A Bispecific, Tetravalent Antibody Targeting Inflammatory and Pruritogenic Pathways in Atopic Dermatitis.

Inhibition of IL-4/IL-13 signaling has dramatically improved the treatment of atopic dermatitis (AD). However, in many patients, clinical responses are slow to develop and remain modest. Indeed, some symptoms of AD are dependent on IL-31, which is only partially reduced by IL-4/IL-13 inhibition. Thus, there is an unmet need for AD treatments that concomitantly block IL-4/IL-13 and IL-31 pathways. We engineered NM26-2198, a bispecific tetravalent antibody designed to accomplish this task. In reporter cell lines, NM26-2198 concomitantly inhibited IL-4/IL-13 and IL-31 signaling with a potency comparable with that of the combination of an anti-IL-4Rα antibody (dupilumab) and an anti-IL-31 antibody (BMS-981164). In human PBMCs, NM26-2198 inhibited IL-4-induced upregulation of CD23, demonstrating functional binding to FcγRII (CD32). NM26-2198 also inhibited the secretion of the AD biomarker thymus and activation-regulated chemokine (TARC) in blood samples from healthy human donors. In male cynomolgus monkeys, NM26-2198 exhibited favorable pharmacokinetics and significantly inhibited IL-31-induced scratching at a dose of 30 mg/kg. In a repeat-dose, good laboratory practice toxicology study in cynomolgus monkeys, no adverse effects of NM26-2198 were observed at a weekly dose of 125 mg/kg. Together, these results justify the clinical investigation of NM26-2198 as a treatment for moderate-to-severe AD.

Establishment of a system to evaluate the therapeutic effect and the dynamics of an investigational drug on ulcerative colitis using human colonic organoids.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with an intractable, recurrent course. The goal of UC therapy is to target mucosal healing because immune-suppressive therapy for UC frequently results in relapse. However, few drugs directly target mucosal healing. We, therefore, aim to evaluate the therapeutic effect of an investigational drug on intestinal epithelial cells in an in vitro UC model using human colonic organoids. METHODS: Colonic organoids were isolated from human colon and cultured. A mixture of cytokines and bacterial components were used to mimic UC in humans. The effect of the investigational drug on colonic organoid was evaluated by microarray analysis and 3D immunofluorescence. The enrichment of stem cells was assessed with a colony formation assay. RESULTS: Inflammatory stimulation resulted in a significant induction of inflammatory-related genes in colonic organoids whereas cell differentiation was suppressed. Treatment with the investigational drug KAG-308 showed reciprocal dynamics of gene expression to inflammatory stimulation, which resulted in not only the suppression of immune response but also the promotion of cellular differentiation towards secretory lineages. Moreover, SPDEF and Reg4 were identified as novel targets for the enrichment of intestinal epithelial stem cells and mucosal healing. CONCLUSIONS: The establishment of in vitro UC model using human colonic organoid could reveal the effects and targets of investigational drugs in intestinal epithelial cells under inflammation conditions. Further maturation of this system might be more efficient to predict the effect on UC, as compared with the use of animal model, for the development of new drugs.

Oral administration of EP4-selective agonist KAG-308 suppresses mouse knee osteoarthritis development through reduction of chondrocyte hypertrophy and TNF secretion.

Osteoarthritis (OA) is one of the world's most common degenerative diseases, but there is no disease-modifying treatment available. Previous studies have shown that prostaglandin E2 (PGE2) and PGE2 receptor 4 (EP4) are involved in OA pathogenesis; however, their roles are not fully understood. Here, we examined the efficacy of oral administration of KAG-308, an EP4-selective agonist, in surgically induced mouse knee OA. Cartilage degeneration and synovitis were significantly inhibited by the KAG-308 treatment. Chondrocyte hypertrophy and expression of tumor necrosis factor alpha (TNF) and matrix metalloproteinase 13 (Mmp13) in the synovium were suppressed in the KAG-308-treated mice. In cultured chondrocytes, hypertrophic differentiation was inhibited by KAG-308 and intranuclear translocation of histone deacetylase 4 (Hdac4) was enhanced. In cultured synoviocytes, lipopolysaccharide (LPS)-induced expression of TNF and Mmp13 was also suppressed by KAG-308. KAG-308 was detected in the synovium and cartilage of orally treated mice. TNF secretion from the synovia of KAG-308-treated mice was significantly lower than control mice. Thus, we conclude that oral administration of KAG-308 suppresses OA development through suppression of chondrocyte hypertrophy and synovitis. KAG-308 may be a potent candidate for OA drug development.

KAG-308, a newly-identified EP4-selective agonist shows efficacy for treating ulcerative colitis and can bring about lower risk of colorectal carcinogenesis by oral administration.

Agonists for EP4 receptor, a prostaglandin E2 receptor subtype, appear to be a promising therapeutic strategy for ulcerative colitis (UC) due to their anti-inflammatory and epithelial regeneration activities. However, the clinical development of orally-available EP4 agonists for mild to moderate UC has not yet been reported. Furthermore, the possibility of an increased risk of colitis-associated cancer (CAC) through direct proliferative effects on epithelial cells via EP4 signaling has not been ruled out. Recently, we identified KAG-308 as an orally-available EP4-selective agonist. Here, we investigated the pharmacological and pharmacokinetic profiles of KAG-308. Then, we compared KAG-308 and sulfasalazine (SASP) for their abilities to prevent colitis and promote mucosal healing in a mouse model of dextran sulfate sodium (DSS)-induced colitis. Finally, the effect of KAG-308 treatment on CAC was evaluated in an azoxymethane (AOM)/DSS-induced CAC mouse model. KAG-308 selectively activated EP4 and potently inhibited tumor necrosis factor-α production in peripheral whole blood and T cells. Oral administration of KAG-308, which showed relatively high bioavailability, suppressed the onset of DSS-induced colitis and promoted histological mucosal healing, while SASP did not. KAG-308 also prevented colorectal carcinogenesis by inhibiting colitis development and consequently decreasing mortality in a CAC model, whereas SASP had marginal effects. In contrast, MF-482, an EP4 antagonist, increased mortality. These results indicated that orally-administered KAG-308 suppressed colitis development and promoted mucosal healing. Moreover, it exhibited preventive effects on colorectal carcinogenesis, and thus may be a new therapeutic strategy for the management of UC that confers a reduced risk of colorectal carcinogenesis.

Effect of inhaled KP-496, a novel dual antagonist of the cysteinyl leukotriene and thromboxane A2 receptors, on a bleomycin-induced pulmonary fibrosis model in mice.

Cysteinyl-leukotrienes (cysLTs) and thromboxane A(2) (TXA(2)) are important mediators in inflammatory lung diseases such as bronchial asthma and idiopathic pulmonary fibrosis (IPF). We examined the effects of inhaled KP-496, a novel dual antagonist of the cysLTs and TXA(2) receptors, on bleomycin-induced IPF in mice. Mice were intravenously injected bleomycin on day 0, and 0.5% of KP-496 was inhaled twice a day (30 min/time) for the entire experimental period. The effects of KP-496 were evaluated by the number of infiltrated cells in bronchoalveolar lavage fluid (BALF), hydroxyl-L-proline content in the lung, and histopathology. Analyses of BALF on days 7 and 21 revealed that inhaled KP-496 significantly decreased total cell numbers, macrophages, neutrophils, and eosinophils on both days. KP-496 significantly decreased hydroxyl-L-proline content in the lung on day 21. Histopathological analyses of lungs on day 21 demonstrated that KP-496 significantly suppressed inflammatory and fibrotic changes. Our results suggested that the suppression of cysLTs and TXA(2) pathways by KP-496 could control airway inflammation and pulmonary fibrosis, and that KP-496 could be a new therapeutic agent for lung diseases with inflammation and fibrogenesis such as IPF and chronic obstructive pulmonary disease.

KIF1Bbeta2, capable of interacting with CHP, is localized to synaptic vesicles.

Kinesin family proteins are microtubule-dependent molecular motors involved in the intracellular motile process. Using a Ca2+ -binding protein, CHP (calcineurin B homologous protein), as a bait for yeast two hybrid screening, we identified a novel kinesin-related protein, KIF1Bbeta2. KIF1Bbeta2 is a member of the KIF1 subfamily of kinesin-related proteins, and consists of an amino terminal KIF1B-type motor domain followed by a tail region highly similar to that of KIF1A. CHP binds to regions adjacent to the motor domains of KIF1Bbeta2 and KIF1B, but not to those of the other KIF1 family members, KIF1A and KIF1C. Immunostaining of neuronal cells showed that a significant portion of KIF1Bbeta2 is co-localized with synaptophysin, a marker protein for synaptic vesicles, but not with a mitochondria-staining dye. Subcellular fractionation analysis indicated the co-localization of KIF1Bbeta2 with synaptophysin. These results suggest that KIF1Bbeta2, a novel CHP-interacting molecular motor, mediates the transport of synaptic vesicles in neuronal cells.