CCR1

In chemotaxis tests, CCX354 has strong action with an IC50 of less than 100 nM[1].

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Researchers compared several well-studied CCR1 antagonists including AZD4818, BX471, CCX354, CP-481715, MLN-3897 and PS899877 for their ability to inhibit binding of [(125)I]-CCL3 in vitro using membranes prepared from RPMI 8226 cells, a human multiple myeloma cell line that endogenously expresses CCR1. In addition, antagonists were assessed for their ability to modulate CCL3-mediated internalization of CCR1 and CCL3-mediated cell migration using RPMI 8226 cells. As many GPCRs signal through β-arrestin-dependent pathways that are separate and distinct from those driven by G-proteins, Researchers also evaluated the compounds for their ability to alter β-arrestin translocation. Key results: There were clear differences between the CCR1 antagonists in their ability to inhibit CCL3 binding to myeloma cells, as well as in their ability to inhibit G-protein-dependent and -independent functional responses. Conclusions and implications: Our studies demonstrate that tissue phenotype seems to be relevant with regards to CCR1. Moreover, it appears that for CCR1 antagonists, inhibition of β-arrestin translocation is not necessarily linked to chemotaxis or receptor internalization[2].

CCX354 demonstrates superior pharmacokinetic properties and in vivo anti-inflammatory activity [1].

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The safety and pharmacokinetic (PK)/pharmacodynamic (PD) profile of the novel CCR1 antagonist CCX354 was evaluated in double-blind, placebo-controlled, single- and multiple-dose phase I studies (1-300 mg/day oral doses). CCX354 was well tolerated and displayed a linear dose-exposure profile, with half-life approaching 7 h at the 300-mg dose. The extent of CCR1 receptor blockade on blood monocytes, which correlated well with plasma concentrations of the drug, was assessed using fluorescently labeled CCL3 binding in whole blood from phase I subjects. High levels of receptor coverage at the 12-h time point were achieved after a single dose of 100 mg CCX354. Preclinical studies indicate that effective blockade of inflammatory cell infiltration into tissues requires ≥90% CCR1 inhibition on blood leukocytes at all times. The comparison of the properties of CCX354 with those published for other CCR1 antagonists has informed the dose selection for ongoing clinical development of CCX354 in rheumatoid arthritis (RA)[3].

Binding assay [2]
Membranes were prepared from HEK_CCR1 or RPMI 8226 cells as previously described (Gilchrist et al., 1998) and stored in aliquots at −80°C until needed. For the competition assays, membranes were suspended at 10 μg mL−1 in HEM buffer comprised of 50 mM HEPES, pH 7.5, 1 mM EDTA and 5 mM MgCl2. Compounds were serially diluted in HEM buffer to a 10× concentration and then added to the membrane mixture. A final concentration of 2 pM [125I]-CCL3 was added and the tubes were incubated at 37°C, shaking, for 2 h. Initial experiments were performed to determine that equilibrium had been reached at the 2 h time point (data not shown). The bound and free radioligands were separated by filtration through Whatman GF/C filter paper soaked in TEM2 buffer comprised of 20 mm Tris-HCl, pH 7.4, 0.5 mM EDTA and 5 mM MgCl2 supplemented with 0.3% polyethyleneimine and 20 mg·mL−1 BSA using a tissue harvester. Filters were washed twice with ice-cold TEM2 buffer and then counted (1 min per sample) using a Packard Gamma Counter. Binding assays were performed in duplicate, and non-specific binding was determined by adding cold CCL3 (100 nM) at the same time as the radioligand to some samples. Data from binding experiments were analysed by non-linear regression analysis to determine IC50 values. For saturation experiments, we used 10 μg mL−1 of membrane prepared from HEK_CCR1 or RPMI 8226 cells and increasing concentrations of [125I]-CCL3 (0–100 pM) with and without cold CCL3 (100 nM) to define the non-specific binding. Binding experiments were carried out at 37°C for 2 h and the KD and Bmax values were determined using GraphPad Prism version 6.0. Experiments were conducted in duplicate and repeated as indicated in Table 2.

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PathHunter β-arrestin translocation assay [2]
To quantitatively assess β-arrestin translocation, we utilized an enzyme fragment complementation format from DiscoveRx Corporation in which CCR1 is fused to a ProLink™ peptide derived from β-galactosidase, and β-arrestin 2 is fused to an N-terminal deletion mutant of β-galactosidase [enzyme acceptor (EA) ]. Following addition of CCL3, the β-arrestin–EA fusion protein binds activated CCR1-ProLink. PathHunter hCCR1_CHO cells were plated in 96-well half volume white plates at 1 × 104 cells per well in Optimem and allowed to attach overnight. Initial experiments established an EC50 of 200 pM for CCL3. Serial dilutions of the antagonists were made in Optimem and added to triplicate wells. The plates were incubated at 37°C with 5% CO2 for 90 min and then increasing concentrations of CCL3 were added and the plates incubated at 37°C with 5% CO2 for an additional 60 min. Detection reagent was added and the plate incubated for 60 min at room temperature. The plates were read using the luminescent setting on a DTX800 multimode plate reader. The pKB values were determined using the Gaddum/Schild equation on GraphPad Prism version 6.0. Experiments were conducted in triplicate and repeated as indicated in Table 3.

Receptor internalization assay [2]
Receptor internalization experiments were performed using flow cytometry. Staining for surface CCR1 and CCR5 was performed as recommended by the manufacturer of the PE-conjugated anti-CCR1 and FITC conjugated anti-CCR5). Briefly, 5 × 106 RPMI 8226 cells were suspended in RPMI 1640 media with 1% FBS and incubated with various concentrations of antagonist. After 15 min, the cells were activated with CCL3 (1 nM) and incubated for 2 h at 37°C. After being washed with RPMI 1640 media with 1% FBS, cells were suspended in PBS with 1% FBS and 10 μL of each mAb was added. Thereafter, cells were incubated for 30 min in the dark at 4°C. Following two washes with PBS with 1% FBS, samples were analysed on a BD FACScalibur flow cytometer using Cell Quest software. At least 10 000 events were acquired. Healthy populations were identified and gated on FITC versus PE plots. For the cell surface expression figure, the percentage of cells in the upper left quadrant (high CCR1/low CCR5) without CCL3 exposure was set to 1.0 and the fold change in fluorescence of the 1 nM CCL3 only (control), and 1 nM CCL3 with increasing concentrations of CCR1 antagonist are shown. To calculate the IC50 values (Table 3), the samples with 1 nM CCL3 (no antagonist) were set to 1.0, and a non-linear regression analysis was run using GraphPad Prism version 6.0. Experiments were repeated as indicated in Table 3.

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Chemotaxis assay [2]
MultiScreen®-Migration Invasion and Chemotaxis filter plates with 8 μm pore size were used for all chemotaxis experiments. We utilized the fluorescent dye Calcein AM to label RMPI 8226 cells. The cells were washed once with HBSS supplemented with 10 mM HEPES (HHBSS) and suspended at 4 × 106 cells mL−1. Calcein AM (2 μM) was added, and the cells were incubated for 30 min at 37°C. Cells were then washed twice with HHBSS before being suspended in HHBSS at 2 × 106 cells mL−1. Labelled cells (50 μL) were added to the top chambers along with antagonists (or vehicle controls). The bottom chambers contained 150 μL control media (HHBSS) or chemoattractant (1 nM CCL3 in control media). The plate was placed in the 37°C incubator for 3 h. After 3 h, 50 μL of the cell-containing media were removed from the bottom chamber and transferred to a 96-well black plate with a clear bottom. This plate was read using EX490/EM520 on a DTX800 multimode plate reader. For chemotaxis, each point was performed in quadruplicate and the number of cells that migrated spontaneously to the chemotaxis buffer was subtracted. The chemotactic index was determined by dividing the number of migrated cells in the presence of the antagonist by the number of cells that migrated spontaneously to CCL3. Experiments were performed in quadruplicate and IC50 values calculated using non-linear regression analysis. For Table 3, the chemotactic index was averaged for triplicate experiments with each compound.

[1]. Compounds for the treatment of osteoporosis and cancers. US 20100113472 A1.

[2]. Identifying bias in CCR1 antagonists using radiolabelled binding, receptor internalization, β-arrestin translocation and chemotaxis assays. Br J Pharmacol. 2014 Nov;171(22):5127-38.

[3]. Pharmacokinetic and pharmacodynamic evaluation of the novel CCR1 antagonist CCX354 in healthy human subjects: implications for selection of clinical dose. Clin Pharmacol Ther. 2011 May;89(5):726-34.

CCX354-C has been used in trials studying the treatment of Rheumatoid Arthritis.

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Background and purpose: Investigators have suggested that the chemokine receptor CCR1 plays a role in multiple myeloma. Studies using antisense and neutralizing antibodies to CCR1 showed that down-regulation of the receptor altered disease progression in a mouse model. More recently, experiments utilizing scid mice injected with human myeloma cells demonstrated that the CCR1 antagonist BX471 reduced osteolytic lesions, while the CCR1 antagonist MLN-3897 prevented myeloma cell adhesion to osteoclasts. However, information is limited regarding the pharmacology of CCR1 antagonists in myeloma cells.[2]

C22H22CLN7O2

451.908782482147

451.152

C, 58.47; H, 4.91; Cl, 7.84; N, 21.70; O, 7.08

1010073-75-2

135565361

Off-white to light yellow solid powder

1.5±0.1 g/cm3

778.4±60.0 °C at 760 mmHg

424.6±32.9 °C

0.0±2.7 mmHg at 25°C

1.724

1.24

1

6

5

32

650

0

ZIMLRKWQDLVPEK-UHFFFAOYSA-N

InChI=1S/C22H22ClN7O2/c1-32-18-13-15(4-5-17(18)23)28-9-11-29(12-10-28)19(31)14-30-22-16(3-2-6-26-22)20(27-30)21-24-7-8-25-21/h2-8,13H,9-12,14H2,1H3,(H,24,25)

1-[4-(4-chloro-3-methoxyphenyl)piperazin-1-yl]-2-[3-(1H-imidazol-2-yl)pyrazolo[3,4-b]pyridin-1-yl]ethanone

CCX354; 1010073-75-2; CCX-354; CCR1 antagonist 1; 22PQS5K5TY; GSK-2941266; CCX354-C; Ethanone, 1-(4-(4-chloro-3-methoxyphenyl)-1-piperazinyl)-2-(3-(1H-imidazol-2-yl)-1H-pyrazolo(3,4-b)pyridin-1-yl)-;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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