CC Chemokine Receptor 2 Antagonist Research Articles

CC chemokine receptor 2 (CCR2) expression promotes diffuse large B-Cell lymphoma survival and invasion

In recent years, CC chemokine receptor 2 (CCR2) has been found to be involved in tumor growth, angiogenesis, epithelial mesenchymal transition, metastasis, and immune escape. CCR2 overexpression was first identified as a poor prognostic predictor in diffuse large B-cell lymphoma (DLBCL) in our published article, but the mechanisms involved remain unknown. In this work, we collected data from another 138 patients with DLBCL data and verified the CCR2 expression level and its relationship to clinicopathological characteristics. Furthermore, we explored the possible mechanisms via in vitro and in vivo experiments. We showed that CCR2 overexpression was an independent prognostic marker and predicted shorter overall survival (OS) and progression-free survival (PFS) in patients with DLBCL. Blockade of CCR2 expression with a CCR2 antagonist inhibited tumor cell proliferation, migration, and anti-apoptosis ability in vitro by affecting the PI3K/Akt signaling pathway and the p38 MAPK signaling pathway. Furthermore, administration of a CCR2 antagonist decreased tumor growth and dissemination of DLBCL cells and increased survival time in the xenograft model. Our study demonstrates that CCR2 expression plays an important role in the development of DLBCL by stimulating cell proliferation, migration, and anti-apoptosis. Therefore, the inhibition of CCR2 may be a potential target for anticancer therapy in DLBCL.The authors show that CC chemokine receptor 2 (CCR2) overexpression is an independent prognostic marker for diffuse large B-cell lymphoma (DLBCL) and predicts overall survival and progression-free survival in these patients. Blockade of CCR2 signaling with a CCR2 antagonist inhibits tumor cell proliferation, migration, and apoptosis inhibition by activating the PI3K/Akt signaling pathway and inhibiting the p38 MAPK signaling pathway. Furthermore, administration of a CCR2 antagonist decreases tumor growth and dissemination of DLBCL cells and increases survival time in a xenograft model.

Monocyte Chemotactic Protein-1 (MCP1) Accumulation in Human Osteoclast Precursor Cultures.

In vitro osteoclast methods require constant treatment with macrophage colony stimulating factor (M-CSF) to support precursor survival and addition of the differentiation agent receptor activator of NF-κB ligand (RANKL). Constant exposure to granulocyte macrophage colony stimulating factor (GM-CSF) suppresses human osteoclast formation in vitro. Addition of the chemokine monocyte chemotactic protein-1 (MCP1) to such cultures dramatically increases osteoclast formation and overcomes GM-CSF mediated suppression. We investigated the effect of M-CSF, GM-CSF and the combination of M-CSF and GM-CSF treatment on the expression of chemokines in human CD14+ cells in culture. Of assayed chemokines, MCP1 was the most abundant in terms of mRNA transcript and protein in M-CSF treated cultures and was suppressed by GM-CSF. MCP1 protein accumulated up to 50 ng/mL in culture medium, greatly exceeding other assayed chemokines. C-C chemokine receptor-2 (CCR2) is the receptor for MCP1: the formation of osteoclast-like cells was inhibited by constant exposure to the CCR2 antagonist RS102895, in part by decreasing expression of RANK, the receptor for RANKL.

Pharmacological Inhibition of CCR2 Signaling Exacerbates Exercise-Induced Inflammation Independently of Neutrophil Infiltration and Oxidative Stress

Although exercise-induced humoral factors known as exerkines benefit systemic health, the role of most exerkines has not been investigated. Monocyte chemoattractant protein-1 (MCP-1) is a representative chemokine whose circulating concentrations increase after exercise, and it is one of the exerkines. MCP-1 is a ligand for CC chemokine receptor 2 (CCR2), which is expressed on monocytes, macrophages, and muscle cells. However, there is no information on the role of CCR2 signaling in exercise. Therefore, to investigate the research question, we administrated CCR2 antagonist or PBS to mice to inhibit CCR2 signaling before and after exercise. Our results showed that CCR2 signaling inhibition promoted exercise-induced macrophage infiltration and inflammation 24 h after exercise in muscle. CCR2 signaling inhibition also exacerbated exercise-induced inflammation immediately after exercise in muscle. However, neutrophil infiltration and oxidative stress had no contribution to exercise-induced inflammation by CCR2 signaling inhibition. CCR2 signaling inhibition also exacerbated exercise-induced inflammation immediately after exercise in kidney, liver, and adipose tissues. To summarize, pharmacological inhibition of CCR2 signaling exacerbated exercise-induced inflammation independently of neutrophil infiltration and oxidative stress.

CCR2 Expression Promotes Diffuse Large B Lymphoma Cell Survival and Invasion

Objective: Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma world wide. It is a phenotypically and genetically heterogeneous disease, accounting for 30-40% of all cases. 50%-70% of patients can be cured by the R-CHOP regimen, but nearly one-third of patients develop relapsed or refractory disease. CC chemokine receptor 2 (CCR2), the high affinity receptor of CC-Chemokine Ligand 2 (CCL2), which is the most representative of the CC chemokine family members, has be regarded to involve in tumor growth, angiogenesis, epithelial mesenchymal transition, metastasis and immune escape etc.. In recent years, the role and mechanism in DLBCL has not been reported yet. Our preliminary study showed that high expression of CCR2 was correlated with clinicopathological characteristics, and an adverse prognostic factor for overall survival (OS) and progression-free survival (PFS) of DLBCL patients. The purpose of this study is to investigate the role of CCR2 expression in DLBCL cells proliferation and migration by in vitro and in vivo.Methods: CCR2 expression were analyzed in human DLBCL cell lines (SUDHL-2, SUDHL-4, SUDHL-6, OCI-Ly8 and OCI-Ly10) by Western blot (WB). SUDHL-2 and OCI-Ly8 cells were incubated with CCR2 antagonist SC-202525 (Santa Crutz Biotechnology), and control cells were left untreated. The proliferation, migration, apoptosis and signaling pathway were detected by CCK8, transwell, flow cytometry (FC) and WB, respectively in vitro. The engraftment, tumor growth, dissemination and survival time were observed in BALB/c nude mice.Results: CCR2 were expressed in all human DLBCL cell lines (relative CCR2 expression was higher in SUDHL-2, SUDHL-4 and OCI-Ly8 than in SUDHL-6 and OCI-Ly10 cell lines). Blockade of CCR2 expression signaling with CCR2 antagonist inhibited tumor cell proliferation, migration and anti-apoptosis ability. The signaling involved in the proliferation and migration of DLBCL cells by activating PI3K/Akt signaling pathway, and induced apoptosis through activation of P38MARK signaling pathway. Expression of CCR2 was also associated with increased engraftment, tumor growth and dissemination, and decreased survival time in xenograft mice. Furthermore, administration of CCR2 antagonist decreased tumor growth and dissemination of DLBCL cells, and increased survival time in the xenograft model.Conclusions: Our study demonstrates that CCR2 plays an important role in the development of DLBCL by stimulating cell proliferation, migration and anti-apoptosis. The inhibition of CCR2 may, therefore, be a potential target for anticancer therapy in DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2

Covalently acting inhibitors constitute a large and growing fraction of approved small-molecule therapeutics as well as useful tools for a variety of in vitro and in vivo applications. Here, we aimed to develop a covalent antagonist of CC chemokine receptor 2 (CCR2), a class A GPCR that has been pursued as a therapeutic target in inflammation and immuno-oncology. Based on a known intracellularly binding CCR2 antagonist, several covalent derivatives were synthesized and characterized by radioligand binding and functional assays. These studies revealed compound 14 as an intracellular covalent ligand for CCR2. In silico modeling followed by site-directed mutagenesis confirmed that 14 forms a covalent bond with one of three proximal cysteine residues, which can be engaged interchangeably. To our knowledge, compound 14 represents the first covalent ligand reported for CCR2. Due to its unique properties, it may represent a promising tool for ongoing and future studies of CCR2 pharmacology.

A unique ligand-steered strategy for CC chemokine receptor 2 homology modeling to facilitate structure-based virtual screening.

CC chemokine receptor 2 (CCR2) antagonists that disrupt CCR2/MCP‐1 interaction are expected to treat a variety of inflammatory and autoimmune diseases. The lack of CCR2 crystal structure limits the application of structure‐based drug design (SBDD) to this target. Although a few three‐dimensional theoretical models have been reported, their accuracy remains to be improved in terms of templates and modeling approaches. In this study, we developed a unique ligand‐steered strategy for CCR2 homology modeling. It starts with an initial model based on the X‐ray structure of the closest homolog so far, that is, CXCR4. Then, it uses Elastic Network Normal Mode Analysis (EN‐NMA) and flexible docking (FD) by AutoDock Vina software to generate ligand‐induced fit models. It selects optimal model(s) as well as scoring function(s) via extensive evaluation of model performance based on a unique benchmarking set constructed by our in‐house tool, that is, MUBD‐DecoyMaker. The model of 81_04 presents the optimal enrichment when combined with the scoring function of PMF04, and the proposed binding mode between CCR2 and Teijin lead by this model complies with the reported mutagenesis data. To highlight the advantage of our strategy, we compared it with the only reported ligand‐steered strategy for CCR2 homology modeling, that is, Discovery Studio/Ligand Minimization. Lastly, we performed prospective virtual screening based on 81_04 and CCR2 antagonist bioassay. The identification of two hit compounds, that is, E859‐1281 and MolPort‐007‐767‐945, validated the efficacy of our model and the ligand‐steered strategy.

1865-P: Dual Chemokine Receptor CCR2/CCR5 Antagonist Cenicriviroc Ameliorates Lipotoxicity-Induced Insulin Resistance and NASH in Mice

Hepatic lipid accumulation drives innate immunity with recruitment of bone marrow-derived macrophages and activation of liver-resident Kupffer cells, leading to insulin resistance and nonalcoholic steatohepatitis (NASH) development. However, treatment options that target macrophage/Kupffer cells recruitment or activation to halt the insulin resistance and NASH remain limited. A C-C chemokine receptor 2 (CCR2) plays a central role in macrophage recruitment. In addition, we reported that a different C-C chemokine receptor, CCR5, promotes insulin resistance and hepatic steatosis by regulating macrophage M1/M2 status. Here, we investigated the effect of cenicriviroc (CVC), a dual CCR2 and CCR5 receptor antagonist in a lipotoxic model of NASH with hepatic fibrosis and insulin resistance. Eight-week-old C57BL/6 mice were fed a high-cholesterol, high-fat (CL) diet or a CL diet containing 0.015% CVC (CL+CVC) for 12 weeks. The liver histology, insulin sensitivity, and fibrogenesis were examined. The CVC administration decreased the increase in plasma ALT levels as well as liver triglyceride, cholesterol, and TBARS levels caused by the CL diet. In addition, CVC improved glucose intolerance and hyperinsulinemia in the CL group and augmented the insulin signal, assessed by IRβ and Akt phosphorylation in the liver. Flow cytometry analysis revealed that mice fed CL+CVC had 72% fewer CD11c+CD206- M1-like macrophages, but 94% more CD11c-CD206+M2-like macrophages than CL group, indicating that CVC caused an M2-dominant shift in macrophages/Kupffer cells in the liver. Furthermore, CVC attenuated hepatic fibrosis by repressing the activation of hepatic stellate cells (HSCs) and decreasing hydroxyproline content. Thus, CVC ameliorated lipotoxicity-induced insulin resistance and NASH with hepatic fibrosis by polarizing M2 macrophage and attenuating HSC activation. Disclosure T. Ota: None. G. Chen: None. M. Nagashimada: None. Funding Japan Ministry of Education, Culture, Sports, Science and Technology

Use of a Conformational-Switching Mechanism to Modulate Exposed Polarity: Discovery of CCR2 Antagonist BMS-741672.

We encountered a dilemma in the course of studying a series of antagonists of the G-protein coupled receptor CC chemokine receptor-2 (CCR2): compounds with polar C3 side chains exhibited good ion channel selectivity but poor oral bioavailability, whereas compounds with lipophilic C3 side chains exhibited good oral bioavailability in preclinical species but poor ion channel selectivity. Attempts to solve this through the direct modulation of physicochemical properties failed. However, the installation of a protonation-dependent conformational switching mechanism resolved the problem because it enabled a highly selective and relatively polar molecule to access a small population of a conformer with lower polar surface area and higher membrane permeability. Optimization of the overall properties in this series yielded the CCR2 antagonist BMS-741672 (7), which embodied properties suitable for study in human clinical trials.

A novel CCR2 antagonist inhibits atherogenesis in apoE deficient mice by achieving high receptor occupancy

CC Chemokine Receptor 2 (CCR2) and its endogenous ligand CCL2 are involved in a number of diseases, including atherosclerosis. Several CCR2 antagonists have been developed as potential therapeutic agents, however their in vivo clinical efficacy was limited. In this report, we aimed to determine whether 15a, an antagonist with a long residence time on the human CCR2, is effective in inhibiting the development of atherosclerosis in a mouse disease model. First, radioligand binding assays were performed to determine affinity and binding kinetics of 15a on murine CCR2. To assess the in vivo efficacy, western-type diet fed apoE−/− mice were treated daily with 15a or vehicle as control. Treatment with 15a reduced the amount of circulating CCR2+ monocytes and the size of the atherosclerotic plaques in both the carotid artery and the aortic root. We then showed that the long pharmacokinetic half-life of 15a combined with the high drug concentrations ensured prolonged CCR2 occupancy. These data render 15a a promising compound for drug development and confirms high receptor occupancy as a key parameter when targeting chemokine receptors.

Pretreatment with a heat-killed probiotic modulates monocyte chemoattractant protein-1 and reduces the pathogenicity of influenza and enterovirus 71 infections

It has been proposed that inactivated probiotics may modulate the host immune system and contribute to mitigation of viral infections. This study demonstrated that administration of heat-killed Enterococcus faecalis, a widely used probiotic, can protect host animals against viral infections. The influenza-mediated morbidity and lung inflammation in E. faecalis-treated mice decreased significantly compared with those of the control mice. Furthermore, we found that the protection is associated with production of monocyte chemoattractant protein-1 (MCP-1). The intratracheal injection of a recombinant mouse MCP-1 protein abrogated the antiviral effects elicited by pretreatment with E. faecalis. CC chemokine receptor 2 (CCR2) is a receptor for MCP-1, and the intraperitoneal administration of a CCR2 antagonist effectively inhibited viral pathogenicity. The reduced pathogenicity was also observed in CCR2-deficient mice. Finally, E. faecalis significantly attenuated neuropathogenicity induced by another RNA virus, enterovirus 71. This study demonstrates that killed probiotics can reduce viral disease severity and identify that the MCP-1 pathway might act as a key mediator in the improved antiviral immune response. Our findings suggest that MCP-1 and its related signaling pathway can serve as critical therapeutic targets for development of new antiviral strategies.

CCR5 deficiency accelerates lipopolysaccharide-induced astrogliosis, amyloid-beta deposit and impaired memory function

Chemokine receptors are implicated in inflammation and immune responses. Neuro-inflammation is associated with activation of astrocyte and amyloid-beta (Aβ) generations that lead to pathogenesis of Alzheimer disease (AD). Previous our study showed that deficiency of CC chemokine receptor 5 (CCR5) results in activation of astrocytes and Aβ deposit, and thus memory dysfunction through increase of CC chemokine receptor 2 (CCR2) expression. CCR5 knockout mice were used as an animal model with memory dysfunction. For the purpose LPS was injected i.p. daily (0.25 mg/kg/day). The memory dysfunctions were much higher in LPS-injected CCR5 knockout mice compared to CCR5 wild type mice as well as non-injected CCR5 knockout mice. Associated with severe memory dysfuction in LPS injected CCR5 knockout mice, LPS injection significant increase expression of inflammatory proteins, astrocyte activation, expressions of β-secretase as well as Aβ deposition in the brain of CCR5 knockout mice as compared with that of CCR5 wild type mice. In CCR5 knockout mice, CCR2 expressions were high and co-localized with GFAP which was significantly elevated by LPS. Expression of monocyte chemoattractant protein-1 (MCP-1) which ligands of CCR2 also increased by LPS injection, and increment of MCP-1 expression is much higher in CCR5 knockout mice. BV-2 cells treated with CCR5 antagonist, D-ala-peptide T-amide (DAPTA) and cultured astrocytes isolated from CCR5 knockout mice treated with LPS (1 μg/ml) and CCR2 antagonist, decreased the NF-ĸB activation and Aβ level. These findings suggest that the deficiency of CCR5 enhances response of LPS, which accelerates to neuro-inflammation and memory impairment.

Role of the monocyte chemoattractant protein-1/C-C chemokine receptor 2 signaling pathway in transient receptor potential vanilloid type 1 ablation-induced renal injury in salt-sensitive hypertension.

Our recent studies indicate that the transient receptor potential vanilloid type 1 (TRPV1) channel may act as a potential regulator of monocyte/macrophage recruitment to reduce renal injury in salt-sensitive hypertension. This study tests the hypothesis that deletion of TRPV1 exaggerates salt-sensitive hypertension-induced renal injury due to enhanced inflammatory responses via monocyte chemoattractant protein-1 (MCP-1)/C-C chemokine receptor 2 (CCR2)-dependent pathways. Wild type (WT) and TRPV1-null mutant (TRPV1(-/-)) mice were subjected to uninephrectomy and deoxycorticosterone acetate (DOCA)-salt treatment for four weeks with or without the selective CCR2 antagonist, RS504393. DOCA-salt treatment increased systolic blood pressure (SBP) to the same degree in both strains, but increased urinary excretion of albumin and 8-isoprostane and decreased creatinine clearance with greater magnitude in TRPV1(-/-) mice compared to WT mice. DOCA-salt treatment also caused renal glomerulosclerosis, tubulointerstitial injury, collagen deposition, monocyte/macrophage infiltration, proinflammatory cytokine and chemokine production, and NF-κB activation in greater degree in TRPV1(-/-) mice compared to WT mice. Blockade of the CCR2 with RS504393 (4 mg/kg/day) had no effect on SBP in DOCA-salt-treated WT or TRPV1(-/-) mice compared to their respective controls. However, treatment with RS504393 ameliorated renal dysfunction and morphological damage, and prevented the increase in monocyte/macrophage infiltration, cytokine/chemokine production, and NF-κB activity in both DOCA-salt hypertensive strains with a greater effect in DOCA-salt-treated TRPV1(-/-) mice compared to DOCA-salt-treated WT mice. No differences in CCR2 protein expression in kidney were found between DOCA-salt-treated WT and TRPV1(-/-) mice with or without RS504393 treatment. Our studies for the first time indicate that deletion of TRPV1 aggravated renal injury in salt-sensitive hypertension via enhancing MCP-1/CCR2 signaling-dependent inflammatory responses.

Klotho gene deficiency causes salt-sensitive hypertension via monocyte chemotactic protein-1/CC chemokine receptor 2-mediated inflammation.

Klotho (KL) is a newly discovered aging suppressor gene. In mice, the KL gene extends the lifespan when overexpressed and shortens the lifespan when disrupted. This study investigated if KL deficiency affects BP and salt sensitivity using KL mutant heterozygous (+/-) mice and wild-type (WT) mice (9 weeks of age, 16 mice per group). Notably, systolic BP in KL(+/-) mice began to increase at the age of 15 weeks, reached a peak level at the age of 17 weeks, and remained elevated thereafter, whereas systolic BP remained consistent in WT mice. High salt (HS) intake further increased BP in KL(+/-) mice but did not affect BP in WT mice. Blockade of CC chemokine receptor 2 (CCR2), involved in monocyte chemotaxis, by a specific CCR2 antagonist (INCB3284) abolished the HS-induced increase in BP in KL(+/-) mice. Furthermore, HS loading substantially increased the expression of monocyte chemotactic protein-1 and the infiltration of macrophages and T cells in kidneys in KL(+/-) mice, and treatment with INCB3284 abolished these effects. Treatment of KL(+/-) mice with INCB3284 also attenuated the increased renal expressions of serum glucocorticoid-regulated kinase 1, thiazide-sensitive NaCl cotransporter, and ATP synthase β along with the renal structural damage and functional impairment induced by HS loading. In conclusion, KL deficiency caused salt-sensitive hypertension and renal damage by CCR2-mediated inflammation.

Monocytes Infiltrate the Pancreas via the MCP-1/CCR2 Pathway and Differentiate into Stellate Cells

Recent studies have shown that monocytes possess pluripotent plasticity. We previously reported that monocytes could differentiate into hepatic stellate cells. Although stellate cells are also present in the pancreas, their origin remains unclear. An accumulation of enhanced green fluorescent protein (EGFP)+CD45– cells was observed in the pancreases and livers of chimeric mice, which were transplanted with a single hematopoietic stem cell isolated from EGFP-transgenic mice and treated with carbon tetrachloride (CCl4). Because the vast majority of EGFP+CD45– cells in the pancreas expressed stellate cell-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin, they were characterized as pancreatic stellate cells (PaSCs). EGFP+ PaSCs were also observed in CCl4-treated mice adoptively transferred with monocytes but not with other cell lineages isolated from EGFP-transgenic mice. The expression of monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) increased in the pancreas of CCl4-treated mice and their respective receptors, C-C chemokine receptor 2 (CCR2) and Ang II type 1 receptor (AT1R), were expressed on Ly6Chigh monocytes isolated from EGFP-transgenic mice. We examined the effect of an AT1R antagonist, irbesartan, which is also a CCR2 antagonist, on the migration of monocytes into the pancreas. Monocytes migrated toward MCP-1 but not Ang II in vitro. Irbesartan inhibited not only their in vitro chemotaxis but also in vivo migration of adoptively transferred monocytes from peripheral blood into the pancreas. Irbesartan treatment significantly reduced the numbers of EGFP+F4/80+CCR2+ monocytic cells and EGFP+ PaSCs in the pancreas of CCl4-treated chimeric mice receiving EGFP+ bone marrow cells. A specific CCR2 antagonist RS504393 inhibited the occurrence of EGFP+ PaSCs in injured mice. We propose that CCR2+ monocytes migrate into the pancreas possibly via the MCP-1/CCR2 pathway and give rise to PaSCs.

CC-Chemokine Ligand 2 Facilitates Conditioned Place Preference to Methamphetamine Through the Activation of Dopamine Systems

Methamphetamine addiction is characterized by drug craving caused by stimulation of the reward system. Because neuroinflammation underlies several neurological disorders, we investigated whether CC-chemokine ligand 2 (CCL2) participates in the methamphetamine dependence using mice. Upregulation of CCL2 but not CC-chemokine receptor 2 (CCR2), a dominant receptor for CCL2, mRNA in both the prefrontal cortex (PFC) and nucleus accumbens (NAC) was observed after methamphetamine (3 mg/kg, s.c.) administration. Using immunohistochemistry, high CCL2 protein levels localized to neurons in the PFC and NAC. In the conditioned place preference (CPP) test, methamphetamine (0.3 - 3 mg/kg, s.c.) induced a CPP, reflecting psychic dependence on methamphetamine, in a dose-dependent manner. The CPP to methamphetamine was attenuated by RS504393 (1 mg/kg, s.c.), a CCR2 antagonist. Moreover, methamphetamine increased phosphorylated tyrosine hydroxylase (pTH) levels in the ventral tegmental area (VTA). Increased levels of pTH in the VTA by methamphetamine was also suppressed by RS504393. Furthermore, intracerebroventricular injection of recombinant CCL2 increased pTH levels in the VTA. Taken together, we demonstrate that activation of dopamine neurons, which enhances reward-system activity, via the CCL2-CCR2 axis plays a crucial role in psychic dependence on methamphetamine. Novel treatments targeting this machinery may be effective for drug addiction.

Monocytes Infiltrate The Pancreas Via MCP-1/CCR2 Signaling and Differentiate Into Pancreatic Stellate Cells During Pancreatic Injury

IntroductionWe previously reported that monocytes could differentiate into hepatic stellate cells during carbon tetrachloride (CCl4) injury. Pancreatic stellate cells (PaSCs), which play a central role in the development of pancreatic fibrosis, share many morphological and functional characteristics with those of hepatic stellate cells. Although PaSCs have been thought to be of mesenchymal origin, some authors have recently reported that they are derived from bone marrow (BM) cells during inflammation. Meanwhile, both monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) are thought to be involved in the development of pancreatic fibrosis. We hypothesized that PaSCs are also derived from monocytes and investigated the mechanism of how monocytes enter the injured pancreas, focusing on MCP-1 and Ang II. MethodsFirst, we generated chimeric mice by transplantation of clonal populations of cells derived from single lineage negative Sca-1+c-kit+CD34- cells or total-nucleated cells, which were sorted from the BM of enhanced green fluorescent protein (EGFP)-transgenic mice, into lethally irradiated C57BL/6 mice. Since 2 months after BM transplantation, the chimeric mice revealing high-level multilineage engraftment were intraperitoneally injected with CCl4 twice a week. Next, we intravenously transferred Ly6Chighc-kit– monocytes, which were isolated from the BM of EGFP-transgenic mice, into non-irradiated CCl4-treated mice. We analyzed the presence of EGFP+ PaSCs in the injured pancreas using immunofluorescence and laser confocal scanning microscopy. Finally, we investigated the effect of Ang II type 1 receptor (AT1R) antagonist, irbesartan, which is also an antagonist of MCP-1 receptor, CC chemokine receptor 2 (CCR2) on the occurrence of monocyte-derived PaSCs in the pancreases of CCl4-treated mice. ResultsAfter 12 weeks of injury by CCl4, EGFP+CD45- cells were detected in the pancreases of chimeric mice. They expressed PaSC-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin. Next, we found EGFP+ PaSCs in CCl4-treated mice with adoptively transferred Ly6Chigh monocytes but not other lineages. MCP-1 and Ang II were highly produced in the pancreas of CCl4-treted mice and their receptors CCR2 and AT1R were respectively expressed on Ly6Chigh monocytes isolated from the BM of EGFP-transgenic mice. Monocytes migrated toward MCP-1, but not Ang II, and irbesartan inhibited their chemotaxis in vitro. Irbesartan treatment significantly reduced the numbers of EGFP+CCR2+F4/80+ macrophages and EGFP+ PaSCs in the pancreas of CCl4-treated mice. We observed that a specific CCR2 antagonist RS504393 inhibited the monocyte migration toward MCP-1 in vitro and the occurrence of EGFP+ PaSCs in the injured mice like irbesartan. ConclusionWe propose that CCR2+ monocytes migrate into the injured pancreas via the MCP-1/CCR2 pathway and differentiate into PaSCs during inflammation. Disclosures:Masuya:Dainippon-Sumitomo Pharmaceutical: Research Funding.

Development of 3D-pharmacophore model followed by successive virtual screening, molecular docking and ADME studies for the design of potent CCR2 antagonists for inflammation-driven diseases

In order to elucidate the essential structural features for CC chemokine receptor 2 (CCR2) antagonism, 3D-pharmacophore hypotheses were built based on a set of known compounds from the literature. The hypotheses were developed with the aid of HypoGen module within Discovery Studio 2.5 program. Multiple validation approaches provided the confidence in utilising the predictive pharmacophore models developed in this study. The most predictive pharmacophore model (Hypo1) was found to be statistically significant along with its ability to predict activities of the known CCR2 antagonists in the training and test set with high correlation coefficient. The best model was then used as a 3D search query in the virtual screening of chemical databases including ChemDiv and MiniMaybridge. Lipinski's rule of five and molecular docking studies were applied to the screened hits for retrieving potential lead compounds. Eight hits showed better in silico CCR2-binding affinities than the reported CCR2 antagonists, along with good absorption, distribution, metabolism and excretion profiles. The current 3D-quantitative structure–activity relationship (QSAR) pharmacophore modelling and molecular docking studies attempt to elucidate QSAR for CCR2 antagonism and identify novel potent CCR2 antagonist scaffolds.

Inhibition of MCP-1/CCR2 signaling pathway is involved in synergistic inhibitory effects of irbesartan with rosuvastatin on vascular remodeling

Additional beneficial effects of angiotensin II type 1 (AT1) receptor blockers beyond AT1 receptor blockade have been highlighted. Irbesartan is reported to act as an antagonist of the monocyte chemoattractant protein-1 (MCP-1) receptor, C-C chemokine receptor 2 (CCR2). We examined the possible synergistic effects of the combination of irbesartan with rosuvastatin on preventing vascular remodeling focusing on the MCP-1/CCR2 pathway. We observed that administration of irbesartan and CCR2 antagonist, propagermanium, at noneffective doses, decreased the neointima with a decrease in PCNA labeling index in the injured mouse femoral artery induced by cuff placement. We also observed that administration of a noneffective dose of rosuvastatin with propagermanium decreased the neointima area, suggesting that the inhibitory effect of rosuvastatin on neointima formation is at least partly attributable to blockade of the MCP-1/CCR2 pathway. Moreover, we demonstrated that the combination of irbesartan with rosuvastatin decreased neointima formation. MCP-1 mRNA level was significantly increased in injured femoral arteries, and administration of irbesartan with rosuvastatin decreased the mRNA levels of MCP-1, TNFα, and IL-1β, and increased PPARγ mRNA expression. These results suggest that the synergistic inhibitory effects of irbesartan with rosuvastatin on neointima formation may involve attenuation of MCP-1/CCR2 signaling.

Orally-Administered Chemokine Receptor CCR2 Antagonist CCX140-B in Type 2 Diabetes: A Pilot Double-Blind, Randomized Clinical Trial

Study background: Inflammatory macrophages expressing the C-C chemokine receptor 2 (CCR2) accumulate in adipose tissue and contribute to insulin resistance. CCX140-B is an orally-administered antagonist of CCR2 expressed on monocytes and macrophages and blocks infiltration of these cells into adipose tissue. A pilot Phase 2 clinical trial was conducted in patients with type 2 diabetes with the primary objective to evaluate the safety and tolerability of CCX140-B. Key secondary objectives included assessment of glycemic parameters, fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c). Methods: This is a randomized, double-blind, clinical trial of CCX140-B in 159 subjects with type 2 diabetes on stable metformin for at least 8 weeks prior to study entry. HbA1c was 6.5 to 10% and FPG 135 to 270 mg/dL at study entry. Randomized subjects received double-blind placebo (N=32), 5 mg CCX140-B (N=63), 10 mg CCX140-B (N=32), or pioglitazone 30 mg (N=32) once daily orally for 4 weeks, with a 4-week follow-up period. Results: CCX140-B was well tolerated. No serious adverse events occurred with CCX140-B. FPG showed a CCX140-B dose-dependent decrease, with 10 mg CCX140-B showing a similar decrease to pioglitazone (leastsquares mean change at week 4 of -16.1 vs. -21.4 mg/dL, respectively). HbA1c least-squares mean changes from baseline to week 4 for the placebo, 5 mg CCX140-B, 10 mg CCX140-B, and pioglitazone groups were -0.09%, -0.09%, -0.23% (p=0.045 vs. placebo), and -0.13% (NS vs. placebo), respectively. No detrimental changes were seen in plasma monocyte chemoattractant protein-1 or blood monocyte counts with CCX140-B. Conclusion: CCX140-B, an orally administered, specific CCR2 antagonist was found to be well tolerated and safe in this Phase 2 clinical trial and showed evidence of a beneficial effect on glycemic parameters. If confirmed, CCX140-B might be beneficial in treatment of patients with type 2 diabetes. (Clinical Trial Registry: clinicaltrials.gov identifier NCT01028963).

Addition of Angiotensin II Type 1 Receptor Blocker to CCR2 Antagonist Markedly Attenuates Crescentic Glomerulonephritis

The monocyte chemoattractant protein-1 (MCP-1)/CC-chemokine receptor 2 (CCR2) pathway plays a critical role in the development of antiglomerular basement membrane (anti-GBM) nephritis. We recently showed angiotensin II (Ang II) infusion in rats activated MCP-1 and transforming growth factor-β1 (TGF-β1), which in turn induced macrophage infiltration of renal tissues. This study was performed to demonstrate that combination therapy with a CCR2 antagonist (CA) and an Ang II type 1 receptor blocker (ARB) ameliorated renal injury in the anti-GBM nephritis model. An anti-GBM nephritis rat model developed progressive proteinuria and glomerular crescent formation, accompanied by increased macrophage infiltration and glomerular expression of MCP-1, angiotensinogen, Ang II, and TGF-β1. Treatment with CA alone or ARB alone moderately ameliorated kidney injury; however, the combination treatment with CA and ARB dramatically prevented proteinuria and markedly reduced glomerular crescent formation. The combination treatment also suppressed the induction of macrophage infiltration, MCP-1, angiotensinogen, Ang II, and TGF-β1 and reversed the fibrotic change in the glomeruli. Next, primary cultured glomerular mesangial cells (MCs) stimulated by Ang II showed significant increases in MCP-1 and TGF-β1 expression. Furthermore, cocultured model consisting of MCs, parietal epithelial cells, and macrophages showed an increase in Ang II-induced cell proliferation and collagen secretion. ARB treatment attenuated these augmentations. These data suggest that Ang II enhances glomerular crescent formation of anti-GBM nephritis. Moreover, our results demonstrate that inhibition of the MCP-1/CCR2 pathway with a combination of ARB effectively reduces renal injury in anti-GBM nephritis.