Development and characterization of a human in vitro resorption assay: demonstration of utility using novel antiresorptive agents.
A human in vitro resorption assay has been developed using osteoclastoma-derived osteoclasts and used to evaluate novel antiresorptive agents including antagonists of the alphavbeta3 integrin, and inhibitors of cathepsin K and the osteoclast ATPase. The potency of novel compounds in the in vitro resorption assay correlates with functional assays for each class of inhibitor: the human alphavbeta3-mediated cell adhesion assay for the vitronectin receptor antagonists (r2 = 0.82), the chick osteoclast vacuolar ATPase enzyme assay for the H+-ATPase inhibitors (r2 = 0.77) and the recombinant human cathepsin K enzyme assay for the cathepsin K inhibitors (r2 = 0.80). Cell suspensions, rich in osteoclasts, are prepared by collagenase digestion of the tumor tissue. These cells can be stored long-term in liquid nitrogen and upon thawing maintain their bone-resorbing phenotype. The cryopreserved cells can be cultured on bovine cortical bone for 24-48 h and resorption can be measured by either confocal microscopy or biochemical assays. The resorptive activity of osteoclasts derived from a number of tumors can be inhibited reproducibly using a number of mechanistically unique antiresorptive compounds. In addition, the measurement of resorption pits by laser confocal microscopy correlates with the release of type I collagen C-telopeptides or N-telopeptides, as measured by enzyme-linked immunosorbent assay. Resorption can be measured reproducibly using a 48-h incubation of osteoclasts on bone slices, or a 24-h incubation with bone particles. This in vitro human osteoclast resorption assay provides a robust system for the evaluation of inhibitors of osteoclastic function that may be developed for the treatment of metabolic bone diseases such as osteoporosis.
James
I E
IE
Department of Bone and Cartilage Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
Lark
M W
MW
Zembryki
D
D
Lee-Rykaczewski
E V
EV
Hwang
S M
SM
Tomaszek
T A
TA
Belfiore
P
P
Gowen
M
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis*; D001859:Bone Neoplasms / Q000737:chemistry*; D001862:Bone Resorption; D056657:Cathepsin K; D002403:Cathepsins / Q000378:metabolism; D002417:Cattle; D002448:Cell Adhesion; D002469:Cell Separation; D004797:Enzyme-Linked Immunosorbent Assay; D005615:Freezing; D018212:Giant Cell Tumor of Bone / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D010641:Phenotype; D015203:Reproducibility of Results
D014408:Biomarkers, Tumor; D002403:Cathepsins; C534130:CTSK protein, human; D056657:Cathepsin K; D000251:Adenosine Triphosphatases