MIP-1α-CCR1 ( Ki = 1 nM ); RANTES-CCR1 ( Ki = 2.8 nM ); MCP-3-CCR1 ( Ki = 5.5 nM )
CC chemokine receptor-1 (CCR1): It can displace CCR1 ligands including macrophage inflammatory protein-1α (MIP-1α), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity, and the Ki values range from 1 nM to 5.5 nM[1]
- Murine CCR1: The non-peptide antagonist BX471 can displace radiolabeled MIP-1α from murine CCR1[2]
- Human CCR1: BX471 can inhibit the ability of MIP-1α/CCL3 to increase Ca²⁺ transients in HEK 293 cells expressing human CCR1[2]

In vitro activity: BX471 is a highly effective functional antagonist due to its capacity to impede several CCR1-mediated processes, such as leukocyte migration, extracellular acidification rate increase, Ca2+ mobilization, and CD11b expression. BX471 exhibits a selectivity for CCR1 that is more than 10,000 times greater than that of 28 G-protein-coupled receptors[1]. With a Ki of 215±46 nM, BX471 can also, in a concentration-dependent manner, replace 125I-MIP-1α/CCL3 binding to mouse CCR1. BX471 attenuates the Ca2+ transients induced by MIP-1α/CCL3 in mouse and human CCR1 at IC50 values of 198±7 nM and 5.8±1 nM, respectively[2]. In isolated blood monocytes, BX471 (0.1–10 μM) exhibits a dose-dependent inhibition of shear-resistant and RANTES-mediated adhesion on IL-1β-activated microvascular endothelium in shear flow. Moreover, BX471 prevents T cells from adhering to activated endothelium via the RANTES pathway[4].

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BX471 is a potent functional antagonist. It can inhibit a series of CCR1-mediated effects, such as Ca²⁺ mobilization, the increase in extracellular acidification rate, CD11b expression, and leukocyte migration. Moreover, BX471 shows a selectivity of more than 10,000-fold for CCR1 when compared with 28 G-protein-coupled receptors[1]
- In HEK cells transfected with murine CCR1, BX471 can displace radiolabeled MIP-1α from murine CCR1. When Fluo-3-loaded HEK 293 cells expressing human and murine CCR1 are pretreated with increasing concentrations of BX471 for 15 minutes and then stimulated with the CCR1 agonist MIP-1α/CCL3, BX471 can inhibit the ability of MIP-1α/CCL3 to increase Ca²⁺ transients in these cells[2]
- In an in vitro flow assay over microvascular endothelium, BX471 can block the firm adhesion of monocytes triggered by RANTES on inflamed endothelium[4]
- For wild-type mice, pretreatment with the specific CCR1 antagonist BX471 can suppress the infiltration of neutrophils and macrophages in a 7-day mouse model of renal ischemia-reperfusion injury[3]

BX471 (4 mg/kg, p.o. or i.v.) has a 60% bioavailability when taken orally in dogs. Moreover, BX471 successfully lowers the disease in a multiple sclerosis rat experimental allergic encephalomyelitis model[1]. BX471 (20 mg/kg, s.c.) rapidly declines to approximately 0.4 μM after two hours, reaching peak plasma levels of 9 μM by about thirty minutes. The drug's plasma levels decrease to 0.1 μM or less after 4 to 8 hours. For ten days, mice given 20 mg/kg of BX471 had a roughly 55% decrease in interstitial CD45 positive leukocytes. The quantity of peripheral blood CCR5-positive CD8 cells is slightly impacted by BX471. In UUO kidneys, BX471 lowers the number of FSP1-positive cells by 65% when compared to vehicle control[2]. Following ischemia-reperfusion injury, pretreatment with BX471 decreases the accumulation of neutrophils and macrophages in the kidney[3]. Efficacy of BX 471 in a Rat EAE Model of Multiple Sclerosis [1]
Before determining the efficacy of BX 471 in a rat EAE model of multiple sclerosis, we tested its ability to inhibit MIP-1α binding to rat CCR1 receptors. Scatchard analysis of competition binding studies with BX 471 demonstrated that the compound was able to inhibit chemokine binding to rat CCR1 with a K i of 121 ± 60 nm (data not shown), which was approximately 100 times less effective for rat CCR1 than for human CCR1. In addition, BX 471 does not inhibit chemokine binding to rat CCR5 (data not shown) and is thus specific for rat CCR1. [1]
From pharmacokinetic studies in rats, we determined that the subcutaneous administration of BX 471 three times a day would give blood drug levels of 1 to 5 μm (data not shown), which we calculated would be about 10–50 times the K i on rat CCR1 receptors and should be sufficient for inhibition of MIP-1α binding. Unfortunately in contrast to the pharmacokinetic data obtained in the dog, BX 471 was poorly orally available in the rat (<20%, data not shown). Based on these studies, a rat EAE model of multiple sclerosis was set up. Animals were dosed subcutaneously three times a day with vehicle or with BX 471 at 5, 20, and 50 mg/kg. The CCR1 antagonist, BX 471, dose-dependently decreased the severity of the disease (Fig. 10 A). At the highest dose, 50 mg/kg, there was a marked reduction in the clinical score that was statistically significant at p = 0.05 (analyzed by analysis of variance) compared with the vehicle control. However, even at the lower two doses of BX 471, 20 mg/kg and 5 mg/kg, there were still noticeable decreases in the clinical score. This is more readily observed by expressing the data as the average accumulated clinical score per treatment group (Fig. 10 B). Statistical analysis of the average accumulated clinical score data by t test revealed that the 50 and 20 mg/kg doses were statistically significant compared with the vehicle control p = 0.003 andp = 0.014, respectively.

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BX471 is effective in reducing disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis[1]
- In mice with unilateral ureter obstruction (UUO), compared with the control group, the UUO kidneys of mice treated with BX471 (from day 0 to 10 and day 6 to 10) show a 40%-60% reduction in interstitial macrophage and lymphocyte infiltration. The treated mice also have a significant decrease in the mRNA levels of CCR1 and CCR5, and FACS analysis reveals a corresponding reduction in CD8⁺/CCR5⁺ T cells. Markers of renal fibrosis, including interstitial fibroblasts, interstitial volume, and the mRNA and protein expression of collagen I, are all significantly reduced in BX471-treated mice compared with vehicle controls. However, the treatment is ineffective when the drug is administered only from day 0 to 5[2]
- In a rat heterotopic heart transplant rejection model, when animals are treated with BX471 combined with a subtherapeutic dose of cyclosporin (2.5 mg/kg) (which alone cannot prolong transplant rejection), the effect of prolonging transplant rejection is much better than that of animals treated with either cyclosporin or BX471 alone[4]
- In a 7-day mouse model of renal ischemia-reperfusion injury, by day 7, the injured kidneys of mice lacking CCR1 contain 35% fewer neutrophils and 45% fewer macrophages than those of wild-type control mice. Additionally, the injured kidneys of CCR1-deficient mice have lower levels of the CCR1 ligands CCL3 (MIP-1α) and CCL5 (RANTES) compared with wild-type controls. Local leukocyte proliferation and apoptosis are observed after injury but are not dependent on CCR1. Moreover, the extent of necrotic and fibrotic damage and the decline in renal function in injured kidneys are similar between wild-type and CCR1-deficient mice[3]

Chemokine Binding Studies [1]
Binding assays were performed by filtration as described previously. Radiolabeled chemokines at a final concentration of approximately 0.1–0.2 nm were used as ligand. HEK293 cells expressing human CCR1 at 8,000 or 300,000 cells per assay point were used as the receptor source. Nonspecific binding was determined in the presence of 100 nm unlabeled chemokine. The binding data were curve-fitted with the computer program IGOR to determine the affinity and number of sites.

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Cytosolic Ca2+ Measurements[1]
HEK293 cells expressing human CCR1 were plated on poly-d-lysine-coated black wall 96-well plates at 80,000 cells/well and were cultured overnight. Cells were then loaded with 4 μm Fluo-3, a calcium-sensitive fluorescence dye, for 60 min at 37 °C in Hanks' balanced salts solution containing 20 mm Hepes, 3.2 mm calcium chloride, 1% fetal bovine serum, 2.5 mm probenecid, and 0.04% pluronic acid. The excess dye was removed by gently washing cells 4 times with assay buffer (Hanks' balanced salts solution containing 20 mmHepes, 2.5 mm probenecid, and 0.1% bovine serum albumin) using a Denley washer. Changes in intracellular free Ca2+ concentration were measured with a FLIPR immediately after the addition of agonist at 37 °C. To examine the antagonistic activity of BX471, the cells were pretreated with the compound for 15 min before the addition of agonist. The intracellular Ca2+ concentration in nm was calculated based on the equation Ca2+= K D (F −F min)/(F max −F) (7). K D is the dissociation constant of the complex of Fluo-3 and Ca2+ (390 nm for Fluo-3). F is the measured fluorescence intensity.F max is the maximal fluorescence intensity determined in the presence of 0.1% triton X-100.F min is the minimum fluorescence intensity determined in the presence of 0.1% Triton X-100 plus 5 mmEGTA.

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BX471 (also known as ZK-811752) is a novel, oral and non-peptide CCR1 (CC chemokine receptor-1) antagonist that has a Ki of 1 nM for human CCR1. It may be helpful in the management of chronic inflammatory conditions. Compared to CCR2, CCR5, and CXCR4, BX471 shows a 250-fold preference for CCR1. When it comes to treating autoimmune disorders, CCR1 is a top therapeutic target.

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Competition binding assay for CCR1 ligands: Cells expressing CCR1 are incubated with radiolabeled CCR1 ligands (such as ¹²⁵I-MIP-1α/CCL3) along with different concentrations of BX471. After a certain period of incubation, the binding reactions are terminated by centrifuging the cells. The specific binding of the radiolabeled ligands to CCR1 is then measured, and the Ki values of BX471 for displacing the ligands are calculated based on the displacement data. Nonspecific binding is determined and subtracted from the total binding to obtain specific binding[2]
- Ca²⁺ mobilization assay: Cells expressing CCR1 (such as HEK 293 cells) are loaded with a Ca²⁺-sensitive fluorescent dye (like Fluo-3). The loaded cells are pretreated with various concentrations of BX471 for 15 minutes. Subsequently, the cells are stimulated with a CCR1 agonist (such as MIP-1α/CCL3). The changes in fluorescence intensity, which represent the changes in intracellular Ca²⁺ concentration, are measured over time to evaluate the inhibitory effect of BX471 on CCR1-mediated Ca²⁺ mobilization[2]

BX471 (0.1–10 μM) inhibits shear-resistant and RANTES-mediated adhesion on IL-1β-activated microvascular endothelium in shear flow in isolated blood monocytes in a dose-dependent manner. Additionally, T cells' RANTES-mediated adhesion to activated endothelium is inhibited by BX471. With a Ki of 215±46 nM, BX471 can also, in a concentration-dependent manner, replace 125I-MIP-1α/CCL3 binding to mouse CCR1. BX471 inhibits the Ca2+ transients induced by MIP-1α/CCL3 in both human and mouse CCR1, with IC50 values of 5.8±1 nM and 198±7 nM, respectively, as concentrations of the compound increase. The ability of BX 471 to block several CCR1-mediated processes, such as leukocyte migration, extracellular acidification rate increase, Ca2+mobilization, and CD11b expression, makes it a strong functional antagonist. BX 471 exhibits a selectivity for CCR1 that is more than 10,000 times greater than that of 28 G-protein-coupled receptors.

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CD11b Expression on Peripheral Blood Mononuclear Cells[1]
CD11b expressed on peripheral blood mononuclear cells in a whole blood assay was measured as described. Briefly, human whole blood was collected by venipuncture into 2.5-ml Vacutainer tubes containing EDTA. The blood was kept at room temperature and used immediately after phlebotomy. The whole blood samples (200 μl) were pretreated with or without 1 μm BX471 at 37 °C for 15 min followed by treatment with or without 100 nm MIP-1α for an additional 15 min. The reaction was terminated by the addition of 1 ml of cold phosphate-buffered salt solution wash. The tubes were centrifuged (200 × g for 7 min at 4 °C), and the supernatant was removed by aspiration. The cell pellet was resuspended in cold phosphate-buffered salt solution, 10 μl of 1 mg/ml heat-aggregated IgG was added, and the tubes were incubated for 10 min at 4 °C. Antibodies CD11b FITC (5 μl) and CD14 PE (20 μl) were added to each assay tube and incubated for 20 min at 4 °C. Finally, 1 ml of ice-cold phosphate-buffered salt solution was added, and the cells were pelleted as above and analyzed by FACScan.

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Leukocyte migration assay: A suitable cell migration system (such as a transwell chamber) is used. The lower chamber contains a chemoattractant that can induce leukocyte migration through CCR1. Different concentrations of BX471 are added to either the upper or both chambers. Leukocytes are placed in the upper chamber, and after incubation for a specific time, the number of leukocytes that migrate to the lower chamber is counted. The inhibitory effect of BX471 on CCR1-mediated leukocyte migration is assessed by comparing the migration numbers in the presence and absence of BX471[1]
- CD11b expression assay: Leukocytes are treated with a CCR1 agonist in the presence or absence of different concentrations of BX471. After incubation, the cells are stained with a fluorescently labeled antibody against CD11b. The expression level of CD11b on the leukocyte surface is detected using flow cytometry. The effect of BX471 on CCR1-mediated CD11b expression is analyzed by comparing the fluorescence intensity in the treated and control groups[1]
- Real-time RT-PCR for chemokine receptor mRNA: Total RNA is extracted from renal tissues (such as obstructed and contralateral kidneys of UUO mice). The RNA is reverse-transcribed into cDNA. Real-time PCR is performed using specific primers for CCR1, CCR2, CCR5, and a housekeeping gene (like GAPDH). The mRNA expression levels of the chemokine receptors are normalized to the expression level of GAPDH. The relative expression levels of the receptors in BX471-treated and control groups are compared to evaluate the effect of BX471 on chemokine receptor mRNA expression[2]
- FACS analysis for leukocyte subsets: Cells are isolated from renal tissues or blood. The cells are stained with fluorescently labeled antibodies against cell surface markers (such as CD45 for leukocytes, CD3 for lymphocytes, F4/80 for macrophages, Gr-1 for neutrophils, CD8 for CD8⁺ T cells, and CCR5). The stained cells are analyzed using flow cytometry. The percentages of different leukocyte subsets (such as CD45⁺ leukocytes, CD3⁺ lymphocytes, F4/80⁺ macrophages, Gr-1⁺ neutrophils, and CD8⁺/CCR5⁺ T cells) in BX471-treated and control groups are compared to assess the effect of BX471 on leukocyte infiltration and subset distribution[2, 3]
- Western blot for collagen I protein: Protein is extracted from renal tissues. The protein samples are separated by SDS-PAGE and transferred to a membrane. The membrane is incubated with a primary antibody against collagen I and then with a secondary antibody conjugated to an enzyme (such as horseradish peroxidase). The protein bands are visualized using a chemiluminescent substrate. The intensity of the collagen I bands is quantified, and the protein expression levels in BX471-treated and control groups are compared to evaluate the effect of BX471 on renal fibrosis[2]
- Immunohistochemical staining: Renal tissue sections are prepared. The sections are deparaffinized, rehydrated, and subjected to antigen retrieval. They are then incubated with primary antibodies against specific markers (such as CD45 for leukocytes, FSP1 for fibroblasts). After washing, the sections are incubated with a secondary antibody conjugated to a fluorescent or enzyme label. The stained sections are observed under a microscope, and the number of positive cells or the extent of staining is quantified to assess leukocyte infiltration and fibroblast accumulation in BX471-treated and control groups[2, 3]
- Naphthol AS-D chloroacetate esterase staining for neutrophils: Renal tissue sections are stained with naphthol AS-D chloroacetate esterase reagent. Neutrophils exhibit positive staining (orange-red granules). The stained sections are observed under a microscope, and the number of positive neutrophils is counted to evaluate the effect of BX471 on neutrophil infiltration[3]
- TUNEL assay for apoptosis: Renal tissue sections are prepared and processed for TUNEL staining using a TUNEL kit. The sections are incubated with terminal deoxynucleotidyl transferase and fluorescently labeled dUTP. Apoptotic cells show positive fluorescence. The number of TUNEL-positive cells is counted to assess the effect of CCR1 deficiency (related to BX471's target) on renal cell apoptosis[3]
- Ki67 staining for cell proliferation: Renal tissue sections are stained with a primary antibody against Ki67 (a cell proliferation marker) and then with a secondary antibody. Ki67-positive cells (cells in the proliferative phase) are visualized under a microscope. The number of positive cells is counted to evaluate the effect of CCR1 deficiency on renal cell proliferation[3]

Pharmacokinetics of BX 471 in dogs [1] This study investigated the oral bioavailability of BX 471 in conscious dogs. BX 471 was dissolved in 40% cyclodextrin saline at a dose of 4 mg/kg and administered to fasting male beagle dogs via cephalic intravenous bolus or gavage. Plasma samples were prepared and the concentration of the compound in plasma was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS). As shown in Figure 9, BX 471 reached peak plasma concentrations approximately 2 hours after oral administration and remained at measurable concentrations for up to 6 hours. The volume of distribution of BX 471 (0.5 L/kg) was close to that of body fluids (0.6 L/kg), indicating that the compound was mainly distributed in body fluids (Table III). The clearance rate in dogs was low, at 2 ml/min/kg (less than 10% of total hepatic blood flow), resulting in a moderate terminal half-life of 3 hours (Figure 9 and Table III). For dogs administered orally, the half-life of BX 471 is approximately 3 hours. Analysis of the area under the curve (AUC) using TOPFIT software to calculate the percentage of oral bioavailability indicates that BX 471 is an orally absorbed drug in fasting dogs with an oral bioavailability of approximately 60% (Figure 9 and Table III). In dogs, BX471 is orally active with an oral bioavailability of 60% [1]. In mice, after a single subcutaneous injection of 20 mg/kg BX471 (dissolved in 40% cyclodextrin), the plasma concentration of BX471 changed over time, showing certain pharmacokinetic characteristics (specific parameters such as half-life are not described in detail in the literature) [2].

Effects of BX 471 on systemic toxicity [1]
To demonstrate that the antagonistic effect of BX 471 on CCR1 was not caused by the cytotoxicity of the compound, we treated HEK293 cells transfected with THP-1 or CCR1 at a concentration of up to 10 μM of BX 471 for 24 hours and monitored cytotoxicity by WST-1 staining. No obvious toxicity was observed (data not shown). We further tested the toxicity of BX 471 in vivo by performing a series of serum diagnostic tests, including liver and kidney function tests and blood electrolyte tests, on rabbits that were given BX 471 at a dose of 20 mg/kg/day for 30 consecutive days. All test results were within the normal range (data not shown). The results indicate that the inhibition of CCR1 activation by BX 471 was not due to cytotoxicity and that long-term use of the drug had no adverse effects on the normal physiological functions of animals. Information on the median lethal dose, hepatotoxicity, nephrotoxicity, drug interactions, and plasma protein binding rate of BX471 was not provided in the literature [1, 2, 3, 4].

[1]. Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1. J Biol Chem. 2000 Jun 23;275(25):19000-8.

[2]. A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation. J Clin Invest. 2002 Jan;109(2):251-9.

[3]. Chemokine receptor CCR1 regulates inflammatory cell infiltration after renal ischemia-reperfusion injury. J Immunol. 2008 Dec 15;181(12):8670-6.

[4]. A non-peptide functional antagonist of the CCR1 chemokine receptor is effective in rat heart transplant rejection. J Biol Chem. 2001 Feb 9;276(6):4199-204.

CC chemokine receptor 1 (CCR1) is a major target for the treatment of autoimmune diseases. Through high-throughput screening and chemical optimization, we identified a novel non-peptide CCR1 antagonist, RN-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl]urea hydrochloride (BX 471). Competitive binding assays showed that BX 471 can replace CCR1 ligands macrophage inflammatory protein-1α (MIP-1α), RANTES, and monocyte chemoattractant protein-3 (MCP-3) with high affinity (K_i ranging from 1 nM to 5.5 nM). BX 471 is a potent functional antagonist that inhibits multiple CCR1-mediated effects, including Ca²⁺ mobilization, increased extracellular acidification, CD11b expression, and leukocyte migration. BX 471 is more than 10,000 times more selective for CCR1 than 28 G protein-coupled receptors. Pharmacokinetic studies have shown that BX 471 is orally active with a bioavailability of 60% in dogs. In addition, BX 471 effectively alleviates disease symptoms in a rat model of experimental allergic encephalomyelitis (multiple sclerosis). This study is the first to demonstrate the efficacy of a non-peptide chemokine receptor antagonist in an animal model of an autoimmune disease. In summary, we have discovered a potent, selective and orally effective CCR1 antagonist that may help treat chronic inflammatory diseases. [1] The expression of chemokines and their receptors is thought to be associated with leukocyte infiltration and progressive renal fibrosis following unilateral ureteral obstruction (UUO). We hypothesize that blocking the chemokine receptor CCR1 with the non-peptide antagonist BX471 can reduce leukocyte infiltration and renal fibrosis following UUO. In mice with unexplained renal fibrosis (UUO) treated with BX471 (days 0-10 and 6-10), interstitial macrophage and lymphocyte infiltration was reduced by 40-60% compared to the control group. CCR1 and CCR5 mRNA levels were also significantly reduced in the treatment group, and FACS analysis showed a corresponding decrease in CD8+/CCR5+ T cell numbers. Compared to the vector control group, BX471 treatment significantly reduced renal fibrosis markers such as interstitial fibroblasts, interstitial volume, and type I collagen mRNA and protein expression. Conversely, administration only on days 0-5 was ineffective. In conclusion, blocking CCR1 significantly reduced cell infiltration and renal fibrosis following UUO. Importantly, delayed administration was also effective. Therefore, we conclude that CCR1 blockade may represent a novel therapeutic strategy for reducing cell infiltration and renal fibrosis, which are major factors contributing to the progression of end-stage renal failure. [2] Neutrophils and macrophages rapidly infiltrate the kidneys after renal ischemia-reperfusion injury, but the specific molecular recruitment mechanisms of these cell types have not been fully elucidated. This study uses genetic and pharmacological evidence to demonstrate that the chemokine receptor CCR1 plays a positive role in macrophage and neutrophil infiltration in a 7-day mouse model of renal ischemia-reperfusion injury. By day 7, the number of neutrophils and macrophages in the damaged kidneys of CCR1-deficient mice was reduced by 35% and 45%, respectively, compared to wild-type control mice. Pretreatment of wild-type mice with the specific CCR1 antagonist BX471 also inhibited neutrophil and macrophage infiltration in this model. Compared to wild-type control mice, the levels of CCR1 ligands CCL3 (MIP-1α) and CCL5 (RANTES) in the damaged kidneys of CCR1-deficient mice were also reduced, suggesting that these inflammatory chemokines originate from leukocytes and that a CCR1-dependent positive feedback loop for leukocyte infiltration exists in this model. Local leukocyte proliferation and apoptosis were detected after injury, but these processes were not dependent on CCR1. In addition, the degree of necrosis and fibrosis of the damaged kidneys and the decline in renal function were similar in wild-type mice and CCR1-deficient mice. Therefore, in a mouse model of renal ischemia-reperfusion injury, CCR1 appears to regulate the migration of macrophages and neutrophils to the kidneys, but this activity does not appear to affect tissue damage. [3]

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Chemokines such as RANTES appear to play a role in organ transplant rejection. Since RANTES is a potent agonist of the chemokine receptor CCR1, we investigated the efficacy of the CCR1 receptor antagonist BX471 in a rat model of heterotopic heart transplant rejection. Animals were treated with BX471 in combination with a subtherapeutic dose of cyclosporine (2.5 mg/kg). Although treatment with cyclosporine or BX471 alone did not effectively prolong transplant rejection, its efficacy was far superior to that of treatment with cyclosporine or BX471 alone. We investigated the mechanism of action of CCR1 antagonists through in vitro microvascular endothelial cell flow experiments and found that the antagonist could block the strong adhesion of RANTES-induced monocytes to inflammatory endothelial cells. These data together indicate that CCR1 plays an important role in allogeneic transplant rejection. [4]

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BX471 is a novel non-peptide CCR1 antagonist. CCR1 is a key target for the treatment of autoimmune diseases. This study is the first to demonstrate that non-peptide chemokine receptor antagonists are effective in animal models of autoimmune diseases, suggesting that BX471 may be beneficial for the treatment of chronic inflammatory diseases [1]
- The expression of chemokines and their receptors is considered to be associated with leukocyte infiltration and progressive renal fibrosis after unilateral ureteral obstruction (UUO). Using BX471 to block CCR1 can significantly reduce cell aggregation and renal fibrosis after UUO, and delayed treatment is also effective. Therefore, CCR1 blockade may be a novel therapeutic strategy to reduce cell infiltration and renal fibrosis, which are important factors leading to end-stage renal failure [2]. Neutrophils and macrophages rapidly infiltrate the kidneys after renal ischemia-reperfusion injury, but the specific molecular recruitment mechanisms of these cell types have not been fully elucidated. BX471, as a specific CCR1 antagonist, can modulate the migration of macrophages and neutrophils to the kidneys in a mouse model of renal ischemia-reperfusion injury, but this effect does not appear to affect tissue damage [3]. Chemokines such as RANTES appear to play a role in organ transplant rejection. Since RANTES is a potent agonist of the chemokine receptor CCR1, BX471, as a CCR1 antagonist, may play an important role in allogeneic transplant rejection, especially when used in combination with subtherapeutic doses of cyclosporine [4].
- Compared with the wild-type control group, the levels of CCR1 ligands CCL3 (MIP-1α) and CCL5 (RANTES) in the damaged kidneys of CCR1-deficient mice were lower, suggesting that leukocytes are the source of these inflammatory chemokines and that there is a CCR1-dependent leukocyte infiltration positive feedback loop in the renal ischemia-reperfusion injury model [3].

C21H24CLFN4O3

434.89

434.152

C, 58.00; H, 5.56; Cl, 8.15; F, 4.37; N, 12.88; O, 11.04

217645-70-0

BX471 hydrochloride; 288262-96-4

512282

White to off-white solid powder

1.3±0.1 g/cm3

593.5±50.0 °C at 760 mmHg

312.8±30.1 °C

0.0±1.7 mmHg at 25°C

1.617

2.77

2

5

6

30

591

1

O=C(N)NC1=CC(Cl)=CC=C1OCC(N2[C@H](C)CN(CC3=CC=C(F)C=C3)CC2)=O

XQYASZNUFDVMFH-CQSZACIVSA-N

InChI=1S/C21H24ClFN4O3/c1-14-11-26(12-15-2-5-17(23)6-3-15)8-9-27(14)20(28)13-30-19-7-4-16(22)10-18(19)25-21(24)29/h2-7,10,14H,8-9,11-13H2,1H3,(H3,24,25,29)/t14-/m1/s1

[5-chloro-2-[2-[(2R)-4-[(4-fluorophenyl)methyl]-2-methylpiperazin-1-yl]-2-oxoethoxy]phenyl]urea

ZK811752; ZK 811752; ZK-811752; BX471; [5-chloro-2-[2-[(2R)-4-[(4-fluorophenyl)methyl]-2-methylpiperazin-1-yl]-2-oxoethoxy]phenyl]urea; 76K17ZG4ZN; BX 471; BX-471

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.75 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.78 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.78 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5 mg/mL (11.50 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)