Pharmacological characterization of GLPG1972/S201086, a potent and selective small-molecule inhibitor of ADAMTS5.

OBJECTIVE: A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) is a key enzyme in degradation of cartilage in osteoarthritis (OA). We report the pharmacological characterization of GLPG1972/S201086, a new, potent and selective small-molecule ADAMTS5 inhibitor. METHODS: Potency and selectivity of GLPG1972/S201086 for ADAMTS5 were determined using fluorescently labeled peptide substrates. Inhibitory effects of GLPG1972/S201086 on interleukin-1α-stimulated glycosaminoglycan release in mouse femoral head cartilage explants and on interleukin-1ß-stimulated release of an ADAMTS5-derived aggrecan neoepitope (quantified with ELISA) in human articular cartilage explants were determined. In the destabilization of the medial meniscus (DMM) mouse and menisectomized (MNX) rat models, effects of oral GLPG1972/S201086 on relevant OA histological and histomorphometric parameters were evaluated. RESULTS: GLPG1972/S201086 inhibited human and rat ADAMTS5 (IC50 ± SD: 19 ± 2 nM and <23 ± 1 nM, respectively), with 8-fold selectivity over ADAMTS4, and 60->5,000-fold selectivity over other related proteases in humans. GLPG1972/S201086 dose-dependently inhibited cytokine-stimulated aggrenolysis in mouse and human cartilage explants (100% at 20 µM and 10 µM, respectively). In DMM mice, GLPG1972/S201086 (30-120 mg/kg b.i.d) vs vehicle reduced femorotibial cartilage proteoglycan loss (23-37%), cartilage structural damage (23-39%) and subchondral bone sclerosis (21-36%). In MNX rats, GLPG1972/S201086 (10-50 mg/kg b.i.d) vs vehicle reduced cartilage damage (OARSI score reduction, 6-23%), and decreased proteoglycan loss (∼27%) and subchondral bone sclerosis (77-110%). CONCLUSIONS: GLPG1972/S201086 is a potent, selective and orally available ADAMTS5 inhibitor, demonstrating significant protective efficacy on both cartilage and subchondral bone in two relevant in vivo preclinical OA models.

Phasic production of urinary pyridinium crosslinks in mice: the effect of ovariectomy.

Considerable sample to sample variability in deoxypyridinoline crosslink/creatinine (Dpd/CREAT) ratio values was confirmed when twice-weekly sampling for 77 days was performed in C57 mice. Analysis of samples from individual mice indicated that, in the majority of mice in a given group (54-67%), phasic changes occurred with periodic peaks as much as 4-5 times basal values. Alignment of peaks in the individual time courses of mice revealed a clear cyclic crosslink production (periodicity 12-14 days) for the population, although not all mice gave a peak in every case. Ovariectomy (OVX) (compared to sham-operated mice) increased mean values of crosslink production by either C57 or C57 x 129 F1 mice from about 10 days after operation with highest values between 21 and 35 days, and then a decrease in the difference between sham and OVX, particularly in C57 mice. Analysis of both time courses for individual mice and distribution curves of the data from groups of mice indicated that OVX consistently increased basal crosslink values (6-9 ratio units) with phasic peak values superimposed. The peak alignment analysis reinforced this observation. The influence of the background variability was avoided by pooling data over one cycle time and the use of nonparametric statistics, and the effect of OVX was analyzed in several strains of mice. Crosslink levels and phasic production declined with age but were also influenced by manipulation of the mice, suggesting that experimental conditions should be rigorously controlled when this urinary crosslink measure is used as a parameter in mouse models of OVX-induced osteoporosis.

Mechanisms involved in prostaglandin-induced increase in bone resorption in neonatal mouse calvaria.

Prostaglandins (PG) E1, E2 and F2alpha induce bone resorption in isolated neonatal parietal bone cultures, and an associated increase in interleukin-6 (IL-6) production. Indomethacin had little effect on the response to PGE2, or the relatively non-selective EP receptor agonists 11-deoxy PGE1 and misoprostol, but blocked the effects of PGF2alpha and the F receptor agonist fluprostenol, indicating an indirect action via release of other prostaglandins. It is more likely that there is positive autoregulation of prostaglandins production in this preparation mediated via stimulation of F receptors. The effects of selective EP receptor agonists sulprostone (EP1,3) and 17-phenyl trinor PGE2(EP1), indicated the involvement of EP2 and/or EP4 receptors, which signal via cAMP. The relatively weak increase in IL-6 production by misoprostol (with respect to resorption) suggests that these responses are controlled by different combination of EP2 and EP4 receptors. The PKA activator, forskolin, induced small increases in bone resorption at lower concentrations (50-500 ng/ml) but a reversal of this effect, and inhibition of resorption induced by other stimuli (PTH, PGE2), at higher concentrations (0.5-5 microg/ml). IL-6 production was markedly increased only at the higher concentrations. The inhibitory effect of forskolin may be a calcitonin-mimetic effect. PMA induced both resorption and IL-6 production which were both blocked by indomethacin, indicating a role for PKC in the control of prostaglandin production.

Increases in osteocalcin after ovariectomy are amplified by LPS injection: strain differences in bone remodelling.

1. LPS (Escherichia coli serotype 0111:B4, 300 micrograms/mouse IP) increases serum osteocalcin in normal female C57B16 mice from 2 to 6 hr after its injection, with peak levels at 2-4 hr after LPS. 2. Both basal and LPS-stimulated serum osteocalcin were markedly inhibited by dexamethasone (10 mg/kg IP). 3. When observed 3 hr after LPS injection, serum osteocalcin was increased by ovariectomy (OVX) (with respect to sham-operated mice) and this increase was amplified in LPS-treated mice. This increase in osteocalcin was maximal 14 days after OVX, whereas urinary deoxypyridinoline cross-link levels were increased at all observation times (11-28 days). 4. All these changes were also observed in Balb/c mice but their magnitudes were consistently lower than those in C57B16 mice. 5. We propose that, (1) osteocalcin is a useful marker of bone remodelling in mice and the precision of measurement of changes in its levels after OVX is increased by LPS treatment and (2) C57B16 mice give greater magnitude and more consistent changes in both serum osteocalcin and urinary deoxypyridinoline cross-links after OVX, and may be a better strain for development of an in vivo model of post-menopausal osteoporosis.

Effects of SK&F 86002 on cytokine-stimulated IL6 production in cultured neonatal mouse calvaria and SaOS2 osteoblastic cells: the role of prostaglandins and other mechanisms of action.

The cytokine-suppressant anti-inflammatory drug (CSAID) SK&F 86002 inhibits bone resorption in vitro and this effect cannot be totally explained by its inhibition of IL 1 beta and TNF alpha release. IL6 is another cytokine important in the mechanisms of bone resorption and could be a target for the actions of SK&F 86002. IL6 release and resorption (45Ca release) were induced by IL 1 beta in neonatal mouse calvaria bones in culture. Both indomethacin (5 x 10(-8)-5 x 10(-6) M) and SK&F 86002 (5 x 10(-7)-10(-5) M) markedly inhibited the IL6 release and totally inhibited resorption at all concentrations tested. This may result from inhibition of prostaglandin production by both compounds. In human osteoblastic cells (SaOS2) both basal and TNF alpha-stimulated IL6 production were inhibited in a concentration-related manner by SK&F 86002 but not by indomethacin. The effect of SK&F 86002 was greatest in 6 h cultures where relatively low levels of IL6 are produced and progressively less in 24 and 48 h cultures which produce higher levels of IL6. This is unlikely to be an indirect effect via inhibition of IL 1 beta production as no IL 1 beta (< 0.3 pg/ml) was detected in non-stimulated or stimulated culture supernatants. Therefore, SK&F 86002 may inhibit IL6 production in osteoblastic cells via a more direct mechanism, possibly involving inhibition of the p38 MAP kinase, the mechanism proposed for its inhibition of IL 1 beta and TNF alpha release.