Cell experiment:

Transfected mesangial cells (MCs) are serum restricted for 24 h, after which the medium is replaced by serum-free DMEM containing normal glucose (NG; 5.6 mM), NG+Mannitol (NG+M; 24.4 mM), or high glucose (HG; 30 mM). In addition, nontransfected MCs are cultured under NG, NG+M, or HG with or without RS102895 or anti-TGF-1 antibody (25 μg/mL). Nontransfected MCs are also exposed to medium containing recombinant human MCP-1 (10 ng/mL) or recombinant human TGF-1 (2 ng/mL). At 24 h after the media change, cells are harvested and the conditioned culture media are collected[2].

Animal experiment:

Rats[3]CCR2 antagonist RS102895 is dissolved in DMSO. Rats receive daily intrathecal injection of either RS102895 (3 g/L) 10 μL or 10 % DMSO 10 μL between 9 and 20 days after operation. All rats are randomLy divided into five groups (n = 10 per group): Sham group, Sham + RS102895 group, BCP group, BCP + RS102895 group, and BCP + DMSO group. Rats are sacrificed 20 days after operation and the tissue samples from the L4-L5 spinal cord segments are rapidly removed and immediately frozen in liquid nitrogen and stored at ?80℃ until use for RT-PCR and Western blot[3].

RS102895 is a potent CCR2 antagonist, with an IC50 of 360 nM, and shows no effect on CCR1. CCR2|360 nM (IC50)|CCR1|17800 nM (IC50)|Human α1a receptor|130 nM (IC50)|Human α1d receptor|320 nM (IC50)|5HT-1a receptor|470 nM (IC50)

RS102895 is a potent CCR2 antagonist, with an IC50 of 360 nM, and shows no effect on CCR1. RS102895 also inhibits human α1a and α1d receptors, rat brain cortex 5HT1a receptor in cells with IC50s of 130, 320, 470 nM, respectively. RS102895 suppresses wild type and D284N mutant MCP-1 receptor (IC50, 550 nM and 568 nM, respectively), less potently inhibits D284A MCP-1 receptor (IC50, 1892 nM), and has no effects on E291A, E291Q, D284A/E291A or D284N/E291Q (IC50, >100,000 nM)[1]. RS102895 ameliorates the increased extracellular matrix (ECM) protein expression by inhibition of CCR2 at 10 μM, and obviously blocks fibronectin and type IV collagen protein expression in high glucose (HG)-stimulated mesangial cells (MCs) at 1 or 10 μM. RS102895 (10 μM) also abrogates the increased TGF-1 levels in MCs treated with MCP-1[2].

RS102895 (3 g/L) causes progressive decrease in pain threshold in rats with bone cancer pain (BCP) at day 3-9 after surgery via intrathecal injection, but the pain threshold increases after 12 days. RS102895 also potently reverses the pattern of NR2B, nNOS, and SIGIRR expression in spinal cord[3].

[1]. Mirzadegan T, et al. Identification of the binding site for a novel class of CCR2b chemokine receptor antagonists: binding to a common chemokine receptor motif within the helical bundle. J Biol Chem. 2000 Aug 18;275(33):25562-71. [2]. Park J, et al. MCP-1/CCR2 system is involved in high glucose-induced fibronectin and type IV collagen expression in cultured mesangial cells. Am J Physiol Renal Physiol. 2008 Sep;295(3):F749-57. [3]. Ren F, et al. Analgesic Effect of Intrathecal Administration of Chemokine Receptor CCR2 Antagonist is Related to Change in Spinal NR2B, nNOS, and SIGIRR Expression in Rat with Bone Cancer Pain. Cell Biochem Biophys. 2015 Jun;72(2):611-6.