Patent Application
20070031333
1. Field of the Invention
This invention disclosure addresses development of new imaging technology for noninvasive quantitative measurement of apoptosis in vivo. Apoptosis is genetically defined cell death. Understanding the role of apoptosis is critical to the development of new molecular-based therapeutic modalities. An apoptosis-imaging agent could be used to modify therapy for cancer patients, and to monitor cardiac transplant patients. Annexin V is a protein that binds to exposed phosphatidylserine on dead or dying cells. This invention disclosure describes a method to label Annexin-V with a positron emitter (F-18) appropriate for PET scanning. Potential advantages of PET include improved spatial resolution and quantitation, which will be important for monitoring early cancers and for cardiac studies.
2. Description of the Related Art
Cancer cells survive, in part, due to specific signaling pathways that prevent apoptosis in cells harboring protooncogene or suppressor oncogene mutations. A current goal of cancer research is to develop novel therapeutics that will specifically target mutated gene products that are responsible for cancer cell growth and resistance to apoptosis. Thus, many new anti-tumor agents block intracellular survival signaling pathways which then permits apoptotic cell death. Since current chemotherapy dosages are based on the maximally tolerated dose, both novel therapeutics and current chemotherapy agents would benefit from direct measure of tumor cell kill to achieve the biologic goal without causing excess toxicity.
Apoptosis is a genetically defined cell death that involves activation of a core enzymatic machine consisting of cysteine proteases, called caspases. An early molecular event in apoptosis is the flipping of phosphatidylserine (PS) from the inner lipid bilayer to the outer layer of the plasma membrane. Annexin V binds PS with extremely high affinity (Kd=7 nmol/L), as well as specificity, and has been used in vitro to identify apoptotic cells. Technetium-99m labeled Annexin V (Apomate™) is currently in early clinical trials for the detection of apoptosis by Single Photon Emission Computed Tomography (SPECT) in cardiac allograft rejection. Apomate™ as a marker of tumor cell death is also being explored.
In Positron Emission Tomography (PET), the simultaneous detection of the two coincident 511 keV gamma rays from the annihilation of a positron permits greater sensitivity and spatial resolution than that obtained with SPECT and provides truly quantitative imaging. Annexin V labeled with the positron emitter F-18 will be capable of detecting tumor cell death, thereby serving as an early predictor of clinical response to anti-tumor therapy. It is believed that the inherent advantages of PET would make 18F-Annexin V a better imaging agent than Apomate™ for quantifying the amount of cell death in tumors, especially in small metastatic lesions.
Traditional anti-cancer drug development focused on agents that directly killed cells; thus end-points of clinical trials required tumor regression. New anti-cancer therapies target specific molecular pathways. These molecular targeting agents are used to amplify the effect of other anti-cancer drugs. Investigators who are interested in new drug development need to determine whether a novel agent affects the target in a specified fashion. Traditional tests of novel agents measure tumor regression 6 to 12 weeks following the therapy. Tumor regression with this time frame is the result of some combination of increased cell death and decreased cancer growth. It is therefore difficult with traditional regression techniques to assess the effect of cell death alone on tumor response. Early response detection methods are therefore needed in order to determine the effectiveness of novel anti-tumor agents.
The invention provides a method for noninvasive measurement of apoptosis. 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 one of 4-[18F]fluorophenacyl-bromide (FPB), N-succinimidyl-8-[(4′-[18F]fluorobenzyl)amino] suberate (SFBS), or N-succinimidyl-4-[18F]fluorobenzoate (SFB). When the produced prosthetic group is SFB, the step of conjugating the SFB to the Annexin V may include the steps of placing the SFB into a methylene chloride solution, evaporating the methylene chloride solution to dryness using a stream of nitrogen to produce a residue, adding a solution of Annexin V to the residue, and incubating a result of the adding step.
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 99mTc-Annexin measured both by region of interest (ROI) analysis and biodistribution data. Higher 99mTc-Annexin uptake in cardiac allografts compared to control cardiac isografts in rats has also been observed. In early clinical studies, cardiac SPECT imaging with 99mTc-Annexin has been used to visualize cardiac transplant rejection. It has been reported that 99mTc-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 99mTc-Annexin V after chemotherapy in B-cell lymphomas, breast cancer tumors, and hepatomas in rodents by imaging 1-2 hours post-injection of 99mTc-Annexin V. SPECT imaging studies of Apomate™ (Theseus Imaging Corporation's kit for the preparation of 99mTc 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 18F-Annexin V. Since useful images of chemotherapy induced tumor cell death were obtained at 2 hours post-injection of 99mTc-labeled Annexin V, the 110 minute half-life of 18F should be sufficiently long-lived to allow 18F 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
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 18F labeled small molecule containing a protein conjugating group is synthesized and purified by chromatographic methods to produce a high specific activity prosthetic group. The 18F 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 18F labeled prosthetic group, the amount of protein used, and the efficiency of the conjugation reaction.
Several 18F 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 18F 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 18F labeled prosthetic group was that of 4-[18F] 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 (<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 18F 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 [18F]fluorobenzylamine; however, this can also produce a [18F]fluorobenzylamide dimer which is unreactive with protein. Referring to
Scheme 1 produces [18F]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 [18F]fluoride and kryptofix-222 in DMSO to give 4-[18F]fluorobenzaldehyde (2) which is then oxidized to 4-[18F]fluorobenzoic acid L3), followed by formation of the activated-ester [18F]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 [18F]SFB. Referring to
Referring to
Referring to
An exemplary synthesis of no-carrier-added (n.c.a.) N-Succinimidyl 4-[18F]Fluorobenzoate ([18F]SFB) has been carried out as described below: The aqueous [18F]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 18F— 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-[18F]fluorobenzoate to 4-[18F]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-[18F]fluorobenzoic acid, is blown dry with a stream of nitrogen and the 4-[18F]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-[18F]Fluorobenzoate ([18F]SFB) is followed by the addition of 10 mL of a 20% solution of tetrabutylammonium hydroxide to the acetonitrile solution of 4-[18F]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 [18F]SFB) prior to the elution of the desired [18F]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, [18F]SFB prepared by this method has not ever been used to conjugate any proteins.
To improve the yield of [18F]SFB, the synthesis of [18F]SFB from 4-[18F]fluorobenzoate has been performed using another reported procedure. In this method, the acetonitrile solution of 4-[18F]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-[18F]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 [18F[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 [18F]SFB is approximately 14 minutes, and the decay corrected yield of [18F]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 [18F]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 [18F]SFB eluted with 2.0 mL methylene chloride.
The following exemplary conjugation of [18F]SFB to Annexin V utilizes conditions previously reported for the conjugation of [18F]SFB to antibody fragments. The methylene chloride solution of [18F]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 [18F]SFB to Annexin V.
*Entire 2.0 mL solution of [18F]SFB concentrated for this reaction.
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 18F-Annexin V, the amount of Annexin V and thus the reaction volume must be small. Good yields of 18F-Annexin V can be produced in a 25 μL volume (125 μg); however, when starting with a large volume of [18F]SFB in methylene chloride, yields can be reduced because of the difficulty in concentrating a large volume of [18F]SFB into a small area where it can be readily dissolved in 25 μL.
The binding activity of [18F]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. [18F]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 CaCl2. To one tube is added 4.2×108 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).
Results:
Percent binding to cells were 50-52%, n=4;
Reported values for 99mTc-Annexin V are 75-85%.
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.
The following publications are incorporated by reference herein:
This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 60/437,719, entitled “Novel F-18 Labeled Annexin V, Synthesis Thereof, and Use Thereof”, filed Jan. 2, 2003, the contents of which are incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 60437719 | Jan 2003 | US |
