INCB3344 is an effective, specific and orally bioavailable CCR2 antagonist with IC50 values of 9.5 nM (mCCR2) and 5.1 nM (hCCR2) in binding antagonism and 7.8 nM (mCCR2) and in 3.8 nM (hCCR2) antagonism of chemotaxis activity.

CCR2 (mouse):9.5 nM, CCR2 (human):5.1 nM

INCB3344的药理活性首先通过利用小鼠单核细胞系WEHI-274.1，通过全细胞结合试验，评估其抑制CCL2与CCR2结合的能力来进行表征。在该试验中，INCB3344的结合IC50被确定为10 nM，而在90 nM的浓度下观察到>90%的结合抑制[1]。INCB3344还是对大鼠和猕猴CCR2的有效拮抗剂，其结合拮抗IC50值分别为7.3和16 nM，化学趋化活性拮抗的IC50值分别为2.7和6.2 nM。此外，INCB3344对超过50种离子通道、转运蛋白、趋化因子受体及其他选定的GPCRs展示出>1 μM的IC50值。它也是一种选择性的mCCR2拮抗剂，对最与mCCR2同源的两种趋化因子受体，即murine CCR1和murine CCR5表现出>1 μM和>3 μM的IC50值[4]。

INCB3344能够防止去氧皮质酮醋酸酯(DOCA)/盐诱导的血管CCR2表达变化。在一系列独立实验中，从DOCA/盐处理周期的第7天到第21天接受INCB3344的小鼠主动脉中CCR2表达提高约1.5倍，与安慰剂动物相比明显较低；但是，这一CCR2表达水平显著低于仅接受载体处理组的水平。同样，接受INCB3344的小鼠中DOCA/盐处理引起的其受体配体CCL2表达增加被减弱。相比之下，接受载体或INCB3344的DOCA/盐处理小鼠中CCL7、CCL8和CCL12的水平均有类似程度的提高[2]。通过静脉给药给CD-1小鼠时，INCB3344表现出高清除率和适中的分布体积，导致半衰期短，仅为1小时。尽管清除率高，但通过口服给药仍能获得良好的暴露效果，10 mg/kg剂量的AUC为2664 nM h。口服生物利用度为47%。相比之下，以相同剂量口服给Balb/c小鼠时获得略好的口服暴露（AUC=3888 nM h）[4]。

Cells (5×10^5) in RPMI 1640 (VWR), +0.1% BSA+20 mM HEPES (VWR), are added to various concentrations of INCB3344 in RPMI 1640 followed immediately by the addition of 150 pM 125I-labeled mCCL2 (mouse CCL2(JE)) and incubated for 30 min at room temperature (RT). For the nonspecific control, 0.3 μM mCCL2 is added in place of INCB3344. Cells are then harvested through 1.2-μm polyvinylidene difluoride filters, the filters are air-dried, and binding is determined by counting in a gamma counter. Antagonist activity is reported as the inhibitor concentration required for IC50 of specific binding. Specific binding is defined as the total binding minus the nonspecific binding and typically represents 97% of the total binding [2].

WEHI-274.1 cells (5×10^5) in RPMI 1640 (VWR) with or without various concentrations of INCB3344 in RPMI 1640 are loaded in the wells on top of an 8-μm polycarbonate filter in a 96-well-modified Boyden chamber. Beneath the filter, 30 nM mCCL2 with or without INCB3344 or media is placed in a corresponding 96-well plate. The sealed chambers are incubated for 45 min at 37°C, 5% CO2. Filters are washed, stained with Wright-Giemsa, and the number of cells that migrate toward mCCL2 in the bottom chamber counted by microscopy. The ability of INCB3344 to antagonize CCR2-mediated chemotaxis is reported as the inhibitor concentration required for IC50 values of specific migration to mCCL2. Specific migration is defined as the total migration minus the background migration. A similar assay is used to determine the impact of INCB3344 on CCR1-mediated chemotaxis of WEHI-274.1 cells, by using mouse MIP-1α as a ligand. In addition C5a, FMLP and RANTES are similarly tested in the presence of INCB3344 for migration of WEHI-274.1 cells. For the studies on the impact of INCB3344 on CCR5-mediated chemotaxis, murine T cells are used as the cell system with mouse MIP-1β as the ligand [2].

In a subset of experiments, DOCA/salt-treated mice are further randomly assigned to receive the CCR2 antagonist, INCB3344 (30 mg/kg per day) or vehicle (10% DMSO/0.9% carboxymethylcellulose) via daily intraperitoneal injections commencing 10 days after induction of hypertension and continuing until the end of the 21-day treatment period. The normotensive control group for these experiments consists of sham-treated mice that receive a vehicle from days 10 to 21 [3]. Adult male Sprague-Dawley rats (200-250 g) are used. After t=0 baseline measurement, rats are lightly anesthetized under an isoflurane/oxygen (5%; 2 L/min) flow and 25 μL of either saline (vehicle), 1 μg of CCL2 and/or 1 mM of INCB3344 is administered intrathecally between L5 and L6 vertebrae. Animals are tested once at 30, 60, 90, 120, and 240 min following drug administration. The percentage of maximal potential effect is calculated for every time point [4].