Patent Application: US-201213465301-A

Abstract:
the present invention discloses magnetic resonance compatible contrast agents for water - poor structures , such as bone and tissue calcification . in particular , the present invention discloses bisphosphonate - based magnetic resonance imaging contrast agents specific for hydroxyapatite , the calcium salt most commonly associated with malignant calcification .

Description:
in a present invention , a synthetic strategy is developed for bp - based mri contrast agents particularly for water - poor structure such as bone lesions and tissue calcification , and more particularly for breast cancer microcalcification . bp - based mri contrast agents are designed in which the small molecule bps , a targeting ligand is engineered to contain a primary amine for conjugation , and is optimized for binding affinity and physicochemical properties independent of the desired functional molecules . functional molecules are conjugated covalently to the targeting ligands with linkers that provide adequate isolation of the two functions . the bp - based mri contrast agents of present invention are prepared according to the methods known in the art , as illustrated in general in fig1 - 6 and described for specific compounds in examples 1 - 6 . products are characterized by analytical hplc , nmr and lcms , and are obtained in typical yields of 50 - 60 %. fig1 of present invention describe a synthetic scheme for metal hepta coordinated bp - based mri contrast agents in which bps can be added in a pre - loaded strategy after metal chelation by dota . linker with terminal primary amine and carboxylic acid functionality is conjugated with dota ( tbu ) 3 - nhs and subsequent removal of protecting groups on carboxylic moiety results in intermediate for metal chelation . metal chelation is performed by reaction with metal chloride . carboxylic acid functional group on dota pre - loaded with metal is activated and conjugated with primary amine functional group of bp to results in bp - based mri contrast agents . in one aspect of present invention , a method for synthesizing a bp - based mri contrast agent is provided . the method involves steps of : ( a ) starting synthesis with an organic chelating ligand selected from the group of : where in one embodiment r is t - butyl ester , ester or hydrogen , ( b ) reacting an organic chelating ligand with a linker having a primary amine and a carboxylic moiety at opposing ends , ( c ) treating the carboxylic moiety with oxalyl chloride to form an acid chloride at the carboxylic moiety , ( d ) reacting said acid chloride in one pot with trialkyl phosphite and dialkyl phosphite to form a alkylester protected bp , ( e ) deprotecting one or more carboxylic acid ester of an organic chelating ligand to yield one or more carboxylic acid functionality , ( f ) chelating a metal ion to result in a metal chelate , where the linker separate the metal chelate and the alkylester protected bp , and ( g ) deprotecting one or more bp ester of the alkylester protected bp to results in the bp - based mri contrast agent . in some embodiments , linker is independently selected from amino acid , alkane , polyethylene glycol and polypropylene glycol . in some embodiments , amino acid is natural amino acid . in some embodiments , amino acid is unnatural amino acid . in some embodiments , an alkane is c1 - c20 straight chain carbon unit . in some embodiments , polyethylene glycol is 6 to 20 ethylene glycol unit . in some embodiments , polypropylene glycol is 6 to 20 propylene glycol unit . in some embodiments , alkyl is methyl , ethyl or propyl . in some embodiments , metal ion is y , in , gd , eu , or a lanthanide . in an another aspect of present invention , a method for synthesizing a bp - based mri contrast agent is provided . the method involves steps of : ( a ) starting synthesis with an organic chelating ligand selected from the group of where in one embodiment r is t - butyl ester , ester or hydrogen , and ( b ) reacting an organic chelating ligand with a linker having a primary amine and a carboxylic moiety at opposing ends , ( c ) deprotecting one or more carboxylic acid ester of the organic chelating ligand to yield one or more carboxylic acid functionality , ( d ) chelating a metal ion to result in a metal chelate , where a metal chelate having a carboxylic moiety , and ( e ) reacting an amino bp with carboxylic moiety of a metal chelate to form an amide bond to results in bp - based mri contrast agent . in some embodiments , linker is independently selected from amino acid , alkane , polyethylene glycol and polypropylene glycol . in some embodiments , amino acid is natural amino acid . in some embodiments , amino acid is unnatural amino acid . in some embodiments , an alkane is c1 - c20 straight chain carbon unit . in some embodiments , polyethylene glycol is 6 to 20 ethylene glycol unit . in some embodiments , polypropylene glycol is 6 to 20 propylene glycol unit . in some embodiments , bp is independently selected from alendronate , neridronate , pamidronate , risedronate , tiludronate and zoledronate . in some embodiments , metal ion is y , in , gd , flu , or a lanthanide . in an another aspect of present invention , a method for synthesizing a bp - based mri contrast agent is provided . the method involves steps of : ( a ) starting synthesis with an organic chelating ligand selected from the group of where in one embodiment r is t - butyl ester , ester or hydrogen , ( b ) reacting an amino bp with an organic chelating ligand to form an amide bond between a bp and an organic chelating ligand , ( c ) deprotecting one or more carboxylic acid ester of an organic chelating ligand to yield one or more carboxylic acid functionality , and ( d ) chelating a metal ion to one or more carboxylic acid ester of the organic chelating ligand to result in bp - based mri contrast agent . in some embodiments , linker separates an organic chelating ligand and the bp . in some embodiments , linker is independently selected from amino acid , alkane , polyethylene glycol and polypropylene glycol . in some embodiments , amino acid is natural amino acid . in some embodiments , amino acid is unnatural amino acid . in some embodiments , an alkane is c1 - c20 straight chain carbon unit . in some embodiments , polyethylene glycol is 6 to 20 ethylene glycol unit . in some embodiments , polypropylene glycol is 6 to 20 propylene glycol unit . in some embodiments , bp is independently selected from alendronate , neridronate , pamidronate , risedronate , tiludronate and zoledronate . in some embodiments , metal ion is y , in , gd , eu , or a lanthanide . in an another aspect , the present invention provides a contrast agent represented in general formula [ ii ], and pharmaceutically acceptable salts , hydrates and solvents thereof : in one embodiment , bisphosphonate is independently selected from alendronate , etidronate , ibandronate , incadronate , neridronate , olpadronate , phosphonate , pamidronate , risedronate , tiludronate and zoledronate . in some embodiments , linker is independently selected from amino acid , alkane , polyethylene glycol and polypropylene glycol . in some embodiments , amino acid is natural amino acid . in some embodiments , amino acid is unnatural amino acid . in some embodiments , an alkane is c1 - c20 straight chain carbon unit . in some embodiments , polyethylene glycol is 6 to 20 ethylene glycol unit . in some embodiments , polypropylene glycol is 6 to 20 propylene glycol unit . in an another aspect , the present invention provides a contrast agent for mri having a formula selected from the group of : in one embodiment , bisphosphonate is independently selected from alendronate , etidronate , ibandronate , incadronate , neridronate , olpadronate , phosphonate , pamidronate , risedronate , tiludronate and zoledronate . in some embodiments eu is loaded for paracest contrast agent . in some embodiments , y is loaded for hyperpolarized mri contrast agent . in some embodiments , linker is independently selected from amino acid , alkane , polyethylene glycol and polypropylene glycol . in some embodiments , amino acid is natural amino acid . in some embodiments , amino acid is unnatural amino acid . in some embodiments , an alkane is c1 - c20 straight chain carbon unit . in some embodiments , polyethylene glycol is 6 to 20 ethylene glycol unit . in some embodiments , polypropylene glycol is 6 to 20 propylene glycol unit . the bp - based mri contrast agents generated by methods of present invention can be used for many medical and non medical application that would benefit from mri of water - poor structure such as bone lesions and tissue calcification , but none is of immediate need than breast cancer detection . in the general population , breast cancer screening employs x - ray mammography { van ongeval , 2006 }. in 30 % to 50 % of cases , microcalcification is the hallmark for the presence of cancer { morgan , 2005 }, although x - ray mammography cannot distinguish the chemical form of the calcium salts present , and therefore relies on the pattern of crystal deposition { stomper , 2003 }. however , breast cancer calcifications are of two major types . type i crystals , found more frequently in benign ductal cysts , are birefringent and colorless , and are composed of calcium oxalate { morgan , 2005 }. type ii crystals , most often seen in proliferative lesions and associated with breast cancer cells , are composed of calcium hydroxyapatite ( ha ), and are non - birefringent and basophilic { haka , 2002 }. because of the relatively low sensitivity and specificity of x - ray mammography , mri has become the standard of care for screening women at high genetic risk of the disease { saslow , 2007 }. yet , the sensitivity and specificity of mr in this setting , estimated to be 80 % and 90 %, respectively { lehman , 2007 }, are still not high enough for maximal positive - and negative - predictive value . ha microcalcifications are a hallmark of malignant breast cancer but cannot be detected by current clinical mri . the major medical application of present invention is in the high sensitivity mri detection of tissue calcification , especially microcalcification in breast cancer , without the need for ionizing radiation . present invention demonstrates an application of ute sequences for mri of contrast agents bound to calcifications in - vivo and in - vitro . relaxivity properties and adsorption affinities of the complexes are tested using ha as a model of the calcification and bone surface , over other calcium salts , such as , ca - oxalate ( co ), ca - pyrophosphate ( cpp ), ca - phosphate ( cp ) and ca - carbonate ( cc ) salts . for in - vitro detection of ha by mri , after a short incubation time with [ gd 3 + - dota ]- thr - pam - na ( scheme 1 ), ute mri , but not conventional gradient echo ( gre ) sequence mri is able to detect ha crystals with high sensitivity . signal enhancement is dependent on the concentration of [ gd 3 + - dota ]- thr - pam - na incubated with the ha crystals , with incubation concentrations as low as 1 μm resulting in detectable signal enhancement . signal enhancement is also dependent on relaxation time ( tr ), with tr ≈ 200 msec providing the lowest background from bulk water and the highest signal enhancement of the ha crystals . to determine the selectivity and specificity of [ gd 3 + - dota ]- thr - pam - na for ha , a major mineral component of calcifications and normal bone , over other calcium salts , in the present invention an incubation of equal quantity each of ca - hydroxyapatite ( ha ), ca - pyrophosphate ( cpp ), ca - phosphate ( cp ), ca - oxalate ( co ) and ca - carbonate ( cc ) salts with [ gd 3 + - dota ]- thr - pam - na in phosphate buffered saline ( pbs ) is performed . ute mri is taken before and after washing crystals , [ gd 3 + - dota ]- thr - pam - na has more than three fold higher specificity for ha over other calcium salts found in the body , and permits mri detection of ha with good sensitivity . for in - vivo detection of subcutaneously implanted ha crystals as a model of breast cancer microcalcification , in present invention , mice with subcutaneously implanted ha slurries ( in pbs ) are imaged by both ute mri and microct after intravenous ( iv ) injection of [ gd 3 + - dota ]- thr - pam - na . after a minimum of 4 h of clearance , and consistent with the in - vitro results , ute mri provides a sensitive detection of ha crystals in - vivo , with the signal enhancement corresponding to the location of the x - ray dense crystals by microct . of note , the crystals are invisible by ute mri pre - injection of the contrast agent . 1 . preparation of [ gd 3 + - dota ]- thr - pam - na ( scheme 1 ; fig7 ) to a solution of threonine ( 0 . 19 mmol ) in 0 . 1 ml water and dimethylformamide ( dmf ; 0 . 4 ml ) at 0 ° c ., is added triethylamine ( tea ; 0 . 38 mmol ) followed by dropwise addition of dota ( tbu ) 3 - nhs ester ( 0 . 12 mmol ) in dimethylformamide ( dmf ; 0 . 5 ml ) for 10 min with stirring . after 10 min , the ice bath is removed and stirring continued at room temperature ( rt ) for 16 h . the reaction mixture is poured over 2 ml ice - cold water and purified by preparative hplc . dota ( tbu ) 3 - thr ( 0 . 10 mmol ) is taken in trifluoroacetic acid ( tfa ; 1 ml ). the solution is stirred at rt for 2 . 5 h then the acid is removed by a n 2 stream . after lyophilization , an intermediate dota ( cooh ) 3 - thr is obtained without further purification as a white powder . the chelation of gd is performed by adding 0 . 10 ml of 1 m gdcl 3 ( 0 . 10 mmol ) in water to a solution of 0 . 10 mmol of dota ( cooh ) 3 - thr in 0 . 9 ml of 0 . 5 m acetic acid buffer ( hac / ac − ), ph 5 . 5 . the reaction mixture is stirred at rt for 12 h and purification by preparative hplc results in an intermediate [ gd 3 + - dota ]- thr . me - pam { bhushan , 2007 } ( 0 . 01 mmol ), o -( 6 - chlorobenzotriazol - 1 - yl )- n , n , n ′, n ′- tetramethyluronium hexafluorophosphate ( hctu ; 0 . 01 mmol ), and n - methylmorpholine ( nmm ; 0 . 01 mmol ) are added at rt under n 2 atmosphere to 0 . 01 mmol [ gd 3 + - dota ]- thr in anhydrous dimethylsulfoxide ( dmso ; 0 . 5 ml ). after stirring for 1 h at rt , the reaction mixture is poured over 3 ml ice - cold water and purification by preparative hplc results in an intermediate [ gd 3 + - dota ]- thr - pam - me . trimethylsilyl bromide ( me 3 sibr ; 0 . 04 mmol ) is added slowly to a solution of [ gd 3 + - dota ]- thr - pam - me ( 0 . 01 mmol ) in dry dimethylformamide ( dmf ; 0 . 1 ml ) at 0 ° c . under nitrogen atmosphere . the reaction mixture is vortexed at rt for 12 h . methanolic naoh is added to adjust ph between 4 and 4 . 2 , vortexing for 30 min at rt followed by preparative hplc purification results in the product [ gd 3 + - dota ]- thr - pam - na . 2 . preparation of [ gd 4 + - dota ]-( peg ) 8 - pam - na ( scheme 2 ; fig8 ) to a solution of amino -( peg ) 8 - cooh ( 0 . 19 mmol ) in 0 . 1 ml water and dimethylformamide ( dmf ; 0 . 4 ml ) at 0 ° c ., is added triethylamine ( tea ; 0 . 38 mmol ) followed by dropwise addition of dota ( tbu ) 4 - nhs ester ( 0 . 12 mmol ) in dimethylformamide ( dmf ; 0 . 5 ml ) for 10 min with stirring . after 10 min , the ice bath is removed and stirring is continued at rt for 16 h . the reaction mixture is poured over 2 ml ice - cold water and an intermediate dota ( tbu ) 4 -( peg ) 8 is purified by preparative hplc . dota ( tbu ) 4 -( peg ) 8 ( 0 . 10 mmol ) is taken in trifluoroacetic acid ( tfa ; 1 ml ). the solution is stirred at rt for 2 . 5 h then the acid is removed by a n 2 stream . after lyophilization , an intermediate dota ( cooh ) 4 -( peg ) 8 is obtained without further purification as a white powder . the chelation of gd is performed by adding 0 . 15 ml of 1 m gdcl 3 ( 0 . 10 mmol ) in water to a solution of 0 . 10 mmol of dota ( cooh ) 4 -( peg ) 8 in 0 . 85 ml of 0 . 5 m acetic acid buffer ( hac / ac − ), ph 5 . 5 . the reaction mixture is stirred at rt for 12 h and an intermediate [ gd 4 + - dota ]-( peg ) 5 is purified by preparative hplc . me - pam ( 0 . 01 mmol ), o -( 6 - chlorobenzotriazol - 1 - yl )- n , n , n ′, n ′- tetramethyluronium hexafluorophosphate ( hctu ; 0 . 01 mmol ), and n - methylmorpholine ( nmm ; 0 . 01 mmol ) are added at rt under n 2 atmosphere to 0 . 01 mmol [ gd 4 + - dota ]-( peg ) 8 in 1 ml anhydrous dimethylsulfoxide ( dmso ; 0 . 5 ml ). after stirring for 1 h at rt , the reaction mixture is poured over 3 ml ice - cold water and is purified by preparative hplc to obtain an intermediate [ gd 4 + - dota ]-( peg ) 8 - pam - me . trimethylsilyl bromide ( me 3 sibr ; 0 . 04 mmol ) is added slowly to a solution of [ gd 4 + - dota ]-( peg ) 8 - pam - me ( 0 . 01 mmol ) in dry dimethylformamide ( dmf ; 0 . 1 ml ) at 0 ° c . under nitrogen atmosphere . the reaction mixture is vortexed at rt for 12 h . methanolic naoh is added to adjust ph between 4 and 4 . 2 , being vortexed for 30 min at rt and the product [ gd 4 + - dota ]-( peg ) 8 - pam - na is purified by preparative hplc . 3 . preparation of [ gd 3 + - dota ]- thr - bp - me ( scheme 3 ; fig9 ) dota ( tbu ) 3 - thr ( 0 . 02 mmol ) is taken in tetrahydrofuran ( thf ; 1 ml ) at 0 ° c . under nitrogen atmosphere , is added dimethylformamide ( dmf ; 5 μl ) and 0 . 04 mmol of 2 m solution of oxalyl chloride in tetrahydrofuran ( thf ). the solution is stirred at rt for 1 h and after that solvent is removed to get solid dota ( tbu ) 3 - thr - ci which is used for next step reaction . to dota ( tbu ) 3 - thr - cl ( 0 . 02 mmol ), is added dropwise trimethyl phosphite ( 0 . 025 mmol ) at 0 ° c . under nitrogen atmosphere for 5 minutes and is stirred at rt for about 30 minutes . to the above reaction mixture , is added dropwise dimethyl phosphite ( 0 . 025 mmol ) at 0 ° c . under nitrogen atmosphere for 5 minutes and is stirred at rt for about 30 minutes then is added 2 ml cold water and an intermediate dota ( tbu ) 3 - thr - bp - me is purified by preparative hplc . dota ( tbu ) 3 - thr - bp - me ( 0 . 01 mmol ) is taken in trifluoroacetic acid ( tfa ; 1 ml ). the solution is stirred at rt for 2 . 5 h then the acid is removed by a n 2 stream . after lyophilization , an intermediate dota ( cooh ) 3 - thr - bp - me is obtained without further purification as a white powder . the chelation of gd is performed by adding 0 . 10 ml of 1 m gdcl 3 ( 0 . 01 mmol ) in water to a solution of 0 . 01 mmol of dota ( cooh ) 3 - thr - bp - me in 0 . 9 ml of 0 . 05 m acetic acid buffer ( hac / ac − ), ph 5 . 5 . the reaction mixture is stirred at rt for 12 h and product [ gd 3 + - dota ]- thr - bp - me is purified by preparative hplc . 4 . preparation of [ gd 4 + - dota ]-( peg ) 8 - bp - me ( scheme 4 ; fig1 ) dota ( tbu ) 4 -( peg ) 8 ( 0 . 02 mmol ) is taken in tetrahydrofuran ( thf ; 1 ml ) at 0 ° c . under nitrogen atmosphere , is added dimethylformamide ( dmf ; 5 μl ) and 0 . 04 mmol of 2 m solution of oxalyl chloride in tetrahydrofuran ( thf ). the solution is stirred at rt for 1 h and after that solvent is removed to get solid dota ( tbu ) 4 -( peg ) 8 - cl which is used for next step reaction . to dota ( tbu ) 4 -( peg ) 8 - cl ( 0 . 02 mmol ), is added dropwise trimethyl phosphite ( 0 . 025 mmol ) at 0 ° c . under nitrogen atmosphere for 5 minutes and is stirred at rt for about 30 minutes . to the above reaction mixture is added dropwise dimethyl phosphite ( 0 . 025 mmol ) at 0 ° c . under nitrogen atmosphere for 5 minutes and is stirred at rt for about 30 minutes then is added 2 ml cold water and an intermediate dota ( tbu ) 4 -( peg ) 8 - bp - me is purified by preparative hplc . dota ( tbu ) 4 -( peg ) 8 - bp - me ( 0 . 01 mmol ) is taken in trifluoroacetic acid ( tfa ; 1 ml ). the solution is stirred at rt for 2 . 5 h then the acid is removed by a n 2 stream . after lyophilization , an intermediate dota ( cooh ) 4 -( peg ) 8 - bp - me is obtained without further purification as a white powder . the chelation of gd is performed by adding 0 . 15 ml of 1 m gdcl 3 ( 0 . 01 mmol ) in water to a solution of 0 . 01 mmol of dota ( cooh ) 4 -( peg ) 8 - bp - me in 0 . 85 ml of 0 . 05 m acetic acid butter ( hac / ac − ), ph 5 . 5 . the reaction mixture is stirred at rt for 12 h and product [ gd 4 + - dota ]-( peg ) 8 - bp - me is purified by preparative hplc . pamidronic acid / me - pam ( 0 . 01 mmol ), o -( 6 - chlorobenzotriazol - 1 - yl )- n , n , n ′, n ′- tetramethyluronium hexafluorophosphate ( hctu ; 0 . 01 mmol ), and n - methylmorpholine ( nmm ; 0 . 01 mmol ) are added at rt under n 2 atmosphere to 0 . 01 mmol dota ( tbu ) 3 - thr in anhydrous dimethylsulfoxide ( dmso ; 0 . 5 ml ). after stirring for 1 h at rt , the reaction mixture is poured over 3 ml ice - cold water and an intermediate dota ( tbu ) 3 - thr - pam / dota ( tbu ) 3 - thr - pam - me is purified by preparative hplc . trimethylsilyl bromide ( me 3 sibr ; 0 . 04 mmol ) is added slowly to a solution of dota ( tbu ) 3 - thr - pam - me ( 0 . 01 mmol ) in dry dimethylformamide ( dmf ; 0 . 1 ml ) at 0 ° c . under nitrogen atmosphere . the reaction mixture is vortexed at rt for 12 h . methanol / water ( 4 / 1 ) are added , being vortexed for 30 min at rt and an intermediate dota ( tbu ) 3 - thr - pam is purified by preparative hplc . dota ( tbu ) 3 - thr - pam ( 0 . 01 mmol ) is taken in trifluoroacetic acid ( tfa ; 1 ml ). the solution is stirred at rt for 2 . 5 h then the acid is removed by a n 2 stream . after lyophilization , an intermediate dota ( cooh ) 3 - thr - pam is obtained without further purification as a white powder . the chelation of gd is performed by adding 0 . 10 ml of 1 m gdcl 3 ( 0 . 01 mmol ) in water to a solution of 0 . 01 mmol of dota ( cooh ) 3 - thr - pam in 0 . 9 ml of 0 . 05 m acetic acid buffer ( hac / ac − ), ph 5 . 5 . the reaction mixture is stirred at rt for 12 h and product [ gd 3 + - dota ]- thr - pam - na is purified by preparative hplc . 6 . preparation of [ gd 4 + - dota ]-( peg ) 8 - pam - na ( scheme 6 ; fig1 ) pamidronic acid / me - pam ( 0 . 01 mmol ), o -( 6 - chlorobenzotriazol - 1 - yl )- n , n , n ′, n ′- tetramethyluronium hexafluorophosphate ( hctu ; 0 . 01 mmol ), and n - methylmorpholine ( nmm ; 0 . 01 mmol ) are added at rt under n 2 atmosphere to 0 . 01 mmol dota ( tbu ) 4 -( peg ) 8 in anhydrous dimethylsulfoxide ( dmso ; 0 . 5 ml ). after stirring for 1 h at rt , the reaction mixture is poured over 3 ml ice - cold water and an intermediate dota ( tbu ) 4 -( peg ) 8 - pam / dota ( tbu ) 4 -( peg ) 8 - pam - me is purified by preparative hplc . trimethylsilyl bromide ( me 3 sibr ; 0 . 04 mmol ) is added slowly to a solution of dota ( tbu ) 4 -( peg ) 8 - pam - me ( 0 . 01 mmol ) in dry dimethylformamide ( dmf ; 0 . 1 ml ) at 0 ° c . under nitrogen atmosphere . the reaction mixture is vortexed at rt for 12 h . methanol / water ( 4 / 1 ) are added , being vortexed for 30 min at rt and an intermediate dota ( tbu ) 4 -( peg ) 8 - pam is purified by preparative hplc . dota ( tbu ) 4 -( peg ) 8 - pam ( 0 . 01 mmol ) is taken in trifluoroacetic acid ( tfa ; 1 ml ). the solution is stirred at rt for 2 . 5 h then the acid removed by a n 2 stream . after lyophilization , an intermediate dota ( cooh ) 4 -( peg ) 8 - pam is obtained without further purification as a white powder . the chelation of gd is performed by adding 0 . 15 ml of 1 m gdcl 3 ( 0 . 01 mmol ) in water to a solution of 0 . 01 mmol of dota ( cooh ) 4 -( peg ) 8 - pam in 0 . 85 ml of 0 . 05 m acetic acid buffer ( hac / ac − ), ph 5 . 5 . the reaction mixture is stirred at rt for 12 h and product [ gd 4 + - dota ]-( peg ) 8 - pam - na is purified by preparative hplc . mri can be performed on a 1 . 5 t ge signa clinical scanner equipped with a custom low - pass birdcage coil ( 10 cm length , 6 cm diameter ). the custom ute sequence is based on previous work in the field { irarrazabal , 1995 ; song , 1998 }. 8 . in - vitro ute and gre mri of ha crystals bound by [ gd 3 + - dota ]- thr - pam - na 1 mm of [ gd 3 + - dota ]- thr - pam - na is added to 5 mg of ha crystals in 50 μl pbs ( ph 7 . 4 ) and is vortexed for 1 h at rt in a 1 . 5 ml eppendorf tube . 5 mg ha in 1 mm of [ gd 3 + - dota ]- thr and 50 μl pbs is used as a control . mri , pre - and post - washing with 4 × 500 μl pbs , are acquired using an ute sequence ( tr = 200 msec , te = 100 μsec ) or conventional gre sequence ( tr = 200 msec , te = 1 . 8 msec ). other acquisition parameters includes fov = 6 cm , slice thickness = 5 mm , matrix size = 256 × 256 , nex = 4 . 9 . contrast agent concentration and tr dependence of ute mri signals 5 mg ha is placed in 1 . 5 ml plastic eppendorf tubes , then 0 , 0 . 1 , 1 , 10 , or 100 μm [ gd 3 + - dota ]- thr - pam - na in 50 μl pbs is added to each . after vortexing 1 h at rt , the crystals are washed with 4 × 500 μl pbs and ute mri acquisition is performed using a fixed te = 100 μsec and varying tr of 17 , 50 , 200 , 500 msec . other acquisition parameters includes fov = 11 cm , slice thickness = 10 mm , matrix size = 256 × 256 , nex = 2 . 5 mg of ha or the phosphate , oxalate , carbonate , or pyrophosphate salts of calcium is placed in 1 . 5 ml eppendorf tube and is incubated with 10 μm [ gd 3 + - dota ]- thr - pam - na in 50 μl pbs for 1 h at rt with continuous vortexing . ute mri acquisition is performed pre - and post - washing with 4 × 500 μl pbs using tr = 200 msec and te = 100 μsec . other acquisition parameters includes fov = 9 cm , slice thickness = 10 mm , matrix size = 256 × 256 , nex = 2 . 50 mg of ha crystals is taken in 3004 pbs , is implanted subcutaneously at right flank of anesthetized mice . ute mri is taken after implantation of ha crystal using tr / te = 200 msec / 100 μsec , fov = 6 cm , slice thickness = 5 mm , matrix size = 256 × 256 . 4 μmol of [ gd 3 + - dota ]- thr - pam - na in 300 μl saline is injected intravenously . after 4 h of clearance , an ute mri is taken with same parameters . 1 . caravan , p . strategies for increasing the sensitivity of gadolinium based mri contrast agents . chem . soc . rev . 35 , 512 - 523 ( 2006 ). 2 . caravan , p ., ellison , j . j ., mcmurry , t . j . & amp ; lauffer , r . b . gadolinium ( iii ) chelates as mri contrast agents : structure , dynamics , and applications . chem . rev . 99 , 2293 - 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risk women : prospective multi - institution breast cancer screening study . radiology 244 , 381 - 388 ( 2007 ). 19 . bhushan , k . r ., tanaka , e . & amp ; frangioni , j . v . synthesis of conjugatable bisphosphonates for molecular imaging of large animals . angew . chem . int . ed . engl . 46 , 7969 - 7971 ( 2007 ).