Inhibition of CC chemokine receptor 1 ameliorates osteoarthritis in mouse by activating PPAR-γ.

BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage destruction and inflammation. CC chemokine receptor 1 (CCR1), a member of the chemokine family and its receptor family, plays a role in the autoimmune response. The impact of BX471, a specific small molecule inhibitor of CCR1, on CCR1 expression in cartilage and its effects on OA remain underexplored. METHODS: This study used immunohistochemistry (IHC) to assess CCR1 expression in IL-1ß-induced mouse chondrocytes and a medial meniscus mouse model of destabilization of the medial meniscus (DMM). Chondrocytes treated with varying concentrations of BX471 for 24 h were subjected to IL-1ß (10 ng/ml) treatment. The levels of the aging-related genes P16INK4a and P21CIP1 were analyzed via western blotting, and senescence-associated ß-galactosidase (SA-ß-gal) activity was measured. The expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), aggrecan (AGG), and the transcription factor SOX9 were determined through western blotting and RT‒qPCR. Collagen II, matrix metalloproteinase 13 (MMP13), and peroxisome proliferator-activated receptor (PPAR)-γ expression was analyzed via western blot, RT‒qPCR, and immunofluorescence. The impact of BX471 on inflammatory metabolism-related proteins under PPAR-γ inhibition conditions (using GW-9662) was examined through western blotting. The expression of MAPK signaling pathway-related molecules was assessed through western blotting. In vivo, various concentrations of BX471 or an equivalent medium were injected into DMM model joints. Cartilage destruction was evaluated through Safranin O/Fast green and hematoxylin-eosin (H&E) staining. RESULTS: This study revealed that inhibiting CCR1 mitigates IL-1ß-induced aging, downregulates the expression of iNOS, COX-2, and MMP13, and alleviates the IL-1ß-induced decrease in anabolic indices. Mechanistically, the MAPK signaling pathway and PPAR-γ may be involved in inhibiting the protective effect of CCR1 on chondrocytes. In vivo, BX471 protected cartilage in a DMM model. CONCLUSION: This study demonstrated the expression of CCR1 in chondrocytes. Inhibiting CCR1 reduced the inflammatory response, alleviated cartilage aging, and retarded degeneration through the MAPK signaling pathway and PPAR-γ, suggesting its potential therapeutic value for OA.

Bardoxolone methyl prevents metabolic dysfunction-associated steatohepatitis by inhibiting macrophage infiltration.

BACKGROUND AND PURPOSE: Bardoxolone methyl (2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester, CDDO-Me) is a potent activator of nuclear factor erythroid 2-related factor 2 (Nrf2), which induces the expression of antioxidative-associated genes. CDDO-Me exerts protective effects against chronic inflammatory diseases in the kidneys and lungs. However, its pharmacological effects on metabolic dysfunction-associated steatohepatitis (MASH) caused by fat accumulation remain unknown. In this study, we examined the hepatoprotective effects of CDDO-Me in a diet-induced MASH mouse model and elucidated its pharmacological mechanisms using RNA-seq analysis. EXPERIMENTAL APPROACH: CDDO-Me was orally administered to mice fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD), and histological, biochemical, and transcriptomic analyses were performed on livers of mice that developed MASH. KEY RESULTS: CDDO-Me administration induced the expression of antioxidant genes and cholesterol transporters downstream of Nrf2 and significantly prevented the symptoms of MASH. Whole-transcriptome analysis revealed that CDDO-Me inhibited the inflammatory pathway that led to phagocyte recruitment, in addition to activating the Nrf2-dependent pathway. Among inflammatory pathways, CC chemokine ligands (CCL)3 and CCL4, which are downstream of NF-κB and are associated with the recruitment of macrophages expressing CC chemokine receptors (CCR)1 and CCR5, were released into the blood in MASH mice. However, CDDO-Me directly inhibited the expression of CCL3-CCR1 and CCL4-CCR5 in macrophages. CONCLUSIONS AND IMPLICATIONS: Overall, we revealed the potent hepatoprotective effect of CDDO-Me in a MASH mouse model and demonstrated that its pharmacological effects were closely associated with a reduction of macrophage infiltration, through CCL3-CCR1 and CCL4-CCR5 inhibition, in addition to Nrf2-mediated hepatoprotective effects.

Novel Findings on CCR1 Receptor in CNS Disorders: A Pathogenic Marker Useful in Controlling Neuroimmune and Neuroinflammatory Mechanisms in Parkinson's Disease.

Parkinson's disease (PD) is recognized as the second most common neurodegenerative disease worldwide. Even if PD etiopathogenesis is not yet fully understood, in recent years, it has been advanced that a chronic state of inflammation could play a decisive role in the development of this pathology, establishing the close link between PD and neuroinflammation. In the broad panorama of inflammation and its several signaling pathways, the C-C chemokine receptor type 1 (CCR1) could play a key pathogenic role in PD progression, and could constitute a valuable target for the development of innovative anti-PD therapies. In this study, we probed the neuroprotective properties of the CCR1 antagonist BX471 compound in a mouse model of MPTP-induced nigrostriatal degeneration. BX471 treatments were performed intraperitoneally at a dose of 3 mg/kg, 10 mg/kg, and 30 mg/kg, starting 24 h after the last injection of MPTP and continuing for 7 days. From our data, BX471 treatment strongly blocked CCR1 and, as a result, decreased PD features, also reducing the neuroinflammatory state by regulating glial activation, NF-κB pathway, proinflammatory enzymes, and cytokines overexpression. Moreover, we showed that BX471's antagonistic action on CCR1 reduced the infiltration of immune cells, including mast cells and lymphocyte T activation. In addition, biochemical analyses carried out on serum revealed a considerable increase in circulating levels of CCR1 following MPTP-induced PD. In light of these findings, CCR1 could represent a useful pathological marker of PD, and its targeting could be a worthy candidate for the future development of new immunotherapies against PD.

Met-RANTES preserves the blood-brain barrier through inhibiting CCR1/SRC/Rac1 pathway after intracerebral hemorrhage in mice.

BACKGROUND: C-C chemokine receptor type 1 (CCR1) and its endogenous ligand, CCL5, participate in the pathogenesis of neuroinflammatory diseases. However, much remains unknown regarding CCL5/CCR1 signaling in blood-brain barrier (BBB) permeability after intracerebral hemorrhage (ICH). METHODS: A total of 250 CD1 male mice were used and ICH was induced via autologous whole blood injection. Either Met-RANTES, a selective CCR1 antagonist, or Met-RANTES combined with a Rac1 CRISPR activator was administered to the mice 1 h after ICH. Post-ICH assessments included neurobehavioral tests, brain water content, BBB integrity, hematoma volume, Western blot, and immunofluorescence staining. The CCR1 ligand, rCCL5, and SRC CRISPR knockout in naïve mice were used to further elucidate detrimental CCL5/CCR1/SRC signaling. RESULTS: Brain endogenous CCR1 and CCL5 were upregulated after ICH in mice with a peak at 24 h, and CCR1 was expressed in endothelial cells, astrocytes, and neurons. Met-R treatment reduced brain edema and neurobehavioral impairment, as well as preserved BBB integrity and tight junction protein expression in ICH mice. Met-R treatment decreased expression of p-SRC, Rac1, albumin, and MMP9, but increased claudin-5, occludin, and ZO-1 tight junction proteins after ICH. These effects were regressed using the Rac1 CRISPR activator. Administration of rCCL5 in naïve mice increased expression of p-SRC, Rac1, albumin, and MMP9, but decreased levels of claudin-5, occludin, and ZO-1 tight junction proteins. These effects in naïve mice were reversed with SRC CRISPR (KO). CONCLUSIONS: Our findings demonstrate that CCR5 inhibition by Met-R improves neurological deficits after ICH by preserving BBB integrity through inhibiting CCR1/SRC/Rac1 signaling pathway in mice. Thus, Met-R has therapeutic potential in the management of ICH patients.

Possible involvement of crosstalk between endometrial cells and mast cells in the development of endometriosis via CCL8/CCR1.

BACKGROUND: The density and the activity of mast cells are associated with endometriosis. However, the role of mast cells on the pathogenesis of endometriosis remains unclear. Our study aims to investigate whether endometrial cells interact with mast cells and the involvement of their crosstalk in the development of endometriosis. METHODS: The transwell assay was applied to investigate the effect of mast cells on the migratory ability of human primary endometrial cells. Mast cells were cocultured with endometrial epithelial and stromal cells respectively and total RNAs were isolated and subjected to mRNA sequencing. Next, the transwell assay, CCK-8, and tube formation were applied to study the role of CCL8 on the endometrial and endothelial cells in vitro. The mouse model was also established to confirm the role of CCL8 in the development and angiogenesis of endometriosis. RESULTS: CCL8 was up-regulated in mast cells when cocultured with endometrial cells. CCL8 was highly expressed in the ectopic endometrium and the serum of patients with endometriosis. CCL8 promoted the migratory ability of endometrial epithelial and stromal cells and increased the proliferation, migration, and tube formation of endothelial cells. CCR1, the receptor of CCL8, was over-expressed in the ectopic endometrium and colocalized with blood vessels in ovarian endometriomas. The inhibition of CCR1 suppressed the development and angiogenesis of endometriosis in vivo. CONCLUSION: The crosstalk between endometrial cells and mast cells in the development of endometriosis via CCL8/CCR1 was demonstrated, thereby providing a new treatment strategy for endometriosis.

KSHV enhances mesenchymal stem cell homing and promotes KS-like pathogenesis.

Kaposi's sarcoma (KS) tends to occur in injured or inflamed sites of the body, which is described as the "Koebner phenomenon". KS is also unique in its extraordinary angio-hyperplastic inflammatory phenotype. Recently, evidence has accrued indicating that KS may derive from KSHV-infected mesenchymal stem cells (MSCs), which possess enhanced migration and homing ability. Inspired by these findings, we hypothesized that KS may arise from KSHV-infected MSCs that chemotactically migrate to preexisting inflammatory or injured sites. Here we report that KSHV infection of human MSCs significantly up-regulated expression of several chemokine receptors and enhanced cell migration ability in vitro. Furthermore, using a wound mouse model, we demonstrated that KSHV infection dramatically promotes MSCs migrating and settling in the wound sites. In addition, two mice in the KSHV-infected group showed purpura and tumors with KS-like features. Taken together, KSHV-enhanced MSC migration ability and inflammatory microenvironment play crucial roles in KS development.

Rapid identification of highly potent human anti-GPCR antagonist monoclonal antibodies.

Complex cellular targets such as G protein-coupled receptors (GPCRs), ion channels, and other multi-transmembrane proteins represent a significant challenge for therapeutic antibody discovery, primarily because of poor stability of the target protein upon extraction from cell membranes. To assess whether a limited set of membrane-bound antigen formats could be exploited to identify functional antibodies directed against such targets, we selected a GPCR of therapeutic relevance (CCR1) and identified target binders using an in vitro yeast-based antibody discovery platform (AdimabTM) to expedite hit identification. Initially, we compared two different biotinylated antigen formats overexpressing human CCR1 in a 'scouting' approach using a subset of the antibody library. Binders were isolated using streptavidin-coated beads, expressed as yeast supernatants, and screened using a high-throughput binding assay and flow cytometry on appropriate cell lines. The most suitable antigen was then selected to isolate target binders using the full library diversity. This approach identified a combined total of 183 mAbs with diverse heavy chain sequences. A subset of clones exhibited high potencies in primary cell chemotaxis assays, with IC50 values in the low nM/high pM range. To assess the feasibility of any further affinity enhancement, full-length hCCR1 protein was purified, complementary-determining region diversified libraries were constructed from a high and lower affinity mAb, and improved binders were isolated by fluorescence-activated cell sorting selections. A significant affinity enhancement was observed for the lower affinity parental mAb, but not the high affinity mAb. These data exemplify a methodology to generate potent human mAbs for challenging targets rapidly using whole cells as antigen and define a route to the identification of affinity-matured variants if required.

The blockade of CC chemokine receptor type 1 influences the level of nociceptive factors and enhances opioid analgesic potency in a rat model of neuropathic pain.

A growing body of evidence has indicated that the release of nociceptive factors, such as interleukins and chemokines, by activated immune and glial cells has crucial significance for neuropathic pain generation and maintenance. Moreover, changes in the production of nociceptive immune factors are associated with low opioid efficacy in the treatment of neuropathy. Recently, it has been suggested that CC chemokine receptor type 1 (CCR1) signaling is important for nociception. Our study provides evidence that the development of hypersensitivity in rats following chronic constriction injury (CCI) of the sciatic nerve is associated with significant up-regulation of endogenous CCR1 ligands, namely, CCL2, CCL3, CCL4, CCL6, CCL7 and CCL9 in the spinal cord and CCL2, CCL6, CCL7 and CCL9 in dorsal root ganglia (DRG). We showed that single and repeated intrathecal administration of J113863 (an antagonist of CCR1) attenuated mechanical and thermal hypersensitivity. Moreover, repeated administration of a CCR1 antagonist enhanced the analgesic properties of morphine and buprenorphine after CCI. Simultaneously, repeated administration of J113863 reduced the protein levels of IBA-1 in the spinal cord and MPO and CD4 in the DRG and, as a consequence, the level of pronociceptive factors, such as interleukin-1ß (IL-1ß), IL-6 and IL-18. The data obtained provide evidence that CCR1 blockade reduces hypersensitivity and increases opioid-induced analgesia through the modulation of neuroimmune interactions.

CCR1 Activation Promotes Neuroinflammation Through CCR1/TPR1/ERK1/2 Signaling Pathway After Intracerebral Hemorrhage in Mice.

The activation of C-C chemokine receptor type 1 (CCR1) has been shown to be pro-inflammatory in several animal models of neurological diseases. The objective of this study was to investigate the activation of CCR1 on neuroinflammation in a mouse model of intracerebral hemorrhage (ICH) and the mechanism of CCR1/tetratricopeptide repeat 1 (TPR1)/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway in CCR1-mediated neuroinflammation. Adult male CD1 mice (n = 210) were used in the study. The selective CCR1 antagonist Met-RANTES was administered intranasally at 1 h after autologous blood injection. To elucidate potential mechanism, a specific ERK1/2 activator (ceramide C6) was administered prior to Met-RANTES treatment; CCR1 activator (recombinant CCL5, rCCL5) and TPR1 CRISPR were administered in naïve mouse. Neurobehavioral assessments, brain water content, immunofluorescence staining, and western blot were performed. The endogenous expressions of CCR1, CCL5, TPR1, and p-ERK1/2 were increased in the brain after ICH. CCR1 were expressed on microglia, neurons, and astrocytes. The inhibition of CCR1 with Met-RANTES improved neurologic function, decreased brain edema, and suppressed microglia/macrophage activations and neutrophil infiltration after ICH. Met-RANTES treatment decreased expressions of CCR1, TPR1, p-ERK, TNF-α, and IL-1ß, which was reversed by ceramide C6. The brain CCR1 activation by rCCL5 injection in naïve mouse resulted in neurological deficits and increased expressions of CCR1, TPR1, p-ERK, TNF-α, and IL-1ß. These detrimental effects of rCCL5 were reversed by TPR1 knockdown using TPR1 CRISPR. Our study demonstrated that CCR1 activation promoted neuroinflammation through CCR1/TPR1/ERK1/2 signaling pathway after ICH in mice. CCR1 inhibition with Met-RANTES attenuated neuroinflammation, thereby reducing brain edema and improving neurobehavioral functions. Targeting CCR1 activation may provide a promising therapeutic approach in the management of ICH patients.

Identification of novel azaindazole CCR1 antagonist clinical candidates.

Exploring various cyclization strategies, using a submicromolar pyrazole HTS screening hit 6 as a starting point, a novel indazole based CCR1 antagonist core was discovered. This report presents the design and SAR of CCR1 indazole and azaindazole antagonists leading to the identification of three development compounds, including 19e that was advanced to early clinical trials.

Knockdown of FOXP1 promotes the development of lung adenocarcinoma.

Lung cancer is one of the most common cancers in the world, which accounts for about 27% of all cancer deaths. However, the mechanisms underlying the pathogenesis of lung cancer cells remain largely elusive. In this study, we examined the role of the Forkhead box protein P1 (FOXP1) in lung cancer development. Our Oncomine analysis shows that FOXP1 is downregulated in lung adenocarcinoma compared with normal lung tissue. Knockdown of FOXP1 promotes the growth and invasion of PC9 and A549 cells by regulating genes of chemokine signaling molecules, including CCR1, ADCY5, GNG7, VAV3, and PLCB1. Simultaneous knockdown of CCR1 and FOXP1 attenuated FOXP1 knockdown-induced increase of lung cancer cell growth. Finally, knockdown of FOXP1 in PC9 cells promotes the tumorigenesis via CCR1 signaling in xenograft mouse model. Taken together, our data suggest that FOXP1 plays important roles in preventing lung adenocarcinoma development via suppressing chemokine signaling pathways.

Discovery and optimization of pyrazole amides as antagonists of CCR1.

A HTS screen for CCR1 antagonists afforded a novel sub-micromolar hit 5 containing a pyrazole core. In this report the design, optimization, and SAR of novel CCR1 antagonists based on a pyrazole core motif is presented. Optimization led to the advanced candidate compounds (S)-16q and (S)-16r with 250-fold improved CCR1 potency, excellent off-target selectivity and attractive drug-like properties.

Pyrrolone Derivatives as Intracellular Allosteric Modulators for Chemokine Receptors: Selective and Dual-Targeting Inhibitors of CC Chemokine Receptors 1 and 2.

The recent crystal structures of CC chemokine receptors 2 and 9 (CCR2 and CCR9) have provided structural evidence for an allosteric, intracellular binding site. The high conservation of residues involved in this site suggests its presence in most chemokine receptors, including the close homologue CCR1. By using [3H]CCR2-RA-[ R], a high-affinity, CCR2 intracellular ligand, we report an intracellular binding site in CCR1, where this radioligand also binds with high affinity. In addition, we report the synthesis and biological characterization of a series of pyrrolone derivatives for CCR1 and CCR2, which allowed us to identify several high-affinity intracellular ligands, including selective and potential multitarget antagonists. Evaluation of selected compounds in a functional [35S]GTPγS assay revealed that they act as inverse agonists in CCR1, providing a new manner of pharmacological modulation. Thus, this intracellular binding site enables the design of selective and multitarget inhibitors as a novel therapeutic approach.

Involvement of Macrophage Inflammatory Protein-1 Family Members in the Development of Diabetic Neuropathy and Their Contribution to Effectiveness of Morphine.

Current investigations underline the important roles of C-C motif ligands in the development of neuropathic pain; however, their participation in diabetic neuropathy is still undefined. Therefore, the goal of our study was to evaluate the participation of macrophage inflammatory protein-1 (MIP-1) family members (CCL3, CCL4, CCL9) in a streptozotocin (STZ)-induced mouse model of diabetic neuropathic pain. Single intrathecal administration of each MIP-1 member (10, 100, or 500 ng/5 µl) in naïve mice evoked hypersensitivity to mechanical (von Frey test) and thermal (cold plate test) stimuli. Concomitantly, protein analysis has shown that, 7 days following STZ injection, the levels of CCL3 and CCL9 (but not CCL4) are increased in the lumbar spinal cord. Performed additionally, immunofluorescence staining undoubtedly revealed that CCL3, CCL9, and their receptors (CCR1 and CCR5) are expressed predominantly by neurons. In vitro studies provided evidence that the observed expression of CCL3 and CCL9 may be partially of glial origin; however, this observation was only partially possible to confirm by immunohistochemical study. Single intrathecal administration of CCL3 or CCL9 neutralizing antibody (2 and 4 µg/5 µl) delayed neuropathic pain symptoms as measured at day 7 following STZ administration. Single intrathecal injection of a CCR1 antagonist (J113863; 15 and 20 µg/5 µl) also attenuated pain-related behavior as evaluated at day 7 after STZ. Both neutralizing antibodies, as well as the CCR1 antagonist, enhanced the effectiveness of morphine in STZ-induced diabetic neuropathy. These findings highlight the important roles of CCL3 and CCL9 in the pathology of diabetic neuropathic pain and suggest that they play pivotal roles in opioid analgesia.

Efficacy, safety and tolerability of the CCR1 antagonist BAY 86-5047 for the treatment of endometriosis-associated pelvic pain: a randomized controlled trial.

INTRODUCTION: Antagonism of CC chemokine receptor type 1 (CCR1) may provide a novel treatment approach for women with symptomatic endometriosis. Studies of CCR1 antagonists in these patients have not been reported. MATERIAL AND METHODS: Women (n = 110; 18-45 years) with symptomatic endometriosis were randomized to BAY 86-5047 or placebo for 12 weeks. Pelvic pain was assessed using the visual analogue scale (VAS) and women recorded the intake of pain medication in a diary. The primary efficacy outcome was a composite of the absolute change in VAS score and the cumulative change in consumption of analgesics between baseline and the end of treatment. Safety assessments included adverse events, blood and urine evaluation and electrocardiography. RESULTS: Mean VAS scores decreased from 64.8 mm at baseline to 49.2 mm at week 12 in the BAY 86-5047 group and from 67.2 mm to 47.8 mm in the placebo group. The proportion of women using analgesics decreased from 33.9% to 11.5% or from 44.4% to 15.4% for patients who received BAY 86-5047 or placebo, respectively. There was no significant difference between the two treatment groups in terms of change in VAS scores (p = 0.45) or intake of analgesics (p = 0.82). A three-step sensitivity analysis failed to show superiority of BAY 86-5047 over placebo (p = 0.67). BAY 86-5047 was well tolerated and no significant safety concerns arose during the study. CONCLUSIONS: Based on these results, BAY 86-5047 is unlikely to be useful in the treatment of women with endometriosis-associated pelvic pain.

Pharmacological Blockade of the Chemokine Receptor CCR1 Protects Mice from Systemic Candidiasis of Hematogenous Origin.

Systemic candidiasis is a leading cause of nosocomial bloodstream infection with a high mortality rate despite treatment. Immune-based strategies are needed to improve outcomes. We previously reported that genetic deficiency in the chemokine receptor CCR1 improves survival and ameliorates tissue damage in Candida-infected mice. Here, we found that treatment of immunocompetent Candida-infected mice with the CCR1-selective antagonist BL5923 improves survival, decreases the kidney fungal burden, and protects from renal tissue injury.

Treatment for rheumatoid arthritis by chemokine blockade.

Chemokines induce migration of inflammatory cells. In the synovial tissue of rheumatoid arthritis (RA), abundant chemokines are expressed, which contribute migration of lymphocytes and monocytes/macrophages, stimulation of synovial cells, and angiogenesis. Blockade of CCL2, CCL3, CCL5, CCR1, CCR9, CXCL2, CXCL5, CXCL13, CXCL16, CXCR3, CXCR4, CXCR7, and CX3CL1 showed improvement of arthritis of animal models. Moreover, CCR1 antagonist and anti-CXCL10 antibody reduced arthritis of patients with RA. Chemokine is a promising target for RA therapy.

CCR1 antagonism attenuates T cell trafficking to omentum and liver in obesity-associated cancer.

Obesity is a global health problem presenting serious risk of disease fuelled by chronic inflammation, including type 2 diabetes mellitus, cardiovascular disease, liver disease and cancer. Visceral fat, in particular the omentum and liver of obese individuals are sites of excessive inflammation. We propose that chemokine-mediated trafficking of pro-inflammatory cells to the omentum and liver contributes to local and subsequent systemic inflammation. Oesophagogastric adenocarcinoma (OAC) is an exemplar model of obesity and inflammation driven cancer. We have demonstrated that T cells actively migrate to the secreted factors from the omentum and liver of OAC patients and that both CD4(+) and CD8(+) T cells bearing the chemokine receptor CCR5 are significantly more prevalent in these tissues compared to matched blood. The CCR5 ligand and inflammatory chemokine MIP-1α is also secreted at significantly higher concentrations in the omentum and liver of our OAC patient cohort compared to matched serum. Furthermore, we report that MIP-1α receptor antagonism can significantly reduce T cell migration to the secreted factors from OAC omentum and liver. These novel data suggest that chemokine receptor antagonism may have therapeutic potential to reduce inflammatory T cell infiltration to the omentum and liver and in doing so, may ameliorate pathological inflammation in obesity and obesity-associated cancer.

Involvement of CC Chemokine Receptor 1 and CCL3 in Acute and Chronic Inflammatory Pain in Mice.

Chemokines are chemotactic cytokines whose involvement in nociceptive processing is being increasingly recognized. Based on the previous description of the involvement of CC chemokine receptor type 1 (CCR1) in pathological pain, we have assessed the participation of CCR1 and its endogenous ligands CCL3 and CCL5 in hyperalgesia and allodynia in mice after acute inflammation with carrageenan and chronic inflammation with complete Freund's adjuvant (CFA). The subcutaneous administration of the CCR1 antagonist J113863 (3-30 mg/kg; 30 min. before) dose dependently inhibited carrageenan- and CFA-evoked thermal hyperalgesia and mechanical allodynia produced by CFA, but not by carrageenan. The maximal dose of J113863 did not modify the increase in paw thickness induced by carrageenan or CFA. An almost ten times augmentation of CCL3 levels was detected by ELISA assays in both carrageenan and CFA paws, but not in spinal cords of inflamed mice, whereas CCL5 concentrations remained unaltered. Accordingly, a marked increase of CCL3 mRNA expression was observed in inflamed paws, with CCL3 protein detected in neutrophils and macrophages by immunohistochemical experiments. The intraplantar administration of an anti-CCL3 antibody (0.3-3 µg) blocked thermal hyperalgesia in carrageenan- and CFA-inflamed mice as well as CFA-evoked mechanical allodynia. Our data suggest that the increased concentrations of CCL3 present in inflamed tissues can be involved in acute and chronic inflammatory hyperalgesia as well as in chronic mechanical allodynia, and that these hypernociceptive symptoms can be counteracted by its neutralization with an antibody or by the blockade of CCR1 receptors.

Metabolism, Excretion and Pharmacokinetics of MLN3897, a CCR1 Antagonist, in Humans.

UNLABELLED: MLN3897 is a small molecule antagonist of the C-C chemokine receptor-1. Since preclinical studies showed that the molecule was metabolized into two halves, the metabolism, excretion, and pharmacokinetics of MLN3897 were investigated in humans using MLN3897 14C-radiolabeled either on the chlorophenyl (CP) or the tricyclic (TC) half of MLN3897 after an oral dose. OBJECTIVE: To evaluate the mass balance, metabolism and pharmacokinetics of MLN3897 in two cohorts of six randomized healthy subjects. METHOD: After receiving informed consent, subjects were dosed after an overnight fast of 10-hours followed by at least 4- hours after dosing on day-1. Each cohort received a single 29 mg oral dose of either the CP or the TC as an oral solution in water. Serial blood samples, urine and feces were collected over a 10-day period post-dose. RESULTS: For both radiolabeled moieties, 55-59% of the dose was recovered in feces and 32% recovered in urine. MLN3897 was metabolized extensively in humans, with minor amounts of intact MLN3897 detected in the urine and feces. N-oxidation of the tricyclic moiety (M28) and N-dealkylation of the piperidinyl moiety were the primary metabolic pathways leading to further formation of the carboxylic acid metabolite (M19) and the (4-(4-chlorophenyl)-3,3- dimethylpiperidin-4-ol) metabolite (M40). Oxidative metabolites M11, M19, M28, M44 were present at >10% of the total circulating radioactivity and also at >25% of MLN3897 exposure. Metabolites resulting from the chlorophenyl-labeled moiety (M40) had significantly more systemic exposure compared to the tricyclic-labeled moiety (M19).