A metabolically-stabilized phosphonate analog of lysophosphatidic acid attenuates collagen-induced arthritis

Abstract

Rheumatoid arthritis (RA) is a destructive arthropathy with systemic manifestations, characterized by chronic synovial inflammation. Under the influence of the pro-inflammatory milieu synovial fibroblasts (SFs), the main effector cells in disease pathogenesis become activated and hyperplastic while releasing a number of signals that include pro-inflammatory factors and tissue remodeling enzymes. Activated RA SFs in mouse or human arthritic joints express significant quantities of autotaxin (ATX), a lysophospholipase D responsible for the majority of lysophosphatidic acid (LPA) production in the serum and inflamed sites. Conditional genetic ablation of ATX from SFs resulted in attenuation of disease symptoms in animal models, an effect attributed to diminished LPA signaling in the synovium, shown to activate SF effector functions. Here we show that administration of 1-bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl-phosphonate (BrP-LPA), a metabolically stabilized analog of LPA and a dual function inhibitor of ATX and pan-antagonist of LPA receptors, attenuates collagen induced arthritis (CIA) development, thus validating the ATX/LPA axis as a novel therapeutic target in RA.

Figure 1. BrP-LPA inhibits the ATX-mediated hydrolysis of natural LPC substrates in vitro.

A–C. Steady-state hydrolysis of LPC (16:0, 18:0, 18:1 respectively) by recombinant ATX as measured with the TOOS activity assay. D–E. ATX activity inhibition dose-response curves in the presence of various BrP-LPA concentrations as measured with the TOOS assay using as substrates 50 μΜ of 16:0 (D) and 18:0 LPC (E). F–G. Cornish-Bowden (F) and Lineweaver-Burk (G) plots show that BrP-LPA is a competitive ATX inhibitor. H. The kinetic parameters (k_m, V_max) obtained by the incubation of ATX at various concentrations of 16:0 LPC (100–400 μΜ) in the absence or presence of BrPLPA (0–10 μΜ). All presented values are the means (±std) of two independent experiments.

Figure 2. BrP-LPA inhibits ATX activity ex vivo.

A. Effect of BrP-LPA on plasma ATX/lysoPLD hydrolysis of exogenous added 50 μΜ LPC (14:0, 16:0, 18:0). B. Correlation of recovered BrP-LPA in whole blood with the added BrP-LPA. C. Inhibition of endogenous ATX/lysoPLD activity in whole blood ex vivo after the addition of increasing BrP-LPA concentrations (0–10 μΜ). ATX activity was measured in the presence of 1 mM LPC with the TOOS assay. D. LPC levels and E. LPA levels measured in whole blood ex vivo in the absence/presence of different BrP-LPA concentrations (0.03–10 μΜ). F. Per cent residual levels of the indicated LPA species in the presence of increasing amounts of BrP-LPA. Solid lines, best fits of averaged data points. * indicates significant (p<0.05), *** indicates significant (p<0.001) decrease relative to control group.

Figure 3. BrP-LPA inhibits ATX activity in vivo.

A. Plasma BrP-LPA pharmacokinetic profile following i.p administration of 10 mg/Kg BrP-LPA in female mice. B. Plasma per cent (%) residual ATX activity measured in the presence of 1 mM LPC with the TOOS assay and C. Plasma % residual total LPA levels at different time points after i.p. administration of BrP-LPA. D. Plasma concentration of different LPA and E. LPC species at different time points following BrP-LPA i.p administration. The time point 0 refers to the vehicle control. The presented values are the means (±std) of two independent experiments. *(p<0.05) and **(p<0.01) indicate a significant decrease relative to control group.

Figure 4. BrP-LPA attenuates collagen induced arthritis.

A. Schematic representation of immunization and drug administration schemes. DBA/1 mice subjected to CIA were treated intraperitoneally with vehicle, BrP-LPA (10 mg/kg), or dexamethasone (Dex, 3 mg/kg) twice a week. B–C. Time dependent clinical scores (± SEM) after primary immunization with CII. Data are pooled from two different experiments (n = 10). D. Representative sections of the forelimb and hindlimb joints of vehicle-treated and BrP-LPA-treated mice, stained with H&E. E. Quantification of disease severity in vehicle-treated and BrP-LPA-treated mice. Joint sections were assessed histologically in a blinded manner by three independent examiners. Data are shown as mean joint values per mouse ± SD. F. Humoral response to collagen in vehicle-treated or BrP-LPA-treated mice. Levels of CII-specific Abs were determined by ELISA in mouse sera. Results show the mean ± SEM values. *(p<0.05) and **(p<0.01) indicate a significant decrease relative to control group.