Discovery of a novel orally active PDE-4 inhibitor effective in an ovalbumin-induced asthma murine model.

Phosphodiesterase-4 (PDE-4) is responsible for metabolizing adenosine 3',5'-cyclic monophosphate that reduces the activation of a wide range of inflammatory cells including eosinophils. PDE-4 inhibitors are under development for the treatment of respiratory diseases such as asthma and chronic obstructive pulmonary disease. Herein, we report a novel PDE-4 inhibitor, PDE-423 (3-[1-(3-cyclopropylmethoxy-4-difluoromethoxybenzyl)-1H-pyrazol-3-yl]-benzoic acid), which shows good in vitro and in vivo oral activities. PDE-423 exhibited in vitro IC(50)s of 140 nM and 550 nM in enzyme assay and cell-based assay, respectively. In vivo study using ovalbumin-induced asthmatic mice revealed that PDE-423 reduced methacholine-stimulated airway hyperreactivity in a dose-dependent manner by once daily oral administration (ED(50)=18.3 mg/kg), in parallel with decreased eosinophil peroxidase activity and improved lung histology. In addition, PDE-423 was effective in diminishing lipopolysaccharide-induced neutrophilia in vivo as well as in vitro. Oral administration of PDE-423 (100 mg/kg) had no effect on the duration of xylazine/ketamine-induced anesthesia and did not induce vomiting incidence in ferrets up to the dose of 1000 mg/kg. The present study indicates that a novel PDE-4 inhibitor, PDE-423, has good pharmacological profiles implicating this as a potential candidate for the development of a new anti-asthmatic drug.

Protective effects of pyrrolidine dithiocarbamate against airway inflammation in the ovalbumin-induced mouse model.

Pyrrolidine dithiocarbamate (PDTC) is known to exert anti-tumor and anti-inflammatory effects. However, the effects of PDTC against airway inflammation and its underlying mechanisms have not been reported. In the present study, we examined the protective effects of PDTC in a murine model of asthma induced by ovalbumin. PDTC reduced the number of infiltrating inflammatory cells in concert with reduced eosinophil peroxidase (EPO) activity in bronchoalveolar lavage fluid. In parallel, PDTC decreased airway hyperresponsiveness in a dose dependent manner. All these effects were correlated with heme oxygenase-1 (HO-1) mRNA and protein induction, and reversed by ZnPP, a HO-1 inhibitor. In addition, PDTC reduced the secretion of Th(2) cytokines such as IL-4 and IL-5, whereas ZnPP blocked the inhibitory effects of PDTC on Th(2) cytokine secretion. These results suggest that PDTC protects against airway inflammation at least in part via HO-1 induction, and that inhibitory action on Th(2) cytokines may be associated with the protective mechanism of PDTC.

KR-62436, 6-{2-[2-(5-cyano-4,5-dihydropyrazol-1-yl)-2-oxoethylamino]ethylamino}nicotinonitrile, is a novel dipeptidyl peptidase-IV (DPP-IV) inhibitor with anti-hyperglycemic activity.

Dipeptidyl peptidase-IV (DPP-IV) is involved in the inactivation of glucagon-like peptide-1 (GLP-1), a potent insulinotropic peptide. Thus, DPP-IV inhibition can be an effective approach to treat type 2 diabetes mellitus by potentiating insulin secretion. This study describes the biological effects of a new DPP-IV inhibitor, KR-62436 (6-{2-[2-(5-cyano-4,5-dihydropyrazol-1-yl)-2-oxoethylamino]ethylamino}nicotinonitrile) in vitro and in vivo. KR-62436 inhibited rat plasma DPP-IV, porcine kidney DPP-IV as well as human DPP-IV (Caco-2) with IC50 values of 0.78, 0.49, 0.14 microM, respectively. In addition, the compound (10 microM) almost completely inhibited DPP-IV-mediated degradation of GLP-1 in vitro. KR-62436 inhibited the enzyme in a competitive manner, and exhibited selectivity against several proteases including proline-specific proteases. In vivo efficacy of the compound was examined by using normal C57BL/6J mice and ob/ob mice, a type 2 diabetes animal model. Administration of KR-62436 to C57BL/6J mice either orally or subcutaneously resulted in the suppression of plasma DPP-IV activity, increase in intact GLP-1 and insulin levels in plasma. Furthermore, the plasma glucose concentrations during oral glucose tolerance test (OGTT) were reduced upon oral administration of KR-62436. This study demonstrates that KR-62436 could be a good lead compound for further development as a new anti-diabetic agent.

Roflumilast inhibits lipopolysaccharide-induced inflammatory mediators via suppression of nuclear factor-kappaB, p38 mitogen-activated protein kinase, and c-Jun NH2-terminal kinase activation.

Roflumilast, a potent and selective phosphodiesterase 4 (PDE4) inhibitor, has been demonstrated to be an effective anti-inflammatory agent in airway inflammatory diseases. In the present study, we investigated the mechanism of anti-inflammatory effects of roflumilast in murine macrophage cell line RAW264.7 cells. Roflumilast inhibited NO, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta production via suppression of their gene expressions in lipopolysaccharide (LPS)-stimulated macrophages. To elucidate the mechanism by which roflumilast inhibits the production of inflammatory mediators, we examined the effect of roflumilast on the activation of nuclear factor-kappaB (NF-kappaB) in these cells. Roflumilast inhibited the DNA binding activity of NF-kappaB by preventing inhibitor kappaBalpha phosphorylation and degradation. The phosphorylation of mitogen-activated protein (MAP) kinases, including stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase, was also markedly inhibited by roflumilast. Similar to the effects of roflumilast, treatment of either SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole] or SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone], specific inhibitors of p38 MAP kinase and JNK, respectively, suppressed NO, TNF-alpha, and IL-1beta production. Consistent with in vitro results, administration of roflumilast recovered the survival rate of LPS-treated mice, with concurrent suppression of plasma levels of nitrite/nitrate, TNF-alpha, and IL-1beta. These results suggest that the inhibitory activity of roflumilast on the production of inflammatory mediators seems to be mediated via inhibition of NF-kappaB, p38 MAP kinase, and JNK activation in macrophages.

Inhibition of dipeptidyl peptidase IV by novel inhibitors with pyrazolidine scaffold.

Inhibition of dipeptidyl peptidase IV (DPP-IV) activity has been reported to improve nutrient-stimulated insulin secretion through the stabilization of glucagon-like peptide (GLP-1). In the present study, we identified novel DPP-IV inhibitors of pyrazolidine derivatives (Compounds 1 and 2) and characterized their biological effects in vitro and in vivo. Compound 1, an isoleucine pyrazolidide with a phenyl urea group, inhibited rat plasma DPP-IV, porcine kidney DPP-IV, as well as human Caco-2 DPP-IV with IC(50) values of 1.70, 2.26, and 2.02 microM, respectively. Because of the poor pharmacokinetic properties of Compound 1, further optimization was carried out, leading to the discovery of Compound 2, which had similar in vitro activities. Compound 2 acted as a selective and competitive inhibitor of DPP-IV. MALDI-TOF mass spectrometric analysis proved that the compound (20 microM) effectively blocked the degradation of active GLP-1 peptide by 61%. Although similar in in vitro potency, marked improvement of in vivo efficacy and pharmacokinetic properties was seen with Compound 2. Oral administration of Compound 2 resulted in potent and rapid inhibition of circulating DPP-IV in C57BL/6J mice, with ED(50) values of 26mg/kg (s.c.) and 42mg/kg (p.o.). In addition, this compound improved glucose tolerance in ob/ob mice, as determined by an oral glucose tolerance test (OGTT). These results indicate that Compound 2 is a potent and selective DPP-IV inhibitor with oral anti-hyperglycemic activity in vivo.

Synthesis and evaluation of pyrazolidine derivatives as dipeptidyl peptidase IV (DP-IV) inhibitors.

A new series of pyrazolidine derivatives was synthesized and evaluated for their ability to inhibit dipeptidyl peptidase IV (DP-IV). Compound 9i was the most active in this series, exhibited IC50 value of 1.56 microM and ED50 value of 80 mg/kg (in vivo DP-IV inhibition; po).

Fine localization of Nefl and Nef3 and its exclusion as candidate gene for lens rupture 2(lr2).

Cataract causing lr2 gene is found in the CXSD mouse, which is a recombinant inbred strain of BALB/c and STS mice. For the process of positional cloning of lr2, several candidate genes were selected in the middle region of chromosome 14, but most of them were excluded by combination of recombination and homozygosity mapping. Components of neurofilament proteins, neurofilament light polypeptide (Nefl) and neurofilament3 medium (Nef3), were linked to D14Mit87 which was not separated from the lr2 locus in the homozygosity mapping. When the expression levels of Nefl and Nef3 in eyes were compared in CXSD and BALB/c mice, there were no differences in expression levels. The cDNA sequences of the two genes from CXSD, BALB/c and STS mice were subsequently compared. Several nucleotide differences in cDNA sequences were detected between the mice strains but the majority of the changes were silent mutations that did not alter the amino acids. The sole amino acid difference, E567K in the glutamate rich region of Nfm, between BALB/c and CXSD was found to be a simple genetic polymorphism because the same substitution existed in STS, a non-cataract mouse strain. Therefore we excluded Nefl and Nef3 from the candidate genes for lr2 based on expression and mutation analyses.

Discovery of a novel protein tyrosine phosphatase-1B inhibitor, KR61639: potential development as an antihyperglycemic agent.

Protein tyrosine phosphatase-1B (PTP-1B), a negative regulator of insulin signaling, may be an attractive therapeutic target for type 2 diabetes mellitus. High throughput screening (HTS) for PTP-1B inhibitors using compounds from the Korea Chemical Bank identified several hits (active compounds). Among them, a hit with 1,2-naphthoquinone scaffold was chosen for lead development. KR61639, [4-[1-(1H-indol-3-yl)-3,4-dioxo-3,4-dihydro-naphthalen-2-ylmethyl]-phenoxy]-acetic acid tert-butyl ester, inhibited human recombinant PTP-1B with an IC(50) value of 0.65 microM in a noncompetitive manner. KR61639 showed modest selectivity over several phosphatases and increased insulin-stimulated glycogen synthesis in HepG2 cells and stimulated 2-deoxyglucose uptake in 3T3/L1 adipocytes. In addition, in vivo study using ob/ob mouse demonstrated that KR61639 exerted a hypoglycemic action when given orally. Thus, KR61639 may be a good starting point for lead optimization in developing a novel antidiabetic agent.

KR-31378 ameliorates atherosclerosis by blocking monocyte recruitment in hypercholestrolemic mice.

The recruitment of monocytes into the artery wall is a crucial early step in atherogenesis. A novel compound, KR-31378, has been shown to be a neuroprotective agent for ischemia-reperfusion damage in rat brain via its potent antioxidant and antiapoptotic actions. Here, we report the effects of this compound on atherogenesis and possible mechanisms of action. In Ldlr knockout mice fed with a high-fat, high-cholesterol diet, treatment with KR-31378 significantly inhibited fatty streak formation and macrophage accumulation. To address the possibility that KR-31378 may influence the initial stages of atherogenesis, we examined its effect on the adhesion and migration of monocytes to endothelial cells stimulated with tumor necrosis factor-alpha. KR-31378 decreased the adhesion in a dose-dependent manner. The observed decreases in cell adhesion and migration correlated with KR-31378-mediated down-regulation of vascular cell adhesion molecule-1 (VCAM-1) and interleukin (IL)-8. Nuclear factor-kappaB (NF-kappaB) is known to regulate the expression of adhesive and chemotactic molecules including VCAM-1 and IL-8. Indeed, transient transfection experiments, electrophoretic mobility shift assay, and IkappaB degradation assay showed that KR-31378 decreased NF-kappaB activation. These results indicate that KR-31378 potently reduces fatty streak formation by inhibiting NF-kappaB-dependent cellular adhesion and chemotactic molecule expression, which are crucial to monocyte infiltration into the arterial wall during the early stages of atherogenesis.

Impaired D2 dopamine receptor function in mice lacking type 5 adenylyl cyclase.

Dopamine receptor subtypes D1 and D2, and many other seven-transmembrane receptors including adenosine receptor A2A, are colocalized in striatum of brain. These receptors stimulate or inhibit adenylyl cyclases (ACs) to produce distinct physiological and pharmacological responses and interact with each other synergistically or antagonistically at various levels. The identity of the AC isoform that is coupled to each of these receptors, however, remains unknown. To investigate the in vivo role of the type 5 adenylyl cyclase (AC5), which is preferentially expressed in striatum, mice deficient for the AC5 gene were generated. The genetic ablation of the AC5 gene eliminated >80% of forskolin-induced AC activity and 85-90% of AC activity stimulated by either D1 or A2A receptor agonists in striatum. However, D1- or A2A-specific pharmaco-behaviors were basically preserved, whereas the signal cascade from D2 to AC was completely abolished in AC5(-/-), and motor activity of AC5(-/-) was not suppressed by treatment of cataleptic doses of the antipsychotic drugs haloperidol and sulpiride. Interestingly, both haloperidol and clozapine at low doses remarkably increased the locomotion of AC5(-/-) in the open field test that was produced in part by a common mechanism that involved the increased activation of D1 dopamine receptors. Together, these results suggest that AC5 is the principal AC integrating signals from multiple receptors including D1, D2, and A2A in striatum and the cascade involving AC5 among diverse D2 signaling pathways is essential for neuroleptic effects of antipsychotic drugs.