CCR1
AZD 4818 targets CCR1, a chemokine receptor involved in the recruitment of inflammatory cells. It is a highly potent and selective CCR1 antagonist with a pIC50 of 8.6. By inhibiting CCR1, it blocks the chemotaxis of human monocytes to MIP-1α. This modulates aberrant immune responses implicated in conditions such as COPD and systemic lupus erythematosus (SLE).

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].
In vitro, AZD 4818 inhibits chemotaxis of human monocytes to MIP-1α in a concentration-dependent manner. It is a highly potent and selective CCR1 antagonist with a pIC50 of 8.6. The compound's ability to inhibit CCR1-mediated chemotaxis makes it a valuable tool for studying inflammatory diseases.

In vivo, AZD 4818 is used for the treatment of chronic obstructive pulmonary disease (COPD). By selectively inhibiting CCR1 signaling, it aims to modulate aberrant immune responses. It may also have applications in other inflammatory conditions such as systemic lupus erythematosus (SLE).

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.
The activity of AZD 4818 can be assessed using cell-based chemotaxis assays. Human monocytes are placed in a chamber with a membrane separating them from a solution containing MIP-1α. The number of cells that migrate across the membrane in the presence or absence of varying concentrations of AZD 4818 is counted. The IC50 for inhibition of chemotaxis is determined from dose-response curves.

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 (GraphPad Prism version 6.0). For Table 3, the chemotactic index was averaged for triplicate experiments with each compound.
To evaluate the cellular effects of AZD 4818, monocytes or other immune cells are treated with the compound, and the inhibition of MIP-1α-induced signaling is measured. This can include the measurement of calcium mobilization, MAP kinase activation, or other downstream signaling events. The effects on cell migration are assessed using chemotaxis assays.

In vivo studies with AZD 4818 typically involve administration to animals or humans via oral or parenteral routes. In models of COPD or other inflammatory diseases, the compound's effects on inflammation, immune cell infiltration, and disease progression are assessed.

AZD 4818 has a molecular formula of C27H32Cl2N2O7 and a molecular weight of 567.46. Its CAS number is 1003566-93-5. The compound is a chemokine CCR1 antagonist. It is soluble in DMSO and other organic solvents. The purity is typically >98%.

Specific toxicology data for AZD 4818 are not detailed in the available literature. However, as an immune modulator, it may have effects on the immune system. As with all research compounds, standard safety precautions should be taken during handling.

[1]. AZD-4818, a chemokine CCR1 antagonist: WO2008103126 and WO2009011653. Expert Opin Ther Pat. 2009;19(11):1629-1633.

[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.

AZD4818 has been used in clinical trials for the treatment of chronic obstructive pulmonary disease (COPD). We confirmed using HEK_CCR1Gqi5 cell membranes that AZD-4818, BX471, CCX354, CP481715, and PS899877 are all potent inhibitors of [125I]-CCL3 binding (Figure 1; Table 2). For BX471, CP481715, and PS899877, our results are similar to previous reports in HEK cells (Anders et al., 2002; Vallet et al., 2007; Merritt et al., 2010). We then detected these compounds using RPMI 8226 cell membranes (Figure 2; Table 2). While two compounds exhibited comparable potency to the cell membranes of both cell lines (BX471 and PS899877), other compounds showed a preference for the recombinant HEK_CCR1 system. For AZD4818, CCX354, and CP481715, when tested using a Welch-corrected unpaired t-test, their affinity for the HEK_CCR1 cell membrane was significantly higher than that for the RPMI 8226 cell membrane (P < 0.05). Furthermore, the potency ranking varied significantly across different cell membranes. Using HEK_CCR1 cell membranes, we found that MLN3879 > CCX354 ≥ AZD4818 > CP481715 = BX471 > PS899877; while using RPMI 8226 cell membranes, we found that MLN3879 > BX471 > CP481715 ≥ PS899877 > AZD4818 > CCX354. [1]
AZD 4818 is a highly potent and selective CCR1 antagonist used in research on inflammatory diseases. It inhibits chemotaxis of human monocytes to MIP-1α. It has been investigated for the treatment of COPD and other inflammatory conditions. It is not a clinically approved drug.

C27H32CL2N2O7

567.46

566.159

C, 57.15; H, 5.68; Cl, 12.50; N, 4.94; O, 19.74

1003566-93-5

1003566-93-5

24955007

White to off-white solid powder

4.317

3

8

9

38

838

1

CC(C(O)=O)(OC1=C(Cl)C=C(C(NC)=O)C(OC[C@@H](O)CN2CCC3(CC4=CC(Cl)=CC=C4O3)CC2)=C1)C

HVTUHSABWJPWNK-SFHVURJKSA-N

InChI=1S/C27H32Cl2N2O7/c1-26(2,25(34)35)37-23-12-22(19(11-20(23)29)24(33)30-3)36-15-18(32)14-31-8-6-27(7-9-31)13-16-10-17(28)4-5-21(16)38-27/h4-5,10-12,18,32H,6-9,13-15H2,1-3H3,(H,30,33)(H,34,35)/t18-/m0/s1

2-[2-chloro-5-[(2S)-3-(5-chlorospiro[3H-1-benzofuran-2,4'-piperidine]-1'-yl)-2-hydroxypropoxy]-4-(methylcarbamoyl)phenoxy]-2-methylpropanoic acid

AZD4818; AZD-4818; AZD-4818; 1003566-93-5; UNII-S5UP6P540K; S5UP6P540K; 2-(2-Chloro-5-(((2S)-3-(5-chloro-2,3-dihydrospiro(benzofuran-2,4'-piperidin)-1'-yl)-2-hydroxypropyl)oxy)-4-((methylamino)carbonyl)phenoxy)-2-methylpropanoic acid; AZD-4818(AZD4818); AZD 4818

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)

DMSO: ~50 mg/mL (~88.1 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.41 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: ≥ 2.5 mg/mL (4.41 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 25.0 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: ≥ 2.5 mg/mL (4.41 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 25.0 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.)