Preliminary Biological Evaluation of Novel 99mTc-Cys-Annexin A5 as a Apoptosis Imaging Agent

A novel annexin A5 derivative (cys-annexin A5) with a single cysteine residue at its C-terminal has been developed and successfully labeled in high labeling yield with 99mTc by a ligand exchange reaction. Like the 1st generation 99mTc-HYNIC-annexin A5, the novel 99mTc-cys-annexin A5 derivative shows in normal mice mainly renal and, to a lesser extent, hepatobiliary excretion. In rat models of hepatic apoptosis there was 283% increase in hepatic uptake of 99mTc-cys-annexin A5 as compared to normal mice. The results indicate that the novel 99mTc-cys-annexin A5 is a potential apoptosis imaging agent.


Introduction
Apoptosis or programmed cell death (PCD) plays an important role not only in physiology but also in pathology [1,2]. Therefore, the detection and quantification of apoptosis in vivo are of significant clinical value for diagnosis and assessment of therapeutic efficacy.

OPEN ACCESS
One of the hallmarks of cells going into apoptosis is the externalization of the phospholipid phosphatidylserine (PS) at the cell membrane [3,4]. Annexin A5, 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 a derivative of annexin A5 a suitable candidate for imaging of apoptosis.
Recent studies reveal that after structural modification in the recombinant expression annexin A5 can be directly marked with 99m Tc, such as 99m Tc(CO)3-HIS-cys-AnxV [21], 99m Tc-annexin-V-117 [22] and 99m Tc-His10-annexin V [23]. New annexin A5 molecules labeled by site-specific methods will greatly improve sensitivity for detecting cell death in vivo [24]. Recently, Professor Hua Zichun and his colleagues have developed a novel annexin A5 derivative (cys-annexin A5) with a single cysteine residue at C-terminal [25]. Their findings show that the the detection signal of fluorescein isothiocyanate (FITC)-cys-annexin A5 is greater than that of FITC-wild-type annexin A5.
We herein report the labeling and preliminary in vivo evaluation of the novel site-specific 99m Tc-cys-annexin A5 in normal mice and in rat models of apoptosis induced by cycloheximide. In mice tracer uptake was studied by ex vivo biodistribution experiments and the results were compared to those of the 1st-generation 99m Tc-HYNIC-annexin A5. In a rat model of hepatic apoptosis, tracer uptake was studied by SPECT. Apoptosis was confirmed in situ on liver slices using the terminal deoxynucleotidyl transferase (TdT) dUTP nick endlabeling (TUNEL) assay.

Radiolabeling
Annexin A5 has been labelled with a number of isotopes including 99m Tc, 124 I, 18 F and 68 Ga [18,[26][27][28][29]. Positron emission tomography (PET) with its higher sensitivity, better spatial resolution and the ability to better quantify the radiopharmaceutical uptake is superior to SPECT imaging, and the low uptake of 18 F-annexin A5 in the liver, spleen and kidney might represent an advantage over 99m Tc-annexin A5 [30], however production of 18 F-annexin A5 depends on the availability of an expensive on-site cyclotron for the production of fluorine-18 (t1/2 = 109.8 min). 68 Ga has suitable physical properties for PET imaging. Recently, it was reported that 68 Ga-annexin A5 has a similar biodistribution in mice compared with other 99m Tc-annexin A5 [29]. In the present study, site-specific labeling of cys-annexin A5 with 99m Tc by a ligand exchange reaction. In the presence of disodium edetate, 99m Tc-cys-annexin A5 was labeled with Na 99m TcO 4 by reduction with stannous chloride.
HPLC analysis revealed cys-annexin A5 and 99m Tc-cys-annexin A5 that were eluted at a same retention time of 9.8 m, whereas 99m Tc-colloidal and the formation of free technetium (Na 99m TcO 4 ) eluted at a retention times of 12.5 m and 15.9 m, respectively ( Figure 1). For each radiolabeled complex, the single peak in the HPLC-chromatogram clearly shows the formation of only one complex and excludes the possibility of residual Na 99m TcO 4 or other components. According the HPLC analysis, the radiochemical purity of 99m Tc-cys-annexin A5 was greater than 95%. The radiolabeled compounds were used immediately after the formulation for both in vitro and in vivo studies.

Blood Kinetics Studies
Pharmacokinetic parameters were listed in Table 1. Figure 2 shows the blood clearance of 99m Tc-cys-annexin A5 in the mice 4 h post injection. Pharmacokinetics of 99m Tc-cys-annexin A5 comply with the two-compartment model with the pharmacokinetic equation of C = 5.972e −0.123t + 0.877e −0.005t . The values of CL and AUC were 0.084 and 238, respectively. In the early phase, the blood clearance of 99m Tc-cys-annexin A5 was fast. After 1 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.

Imaging of Rat Model of Apoptosis
There were three rats treatment with cycloheximide to induce liver apoptosis and two rats as the control group. Figure 4 shows the planar images of normal and cycloheximide (CHX)-treated rats. 99m Tc-cys-annexin A5 tracer uptake in the liver was increased with CHX treatment (indicated by arrows). Biodistribution of 99m Tc-cys-annexin A5 in treated rats indicated that liver, spleen and kidney uptakes were 0.28 ± 0.01%ID/g,1.22 ± 0.03%ID/g and 5.21 ± 0.02%ID/g, respectively, at 190 m p.i. The uptakes in liver, spleen and kidney of control rats were 0.10 ± 0.005%ID/g, 0.25 ± 0.01%ID/g and 5.50 ± 0.03%ID/g, respectively, at 190 m p.i. The uptake ratios (treated/control) of liver, spleen and kidney were 2.83, 4.94 and 0.95, respectively, at 190 m p.i. There were no differences in the blood pool activity between treated and control rats. The changes observed in 99m Tc-cys-annexin A5 biodistribution as measured via SPECT correlated with the increase in cell death observed using TUNEL histochemistry. Photomicrographs of the corresponding 5 µm liver section after TUNEL staining of CHX-treated rat (1st column) and control (2nd column) 6 h after CHX treatment. TUNEL-positive nuclei (green nuclei, indicated by arrows) are diffused in treated, but little in control mice and indicate apoptosis. Rat treated with CHX Untreated rat liver

Toxicity Test
The toxicity test was evaluated by the death and 48 h survival of the mice, which were injected with 0.5 mL 99m Tc-cys-annexin A5 (3.7MBq), respectively. Saline-injected (of the same volume) mouse group was used as the control group. As expected, the mice showed no signs of toxicity through the overall study period.
In summary, 99m Tc-cys-annexin A5 is a promising imaging agent in the detection of apoptosis and early assessment of tumor therapeutic efficacy. We plan to develop a lyophilized kit of cys-annexin A5 for 99m Tc-labeling for clinical use.

General
All analytical chemical reagents employed were purchased from commercial sources and used without further purification. cys-Annexin A5 was supplied by Jiangsu Target Pharma Laboratories Inc. (Changzhou, China). Na 99m TcO 4 was supplied by Jiangsu Institute of Nuclear Medicine (Wuxi, China) HPLC was performed on a Waters 600-type high-performance liquid chromatography (Milford, MA, USA) equipped with a dural λ absorbance detector (Waters 2487), binary HPLC pump (Waters 1525) and Cd(Te) detector equipped with a scintillation analyzer (Perkin Elmer, Waltham, MA, USA). A Packard-multi-prias γ counter (made in USA), a fluorescence microscope (Olympas X51, Tokyo, Japan) and a Philips SKY Light single photon emission computed tomography instrument (SPECT) (San Francisco, CA, USA) were used. The animal experiments in this study were approved by the Animal Care and Ethnics Committee of Jiangsu Institute of Nuclear Medicine.

Quality Control of 99m Tc-cys-Annexin A5
The radiochemical purity (RCP) and radiolabeling yield (RLY) of 999m Tc-cys-annexin v was determined by HPLC. The RCP of 99m Tc-cys-annexin A5 was determined a Waters 600-type high-performance liquid chromatography. The sample was passed through a millipore filter carefully and injected into the HPLC column (TSK-GEL swG2000SWXL, 300 × 7.8 mm 5 µm, Tosoh Bioscience Co., Ltd, Shanghai, China). The absorbance was measured on the UV detector at 278nm. 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).

Biodistribution in Normal Mice of 99m Tc-cys-Annexin A5
Thirty-five ICR mice were randomly divided into seven groups and injected via the tail vein with 99m Tc-cys-annexin A5 in the volume of 0.2 mL and activity of approximately 3.7 MBq. Groups of mice were sacrificed at 5, 15, 30, 60, 120, 180 and 240 min after injection. The organs of interest (brain, heart, liver, lung, kidney, spleen and muscle etc.) were dissected and weighed, as well as 100 μL blood were taken from carotid artery. The activity for each sample was determined by a γ counter. Distribution of the radioactivity in different tissues and organs was calculated and expressed as percentage of injection dose per gram (%ID/g).

Animal Model of Apoptosis
Three male SD rats (288 ± 1g) were treated IV with 5 mg/kg cycloheximide to induce liver apoptosis. Two male SD rats (289 g and 291 g) were treated IV with saline as the control group. 3 h after the treatment, rats were injected via the tail vein with 0.2 mL (18.5 MBq) 99m Tc-cys-annexin A5 and 3 h later imaged with SPECT for 10 min. The organs of interest (liver, spleen and kidney) were dissected and weighed at the end of the scan. Liver samples were divided into 2 parts. Then, using aliquots of the liver, formalin-fixed paraffin-embedded specimens were prepared for Terminal deoxynucleotidyl Transferase dUTP nick end labeling (TUNEL) staining.

TUNEL Staining
Because our imaging studies were designed to determine the uptake and biodistribution of 99m Tc-cys-annexin A5 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 deoxynucleotide transferase 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 a Olympus fluorescence microscope.

Conclusions
Cys-annexin A5, a novel annexin A5 derivative with a single cysteine residue at the C-terminal, could be labeled with 99m Tc in high yields and high radiochemical purity. In normal mice, 99m Tc-cys-annexin A5 was rapidly cleared from the blood and excreted mainly through the renal pathway. Hepatic uptake of 99m Tc-cys-annexin A5 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 99m Tc-cys-annexin A5 is a potential apoptosis imaging agent and further optimization of the cys-annexin A5 kit is nevertheless desirable in order to improve the potential clinical usefulness of this new technique.