In vivo and ex vivo cetuximab sensitivity assay using three-dimensional primary culture system to stratify KRAS mutant colorectal cancer.

In clinic, cetuximab, an anti-EGFR antibody, improves treatment outcomes in colorectal cancer (CRC). KRAS-mutant CRC is generally resistant to cetuximab, although difference of the sensitivity among KRAS-mutants has not been studied in detail. We previously developed the cancer tissue-originated spheroid (CTOS) method, a primary culture method for cancer cells. We applied CTOS method to investigate whether ex vivo cetuximab sensitivity assays reflect the difference in sensitivity in the xenografts. Firstly, in vivo cetuximab treatment was performed with xenografts derived from 10 CTOS lines (3 KRAS-wildtype and 7 KRAS mutants). All two CTOS lines which exhibited tumor regression were KRAS-wildtype, meanwhile all KRAS-mutant CTOS lines grew more than the initial size: were resistant to cetuximab according to the clinical evaluation criteria, although the sensitivity was quite diverse. We divided KRAS-mutants into two groups; partially responsive group in which cetuximab had a substantial growth inhibitory effect, and resistant group which exhibited no effect. The ex vivo signaling assay with EGF stimulation revealed that the partially responsive group, but not the resistant group, exhibited suppressed ERK phosphorylation ex vivo. Furthermore, two lines from the partially responsive group, but none of the lines in the resistant group, exhibited a combinatory effect of cetuximab and trametinib, a MEK inhibitor, ex vivo and in vivo. Taken together, the results indicate that ex vivo signaling assay reflects the difference in sensitivity in vivo and stratifies KRAS mutant CTOS lines by sensitivity. Therefore, coupling the in vivo and ex vivo assays with CTOS can be a useful platform for understanding the mechanism of diversity in drug sensitivity.

Cyclooxygenase-2/prostaglandin E2 accelerates the healing of gastric ulcers via EP4 receptors.

We examined the involvement of cyclooxygenase (COX)-1 as well as COX-2 in the healing of gastric ulcers and investigated which prostaglandin (PG) EP receptor subtype is responsible for the healing-promoting action of PGE2. Male SD rats and C57BL/6 mice, including wild-type, COX-1(-/-), and COX-2(-/-), were used. Gastric ulcers were produced by thermocauterization under ether anesthesia. Gastric ulcer healing was significantly delayed in both rats and mice by indomethacin and rofecoxib but not SC-560 given for 14 days after ulceration. The impaired healing was also observed in COX-2(-/-) but not COX-1(-/-) mice. Mucosal PGE2 content increased after ulceration, and this response was significantly suppressed by indomethacin and rofecoxib but not SC-560. The delayed healing in mice caused by indomethacin was significantly reversed by the coadministration of 11-deoxy-PGE1 (EP3/EP4 agonist) but not other prostanoids, including the EP1, EP2, and EP3 agonists. By contrast, CJ-42794 (selective EP(4) antagonist) significantly delayed the ulcer healing in rats and mice. VEGF expression and angiogenesis were both upregulated in the ulcerated mucosa, and these responses were suppressed by indomethacin, rofocoxib, and CJ-42794. The expression of VEGF in primary rat gastric fibroblasts was increased by PGE2 or AE1-329 (EP4 agonist), and these responses were both attenuated by coadministration of CJ-42794. These results confirmed the importance of COX-2/PGE2 in the healing mechanism of gastric ulcers and further suggested that the healing-promoting action of PGE2 is mediated by the activation of EP4 receptors and is associated with VEGF expression.

Effects of TAK-427 on acute nasal symptoms and nasal obstruction in guinea pig model of experimental allergic rhinitis.

TAK-427 (2-[6-[[3-[4-(diphenylmethoxy)piperidino]propyl]amino]imidazo[1,2-b]pyridazin-2-yl]-2-methylpropionic acid dihydrate) is a novel anti-allergic agent that has both histamine H1-receptor antagonist and anti-inflammatory activities. In this study, we evaluated the efficacy of TAK-427 on acute nasal responses and nasal obstruction using various guinea pig models of allergic rhinitis. TAK-427 inhibited the histamine-induced nasal reactions with an ID50 value of 0.633 mg/kg, p.o. TAK-427 (0.1-10 mg/kg, p.o.) and most histamine H1-receptor antagonists tested inhibited the increase in intranasal pressure, nasal hypersecretion, sneezing and nasal itching caused by a single antigen challenge in sensitized guinea pigs. In addition, TAK-427 (0.3, 30 mg/kg, p.o.) significantly inhibited the development of nasal obstruction when sensitized guinea pigs were repeatedly challenged via inhalation with Japanese cedar pollen, whereas the histamine H1-receptor antagonist, azelastine (1 mg/kg, p.o.), and ketotifen (1 mg/kg, p.o.) were without effect. These results suggest that TAK-427 might not only suppress acute nasal symptoms but also ameliorate nasal obstruction via the effects other than those as a histamine H1-receptor antagonist.

Synthesis of eosinophil infiltration inhibitors with antihistaminic activity.

A series of [1, 2, 4]triazolo[1, 5-b]pyridazines (5) and imidazo[1, 2-b]pyridazines (6) having cyclic amines was synthesized and evaluated for antihistaminic activity and inhibitory effect on eosinophil infiltration. When a piperidine or a piperazine containing a benzhydryl group and a suitable spacer was incorporated at the 6-position, the fused pyridazines were found to exhibit both antihistaminic activity and an inhibitory effect on eosinophil chemotaxis. Above all, 6a showed potent antihistaminic activity, but little blockade of central H(1) receptors in contrast with its complete blockade of peripheral H(1) receptors as determined by an ex vivo binding assay. Furthermore, 6a inhibited eosinophil infiltration of the skin caused by a topical antigen challenge in sensitized guinea pigs, while an antihistamine terfenadine was not effective. After the pharmacokinetic study, 6a was found to be rapidly hydrolyzed to 6o, which was also orally active. Compound 6o, 2-[6-[[3-[4-(diphenylmethoxy)piperidino]propyl]amino]imidazo[1, 2-b]pyridazin-2-yl]-2-methylpropionic acid dihydrate (TAK-427), having both antihistaminic and antiinflammatory activity, is currently undergoing clinical trials as a therapeutic agent for atopic dermatitis and allergic rhinitis.

Inhibition of allergic dermal inflammation by the novel imidazopyridazine derivative TAK-427 in a guinea pig experimental model of eczema.

Antigen challenge by patch ovalbumin emulsion induced an eczema-like skin lesion in epicutaneously sensitized guinea pigs. Diseased skin sites were macroscopically characterized by manifestations of dermatitis, such as erythema, edema, and papules, and microscopically characterized by acanthosis, spongiosis, and dermal infiltration by eosinophils. Using such lesions as a model of eczema, we evaluated the potential value of TAK-427 [2-[6-[[3-[4-(diphenylmethoxy)piperidino]propyl]amino] imidazo[1,2-b]pyridazin-2-yl]-2-methylpropionic acid dihydrate] as a therapeutic agent for atopic dermatitis by comparing it with dexamethasone and antihistamines. TAK-427 (0.3-30 mg/kg, p.o.) and dexamethasone (3 and 10 mg/kg, p.o.) inhibited eosinophil infiltration into the skin and ameliorated the dermatitis manifestations and epidermal damage. By contrast, none of the antihistamines tested (azelastine, ketotifen, terfenadine, and cetirizine) suppressed the eosinophil infiltration or dermatitis manifestations. To elucidate the mechanism by which TAK-427 inhibited the development of eczema, we investigated cytokine expression in the affected skin. Both TAK-427 and dexamethasone suppressed the increased mRNA expression of interleukin (IL)-13, granulocyte-macrophage colony-stimulating factor, IL-1alpha, tumor necrosis factor-alpha, interferon-gamma, and IL-8, but not IL-10, suggesting that TAK-427 inhibits allergic inflammation of the skin leading to the development of eczema by inhibiting the expression of proinflammatory cytokines after antigen challenge.

Increased expression of the human Ca2+-activated Cl- channel 1 (CaCC1) gene in the asthmatic airway.

Mucus overproduction is a clinical feature of asthma. Ca2+-activated Cl- channel 1 (CaCC1) has been identified as a protein that is expressed in intestinal epithelia and that plays an important role in fluid and electrolyte transport. Recently, its mouse counterpart, gob-5, was identified as a key molecule in the induction of murine asthma through mucus overproduction. To elucidate the relationship of CaCC1 to human asthma, we examined CaCC1 expression using real-time quantitative polymerase chain reaction analysis in bronchial tissues from patients with asthma and normal control subjects. The expression of CaCC1 was significantly upregulated in patients with bronchial asthma compared with control subjects. In situ hybridization and immunohistochemical analysis demonstrated that CaCC1 is located in the bronchial epithelium, especially in mucus-producing goblet cells. In vitro transfection of a CaCC1 expression vector into the human mucoepidermoid cell line, NCI-H292, increased mucus production and induced the MUC5AC gene. These results suggest that CaCC1 plays a direct role in mucus production and differentiation in goblet cells and may contribute to the pathogenesis of asthma through its mucus-inducing activity.