Identification of novel quinazolinedione derivatives as RORγt inverse agonist.

Novel small molecules were synthesized and evaluated as retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists for the treatment of inflammatory and autoimmune diseases. A hit compound, 1, was discovered by high-throughput screening of our compound library. The structure-activity relationship (SAR) study of compound 1 showed that the introduction of a chlorine group at the 3-position of 4-cyanophenyl moiety increased the potency and a 3-methylpentane-1,5-diamide linker is favorable for the activity. The carbazole moiety of 1 was also optimized; a quinazolinedione derivative 18i suppressed the increase of IL-17A mRNA level in the lymph node of a rat model of experimental autoimmune encephalomyelitis (EAE) upon oral administration. These results indicate that the novel quinazolinedione derivatives have great potential as orally available small-molecule RORγt inverse agonists for the treatment of Th17-driven autoimmune diseases. A U-shaped bioactive conformation of this chemotype with RORγt protein was also observed.

Discovery of orally efficacious RORγt inverse agonists, part 1: Identification of novel phenylglycinamides as lead scaffolds.

A series of novel phenylglycinamides as retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists were discovered through optimization of a high-throughput screen hit 1. (R)-N-(2-((3,5-Difluoro-4-(trimethylsilyl)phenyl) amino)-1-(4-methoxyphenyl)-2-oxoethyl)-3-hydroxy-N-methylisoxazole-5-carboxamide (22) was identified as one of the best of these compounds. It displayed higher subtype selectivity and specificity over other nuclear receptors and demonstrated in vivo potency to suppress the transcriptional activity of RORγt in a mouse PD (pharmacodynamic) model upon oral administration.

Discovery of orally efficacious RORγt inverse agonists. Part 2: Design, synthesis, and biological evaluation of novel tetrahydroisoquinoline derivatives.

A series of tetrahydroisoquinoline derivatives were designed, synthesized, and evaluated for their potential as novel orally efficacious retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists for the treatment of Th17-driven autoimmune diseases. We carried out cyclization of the phenylglycinamide core by structure-based drug design and successfully identified a tetrahydroisoquinoline carboxylic acid derivative 14 with good biochemical binding and cellular reporter activity. Interestingly, the combination of a carboxylic acid tether and a central fused bicyclic ring was crucial for optimizing PK properties, and the compound 14 showed significantly improved PK profile. Successive optimization of the carboxylate tether led to the discovery of compound 15 with increased inverse agonistic activity and an excellent PK profile. Oral treatment of mice with compound 15 robustly and dose-dependently inhibited IL-17A production in an IL23-induced gene expression assay.

Expression of Mas-related gene X2 on mast cells is upregulated in the skin of patients with severe chronic urticaria.

BACKGROUND: Wheal reactions to intradermally injected neuropeptides, such as substance P (SP) and vasoactive intestinal peptide, are significantly larger and longer lasting in patients with chronic urticaria (CU) than in nonatopic control (NC) subjects. Mas-related gene X2 (MrgX2) has been identified as a receptor for basic neuropeptides, such as SP and vasoactive intestinal peptide. Mast cell (MC) responsiveness to eosinophil mediators contributes to the late-phase reaction of allergy. OBJECTIVE: We sought to compare the frequency of MrgX2 expression in skin MCs from patients with CU and NC subjects and to identify the receptor for basic eosinophil granule proteins on human skin MCs. METHODS: MrgX2 expression was investigated by using immunofluorescence in skin tissues from NC subjects and patients with severe CU and on skin-derived cultured MCs. MrgX2 expression in human MCs was reduced by using a lentiviral small hairpin RNA silencing technique. Ca(2+) influx was measured in CHO cells transfected with MrgX2 in response to eosinophil granule proteins. Histamine and prostaglandin D2 levels were measured by using enzyme immunoassays. RESULTS: The number of MrgX2(+) skin MCs and the percentage of MrgX2(+) MCs in all MCs in patients with CU were significantly greater than those in NC subjects. Eosinophil infiltration in urticarial lesions was observed in 7 of 9 patients with CU. SP, major basic protein, and eosinophil peroxidase, but not eosinophil-derived neurotoxin, induced histamine release from human skin MCs through MrgX2. CONCLUSION: MrgX2 might be a new target molecule for the treatment of wheal reactions in patients with severe CU.

Impact of Concomitant Thiopurine on the Efficacy and Safety of Filgotinib in Patients with Ulcerative Colitis: Post hoc Analysis of the Phase 2b/3 SELECTION Study.

BACKGROUND AND AIMS: SELECTION is the first study to assess the impact of concomitant thiopurine and other immunomodulator [IM] use on the efficacy and safety of a Janus kinase inhibitor, filgotinib, in patients with ulcerative colitis. METHODS: Data from the phase 2b/3 SELECTION study were used for this post hoc analysis. Patients were randomized [2:2:1] to two induction studies [biologic-naive, biologic-experienced] to filgotinib 200 mg, 100 mg, or placebo. At week 10, patients receiving filgotinib were re-randomized [2:1] to continue filgotinib or switch to placebo until week 58 [maintenance]. Outcomes were compared between subgroups with and without concomitant IM use. RESULTS: At week 10, a similar proportion of patients in +IM and -IM groups treated with filgotinib 200 mg achieved Mayo Clinic Score [MCS] response [biologic-naive: 65.8% vs 66.9%; biologic-experienced: 61.3% vs 50.5%] and clinical remission [biologic-naive: 26.0% vs 26.2%; biologic-experienced: 11.3% vs 11.5%]. At week 58, a similar proportion of patients in +IM and -IM groups treated with filgotinib 200 mg achieved MCS response [biologic-naive: 74.2% vs 75.0%; biologic-experienced: 45.5% vs 61.4%] and clinical remission [biologic-naive: 51.6% vs 47.4%; biologic-experienced: 22.7% vs 24.3%]. The probability of protocol-specified disease worsening during the maintenance study in patients treated with filgotinib 200 mg did not differ between +IM and -IM groups [p = 0.6700]. No differences were observed in the incidences of adverse events between +IM and -IM groups in induction/maintenance studies. CONCLUSIONS: The efficacy and safety profiles of filgotinib treatment in SELECTION did not differ with or without concomitant IM use.

Design and biological evaluation of imidazo[1,2-a]pyridines as novel and potent ASK1 inhibitors.

Imidazo[1,2-a]pyridine derivatives were designed, synthesized, and evaluated as inhibitors of the apoptosis signal-regulating kinase 1 (ASK1). These were based on a benzothiazole derivative that was discovered from high-throughput screening of our compound library. As a result, we identified potent, selective, and orally bioavailable ASK1 inhibitors for wide range of therapeutic targets.

Diagnostic Features of Perianal Fistula in Patients With Crohn's Disease: Analysis of a Japanese Claims Database.

Background: Perianal fistula (PAF) is a disabling complication of Crohn's disease (CD) which greatly impacts the quality of life. To address a scarcity of data in Asian populations, we determined the prevalence of CD-associated PAF in Japan, the order of diagnosis, and medical history of patients. Methods: A retrospective, longitudinal, observational cohort study was conducted, using an employer-based health insurance claims database. The study included patients diagnosed with CD and/or PAF from October 2013 to September 2019. Results: The age- and gender-adjusted prevalence rates of CD-associated PAF increased from 10.33 per 100 000 in 2014, to 13.68 per 100 000 in 2019. Among patients with CD-associated PAF, 15.7% were diagnosed with PAF after diagnosis of CD, 68.6% were diagnosed with PAF before diagnosis with CD, and 15.7% were diagnosed with CD and PAF within the same month. Of the patients diagnosed with CD after PAF, approximately 30% were diagnosed with PAF by the age of 20 years, whereas less than 10% of PAF patients without CD were diagnosed with PAF by the age of 20 years. Conclusions: The study reveals the prevalence of CD-associated PAF in Japan and that most individuals were diagnosed with CD after the diagnosis of PAF. Crohn's disease may be underdiagnosed in patients with PAF; patients diagnosed with PAF at a young age should be monitored to allow timely diagnosis of CD.

Anti-inflammatory therapy by ibudilast, a phosphodiesterase inhibitor, in demyelination of twitcher, a genetic demyelination model.

BACKGROUND: Twitcher mouse (twi/twi) is an authentic murine model of Krabbe's disease. Accumulation of psychosine, resulting in apoptosis of oligodendrocytes and subsequent demyelination, is a cardinal event to the pathogenesis of this disease. Moreover, recruitment of inflammatory cells plays a significant role in the pathological process in the twi/twi central and peripheral nervous systems. In this study, we investigated the 1) the relationship between tumor necrosis factor-alpha (TNFalpha), pro-inflammatory cytokine, and the progression of this disease and 2) effect of the anti-inflammatory therapy by ibudilast, a phosphodiesterase inhibitor. METHODS: We quantified the expression level of TNFalpha and TNF-receptor mRNA in twi/twi using semi-quantitative RT-PCR. The relationship between TNFalpha expression, apoptosis of oligodendrocytes and demyelination was studied with immunohistochemistry and TUNEL method. We then treated twi/twi with a daily intraperitoneal injection of ibudilast (10 mg/kg), which suppress TNFalpha production in the brain. RESULTS: We found that TNFalpha-immunoreactive microglia/macrophages appeared in the twi/twi brain and that the mRNA levels of TNFalpha and TNF-receptor 1 was increased with the progression of demyelination. The distribution profile of TNFalpha-immunoreactive microglia/macrophages overlapped that of TUNEL-positive oligodendrocytes in the twi/twi brain. When twi/twi was treated with ibudilast from PND30, the number of oligodendrocytes undergoing apoptosis was markedly reduced and demyelination was milder. Obvious improvement of clinical symptom was noted in two of five. The failure of constant clinical improvement by ibudilast may result from hepatotoxicity and/or the inhibition of proliferation of NG2-positive oligodendrocyte precursors. CONCLUSION: We conclude that anti-inflammatory therapy by a phosphodiesterase inhibitor can be considered as a novel alternative therapy for Krabbe's disease.

Pronounced adipogenesis and increased insulin sensitivity caused by overproduction of prostaglandin D2 in vivo.

Lipocalin-type prostaglandin (PG) D synthase is expressed in adipose tissues and involved in the regulation of glucose tolerance and atherosclerosis in type 2 diabetes. However, the physiological roles of PGD(2) in adipogenesis in vivo are not clear, as lipocalin-type prostaglandin D synthase can also act as a transporter for lipophilic molecules, such as retinoids. We generated transgenic (TG) mice overexpressing human hematopoietic PGDS (H-PGDS) and investigated the in vivo functions of PGD(2) in adipogenesis. PGD(2) production in white adipose tissue of H-PGDS TG mice was increased approximately seven-fold as compared with that in wild-type (WT) mice. With a high-fat diet, H-PGDS TG mice gained more body weight than WT mice. Serum leptin and insulin levels were increased in H-PGDS TG mice, and the triglyceride level was decreased by about 50% as compared with WT mice. Furthermore, in the white adipose tissue of H-PGDS TG mice, transcription levels of peroxisome proliferator-activated receptor gamma, fatty acid binding protein 4 and lipoprotein lipase were increased approximately two-fold to five-fold as compared with those of WT mice. Finally, H-PGDS TG mice showed clear hypoglycemia after insulin clamp. These results indicate that TG mice overexpressing H-PGDS abundantly produced PGD(2) in adipose tissues, resulting in pronounced adipogenesis and increased insulin sensitivity. The present study provides the first evidence that PGD(2) participates in the differentiation of adipocytes and in insulin sensitivity in vivo, and the H-PGDS TG mice could constitute a novel model mouse for diabetes studies.

In vivo and in vitro effects of SAR 943, a rapamycin analogue, on airway inflammation and remodeling.

No current therapy is considered to be satisfactory for severe asthma, and alternative approaches are still required for what is a major unmet medical need. In this study, we compared the effect of a rapamycin derivative, SAR 943, with budesonide, using a murine model of lung inflammation and remodeling. Allergen challenge of ovalbumin-sensitized BALB/c mice induced an increase in the levels of interleukin-5 and interleukin-4; numbers of eosinophil, neutrophil, and lymphocyte; cellular fibronectin; lung epithelial cell proliferation and mucus hypersecretory phenotype; as well as hyperreactivity to methacholine. Both SAR 943 and budesonide, when given intranasally 1 hour before and 24 hours after the aerosol challenge, inhibited all of these parameters with a similar potency (effective dose 50% of 1 mg/kg). In primary cultured smooth muscle cells from human airways, SAR 943 dose dependently inhibited epidermal growth factor-induced proliferation but did not affect the basal cell proliferation. Neither the basal nor stimulated proliferation of a human bronchial epithelial cell line (16HBE14o-) was affected by SAR 943. In conclusion, SAR 943 is as effective as budesonide in inhibiting both lung inflammation and remodeling in a murine model of asthma. Hence, this class of compound could offer beneficial effects in patients with severe asthma.

Pronounced eosinophilic lung inflammation and Th2 cytokine release in human lipocalin-type prostaglandin D synthase transgenic mice.

PGD(2) is a major lipid mediator released from mast cells, but little is known about its role in the development of allergic reactions. We used transgenic (TG) mice overexpressing human lipocalin-type PGD synthase to examine the effect of overproduction of PGD(2) in an OVA-induced murine asthma model. The sensitization of wild-type (WT) and TG mice was similar as judged by the content of OVA-specific IgE. After OVA challenge, PGD(2), but not PGE(2), substantially increased in the lungs of WT and TG mice with greater PGD(2) increment in TG mice compared with WT mice. The numbers of eosinophils and lymphocytes in the bronchoalveolar lavage (BAL) fluid were significantly greater in TG mice than in WT mice on days 1 and 3 post-OVA challenge, whereas the numbers of macrophages and neutrophils were the same in both WT and TG mice. The levels of IL-4, IL-5, and eotaxin in BAL fluid were also significantly higher in TG mice than in WT mice, although the level of IFN-gamma in the BAL fluid of TG mice was decreased compared with that in WT mice. Furthermore, lymphocytes isolated from the lungs of TG mice secreted less IFN-gamma than those from WT mice, whereas IL-4 production was unchanged between WT and TG mice. Thus, overproduction of PGD(2) caused an increase in the levels of Th2 cytokines and a chemokine, accompanied by the enhanced accumulation of eosinophils and lymphocytes in the lung. These results indicate that PGD(2) plays an important role in late phase allergic reactions in the pathophysiology of bronchial asthma.

Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages.

We cloned the cDNA for mouse microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and expressed the recombinant enzyme in Escherichia coli. The membrane fraction containing recombinant mPGES-1 catalyzed the isomerization of PGH2 to PGE2 in the presence of GSH with K(m) values of 130 microM for PGH2 and 37 microM for GSH, a turnover number of 600 min(-1), and a k(cat)/K(m) ratio of 4.6 min(-1) microM(-1). Recombinant mPGES-1 was purified and used to generate a polyclonal antibody highly specific for mPGES-1. The antibody showed a single band on Western blotting of microsomal fractions from lipopolysaccharide-treated mouse peritoneal macrophages. Northern and Western blotting analyses revealed that mPGES-1 was induced together with cyclooxygenase-2 in mouse macrophages after treatment of the cells with lipopolysaccharide. Confocal immunofluorescence microscopy revealed that both mPGES-1 and cyclooxygenase-2 were colocalized in the lipopolysaccharide-treated macrophages. Taken together, these results demonstrate that mPGES-1 is an efficient downstream enzyme for the production of PGE2 in the activated macrophages treated by lipopolysaccharide.

Regulation of lipocalin-type prostaglandin D synthase gene expression by Hes-1 through E-box and interleukin-1 beta via two NF-kappa B elements in rat leptomeningeal cells.

The promoter function of the rat lipocalin-type prostaglandin D synthase (L-PGDS) gene was characterized in primary cultures of leptomeningeal cells prepared from the neonatal rat brain. Luciferase reporter assays with deletion and site-directed mutation of the promoter region (-1250 to +77) showed that an AP-2 element at -109 was required for activation and an E-box at +57, for repression. Binding of nuclear factors to each of these cis-elements was demonstrated by an electrophoretic mobility shift assay. Several components of the Notch-Hes signaling pathway, Jagged, Notch1, Notch3, and Hes-1, were expressed in the leptomeningeal cells. Human Hes-1 co-expressed in the leptomeningeal cells bound to the E-box of the rat L-PGDS gene, and repressed the promoter activity of the rat L-PGDS gene in a dose-dependent manner. The L-PGDS gene expression was up-regulated slowly by interleukin-1 beta to the maximum level at 24 h. The reporter assay with deletion and mutation revealed that two NF-kappa B elements at -1106 and -291 were essential for this up-regulation. Binding of two NF-kappa B subunits, p65 and c-Rel, to these two NF-kappa B elements occurred after the interleukin-1 beta treatment. Therefore, the L-PGDS gene is the first gene identified as the target for the Notch-Hes signal through the E-box among a variety of genes involved in the prostanoid biosynthesis, classified to the lipocalin family, and expressed in the leptomeninges. Moreover, the L-PGDS gene is a unique gene that is activated slowly by the NF-kappa B system.

PPARgamma coactivator 1beta/ERR ligand 1 is an ERR protein ligand, whose expression induces a high-energy expenditure and antagonizes obesity.

A well balanced body energy budget controlled by limitation of calorie uptake and/or increment of energy expenditure, which is typically achieved by proper physical exercise, is most effective against obesity and diabetes mellitus. Recently, peroxisome proliferator-activated receptor (PPAR) gamma, a member of the nuclear receptor, and its cofactors have been shown to be involved in lipid metabolism and in the control of energy expenditure. Here we show that PPARgamma coactivator 1 (PGC-1) beta functions as ERRL1 (for ERR ligand 1), which can bind and activate orphan ERRs (estrogen receptor-related receptors) in vitro. Consistently, PGC-1beta/ERRL1 transgenic mice exhibit increased expression of the medium-chain acyl CoA dehydrogenase, a known ERR target and a pivotal enzyme of mitochondrial beta-oxidation in skeletal muscle. As a result, the PGC-1beta/ERRL1 mice show a state similar to an athlete; namely, the mice are hyperphagic and of elevated energy expenditure and are resistant to obesity induced by a high-fat diet or by a genetic abnormality. These results demonstrate that PGC-1beta/ERRL1 can function as a protein ligand of ERR, and that its level contributes to the control of energy balance in vivo, and provide a strategy for developing novel antiobesity drugs.