Structure-activity relationship study, target identification, and pharmacological characterization of a small molecular IL-12/23 inhibitor, APY0201.

Interleukin-12 (IL-12) and IL-23 are proinflammatory cytokines and therapeutic targets for inflammatory and autoimmune diseases, including inflammatory bowel diseases, psoriasis, rheumatoid arthritis, and multiple sclerosis. We describe the discovery of APY0201, a unique small molecular IL-12/23 production inhibitor, from activated macrophages and monocytes, and demonstrate ameliorated inflammation in an experimental model of colitis. Through a chemical proteomics approach using a highly sensitive direct nanoflow LC-MS/MS system and bait compounds equipped with the FLAG epitope associated regulator of PIKfyve (ArPIKfyve) was detected. Further study identified its associated protein phosphoinositide kinase, FYVE finger-containing (PIKfyve), as the target protein of APY0201, which was characterized as a potent, highly selective, ATP-competitive PIKfyve inhibitor that interrupts the conversion of phosphatidylinositol 3-phosphate (PtdIns3P) to PtdIns(3,5)P2. These results elucidate the function of PIKfyve kinase in the IL-12/23 production pathway and in IL-12/23-driven inflammatory disease pathologies to provide a compelling rationale for targeting PIKfyve kinase in inflammatory and autoimmune diseases.

Oral treatment with a novel small molecule alpha 4 integrin antagonist, AJM300, prevents the development of experimental colitis in mice.

BACKGROUND AND AIMS: Inhibition of lymphocyte trafficking by treatment with an anti-α4 integrin antibody has been clinically validated as a therapeutic approach for inflammatory bowel disease (IBD), and the orally effective 'anti-α4 integrin therapy' may be more convenient in clinical practice. The aim of this study was to investigate the pharmacological profile and anti-inflammatory effect of a novel, orally active small molecule α4 integrin antagonist, AJM300. METHODS: The binding specificity/potency of HCA2969 (the active metabolite of AJM300) were investigated in vitro. The pharmacodynamics for α4 integrin antagonism of AJM300 was investigated in mice. The anti-inflammatory effect of AJM300 fed in a diet and the anti-α4 integrin monoclonal antibody was evaluated in a mouse colitis model induced by transfer of IL-10 deficient T cells. RESULTS: HCA2969 selectively inhibited the in vitro binding of α4 integrin (α4ß7/α4ß1) to the cell adhesion molecules. Oral treatment with AJM300 dose-dependently inhibited lymphocyte homing to Peyer's patches and increased the peripheral lymphocyte count in the same dose range. AJM300 dose-dependently prevented the development of experimental colitis in mice. A significant inhibition of colon weight increase was accompanied by inhibition of T-cell infiltration into the lamina propria of colon. The maximum efficacy of AJM300 (1% diet) was comparable to that achieved by the saturated α4 integrin blockade with antibody. CONCLUSIONS: Oral treatment with the selective small molecule α4 integrin antagonist (AJM300) prevented the development of colitis and its efficacy was comparable to that of the anti-α4 integrin antibody.

Periductal induction of high endothelial venule-like vessels in type 1 autoimmune pancreatitis.

OBJECTIVES: Type 1 autoimmune pancreatitis (AIP) is histologically characterized by dense lymphoplasmacytic infiltration and marked storiform fibrosis, manifestations associated with pancreatic ducts. Such periductal lymphocyte recruitment is thought to be elicited by dysregulation of mechanisms governing physiological lymphocyte homing. The present study was undertaken to determine whether vascular addressins including peripheral lymph node addressin and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) play a role in type 1 AIP histogenesis. METHODS: Tissue sections of type 1 AIP and tumor-associated non-AIP chronic pancreatitis, as well as normal pancreas, were subjected to immunohistochemical analysis using vascular addressin-related antibodies. RESULTS: The number of periductal mouse endothelial cell antigen 79-positive high endothelial venule (HEV)-like vessels was increased in type 1 AIP relative to that seen in non-AIP chronic pancreatitis, whereas the number of MAdCAM-1-positive HEV-like vessels did not differ between the 2 conditions. Mouse endothelial cell antigen 79 antigens are expressed on duct-forming epithelial cells not only in pancreas but also in salivary glands, which often harbor extrapancreatic lesions in type 1 AIP. CONCLUSIONS: Type 1 AIP can be characterized by periductal induction of MECA-79-positive HEV-like vessels. MECA-79-positive 6-sulfo sialyl Lewis X-related carbohydrate antigens expressed on duct-forming epithelial cells could be associated with type 1 AIP pathogenesis.

Lymphocyte recruitment via high endothelial venules in lymphoid stroma of Warthin's tumour.

AIMS: Warthin's tumour is composed of bilayered oncocytic epithelium and organised lymphoid stroma, which resembles mucosa-associated lymphoid tissue (MALT); however, the histogenesis of the lymphoid stroma is not fully understood. We hypothesised that lymphocytes consisting of the stroma are recruited via high endothelial venules (HEVs) by the mechanism operating in normal lymphocyte homing in secondary lymphoid organs. The aim of this study was to determine immunohistochemically the molecules expressed on these HEVs. METHODS: Tissue sections of Warthin's tumour (n = 10) were immunostained for vascular addressin-related antigens including peripheral lymph node addressin (PNAd) and mucosal addressin cell adhesion molecule 1 (MAdCAM-1). An L-selectin·IgM chimera in situ binding assay was also carried out. Triple immunostaining for PNAd, CD3, and CD20/CD79α was performed to determine which lymphocyte subsets are closely associated with these HEVs. RESULTS: HEVs in the lymphoid stroma of Warthin's tumour express PNAd, which is detected by MECA-79 as well as recently developed monoclonal antibodies S1 and S2. These HEVs were bound by L-selectin·IgM chimeras in a calcium-dependent manner, and numbers of lymphocytes, particularly T cells, attached to these HEVs. CONCLUSIONS: The lymphoid stroma of Warthin's tumour is most likely developed by lymphocytes recruited via HEVs.

GlcNAc6ST-1-mediated decoration of MAdCAM-1 protein with L-selectin ligand carbohydrates directs disease activity of ulcerative colitis.

BACKGROUND: A diffuse lymphocyte infiltrate is 1 of the characteristic features of ulcerative colitis (UC). Such lymphocyte recruitment requires lymphocyte rolling mediated by L-selectin ligand carbohydrates (6-sulfo sialyl Lewis X-capped O-glycans) and/or mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expressed on high endothelial venule (HEV)-like vessels. The present study was undertaken to elucidate the role of MAdCAM-1 posttranslationally modified ("decorated") with L-selectin ligand carbohydrates in UC pathogenesis and consequent clinical outcomes. METHODS: Biopsy specimens composed of active and remission phases of UC as well as normal colonic mucosa were immunostained for CD34, MAdCAM-1, and MECA-79, and the immunostained sections were quantitatively analyzed. Reverse-transcriptase polymerase chain reaction (RT-PCR) was carried out to evaluate transcripts of MAdCAM-1 and N-acetylglucosamine-6-O-sulfotransferases (GlcNAc6STs). CHO and Lec2 cells transfected with CD34 and MAdCAM-1 together with enzymes involved in L-selectin ligand carbohydrate biosynthesis were analyzed by immunofluorescence, FACS, and Western blotting to characterize the biochemical properties of GlcNAc6STs. RESULTS: The number of MAdCAM-1(+) vessels was increased in UC, with no significant difference between active and remission phases. An increased ratio of MECA-79(+) to MAdCAM-1(+) vessels with preferential GlcNAc6ST-1 transcripts was observed in the active phase of UC compared to the remission phase. MAdCAM-1 protein was colocalized with L-selectin ligand carbohydrates at the luminal surface of HEV-like vessels in situ. GlcNAc6ST-1 preferentially utilizes MAdCAM-1 as a scaffold protein for GlcNAc-6-O-sulfation in L-selectin ligand carbohydrate biosynthesis. CONCLUSIONS: UC disease activity is not regulated by expression of MAdCAM-1 protein itself, but rather by GlcNAc6ST-1-mediated decoration of MAdCAM-1 protein with L-selectin ligand carbohydrates.

Effect of a humanized monoclonal antibody to von Willebrand factor in a canine model of coronary arterial thrombosis.

The purpose of this study was to investigate the antithrombotic effect and bleeding time prolongation of AJW200, a humanized monoclonal antibody to von Willebrand factor (vWF), in a canine model of coronary arterial thrombosis. AJW200 significantly inhibited cyclic flow reductions, as well as botrocetin-induced platelet aggregation, at 0.1 mg/kg. A significant prolongation of bleeding time was observed at 0.3-1 mg/kg. Approximately 50% occupancy of vWF (approximately 0.7 microg/ml AJW200 in plasma) and 80-100% occupancy (approximately 20 microg/ml AJW200 in plasma) were needed for the antithrombotic effect and the extensive prolongation of bleeding time, respectively. On the contrary, the minimal effective dose of abciximab (0.8 mg/kg) was associated with a significant prolongation of bleeding time. These results suggest that the pharmacological blockade of platelet glycoprotein (GP) Ib-vWF interaction with AJW200 results in a safer antithrombotic profile than platelet GPIIb/IIIa blockade with abciximab in dogs.

Pharmacokinetics and pharmacodynamics of AJW200, a humanized monoclonal antibody to von Willebrand factor, in monkeys.

The interaction between platelet glycoprotein Ib and von Willebrand factor (vWF) plays a crucial role in platelet-mediated thrombus formation under high-shear-stress conditions. The aim of this study was to investigate the antiplatelet profile of a humanized anti-vWF monoclonal antibody, AJW200. In vitro studies were performed with a modified cone-and-plate viscometer and human platelets. AJW200 inhibited high-shear-stress-induced platelet adhesion, aggregation, and thrombin generation, but it did not have such effects under low-shear-stress conditions. Although abciximab inhibited platelet aggregation under both shear stress conditions, it did not inhibit platelet adhesion and thrombin generation. In addition, the pharmacokinetics and pharmacodynamics of AJW200 were evaluated in cynomolgus monkeys. Sustained inhibition of ristocetin-induced platelet aggregation was observed over 24 hours, 6 days, and 2 weeks after a single bolus injection of 0.3, 1, and 3 mg/kg, respectively. Moderate prolongation of the bleeding time was observed at the doses of 1 and 3 mg/kg. Abciximab markedly prolonged the bleeding time at 0.4 mg/kg, at which concentration complete inhibition of ADP-induced platelet aggregation was observed. These results suggest that glycoprotein Ib-vWF blockade with AJW200 results in a sustained antiplatelet effect without extensive prolongation of the bleeding time, probably due to a shear-stress-dependent inhibitory action.