Randomized controlled studies on the efficacy of antiarthritic agents in inhibiting cartilage degeneration and pain associated with progression of osteoarthritis in the rat

Abstract

Background: As an initial step in the development of a local therapeutic to treat osteoarthritis (OA), a number of agents were tested for their ability to block activation of inflammation through nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), subchondral bone changes through receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclastogenesis, and proteolytic degradation through matrix metalloproteinase (MMP)-13 activity. Candidates with low toxicity and predicted efficacy were further examined using either of two widely accepted models of OA joint degeneration in the rat: the monoiodoacetic acid (MIA) model or the medial meniscal tear/medial collateral ligament tear (MMT/MCLT) model.

Methods: Potential therapeutics were assessed for their effects on the activation of nuclear factor (NF)-κB, RANKL-mediated osteoclastogenesis, and MMP-13 activity in vitro using previously established assays. Toxicity was measured using HeLa cells, a synovial cell line, or primary human chondrocytes. Drugs predicted to perform well in vivo were tested either systemically or via intraarticular injection in the MIA or the MMT/MCLT model of OA. Pain behavior was measured by mechanical hyperalgesia using the digital Randall-Selitto test (dRS) or by incapacitance with weight bearing (WB). Joint degeneration was evaluated using micro computed tomography and a comprehensive semiquantitative scoring of cartilage, subchondral bone, and synovial histopathology.

Results: Several agents were effective both in vitro and in vivo. With regard to pain behavior, systemically delivered clonidine was superior in treating MIA-induced changes in WB or dRS, while systemic clonidine, curcumin, tacrolimus, and fluocinolone were all somewhat effective in modifying MMT/MCLT-induced changes in WB. Systemic tacrolimus was the most effective in slowing disease progression as measured by histopathology in the MMT/MCLT model.

Conclusions: All of the agents that demonstrated highest benefit in vivo, excepting clonidine, were found to inhibit MMP-13, NF-κB, and bone matrix remodeling in vitro. The MIA and MMT/MCLT models of OA, previously shown to possess inflammatory characteristics and to display associated pain behavior, were affected to different degrees by the same drugs. Although no therapeutic was remarkable across all measures, the several which showed the most promise in either model merit continued study with alternative dosing and therapeutic strategies.

Fig. 1

a–h Tumor necrosis factor (TNF)-stimulated nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activity relative to media alone, with or without drug. The ratio of the NF-κB activity of viable cells to the activity detected without exposure to TNF-α (fold stimulation above basal untreated levels of activity with or without drug). Controls: Media = untreated HeLa cells; Media + TNF = cells treated with TNF-α; Media + Enbrel = cells treated with TNF-α in the presence of Enbrel, a known TNF inhibitor; Test = cells treated with TNF-α in the presence of different concentrations of drug. Shown are resultsFollowing one-way analysis of variance with the drugs tested in vivo (i.e., clonidine, curcumin, fluocinolone, meloxicam, tacrolimus, tranilast, triamcinolone hexacetonide, and withaferin). Following one-way analysis of variance, pairwise comparisons with the media TNF control were made using a standard two-tailed t test. *p ≤ 0.05. Additional data is provided in Additional file 1

Fig. 2

a–i The effect of tested drugs (alendronate, clonidine, curcumin, fluocinolone, meloxicam, tacrolimus, tranilast, triamcinolone hexacetonide, and withaferin) on osteoclast differentiation and resorption. The lowest concentrations tested are shown; additional data is provided in Additional file 1. Following one-way analysis of variance, pairwise comparisons with the tumor necrosis factor control were made using a standard two-tailed t test. *p ≤ 0.05, **p ≤ 0.001. RANK receptor activator of NF-κB

Fig. 3

a–d The effect of tested drugs (alendronate, clonidine, curcumin, fluocinolone, meloxicam, tacrolimus, tranilast, triamcinolone hexacetonide, and withaferin) on matrix metalloproteinase (MMP)-13 activity of the chondrogenic pellets. The lowest concentrations tested are shown. Additional data is provided in Additional file 1. Following one-way analysis of variance, pairwise comparisons with the tumor necrosis factor (TNF) control were made using a standard two-tailed t test. *p ≤ 0.05

Fig. 4

Representative frontal images from the intraarticular (IA) delivery studies in the monoiodoacetic acid (MIA; original magnification × 16) and medial meniscal tear/medial collateral ligament tear (MMT/MCLT; original magnification × 25) rat models (28 and 21 days postinjury, respectively). Shown are images from animals treated with saline, 4.5 mg of clonidine, 150 mg of triamcinolone, 30 ng of tacrolimus, 15 ng of fluocinolone, 100 mg of meloxicam, or 30 mg of curcumin. MIA: M medial, L lateral, S synovium, large arrows affected cartilage surface, small arrows osteophyte. MMT/MCLT: M marrow, large arrows affected cartilage surface, small arrows osteophyte. Top right: Comparisons of mean cartilage matrix damage and total joint scores with matrix (a–d). a Trial 2 MIA systemic study (n = 10 per group). b Trial 3 MIA IA study (n = 10 per group). c Trial 1 MMT/MCLT systemic study (n = 8). d Trial 2 MMT/MCLT IA study (n = 10). Toluidine blue–stained sections from knees of animals treated with test compounds were analyzed for proteoglycan and cartilage matrix loss, subchondral bone resorption, sclerosis, and osteophyte formation as well as synovitis (see Additional file 1). Femoral and tibial cartilage degeneration scores were summed for total cartilage scores (blue). Total joint scores also included osteophyte analysis (red)

Fig. 5

Micro computed tomographic (μCT) analysis of knee joints from rats treated with monoiodoacetic acid (MIA) in trial 2 (n = 5). Vehicle ipsilateral is the injured joint, and vehicle contralateral is the untreated control joint, of the vehicle-treated animal. Comparison of joints analyzed by μCT from five animals of each group in MIA trial 2. Note that only four knees from the clonidine group were analyzed. a Relative bone volume fraction (bone volume/total volume). b Connectivity density (1/mm³). c Trabecular thickness (distance in millimeters). d Trabecular spacing (distance in millimeters). One-way analysis of variance was used to look for differences between groups. If p ≤ 0.05, then Bonferroni’s post hoc test was performed to identify significant differences between groups