Sabine Daufeldt

Pharmacological effect of tetracyclines on proteoglycanases from interleukin-1 -treated articular cartilage

Based on previous in vivo and in situ studies showing that tetracyclines possess antidegenerative effects on cartilage in conjunction with a reduced proteoglycan (PG) loss from the extracellular matrix, we investigated the effects of doxycycline, minocycline and tetracycline on the degradation and biosynthesis of PGs by bovine articular cartilage explants, both in vitro and in situ. Doxycycline, minocycline and tetracycline dose dependently, although weakly, inhibited PG degrading matrix metalloproteinases (MMPs) in vitro, when tested at concentrations ranging from 1 to 100 microM. Ro 31-4724 proved to be a potent inhibitor of MMP proteoglycanases (IC50 value 1.5 nM). Only at a concentration of 100 microM did doxycycline and minocycline significantly inhibit the interleukin-1 (IL-1)-induced augmentation of PG loss from cartilage explants into the nutrient media. The tetracyclines did not modulate the IL-1-mediated reduced aggregability of PGs, whereas 10 microM Ro 31-4724 partially restored the aggregability of PGs ex vivo. Tetracycline even at this high concentration was ineffective. Compared to the effects of the MMP inhibitor Ro 31-4724, treatment with tetracyclines at therapeutic serum levels of 1 or 10 microM was minimal, with little or no effect on cartilage proteoglycanases and PG biosynthesis. In our experiments, tetracyclines and Ro 31-4724 at doses evaluated had no cytotoxic effects on chondrocytes.

Pharmacological influence of antirheumatic drugs on proteoglycanases from interleukin-1 treated articular cartilage

The purpose of this study was to examine whether drugs used in the treatment of arthritic disorders possess any inhibitory potential on the proteoglycanolytic activities of matrix metalloproteinases (MMPs), and to determine whether drugs which inhibit these enzymes also modulate the biosynthesis and release of proteoglycans (PGs) from interleukin-1-(IL-1) treated articular cartilage explants. The cartilage-bone marrow extract and the glycosaminoglycan-peptide complex (DAK-16) dose-dependently inhibited MMP proteoglycanases in vitro when tested at concentrations ranging from 0.5 to 55 mg/mL, displaying an IC50 value of 31.78 mg/mL and 10.64 mg/mL (1.9 x 10[-4] M) respectively. (R,S)-N-[2-[2-(hydroxyamino)-2-oxoethyl]-4-methyl-1-oxopentyl++ +]-L-leucyl-L-phenylalaninamide (U-24522) proved to be a potent inhibitor of MMP proteoglycanases (IC50 value 1.8 x 10[-9] M). None of the other tested drugs, such as possible chondroprotective drugs, nonsteroidal anti-inflammatory drugs (NSAIDs), disease modifying antirheumatic drugs (DMARDs), glucocorticoids and angiotensin-converting enzyme inhibitors tested at a concentration of 10(-4) M displayed any significant inhibition. Only U-24522, tested at a concentration ranging from 10(-4) to 10(-6) M, significantly inhibited the IL-1-induced augmentation of PG loss from cartilage explants into the nutrient media, whereas DAK-16 and the cartilage-bone marrow extract were ineffective. DAK-16 and the cartilage-bone marrow extract did not modulate the IL-1-mediated reduced biosynthesis and aggregability of PGs by the cartilage explants. The addition of 10(-5) M U-24522, however, partially maintained the aggregability of PGs ex vivo. In our experiments, both possible chondroprotective drugs as well as U-24522 demonstrated no cytotoxic effects on chondrocytes.

Effects of the hydroxamic acid derivate Ro 31-4724 on the metabolism and morphology of interleukin-1-treated cartilage explants.

Matrix metalloproteinases (MMPs) belong to the key enzymes of the proteolytic destruction of cartilage matrix during chronic rheumatic diseases. Our work focused on the inhibitory potential of the hydroxamate Ro 31-4724 on the activity of MMP-proteoglycanases as well as on the viability, morphology and proteoglycan metabolism of interleukin-1 (IL-1)-treated bovine articular cartilage explants. The in vitro activity of MMP-proteoglycanases as well as the release of proteoglycans from IL-1-treated cartilage explants were significantly and concentration-dependently inhibited by Ro 31-4724 tested at concentrations ranging from 1 nmol/l to 10 mumol/l. Histopathological evaluation of sections from cartilage explants treated with this drug revealed no microscopically discernible alterations, and did not show any cytotoxic effects of Ro 31-4724. In addition, Ro 31-4724 had no effect on the rate of proteoglycan biosynthesis by IL-1-treated cartilage explants and increased the percentage of newly synthesized proteoglycans to form macromolecular aggregates. In conclusion, Ro 31-4724 displayed MMP-proteoglycanase inhibitory activity both in vitro and ex vivo and proved to be not harmful to the morphology, viability and proteoglycan biosynthesis of bovine articular cartilage explants.