Inhibitors of spermidine synthase for the treatment of osteoarthritis and cartilage rehabilitation

Abstract

This invention provides a method for the treatment of a subject in need of treatment for osteoarthritis comprising administering to said subject an amount of an inhibitor of spermidine biosynthesis sufficient to effect a substantial inhibition of spermidine biosynthesis. This invention also provides the use of an inhibitor of spermidine biosynthesis in the treatment of a subject in need of treatment of osteoarthritis in an amount sufficient to effect a substantial inhibition of spermidine biosynthesis This invention further provides a method of preparing a therapeutic composition for the treatment of a subject in need of a treatment for osteoarthritis and the invention further provides a method identifying an inhibitor of spermidine biosynthesis, whereby the inhibitor is a spermidine synthesis inhibitor.

1 - 13 . (canceled)  
     
     
         14 . A method of identifying an inhibitor of spermidine biosynthesis, whereby the inhibitor is a spermidine synthase inhibitor and whereby the identification is performed by the steps of: 
 a. obtaining a candidate spermidine synthase inhibitor;    b. evaluating the effect of said candidate inhibitor as compared to a control on any one of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis by an evaluating method    
     
     
         15 . The method of  claim 14  wherein the evaluating method comprises the steps of: 
 i. providing a test system comprising DNA encoding spermidine synthase;  
 ii. contacting said system with the said test candidate spermidine synthase inhibitor under conditions which normally lead to expression of spermidine; and  
 iii. determining the effect of the test candidate inhibitor on an end-point indication as compared to a control, wherein said effect is indicative of inhibition of any one of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis by the test candidate inhibitor.  
 
     
     
         16 . The method according to  claim 15 , wherein said test system is an in vitro transfected cell culture comprising an exogenously expressed spermidine synthase.  
     
     
         17 . The method according to  claim 16 , wherein said transfected cell culture is a culture of RCJ3.1C5.18 cells stably transfected with pCMVneo expression vector comprising a nucleic acid sequence coding for the spermidine synthase protein.  
     
     
         18 . The method according to  claim 15 , wherein said test system is an ex vivo bone culture comprising an endogenously expressed spermidine synthase.  
     
     
         19 . The method according to  claim 18 , wherein said bone culture is an embryonic bone culture.  
     
     
         20 . The method according to  claim 15 , wherein said test system is an in vivo test system comprising an animal model.  
     
     
         21 . The method according to  claim 20 , wherein said end point indication is development of arthritis.  
     
     
         22 . The method according to  claim 21 , wherein the development of arthritis is determined by paw thickness of said animal, wherein less increase of the size of the paw as compared to a control is indicative of inhibition of any one of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis and development of arthritis by said test candidate inhibitor.  
     
     
         23 . The method according to any one of  claims 20  to  22 , wherein said animal model is a transgenic mouse.  
     
     
         24 . The method according to  claim 20 , wherein said in vivo test system is an arthritic mammalian model expressing endogenous spermidine synthase.  
     
     
         25 . The method according to  claim 24 , wherein said end point indication is development of arthritis.  
     
     
         26 . The method according to  claim 25 , wherein the development of arthritis is determined by paw thickness of said arthritic mammal, wherein less increase of the size of the paw as compared to a control is indicative of inhibition of any one of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis, and development of arthritis by said test candidate inhibitor.  
     
     
         27 . The method according to any one of  claims 24  to  26 , wherein said arthritic mammal is an arthritic rat.  
     
     
         28 . The method according to  claim 14 , wherein obtaining a candidate spermidine synthase inhibitor is by selecting an inhibitor from the group consisting of adenosyl spermidine, AdoDATO, DCHA, trans-4-methylcyclohexylamine (4MCHA), cyclohexylamine, methylglyoxal bis (cyclopentylamidinohydrazone) (MGBP), 2-mercaptopropylamine, N-chlorosulfonyldicyclohexylamine, 5′-((3-aminopropyl) amino)-5′-deoxyadenosine, 1-aminooxyl-3-aminopropane, 5′-(isobutylthio) adenosine, 5′-(methylthio) adenosine and any functional homologs and analogs thereof.  
     
     
         29 . The method according to  claim 14 , wherein obtaining a candidate spermidine synthase inhibitor is performed by a screening method for a substance which is an inhibitor of spermidine synthase, which screening method comprises the steps of: 
 i. providing a mixture comprising spermidine synthase;    ii. contacting said mixture with a test substance under conditions which normally lead to biosynthesis of spermidine; and    iii. determining the effect of the test substance on an end-point indication, whereby inhibition of said end point is indicative of inhibition of spermidine synthase by the test substance.    
     
     
         30 . The method according to  claim 29 , wherein the end point indication is the presence of a product of spermidine synthase catalytic reaction.  
     
     
         31 . The method according to  claim 30 , wherein the presence of a product of spermidine synthase catalytic reaction leads to any one of fluorescent or radioactive detectable signals.