MIP-1α-CCR1 ( Ki = 1 nM ); RANTES-CCR1 ( Ki = 2.8 nM ); MCP-3-CCR1 ( Ki = 5.5 nM )

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 proportion of FSP1-positive cells by 65% when compared to the vehicle control. Pretreatment with BX471 decreases the accumulation of neutrophils and macrophages in the kidney following ischemia-reperfusion injury.Orally active, BX 471 (4 mg/kg, p.o. or i.v.) has a 60% bioavailability in dogs. Additionally, in a rat model of experimental allergic encephalomyelitis associated with multiple sclerosis, BX 471 effectively lowers disease.

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.

In summary, dermal microvascular endothelial cells cultured to confluence in Petri dishes are stimulated with IL-1β (10 ng/mL) for a duration of 12 hours, and immediately before the assay, they are pre-incubated with RANTES (10 nM) for 30 minutes at 37°C. The plates are mounted on the stage of an Olympus IMT-2 inverted microscope with ×20 and ×40 phase-contrast objectives, and they are assembled as the lower wall of a parallel wall flow chamber. Separated human blood monocytes are resuspended at a density of 5×10⁵ cells/mL in assay buffer (HBSS) that has 0.5% human serum albumin, 10 mM HEPES, and a pH of 7.4. Addition of 1 mM Mg²⁺ and 1 mM Ca²⁺ occurs shortly before the assay. Cell suspensions are perfused into the flow chamber for five minutes at a rate of 1.5 dyn/cm² while being maintained in a heating block at 37°C for the assay. Monocytes undergoing inhibition experiments are first preincubated for 10 minutes at 37°C with either a Me₂SO control or BX471 at varying concentrations (0.1–10 μM). Expressed as cells/mm², the number of firmLy adherent cells after 5 min is quantified in multiple fields (at least five per experiment) through image analysis using a JVC SR L 900 E video recorder and a long integration JVC 3CCD video camera. Primary adhesion, or the direct interactions between monocytes and endothelium, is the only type of adhesion that is examined.

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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.

EAE Study in Lewis Rats [1]
Male Lewis rats were immunized subcutaneously into both hind footpads with 50 μl of a guinea pig spinal cord homogenate. The guinea pig spinal cord homogenate was prepared by homogenizing guinea pig (male Hartley) whole spinal cords and adjusting the concentration to 1 g/ml with 0.9% physiological saline. This homogenate was then diluted (1:1) with complete Freund's adjuvant containing 1 mg/ml Mycobacterium tuberculosis. One day after immunization the animals were injected subcutaneously 3 times/day with increasing doses of the CCR1 antagonist BX471 (5, 20, and 50 mg/kg) dissolved in 40% cyclodextrin insaline solution or with the vehicle as a control. There were 10 animals per treatment group. Rats were weighed, and clinical symptoms were evaluated on a daily basis throughout the study and scored as follows: 0, no symptoms; 1, complete tail paralysis; 2, paraparesis, abnormal gait; 3, paralysis of one hind limb; 4, paralysis of both hind limbs; 5, moribund or dead. Clinical score data were analyzed using an analysis of variance and Fisher's least significant difference.

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Male beagle dogs that have been fattened (n = 3 per treatment group) are administered BX471 orally or intravenously (IV) at a dose of 4 mg/kg through the cephalic vein. 40% aqueous cyclodextrin is used as a vehicle in which to dissolve the compound. An in-dwelling catheter is used to draw blood serially from the jugular vein at predetermined intervals up to six hours after dosing. One use for EDTA is as an anticoagulant. The samples undergo a centrifugation process (1000× g for 10 min at 4°C), and the plasma is kept frozen until HPLC-MS (electrospray mode operated under a positive ion mode) is used to analyze the drug levels. Four parts ice-cold methanol containing a fixed amount of an internal standard are added to one part plasma to thaw and denature the samples. Following a centrifugation at 5000× g to remove the resultant protein precipitate, the supernatants are immediately analyzed. The BX471 plasma calibration standards are prepared across the quantification range concurrently, processed, and analyzed in the same way. Utilizing an electrospray inlet operated at 3.57 kV, a FISONS, VG Platform single quadrupole instrument is employed in these analyses. After a brief isocratic elution procedure (35% methanol, 65% water containing 0.1% trifluoroacetic acid), chromatographic separation is achieved using a YMC AQ octadecyl silane reversed phase column (4.6×250 mm). The mass spectrometer receives 50 μL/min of infusion by splitting the total column flow (1 mL/min) post-column. After injecting 50 microliters of solution onto the column, the chromatograms are obtained over a total run time of 7.5 minutes per sample. A solitary ion positive ionization mode is used to gather the ions. Ion current ratios between the internal standard peak and the analyte in the plasma standards are plotted over the quantification range to create a calibration curve for quantification. Inferred from the area under the curve measurements is the percentage of oral availability. WinNonLin version 3.0 is utilized to compute pharmacokinetic parameters.

Pharmacokinetics of BX 471 in Dogs [1]
The oral bioavailability of BX 471 was examined in conscious dogs. BX 471 was administered to fasted male beagle dogs at 4 mg/kg in a vehicle of 40% cyclodextrin in saline by bolus intravenous injection via the cephalic vein or by oral gavage. The plasma samples were prepared, and compound concentrations in the plasma were determined by HPLC-MS. As shown in Fig.9, BX 471 reached peak plasma levels approximately 2 h after oral dosing and maintained measurable concentrations for up to 6 h. BX 471 exhibits a volume of distribution (0.5 l/kg) close to the volume of body water (0.6 l/kg), suggesting that the compound is confined primarily to the aqueous volume (Table III). Low clearance, 2 ml/min/kg (which represents less than 10% of the total liver blood flow) in the dog resulted in a moderate terminal half-life of 3 h (Fig. 9 and Table III). For dogs that were orally dosed, the half-life for BX 471 was approximately 3 h. Calculations of percent oral availability using area under curve measurements obtained from analysis using TOPFIT software indicated that BX 471 is an orally absorbed drug in fasted dogs with an oral bioavailability of approximately 60% (Fig.9 and Table III).

Effect of BX 471 on General Toxicity [1]
To demonstrate that CCR1 antagonism by BX 471 was not due to the cellular toxicity of the compound, THP-1- or CCR1-transfected HEK293 cells were treated with BX 471 at concentrations up to 10 μm for 24 h, and cellular toxicity was monitored by measuring WST-1 staining. No significant toxicity was observed (data not shown). The toxicity for BX 471 was further examined in vivo by a battery of serum diagnostic tests including hepatic and renal function tests and blood electrolytes on rabbits that had been dosed with BX 471 at 20 mg/kg/day for 30 days. The test results all fell within the normal range (data not shown). The results suggest that the inhibition of BX 471 on CCR1 activation was not due to cellular toxicity and that chronic treatment with the drug had no adverse effects on the normal physiology of the animals.

[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.

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases. [1]

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The expression of chemokines and their receptors is thought to contribute to leukocyte infiltration and progressive renal fibrosis after unilateral ureter obstruction (UUO). We hypothesized that blocking the chemokine receptor CCR1 using the nonpeptide antagonist BX471 could reduce leukocyte infiltration and renal fibrosis after UUO. UUO kidneys from BX471-treated mice (day 0-10 and day 6-10) revealed a 40-60% reduction of interstitial macrophage and lymphocyte infiltrate compared with controls. Treated mice also showed a marked reduction of CCR1 and CCR5 mRNA levels, and FACS analysis showed a comparable reduction of CD8+/CCR5+ T cells. Markers of renal fibrosis, such as interstitial fibroblasts, interstitial volume, mRNA and protein expression for collagen I, were all significantly reduced by BX471-treatment compared with vehicle controls. By contrast treatment was ineffective when the drug was supplied only from days 0 to 5. In summary, blockade of CCR1 substantially reduces cell accumulation and renal fibrosis after UUO. Most interestingly, late onset of treatment is also effective. We therefore conclude that CCR1 blockade may represent a new therapeutic strategy for reducing cellular infiltration and renal fibrosis as major factors in the progression to end-stage renal failure.[2]

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Neutrophils and macrophages rapidly infiltrate the kidney after renal ischemia-reperfusion injury, however specific molecular recruitment mechanisms have not been fully delineated for these cell types. Here we provide genetic and pharmacologic evidence supporting a positive role for the chemokine receptor CCR1 in macrophage and neutrophil infiltration in a 7 day mouse model of renal ischemia-reperfusion injury. By day 7, injured kidneys from mice lacking CCR1 contained 35% fewer neutrophils and 45% fewer macrophages than injured kidneys from wild-type control mice. Pretreatment of wild-type mice with the specific CCR1 antagonist BX471 also suppressed neutrophil and macrophage infiltration in the model. Injured kidneys from mice lacking CCR1 also had reduced content of the CCR1 ligands CCL3 (MIP-1alpha) and CCL5 (RANTES) compared with injured kidneys from wild-type controls, suggesting a leukocyte source for these inflammatory chemokines and existence of a CCR1-dependent positive feedback loop for leukocyte infiltration in the model. Local leukocyte proliferation and apoptosis were detected after injury, but were not dependent on CCR1. Also, the extent of necrotic and fibrotic damage and decline in renal function in injured kidneys was similar in wild-type and CCR1-deficient mice. Thus, CCR1 appears to regulate trafficking of macrophages and neutrophils to kidney in a mouse model of renal ischemia-reperfusion injury, however this activity does not appear to affect tissue injury.[3]

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Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection. [4]

C21H25CL2FN4O3

471.35

470.129

C, 53.51; H, 5.35; Cl, 15.04; F, 4.03; N, 11.89; O, 10.18

288262-96-4

BX471; 217645-70-0

5311124

White to yellow solid powder

4.346

3

5

6

31

591

1

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

FRUCNQBAWUHKLS-PFEQFJNWSA-N

InChI=1S/C21H24ClFN4O3.ClH/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);1H/t14-;/m1./s1

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: ≥ 3 mg/mL (6.36 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 30.0 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: ≥ 3 mg/mL (6.36 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 30.0 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: ≥ 3 mg/mL (6.36 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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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