Potent antiarthritic properties of a glucocorticoid derivative, NCX-1015, in an experimental model of arthritis

Abstract

Here, we describe the improved antiarthritic properties of a nitric oxide-releasing derivative of prednisolone that includes a sparing of the effects on bone. Glucocorticoids are widely used in the treatment of chronic inflammatory pathologies, but their use is often accompanied by side effects, including osteoporosis. Recently, a new steroid able to release low levels of nitric oxide showed potent inhibition of leukocyte trafficking and chemokine generation in models of acute inflammation. The objective of this study was to assess the anti-inflammatory activity of this nitric-oxide releasing glucocorticoid, nitro-prednisolone (NCX-1015), in parallel with the parent compound prednisolone and a control molecule lacking an NO group, (NCX-1016), in a model of rat collagen-induced arthritis. Dosing of rats with NCX-1015 (0.4-4 micromol/kg, i.p.) greatly reduced all parameters of inflammation. A significant but inferior anti-inflammatory effect also was obtained with prednisolone. Collagen-induced arthritic rats had elevated pyridinoline values (> 60% over naïve rats), indicating bone and cartilage erosion; this increase was prevented by NCX-1015 but not by prednisolone or NCX-1016 treatment. In vitro, prednisolone (1 nM), but not NCX-1015, elevated bone resorbing activity of rat primary osteoclasts. In conclusion, NCX-1015 is a steroid derivative with a potential for the treatment of chronic inflammatory pathologies and that has milder side effects anticipated on the bone compartment.

Figure 1

Modulation of CIA in the rat by prednisolone and the GC derivatives. (A) Chemical structure of prednisolone and the chains added to position 21 to yield either NCX-1015 or NCX-1016. (B–D) Rats were treated daily i.p. with NCX-1015 (▴, 4 μmol/kg; ▵, 0.4 μmol/kg), prednisolone (●, 4 μmol/kg; ○, 0.4 μmol/kg), NCX-1016 (♦, 4 μmol/kg) or vehicle (■, 100 μl peanut oil) from day 12 to day 18 after collagen II injection. A group of naïve rats (□) also was used. During the development of the arthritis, plethysmometry was used to assess paw volume (B), and rats' paws and ankles were individually assessed and scored (C), as described in Materials and Methods. After killing the animals (day 18), anklebone thickness was measured with calipers (D). Data are expressed as means ± SEM (n = 10 per group). *, P ≤ 0.05 vs. vehicle-treated group and #, P ≤ 0.05 vs. naïve animals.

Figure 2

Histological analysis of joint morphology. Light micrographs of rat phalangial articulations in naïve (A) and CIA rats (B–D) after hematoxylin and eosin staining. (A) Naïve rat with normal articulation containing intact cartilage, bone, and synovium. (B) Representative CIA rat with ulceration of the cartilage and pronounced synovitis but intact bone compartment. (C) Effect of prednisolone (4 μmol/kg; 12–18 days) with reduced synovitis and cartilage ulceration, as seen at day 18 postCIA. (D) Articulation of a CIA rat treated with NCX-1015 (4 μmol/kg; 12–18 days) shows almost no sign of synovitis or adherent cell to the cartilage and appears essentially normal. B, bone; S, synovium; C, cartilage. Pictures are representative of nine distinct rats per group.

Figure 3

Effect of CIA and GC treatment on serum levels of cytokines and pyridinoline. (A–C) Prednisolone (Pred), NCX-1015, NCX-1016, or vehicle (V) treatment was the same as in Fig. 1 (i.p. from day 12 at the indicated doses in μmol/kg), whereas in D, drugs or vehicle (V) were given orally. A group of naïve rats also was used. (A) IL-1β, (B) IL-6 and (C and D) pyridinoline concentration as measured by using specific ELISA. Data are expressed as means ± SEM (n = 10 per group). *, P ≤ 0.05 vs. vehicle-treated group and #, P ≤ 0.05 vs. naïve animals.

Figure 4

Bone resorption measured in rat primary osteoclasts. A mixed population of rat bone cells was incubated overnight with prednisolone (Pred), NCX-1015, NCX-1016, or vehicle (V). Osteoclast activity was measured as described in Materials and Methods. (A) Osteoclast bone resorptive activity after 18-h incubation with increasing concentrations of prednisolone. *, P < 0.05 vs. vehicle (dose 0). (B) NCX-1016 mimicked prednisolone effect, whereas NCX-1015 was inactive. *, P < 0.05 vs. vehicle-treated cells. (C) Addition of the NO-donor NOC-18 (1 μM) to 1 nM prednisolone or NCX-1016 abolished the increase in bone resorption. (D) In the presence of the guanylate cyclase inhibitor (ODQ, 5 μM), 1 nM NCX-1015 stimulated osteoclast activity. All results are the means ± SEM of at least three to four experiments performed in triplicate.