Moon Ho Son

Potentiation of cadmium-induced cytotoxicity by sulfur amino acid deprivation through activation of extracellular signal-regulated kinase1/2 (ERK1/2) in conjunction with p38 kinase or c-jun N-terminal kinase (JNK): Complete inhibition of the potentiated toxicity by U0126 an ERK1/2 and p38 kinase inhibitor

The mechanisms of cadmium-induced toxicity may include oxidative stress, altered redox homeostasis, and injuries to organelles. The current study was designed to study the effect of decreased cellular glutathione (GSH) content by sulfur amino acid deprivation on cadmium toxicity and to identify the signaling pathways responsible for the cytotoxicity. GSH content was increased by cadmium in H4IIE cells prior to cell death, which was prevented by excess GSH or cysteine. Cell viability, however, was not improved by GSH or cysteine complexation of cadmium. Cadmium-induced cytotoxicity was 40-fold potentiated in cells with decreased GSH by sulfur amino acid deprivation. Cadmium in combination with decreased GSH markedly increased apoptotic cell death. Mitogen-activated protein kinases including extracellular signal-regulated kinase 1/2, p38 kinase and c-Jun N-terminal kinase (JNK) were all activated 1-12 hr after sulfur amino acid deprivation. U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), which inhibited activation of extracellular signal-regulated kinase1/2 and p38 kinase in cells under sulfur amino acid deprivation, completely prevented potentiation in Cd-induced cytotoxicity and apoptosis. Potentiation of cadmium toxicity by sulfur amino acid deprivation was prevented in part by either PD98059 or SB203580, or in cells stably expressing dominant negative mutant of JNK1, and to greater extents by PD98059 in combination with either SB203580 or JNK1(-) transfection. These results demonstrated that decreased cellular GSH content potentiated cytotoxicity induced by cadmium at the level of human exposure, and that the potentiation of cytotoxicity resulted from activation of extracellular signal-regulated kinase1/2 in conjunction with p38 kinase or JNK.

A novel dipeptidyl peptidase IV inhibitor DA-1229 ameliorates streptozotocin-induced diabetes by increasing β-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 β-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 β-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 β-cell mass. The number of replicating β-cells and that of scattered small β-cell unit representing β-cell neogenesis were significantly increased in STZ-DA mice compared to STZ-NC mice, explaining increased β-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 β-cell mass.

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

A series of β-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.

PAR-5359, a well-balanced PPARα/γ dual agonist, exhibits equivalent antidiabetic and hypolipidemic activities in vitro and in vivo

Peroxisome proliferator-activated receptor (PPAR) alpha and gamma are key regulators of lipid homeostasis and insulin resistance. In this study, we characterize the pharmacological profiles of PAR-5359, a dual agonist of PPARalpha and gamma with well-balanced activities. In transient transactivation assay, PAR-5359 (3-(4-(2[4-(4chloro-phenyl)-3,6-dihydro-2H-pyridin-1-yl]-ethoxy)-phenyl)-(2S)-ethoxy-propionic acid) significantly activated human and mouse PPARalpha and gamma without activating PPARdelta. In functional assays using human mesenchymal stem cells and human hepatoma HepG2 cells, PAR-5359 significantly induced adipocyte differentiation and human ApoA1 secretion, which coincided with its transactivation potencies against the corresponding human receptor subtypes. Interestingly, PAR-5359 showed equivalent potencies against the mouse receptor subtypes (alpha and gamma; 2.84 microM and 3.02 microM, respectively), which suggests the possibility that PAR-5359 could simultaneously activates each subtype of receptors subtype in under physiological conditions. In an insulin-resistant ob/ob mouse model, PAR-5359 significantly reduced plasma insulin levels, improved insulin sensitivity (HOMA-IR), and completely normalized plasma glucose levels. In a severe diabetic db/db mouse model, PAR-5359 dose-dependently reduced the plasma levels of glucose (ED(30) = 0.07 mg/kg). Furthermore, it lowered plasma levels of non HDL- (ED(30) = 0.13 mg/kg) and total cholesterol (ED(30) = 0.03 mg/kg) in high cholesterol diet-fed rats for 4 days treatment. These results suggest that PAR-5359 has the balanced activities for PPARalpha and PPARgamma in vivo as well as in vitro. And its balanced activities may render PAR-5359 as a pharmacological tool in elucidating the complex roles of PPARalpha/gamma dual agonists.

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

Peroxisome proliferator-activated receptor (PPAR) γ 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 PPARγ. 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 PPARγ with an EC50 of 41 nM and a maximal response of 37% relative to the full agonist rosiglitazone without activating human PPARδ. PAR-1622 was 56 folds more selective for human PPARγ than for human PPARα (EC50, 2304 nM), which means that it is a selective partial activator of PPARγ. PAR-1622 also showed a partial activator against mouse PPARγ with an EC50 of 427 nM and a maximal response was 57% of that of rosiglitazone. INT-131, a selective PPARγ partial agonist in clinical stage, also was a partial activator against human PPARγ with an EC50 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 PPARγ and has excellent antihyperglycemic activities and a broad safety profile for fluid retention.

Role of glucose utilization in the restoration of hypophysectomy-induced hepatic cytochrome P450 2E1 by growth hormone in rats.

Growth hormone and insulin are the primary determinants for cytochrome P450 2E1 (CYP2E1) expression. The role of glucose on the induction of CYP2E1 by hypophysectomy and on the restorative effect by growth hormone was investigated in the rat liver. Western and Northern blot analyses revealed that hypophysectomy induced CYP2E1 by 5-fold at 1-4 weeks, relative to control, with a concomitant increase in CYP2E1 mRNA. Hypophysectomized rats (HXR) showed a 20% reduction in the plasma glucose level. Hypophysectomy-induced increase in the CYP2E1 mRNA was completely abolished by glucose feeding in drinking water (10%) for 7 days. Treatment of HXR with hGH (2 I.U./kg, twice a day, for 7 days) inhibited the increases in CYP2E1 protein and mRNA levels with restoration of the plasma glucose level. In contrast to the effect of human growth hormone (hGH) on CYP2E1 in HXR with free access to foods, CYP2E1 expression failed to be restored by hGH in starving HXR. However, glucose feeding of starving HXR abolished the induction of CYP2E1. Effects of hypophysectomy and hGH treatment were studied in streptozotocin-induced diabetic rats. Insulin, but not hGH, prevented an increase in CYP2E1 mRNA in diabetic rats. The hepatic CYP2E1 induction in hypophysectomized diabetic rats was inhibited by hGH treatment, indicating that the hGH effect on CYP2E1 expression did not involve insulin production. These results provide evidence that the induction of hepatic CYP2E1 by hypophysectomy may result from reduced glucose utilization, and that the effect of hGH on CYP2E1 expression may be mediated with enhanced glucose utilization, but not with insulin production.

DA-125, a novel anthracycline derivative showing high-affinity DNA binding and topoisomerase II inhibitory activities, exerts cytotoxicity via c-Jun N-terminal kinase pathway

Abstract. Purpose: DA-125 [(8S,10S)-8-(3-Aminopropanoyloxyacetyl)-10- [(2,6-dideoxy-2-fluoro-α-L-talopyranosyl)oxy]- 7,8,9,10-tetrahydro-6,8,11-trihydroxy- 1-methoxy-5,12-naphthacene-dione hydrochloride] is a novel anthracycline derivative with anticancer activity. In the present study, we compared the cytotoxicity of DA-125 with that of doxorubicin in H4IIE rat hepatoma cells and investigated the mechanistic basis. Because activation of MAP kinases, in particular c-Jun N-terminal kinase (JNK), is implicated in apoptotic cell death, the signaling pathways responsible for DA-125-induced apoptosis were studied. Methods: Cytotoxicity and apoptosis were measured in H4IIE cells and cells were stably transfected with a dominant-negative mutant of JNK1 (JNK1–) by MTT and TUNEL assays. Inhibition of topoisomerase II activity was determined in vitro. Drug accumulation and DNA binding affinity were determined by fluorescence spectroscopy. Results: The cytotoxicity of DA-125 was greater than that of doxorubicin (IC50 11.5 vs 70 µM). DA-125 induced apoptosis with 30-fold greater potency than doxorubicin. Inhibition of topoisomerase II by DA-125 was fourfold greater. The presence of excess β-alanine, a DA-125 moiety, failed to alter cytotoxicity and accumulation of DA-125, indicating that the improved cytotoxicity of DA-125 did not result from the β-alanine moiety. Greater cellular accumulation of DA-125 correlated with its high-affinity DNA binding. Although neither PD98059 nor SB203580 altered the degree of cytotoxicity induced by DA-125, JNK1– cells exhibited about a twofold greater viability than control cells. DA-125-induced apoptosis was also decreased in JNK1–-transfected cells. Conclusions: DA-125 potently inhibited topoisomerase II activity and induced apoptosis by a high rate of prooxidant production. DA-125 exhibited high-affinity DNA binding with improved cellular drug accumulation. Apoptosis induced by DA-125 involved the pathway of JNK1, but not ERK1/2 or p38 kinase

Potentiation of arsenic-induced cytotoxicity by sulfur amino acid deprivation (SAAD) through activation of ERK1/2, p38 kinase and JNK1: the distinct role of JNK1 in SAAD-potentiated mercury toxicity.

Sulfur amino acid deficiency occurs in certain pathophysiological situations (e.g. protein-calorie malnutrition). Previous studies revealed that sulfur amino acid deprivation (SAAD) activated MAP kinases and potentiated cadmium-induced cytotoxicity by activation of ERK1/2 in conjunction with p38 kinase or JNK. The present study was designed to determine susceptibility of cells to a variety of heavy metals in combination with SAAD. Viability was assessed in H4IIE cells treated with sodium arsenite, mercuric chloride, sodium selenite, lead acetate, chromium trioxide or manganese chloride. SAAD potentiated the cytotoxicity of H4IIE cells by arsenic or mercury (i.e. EC50, 19 and 5 microM in SAAD vs. 401 and 42 microM in control medium, respectively). TUNEL assays revealed that the potentiated arsenic or mercury toxicity involved apoptotic cell death. Lead or selenite moderately elicited cell death, which was not enhanced by SAAD. Chromium or manganese caused no significant cytotoxicity. Treatment of cells with U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] an ERK1/2 inhibitor or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] a p38 kinase inhibitor effectively prevented SAAD-potentiated arsenic toxicity. The potentiated arsenic toxicity was also inhibited in cells stably expressing a dominant negative mutant of c-Jun N-terminal kinase 1 [JNK1(-)]. The inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase failed to prevent mercury-induced toxicity enhanced by SAAD. JNK1(-) cells were minimally susceptible to mercury in SAAD medium. These results demonstrated that SAAD potentiated cytotoxicity induced by arsenic or mercury and that activation of ERK1/2, p38 kinase and JNK1 was responsible for the potentiated arsenic toxicity, whereas the mercury toxicity enhanced by SAAD was mediated with the activity of JNK1.

Dipeptidyl peptidase-4 inhibitor with β-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 β-amino amide containing piperazine derivatives have been prepared and evaluated as a inhibitor of DPP4. Finally compound 5m was selected for further evaluation.

Design, synthesis, and evaluation of novel aryl-tetrahydropyridine PPARα/γ 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.