Abstract Background The development of anti-TNFα immunotherapies has led to great advances in quality of life for Crohn’s and colitis patients and has allowed a reduction in steroid use for these diseases. However, a significant proportion of IBD patients is resistant to anti-TNFα agents, or becomes resistant to such treatments over time. Preclinical research and clinical trials have suggested that CCR9 antagonism can effectively reduce colon inflammation. CCR9 is expressed on a subset of T cells dedicated to monitoring the immune status of the intestine. When activated by its only known chemokine ligand, CCL25/TECK, CCR9 triggers the adhesion of gut-specific T cells to vascular endothelium and the subsequent ingress of these cells to intestinal tissues. Antagonism of this receptor inhibits the accumulation of T cells within inflamed intestinal epithelium and lamina propria. Since anti-TNFα and CCR9 antagonists act through entirely different mechanisms, we asked whether these two approaches might synergise and expand the effectiveness and scope of each therapy. Methods We used a piroxicam-accelerated version of the MDR1a−/− spontaneous colitis model in mice. In this model, 7week MDR1a−/− mice (or WT FVB controls) receive piroxicam in powdered chow for 10 days. Mice are monitored both during and after the piroxicam feeding period, for a total of 21 days. Treatment groups (eight mice each) include an antibody (a saturating dose of either anti-TNFα or its isotype-matched control) and a CCR9 antagonist (or its vehicle alone as a control). Body weight and diarrhoea score were recorded throughout the entire 21 days period, after which colons were obtained from sacrificed animals for weighing, length measurement and imaging. Results We have found that combined dosing of piroxicam-fed MDR1a−/− mice with a CCR9 antagonist and an anti-TNFα MAb significantly reverses loss of body weight, diarrhoea score and colon inflammation. The combined dosing was more effective than anti-TNFα or CCR9 antagonist alone, as measured by colon length/weight ratio, neutrophil accumulation in the colonic epithelium and reversal of the loss in body weight characteristic of this model. Conclusion These data suggest that combining a CCR9 antagonist with existing anti-TNFα treatment regimens may extend the benefits of anti-TNFα immunotherapy to a larger class of patients suffering from IBD and may prolong the effectiveness of anti-TNFα immunotherapy for those patients already undergoing such treatments.
e13580 Background: Glioblastoma (GBM) is the most common form of malignant brain cancer. Despite aggressive therapy, consisting of radiotherapy, surgical resection, and chemotherapeutic treatment, mean survival from time of diagnosis remains little more than one year. The chemokine receptor CXCR7 is highly expressed on both human glioma cells and tumor-associated vasculature, and may play a key role in tumor growth and survival. Methods: The in vitro activity of the CXCR7 antagonist CCX662 was determined using radioligand binding assays and trans-endothelial migration assays. The therapeutic effects of CCX662 were investigated in the ENU-induced rat model of GBM in combination with radiotherapy. Results: CCX662 is a highly potent and selective small molecule inhibitor of CXCR7. CCX662 inhibits the binding of 125 I-CXCL12 to CXCR7 with an IC 50 of 9 nM in buffer, and displays minimal serum shift with an IC 50 of 18 nM in 100% human serum. CCX662 inhibits the CXCR4-directed trans-endothelial migration of CXCR4+/CXCR7+ NC37 cells towards CXCL12 (SDF1) with an IC 50 of 106 nM. CCX662 also potently inhibits binding of 125 I-CXCL12 to rat CXCR7 in the presence of 100% rat serum with an IC 50 of 14 nM. In vivo inhibition of CXCR7 with CCX662, in concert with radiotherapy, results in a significant extension of survival time in the ENU-induced rat model of GBM. The median survival time for rats (reflective of GBM tumor progression) treated with the combination of irradiation and CCX662 was 234 days compared with 160 days for untreated rats (p<0.01) or compared with 174 days for rats given irradiation plus vehicle (p<0.01). As expected for a non-cytotoxic drug, dose-range finding 14-day toxicology studies in rats and dogs have demonstrated large (> 10 and 25-fold, respectively) safety margin relative to highly effective levels of the drug. Conclusions: CCX662 is a highly selective and potent small molecule inhibitor of CXCR7 with profound, therapeutic benefit in an aggressive rodent model of GBM. These data indicate that inhibition of CXCR7, using CCX662, may be a promising strategy for the treatment of glioblastoma.
Chemokine (C-C motif) receptor 2 (CCR2) is central for the migration of monocytes into inflamed tissues. The novel CCR2 antagonist CCX140-B, which is currently in two separate phase 2 clinical trials in diabetic nephropathy, has recently been shown to reduce hemoglobin A1c and fasting blood glucose levels in type 2 diabetics. In this report, we describe the effects of this compound on glycemic and renal function parameters in diabetic mice. Since CCX140-B has a low affinity for mouse CCR2, transgenic human CCR2 knockin mice were generated and rendered diabetic with either a high-fat diet (diet-induced obesity) or by deletion of the leptin receptor gene (db/db). CCX140-B treatment in both models resulted in decreased albuminuria, which was associated with decreased glomerular hypertrophy and increased podocyte density. Moreover, treatment of diet-induced obese mice with CCX140-B resulted in decreased levels of fasting blood glucose and insulin, normalization of homeostatic model assessment of insulin resistance values, and decreased numbers of adipose tissue inflammatory macrophages. Unlike other CCR2 antagonists, CCX140-B had no effect on plasma levels of the CCR2 ligand CCL2 or on the numbers of blood monocytes. These results support the ongoing evaluation of this molecule in diabetic subjects with impaired renal function.
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 Crohn9s 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.
The anaphylatoxin C5a and its receptor C5aR (CD88) are complement pathway effectors implicated in renal diseases, including ANCA-associated vasculitis. We investigated the kidney expression of C5aR and a second C5a receptor C5L2 by using immunohistochemistry and in situ hybridization on formalin-fixed, paraffin-embedded human and mouse kidney. C5aR was detected on interstitial macrophages and in multiple tubular regions, both distal and proximal; C5L2 had a similar expression pattern. The 5/6 nephrectomy model of chronic kidney injury exhibited increased C5aR expression by infiltrating cells within the fibrotic regions. Functional assessment of myeloid C5aR in vitro revealed that C5a induced the expression of chemokines and remodeling factors by macrophages, including CCL-3/-4/-7,-20, MMP-1/-3/-8/-12, and F3, and promoted survival by blocking neutrophil apoptosis. C5a activity was C5aR dependent, as demonstrated by reversal with the C5aR inhibitor avacopan. Collectively, these results suggest that myeloid C5aR may induce excessive inflammation in the kidney via immune cell recruitment, extracellular matrix destruction, and remodeling, resulting in fibrotic tissue deposition.
Necrotizing and crescentic GN (NCGN) with a paucity of glomerular immunoglobulin deposits is associated with ANCA. The most common ANCA target antigens are myeloperoxidase (MPO) and proteinase 3. In a manner that requires activation of the alternative complement pathway, passive transfer of antibodies to mouse MPO (anti-MPO) induces a mouse model of ANCA NCGN that closely mimics human disease. Here, we confirm the importance of C5aR/CD88 in the mediation of anti-MPO-induced NCGN and report that C6 is not required. We further demonstrate that deficiency of C5a-like receptor (C5L2) has the reverse effect of C5aR/CD88 deficiency and results in more severe disease, indicating that C5aR/CD88 engagement enhances inflammation and C5L2 engagement suppresses inflammation. Oral administration of CCX168, a small molecule antagonist of human C5aR/CD88, ameliorated anti-MPO-induced NCGN in mice expressing human C5aR/CD88. These observations suggest that blockade of C5aR/CD88 might have therapeutic benefit in patients with ANCA-associated vasculitis and GN.
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.
