on the basis of a study of a series of alkyl ether analogs of pyropheophorbide - a , we developed a relatively long wavelength absorbing photosensitizer , the 3 -( 1 - hexyloxy ) ethyl - derivative of pyropheophorbide - a 1 ( hpph ). this compound is tumor - avid and currently in phase i / ii human clinical trials at the roswell park cancer institute . we investigated the utility of this compound as a “ vehicle ” by conjugation with mono - or di - bisaminoethanethiols ( n 2 s 2 ligand ). the results obtained from in vivo biodistribution experiments indicated that the tumor / non - tumor uptake ratio of the drug depends on the time and tumor size . with time , the clearance of the hpph - based compounds from tumor was found to be slower than from most of the non - tumor tissues . however , the short 6 h half - life of 99m tc was found to be incompatible with 24 - hour imaging time , suggesting that the use of a longer - lived isotope could provide a useful scanning agent . another approach for developing an improved tumor - imaging agent could be to replace hpph with those compounds that exhibit significantly higher tumor to non - tumor ratio . the synthesis of the related long - lived radionuclide could generate improved imaging and therapeutic ( pdt ) agents . a compound that effectively functions both as an imaging agent and a photosensitizer creates an entirely new paradigm for tumor diagnosis and therapy . after peripheral intravenous injection of this compound , a patient can be scanned with a scanner . the location of the tumor site ( s ) can thus be defined , and , while the patient remains in the scanner , an interventional nuclear scientist can transcutaneously insert ultra - slim needles that can act as introducers for light - transmission fibers into the lesion ( s ). because each fiber diameter is & lt ; 400 microns , the introducer needles would produce negligible tissue damage . a light source can be coupled to the fibers , and pdt of the lesion ( s ) can be commenced , without any significant injury to other organs . because the same molecule represents the contrast medium and the therapeutic medium , the lesion ( s ) can be continuously imaged during needle / fiber placement , without any ambiguity in terms of localization or “ misregistration ” of separate diagnostic / therapeutic images . this paradigm would make the low - toxicity and high efficacy of pdt available to virtually any location from the skull base to the floor of the pelvis . positron emission tomography ( pet ) is a technique that permits non - invasive use of positron labeled molecular imaging probes to image and assay biochemical processes at cellular function in living subject . compared to single - photon - emission - computed tomography ( spect ), to produce tomographic images , pet is at least tenfold more sensitive . the short half - lives of the most commonly used positron emitting nuclides are not suitable for drugs with biological half lives in days . however , iodine - 124 is a positron emitter with a half - life of 4 . 2 days and is suitable for labeling probes with biological half lives of few days . this isotope has not been widely used because of the limited availability and complex decay scheme including several high - energy gamma rays . pentlow et al . were the first to show that quantitative imaging with 124 i is possible . in our attempt to develop an efficient bifunctional diagnostic / therapeutic agent , we initially synthesized and evaluated certain pyropheophorbide analogs ( derived from chlorophyll - a )- n 2 s 2 — 99m tc conjugates ( 23 ). the in vivo biodistribution results suggested that the short 6h half - life of 99m tc is incompatible with the 24 h imaging time ( the time for maximum uptake of the drug and therapy ), suggesting that the use of a longer - lived isotope could provide a useful scanning agent . therefore , our objective was to introduce 124 i positron emitter to certain tumor - avid porphyrin - based photosensitizers containing iodobenzyl functionalities and investigate their utility in tumor imaging and photodynamic therapy . there are several methods for labeling the compounds with iodine isotopes . conversion of the cold iodo - to radioactive iodo - is possible , but the specific activity of the resulting product is low . it has been shown that in general iodine substituted at aliphatic chain is less stable than that present in aromatic structures . therefore , we prepared a series of aromatic alkyl ethers and evaluated them for in vitro ( rif cells ) and in vivo efficacy ( rif cells ). among a series of alkyl ether analogs with variable carbon units containing an iodophenyl group , the 3 - devinyl - 3 -( 1 ′- 3 ″- iodobenzyloxy ) ethyl pyropheophorbide - a ( scheme 1 ) in preliminary screening was found to be as effective as hpph , a photosensitizer developed in our laboratory , and is at phase ii human clinical trials . where r is — cooh , — co 2 r 3 , — conhr 4 , monosaccharide , disaccharide , polysaccharide , folic acid residue , or integrin antagonist ; r 1 , when present , is c 1 - c 12 alkyl , r 3 is c 1 - c 12 alkyl and r 4 completes an amino acid residue . methyl - 3 - devinyl - 3 -{ 1 ′-( 3 - iodobenzyloxy ) ethyl pyropheophorbide a : as seen in fig3 , pyropheophorbide - a 1 was obtained from chlorophyll - a by following the literature procedure . it was reacted with hbr / acetic acid and the intermediate unstable bromo - derivative was immediately reacted with 3 - iodobenzylalcohol under nitrogen atmosphere at room temperature for 45 min . after the standard work - up , the reaction mixture was purified by column chromatography ( alumina gr . iii , eluted with dichloromethane ) and the desired iodo - derivative 2 was isolated in 70 % yield . uv - vis ( ch 2 cl 2 ): 662 ( 4 . 75 × 10 4 ), 536 ( 1 . 08 × 10 4 ), 505 ( 1 . 18 × 10 4 ). 410 ( 1 . 45 × 10 5 ). 1 h - nmr ( cdcl 3 ; 400 mhz ): δ 9 . 76 , 9 . 55 and 8 . 56 ( all s , 1h , meso - h ); 7 . 76 ( s , 1h , arh ); 7 . 64 ( d , j = 6 . 8 , 1h , arh ); 7 . 30 ( d , j = 8 . 0 , 1h , arh ); 7 . 05 ( t , j = 8 . 2 , 1h , arh ); 6 . 00 ( q , j = 6 . 9 , 1h , 3 1 - h ); 5 . 20 ( dd ( abx pattern ), j = 19 . 6 , 60 . 0 , 2h , 13 2 - ch 2 ); 4 . 70 ( d , j = 12 . 0 , 1h , och 2 ar ); 4 . 56 ( dd , j = 3 . 2 , 11 . 6 , 1h , och 2 ar ); 4 . 48 - 4 . 53 ( m , 1h , 18 - h ); 4 . 30 - 4 . 33 ( m , 1h , 17 - h ); 3 . 72 ( q , j = 8 . 0 , 2h , 8 - c h 2 ch 3 ); 3 . 69 , 3 . 61 , 3 . 38 and 3 . 21 ( all s , all 3h , for 17 3 - co 2 ch 3 and 3 × ring ch 3 ); 2 . 66 - 2 . 74 , 2 . 52 - 2 . 61 and 2 . 23 - 2 . 37 ( m , 4h , 17 1 and 17 2 - h ); 2 . 18 ( dd , j = 2 . 8 , 6 . 4 , 3h , 3 2 - ch 3 ); 1 . 83 ( d , j = 8 . 0 , 3h , 18 - ch 3 ); 1 . 72 ( t , j = 7 . 6 , 3h , 8 - ch 2 c h 3 ); 0 . 41 ( brs , 1h , nh ); — 1 . 71 ( brs , 1h , nh ). mass : calculated for c 41 h 43 n 4 o 4 i : 782 . found : 805 ( m + + na ). 1 h - nmr ( cdcl 3 ; 600 mhz ): δ 9 . 76 , 9 . 54 and 8 . 55 ( all s , 1h , meso - h ); 7 . 43 ( m , 2h , arh ); 7 . 36 ( m , 2h , arh ); 6 . 01 ( q , j = 6 . 7 , 1h , 3 1 - h ); 5 . 20 , dd ( abx pattern ), j = 19 . 1 , 87 . 9 , 2h , 13 2 - ch 2 ); 4 . 78 ( dd , j = 5 . 4 , 11 . 9 , 1h , och 2 ar ); 4 . 61 ( dd , j = 1 . 7 , 12 . 0 , 1h , och 2 ar ); 4 . 50 ( q , j = 7 . 4 , 1h , 18 - h ); 4 . 32 ( d , j = 8 . 8 , 1h , 17 - h ); 3 . 72 ( q , j = 7 . 8 , 2h , 8 - c h 2 ch 3 ); 3 . 69 , 3 . 61 , 3 . 37 and 3 . 18 ( all s , all 3h , for 17 3 - co 2 ch 3 and 3 × ring ch 3 ); 2 . 66 - 2 . 75 , 2 . 52 - 2 . 61 and 2 . 23 - 2 . 37 ( m , 4h , 17 1 and 17 2 - h ); 2 . 16 ( m , 3h , 3 2 - ch 3 ); 1 . 83 ( d , j = 7 . 2 , 3h , 18 - ch 3 ); 1 . 72 ( t , j = 7 . 6 , 3h , 8 - ch 2 c h 3 ); 0 . 45 ( brs , 1h , nh ); 0 . 19 ( s , 9h , tert - butyltin ); − 0 . 59 ( brs , 1h , nh ). mass : calculated for c 45 h 52 n 4 o 4 sn : 831 . found : 854 ( m + + na ). the trimethyltin analog 3 ( 50 μg ) obtained by reacting 2 with hexamethyldistannane and bis -( triphenylphosphine ) palladium ( ii ) dichloride in 1 , 4 - dioxane ( see fig3 ) was dissolved in 100 μl of 10 % acetic acid in methanol . na 124 i was added in 0 . 1n naoh . the solution was mixed and an iodogen bead was added . the reaction mixture was incubated at room temperature for 30 minutes and the reaction product was purified using hplc ( fig1 ). the labeled product was collected . the hplc chromatogram of the purified product is shown in fig2 . for evaluating in vitro photosensitizing efficacy of 3 - iodobenxyloxyethyl - pyropheophorbide - a 2 , rif tumor cells were grown in alpha - dmem with 10 % fetal calf serum , penicillin and streptomycin . cells were maintained in 5 % co 2 , 95 % air and 100 % humidity . for determining the pdt efficacy , these cells were plated in 96 - well plates and a density of 1 × 10 4 cells well in complete media . after overnight incubation to allow the cells to attach , the hpph and the related cold - iodo derivative 2 were individually added at variable concentrations . after 3 hr incubation in the dark at 37 ° c ., the cells were washed once with pbs , and irradiated with light . after light treatment , the cells were washed once and placed in complete media and incubated for 48 hrs . then 10 μl of a 4 - mg / ml solution of mtt was added to each well . after incubating for 4 hr at 37 ° c . the mtt + media were removed and 100 μl dmso was added to solubilize the formazin crystals . the 96 - well plate was read on a microtiter plate reader at an absorbance of 560 nm . the optimal cell kill was obtained at a concentration of 1 . 0 μm . the results were plotted as percent survival of the corresponding dark ( drug no light ) control for each compound tested . ( fig4 ) each data point represents the mean from 3 separate experiments , and the error bars are the standard deviation . each experiment used 5 replicate wells . methyl - 3 - iodo - benzyloxy - ethyl ) pyropheophorbide - a : the in vitro photosensitizing efficacy of hpph and the iodo - benzyloxyethyl - pyropheophorbide - a 2 as seen in fig3 , was compared at variable experimental conditions and the results are summarized in fig4 . as can be seen both photosensitizers produced similar efficacy at 0 . 6 μm drug concentration . however , at lower concentration 0 . 3 μm the iodo - analog 2 was found to be slightly more effective . the in vivo efficacy was determined in c3h mice bearing rif tumors ( 5 mice / group ). the tumors were exposed to light at 665 nm ( in vivo absorption ) with a laser light ( 135 j / cm 2 ) for 30 minutes . the tumor - regrowth was measured everyday ( for details see ‘ methods ’ part of the project ). as can be seen from fig5 , the 3 - devinyl - 3 -( 1 ′- iodobenzyloxy ) ethyl analog was quite effective at a dose of 1 . 0 and 1 . 5 μmol / kg . at lower doses ( 0 . 25 and 0 . 50 μmole / kg ), tumor re - growth was observed at 10 and 15 days post - injection . further studies to optimize the treatment conditions at variable fluence and fluence - rates and time intervals are currently in progress . in initial experiments , the i - 124 labeled photosensitizer 2 at variable radioactive doses ( 35 , 50 and 100 μci ) was injected in three sets of c3h mice ( 3 mice / group , bearing rif tumors at the shoulder ) respectively and the images were taken with a small animal pet scanner at 24 , 48 and 72 h time intervals ( fig6 images a , b , and c ). in all radioactive doses , the best images were obtained at 48 h post injection of the drug . however , as expected , the presence of the compound in some other organs , especially in liver was evident . after pet imaging at 48 h post - injection , a group of mice ( 3 mice / group ) were sacrificed and the biodistribution of the i - 124 pet agent in selected organs / gram were determined . the results are summarized in table 1 . the imaging of specific molecular targets that are associated with cancer should allow earlier diagnosis and better management of oncology patients . positron emission tomography ( pet ) is a highly sensitive non - invasive technology that is ideally suited for pre - clinical and clinical imaging of cancer biology , in contrast to anatomical approaches . by using radiolabelled tracers , which are injected in nonpharmacological doses , three - dimensional images can be reconstructed by a computer to show the concentration and location ( s ) of the tracer of interest . compared to other positron emitters , i - 124 has advantage due to its longer half - life ( 4 . 2 days ). our invention reports a first example for the preparation of i - 124 labelled photosensitizers related to chlorines and bacteriochlorins with long wavelength absorption in the range of 660 - 800 nm . we have also shown the utility of these tumor - avid compounds for tumor detection and therapy . our approach also provides an opportunity to develop target - specific bifunctional agents by targeting certain receptors known to have over - expression in tumors , and these studies are currently in progress .
this invention describes a first report on the synthesis of certain 124 i - labelled photosensitizers related to chlorines and bacteriochlorins with long wavelength absorption in the range of 660 - 800 nm . in preliminary studies , these compounds show a great potential for tumor detection by positron emission tomography and treatment by photodynamic therapy . the development of tumor imaging or improved photodynamic therapy agent itself represent an important step , but a dual function agent provides the potential for diagnostic body scan followed by targeted therapy .