Small molecule antagonist of C-C chemokine receptor 1 (CCR1) reduces disc inflammation in the rabbit model

Abstract

Background context: Targeting chemokines or chemokine receptors is a promising treatment strategy for diseases with chronic inflammation such as rheumatoid arthritis and discogenic pain. Identifying specific molecules and determining their effectiveness in animal models are the first steps in developing these treatments. Macrophage markers have been detected in the intervertebral disc tissues of patients with disc degenerative disease and discogenic pain and in different animal models. Macrophage recruitment into the disc may play a role in initiation of inflammation and if unresolved may lead to chronic inflammation and subsequent back pain.

Purpose: The objectives of these studies are to (1) identify chemokine receptor antagonists that can block macrophage migration induced by disc cells in vitro and (2) determine if intradiscal treatment with these antagonists can reduce disc inflammation and degeneration in vivo.

Study design: In vitro migration assays were used to test effectiveness of chemokine receptor antagonists to block macrophage migration induced by disc cells. The rabbit annular puncture model was used to test for anti-inflammatory and regenerative effects of chemokine receptor antagonist treatment in vivo.

Methods: In vitro - THP-1 human monocytic cell line and freshly isolated rabbit primary splenocytes were assayed for migration using 3 µm Corning Transwell inserts with conditioned media of interleukin (IL)-1β treated human or rabbit disc cells. Inhibition of macrophage migration was evaluated using different concentrations of small molecule antagonists of C-C chemokine receptor (CCR)1 and CCR2. In vivo - New Zealand White rabbits (n=40) underwent disc puncture and intradiscal treatment with saline, CCR1 or CCR2 antagonists within the same procedure. X-ray and magnetic resonance (MR) images and serum samples were taken for disc height, MRI grade and IL-8 serum level analyses. Intervertebral discs were isolated for RNA analysis of inflammatory and disc phenotypic markers and for immunohistochemical analysis of macrophage marker, RAM11. The outcome measures were compared between the three treatment groups. These studies were funded by a research grant from AO Foundation, Switzerland (Project no S-14-86A; 120000 CHF). CCR1 and CCR2 antagonists were kindly provided by ChemoCentryx (Mountain View, CA).

Results: In vitro migration assays showed that THP-1 migration induced by disc cells was blocked by CCR2 antagonist more effectively than CCR1 antagonist, while rabbit splenocyte migration was inhibited by CCR1 antagonist and not the other. In the rabbit annular puncture model, rabbit discs treated with CCR1 antagonist had significantly better MRI grades than those treated with CCR2 antagonist at 6 weeks post-treatment. Gene expression studies demonstrate that discs treated with CCR1 or CCR2 antagonists expressed less inflammatory markers than saline-treated discs at 3 weeks post-treatment. Although CCR2 antagonist treatment did not reduce inflammatory marker expression at 6 weeks, discs treated with CCR1 antagonist expressed less inflammatory markers and also a higher ratio of collagen type 2 to collagen type 1 genes indicating favorable disc matrix production. There were no significant differences between all three treatment groups in regards to disc height indexes, IL-8 serum levels or macrophage marker detection.

Conclusions: These studies have identified that small molecule antagonists against CCR2 and CCR1 were respectively effective in blocking THP-1 and rabbit splenocyte migration induced by disc cells in vitro. Further, both CCR2 and CCR1 antagonist intradiscal treatments were effective in reducing disc inflammation at an early time point of 3 weeks. Lastly, only CCR1 antagonist demonstrated anti-inflammatory effects and better MRI grades at 6 weeks.

Clinical significance: Our preclinical studies demonstrate that CCR1 and CCR2 antagonist delivery through intradiscal injection is sufficient to reduce disc inflammation at early time points, whereas CCR1 antagonists had longer term anti-inflammatory effects. Clinical studies have found that CCR1 antagonist was safe, tolerable and clinically active in reducing inflammation in rheumatoid arthritis patients. These studies suggest that CCR1 antagonist may be a promising biological treatment to reduce disc inflammation that translates to back pain relief.

Keywords: Animal model; Cell migration; Chemokine; Chemokine receptor; Disc degeneration; Disc inflammation; Small molecule antagonist.