In vitro and in vivo models for screening compounds to prevent glucocorticoid-induced bone destruction

Abstract

The present invention demonstrates that glucocorticoid-induced bone disease is due to changes in the birth and death rate of bone cells using a murine model of glucocorticoid excess as well as bone biopsy specimens obtained from patients with glucocorticoid-induced osteoporosis. This invention demonstrates that glucocorticoid administration increases apoptosis of mature osteoblasts and osteocytes and decreases bone formation rate and bone mineral density accompanied by defective osteoblastogenesis and osteoclastogenesis in the bone marrow.

1 . A method of screening for compounds that reduce the bone deteriorating effects of glucocorticoids, comprising the steps of: 
 (a) contacting osteoblast and osteocyte cells with either a glucocorticoid alone or said glucocorticoid in combination with a test compound; and    (b) comparing the number of osteoblast and osteocyte cells undergoing apoptosis following treatment with said glucocorticoid alone or following treatment with said glucocorticoid in combination with said test compound, wherein a lower number of apoptotic cells following treatment with said glucocorticoid in combination with said test compound than with said glucocorticoid alone indicates that the test compound reduces the bond deteriorating effects of said glucocorticoid.    
     
     
         2 . The method of  claim 1 , wherein said contacting is selected from the group consisting of in vitro cell cultures and in vivo murine animal model.  
     
     
         3  (canceled)  
     
     
         4 . The method of  claim 1 , wherein said test compound has little effect on the anti-inflammatory properties of said glucocorticoid, further comprising the step of: 
 (c) comparing the anti-inflammatory response of said glucocorticoid in combination with said test compound to the anti-inflammatory response of said glucocorticoid alone, wherein essentially equivalent anti-inflammatory responses of said glucocorticoid alone and said glucocorticoid in combination with said test compound indictes that the test compound both reduces the bone deteriorating effects while retaining the anti-inflammatory properties of said glucocorticoid.    
     
     
         5 . The method of  claim 4 , wherein said contacting is in an in vivo murine animal model.  
     
     
         6 . The method of  claim 4 , wherein said anti-inflammatory response is determined by models of inflammation selected from the group consisting of the adjuvant-induced arthritis model and hindlimb inflammation model.  
     
     
         7 . A method of screening for glucocorticoid analogs that possess decreased apoptotic properties towards osteoblast and osteocyte cells, comprising the steps of: 
 (a) contacting said cells with either a glucocorticoid or a glucocorticoid analog; and    (b) comparing the number of apoptotic cells following treatment with said glucocorticoid or said glucocorticoid analog, wherein a lower number of apoptotic cells following treatment with said glucocorticoid analog than with said glucocorticoid is indicative of a glucocorticoid analog that possess decreased apoptotic properties towards said cells.    
     
     
         8 . The method of  claim 7 , wherein said contacting is selected from the group consisting of in vitro cell cultures and in vivo murine animal model.  
     
     
         9  (canceled)  
     
     
         10 . The method of  claim 7 , wherein said glucocorticoid analog retains anti-inflammatory properties, further comprising the step of: 
 comparing the anti-inflammatory response of said glucocorticoid in combination with a test compound to the anti-inflammatory response of said glucocorticoid alone, wherein essentially equivalent anti-inflammatory response of said glucocorticoid alone and said glucocorticoid in combination with said test compound indicates that the glucocorticoid analog possesses decreased apoptotic properties while retaining anti-inflammatory properties.    
     
     
         11 . The method of  claim 10 , wherein said contacting is in an in vivo murine animal model.  
     
     
         12 . The method of  claim 11 , wherein said anti-inflammatory response is determined by models of inflammation selected from the group consisting of the adjuvant-induced arthritis model and hindlimb inflammation model.  
     
     
         13 . A method of screening for glucocorticoid analogs that stimulate bone development, comprising the steps of: 
 (a) contacting osteoblast and osteocyte cells with either a glucocorticoid or a test compound; and    (b) comparing the number of said cells undergoing apoptosis following treatment with said glucocorticoid and said test compound, wherein a lower number of apoptotic cells following treatment with said test compound than with said glucocorticoid is indicative of a compound that stimulates bone development.    
     
     
         14 . The method of  claim 13 , wherein said contacting is selected from the group consisting of in vitro cell cultures and in vivo murine animal model.  
     
     
         15 . The method of  claim 13 , wherein determination of said apoptosis is slected from the group consisting of TUNEL, DNA fragmentation and immunohistochemical analysis.  
     
     
         16 . A method of screening for compounds that increase bone mineral density, comprising the steps of: 
 (a) contacting osteoblast and osteocyte cells with either a glucocorticoid or a test compound; and    (b) comparing the number of said cells undergoing apoptosis following treatment with said glucocorticoid and said test compound, wherein a lower number of apoptotic cells following treatment with said test compound than with said glucocorticoid is indicative of a compound that stimulates bone development.    
     
     
         17 . The method of  claim 16 , wherein said contacting is selected from the group consisting of in vitro cell cultures and in vivo murine animal model.  
     
     
         18 . The method of  claim 16 , wherein determination of said apoptosis is selected from the group consisting of TUNEL, DNA fragmentation and immunohistochemical analysis.