NC mice, suggesting that DA-1229-mediated amelioration of established diabetes was due to beneficial effect of DA-1229 on ß-cell mass. The number of replicating ß-cells and that of scattered small ß-cell unit representing ß-cell neogenesis were significantly increased in STZ-DA mice compared to STZ-NC mice, explaining increased ß-cell mass by DA-1229. The expression of PDX-1, a downstream mediator of GLP-1 action, was increased in islets of STZ-DA mice compared to STZ-NC mice. These results suggest a therapeutic potential of DA-1229 in diabetes, particularly that associated with decreased ß-cell mass.

PAM-1616, a selective peroxisome proliferator-activated receptor γ modulator with preserved anti-diabetic efficacy and reduced adverse effects.

Peroxisome proliferator-activated receptor (PPAR) γ is known to be a key regulator of insulin resistance. PAM-1616 is a novel, non-thiazolidinedione small molecule compound synthesized in Dong-A Research Center. In this study, we characterized the pharmacological and safety profiles of PAM-1616 as a selective PPARγ modulator. PAM-1616 selectively binds to human PPARγ (IC(50), 24.1±5.6 nM) and is a partial agonist for human PPARγ with an EC(50) of 83.6±43.7 nM and a maximal response of 24.9±7.1% relative to the full agonist, rosiglitazone. PAM-1616 was selective for human PPARγ than for human PPARα (EC(50), 2658±828 nM) without activating human PPARδ, which makes it a selective modulator of PPARγ. Treatment of high fat diet-induced obese C57BL/6J mice with PAM-1616 for 21 days improved HOMA-IR. Furthermore, PAM-1616 significantly improved hyperglycemia in db/db mice with little side effect when orally administered at a dose of 1 mg/kg/day for 28 days. Intriguingly, PAM-1616 was seen to increase the gene expression of inducible glucose transporter (GLUT4), while it partially induced that of a fatty acid carrier, aP2 in 3T3-L1 adipocytes, and it also showed partial recruitment of an adipogenic cofactor, TRAP220 as compared to rosiglitazone. PAM-1616 did not cause a significant increase in plasma volume of ICR mice when orally administered at a dose of 10 mg/kg/day for 9 days. PAM-1616 increased the expression of fluid retention-inducing genes such as serum/glucocorticoid-regulated kinase (SGK)-1 to a lesser extent as compared to rosiglitazone in human renal epithelial cells. These results suggest that PAM-1616 acts as a selective modulator of PPARγ with excellent antihyperglycemic property. The differential modulation of target gene by PAM-1616 might contribute to the improved side effect profiles.

PAR-1622 is a selective peroxisome proliferator-activated receptor gamma partial activator with preserved antidiabetic efficacy and broader safety profile for fluid retention.

Peroxisome proliferator-activated receptor (PPAR) gamma is known to be a key regulator of insulin resistance. PAR-1622 is a novel small molecule compound synthesized in Dong-A research center. In this study, we characterized the pharmacological profiles of PAR-1622, a selective partial activator of PPARgamma. In transient transactivation assays, PAR-1622 [(S)-2-ethoxy-3(4-(5-(4-(5-(methoxymethyl)isoxazol-3-yl)phenyl)-3-methylthiophen-2-yl)methoxy)phenyl)propanoic acid] showed a partial activator against human PPARgamma with an EC(50) of 41 nM and a maximal response of 37% relative to the full agonist rosiglitazone without activating human PPARdelta. PAR-1622 was 56 folds more selective for human PPARgamma than for human PPARalpha (EC(50), 2304 nM), which means that it is a selective partial activator of PPARgamma. PAR-1622 also showed a partial activator against mouse PPARgamma with an EC(50) of 427 nM and a maximal response was 57% of that of rosiglitazone. INT-131, a selective PPARgamma partial agonist in clinical stage, also was a partial activator against human PPARgamma with an EC(50) of 83 nM and a maximal response achieved by INT-131 was 49% of that observed with full agonist rosiglitazone. In functional assays using human mesenchymal stem cells, PAR-1622 induced adipocyte differentiation, which was 3-fold more potent with a comparable maximum response compared to INT-131. Furthermore, PAR-1622 significantly improved hyperglycemia in db/db when orally administered at a dose of 1 mg/kg/day for 5 days. In hemodilution assays with Evans Blue, rosiglitazone significantly increased the plasma volume in ICR mice that were orally administered 30 mg/kg/day for 9 days; however, PAR-1622 showed no significant effects on plasma volume, similar to INT-131. These results suggest that PAR-1622 is a selective partial activator of PPARgamma and has excellent antihyperglycemic activities and a broad safety profile for fluid retention.

Design, synthesis, and evaluation of novel aryl-tetrahydropyridine PPARalpha/gamma dual agonists.

Aryl-tetrahydropyridine derivatives were prepared and their PPARalpha/gamma dual agonistic activities were evaluated. Among them, compound (S)-5b was identified as a potent PPARalpha/gamma dual agonist with an EC(50) of 1.73 and 0.64 microM in hPPARalpha and gamma, respectively. In diabetic (db/db) mice, compound (S)-5b showed good glucose lowering efficacy and favorable pharmacokinetic properties.