Patent Application: US-74068803-A

Abstract:
a method for noninvasive measurement of apoptosis is described . the method includes the steps of labeling annexin v with a positron emitter , injecting the labeled annexin v into a target cell group , obtaining an image of the target cell group using a positron emission tomography scanner , and evaluating the image to determine an amount of cell death within the target cell group . the target cell group may be a lesion or a suspected tumor . the positron emitter may be f - 18 . the step of labeling annexin v with f - 18 may include the steps of selecting an f - 18 labeled small molecule containing a protein conjugating group , synthesizing and purifying the selected molecule , producing a high specific activity prosthetic group as a result of the synthesizing and purifying step , and conjugating the prosthetic group to the annexin v . the produced prosthetic group may be n - succinimidyl - 4 - fluorobenzoate .

Description:
there are two primary processes that lead to cell death , necrosis and apoptosis . necrosis is caused by gross disruption of the cell membrane and is often the result of significant osmotic , mechanical , or chemical damage . molecular identification of necrosis is not well defined due to the chaotic nature of cell death . the most common histologic feature of necrosis is perinecrotic inflammation . apoptosis is a genetically defined cell death that involves activation of a core enzymatic machine consisting of cysteine proteases , called caspases . in most cases , the activation of caspases represents irreversible progression to cell death . the process of apoptosis occurs during normal growth and differentiation to eliminate cells that are no longer necessary for tissue function . tumor cells are believed to contain a block in differentiation , therefore they may be rendered more susceptible than normal cells to the induction of apoptosis . several studies have shown that apoptosis can be detected on an organ level by imaging . hepatic apoptosis caused by intravenous injection of antifas antibody in mice was detected by an increase in hepatic uptake of 99m tc - annexin measured both by region of interest ( roi ) analysis and biodistribution data . higher 99m tc - annexin uptake in cardiac allografts compared to control cardiac isografts in rats has also been observed . in early clinical studies , cardiac spect imaging with 99m tc - annexin has been used to visualize cardiac transplant rejection . it has been reported that 99m tc - annexin v has rapid blood clearance in humans , and it has been stated that this rapid clearance should permit diagnostic images on transplant rejection within 2 hours post - injection . the relatively small size of tumors compared to whole organs makes detection of apoptosis in tumors by external imaging more challenging . several research groups reported significant increases in the localization of 99m tc - annexin v after chemotherapy in b - cell lymphomas , breast cancer tumors , and hepatomas in rodents by imaging 1 - 2 hours post - injection of 99m tc - annexin v . spect imaging studies of apomate ™ ( theseus imaging corporation &# 39 ; s kit for the preparation of 99m tc labeled annexin v ) have begun in patients with lymphomas , sarcomas , breast and lung cancers . in several studies , patients who showed increased tumor signal with apomate ™ after chemotherapy demonstrated better therapeutic outcomes than patients whose tumors demonstrated little uptake . in these studies imaging was performed at two and 24 hours post - injection of apomate ™. the sensitivity of a dedicated pet camera is 20 - 100 times greater than spect and the 4 - 5 mm spatial resolution of pet is superior to the 8 - 1 3 mm spatial resolution of spect ; therefore , a strong case can be made that imaging cell death in tumors could best be done with pet imaging using a labeled compound such as 18 f - annexin v . since useful images of chemotherapy induced tumor cell death were obtained at 2 hours post - injection of 99m tc - labeled annexin v , the 110 minute half - life of 18 f should be sufficiently long - lived to allow 18 f labeled annexin v to provide corresponding pet images . pet cameras can rigorously correct for the effect of gamma - ray attenuation , and thereby provide quantitative measurements of radiotracer concentration in vivo . the recent availability of high spatial resolution animal pet scanners has led to strong interest in pet ligands for rational drug development . referring to fig5 , a flow chart 500 illustrates a method of quantifying apoptosis using pet scanning images and f - 18 labeled annexin v according to a preferred embodiment of the present invention . the first step 505 is to label the annexin v using a positron emitter , such as f - 18 , as further described below . the second step 510 is to inject the f - 18 labeled annexin v into a lesion or suspected tumor , or into any target cell group for which it is desired to quantify an amount of apoptosis . then , in step 515 , a pet scanner is used to produce an image of the target cell group . finally , in step 520 , the image is analyzed to determine an amount of cell death in the target cell group . the use of radiolabeled agents to describe tumor biochemistry in vivo requires that the radiolabel be prepared with high specific activity . in other words , the ratio of radiolabeled target - binding agent to non - radiolabeled target - binding agents needs to be as high as possible . methods used to achieve high specific activity are different when labeling proteins ( e . g ., annexin ), as compared to when small non - protein molecules ( e . g ., glucose ) are labeled . when radiolabeling small non - protein molecules , high specific activity is achieved by starting with high specific activity radionuclide , using a no - carrier - added labeling reaction , followed by a chromatographic separation of labeled compound from free radionuclide and unlabeled precursor . when preparing high specific activity radiolabeled proteins , the changes in the properties of the protein caused by the radiolabel ( charge , lipophilicity , polarity , size ) are too small to permit a chromatographic separation of radiolabeled and non - labeled protein ; therefore , specific activity is determined by the number of moles of radionuclide and protein as well as the efficiency of the labeling reaction . high specific activity labeling with fluorine requires different methods than high specific labeling with iodine . proteins can be radiolabeled with iodine via direct electrophilic radioiodination of tyrosine groups . with this method , the specific activity of the radiolabeled protein is controlled by the amount of protein present , because the efficiency of the labeling reaction is usually very high . unfortunately , the fluorine equivalent ( i . e ., electrophilic fluorination ) requires excess nonradioactive fluoride and thus can only produce low specific activity labeled protein . to prepare high specific activity fluorinated proteins , researchers have taken an approach analogous to the bolton - hunter method of protein radioiodination . in this approach an 18 f labeled small molecule containing a protein conjugating group is synthesized and purified by chromatographic methods to produce a high specific activity prosthetic group . the 18 f labeled prosthetic group is then conjugated to the desired protein . with this two - step approach , the specific activity of the labeled protein depends on the specific activity of the 18 f labeled prosthetic group , the amount of protein used , and the efficiency of the conjugation reaction . several 18 f labeled prosthetic groups appear in the literature , and are summarized in table 2 . deciding which group to use for conjugation with annexin v depends on many factors including yield , specific activity , speed and ease of synthesis and purification of the 18 f labeled prosthetic group . in addition , the protein conjugation group on the prosthetic molecule must be matched with available reactive groups on the protein . the higher the number of reactive moieties on the protein , the more likely the conjugation reaction will proceed with high efficiency ; however , the possibility of multiple conjugations per single protein molecule also increases . although multiple conjugations would tend to increase yield , too many conjugations might reduce biologic activity due to steric blockage of the ps binding site on annexin v . one of the earliest syntheses of an 18 f labeled prosthetic group was that of 4 -[ 18 f ] fluorophenacyl bromide ( fpb ). fpb was prepared in three steps with a total yield of 28 - 40 % in 75 min . and attached to human serum albumin ( hsa ) and fibrinogen at 47 ° c . in 95 % and 25 - 30 % yields respectively . a quicker higher yield three step synthesis (& lt ; 35 min ., 65 % yield ) was later reported . unfortunately , labeling of hsa at room temperature ( temperature less likely to denature proteins ) with fpb prepared by the latter route was much lower ( 7 %). higher protein labeling yields ( 70 %) were achieved by adding more thiol groups to hsa by pretreatment with 2 - iminothiolane . however , treatment with 2 - iminothiolane also increased the amount of protein crosslinking ( intra - and intermolecular ) from the formation of non - native disulfide bonds . several investigators have prepared 18 f labeled prosthetic groups containing n - succinimidyl ester groups that react with lysine amines to form an amide bond . proteins generally have more lysines than free thiol groups and therefore are better targets for labeling proteins with high efficiency . an early method utilized disuccinimidyl suberate to attach an activated ester to [ 18 f ] fluorobenzylamine ; however , this can also produce a [ 18 f ] fluorobenzylamide dimer which is unreactive with protein . referring to fig1 , a three - step synthesis of n - succinimidyl - 4 -[ 18 f ] fluorobenzoate ( sfb ) is outlined in scheme 1 . scheme 1 produces [ 18 f ] sfb in a yield of 25 % in a total synthesis time of 100 minutes . in this synthesis , 4 ( trimethylammonium triflate ) benzaldehyde ( 1 ) is reacted with [ 18 f ] fluoride and kryptofix - 222 in dmso to give 4 -[ 18 f ] fluorobenzaldehyde ( 2 ) which is then oxidized to 4 -[ 18 f ] fluorobenzoic acid l3 ), followed by formation of the activated - ester [ 18 f ] sfb . during the final step careful exclusion of air is necessary to prevent precipitation of dicyclohexyl urea ( dcu ) which can clog the hplc system used in the final purification of [ 18 f ] sfb . referring to fig2 , in a refinement of this route ( scheme 2 ), higher reaction temperatures ( 150 ° c .) were used , and disuccinimidyl carbonate was used in the final step to increase the yield of [ 18 f ] sfb to 34 % in a total synthesis time of 55 minutes and eliminate the formation of dcu . [ 18 f ] sfb prepared by either route gave 18 f labeled f ( ab ′) 2 fragments in a 50 % yield after room temperature incubation with [ 18 f ] sfb for 15 - 20 minutes . referring to fig3 , an alternate three - step synthesis of [ 18 f ] sfb ( scheme 3 ) has been reported . scheme 3 gives higher yields ( 65 - 80 %) in a total synthesis time of one hour . in this synthesis , ethyl 4 ( trimethylammonium triflate ) benzoate ( 4 ) is reacted with [ 18 f ] fluoride and kryptofix - 222 in dimethylacetamide to give ethyl 4 -[ 18 f ] fluorobenzoate ( o ) which is then hydrolyzed to 4 [ 18 f ] fluorobenzoic acid ( 3 ), followed by formation of the activated ester [ 18 f ] sfb as in scheme 2 . referring to fig4 , a single - step synthesis of n - succinimidyl - 4 [ 18 f ]( fluoromethyl ) benzoate ( 7 ) has been reported ( i . e ., scheme 4 ). in this synthesis n - succinimidyl 4 -[( nitrobenzenesulfonyl ) oxymethyl ] benzoate ( z ) is reacted with [ 18 f ] fluoride and kryptofix - 222 in acetone for 5 minutes and then purified by hplc . while this one step synthesis is convenient , the yields of 6 are low ( 10 - 15 %) and a careful hplc purification is necessary to remove all the side products . an exemplary synthesis of no - carrier - added ( n . c . a .) n - succinimidyl 4 -[ 18 f ] fluorobenzoate ([ 18 f ] sfb ) has been carried out as described below : the aqueous [ 18 f ] fluoride solution is placed in a 13 × 100 mm borosilicate tube , 8 μl of im potassium carbonate is added , and the tube is placed in a 95 ° c . oil bath . water is evaporated under a stream of nitrogen until the volume is reduced to 50 - 100 μl . the radioactivity is counted and the time is recorded as the starting time of the synthesis . then the aqueous solution of 18 f — is added to a reactavial containing 500 μl dry acetonitrile , 5 . 0 mg krytofix - 222 , and 8 μl of 1m potassium carbonate . this mixture is evaporated to dryness at 95 ° c . under a stream of nitrogen . additional dry acetonitrile ( 300 μl ) is added to the vial and the azeotropic distillation is repeated . the addition of acetonitrile and evaporation under nitrogen is then repeated twice more . to the dry residue is added 10 mg of ethyl 4 -( trimethylammonium triflate ) benzoate dissolved in 250 μl anhydrous dimethyl acetamide , followed by heating at 150 ° c . for 10 minutes . the next step , hydrolysis of the ethyl ester group of ethyl 4 -[ 18 f ] fluorobenzoate to 4 -[ 18 f ] fluorobenzoic acid , is accomplished by the addition of 500 μl of 1 m naoh and stirring for 8 minutes at 95 ° c . the reaction is then acidified with 650 μl of 1 m hcl and diluted with water to a final volume of 10 ml . the solution is drawn into a syringe with a luer lock fitting and passed through an activated c - 18 sep - pak . polar material is removed from the column by elution with 2 . 0 ml 0 . 01m hcl . the sep - pak column , still retaining 4 -[ 18 f ] fluorobenzoic acid , is blown dry with a stream of nitrogen and the 4 -[ 18 f ] fluorobenzoic acid ( 3 ) is eluted with 2 . 5 ml acetonitrile . the decay corrected yield at this point may range from 56 - 80 %. when this procedure to prepare n - succinimidyl 4 -[ 18 f ] fluorobenzoate ([ 18 f ] sfb ) is followed by the addition of 10 ml of a 20 % solution of tetrabutylammonium hydroxide to the acetonitrile solution of 4 -[ 18 f ] fluorobenzoic acid , followed by evaporation to dryness at 95 ° c . under a stream of nitrogen and drying by azeotropic distillation with three additions of 400 μl acetonitrile , followed by the addition of a solution of 15 mg bis - n - hydroxysuccinimidyl carbonate in 300 μl acetonitrile to the dry residue , sealing the vial and heating the vial at 150 ° c . for 8 minutes , hplc analysis may show an unsymmetrical peak . further resolution of this peak by increasing the percentage of water in the hplc eluate may show that as much as 50 % of applied radioactivity eluting from the hplc column elutes in a broad peak ( not [ 18 f ] sfb ) prior to the elution of the desired [ 18 f ] sfb peak . acetonitrile / water or methanol / water may be used as the chromatography solvent . in general , acetonitrile / water mixtures give better resolution with reverse phase c18 columns than does methanol / water . also note that as shown in table 2 , [ 18 f ] sfb prepared by this method has not ever been used to conjugate any proteins . to improve the yield of [ 18 f ] sfb , the synthesis of [ 18 f ] sfb from 4 -[ 18 f ] fluorobenzoate has been performed using another reported procedure . in this method , the acetonitrile solution of 4 -[ 18 f ] fluorobenzoate that is eluted from the sep - pak is transferred to a round bottom flask and evaporated on a rotary evaporator using reduced pressure from a water aspirator and a room temperature heating bath . the residue is dried by multiple additions of either acetonitrile or acetone followed by evaporation on the rotary evaporator . the residue is reconstituted in acetonitrile , transferred to a reactavial , and evaporated to about 50 μl . to the reactavial is added 100 μl of a 0 . 1m solution of pyridine in acetonitrile and 100 μl of a 0 . 1m solution of disuccinimidylcarbonate in acetonitrile . the vial is sealed and heated at 150 ° c . for 6 - 8 minutes , cooled and purified by hplc . this method may be modified by replacing the pyridine , which acts both as a base and as an acylation catalyst , with dimethylaminopyridine , which is a better acylation catalyst . to the residue of 4 -[ 18 f ] fluorobenzoate is added 50 μl of a 0 . 1m solution of dimethylaminopyridine in acetonitrile and 200 μl of a 0 . 1m solution of disuccinimidylcarbonate in acetonitrile . the vial is sealed and heated at 150 ° c . for 6 - 8 minutes , cooled and 700 μl water added to make the acetonitrile / water ratio similar to the hplc eluate used to purify the [ 18 f [ sfb . the addition of water causes a precipitate to form , and the suspension is transferred to a microfuge tube and centrifuged for three minutes to settle the solid . the supernatant is removed and injected onto an radio - hplc fitted with a delta - pac c18 , 3 micron , 3 . 9 × 150 mm column ( waters ), and a variable wavelength uv detector set for 236 nm and eluted with a solution consisting of 80 % water / 20 % acetonitrile + 0 . 1 % glacial acetic acid at a flow of 1 . 2 ml / min . on this system the retention time of [ 18 f ] sfb is approximately 14 minutes , and the decay corrected yield of [ 18 f ] sfb may range from approximately 52 - 55 % in 2 - 3 hours preparation time . once the prosthetic group is synthesized and purified , it is then conjugated to annexin v . in an exemplary preparation for the conjugation , the [ 18 f ] sfb is dissolved in the hplc eluate ( 80 % water / 20 % acetonitrile + 0 . 1 % glacial acetic acid ), then diluted to a volume of 10 ml with water , drawn into a syringe and loaded onto an activated waters sep - pak . the sep - pak is blown dry using a stream of nitrogen , and the retained [ 18 f ] sfb eluted with 2 . 0 ml methylene chloride . the following exemplary conjugation of [ 18 f ] sfb to annexin v utilizes conditions previously reported for the conjugation of [ 18 f ] sfb to antibody fragments . the methylene chloride solution of [ 18 f ] sfb is added to a 1 . 5 ml microfug tube and evaporated to dryness under a stream of nitrogen . to the residue in the tube is added 25 - 100 μl of a solution of annexin v ( 5 μg / μl ) in 0 . 1 m borate buffer , ph 8 . 5 . this is then incubated for 20 minutes . the reaction was diluted to 100 μl with 0 . 1 m phosphate buffer , ph 7 . 4 , and injected onto a tsk - 2000 size exclusion column and eluted with 0 . 1m phosphate buffer , ph 7 . 4 at a flow rate of 1 . 0 ml / min . the retention time of annexin v is 10 minutes . see table 1 below for a tabulation of experimental results of conjugating [ 18 f ] sfb to annexin v . as shown in table 1 , the highest experimental radiolabeling yields occur using an annexin v concentration of 5 μg / μl . this requires that to produce high specific activity 18 f - annexin v , the amount of annexin v and thus the reaction volume must be small . good yields of 18 f - annexin v can be produced in a 25 μl volume ( 125 μg ); however , when starting with a large volume of [ 18 f ] sfb in methylene chloride , yields can be reduced because of the difficulty in concentrating a large volume of [ 18 f ] sfb into a small area where it can be readily dissolved in 25 μl . the binding activity of [ 18 f ] sfb - annexin v to cells expressing phosphatidylserine ( ps ) has been performed using red blood cells ( rbcs ). normal rbcs have very few binding sites for annexin ; however , rbcs from commercial preserved whole blood ( e . g ., coulter 4 cplus normal control ) have high levels of exposed ps . [ 18 f ] sfb - annexin v at a concentration of 12 nmol / l final concentration is added to each of two tubes containing 1 ml of buffer hnkgb ( 10 mm hepes - na ph 7 . 4 , 136 mm nacl , 2 . 7 mm kcl , 5 mm glucose , and 1 mg / ml bsa ) plus 2 . 5 mm cacl 2 . to one tube is added 4 . 2 × 10 8 rbcs . both tubes are incubated at room temperature for 30 minutes , then centrifuged for 3 minutes at 2000 × g . the supernatants are removed and both the supernatants from both tubes and cells remaining in the single tube are counted . the percentage of radioactivity bound to the cells is calculated from 100 × ( 1 -( supernatant counts in the presence of cells )/ supernatant counts in the absence of cells ). while the present invention has been described with respect to what is presently considered to be the preferred embodiments , it is to be understood that the invention is not limited to the disclosed embodiments . to the contrary , the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims . the scope of the following claims is to be accorded the broadest interpretation so as to encompass all such modifications and equivalent structures and functions . 1 . martin s j , reutelingsperger c p , mcgahon a j , rader j a , van schie r c , laface d m , green d r . early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus : inhibition by overexpression of bcl - 2 and abl . j exp med . 1995 ; 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