Preliminary Biological Evaluation of 18F-FBEM-Cys-Annexin V a Novel Apoptosis Imaging Agent

A novel annexin V derivative (Cys-Annexin V) with a single cysteine residue at its C-terminal has been developed and successfully labeled site-specifically with 18F-FBEM. 18F-FBEM was synthesized by coupling 18F-fluorobenzoic acid (18F-FBA) with N-(2-aminoethyl)maleimide using optimized reaction conditions. The yield of 18F-FBEM-Cys-Annexin V was 71.5% ± 2.0% (n = 4, based on the starting 18F-FBEM, non-decay corrected). The radiochemical purity of 18F-FBEM-Cys-Annexin V was >95%. The specific radioactivities of 18F-FBEM and 18F-FBEM-Cys-Annexin V were >150 and 3.17 GBq/µmol, respectively. Like the 1st generation 18F-SFB-Annexin V, the novel 18F-FBEM-Cys-Annexin V mainly shows renal and to a lesser extent, hepatobiliary excretion in normal mice. In rat hepatic apoptosis models a 3.88 ± 0.05 (n = 4, 1 h) and 10.35 ± 0.08 (n = 4, 2 h) increase in hepatic uptake of 18F-FBEM-Cys-Annexin V compared to normal rats was observed after injection via the tail vein. The liver uptake ratio (treated/control) at 2 h p.i. as measured via microPET correlated with the ratio of apoptotic nuclei in liver observed using TUNEL histochemistry, indicating that the novel 18F-FBEM-Cys-Annexin V is a potential apoptosis imaging agent.


Introduction
Apoptosis plays an important role, not only in physiology but also in pathology [1,2]. Dysregulation of apoptosis is associated with many diseases such as cancer, autoimmunity and neurodegenerative disorders. Therefore, it has significant clinical value of the detection and quantification of apoptosis in vivo for diagnosis and assessment of therapeutic efficacy. One of the early charateristics of apoptosis is the externalization of the phospholipid phosphatidylserine (PS) at the cell membrane [3,4]. Annexin V, a 36-kDa human protein, shows Ca 2+ -dependent binding to negatively charged phospholipid surfaces and was discovered as a vascular anticoagulant protein [5,6]. The anticoagulant activity is based on the high-affinity for PS. These characteristics make annexin V derivatives suitable candidates for imaging of apoptosis. Several annexin V tagged with bifunctional chelators (BFC) have been labeled with 99m Tc for single photon emission computed tomography (SPECT) imaging of apoptosis in vivo [7][8][9][10][11]. However, conjugation of BFC to annexin V for labeling with 99m Tc is usually done by targeting an amino group of one of the 21 lysine residues using BFC, but this method is rather non-specific as any of the -NH2 groups could be targeted. Recent studies have revealed that after structural modification in the recombinant expression annexin V can be directly marked with 99m Tc, giving derivatives such as 99m Tc(CO)3-HIS-cys-Anx V [12], 99m Tc-annexin V-117 [13] and 99m Tc-His10-annexin V [14]. These new annexin V molecules labeled by site-specific methods greatly improve sensitivity for detecting cell death in vivo [15]. Our group has reported a site-specific 99m Tc labeling method of a novel annexin V derivative (Cys-Annexin V) with a single cysteine residue at C-terminal [16]. 99m Tc-Cys-Annexin V is a potential SPECT imaging agent of apoptosis. However, because of its higher sensitivity, better spatial resolution and quantification properties a positron emission tomography (PET) analog would be very desirable. Some groups have reported the labeling of annexin V with N-succinimidy-4-18 F-fluorobenzoate ( 18 F-SFB) for PET imaging of apoptosis [17][18][19], however this labeling method is non-specific, as the 18 F-SFB reacts with any available NH2 group in the protein. Thiol-reactive agents such as N-substituted maleimides can be used to modify proteins on the cysteine group [20]. 18 F-N-[2-(4-Fluorobenzamido)ethyl]maleimide ( 18 F-FBEM) was used to label thiol-containing proteins as a novel site-specific labeling prosthetic group [21][22][23]. We report herein the labeling and preliminary in vivo evaluation of the novel 18 F-FBEM-Cys-Annexin V in normal mice and in rat models of apoptosis induced by cycloheximide. In mice the tracer uptake was studied by dynamic microPET imaging and microPET in a rat model of hepatic apoptosis. Apoptosis was confirmed in situ on liver slices using the terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay.

Radiolabeling
Annexin V has been labeled non-specifically with a number of isotopes, including 99m Tc, 124 I, 18 F and 68 Ga [11,17,18,24,25]. Site-specific labeling of annexin V can help improve its sensitivity for detecting cell death in vivo. To take advantage of the higher resolution and more accurate quantification of PET, labeling annexin V with short half-life positron-emitters such as 18 F is of particular interest. In this study site-specific labeling of Cys-Annexin V with 18 F-FBEM as prosthetic group is presented.
As see in Figure 1, Cys-Annexin V and 18 F-FBEM-Cys-Annexin V were eluted at retention times of 8.6 min and 9.1 min, respectively, whereas 18 F-FBEM eluted at a retention time of 15.9 min. According to HPLC analysis, the radiochemical purity of 18 F-FBEM-Cys-Annexin V was above 95%. The total synthesis time for 18

In Vitro Stability of 18 F-FBEM-Cys-Annexin V
To determine the radioactive decomposed side products which may accumulate in non-target organs, the stability of 18 F-FBEM-Cys-Annexin V was studied. The results of the stability of 18 F-FBEM-Cys-Annexin V in (A) phosphate buffered saline (PBS, 0.1 mol/L, pH 7.2) and (B) human serum, respectively, are presented in Figure 2. The results show that the 18 F-FBEM-Cys-Annexin V is more stable during biodistribution and PET imaging studies.

Blood Kinetics Studies
Pharmacokinetic parameters, obtained using the DAS 2.1.1 pharmacokinetic calculation program, are listed in Table 1. Figure 3 shows the blood clearance of 18  phase (t1/2α) and half-life of elimination phase (t1/2β) were 11.261 and 134.62 min, respectively, which showed that 18 F-FBEM-Cys-Annexin V can be absorbed quickly and eliminated slowly. Biological availability was represented by area under concentration-time curve (AUC) and the values of clearance (CL) and AUC were 0.031 and 643, respectively.  In the early phase, the blood clearance of 18 F-FEBM-Cys-Annexin V was slow. After 2 h, the radioactivity concentration of the tracer agent in blood reaches an equilibrium which coincides with the pharmacokinetic parameters CL, AUC and the pharmacokinetic curves.

Dynamic MicroPET Images of Normal ICR Mice
Representative time-activity curves of the major organs (kidneys, liver and heart) were derived from 60-min dynamic microPET scans after intravenous administration of 18 F-FBEM-Cys-Annexin V tracers ( Figure 4). The radioactivity kinetics were calculated from a region-of interest analysis of the dynamic small animal PET scans over the heart (squares; mainly representing the cardiac blood pool), kidney (triangles) and liver (diamonds). 18 F-FBEM-Cys-Annexin V was excreted mainly through the kidneys, as evidenced by the higher renal uptake at early time points and excretion via the bladder. The kidney uptake reached a peak (11%ID/g) at 13 min after injection and then decreased to 1.43%ID/g at 60 min p.i.

Imaging of Rat Model of Apoptosis
Four rats were treated with cycloheximide to induce liver apoptosis and two rats were used as the control group. Figure 5 shows the representative coronal microPET images of cycloheximide (CHX)-treated and normal rats at different times after intravenous injection of 8.2 MBq 18 F-FBEM-Cys-Annexin V. 18 F-FBEM-Cys-Annexin V tracer uptake in the liver (arrow) was increased with CHX treatment. The uptake ratios (treated/control) of liver were 3.88 ± 0.05 (n = 4) and 10.35 ± 0.08 (n = 4), respectively, at 1 h and 2 h p.i. There were no differences in the blood pool activity between treated and control rats. TUNEL-staining images of liver sections were shown in Figure 6. These images show that the number of apoptotic nuclei in CHX-treated liver was more compared to that in non-treatment control rats. The uptake ratio (treated/control) of liver at 2 h p.i. as measured via microPET correlated with the ratio (treated/control) of apoptotic nuclei in liver observed using TUNEL histochemistry.

General Information
Unless otherwise specified, all reagents were of analytical grade and were obtained from commercial sources. Cys-Annexin V was supplied by Jiangsu Target Pharma Laboratories Inc. (Changzhou, China). 18 F fluoride was obtained from a cyclotron (HM67, Sumitomo Heavy Industries, Ltd, Tokyo, Japan) located at the Jiangsu Institute of Nuclear Medicine by proton irradiation of 18 O-enriched water. A Waters high-performance liquid chromatography (HPLC) system (Waters, Milford, MA, USA) equipped with a Waters 2998 photodiode array detector (PDA) and a semi-preparative C18 HPLC column (250 × 10 mm, 5 μm, CHROM-MATRIX BIO-TECH) was used for 18 F-FBEM purification. The flow rate was 3 mL/min, and the mobile phase changed from 95% solvent A (0.1% trifluoroacetic acid in water) and 5% solvent B (0.1% trifluoroacetic acid in acetonitrile) (0-2 min). The mobile phase was gradually changed to 35% solvent A and 65% solvent B at 32 min. The UV absorbance was monitored at 218 nm, and the UV spectrum was checked with the PDA detector.
Analytical HPLC was performed on Waters Breeze system with a TSK-GEL column (swG2000SWXL, 300 × 7.8 mm 5 µm, Tosoh Bioscience Co., Ltd, Shanghai, China). The absorbance was measured on the UV detector at 278 nm. Radioanalysis of the labeled compound was conducted using a Cd (Te) detector. The flow rate was adjusted to 0.8 mL/min and the isocratic mobile phase was 0.05 mol/L phosphate buffer (pH = 7.0).
A microPET system (Inveon, Siemens Co. Knoxville, TN, USA) and a fluorescence microscope (X51, Olympus, Tokyo, Japan) were used. The animal experiments in this study were approved by the Animal Care and Ethnics Committee of Jiangsu Institute of Nuclear Medicine.

Preparation of 18 F-N-[2-(4-Fluorobenzamido)ethyl]maleimide
18 F-N-[2-(4-Fluorobenzamido)ethyl]maleimide ( 18 F-FBEM) was prepared as previously described using a semi-automatic method with some modifications [22,26]. Briefly, the precursor, ethyl 4-(trimethylammonium) benzoate trifluoromethanesulfonic acid salt (10 mg, 12 µmol) in anhydrous acetonitrile (1.0 mL) was heated at 100 °C for 10 min in a sealed vial with 18.5 GBq [ 18 F]fluoride in the presence of dried Kryptofix2.2.2 (15 mg 6 µmol) and K2CO3 (5 mg, 3.6 mmol). The intermediate was hydrolyzed with NaOH (0.5 M, 0.5 mL) at 90 °C for 5 min. After acidification with 7.5 mL 0.1 M HCl, the solution was loaded onto an activated C18 Sep-Pak column (Waters). The cartridge was then eluted with 3 mL acetonitrile and the eluate was subsequently evaporated at room temperature with a stream of nitrogen to obtain 18 F-fluorobenzoic acid ( 18 F-FBA). 18 F-FBA was treated with N-(2-aminoethyl)maleimide (MAL, 15 mg, 59 µmol), diethyl cyanophosphonate (20 µL, 99 µmol), and N,N-diisopropylethylamine (40 µL, 240 µmol) in anhydrous acetonitrile (0.5 mL). The resulting solution was heated at 75 °C for 7 min. The reaction was quenched by adding water (8.5 mL) and loaded onto an activated C18 Sep-Pak column. The cartridge was eluted with 1 mL ethanol which was then loaded on to the semi-preparative HPLC. The radioactive peak eluting at ~18 min was collected and passed through a C18 Sep-Pak column which was activated by EtOH/water. The cartridge was washed with 20 mL water and then eluted with 1 mL CH2Cl2. The organic layer was evaporated to dryness at room temperature under a stream of nitrogen and utilized for further Cys-Annexin V labeling. The total synthesis time for 18 F-FBEM was about 100 min and 428 ± 65 MBq (n = 4) radiochemically pure 18 F-FBEM was obtained from 18.5 GBq 18 F-fluoride.

In Vitro Stability of 18 F-FBEM-Cys-Annexin V
The in vitro stabilities of freshly prepared 18 F-FBEM-Cys-Annexin V were performed in PBS (0.1 mol/L, pH 7.2) and human serum, respectively, for different time intervals (0-6 h) at 37 °C in a water bath.)

Blood Kinetics Studies of 18 F-FBEM-Cys-Annexin V in Normal Mice
Five ICR mice were injected via the tail vein with 18 F-FBEM-Cys-Annexin V (0.2 mL) and activity of approximately 3.7 MBq. Ten μL of blood were taken from tails at 5, 15, 30, 45, 60, 90 and 120 min after injection. The activity for each sample was determined by a γ-counter and expressed as percentage of injection dose per gram (%ID/g).

Dynamical MicroPET Images of Normal Mice
Four ICR mice were anesthetized with 1%-2% isoflurane, positioned prone, immobilized, and injected via the tail vein with 3.7 MBq 18 F-FBEM-Cys-Annexin V (0.2 mL) and imaged dynamically for 1 h. The images were reconstructed using a two dimensional ordered-subset expectation maximization (2D OSEM) algorithm without correction for attenuation or scattering. For each scan, regions of interest (ROIs) were drawn over the liver and major organs using the vendor-supplied software (ASI Pro 5.2.4.0) on decay-corrected whole-body coronal images. The radioactivity concentrations (accumulation) within the liver, heart and kidneys were obtained from mean pixel values within the multiple ROI volume and then converted to megabecquerel per milliliter per minute using the calibration factor determined for the Inveon PET system. These values were then divided by the administered activity to obtain (assuming a tissue density of 1 g/mL) an image-ROI-derived percent injected dose per gram (%ID/g).

MicroPET Images of Rat Model of Apoptosis
Four male SD rats (258 ± 2 g) were treated IV with 10 mg/kg cycloheximide to induce liver apoptosis. Two male SD rats (262 g and 256 g) were treated IV with saline as the control group. 3 h after treatment, the rats were anesthetized with 1%-2% isoflurane and were injected via the tail vein with 8.2 MBq 18 F-FBEM-Cys-Annexin V (0.2 mL). Ten-minute static scans were acquired at 1 and 2 h after injection with a MicroPET (Inveon, Siemens), respectively, which was from 1 h to 1 h and 10 min or from 2 h to 2 h and 10 min. Immediately after MicroPET imaging, the livers were dissected. Then, using the livers, formalin-fixed paraffin-embedded specimens were prepared for Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining.

TUNEL Staining
Because our imaging studies were designed to determine the uptake and biodistribution of 18 F-FBEM-Cys-AnnexinV after chemically induced apoptosis, it was important to confirm apoptosis in the livers of treated rats by independent methods that provide quantitative results. A marker of apoptosis was scored by performing a TUNEL assay that measures DNA fragmentation, a characteristic feature of apoptosis. Terminal deoxynucleotide transferase adds labeled nucleotides to the 3' termini at double-stranded breaks in the fragmented DNA. TUNEL assays were performed according to the manufacturer's instructions, using the fluorescein-conjugated Colorimetric TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, slices were freed of paraffin through xylene and graded EtOH washes and then incubated with proteinase K (Beyotime Institute of Biotechnology, 2 mg/mL in 10 mmol/L Tris, pH 8.0). After proteinase digestion, the slides were equilibrated in pH 7.4 buffer, the terminal deoxynucleotidetransferase enzyme and Biotin-dUTP labeling mix (Beyotime Institute of Biotechnology) were added, and the slides were incubated at 37 °C for 1 h in a humid chamber. The number of TUNEL-positive cells was counted on 10 randomly selected ×100 fields for each section by use of an Olympus fluorescence microscope.

Conclusions
Cys-annexin V, a novel annexinVderivative with a single cysteine residue at the C-terminal, could be site-specifically labeled with 18 F-FBEM in high yields and high radiochemical purity. In normal mice, 18 F-FBEM-Cys-Annexin V was excreted mainly through the renal pathway. Hepatic uptake of 18 F-FBEM-Cys-Annexin V was significantly increased in the rats treated with CHX compared to controls, which correlated well with the increase in cell death observed using TUNEL histochemistry. These results indicate that the novel 18 F-FBEM-Cys-Annexin V is a potential apoptosis imaging agent and further study is needed.