Non-isotopic detection of osteoplastic activity in vivo using modified bisphosphonates

Abstract

The present invention is directed to a non-isotopic methods for the in vitro and in vivo detection of hydroxyapatite-positive cells and structures.

1 . A contrast agent represented in the general formula [I] or pharmaceutically acceptable salts thereof:  
       
         
           
           
               
               
           
         
       
       wherein 
 D represents a fluorescent moiety;  
 R represents a linlking group that covalently links the dye (D) and bisphosphonate moiety;  
 R 1  represents H, —OH, or a halogen; and  
 R 2  represents, independently for each occurrence, a free electron pair, hydrogen, or a pharmaceutically acceptable counterion.  
 
     
     
         2 . The agent of  claim 1 , wherein R is an amine substituted lower-alkyl which forms an amide bond with a pendant group of the fluorescent moiety.  
     
     
         3 . The agent of  claim 2 , wherein R is —(CH 2 ) L —NH—, where L is an integer from 1 to 6.  
     
     
         4 . The agent of  claim 1 ,  2  or  3  wherein R′ is —OH or —Cl.  
     
     
         5 . The agent of  claim 1 ,  2  or  3 , wherein D is a near-infrared fluorescent moiety.  
     
     
         6 . The agent of  claim 5 , wherein D is a near-infrared fluorescent dye.  
     
     
         7 . The agent of  claim 5 , wherein D is a polysulfonated indocyanine dye.  
     
     
         8 . The agent of  claim 1 , wherein the fluorescent moiety is represented by the formula [II]:  
       
         
           
           
               
               
           
         
       
       wherein 
 L 1 -L 7  are each, independently, a substituted or unsubstituted methine, provided that one of L 1 -L 7  is substituted with the linker group R which is attached to the bisphosphonate,  
 R 3 , independently for each occurrence, is a substituted or unsubstituted alkyl;  
 A and B are each, independently, 5-7 membered substituted or unsubstituted aromatic rings;  
 X and Y are the same or different and each is a group of the formula —O—, —S—, —CH═CH— or —C(R 4 ) 2 —;  
 R 4 , independently for each occurrence, is a hydrogen or substituted or unsubstituted lower alkyl; and  
 r is 0, 1 or 2.  
 
     
     
         9 . The agent of  claim 8 , wherein the fluorescent moiety has one or more of the following features: (a) is free of a carboxylic acid group in a molecule; (b) r is 1; (c) includes 4 or more sulfonic acid groups; (d) includes 10 or less sulfonic acid groups; (e) A and B are, independently, benzo or naphtho rings; and (f) X and Y are —C(CH 3 ) 2 —.  
     
     
         10 . The agent of  claim 1 , wherein the fluorescent moiety is represented by the formula [III] 
       
         
           
           
               
               
           
         
       
       wherein 
 X and Y are the same or different and each is a group of the formula —O—, —S—, —CH═CH— or —C(R 4 ) 2 —;  
 R 4 , independently for each occurrence, is a hydrogen or substituted or unsubstituted lower alkyl; and  
 R 6 , independently for each occurrence, is hydrogen or —SO 3 R 7 ;  
 R 7 , independently for each occurrence, is hydrogen or a pharmaceutically acceptable counter ion;  
 m is 0, 1, 2, 3, 4 or 5.  
 
     
     
         11 . The agent of any of the preceding claims, wherein the near-infrared fluorescent moiety has an extinction coefficient of at least 100,000 M −1  cm −1  in aqueous mediums.  
     
     
         12 . The agent of any of the preceding claims, wherein the near-infrared fluorescent moiety has a quantum efficiency, Φ F , of at least 25%.  
     
     
         13 . The agent of any of the preceding claims, having an LD 50  of 100 mg/Kg or greater humans.  
     
     
         14 . The agent of any of the preceding claims, having a half-life in the human body of at least 10 minutes.  
     
     
         15 . A contrast agent comprising a bisphosphonate covalently linked to a near-infrared fluorescent moiety, wherein the contrast agent (a) has an extinction coefficient of at least 100,000 M −1  cm −1  in aqueous medium, (b) has an LD 50  of at least 100 mg/Kg in humans, and (c) has a half-life in the human body of at least 10 minutes.  
     
     
         16 . The contrast agent of  claim 15 , wherein the bisphosphonate is selected from the group consisting of alendronate, clodronate. EB-1053, etidronate, ibandronate, incadronate, neridronate, olpadronate, phosphonate, palmidronate, risedronate, tiludronate, YH 529 and zoledronate.  
     
     
         17 . A method for manufacturing a composition of for in vivo imaging comprising formulating a contrast agent of any of the preceding claims in a pharmaceutically acceptable excipient.  
     
     
         18 . A kit for in vivo imaging comprising a contrast agent of any of the preceding claims in association with instructions for administering the contrast agent to a patient.  
     
     
         19 . A method for in vivo imaging of tissue with exposed hydroxyapatite, comprising, 
 (i) administering to the animal a contrast agent of any of claims  1 - 16  in an amount sufficient to render hydroxyapatite-containing tissue detectable by a fluorescent detector;    (ii) obtaining a fluorescent image of the animal, or at least a portion thereof, at a wave length(s) which detects the contrast agent;    (iii) constructing an image of the animal including the pattern of distribution of the contrast agent.    
     
     
         20 . The method of  claim 19 , for evaluating a bone for its condition and/or biomechanical property.  
     
     
         21 . The method of  claim 20 , for determining bone matrix density.  
     
     
         22 . The method of  claim 20 , for detecting changes in bone matrix volume.  
     
     
         23 . The method of  claim 20 , as part of a protocol for diagnosing osteoporosis.  
     
     
         24 . The method of  claim 20 , for determining at least one of anisotropic elastic constants, bone strength, or fracture risk.  
     
     
         25 . The method of  claim 19 , for diagnostic detection of bone diseases accompanied with abnormality of calcium hydroxyapatite.  
     
     
         26 . The method of  claim 25 , for detecting the presence of osteoblastic metastase.  
     
     
         27 . The method of  claim 19 , for detecting of vessel micro-calcification.  
     
     
         28 . The method of any of claims  19 - 27 , wherein the animal is a human.