The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclasts.

We have compared the effects of a general matrix metalloproteinase (MMP) inhibitor (CT435) with those of a concentration-dependent specific gelatinase inhibitor (CT543; Ki < 20 nM) on bone resorption in vitro. The test systems consisted of measuring: (i) the release of 45Ca2+ from prelabelled mouse calvarial explants; (ii) the release of 45Ca2+ from prelabelled osteoid-free calvarial explants co-cultured with purified chicken osteoclasts; and (iii) lacunar resorption by isolated rat osteoclasts cultured on ivory slices. Both CT435 and CT543 dose-dependently inhibited the release of 45Ca2+ from neonatal calvarial bones stimulated by either parathyroid hormone or 1,25-dihydroxyvitamin D3. Moreover, CT543 produced a 40% inhibition at a concentration (10(-8) M) selective for the inhibition of human gelatinases A and B. CT435 (10(-5) M) and CT543 (10(-5) M) partially inhibited the release of 45Ca2+ from osteoid-free calvarial explants by chicken osteoclasts with a maximum of approximately 25% for unstimulated cultures, and approximately 36% for cultures stimulated by interleukin-1 alpha (IL-1 alpha; 10(-10) M). Neither inhibitor prevented lacunar resorption on ivory by unstimulated rat osteoclasts, but the compounds produced a partial reduction in both the number and total surface area of lacunae in IL-1 alpha-stimulated cultures, with maximal action at 10(-5) M. Neither of the inhibitors affected protein or DNA synthesis, nor the IL-1 alpha-stimulated secretion of the lysosomal enzyme beta-glucuronidase. Immunocytochemistry demonstrated that isolated rabbit osteoclasts constitutively expressed gelatinase A and synthesized gelatinase B, collagenase and stromelysin, as well as the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) following IL-1 alpha stimulation. These experiments have shown that in addition to collagenase, gelatinases A and B are likely to play a significant role in bone resorption. They further suggest that MMPs produced by osteoclasts are released into the sub-osteoclastic resorption zone where they participate in bone collagen degradation.

Comparative biodistributions of indium-111-labelled macrocycle chimeric B72.3 antibody conjugates in tumour-bearing mice.

A novel 111In ligand (a C-functionalised derivative of 1,4,7-triazacyclononanetriacetic acid), termed 9N3, was covalently attached to chimeric B72.3, labelled with 111In and compared with 111In-labelled chimeric B72.3 diethylenetriaminepentaacetic acid (DTPA) cyclic anhydride conjugate (cDTPA) and a C-linked derivative of DTPA (CT-DTPA) in athymic mice bearing human colon carcinoma xenografts. Significant differences in biodistribution were observed between 9N3 and cDTPA conjugates especially in the tumour uptake and blood, liver, femur and colon levels at 24, 48 and 144 h. Significantly higher tumour uptake was observed for 111In-cB72.3-9N3 compared with 111In-cB72.3-cDTPA at all time points. Radiolocalisation (RI) indices increased with time for the 9N3 conjugate but remained constant for the cDTPA conjugate. The biodistribution of 111In-labelled cB72.3-CT-DTPA was similar to that of 111In-labelled cB72.3-9N3 except for elevated kidney levels. A 12N4 macrocycle (a C-functionalised derivative of 1,4,7,10-tetraazacyclododecanetetraacetic acid) was also tested for its ability to chelate 111In and its biodistribution examined. Labelled conjugates with this macrocycle were more difficult to prepare in a stable form but gave a very similar biodistribution to the 9N3 macrocycle conjugate. Macrocycle-antibody conjugates of this type offer considerable promise for tumour imaging in patients.

Synthesis and biophysical studies of short oligodeoxynucleotides with novel modifications: a possible approach to the problem of mixed base oligodeoxynucleotide synthesis.

The syntheses of 1,2-dideoxy-D-ribofuranose and 1,2-dideoxy-1-phenyl-beta-D-ribofuranose are described. Oligodeoxynucleotides containing these analogues have been synthesised and hybridized to their complementary strands. Hypochromicity studies have shown that these duplices are less stable than either the totally complementary duplex or those containing A.C and G.T mismatches.

A rapid deprotection procedure for phosphotriester DNA synthesis.

An equimolar solution of aldoxime and tetramethylguanidine at 70 degrees C removes both the base and phosphate protection from oligonucleotides prepared by solid phase phosphate triester technology. The rate of cleavage from the succinyl linkage commonly used for solid phase synthesis is also increased. The method is simpler, faster and more easily automated than existing methods.

Improvements to solid phase phosphotriester synthesis of deoxyoligonucleotides.

A solution of benzenesulphonic acid (3%, w/v) in a dimethylformamide and dichloromethane mixture (9:1, v/v) is shown to be a very effective reagent for the detritylation of deoxyoligonucleotides attached to a solid support. The levels of depurination with this reagent were lower than those observed with other reagents such as trichloroacetic acid. Coupling reactions are described using above ambient temperatures with no detectable increase in side products. Both procedures have been successfully incorporated into an automated system, which can compete with the rate of synthesis by the phosphite approach.

Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin.

DNA complementary to calf stomach mRNA has been synthesised and inserted into the Pst1 site of pAT153 by G-C tailing. Clones containing sequences coding for prochymosin were recognised by colony hybridisation with cDNA extended from a chemically synthesised oligodeoxynucleotide primer, the sequence of which was predicted from the published amino acid sequence of calf prochymosin. Two clones were identified which together contained a complete copy of prochymosin mRNA. The nucleotide sequence is in substantial agreement with the reported amino acid sequence of prochymosin and shows that this protein has a mol.wt. of 40431 and chymosin a mol.wt. of 35612. The sequence also indicates that prochymosin is synthesised as a precursor molecule, preprochymosin, having a 16 amino acid hydrophobic leader sequence analogous to that reported for other secreted proteins.