INHIBITION OF BONE-RESORPTION BY SELECTIVE INACTIVATORS OF CYSTEINE PROTEINASES

Inactivators of cysteine proteinases (CPs) were tested as inhibitors o f bone resorption in vitro and in vivo. The following four CP inactiva tors were tested: Ep475, a compound with low membrane permeability whi ch inhibits cathepsins B, L, S, H, and calpain; Ep453, the membrane-pe rmeant prodrug of Ep475; CA074, a compound with low membrane permeabil ity which selectively inactivates cathepsin B; and CA074Me, the membra ne-permeant prodrug of CA074. The test systems consisted of 1) monitor ing the release of radioisotope from prelabelled mouse calvarial expla nts and 2) assessing the extent of bone resorption in an isolated oste oclast assay using confocal laser microscopy. Ep453, Ep475, and CA074M e inhibited both stimulated and basal bone resorption in vitro while C A074 was without effect; the inhibition was reversible and dose depend ent. None of the inhibitors affected protein synthesis, DNA synthesis, the PTH-enhanced secretion of beta-glucuronidase, and N-acetyl-beta-g lucosaminidase, or the spontaneous release of lactate dehydrogenase. E p453, Ep475, and CA074Me dose-dependently inhibited the resorptive act ivity of isolated rat osteoclasts cultured on bone slices with a maxim al effect at 50 mu M. The number of resorption pits and their mean vol ume was reduced, whilst the mean surface area remained unaffected. Aga in, CA074 was without effect. Ep453, Ep475, and CA074Me, but not CA074 , when administered subcutaneously at a dose of 60 mu g/g body weight inhibited bone resorption in vivo as measured by an in vivo/in vitro a ssay, by about 20%. This study demonstrates that cathepsins B, L, and/ or S are involved in bone resorption in vitro and in vivo. Whilst cath epsin L and/or S act extracellularly, and possibly intracellularly, ca thepsin B mediates its effect intracellularly perhaps through the acti vation of other proteinases involved in subosteoclastic collagen degra dation. (C) 1994 Wiley-Liss, Inc.