Kenichi Chiba

E6201 [(3S,4R,5Z,8S,9S,11E)-14-(Ethylamino)-8, 9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione], a Novel Kinase Inhibitor of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase (MEK)-1 and MEK Kinase-1: In Vitro Characterization of Its Anti-Inflammatory and Antihyperproliferative Activities

The goal of this study is to identify a novel inhibitor with anti-inflammatory and antiproliferative properties for the treatment of psoriasis. Compound f152A1 [(3S,5Z,8S,11E)-8,9,16-trihydroxy-14-methoxy-3-methyl-3,4,9,10-tetrahydro-1H-benzo[c][1]oxacyclotetradecine1,7(8H)-dione] was identified as the main active metabolite with strong inhibitory activity against tumor necrosis factor-α (TNFα) transcription in a fraction originated from the fermentation broth of the fungus Curvularia verruculosa. Although active in cell-based assays, f152A1 was unstable in plasma and liver microsome preparations, thus limiting its pharmaceutical utilization. To improve the metabolic properties of f152A1, a medicinal chemistry program was undertaken, resulting in the generation of over 400 analogs of f152A1. Eventually, E6201 [(3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione] was identified as a promising analog in this series. In the present study, we characterized the in vitro activities of E6201 and discovered that the compound inhibits lipopolysaccharide-activated TNFα reporter activity in THP-1-33 cells with an IC50 value of 50 nM and selectively inhibits mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-1 and MEK kinase-1 in cell-free biochemical assays. In addition, E6201 showed inhibitory activity in several other cell-based systems: 1) phosphorylation of c-jun N-terminal kinase and p38 MAPKs; 2) nuclear factor-κB and activated protein-1 activation in various cell types; 3) interleukin (IL)-2 production from human lymphocytes; 4) hyperproliferation of human keratinocytes; 5) IL-8 production from human keratinocytes; and 6) proinflammatory cytokine production from human peripheral blood mononuclear cells. Based on the data presented here, E6201 may be beneficial for treatment of inflammatory and hyperproliferative diseases such as psoriasis through its anti-inflammatory activities on immune cells and antihyperproliferative activities on keratinocytes.

ER-34122, a novel dual 5-lipoxygenase/cyclooxygenase inhibitor with potent anti-inflammatory activity in an arachidonic acid-induced ear inflammation model.

Abstract. Objective and Design: To investigate the effect of ER-34122, a novel pyrazole derivative, on 5-lipoxygenase (LOX) and cyclooxygenase (COX) metabolite production in vitro, ex vivo and in vivo.¶Material: In vitro, lysate of rat basophilic leukemia cells, the microsome fraction of sheep seminal vesicles, human polymorphonuclear leukocytes, human synovial cells, and human monocytes. Ex vivo and in vivo, male Balb/c mice or SD rats.¶Treatment: In ex vivo study, ER-34122 (0.03-1 mg/kg) was orally administered 1 h before withdrawal of blood samples. In carrageenin-induced paw edema, ER-34122 (3–100 mg/kg) and indomethacin (1–10 mg/kg) were orally administered 1 h before carrageenin injection. In arachidonic acid-induced ear inflammation, ER-34122 (0.3–10 mg/kg), zileuton (10–100 mg/kg) and indomethacin (0.3-3 mg/kg) were orally administered 1 h before arachidonic acid application.¶Methods: 5-Hydroxyeicosatetraenoic acid and other eicosanoids were determined by using an HPLC method and enzyme immunoassay, respectively. Rat hind paw edema and mouse ear edema were assessed by measuring paw volume and ear thickness, respectively. Myeloperoxidase (MPO) activity and eicosanoid content of the ear tissue were also determined.¶Results: ER-34122 inhibited both LOX and COX product generation in vitro, and ex vivo. ER-34122 and indomethacin inhibited carrageenin-induced paw edema formation. In the arachidonic acid-induced ear inflammation, ER-34122 inhibited inflammatory responses (edema formation and MPO accumulation) as well as eicosanoids (LTB4, LTC4 and PGE2) generation. A representative LOX inhibitor, zileuton, also inhibited these inflammatory responses, while a COX inhibitor, indomethacin, did not suppress them though it completely inhibited PGE2 generation.¶Conclusions: The anti-inflammatory characteristics of ER-34122 are considered to be superior to those of COX inhibitors such as indomethacin, because in addition to its inhibitory activity on the COX pathway, ER-34122 inhibits LOX products generation, as revealed by the inhibition of edema formation or polymorphonuclear leukocyte infiltration in the arachidonic acid-induced ear inflammation model.

Oral administration of the thrombin receptor antagonist E5555 (atopaxar) attenuates intimal thickening following balloon injury in rats

Thrombin is a powerful agonist for a variety of cellular responses including platelet aggregation and vascular smooth muscle cell (SMC) proliferation. These actions are mediated by a thrombin receptor known as protease-activated receptor-1 (PAR-1). Recently we discovered that 1-(3-tert-butyl-4-methoxy-5-morpholinophenyl)-2-(5,6-diethoxy-7-fluoro-1-imino-1,3-dihydro-2H-isoindol-2-yl)ethanone hydrobromide (E5555, atopaxar) is a potent and selective thrombin receptor antagonist. This study characterized the pharmacological effects of E5555 on SMC proliferation in vitro and in a rat model of intimal thickening after balloon injury in vivo. E5555 selectively inhibited rat aortic SMC proliferation induced by thrombin and thrombin receptor-activating peptide (TRAP) with half maximal inhibitory concentration (IC(50)) values of 0.16 and 0.038 μM, respectively. E5555 did not inhibit rat SMC proliferation induced by basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) at concentrations up to 1μM. In addition, E5555 inhibited human aortic SMC proliferation induced by thrombin at concentrations of 0.3 and 3units/ml with IC(50) values of 0.028 and 0.079 μM, respectively, whereas it did not affect bFGF-induced proliferation at concentrations up to 1μM. Repeated oral administration of 30 mg/kg E5555 (once daily for 16 days) significantly reduced neointimal formation in the balloon-injured rat arterial model. These results suggested that a PAR-1 antagonist could be effective for treating restenosis following vascular intervention in addition to preventing thrombus formation. E5555 could thus have therapeutic potential for restenosis and chronic atherothrombotic disease.

Discovery of a potent, metabolically stabilized resorcylic lactone as an anti-inflammatory lead

With bioactivity-guided phenotype screenings, a potent anti-inflammatory compound f152A1 has been isolated, characterized and identified as the known natural product LL-Z1640-2. Metabolic instability precluded its use for the study on animal disease models. Via total synthesis, a potent, metabolically stabilized analog ER-803064 has been created; addition of the (S)-Me group at C4 onto f152A1 has resulted in a dramatic improvement on its metabolic stability, while preserving the anti-inflammatory activities.

E6201, a Novel Kinase Inhibitor of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase-1 and Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase Kinase-1: In Vivo Effects on Cutaneous Inflammatory Responses by Topical Administration

E6201 [(3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,10-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione)] is a novel anti-inflammatory agent that has potent inhibitory effects on the production of proinflammatory cytokines from leukocytes and antiproliferative activity on keratinocytes. To characterize the in vivo pharmacological activity of E6201, topically administered E6201 was evaluated in several different animal models of dermatitis. E6201 formulated as an ointment or cream showed dose-dependent inhibition of croton oil-induced acute edema formation and neutrophil infiltration into mouse skin. In addition, E6201 cream inhibited the 1-fluoro-2,4-dinitrobenzene-induced contact hypersensitivity reaction mediated by T cells in mice. In this model, E6201 cream also suppressed the migration of neutrophils and lymphocytes into the inflammatory site. Pretreatment with E6201 cream attenuated phorbol-12 myristate 13-acetate-induced ornithine decarboxylase activity, a marker of proliferation in epidermis. Furthermore, E6201 ointment showed inhibitory effects on both mezerein-induced and interleukin (IL)-23-induced epidermal hyperplasia. E6201 also suppressed T cell receptor-stimulated IL-17 production from human T cells. These results indicate that topically administered E6201 may be a useful agent for the prevention and treatment of cutaneous inflammatory and hyperproliferative diseases such as psoriasis.

Effects of ER-34122, a novel dual 5-lipoxygenase/cyclooxygenase inhibitor, on indices of early articular lesion in MRL/MpJ-lpr/lpr mice

Abstract.Objective and Design: To investigate effects of ER-34122, a novel dual 5-lipoxygenase (LOX)/cyclooxygenase (COX) inhibitor, and indomethacin on progression of articular lesions in MRL/MpJ-lpr/lpr (MRL/l) mice.¶Material: 100 male MRL/l mice.¶Treatment: ER-34122 (1-100 mg/kg) and indomethacin (1 mg/kg) were orally administered once a day to MRL/l mice from 6 to 10 or 16 weeks old.¶Methods: Articular lesions were analyzed histopathologically in the early (10 weeks old) or late (16 weeks old) stages of MRL/l mice arthritis. Serum levels of rheumatoid factor were measured by using enzyme-linked immunosorbent assay.¶Results: Articular lesions in the late stage of MRL/l mice arthritis were characterized by cartilage degeneration and pannus formation which were severer than those in the early stage. Polymorphonuclear leukocyte (PMN) infiltration and subsynovial soft tissue edema were observed as characteristic lesions in the early stage. ER-34122 suppressed progression of PMN infiltration, subsynovial soft tissue edema and multiplication of synovial lining cells in the early stage of the arthritis, even though it had no significant effect on other indices of articular lesion, enlargement of lymph nodes and serum levels of rheumatoid factors. On indices of late articular lesion, ER-34122 had no significant beneficial effects. Neither in the early nor late stage, indomethacin, a COX inhibitor, had significant effect on the arthritis at the examined dose.¶Conclusions: These results disclosed that ER-34122, a dual LOX/COX inhibitor, has anti-inflammatory activity in the early stage of the spontaneous arthritis.

Hydroxyindole derivatives as inhibitors of IL-1 generation. I. Synthesis and pharmacological activities of (E)-3-(4-hydroxy-5-methoxyindole-7-yl)-2-methylpropenoic acid derivatives

Summary A series of ( E )-3-(4-hydroxy-5-methoxyindole-7-yl)-2-methylpropenoic acids was prepared and the inhibitory activities of its members on IL-1 generation were evaluated in an in vivo system using the rat carboxymethyl cellulose-lipopolysaccharide (CMC-LPS) air-pouch model. Many compounds in this new series were found to be inhibitors of IL-1 generation. Structure-activity relationships indicated that a methyl substituent at the 1- and 3-positions on the indole ring are important for activity and that a 3,4,5-trimethoxy-substituted 2-phenyl group on the indole ring is suitable to give compounds exhibiting inhibition after oral administration. Among the compounds evaluated, ( E )-3-[1,3-dimethyl-4-hydroxy-5-methoxy-2-(3,4,5-trimethoxyphenyl)indole-7-yl]-2-methylpropenoic acid ( 15m ), which inhibited IL-1 generation by human monocytes stimulated with various reagents such as LPS, opsonized zymosan and immune complexes, showed inhibitory activity in the rat CMC-LPS model after oral administration.

Structure-Based Development of a Protein-Protein Interaction Inhibitor Targeting Tumor Necrosis Factor Receptor-Associated Factor 6.

The interactions between tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and TNF superfamily receptors (TNFRSFs) are promising targets for rheumatoid arthritis (RA) treatment. However, due to the challenging nature of protein-protein interactions (PPIs), a potent inhibitor that surpasses the affinity of the TRAF6-TNFRSF interactions has not been developed. We developed a small-molecule PPI inhibitor of TRAF6-TNFRSF interactions using NMR and in silico techniques. The most potent compound, TRI4, exhibited an affinity higher than those of TNFRSFs and competitively inhibited a TRAF6-TNFRSF interaction. Structural characterization of the TRAF6-TRI4 complex revealed that TRI4 supplants key interactions in the TRAF6-TNFRSF interfaces. In addition, some TRAF6-TRI4 interactions extend beyond the TRAF6-TNFRSF interfaces and increase the binding affinity. Our successful development of TRI4 provides a new opportunity for RA treatment and implications for structure-guided development of PPI inhibitors.

Pharmacological properties of N-methoxy-3-(3,5-ditert-butyl-4-hydroxybenzylidene)-2-pyrrolidone (E-5110), a novel nonsteroidal antiinflammatory agent

The antiinflammatory activity of a novel pyrrolidone derivative E-5110 was investigated using anti-inflammatory, analgesic and antipyretic animal models in comparison to indomethacin (IND) and piroxicam (PIR). The acute antiinflammatory activity of E-5110 on carrageenin paw edema was similar to IND, and half of PIR. E-5110 inhibited the pleural exudate volume and leucocyte infiltration in a reversed passive Arthus reaction more potent than IND. The chronic inflammatory responses in the established adjuvant- and type II collagen-induced arthritis were suppressed by E-5110 similar to IND and PIR. The analgesic potency of E-5110 was similar to IND and PIR, but the antipyretic activity of E-5110 was more potent than that of IND, and slightly more potent than that of PIR. The ulcerogenic effect of E-5110 on rat gastric mucosa was less than that of the reference drugs.

Collagenase production in the exudate of a novel adjuvant-induced air pouch inflammation model in rats.

To elucidate the role of collagenase in tissue destruction in chronic inflammation (e.g., in rheumatoid arthritis), we established a novel air pouch model in rats. Ten ml of air was injected subcutaneously on the dorsa of F344 rats, and 24 hours thereafter, Freunds complete adjuvant was injected into the air pouch. A remarkably high activity of trypsin-activated collagenase was detected dose-dependently in the pouch fluid at 8 days after the challenge injection. Concomitantly with the increase of collagenase activity, the weight of granuloma formed in the pouch decreased, suggesting that collagenase participates in the resorption of granuloma tissues.