Matthew J. Walters

Characterization of CCX282-B, an Orally Bioavailable Antagonist of the CCR9 Chemokine Receptor, for Treatment of Inflammatory Bowel Disease

The chemokine system represents a diverse group of G protein-coupled receptors responsible for orchestrating cell recruitment under both homeostatic and inflammatory conditions. Chemokine receptor 9 (CCR9) is a chemokine receptor known to be central for migration of immune cells into the intestine. Its only ligand, CCL25, is expressed at the mucosal surface of the intestine and is known to be elevated in intestinal inflammation. To date, there are no reports of small-molecule antagonists targeting CCR9. We report, for the first time, the discovery of a small molecule, CCX282-B, which is an orally bioavailable, selective, and potent antagonist of human CCR9. CCX282-B inhibited CCR9-mediated Ca2+ mobilization and chemotaxis on Molt-4 cells with IC50 values of 5.4 and 3.4 nM, respectively. In the presence of 100% human serum, CCX282-B inhibited CCR9-mediated chemotaxis with an IC50 of 33 nM, and the addition of α1-acid glycoprotein did not affect its potency. CCX282-B inhibited chemotaxis of primary CCR9-expressing cells to CCL25 with an IC50 of 6.8 nM. CCX282-B was an equipotent inhibitor of CCL25-directed chemotaxis of both splice forms of CCR9 (CCR9A and CCR9B) with IC50 values of 2.8 and 2.6 nM, respectively. CCX282-B also inhibited mouse and rat CCR9-mediated chemotaxis. Inhibition of CCR9 with CCX282-B results in normalization of Crohns disease such as histopathology associated with the TNFΔARE mice. Analysis of the plasma level of drug associated with this improvement provides an understanding of the pharmacokinetic/pharmacodynamic relationship for CCR9 antagonists in the treatment of intestinal inflammation.

Endothelial expression of CXCR7 and the regulation of systemic CXCL12 levels

The concentration of CXCL12/SDF‐1 in the bloodstream is tightly regulated, given its central role in leucocyte and stem/progenitor cell egress from bone marrow and recruitment to sites of inflammation or injury. The mechanism responsible for this regulation is unknown. Here we show that both genetic deletion and pharmacological inhibition of CXCR7, a high‐affinity CXCL12 receptor, caused pronounced increases in plasma CXCL12 levels. The rise in plasma CXCL12 levels was associated with an impairment in the ability of leucocytes to migrate to a local source of CXCL12. Using a set of complementary and highly sensitive techniques, we found that CXCR7 protein is expressed at low levels in multiple organs in both humans and mice. In humans, CXCR7 was detected primarily on venule endothelium and arteriole smooth muscle cells. CXCR7 expression on venule endothelium was also documented in immunodeficient mice and CXCR7+/lacZ mice. The vascular expression of CXCR7 therefore gives it immediate access to circulating CXCL12. These studies suggest that endothelial CXCR7 regulates circulating CXCL12 levels and that CXCR7 inhibitors might be used to block CXCL12‐mediated cell migration for therapeutic purposes.

Experimental evidence for the use of CCR2 antagonists in the treatment of type 2 diabetes

OBJECTIVE CCR2 inhibition has produced promising experimental and clinical anti-hyperglycemic effects. These results support the thesis that insulin resistance and Type 2 diabetes (T2D) are associated with chronic unresolved inflammation. The aim of this study was to provide a broad analysis of the various physiological changes occurring in mouse models of T2D in connection with pharmacological CCR2 inhibition. MATERIALS/METHODS A mouse-active chemical analogue of the clinical candidate CCX140-B was tested in diet-induced obese (DIO) mice and db/db mice. Measurements included: adipose tissue inflammatory macrophage counts; peripheral blood glucose levels at steady-state and after glucose and insulin challenges; peripheral blood insulin and adiponectin levels; 24-h urine output and urinary glucose levels; pancreatic islet number and size; hepatic triglyceride and glycogen content; and hepatic glucose-6-phosphatase levels. RESULTS In DIO mice, the CCR2 antagonist completely blocked the recruitment of inflammatory macrophages to visceral adipose tissue. The mice exhibited reduced hyperglycemia and insulinemia, improved insulin sensitivity, increased circulating adiponectin levels, decreased pancreatic islet size and increased islet number. It also reduced urine output, glucose excretion, hepatic glycogen and triglyceride content and glucose 6-phosphatase levels. Similar effects were observed in the db/db diabetic mice. CONCLUSIONS These data indicate that pharmacological inhibition of CCR2 in models of T2D can reduce inflammation in adipose tissue, alter hepatic metabolism and ameliorate multiple diabetic parameters. These mechanisms may contribute to the promising anti-diabetic effects seen in humans with at least one CCR2 antagonist.

Abrogation of CC chemokine receptor 9 ameliorates collagen-induced arthritis of mice

IntroductionBiological drugs are effective in patients with rheumatoid arthritis (RA), but increase severe infections. The CC chemokine receptor (CCR) 9 antagonist was effective for Crohn’s disease without critical adverse effects including infections in clinical trials. The present study was carried out to explore the pathogenic roles of chemokine (C-C motif) ligand (CCL) 25 and its receptor, CCR9, in autoimmune arthritis and to study if the CCR9 antagonist could be a new treatment for RA.MethodsCCL25 and CCR9 expression was examined with immunohistochemistry and Western blotting. Concentration of interleukin (IL)-6, matrix metalloproteinase (MMP)-3 and tumor necrosis factor (TNF)-α was measured with enzyme-linked immunosorbent assays. Effects of abrogating CCR9 on collagen-induced arthritis (CIA) was evaluated using CCR9-deficient mice or the CCR9 antagonist, CCX8037. Fluorescence labeled-CD11b+ splenocytes from CIA mice were transferred to recipient CIA mice and those infiltrating into the synovial tissues of the recipient mice were counted.ResultsCCL25 and CCR9 proteins were found in the RA synovial tissues. CCR9 was expressed on macrophages, fibroblast-like synoviocytes (FLS) and dendritic cells in the synovial tissues. Stimulation with CCL25 increased IL-6 and MMP-3 production from RA FLS, and IL-6 and TNF-α production from peripheral blood monocytes. CIA was suppressed in CCR9-deficient mice. CCX8037 also inhibited CIA and the migration of transferred CD11b+ splenocytes into the synovial tissues.ConclusionsThe interaction between CCL25 and CCR9 may play important roles in cell infiltration into the RA synovial tissues and inflammatory mediator production. Blocking CCL25 or CCR9 may represent a novel safe therapy for RA.

CCR9 inhibition does not interfere with the development of immune tolerance to oral antigens

Recent literature indicates that mice deficient in the chemokine receptor CCR9 (CCR9(-/-) mice) are unable to generate oral tolerance. The present report describes how such inability can be overcome by increasing the dose of oral antigen. Pharmacological inhibition of CCR9 did not affect the generation of oral tolerance, regardless of antigen dose. These results highlight the inadequacy of genetic deletion of CCR9 when predicting the effects of pharmacological CCR9 inhibition on intestinal biology.

A Systemically-Administered Small Molecule Antagonist of CCR9 Acts as a Tissue-Selective Inhibitor of Lymphocyte Trafficking

A goal for developers of immunomodulatory drugs has long been a systemically administered small molecule that can selectively inhibit inflammation in specific tissues. The chemokine receptor CCR9 is an attractive target for this approach, as entry of T cells into the small intestine from blood requires interaction between CCR9 and its ligand CCL25. We have tested the ability of a small molecule CCR9 antagonist, CCX8037, to inhibit antigen-mediated T cell accumulation in the intestine. This compound prevented accumulation of gut-imprinted antigen-specific CD8 T cells within epithelium of the small intestine. Interestingly, the antagonist did not affect the robust generation of gut-imprinted CD8 T cells within mesenteric lymph nodes. To distinguish “gut-selective” from “general” T cell inhibition, we tested the drug’s ability to influence accumulation of T cells within skin, a tissue in which CCR9 plays no known role, and we found no appreciable effect. This study demonstrates the feasibility of creating systemically-administered pharmaceuticals capable of tissue-selective immune modulation. This proof of concept is of utmost importance for designing effective treatments against various autoimmune disorders localized to a specific tissue.

CCR9 Antagonists in the Treatment of Ulcerative Colitis

While it has long been established that the chemokine receptor CCR9 and its ligand CCL25 are essential for the movement of leukocytes into the small intestine and the development of small-intestinal inflammation, the role of this chemokine-receptor pair in colonic inflammation is not clear. Toward this end, we compared colonic CCL25 protein levels in healthy individuals to those in patients with ulcerative colitis. In addition, we determined the effect of CCR9 pharmacological inhibition in the mdr1a −/− mouse model of ulcerative colitis. Colon samples from patients with ulcerative colitis had significantly higher levels of CCL25 protein compared to healthy controls, a finding mirrored in the mdr1a −/− mice. In the mdr1a −/− mice, CCR9 antagonists significantly decreased the extent of wasting and colonic remodeling and reduced the levels of inflammatory cytokines in the colon. These findings indicate that the CCR9:CCL25 pair plays a causative role in ulcerative colitis and suggest that CCR9 antagonists will provide a therapeutic benefit in patients with colonic inflammation.

Therapeutic use of a clinical stage CCR2 inhibitor, CCX872, in obesity-associated steatohepatitis

Abstract Background Non-alcoholic fatty liver disease (NAFLD) is a common condition, in which hepatic steatosis is present in up to a third of individuals, whereas the more severe non-alcoholic steatohepatitis (NASH) is seen in about 10%. NAFLD is closely associated with the metabolic syndrome: up to 75% of people with diabetes mellitus, and almost all morbidly obese individuals, have NALFD. Obesity-associated macrophage infiltration of adipose and hepatic tissue is mediated by C-C chemokine receptor type 2 (CCR2), where CCR2+ CD11b+ F4/80+ macrophages contribute to chronic inflammation and insulin resistance. We investigated the efficacy of a small molecule inhibitor of CCR2, CCX872, which is in phase 1 clinical development, for treatment of obesity-associated hepatic steatohepatitis in mice. Methods C57BL/6 mice aged 6 weeks were fed a high-fat diet for 16 weeks. After 8 weeks on this diet, mice were treated with CCX872 (ChemoCentryx, Mountain View, CA, USA) 30 mg/kg once daily or vehicle, both administered subcutaneously. Liver injury was assessed with serum alanine aminotransferase concentration, liver triglyceride content, and flow cytometry of infiltrating cells. Adipose tissue macrophage infiltration was assessed with flow cytometry. Insulin sensitivity was measured by glucose and insulin challenges. Groups were compared with two-tailed Students t tests. Findings Mice treated with CCX872 had better insulin sensitivity (p vs 532·8 [353·6], p=0·028). Hepatic triglyceride accumulation was significantly lower in CCX872-treated mice (mean 159·9 mg/g [SD 2·6] vs 296·6 [98·5], p vs 16·0 [1·9], p=0·03; mean fluorescence intensity [MFI] 6919 [SD 107·7] vs 7365 [162·9], p=0·04) and in omental adipose tissue (9·2% of CD45+ cells [1·9] vs 21·8 [3·7], p=0·02; MFI 6212 [469·2] vs 7952 [379·5], p=0·02). Interpretation Treatment with a clinical-stage small molecule inhibitor of CCR2 improved steatohepatitis and insulin sensitivity in mice kept on a high-fat diet. Funding UK Medical Research Council.

CC Chemokine Receptor 2 Promotes Recruitment of Myeloid Cells Associated with Insulin Resistance in Non-Alcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is a common disease, closely associated with obesity and insulin resistance. We investigated the presence of a subset of myeloid cells associated with metabolic disturbance in the liver of patients with NAFLD and a murine model of obesity-induced liver disease. Gene and protein expression in liver and serum was investigated with RT-PCR or ELISA and correlated to clinical disease. Liver-infiltrating immune cells were isolated from normal or diseased human liver for flow cytometric analysis. In animal experiments, mice were fed a high-fat diet (60% of calories from fat) for 16 wk, or high-fat diet with 30% fructose for 32 wk to induce steatohepatitis and fibrosis. A small molecule inhibitor of CC chemokine receptor 2 (CCR2), CCX872, was administered to some mice. A subset of CD11c+CD206+ immune cells was enriched in human liver tissue, and greater infiltration was observed in NAFLD. The presence of CD11c+CD206+ myeloid cells correlated with systemic insulin resistance. CD11c+CD206+ cells expressed high levels of CCR2, and liver CC chemokine ligand 2 (CCL2) expression was increased in nonalcoholic steatohepatitis and correlated with disease activity. In mice, CCR2 inhibition reduced infiltration of liver CD11b+CD11c+F4/80+ monocytes, which are functional homologs of human CD11c+CD206+ cells, and improved liver injury and glycemic control. A role for CCR2/CCL2 in human NAFLD has long been postulated. These data confirm a role for this chemokine/receptor axis, through mediating adipose and hepatic infiltration of myeloid cells. Inhibition of CCR2 improved hepatic inflammation and fibrosis in murine models of NAFLD. These data confirm the rationale for targeting CCR2 to treat NAFLD. NEW & NOTEWORTHY These data show for the first time that CD11c+CD206+ myeloid cells, previously associated with human adipose tissue inflammation, infiltrate into liver tissue in nonalcoholic fatty liver disease. These cells express CCR2. Inhibition of CCR2 in mice inhibits hepatic inflammation caused by a murine homolog of these myeloid cells and improves experimental liver disease.

Abstract 4290: Potent and selective next generation inhibitors of indoleamine-2,3-dioxygenase (IDO1) for the treatment of cancer

The IDO1 pathway has been proposed to mediate immunosuppressive effects in the tumor microenvironment through its role in the catabolism of tryptophan, resulting in effects on the differentiation and proliferation of T cells. IDO1 inhibition has shown promising clinical benefit as well as exacerbated toxicity in the treatment of melanoma, when combined with the anti-CTLA-4 antibody ipilimumab. We have discovered a novel class of highly selective small molecule inhibitors of IDO1 which surpass the potency of the compounds currently in clinical development. These compounds potently inhibit IDO1 activity in IFN-γ stimulated HeLa cells with single digit nM potency. Importantly, they also retain their potency in the presence of human serum, with IC50 values ranging between 5 and 15 nM in this more physiologically relevant media. Consistent with the role of IDO1+ dendritic cells in the suppression of T cell proliferation, this series of molecules is capable of restoring the proliferative capacity of human T cells (which is inhibited by allogeneic IDO1+ dendritic cells) with EC50 values of 2-3 nM. The molecules exhibit preclinical PK characteristics that are suitable for assessing the contribution of IDO1 to tumor growth in murine models, both alone and in combination with other therapeutic agents. The compounds have high metabolic stability against cultured human hepatocytes and exhibit preclinical PK and ADME characteristics consistent with once-daily dosing in humans. The full preclinical profile of one of these molecules, selected for clinical evaluation, will be the focus of this presentation. In conclusion, we have discovered a novel class of small molecule inhibitors of IDO1, which provides a preclinical basis for the clinical evaluation of a next generation IDO1 inhibitor in combination with other therapeutic agents. Citation Format: Jay P. Powers, Matthew J. Walters, Rajkumar Noubade, Stephen W. Young, Lisa Marshall, Jan Melom, Adam Park, Nick Shah, Pia Bjork, Jordan S. Fridman, Hilary P. Beck, David Chian, Jenny V. McKinnell, Maksim Osipov, Maureen K. Reilly, Hunter P. Shunatona, James R. Walker, Mikhail Zibinsky, Juan C. Jaen. Potent and selective next generation inhibitors of indoleamine-2,3-dioxygenase (IDO1) for the treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4290. doi:10.1158/1538-7445.AM2015-4290