Patent Abstract:
this invention provides a class of dual mode imaging tracer capable of acting both as a fluorescent imaging tracer and a positron emission tomography imaging tracer . tracers in accordance with this invention generally have a fluorescent core with a boron - fluoride element embedded therein . exemplary embodiments of the tracer include 18 f - labeled bodipy compounds and derivative thereof . also provided are tracer kits containing a sterile formulation of a bodipy dye either in a radio - labeled or pre - labeled state , and methods for imaging heart perfusion using the 18 f - labeled dual mode tracers .

Detailed Description:
unless otherwise indicated herein , all terms used herein have the meanings that the terms would have to those skilled in the art of the present invention . practitioners are particularly directed to current textbooks for definitions and terms of the art . it is to be understood , however , that this invention is not limited to the particular methodology , protocols , and reagents described , as these may vary . the term “ alkyl ” herein used means c 1 - c 10 straight or branched chain alkyl or cycloalkyl , for example , methyl , ethyl , n - propyl , i - propyl , n - butyl , i - butyl , sec - butyl , tert - butyl , n - pentyl , i - pentyl , neo - pentyl , tert - pentyl , and the like . substituents for an optionally substituted alkyl include hydroxy , alkoxy ( e . g ., methoxy and ethoxy ), mercapto , alkylthio ( e . g ., methylthio ), cycloalkyl ( e . g ., cyclopropyl , cyclobutyl , cyclopentyl , and cyclohexyl ), halogen ( e . g ., fluoro , chloro , bromo , and iodo ), carboxy , alkoxycarbonyl ( e . g ., methoxycarbonyl and ethoxycarbonyl ), nitro , cyano , haloalkyl ( e . g ., trifluoromethyl ), substituted or unsubstituted amino ( e . g ., methylamino , dimethylamino , and carbamoylamino ), guanidino , phenyl , benzyloxy , and the like . these substituents are able to bind them at one or more of any possible positions . the term “ aryl ” herein used means monocyclic or condensed ring aromatic hydrocarbons . examples of the aryl are phenyl , naphthyl , and the like . substituents for the aromatic ring of in an optionally substituted aryl are , for example , hydroxy , alkoxy , alkyl , halogen , carboxy , alkoxycarbonyl , nitro , cyano , haloalkyl , aryloxy , substituted or unsubstituted amino . these substituents are able to bind to it at one or more of any possible position . the term “ carboxylic acid ” means an organic chemical compound comprising at least one carboxylic acid functional group ( i . e . — c ( o ) oh ). the term “ ester ” includes compounds and moieties which contain a carbon or a heteroatom bound to an oxygen atom which is bonded to the carbon of a carbonyl group . the term “ ester ” includes alkoxycarboxy groups such as methoxycarbonyl , ethoxycarbonyl , propoxycarbonyl , butoxycarbonyl , pentoxycarbonyl , etc . the alkyl , alkenyl , or alkynyl groups are as defined above . as used herein , the term “ hydrate ” means a compound which further includes a stoichiometric or non - stoichiometric amount of water bound by non - covalent intermolecular forces . as used herein , the term “ solvate ” means a compound which further includes a stoichiometric or non - stoichiometric amount of solvent such as water , acetone , ethanol , methanol , dichloromethane , 2 - propanol , or the like , bound by non - covalent intermolecular forces . integrated pet - ct has been widely applied in clinical care as a novel diagnostic imaging tool in patient management owing to its high sensitivity and good resolution . recently , application of pet - ct has been extended to cardiovascular - related diseases and is undergoing rapid expansion in this area ( 6 , 23 - 24 ). these systems permit the integration of the presence of coronary artery calcium and the degree of coronary artery luminal narrowing with the impairment in myocardial vasodilator function . to date , a number of pet mpi probes have been developed for clinical applications . however , many of the tracers may require expensive onsite production and inconvenient on - scanner tracer administration . currently , there is considerable interest in developing novel pet mpi agents with optimal imaging property and longer radioactive half - lives than conventional agents . bodipy dyes constitute a class of fluorophores that have been widely used for the fluorescent labeling of biomolecules ( 25 - 27 ). such dyes feature high stability , high quantum yields and an emission range that can be tuned into the near infrared ( 28 - 29 ). bodipy dyes also typically possess a boron - bound fluorine atom which could provide a site for the incorporation of a [ 18 f ]- fluorine atom , a radionuclide of choice for pet ( 25 - 27 ). in fact , we have discovered the novel methods to produce 18 f - bodipy dyes in high yield . as the reported bodipys may be considered as lipophilic cationic compounds , they may move across phospholipid bilayers similar to the well - studied triphenylphosphonium ( tpp ) ion ( 30 - 31 ). it has long been recognized that lipophilic cations such as tpp + and the fluorescent dye rhodamine derivatives have an affinity to , and accumulate selectively in , the mitochondrial matrix . however , it is heretofore unknown whether bodipy or similar dyes will have the desired properties suitable for myocardial perfusion imaging . in this invention , we unexpectedly discovered that bodipy dyes preferentially accumulate in the heart . we previously disclosed novel methods for efficiently synthesizing and purifying large quantities of 18 f labeled bodipy dyes in co - pending application ser . no . 13 / 549 , 309 , the entire content of which is incorporated herein by reference . using the 18 f - bodipy - 1 probe in bio - distribution study and micropet imaging experiments , we demonstrated for the first time the preferential accumulation of such dyes in the heart in mice . in the micropet study , the heart uptake of 18 f - bodipy - 1 was calculated to be 4 . 38 ± 0 . 46 , 3 . 51 ± 0 . 42 , and 2 . 68 ± 0 . 17 % id / g at 0 . 5 , 2 and 3 h p . i . this demonstrated that 18 f - bodipy - 1 preferentially accumulated in the heart . similarly , rat images indicate rapid blood clearance and clear delineation of the plateau of heart activity for the scanning period . as the rat is much larger than mouse , the uptakes derived from rat imaging are expected to be more accurate than the one from mouse . with the above discoveries , we have uncovered a new class of dual mode tracer for pet imaging in mpi imaging applications . as bodipy dyes provide various positions to be modified , those skilled in the art will readily recognize that various modifications may be made to achieve different hydrophilicity and zeta potentials . such modifications can be guided by side - by - side comparisons with currently available compounds . in summary , we have discovered a new category of catonic compounds that hold great potential for dual mode imaging ( fluorescent and pet ) in imaging applications such as myocardial perfusion imaging . the following examples are provided in order to demonstrate and further illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof . while such examples are typical of those that might be used , other procedures known to those skilled in the art may alternatively be utilized . indeed , those of ordinary skill in the art can readily envision and produce further embodiments , based on the teachings herein , without undue experimentation . all chemicals obtained commercially were of analytical grade and used without further purification . no - carrier - added 18 f - f − was obtained from in - house a siemens rds - 112 negative ion cyclotron . the analytical reversed - phase high performance liquid chromatography ( rp - hplc ) using a vydac protein and peptide column ( 218tp510 ; 5 μm , 250 × 4 . 6 mm ) was performed on a dionex 680 chromatography system with a uvd 170u absorbance detector and model 105s single - channel radiation detector ( carroll & amp ; ramsey associates ). the recorded data were processed using chromeleon version 7 . 1 software . with a flow rate of 1 . 0 ml / min , the mobile phase was changed from 95 % solvent a [ 0 . 1 % trifluoroacetic acid ( tfa ) in water ] and 5 % b [ 0 . 1 % tfa in acetonitrile ( mecn )] ( 0 - 2 min ) to 5 % solvent a and 95 % solvent b at 22 min . uv absorbance was monitored at 218 nm and the identification of the peptides were confirmed based on the uv spectrum using a photodiode array detector . 18 f - bodipy - 1 was synthesized according to our previously reported procedure ( 16 ). in brief , approximately 30 mci azeotropically dried 18 f - fluoride in anhydrous mecn was added to the mixture of bodipy - 1 ( 0 . 37 μmol ) and sncl 4 ( 1 μl ) in 50 μl mecn . the reaction mixture was incubated for 10 min at room temperature . then approximately 1 . 5 mci mixture was taken for hplc purification . the purified radio tracer was rotary evaporated to dryness , reconstituted in normal saline and passed through a 0 . 22 - μm millipore filter into a sterile multidose vial for in vitro and in vivo experiments . animal procedures were performed according to a protocol approved by the institutional animal care and use committee ( iacuc ) of university of southern california . 4 - 6 weeks old female athymic nude mice ( balb / c nu / nu ) and 2 - 3 weeks old male rats were purchased from harlan laboratories ( indianapolis , ind .). the animals were housed in our vivarium for 4 weeks before use . human embryonic kidney 293 ( hek - 293 ) cells were culture in rpmi - 1640 ( containing 5 . 3 mm kcl ) and 10 % fetal bovine serum ( omega scientific , tarzana , calif .). hek - 293 cells were harvested by trypsinization and aliquoted to 1 × 10 6 cells / tube . cells were suspended in 200 μl medium containing different concentration of bodipy - 1 ( 6 . 25 μm , 12 . 5 μm , 25 μm or 50 μm ) and kcl ( 5 . 3 mm , 100 mm or 200 mm ). then the cells were maintained in cell incubator for 1 . 5 h . after incubation , cells were washed twice with cold phosphate - buffered saline ( pbs ) and stained with 100 μl 4 ′- 6 - diamidino - 2 - phenylindole ( dapi , 1 μg / ml ) diluted in pbs . for the quantification of fluorescence by flow cytometry ( cyan analyzer , beckman coulter ), 10 , 000 viable cells ( dapi negative ) were counted and analyzed . the excitation and emission wavelength were 488 nm and 510 - 550 nm respectively . each sample was repeated as triplicate . hek - 293 ( 1 × 10 6 ) cells were suspended in 200 μl medium containing different concentration of kcl ( 5 . 3 mm , 100 mm or 200 mm ) and 1 μci 18 f - bodipy - 1 was added . then the cells were maintained in cell incubator for 1 . 5 h . after incubation , cells were washed twice with cold phosphate - buffered saline ( pbs ). the radioactivity of the cell pellet was counted together with standard solution in a gamma counter . the data were obtained in triplicate . pet scans and image analysis were performed using a micropet r4 rodent model scanner ( siemens medical solutions ) as previously reported ( 17 - 18 ). 18 f - bodipy - 1 was intravenously injected into nude mice ( approximately 100 μci each , n = 3 ) and rats ( approximately 500 μci each , n = 3 ) under isoflurane anesthesia . five min static pet images were then acquired for each scan . the images were reconstructed by 2 - dimensional ordered - subsets expectation maximum ( osem ) algorithm . no attenuation or scatter correction was applied . for each micropet scan , regions of interest ( rois ) were drawn over the normal tissue , and major organs by using vendor software ( asi pro 5 . 2 . 4 . 0 ) on decay - corrected whole - body coronal images . the average radioactivity concentration ( accumulation ) within an organ was obtained from mean pixel values within the multiple roi volume , which were converted to counts / ml / min by using a conversion factor . assuming a tissue density of 1 g / ml , the rois were converted to counts / g / min and then divided by the administered activity to obtain an imaging roi - derived % id / g . the health nude mice were intravenously injected with approximate 50 μci of 18 f - bodipy - 1 . at 3 h after injection , the mouse was sacrificed , then the blood , heart and other major organs were collected , and wet - weighed . the radioactivity in the tissue was measured using a γ counter ( packard , meriden , conn .). the results are presented as percentage injected dose per gram of tissue (% id / g ). values are expressed as means ± sd for a group of three animals . hek - 293 cells were planted in 24 - well plate at density 1 × 10 5 cells / well . 24 h after plantation , cells were incubated with 300 μl medium containing 25 mm bodipy - 1 and different concentration of kcl ( 5 . 3 mm or 150 mm ). then the cells were maintained in cell incubator for 1 . 5 h . after incubation , cells were washed twice with cold phosphate - buffered saline ( pbs ), fixed with 4 % paraformaldehyde ( electron microscopy sciences , hatfield , pa .) and stained with dapi . images were obtained with nikon eclipse 80i fluorescence microscope ( tokyo , japan ). quantitative data was expressed as mean ± sd . means were compared using one - way anova and student &# 39 ; s t - test . p values of & lt ; 0 . 05 were considered statistically significant . the one - step 18 f - fluorination of bodipy - 1 afforded 18 f - bodipy - 1 in 89 . 67 ± 3 . 21 % yield ( n = 4 ) ( fig1 ). counted from the end of bombardment , the total synthesis included hplc purification and product formulation was 62 . 33 ± 7 . 51 min ( n = 4 ). the specific activity ( sa ) to 18 f - bodipy - 1 was estimated to be 48 mci / μmol at the time of injection based on the chemical loading and the radiochemical yield ( rcy ). hek - 293 cell uptake of bodipy - 1 was assessed through facs analysis . the mean fluorescence intensity of cells was elevated with the increase of bodipy - 1 concentration ( fig2 a ). for testing the potassium concentration effect , the relative mean fluorescence intensity of hek - 293 cells in 5 . 3 mm k + ( 25 μm bodipy - 1 ) was set as 1 and the other fluorescence intensity was expressed as the ratios to 5 . 3 mm k + . with the increasing of potassium concentration , the corresponding fluorescence intensity was dropped to 0 . 69 ± 0 . 06 in 100 mm k + and 0 . 39 ± 0 . 01 in 200 mm k + solution ( fig2 b ). fluorescence microscope analysis also showed the decrease of cell fluorescence intensity in 150 mm k + compared to 5 . 3 mm k + ( fig6 ). similarly , the effects of manipulating mitochondrial membrane potential on cellular accumulation of 18 f - bodipy - 1 were assessed through uptake studies on hek - 293 cells using 2 potassium concentration ( medium k + , 100 mm and high k + , 200 mm ). for control experiments , in which the mitochondrial membrane potentials were unaltered , uptake was determined in a near - physiologic buffer ( standard solution , k + concentration : 5 . 3 mm ). the results are depicted in fig2 . the hek - 293 cell uptake of 18 f - bodipy - 1 in standard solution was 2 . 96 ± 0 . 24 % while the cell uptake in medium k + and high k + solution were 1 . 85 ± 0 . 01 % and 1 . 62 ± 0 . 16 %, respectively ( fig2 ). these results clearly demonstrated that uptake of 18 f - bodipy - 1 was electrogenic and driven by the plasma and mitochondrial membrane potentials . hek - 293 cells were incubated with medium containing 25 mm bodipy - 1 and different concentration of kcl ( 5 . 3 mm or 150 mm ). then the cells were maintained in cell incubator for 1 . 5 h . fluorescence microscope analysis showed the decrease of cell fluorescence intensity in 150 mm k + compared to 5 . 3 mm k + . statistic micropet scans were performed on health female nude mice ( n = 3 ) ( 19 - 22 ) and selected coronal images at different time points after injection of 18 f - bodipy - 1 were shown in fig3 a . the heart was clearly visible at each time point examined . quantification of major organ activity accumulation in micropet scans was realized by measuring rois encompassing the entire organ in the coronal orientation . the averaged time - activity curves ( tacs ) of 18 f - bodipy - 1 in heart , liver , kidneys , and muscle were shown in fig3 b . the heart uptake of 18 f - bodipy - 1 was calculated to be 4 . 43 ± 0 . 44 , 3 . 49 ± 0 . 40 , and 1 . 98 ± 0 . 45 % id / g at 0 . 5 , 1 and 2 h p . i . 18 f - bodipy - 1 showed substantially high kidney uptake at 0 . 5 h p . i . ( 12 . 26 ± 1 . 57 % id / g ). the fast clearance of 18 f - bodipy - 1 gave significantly lower kidney uptake at 2 h p . i ., which are 2 . 86 ± 0 . 24 % id / g , respectively . therefore , the fast clearance and high binding to heart tissue of 18 f - bodipy - 1 gave high contrasts . for example , the heart - to - liver and heart - to - muscle reached 3 . 13 ± 0 . 39 , and 3 . 16 ± 0 . 30 at 2 h p . i ., respectively . statistic micropet scans were also performed on health rats ( n = 3 ) and selected coronal images at different time points after injection of 18 f - bodipy - 1 were shown in fig4 a . heart could be also clearly visualized at all time - points examined . due to the larger size of rats , the heart of uptake are consistently lower than those in mouse study , which are 0 . 75 ± 0 . 14 , 0 . 69 ± 0 . 10 , 0 . 70 ± 0 . 16 , and 0 . 61 ± 0 . 14 % id / g at 0 . 5 , 1 , 2 . 5 and 5 h p . i . however , the contrasts are consistent with those in the mouse study . for example , the heart to muscle ratios is 3 . 07 ± 0 . 49 at 2 . 5 h p . i . the biodistribution of 18 f - bodipy - 1 ( 50 μci / mouse ) was examined in health nude mice 3 h p . i . as shown in fig5 , heart uptake was significantly higher than those in blood , muscle and liver . the relative high kidney uptake further confirmed that the probe was cleared from urinal system . the consistence between biodistribution and micropet quantification fully validated the effectiveness of non - invasive micropet cardiac imaging with 18 f - bodipy - 1 . although the present invention has been described in terms of specific exemplary embodiments and examples , it will be appreciated that the embodiments disclosed herein are for illustrative purposes only and various modifications and alterations might be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the following claims . a and r 1 — r 9 are independently selected from the group consisting of hydrogen , halogen , hydroxy , alkoxy , nitro , substituted and unsubstituted animo , alkyl , cycloalky , carboxy , carboxylic acids and esters thereof , cyano , haloalkyl , aryl , including phenyl and aminophenyl , and heteroaryl . the rl - nr 7 r 8 r 9 moiety may be in meta or ortho position within the boron - containing ring . all publication cited herein , including the foregoing are expressly incorporated herein by references for the purpose of describing and disclosing compositions , preparation kits and methodologies that might be used in connection with the invention : 1 . rischpler c , park m j , fung g s , javadi m , tsui b m , higuchi t . advances in pet myocardial perfusion imaging : f - 18 labeled tracers . ann nucl med . 2011 . 2 . beller g a , bergmann s r . myocardial perfusion imaging agents : spect and pet . j nucl cardiol . 2004 ; 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