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. 2004 Jan;63(1):78–83. doi: 10.1136/ard.2002.003624

Treatment with licofelone prevents abnormal subchondral bone cell metabolism in experimental dog osteoarthritis

D Lajeunesse 1, J Martel-Pelletier 1, J Fernandes 1, S Laufer 1, J Pelletier 1
PMCID: PMC1754712  PMID: 14672896

Abstract

Objectives: To determine if treatment with licofelone, a combined 5-lipoxygenase and cyclo-oxygenase inhibitor, in vivo in experimental dog osteoarthritis can modify bone cell metabolism in long term in vitro subchondral osteoblast cell cultures (Ob).

Methods: Group 1 received sectioning of the anterior cruciate ligament (ACL) of the right knee with no active treatment (placebo group). Groups 2 and 3 received sectioning of the ACL of the right knee, and were given licofelone (2.5 or 5.0 mg/kg daily by mouth, respectively) for eight weeks beginning the day after surgery. Primary Ob were prepared from the subchondral bone plate. Levels of phenotypic markers (alkaline phosphatase activity, osteocalcin release), and urokinase plasminogen activator (uPA) and insulin-like growth factor-1 (IGF-I) levels, were evaluated in each group. Lastly, prostaglandin E2 (PGE2) and leucotriene B4 levels were evaluated.

Results: No significant differences in alkaline phosphatase activity or osteocalcin release from Ob between the three groups, under either basal or 1,25(OH)2D3 induction were seen. In contrast, treatment with licofelone reduced uPA and IGF-I levels in Ob. PGE2 levels, which were still raised in the placebo group, were decreased sharply by licofelone. A relationship was found between licofelone treatment and either the reduction in the size of lesions on tibial plateaus or the levels of uPA, IGF-I, or PGE2.

Conclusions: Licofelone treatment prevents and/or delays the abnormal metabolism of subchondral osteoblasts in this model. Licofelone reduced PGE2 levels after long term Ob, suggesting that the reduction in uPA and IGF-I levels is linked, at least in part, to this reduction.

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Figure 1 .

Figure 1

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Evaluation of alkaline phosphatase activity of in vitro osteoblasts after in vivo treatment with placebo or with 2.5 mg/kg or 5.0 mg/kg licofelone daily for eight weeks of dogs with OA. Confluent primary osteoblasts were incubated for their last two days of culture with Ham's F12/DMEM containing 2% charcoal treated FBS in the presence or absence of 50 nM 1,25(OH)2D3. Alkaline phosphatase activity was determined by the hydrolysis of p-nitrophenyl phosphate into p-nitrophenol. Values are the mean (SEM) of seven cell cultures for each experimental group. No statistical differences were seen between the groups.

Figure 2 .

Figure 2

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Evaluation of osteocalcin release of in vitro osteoblasts after in vivo treatment with placebo or with 2.5 mg/kg or 5.0 mg/kg licofelone daily for eight weeks of dogs with OA. Confluent primary osteoblasts were prepared with the same experimental protocol as in fig 1. Osteocalcin release was determined on aliquots of conditioned media by radioimmunoassay. Values are the mean (SEM) of seven cell cultures for each group. No statistical differences were seen between the groups.

Figure 3 .

Figure 3

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Evaluation of urokinase plasminogen activator (uPA) activity in dog osteoblasts. Dogs were treated with placebo or with 2.5 mg/kg or 5.0 mg/kg licofelone for eight weeks before the preparation of primary osteoblast cell cultures. Confluent primary dog osteoblasts were incubated for their last two days of culture in the presence of Ham's F12/DMEM without serum containing 1% ITS mix. uPA activity was determined on aliquots of conditioned media using a chromogenic substrate. Values are the mean (SEM) of seven cell cultures for each group.

Figure 4 .

Figure 4

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Evaluation of insulin-like growth factor-I (IGF-I) levels in dog osteoblasts. The same experimental protocol as in fig 3 was performed. IGF-I levels were determined by a selective ELISA. Values are the mean (SEM) of seven cell cultures for each group.

Figure 5 .

Figure 5

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Evaluation of prostaglandin E2 (PGE2) levels in dog osteoblasts. The same experimental protocol as in fig 3 was performed. PGE2 was determined on aliquots by a selective ELISA. Values are the mean (SEM) of seven cell cultures for each group.

Figure 6 .

Figure 6

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Relationship between the size (surface, mm2) of macroscopic lesions on the tibial plateau and uPA activity, IGF-I or PGE2 levels in dog osteoblasts from dogs treated with placebo or with 2.5 mg/kg or 5.0 mg/kg licofelone daily for eight weeks before the preparation of primary osteoblasts. Individual values for uPA activity are derived from fig 3, IGF-I from fig 4, and PGE2 from fig 5. Values are the mean (SEM) of seven cell cultures for each group.

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