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99mTc-labeled annexin V fragments: a potential SPECT radiopharmaceutical for imaging cell death
Nuclear Medicine and Biology 33 (2006) 635 – 643 www.elsevier.com/locate/nucmedbio
99m
Tc-labeled annexin V fragments: a potential SPECT radiopharmaceutical for imaging cell death
Archana Mukherjeea, Kanchan Kotharia,4, Ge´za To´thb, Erzse´bet Szemenyeib, Hal Dhar Sarmac, Jo´zsef Kfrnyeid, Meera Venkatesha a
Radiopharmaceuticals Division, Radiochemistry and Isotope Group, Bhabha Atomic Research Centre, Mumbai-400085, India b Biological Research Centre of the Hungarian Academy of Sciences, H-6701 Szeged, Hungary c Radiation Biology and Health Science Division, Bhabha Atomic Research Centre, Mumbai-400085, India d Institute of Isotopes Co. Ltd., H1121, Budapest, Hungary Received 10 November 2005; received in revised form 27 February 2006; accepted 16 May 2006
Abstract Introduction: Annexin V is a protein that binds to phosphatidylserine exposed on dying cells. The phosphatidylserine-specific sequence is attributed to a chain on the N-terminal of annexin consisting of 13 amino acid sequence. Radiolabeled annexin V is used for imaging apoptosis. Methods: With an aim to synthesize a probe that can detect cell death akin to annexin V but smaller in size, annexin-13 fragments were derivatized to contain cysteine, cysteine–cysteine and histidine in their sequence at N terminal and were labeled with 99mTc via nitrido and carbonyl precursors. The 99mTc-labeled annexin-13 derivatives were characterized by HPLC and studied for their stability. In vitro and in vivo studies were carried out in apoptotic HL-60 cells and fibrosarcoma tumor-bearing Swiss mice, respectively. Results: The 99mTc complexes were formed in high yields and were found to be stable. HPLC pattern of 99mTc nitrido complex of cysteine– cysteine–annexine 13 (CC-Anx13) and 99mTc carbonyl complex of histdine–annexin 13 (H-Anx13) revealed the formation of single species. In vitro cell uptake studies with 99mTc nitrido complex of cysteine–cysteine–annexin 13 fragment showed 6.5% uptake in apoptotic HL-60 cells. The uptake was found to be specific on testing with apoptotic HL-60 cells. Biodistribution studies of 99mTc nitrido complex with CC-Anx13 in fibrosarcoma tumor-bearing Swiss mice revealed optimum tumor uptake of 0.52 (0.17) %ID/g at 1 h pi. Conclusion: 99mTc(N)-CC-anx13 showed specific uptake in apoptotic tumor cells and warrants further evaluation. D 2006 Elsevier Inc. All rights reserved. Keywords: Annexin V; Apoptosis;
99m
Tc nitrido; Carbonyl precursors
1. Introduction Apoptosis or programmed cell death [1] is an essential life process for a cellular organism. It is an active process of cellular self-destruction which, on one hand, benefits in situations such as malignancies and their treatment while, on the other, leads to a large number of disorders such as organ rejection after transplantation [2], myocardial ischemia or infarct [3] and neurodegenerative diseases [4]. Apoptotic cell death occurs after treatment of cancer by radiation, chemotherapy and photodynamic therapy. The extent and time at which cell death occurs may be important in
4 Corresponding author. Tel.: +91 22 2559 5371; fax: +91 22 2550 5345, +91 22 2551 9613. E-mail address: [email protected] (K. Kothari). 0969-8051/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2006.05.002
elucidating potential clinical information in the management of cancer treatment protocols. The extent of damage after myocardial infarction could also be gauged by estimating the extent of apoptosis. For this reason, considerable efforts have been made in developing and validating methods to image apoptosis in vivo [5,6]. A key feature of programmed cell death is that phophatidylserine is flipped from the internal to the external leaflet of the cell membrane [7]. This can be detected by using annexin V, a protein that binds to phosphatidylserine in the presence of calcium [8–10]. Radionuclide imaging with radiolabeled annexin V is a highly specific technique that enables delineation of apoptotic areas with good resolution [11]. Annexin V has been tagged with several radionuclides such as 99mTc [12–14], 18F [15,16], 64Cu [17] and 123/124I [18–21] to detect cell death in vivo by SPECT or PET imaging.
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Positron emission tomography agents and 123/124I-labeled agents yield superior images with far greater sensitivity than SPECT agents, but have limitations such as high cost and poor logistics for distribution. Hence, there have been demands for design and development of suitable 99mTclabeled agents that can be used more conveniently, economically and universally. 99mTc-HYNIC-annexin V has been used to detect apoptosis in vivo with gamma camera imaging and SPECT [22,23]. Studies with 99mTcHYNIC-annexin V for apoptosis detection have shown a high degree of retention in normal tissues, particularly in the abdomen, that may interfere with the imaging of apoptotic uptake [24 –26]. Most annexin molecules consist of a unique N-terminal region followed by repeated sequences of four units of 60 – 70 amino acids each [27]. It has been inferred that since the annexin proteins generally possess their biospecific sequences on the N-terminal, in the case of annexin V too the phosphatidylserine-specific sequence might be attributed to a chain consisting of 13 amino acids at the N terminal [28–30]. Since small peptides can be synthesized easily and modified suitably to enable radiolabeling to obtain stable products with pharmacokinetics of rapid uptake and quick renal clearance [31], 99mTc-labeled annexin 13 fragments may prove to be a better tool for imaging apoptosis than the whole annexin V [32]. Here, we describe the work carried out towards radiolabeling of annexin 13 fragment with 99mTc through nitrido and tricarbonyl cores, their in vitro evaluation in apoptosis model and in vivo evaluation in animal tumor model of fibrosarcoma. 2. Material and methods Nitrido kit and the coligand (PNP5) were supplied by the IAEA as part of a coordinate research project. Amino acid derivatives and the resin were obtained from SigmaAldrich (St. Louis, MO, USA) or Bachem (Bubendorf, Switzerland). Coupling reagents were acquired from Calbiochem-Novabiochem (L7ufelfingen, Switzerland). All the chemicals, solvents and reagents used were of analytical quality and used without further purification. Sodium borohydride and Na/K tartarate were obtained from Aldrich Chemicals (USA). All chemicals and solvents were of reagent grade and used without further purification. Carbon monoxide in 0.5-L refillable canisters was obtained from M/s Alchemie Gases and Chemicals (Mumbai, India). 99m TcO4 was eluted from a 99Mo/99mTc column generator using normal saline [33]. HPLC analysis was performed using C-18 reversed-phase HiQ Sil (5 Am, 4250 mm), on a Jasco PU 1580 system with a Jasco 1575 tunable UV–visible absorption detector as well as a radiometric detector system. The purity of the peptides was determined by RP-HPLC on a Merck-Hitachi liquid chromatographic system (Merck, Darmstadt, Germany, and Hitachi, Tokyo, Japan) equipped with a Vydac 218TP54 reversed-phase analytical column (2504.6 mm, 5 Am, Separations Group,
Hesperia, CA, USA) and LichroChart 250-4, Lichrospher100 reversed-phase analytical column (Merck). Silica gel 60 F254 precoated aluminum sheets from Merck were used for TLC. Vydac 218TP1010 reversed-phase semipreparative column (25010 mm, 12 Am, Separations Group) was used for semipreparative separations of the peptides. Mass spectra of the peptides were recorded on a Bruker Reflex III MALDI-TOF mass spectrometer. 2.1. Peptide synthesis Peptides CC-Anx13, C-Anx13 and H-Anx13 were synthesized by manual solid-phase techniques [34,35]. The peptide acids were prepared on Merrifield resin (0.9– 1.5 mmol/g substitution). The tert-butyloxycarbonyl-amino acid (Boc-amino acid) resin esters were prepared by the method of Gisin [36]. N a-t-Boc chemistry with N-hydroxybenztriazole and N,NV-dicyclohexylcarbodiimide as coupling reagents was employed for peptide elongation. Stepwise removal of N a-t-Boc protecting group from growing chain of peptides was achieved by 50% trifluoroactic acid (TFA) and 2% anisole in dichloromethane. Peptides were cleaved from the resin with anhydrous HF (10 ml/g resin) in the presence of anisole (1 ml/g resin) for 60 min at 08C. After removal of the scavenger, the peptides were extracted with 30% acetic acid and the filtrate was lyophilized. The crude peptides were purified by RP-HPLC on Vydac 218TP1010 column by gradient elution with water [0.1% (v/v) TFA] (A)/acetonitrile [0.08% (v/v) TFA] (B) solvent system at a flow rate of 4 ml/min. The gradient started from 85% A/15% B and changed to 60% A/40% B in 25 min. The purities of the peptides were assessed by analytical RP-HPLC on Vydac 218TP54 column and by TLC on silica gel 60 F254 precoated aluminum sheets. Water [0.1% (v/v) TFA] (A)/acetonitrile [0.08% (v/v) TFA] (B) gradient solvent system was used for the RP-HPLC analysis. The gradient started from 90% A/10% B and changed to 70% A/30% B in 40 min at a flow rate of 1 ml/min. The TLC solvent systems were as follows: (I) pyridine/isoamyl alcohol/water (1:1:2 v/v) and (II) n-butanol/pyridine/acetic acid/water (15:3:8:10 v/v). Ultraviolet light at k = 254, I2 vapor and ninhydrine were applied to visualize the TLC spots. The mass analysis of the peptide was carried out using MALDI-TOF in the positive ion mode using 2,5-dihydroxy benzoic acid as the matrix. Structure of derivatized annexin V fragments used for radiolabeling studies is given in Fig. 1. 2.2. Radiolabeling studies Labeling of CC-Anx13 and C-Anx13 with 99mTc was carried out via 99mTc-nitrido intermediate, while that of H-Anx13 was carried out via 99mTc-carbonyl core. 2.2.1. Labeling annexin fragments via nitrido intermediate 2.2.1.1. Synthesis of [99mTcN]2+ intermediate. The kit vial, containing succinic dihydrazide (5.0 mg), stannous chloride
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B which gradually changed to 10% A/90% B in 28 min. The gradient remained at 10% A/90% B during 28–30 min. 2.2.2. Labeling of H- anx13 via carbonyl precursor 2.2.2.1. [99mTc-(OH2 )3(CO)3]+ precursor. The precursor was prepared by a modified procedure reported by Alberto et al. [37]. Briefly, NaBH4 (5.5 mg), Na2CO3 (4 mg) and Na/K tartrate (15 mg) were dissolved in 0.5 ml double distilled water in a glass serum vial. The vial was sealed and a needle was introduced through the rubber stopper for facilitating equilibration to atmospheric pressure. Carbon monoxide was purged through the solution for 5 min. After the addition of 1 ml of the generator eluate containing 37–740 MBq (1–20 mCi) of 99mTcO4 , the needle was removed and the vial was heated at 808C for 15 min. After cooling the vial for 10 min and re-equilibration to atmospheric pressure, the pH of the reaction mixture was adjusted to 6 with 300 Al of 1:3 mixture of 0.5 M phosphate buffer (pH 7.5)/1 M HCl.
Fig. 1. Structure of Anx13 derivatives used for labeling with 99mTc: (1) CCAnx13, (2) C-Anx13, (3) H-Anx13.
dihydrate (100 Ag), 1,2-diaminopropane-N,N,NV,NV-tetraacetic acid (5 mg), sodium dihydrogen phosphate (0.5 mg) and disodium hydrogen phosphate (5.8 mg) in freeze dried form, was used for preparing the [99mTcN]+2 intermediate. The kit vial stored at 48C was allowed to attain ambient temperature. One milliliter of freshly eluted 99mTcO4 (37 MBq) was then added to the kit vial, vortexed for 1 min and allowed to stand at room temperature for 20 min.
2.2.2.2. Preparation of 99mTc-(CO)3-H-Anx13. Twenty micrograms (10 Al) of H-Anx13 was added to 0.5 ml of carbonyl precursor at pH 6. The reaction was carried out at 808C for 30 min.
2.2.1.2. Labeling of CC-Anx13 and C-Anx13 via nitrido intermediate. One hundred micrograms each of CC-Anx13 and C-Anx13 in 0.1 ml of saline was added to separate aliquots of 0.9 ml of 99mTc-nitrido intermediate. The reaction mixtures were heated in boiling water bath for 1 h.
2.2.2.3. Characterization of 99mTc-(CO)3-H-Anx13 by HPLC. The radiochemical purity of the precursor and the complex was checked by HPLC using C-18 reversed-phase column and water (A)/acetonitrile (B) with 0.1% TFA as solvent system in gradient mode at a flow rate of 1 ml/min. The gradient system for the analysis of the product started with a linear gradient of 100% A/0% B for 0 –1.5 min which slowly changed to 70% A/30% B in 18 min and further changed to 40% A/60% B from 18 to 21 min. The gradient remained at 40% A/60% B between 21 and 24 min and was then changed to 100% B in 5 min.
2.2.1.3. Labeling C-Anx13 via 99mTc-(N)-PNP intermediate. 2.2.1.3.1. Preparation of 99mTc-(N)-PNP intermediate. Ten microliters of PNP (~10 mg) was dissolved in 1 ml ethanol in nitrogen atmosphere. One hundred microliters of PNP was added to 1 ml of 99mTc-(N)-PNP intermediate along with 0.1 mg (50 Al) of C-Anx13, and the reaction mixture was heated at 1008C for 1 h. 2.2.1.4. Characterization of the nitrido complexes by HPLC technique. Characterization of the different nitrido complexes was carried out by HPLC using C-18 reversed-phase column using gradient elution with water (A)/acetonitrile (B) with 0.1% TFA as solvent system in gradient mode at a flow rate of 1 ml/min. The gradient system for the analysis of the product started with a linear gradient of 90% A/10%
2.3. Stability studies 2.3.1. Stability with time The stability of 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3H-Anx13 was studied at room temperature for 24 h. Radiochemical purity of the complex was checked by HPLC.
Table 1 Analytical data for annexin V fragments No.
Peptides
MALDI-TOF MS [M+H]+ Calculated
Found
1 2 3
CC-Anx13 C-Anx13 H-Anx13
1567 1464 1498
1566.73 1463.81 1497.85
RP-HPLC (kV) 6.44b 4.20c 3.66c
TLCa (R f) (I)
(II)
0.27 0.38 0.40
0.38 0.43 0.23
a Retention factors on silica gel 60 F254 precoated aluminum sheets. Solvent systems: (I) pyridine/isoamyl alcohol/water (1:1:2 v/v), (II) n-butanol/ pyridine/acetic acid/water (15:3:8:10 v/v). b Capacity factor: Vydac 218TP54 column (250.46 cm, d p = 5 Am); the gradient started from 90% A/10% B and changed to 70% A/30% B in 40 min. The flow rate was 1 ml/min; t 0 = 3.22 min; detection at k = 216 nm. c Capacity factors: LichroChart 250-4, Lichrospher100 column (250.46 cm, d p = 5 Am); the gradient started from 90% A/10% B and changed to 60% A/40% B in 30 min. The flow rate was 1 ml/min; t 0 = 2.9 min; detection at k = 216 nm.
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the binding of 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3-HAnx13 to apoptotic cells, the cells treated with camptothecin were harvested at 2z106 cells/tube, washed twice with PBS and resuspended in annexin V binding buffer (Pharmingen, 10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). 99m Tc-(N)-CC-Anx13 and 99mTc-(CO)3-H-Anx13 were added individually at a final concentration of 0.6 Ag/ml to the cell suspension and incubated at room temperature for 30 min. At the end of incubation, the cells were centrifuged at 3000 rpm for 5 min and washed with PBS. The radioactivity associated with the pellet was measured in a
Fig. 2. (A) Probable structure of 99mTc-nitrido complex with CC-Anx13 [99mTc-(N)-CC-Anx13]; (B) biphosphine ligand PNP5 used for the preparation of [99mTc-(N)-PNP]2+ intermediate.
2.3.2. Challenge with cysteine and histidine Stability of 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3-HAnx13 against trans-chelation was checked by challenge studies with cysteine and histidine. One hundred microliters of the complexes was incubated with 10 Al of cysteine (0.1 M) and histidine (0.1 M) in saline at 378C for 1 h. After incubation, the percentage dissociation of 99mTc was measured by HPLC analysis, following the solvent system used for characterization of the complexes. 2.3.3. Serum binding studies Approximately ~10 Ag of 99mTc-(N)-CC-Anx13 and 99m Tc-(CO)3-H-Anx13 was added individually to 0.5 ml of human serum and incubated at 378C for 30 min. The serum proteins were precipitated out by reacting with 0.5 ml of acetonitrile, and the activity bound to the serum protein was measured by counting the activity associated with the precipitate. The supernate was analyzed by HPLC to study serum stability. 2.4. Biological evaluation 2.4.1. Binding studies of 99mTc-(N)-CC-Anx13 with apoptotic cells The biological activity of 99mTc-(N)-CC-Anx13 was evaluated on exponentially growing HL-60 human leukemia cells. HL-60 cells were cultured in RPMI growth medium supplemented with 10% FBS. To induce apoptosis, cells in culture medium were treated with camptothecin dissolved in DMSO to a final concentration of 5 AM and incubated in 5% CO2 incubator at 378C for 6 h. Control cells were incubated with DMSO under similar conditions. To evaluate
Fig. 3. HPLC pattern of (A) 99mTc-nitrido intermediate and (B) asymmetric 99m Tc-nitrido complex with CC-Anx13 [99mTc-(N)-CC-Anx13].
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gamma counter. Control cells were also treated in a similar way. To investigate the specificity of binding, one set of apoptotic HL-60 cells was incubated with 100 of unlabelled annexin 13 fragment prior to the addition of 99m Tc-(N)-CC-Anx13 and 99mTc-(CO)3-H-Anx13 in order to saturate and block any specific binding [38]. All experiments were carried out in triplicate. Induction of apoptosis in HL 60 cells by camptothecin was confirmed by flow cytometric analysis. 2.4.2. Biodistribution studies 99m Tc-(N)-CC-Anx13 was diluted 1:10 in saline, and 0.1 ml (1 Ag) was injected via the tail vein of Swiss mice bearing fibrosarcoma tumor. The animals were sacrificed at 30 min, 1 h and 3 h pi. Organs were excised and counted in flat geometry NaI (Tl) scintillation detector. Percent activity retained in different organs was calculated. Three animals were used for each time point study. All the animal experiments were carried out in compliance with the relevant national laws relating to the conduct of animal experimentation.
639
13.0F0.2 and 14.3F0.2 min (Fig. 4A). When attempts were made to prepare asymmetric complex via 99mTc-(N)(PNP)2+ fragment using diphosphine ligand (Fig. 2B), the complex 99m Tc-(N)-PNP-C-Anx13 could be formed in N 95% yields. But in this case also, HPLC analysis of the complex revealed the formation of two species with retention times of 18.0F0.2 and 24F0.2 min (Fig. 4B). H-Anx13 could be labeled with 99mTc via carbonyl precursor at N 95% yields. The precursor as well as the complex was characterized by HPLC. But the HPLC system used for nitrido complexes was not able to clearly
3. Results 3.1. Peptide synthesis The peptides were synthesized by the manual solid-phase technique using Merrifield resin. The yields of the pure peptides were as follows: C-Anx13, 40%; H-anx13, 47%. The CC-Anx13 had to be purified twice in a row, leading to low yields of 5.9%. The structure of annexin derivatives is depicted in Fig. 1. The results obtained on the weights of the molecule ions by TOF/MS, capacity factors in HPLC and retention factors in TLC of the pure peptides are depicted in Table 1. CC-Anx13 when immersed in boiling water at 1008C for 60 min was found to be stable. This stability was important to carry out the labeling of CC-Anx13 via nitrido intermediate at 1008C [32]. 3.2. Radiolabeling studies On analysis by paper chromatography using ethanol/ chloroform/toluene/ammonium acetate (0.5 M) (5:3:3:0.5 v/v) as mobile phase and Whatman no. 3 paper as support system, the 99mTc-nitrido intermediate remained at the point of spotting (R f ~0) and free pertechnatate migrated with the solvent front (R f ~1). 99mTc-nitrido intermediate could be prepared with 93–95% yields. The ligand CC-Anx13 complexed with the 99mTc-nitrido intermediate in high yields ( N95%) to form a single species as revealed by HPLC analysis (retention time, 11.5 min). The probable structure of 99mTc-nitrido complex with CCAnx13 is given in Fig. 2A. HPLC patterns of 99mTc-nitrido intermediate and 99mTc-(N)-CC-Anx13 are depicted in Fig. 3A and B, respectively. C-Anx13 reacted with nitrido intermediate in N 95% yields. However, HPLC analysis of 99mTc-(N)-C-Anx13 revealed the formation of two species with retention times of
Fig. 4. HPLC pattern of nitrido complexes of 99mTc with C-Anx13. (A) Symmetric complex [99mTc-(N)-C-Anx13], (B) asymmetric complex [99mTc-(N)-PNP-C-Anx13].
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3.3. Stability studies Both 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3-H-Anx13, which were proven to be single species, were found to be stable at room temperature for 24 h. 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3-H-Anx13 were also stable and did not dissociate or transchelate when challenged with cysteine (0.1 M) and histidine (0.1 M) in saline, indicating their potential for further evaluation as apoptosis marker. 3.4. Biological evaluation 3.4.1. In vitro cell uptake studies Apoptosis induction by camptothecin in HL-60 cells was confirmed by flow cytometry. On incubation of camptothecin-treated HL-60 cells with 99mTc-(N)-CC-Anx13 and 99m Tc-(CO)3-H-Anx13 for 30 min, 6.5% and 13% binding were observed, respectively. In the case of 99mTc-(N)-CC-Anx13, similar experimentation in control (nonapoptotic) cells showed merely 1.3% binding, indicating the specific nature of binding to apoptotic cells. This was also confirmed by the decrease in binding to 1.8% when apoptotic cells were incubated with 100-fold of cold CC-anx13 along with 99mTc-(N)-CCAnx13. On the other hand, binding of 99mTc-(CO)3-HAnx13 was seen to be nonspecific wherein an uptake of ~13% was observed in controls (nonapoptotic) as well as apoptotic cells in the presence and absence of cold peptide (Fig. 6). 3.4.2. Biodistribution studies Results of biodistribution studies of 99mTc-(N)-CCAnx13 in tumor-bearing Swiss mice are depicted in Table 2. It is seen that the radioactivity rapidly cleared from blood. Uptake of 99mTc-(N)-CC-Anx13 in tumor was 1.08 (F0.19) %/g at 30 min pi, which decreased to 0.26
Fig. 5. HPLC pattern of (A) carbonyl precursor and (B) complex with H-Anx13 [99mTc-(CO)3-H-Anx13].
99m
Tc-carbonyl
differentiate carbonyl precursor from 99mTc-(CO)3-HAnx13 (retention times, 13.4 and 14 min, respectively). Hence, HPLC was carried out with a slower gradient system. The carbonyl precursor eluted out with a retention time of 8.3 min, while 99mTc-(CO)3-H-Anx13 was eluted out in HPLC with a retention time of 24 min as a single species as seen in Fig. 5A and B, respectively.
Fig. 6. Uptake of 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3-H-Anx13 in (a) untreated HL-60 cells, (b) camptothecin-treated HL-60 cells and (c) camptothecin-treated HL-60 cells in the presence of 100-fold cold CCAnx13.
A. Mukherjee et al. / Nuclear Medicine and Biology 33 (2006) 635 – 643 Table 2 Biodistribution studies of Organ
99m
Tc (N)-CC-Anx13 in tumor-bearing Swiss mice (n = 3) 30 min %/ID
Liver Intestine + Gall bladder Stomach Kidney Heart Lungs Spleen Tumor Muscles Blood Tibia
641
2.70 4.10 0.24 8.92 0.048 0.409 0.03 0.37 2.15 2.07 0.91
(0.66) (0.11) (0.12) (1.02) (0.01) (0.12) (0.01) (0.15) (1.03) (1.08) (0.5)
1h
3h
%ID/g
%/ID
2.81 1.89 0.70 32.4 0.40 2.1 0.21 1.08 0.23 1.37 0.40
1.38 3.32 0.31 6.23 0.05 0.42 0.08 0.24 2.34 0.07 0.015
(0.54) (0.09) (0.39) (5.16) (0.10) (0.6) (0.09) (0.19) (0.11) (0.681) (0.23)
(0.88) (0.83) (0.11) (0.75) (0.02) (0.29) (0.01 ) (0.03) (2.03) (0.06) (0.015)
%ID/g
%/ID
1.11 (0.65) 1.32 (0.315) 0.41 (0.22) 23.04 (2.30) 0.42 (0.04) 2.45 (1.70) 0.19 (0.01) 0.52 (0.17) 0.24 (0.21) 0.05 (0.05) 0.15 (0.1)
1.88 3.74 0.38 9.27 0.01 0.17 0.027 0.14 1.38 0.26 0.809
%ID/g (0.19) (0.35) (0.29) (1.50) (0.01) (0.04) (0.014) (0.02) (0.84) (0.18) (0.21)
1.49 1.54 1.53 35.2 0.3 1.44 0.25 0.26 0.15 0.15 0.32
(0.15) (0.23) (1.23) (6.42) (0.01) (0.27) (0.10) (0.09) (0.11) (0.11) (0.2)
Values in parentheses represent standard deviations.
(F0.09) %/g at 3 h pi. Optimum tumor uptake was 0.52 (F0.17) %/g at 1 h pi. 4. Discussion Annexin V has been labeled with 99mTc via the HYNIC conjugation method for clinical studies to date, and 99mTcHYNIC-annexin V has been the main 99mTc-annexin V radiopharmaceutical [26,38,39]. However, the product has shown high uptake in liver and kidney, and, hence, a need to develop 99mTc-labeled annexin V with better biodistribution profile has been felt [26,40]. The N terminal domain of annexin is considered as the regulatory region of the protein as it contains the major sites for phosphorylation, proteolysis or interactions with other proteins [28]. Annexin V has very high affinity for phospholipids (nanomolar range) and it is attributed to the N terminal domain constituting 13 amino acid sequence [28]. Being a peptide, the N terminal domain constituting 13 amino acid sequence can be easily synthesized and functionalized with a chelating group for labeling with 99mTc, rather than with whole annexin V molecule. Hence, 99mTc-labeled annexin 13 is attractive for exploration as a tool for apoptosis imaging. Hence, the N terminal domain constituting 13 amino acid residue of annexin V was functionalized with cysteine, dicysteine and histidine, and the functionalized annexin 13 fragments were labeled with 99mTc using [99mTc-(N)]2+ and [99mTc-(CO)3(H2O)3]+ precursor to investigate their potential for imaging apoptosis. CC-Anx13 was synthesized for the preparation of asymmetric nitrido complex in the absence of PNP ligand. C-Anx13 is expected to interact with 99mTc-nitrido intermediate to form a symmetric complex with two annexin 13 fragments. Introduction of two biologically active bidentate fragments in a symmetrical manner into a 99mTc-nitrido core is not preferable as this could lead to poor bioavailability. It would be preferable to design a complex with two different bidentate ligands to avoid the contemporary insertion of two biofragments on the same core. 99mTc-(N)-(PNP)-C-Anx13 was hence
prepared with the intention of making such an asymmetrical heterocomplex [41]. Of four different 99mTc-Anx13 complexes studied, although all were formed in high yields of N 95%, it was observed that 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3-HAnx13 were formed as single species, while 99mTc-(N)-CAnx13 and 99mTc-(N)-PNP-C-Anx13 were formed with more than one species. Since complexes with multiple species are generally not acceptable as radiopharmaceuticals, owing to the possibility of differences in their pharmacokinetics [42,43], these complexes were not evaluated for their biological behavior. Hence, in vitro studies were carried out only with 99mTc-(N)-CC-Anx13 and 99mTc(CO)3-H-Anx13 in apoptotic HL60 cells. Among these, 99m Tc-(N)-CC-Anx13 exhibited specific uptake in apoptotic HL60 cells, which could be inferred from poor uptake in untreated HL60 cells as well as in treated cells in the presence of excess cold peptide Anx13. In the case of 99m Tc-(CO)3-H-Anx13, the uptake was higher but not found to be specific. Earlier studies reported on 99mTc carbonyl complex of annexin V mutant have shown high retention of biological activity [44] However, our studies with 99mTc(CO)3-H-Anx13 in apoptotic HL60 cells did not substantiate this finding. One reason could be the loss of biological activity of H-Anx13 fragment after labeling with 99mTc via a carbonyl precursor. Being a peptide, H-Anx may have been more sensitive to loss of activity than high-molecularweight annexin V mutant protein. Imaging apoptosis has many applications with new ones emerging with time. The most widely used application is in cancer treatment [45] for assessing tumor response to novel therapies [46] as tumor often respond to radiation as well as to chemotherapy by direct induction of apoptosis. Again, in the evaluation of the extent of damage to the heart muscle in a patient with myocardial infarction, clinicians welcome the use of an agent to image apoptotic regions. Thus, it is expected that the utility of an apoptosis imaging radiopharmaceutical would be considered important and would grow. In this context, a small molecule that can be easily
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synthesized, labeled and quality controlled would be preferable to a large protein such as annexin V. Economic viability would also be favorable for smaller molecules, as annexin V is expensive. Hence, in our studies the Anx13 peptides were evaluated as possible molecules for imaging apoptosis. Among the four tested complexes,99mTc-(N)-CC-Anx13, which showed the best characteristics, was further evaluated in vivo in tumor-bearing Swiss mice to study its suitability to assess response to cancer therapy. 99mTc-(N)-CC-Anx13 has shown reasonably good tumor uptake. Apart from the above, large molecules do not clear from the body quickly, often accompanied by soft tissue uptake of the radiopharmaceuticals. In the case of 99mTc-HYNIC-annexin V too, earlier studies in normal Balb/c mice have shown high soft tissue retention (spleen, 17 %ID/g; liver, 15.2 %ID/g; stomach, 5.4 %ID/g; and kidney, 187 %ID/g) at 1 h pi [25]. 99m Tc-HYNIC-annexin V has also shown high retention of activity in the abdominal region in murine tumor model, making the product not suitable for evaluation of apoptosis in abdominal regions [11]. In our studies with 99mTc-(N)CC-Anx13 in tumor-bearing Swiss mice, the retention in soft tissues is relatively low in comparison to 99mTcHYNIC-annexin V [25]. Thus, 99mTc-(N)-CC-Anx13 could be a potential agent for apoptosis imaging. 5. Conclusion The N terminal chain of annexin V consisting of 13 amino acids, which is considered to bind to phosphatidylserine exposed on apoptotic cells, could be labeled with 99m Tc using novel chemical approaches in 99mTc radiopharmaceutical chemistry. This study is the first one to report on the use of 99mTc-labeled annexin 13 fragment for apoptosis detection. Among the four different complexes tested, 99m Tc-(N)-CC-Anx13 has shown specific uptake in apoptotic human leukemia HL-60 cells in the in vitro studies. Biodistribution studies of 99mTc-(N)-CC-Anx13 in tumorbearing Swiss mice also yielded encouraging results in tumor uptake and revealed superiority in soft tissue clearance in comparison with 99mTc-HYNIC annexin V. Being a small peptide that can be synthesized in large quantities, 99mTc-(N)-CC-Anx13 warrants further evaluation for imaging apoptosis. Acknowledgments This work was carried out as part of the IAEA CRP project on bDevelopment of 99mTc-based small biomolecules using novel 99mTc cores.Q The work on peptide synthesis was supported by a grant (RET08/2004-DNT) from the National Bureau of Research and Technology in Hungary. The authors are thankful to Dr. Duatti for the supply of the nitrido kit. The authors are also grateful to Dr. V. Venugopal (Director, Radiochemistry and Isotope Group,
Bhabha Atomic Research Centre) and Dr. M.R.A. Pillai (former head, Radiopharmaceuticals Division) for their encouragement and support.
References [1] Boris Z, Bertrand J, Orrenius S. Tumor radiosensitivity and apoptosis. Expt Cell Res 1999;248:10 – 7. [2] Kabelitz D. Apoptosis, graft rejection and transplantation tolerance. Transplantation 1998;65:869 – 75. [3] Mattson MP, Culmsee C, Yu ZF. Apoptosis and antiapoptotic mechanisms in stroke. Cell Tissue Res 2000;301:173 – 87. [4] Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995;267:1456 – 62. [5] Zucker RM, Hunter III ES, Rogers JM. Apoptosis and morphology in mouse embryos by confocal laser scanning microscopy. Methods 1999;18:473 – 80. [6] Hakumaki JM, Poptani H, Sandmair AM, Yia-Herttuala S, Kauppinen RA. 1H MRS detects polyunsaturated fatty acid accumulation during gene therapy of glioma: implications for the in vivo detection of apoptosis. Nat Med 1999;5:1323 – 7. [7] Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 1992;148:2207 – 16. [8] Funakoshi T, Heimark RL, Hendrickson LE, McMullen BA, Fujikawa K. Human placental anticoagulant protein: isolation and characterization. Biochemistry 1987;26:5572 – 8. [9] Reutelingsperger CPM, Hornstra G, Hemker HC. Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord. Eur J Biochem 1985;151:625 – 9. [10] Maurer-Fogy I, Reutelingsperger CPM, Pieters J, Bodo G, Stratowa C, Hauptmann R. Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+-dependent phospholipid-binding protein. Eur J Biochem 1988;174:585 – 92. [11] Subbarayan M, Hafeli UO, Feyes DK, Unnithan J, Emancipator SN, Mukhtar H. A simplified method for preparation of 99mTc-annexin V and its biologic evaluation for in vivo imaging of apoptosis after photodynamic therapy. J Nucl Med 2003;44:650 – 6. [12] Steinmetz ND, Green Am. Chemotherapy induced change in 99mTc HYNIC-annexin V uptake as an early predictor of response to platinum therapy in advanced non-small cell lung cancer. J Nucl Med 2004;45:36P. [13] Post AM, Katsikis PD, Tait JF, Geaghan SM, Strauss HW, Blankenberg FG. Imaging cell death with radiolabeled annexin V in an experimental model of rheumatoid arthritis. J Nucl Med 2002;43:1359 – 65. [14] Blankenberg FG, Naumovski L, Tait JF, Post AM, Strauss HW. Imaging cyclophosphamide-induced intramedullary apoptosis in rats using 99mTc radiolabeled annexin V. J Nucl Med 2001;42:309 – 16. [15] Zijlstra S, Gunawan J, Burchert W. Synthesis and evaluation of a 18F labeled recombinant annexin-V derivative for identification and quantification of apoptotic cells with PET. Appl Radiat Isot 2003;58:201 – 7. [16] Toretsky J, Levenson A, Weinberg IN, Tait JF, Uren A, Mease RC. Preparation of F-18 labeled annexin V: a potential PET radiopharmaceutical for imaging cell death. Nucl Med Biol 2004;31:747 – 52. [17] McQuade P, Jones LA, Vanderheyden JL, Welch MJ. 94mTc and 64Cu labeled annexin V positron-emitting radiopharmaceuticals. J Label Compd Radiopharm 2003;46:S1 – S4. [18] Dekker B, Keen H, Shaw D, Disley L, Hastings D, Hadfield J, et al. Functional comparison of annexin V analogues labeled indirectly and directly with iodine 124. Nucl Med Biol 2005;32:403 – 13. [19] Glaser M, Collingridge DR, Aboagye EO, Hayes LB, Hutchinson OC, Martin SJ, et al. Iodine-124 labeled annexin-V as a potential
A. Mukherjee et al. / Nuclear Medicine and Biology 33 (2006) 635 – 643
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30] [31]
[32]
radiotracer to study apoptosis using positron emission tomography. Appl Radiat Isot 2003;58:55 – 62. Lahorte C, Slegers G, Philippe J, van de Wiele C, Dierckx RA. Synthesis and in vitro evaluation of 123I-labeled human recombinant annexin V. Biomol Eng 2001;17:51 – 3. Russell J, O’Donoghue JA, Finn R, Koziorowski J, Ruan S, Humm JL, et al. Iodination of annexin V for imaging apoptosis. J Nucl Med 2002;43:671 – 7. Blankenberg FG, Katsikis PD, Tait JF, Davis RE, Naumovski L, Ohtsuki K, et al. In vivo detection and imaging of phophatidylserine expression during programmed cell death. Proc Natl Acad Sci U S A 1998;95:6349 – 54. Hoftstra L, Liem IH, Dumont EA, Boersma HH, van Heerde WL, Doevendans PA, et al. Visualization of cell death in vivo in patients with acute myocardial infarction. Lancet 2000;356:209 – 12. Keen HG, Dekker BA, Disley L, Hastings D, Lyons S, Reader AJ, et al. Imaging apoptosis in vivo using 124I annexin V and PET. Nucl Med Biol 2005;32:395 – 402. Blankenberg FG, Katsikis PD, Tait JF, Davis RE, Naumovski L, Ohtsuki K, et al. Imaging of apoptosis (programmed cell death) with 99m Tc annexin V. J Nucl Med 1999;40:184 – 91. Boersma HH, Bennaghmouch A, Heidenal GAK, Kietselaer BLJH, Hofstra L, Stolk LML, et al. Past present and future of annexin A5: from protein discovery to clinical application. J Nucl Med 2005;46: 2035 – 50. Mearly T, Meers P. Calcium dependent annexin binding to phospholipids: stoichiometry, specificity and role of negative charge. Biochemistry 1993;32:11711 – 21. Raynal P, Pollard HB. Annexins: the problem of assessing the biologica role for gene family multifunctional calcium- and phospholipid-binding proteins. Biochim Biophys Acta 1994;1197: 63 – 93. Hayashi H, Owada MK, Sonobe S, Kakunaga T. Characterization of two distinct Ca2+ dependent phospholipid binding proteins of 68 kDa isolation from placenta. J Biol Chem 1989;264:17222 – 30. Pepinsky RB, Sinclair LK, Chow EP, Obrinegreco B. A dimeric form of lipocortin-1 in human placenta. Biochem J 1989;263:97 – 103. Signore A, Annovazzi A, Chianelli M, Corsetti F, Van de Wiele C, Watherhouse RN. Scopinaro, peptide radiopharmaceuticals for diagnosis and therapy. Eur J Nucl Med 2001;28:1555 – 65. Kornyei J, Toth G, Szemenyei E, Duatti A. Development of annexin V fragments for labeling with 99mTc. Report 2nd research coordination meeting on bDevelopment of 99mTc based small biomolecules using novel 99mTc coresQ. Vienna7 IAEA; 2004.
643
[33] Kothari K, Pillai MRA. International symposium on radiochemistry and radiation chemistry, AR-19, 1-2, Bhabha Atomic Research Center, Bombay, India; 2000. [34] Stewart JM, Young JD. Solid phase peptide synthesis. 2nd ed Rockford (IL)7 Pierce Chemical Company; 1984. [35] Tfmbfly C, Kfve´r K, Pe´ter A, Tourwe´ D, Biyeshev D, Benyhe S, et al. Structure–activity on the Phe side chain arrangement of endomorphins using conformationally constrained analogues. J Med Chem 2004;447:735 – 43. [36] Gisin BF. The preparation of Merrifield-resins through total esterification with cesium salts. Helv Chim Acta 1973;56:1476 – 82. [37] Alberto R, Schibli R, Egli A, Schubiger AP, Abram U, Kaden TA. A novel organometallic aqua complex of technetium for the labeling of biomolecules: synthesis of [Tc-99m (OH2)3(CO)3]+ from [(TcO4)-Tc99m] in aqueous solution and its reaction with a bifunctional ligand. J Am Chem Soc 1998;120:7987 – 8. [38] Boersma HH, Stolk LM, Kenis H, Deckers NN, Vanderheyden JL, Guido LH, et al. The ApoCorrect assay: a novel, rapid method to determine the biological functionality of radiolabeled and fluorescent annexin A5. Anal Biochem 2004;327:126 – 34. [39] Kemerink GJ, Liu X, Kieffer D, Kieffer D, Ceyssens S, Mortelmans L, et al. Safety, biodistribution, and dosimetry of 99mTc-HYNICannexin V, a novel human recombinant annexin V for human application. J Nucl Med 2003;44:947 – 52. [40] Glasier M, Collingridge DR, Aboagye EO, Hayes LB, Hutchinson OC, Martin SJ, et al. Iodine-124 labelled annexin V as a potential radiotracer to study apoptosis using positron emission tomography. Appl Radiat Isot 2003;58:55 – 62. [41] Boschi A, Bolzati C, Benini E, Malago E, Uccelli L, Duatti A, et al. A novel approach to the high specific activity labeling of small peptides with the technetium-99m fragment [99mTc(N)(PXP)]2+ (PXP= diphosphine ligand). Bioconjug Chem 2001;12:1035 – 42. [42] Hom R, Katzenellenbogen J. Technetium-99m labeled receptorspecific small molecule radiopharmaceuticals: recent developments and encouraging results. Nucl Med Biol 1997;24:485 – 98. [43] Dilworth JR, Parrot S. The biomedical chemistry of technetium and rhenium. Chem Soc Rev 1998;27(Suppl):43 – 55. [44] Tait JF, Smith C, Gibson DF. Development of annexin V mutants suitable for labeling with Tc (i)-carbonyl complex. Bioconjug Chem 2002;13:1119 – 23. [45] Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995;267:1456 – 62. [46] Blankenberg FG, Strauss HW. Nuclear medicine applications in molecular imaging. J Magn Reson Imaging 2002;16:352 – 61.
99m
Tc-labeled annexin V fragments: a potential SPECT radiopharmaceutical for imaging cell death
Archana Mukherjeea, Kanchan Kotharia,4, Ge´za To´thb, Erzse´bet Szemenyeib, Hal Dhar Sarmac, Jo´zsef Kfrnyeid, Meera Venkatesha a
Radiopharmaceuticals Division, Radiochemistry and Isotope Group, Bhabha Atomic Research Centre, Mumbai-400085, India b Biological Research Centre of the Hungarian Academy of Sciences, H-6701 Szeged, Hungary c Radiation Biology and Health Science Division, Bhabha Atomic Research Centre, Mumbai-400085, India d Institute of Isotopes Co. Ltd., H1121, Budapest, Hungary Received 10 November 2005; received in revised form 27 February 2006; accepted 16 May 2006
Abstract Introduction: Annexin V is a protein that binds to phosphatidylserine exposed on dying cells. The phosphatidylserine-specific sequence is attributed to a chain on the N-terminal of annexin consisting of 13 amino acid sequence. Radiolabeled annexin V is used for imaging apoptosis. Methods: With an aim to synthesize a probe that can detect cell death akin to annexin V but smaller in size, annexin-13 fragments were derivatized to contain cysteine, cysteine–cysteine and histidine in their sequence at N terminal and were labeled with 99mTc via nitrido and carbonyl precursors. The 99mTc-labeled annexin-13 derivatives were characterized by HPLC and studied for their stability. In vitro and in vivo studies were carried out in apoptotic HL-60 cells and fibrosarcoma tumor-bearing Swiss mice, respectively. Results: The 99mTc complexes were formed in high yields and were found to be stable. HPLC pattern of 99mTc nitrido complex of cysteine– cysteine–annexine 13 (CC-Anx13) and 99mTc carbonyl complex of histdine–annexin 13 (H-Anx13) revealed the formation of single species. In vitro cell uptake studies with 99mTc nitrido complex of cysteine–cysteine–annexin 13 fragment showed 6.5% uptake in apoptotic HL-60 cells. The uptake was found to be specific on testing with apoptotic HL-60 cells. Biodistribution studies of 99mTc nitrido complex with CC-Anx13 in fibrosarcoma tumor-bearing Swiss mice revealed optimum tumor uptake of 0.52 (0.17) %ID/g at 1 h pi. Conclusion: 99mTc(N)-CC-anx13 showed specific uptake in apoptotic tumor cells and warrants further evaluation. D 2006 Elsevier Inc. All rights reserved. Keywords: Annexin V; Apoptosis;
99m
Tc nitrido; Carbonyl precursors
1. Introduction Apoptosis or programmed cell death [1] is an essential life process for a cellular organism. It is an active process of cellular self-destruction which, on one hand, benefits in situations such as malignancies and their treatment while, on the other, leads to a large number of disorders such as organ rejection after transplantation [2], myocardial ischemia or infarct [3] and neurodegenerative diseases [4]. Apoptotic cell death occurs after treatment of cancer by radiation, chemotherapy and photodynamic therapy. The extent and time at which cell death occurs may be important in
4 Corresponding author. Tel.: +91 22 2559 5371; fax: +91 22 2550 5345, +91 22 2551 9613. E-mail address: [email protected] (K. Kothari). 0969-8051/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2006.05.002
elucidating potential clinical information in the management of cancer treatment protocols. The extent of damage after myocardial infarction could also be gauged by estimating the extent of apoptosis. For this reason, considerable efforts have been made in developing and validating methods to image apoptosis in vivo [5,6]. A key feature of programmed cell death is that phophatidylserine is flipped from the internal to the external leaflet of the cell membrane [7]. This can be detected by using annexin V, a protein that binds to phosphatidylserine in the presence of calcium [8–10]. Radionuclide imaging with radiolabeled annexin V is a highly specific technique that enables delineation of apoptotic areas with good resolution [11]. Annexin V has been tagged with several radionuclides such as 99mTc [12–14], 18F [15,16], 64Cu [17] and 123/124I [18–21] to detect cell death in vivo by SPECT or PET imaging.
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Positron emission tomography agents and 123/124I-labeled agents yield superior images with far greater sensitivity than SPECT agents, but have limitations such as high cost and poor logistics for distribution. Hence, there have been demands for design and development of suitable 99mTclabeled agents that can be used more conveniently, economically and universally. 99mTc-HYNIC-annexin V has been used to detect apoptosis in vivo with gamma camera imaging and SPECT [22,23]. Studies with 99mTcHYNIC-annexin V for apoptosis detection have shown a high degree of retention in normal tissues, particularly in the abdomen, that may interfere with the imaging of apoptotic uptake [24 –26]. Most annexin molecules consist of a unique N-terminal region followed by repeated sequences of four units of 60 – 70 amino acids each [27]. It has been inferred that since the annexin proteins generally possess their biospecific sequences on the N-terminal, in the case of annexin V too the phosphatidylserine-specific sequence might be attributed to a chain consisting of 13 amino acids at the N terminal [28–30]. Since small peptides can be synthesized easily and modified suitably to enable radiolabeling to obtain stable products with pharmacokinetics of rapid uptake and quick renal clearance [31], 99mTc-labeled annexin 13 fragments may prove to be a better tool for imaging apoptosis than the whole annexin V [32]. Here, we describe the work carried out towards radiolabeling of annexin 13 fragment with 99mTc through nitrido and tricarbonyl cores, their in vitro evaluation in apoptosis model and in vivo evaluation in animal tumor model of fibrosarcoma. 2. Material and methods Nitrido kit and the coligand (PNP5) were supplied by the IAEA as part of a coordinate research project. Amino acid derivatives and the resin were obtained from SigmaAldrich (St. Louis, MO, USA) or Bachem (Bubendorf, Switzerland). Coupling reagents were acquired from Calbiochem-Novabiochem (L7ufelfingen, Switzerland). All the chemicals, solvents and reagents used were of analytical quality and used without further purification. Sodium borohydride and Na/K tartarate were obtained from Aldrich Chemicals (USA). All chemicals and solvents were of reagent grade and used without further purification. Carbon monoxide in 0.5-L refillable canisters was obtained from M/s Alchemie Gases and Chemicals (Mumbai, India). 99m TcO4 was eluted from a 99Mo/99mTc column generator using normal saline [33]. HPLC analysis was performed using C-18 reversed-phase HiQ Sil (5 Am, 4250 mm), on a Jasco PU 1580 system with a Jasco 1575 tunable UV–visible absorption detector as well as a radiometric detector system. The purity of the peptides was determined by RP-HPLC on a Merck-Hitachi liquid chromatographic system (Merck, Darmstadt, Germany, and Hitachi, Tokyo, Japan) equipped with a Vydac 218TP54 reversed-phase analytical column (2504.6 mm, 5 Am, Separations Group,
Hesperia, CA, USA) and LichroChart 250-4, Lichrospher100 reversed-phase analytical column (Merck). Silica gel 60 F254 precoated aluminum sheets from Merck were used for TLC. Vydac 218TP1010 reversed-phase semipreparative column (25010 mm, 12 Am, Separations Group) was used for semipreparative separations of the peptides. Mass spectra of the peptides were recorded on a Bruker Reflex III MALDI-TOF mass spectrometer. 2.1. Peptide synthesis Peptides CC-Anx13, C-Anx13 and H-Anx13 were synthesized by manual solid-phase techniques [34,35]. The peptide acids were prepared on Merrifield resin (0.9– 1.5 mmol/g substitution). The tert-butyloxycarbonyl-amino acid (Boc-amino acid) resin esters were prepared by the method of Gisin [36]. N a-t-Boc chemistry with N-hydroxybenztriazole and N,NV-dicyclohexylcarbodiimide as coupling reagents was employed for peptide elongation. Stepwise removal of N a-t-Boc protecting group from growing chain of peptides was achieved by 50% trifluoroactic acid (TFA) and 2% anisole in dichloromethane. Peptides were cleaved from the resin with anhydrous HF (10 ml/g resin) in the presence of anisole (1 ml/g resin) for 60 min at 08C. After removal of the scavenger, the peptides were extracted with 30% acetic acid and the filtrate was lyophilized. The crude peptides were purified by RP-HPLC on Vydac 218TP1010 column by gradient elution with water [0.1% (v/v) TFA] (A)/acetonitrile [0.08% (v/v) TFA] (B) solvent system at a flow rate of 4 ml/min. The gradient started from 85% A/15% B and changed to 60% A/40% B in 25 min. The purities of the peptides were assessed by analytical RP-HPLC on Vydac 218TP54 column and by TLC on silica gel 60 F254 precoated aluminum sheets. Water [0.1% (v/v) TFA] (A)/acetonitrile [0.08% (v/v) TFA] (B) gradient solvent system was used for the RP-HPLC analysis. The gradient started from 90% A/10% B and changed to 70% A/30% B in 40 min at a flow rate of 1 ml/min. The TLC solvent systems were as follows: (I) pyridine/isoamyl alcohol/water (1:1:2 v/v) and (II) n-butanol/pyridine/acetic acid/water (15:3:8:10 v/v). Ultraviolet light at k = 254, I2 vapor and ninhydrine were applied to visualize the TLC spots. The mass analysis of the peptide was carried out using MALDI-TOF in the positive ion mode using 2,5-dihydroxy benzoic acid as the matrix. Structure of derivatized annexin V fragments used for radiolabeling studies is given in Fig. 1. 2.2. Radiolabeling studies Labeling of CC-Anx13 and C-Anx13 with 99mTc was carried out via 99mTc-nitrido intermediate, while that of H-Anx13 was carried out via 99mTc-carbonyl core. 2.2.1. Labeling annexin fragments via nitrido intermediate 2.2.1.1. Synthesis of [99mTcN]2+ intermediate. The kit vial, containing succinic dihydrazide (5.0 mg), stannous chloride
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B which gradually changed to 10% A/90% B in 28 min. The gradient remained at 10% A/90% B during 28–30 min. 2.2.2. Labeling of H- anx13 via carbonyl precursor 2.2.2.1. [99mTc-(OH2 )3(CO)3]+ precursor. The precursor was prepared by a modified procedure reported by Alberto et al. [37]. Briefly, NaBH4 (5.5 mg), Na2CO3 (4 mg) and Na/K tartrate (15 mg) were dissolved in 0.5 ml double distilled water in a glass serum vial. The vial was sealed and a needle was introduced through the rubber stopper for facilitating equilibration to atmospheric pressure. Carbon monoxide was purged through the solution for 5 min. After the addition of 1 ml of the generator eluate containing 37–740 MBq (1–20 mCi) of 99mTcO4 , the needle was removed and the vial was heated at 808C for 15 min. After cooling the vial for 10 min and re-equilibration to atmospheric pressure, the pH of the reaction mixture was adjusted to 6 with 300 Al of 1:3 mixture of 0.5 M phosphate buffer (pH 7.5)/1 M HCl.
Fig. 1. Structure of Anx13 derivatives used for labeling with 99mTc: (1) CCAnx13, (2) C-Anx13, (3) H-Anx13.
dihydrate (100 Ag), 1,2-diaminopropane-N,N,NV,NV-tetraacetic acid (5 mg), sodium dihydrogen phosphate (0.5 mg) and disodium hydrogen phosphate (5.8 mg) in freeze dried form, was used for preparing the [99mTcN]+2 intermediate. The kit vial stored at 48C was allowed to attain ambient temperature. One milliliter of freshly eluted 99mTcO4 (37 MBq) was then added to the kit vial, vortexed for 1 min and allowed to stand at room temperature for 20 min.
2.2.2.2. Preparation of 99mTc-(CO)3-H-Anx13. Twenty micrograms (10 Al) of H-Anx13 was added to 0.5 ml of carbonyl precursor at pH 6. The reaction was carried out at 808C for 30 min.
2.2.1.2. Labeling of CC-Anx13 and C-Anx13 via nitrido intermediate. One hundred micrograms each of CC-Anx13 and C-Anx13 in 0.1 ml of saline was added to separate aliquots of 0.9 ml of 99mTc-nitrido intermediate. The reaction mixtures were heated in boiling water bath for 1 h.
2.2.2.3. Characterization of 99mTc-(CO)3-H-Anx13 by HPLC. The radiochemical purity of the precursor and the complex was checked by HPLC using C-18 reversed-phase column and water (A)/acetonitrile (B) with 0.1% TFA as solvent system in gradient mode at a flow rate of 1 ml/min. The gradient system for the analysis of the product started with a linear gradient of 100% A/0% B for 0 –1.5 min which slowly changed to 70% A/30% B in 18 min and further changed to 40% A/60% B from 18 to 21 min. The gradient remained at 40% A/60% B between 21 and 24 min and was then changed to 100% B in 5 min.
2.2.1.3. Labeling C-Anx13 via 99mTc-(N)-PNP intermediate. 2.2.1.3.1. Preparation of 99mTc-(N)-PNP intermediate. Ten microliters of PNP (~10 mg) was dissolved in 1 ml ethanol in nitrogen atmosphere. One hundred microliters of PNP was added to 1 ml of 99mTc-(N)-PNP intermediate along with 0.1 mg (50 Al) of C-Anx13, and the reaction mixture was heated at 1008C for 1 h. 2.2.1.4. Characterization of the nitrido complexes by HPLC technique. Characterization of the different nitrido complexes was carried out by HPLC using C-18 reversed-phase column using gradient elution with water (A)/acetonitrile (B) with 0.1% TFA as solvent system in gradient mode at a flow rate of 1 ml/min. The gradient system for the analysis of the product started with a linear gradient of 90% A/10%
2.3. Stability studies 2.3.1. Stability with time The stability of 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3H-Anx13 was studied at room temperature for 24 h. Radiochemical purity of the complex was checked by HPLC.
Table 1 Analytical data for annexin V fragments No.
Peptides
MALDI-TOF MS [M+H]+ Calculated
Found
1 2 3
CC-Anx13 C-Anx13 H-Anx13
1567 1464 1498
1566.73 1463.81 1497.85
RP-HPLC (kV) 6.44b 4.20c 3.66c
TLCa (R f) (I)
(II)
0.27 0.38 0.40
0.38 0.43 0.23
a Retention factors on silica gel 60 F254 precoated aluminum sheets. Solvent systems: (I) pyridine/isoamyl alcohol/water (1:1:2 v/v), (II) n-butanol/ pyridine/acetic acid/water (15:3:8:10 v/v). b Capacity factor: Vydac 218TP54 column (250.46 cm, d p = 5 Am); the gradient started from 90% A/10% B and changed to 70% A/30% B in 40 min. The flow rate was 1 ml/min; t 0 = 3.22 min; detection at k = 216 nm. c Capacity factors: LichroChart 250-4, Lichrospher100 column (250.46 cm, d p = 5 Am); the gradient started from 90% A/10% B and changed to 60% A/40% B in 30 min. The flow rate was 1 ml/min; t 0 = 2.9 min; detection at k = 216 nm.
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the binding of 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3-HAnx13 to apoptotic cells, the cells treated with camptothecin were harvested at 2z106 cells/tube, washed twice with PBS and resuspended in annexin V binding buffer (Pharmingen, 10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). 99m Tc-(N)-CC-Anx13 and 99mTc-(CO)3-H-Anx13 were added individually at a final concentration of 0.6 Ag/ml to the cell suspension and incubated at room temperature for 30 min. At the end of incubation, the cells were centrifuged at 3000 rpm for 5 min and washed with PBS. The radioactivity associated with the pellet was measured in a
Fig. 2. (A) Probable structure of 99mTc-nitrido complex with CC-Anx13 [99mTc-(N)-CC-Anx13]; (B) biphosphine ligand PNP5 used for the preparation of [99mTc-(N)-PNP]2+ intermediate.
2.3.2. Challenge with cysteine and histidine Stability of 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3-HAnx13 against trans-chelation was checked by challenge studies with cysteine and histidine. One hundred microliters of the complexes was incubated with 10 Al of cysteine (0.1 M) and histidine (0.1 M) in saline at 378C for 1 h. After incubation, the percentage dissociation of 99mTc was measured by HPLC analysis, following the solvent system used for characterization of the complexes. 2.3.3. Serum binding studies Approximately ~10 Ag of 99mTc-(N)-CC-Anx13 and 99m Tc-(CO)3-H-Anx13 was added individually to 0.5 ml of human serum and incubated at 378C for 30 min. The serum proteins were precipitated out by reacting with 0.5 ml of acetonitrile, and the activity bound to the serum protein was measured by counting the activity associated with the precipitate. The supernate was analyzed by HPLC to study serum stability. 2.4. Biological evaluation 2.4.1. Binding studies of 99mTc-(N)-CC-Anx13 with apoptotic cells The biological activity of 99mTc-(N)-CC-Anx13 was evaluated on exponentially growing HL-60 human leukemia cells. HL-60 cells were cultured in RPMI growth medium supplemented with 10% FBS. To induce apoptosis, cells in culture medium were treated with camptothecin dissolved in DMSO to a final concentration of 5 AM and incubated in 5% CO2 incubator at 378C for 6 h. Control cells were incubated with DMSO under similar conditions. To evaluate
Fig. 3. HPLC pattern of (A) 99mTc-nitrido intermediate and (B) asymmetric 99m Tc-nitrido complex with CC-Anx13 [99mTc-(N)-CC-Anx13].
A. Mukherjee et al. / Nuclear Medicine and Biology 33 (2006) 635 – 643
gamma counter. Control cells were also treated in a similar way. To investigate the specificity of binding, one set of apoptotic HL-60 cells was incubated with 100 of unlabelled annexin 13 fragment prior to the addition of 99m Tc-(N)-CC-Anx13 and 99mTc-(CO)3-H-Anx13 in order to saturate and block any specific binding [38]. All experiments were carried out in triplicate. Induction of apoptosis in HL 60 cells by camptothecin was confirmed by flow cytometric analysis. 2.4.2. Biodistribution studies 99m Tc-(N)-CC-Anx13 was diluted 1:10 in saline, and 0.1 ml (1 Ag) was injected via the tail vein of Swiss mice bearing fibrosarcoma tumor. The animals were sacrificed at 30 min, 1 h and 3 h pi. Organs were excised and counted in flat geometry NaI (Tl) scintillation detector. Percent activity retained in different organs was calculated. Three animals were used for each time point study. All the animal experiments were carried out in compliance with the relevant national laws relating to the conduct of animal experimentation.
639
13.0F0.2 and 14.3F0.2 min (Fig. 4A). When attempts were made to prepare asymmetric complex via 99mTc-(N)(PNP)2+ fragment using diphosphine ligand (Fig. 2B), the complex 99m Tc-(N)-PNP-C-Anx13 could be formed in N 95% yields. But in this case also, HPLC analysis of the complex revealed the formation of two species with retention times of 18.0F0.2 and 24F0.2 min (Fig. 4B). H-Anx13 could be labeled with 99mTc via carbonyl precursor at N 95% yields. The precursor as well as the complex was characterized by HPLC. But the HPLC system used for nitrido complexes was not able to clearly
3. Results 3.1. Peptide synthesis The peptides were synthesized by the manual solid-phase technique using Merrifield resin. The yields of the pure peptides were as follows: C-Anx13, 40%; H-anx13, 47%. The CC-Anx13 had to be purified twice in a row, leading to low yields of 5.9%. The structure of annexin derivatives is depicted in Fig. 1. The results obtained on the weights of the molecule ions by TOF/MS, capacity factors in HPLC and retention factors in TLC of the pure peptides are depicted in Table 1. CC-Anx13 when immersed in boiling water at 1008C for 60 min was found to be stable. This stability was important to carry out the labeling of CC-Anx13 via nitrido intermediate at 1008C [32]. 3.2. Radiolabeling studies On analysis by paper chromatography using ethanol/ chloroform/toluene/ammonium acetate (0.5 M) (5:3:3:0.5 v/v) as mobile phase and Whatman no. 3 paper as support system, the 99mTc-nitrido intermediate remained at the point of spotting (R f ~0) and free pertechnatate migrated with the solvent front (R f ~1). 99mTc-nitrido intermediate could be prepared with 93–95% yields. The ligand CC-Anx13 complexed with the 99mTc-nitrido intermediate in high yields ( N95%) to form a single species as revealed by HPLC analysis (retention time, 11.5 min). The probable structure of 99mTc-nitrido complex with CCAnx13 is given in Fig. 2A. HPLC patterns of 99mTc-nitrido intermediate and 99mTc-(N)-CC-Anx13 are depicted in Fig. 3A and B, respectively. C-Anx13 reacted with nitrido intermediate in N 95% yields. However, HPLC analysis of 99mTc-(N)-C-Anx13 revealed the formation of two species with retention times of
Fig. 4. HPLC pattern of nitrido complexes of 99mTc with C-Anx13. (A) Symmetric complex [99mTc-(N)-C-Anx13], (B) asymmetric complex [99mTc-(N)-PNP-C-Anx13].
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3.3. Stability studies Both 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3-H-Anx13, which were proven to be single species, were found to be stable at room temperature for 24 h. 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3-H-Anx13 were also stable and did not dissociate or transchelate when challenged with cysteine (0.1 M) and histidine (0.1 M) in saline, indicating their potential for further evaluation as apoptosis marker. 3.4. Biological evaluation 3.4.1. In vitro cell uptake studies Apoptosis induction by camptothecin in HL-60 cells was confirmed by flow cytometry. On incubation of camptothecin-treated HL-60 cells with 99mTc-(N)-CC-Anx13 and 99m Tc-(CO)3-H-Anx13 for 30 min, 6.5% and 13% binding were observed, respectively. In the case of 99mTc-(N)-CC-Anx13, similar experimentation in control (nonapoptotic) cells showed merely 1.3% binding, indicating the specific nature of binding to apoptotic cells. This was also confirmed by the decrease in binding to 1.8% when apoptotic cells were incubated with 100-fold of cold CC-anx13 along with 99mTc-(N)-CCAnx13. On the other hand, binding of 99mTc-(CO)3-HAnx13 was seen to be nonspecific wherein an uptake of ~13% was observed in controls (nonapoptotic) as well as apoptotic cells in the presence and absence of cold peptide (Fig. 6). 3.4.2. Biodistribution studies Results of biodistribution studies of 99mTc-(N)-CCAnx13 in tumor-bearing Swiss mice are depicted in Table 2. It is seen that the radioactivity rapidly cleared from blood. Uptake of 99mTc-(N)-CC-Anx13 in tumor was 1.08 (F0.19) %/g at 30 min pi, which decreased to 0.26
Fig. 5. HPLC pattern of (A) carbonyl precursor and (B) complex with H-Anx13 [99mTc-(CO)3-H-Anx13].
99m
Tc-carbonyl
differentiate carbonyl precursor from 99mTc-(CO)3-HAnx13 (retention times, 13.4 and 14 min, respectively). Hence, HPLC was carried out with a slower gradient system. The carbonyl precursor eluted out with a retention time of 8.3 min, while 99mTc-(CO)3-H-Anx13 was eluted out in HPLC with a retention time of 24 min as a single species as seen in Fig. 5A and B, respectively.
Fig. 6. Uptake of 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3-H-Anx13 in (a) untreated HL-60 cells, (b) camptothecin-treated HL-60 cells and (c) camptothecin-treated HL-60 cells in the presence of 100-fold cold CCAnx13.
A. Mukherjee et al. / Nuclear Medicine and Biology 33 (2006) 635 – 643 Table 2 Biodistribution studies of Organ
99m
Tc (N)-CC-Anx13 in tumor-bearing Swiss mice (n = 3) 30 min %/ID
Liver Intestine + Gall bladder Stomach Kidney Heart Lungs Spleen Tumor Muscles Blood Tibia
641
2.70 4.10 0.24 8.92 0.048 0.409 0.03 0.37 2.15 2.07 0.91
(0.66) (0.11) (0.12) (1.02) (0.01) (0.12) (0.01) (0.15) (1.03) (1.08) (0.5)
1h
3h
%ID/g
%/ID
2.81 1.89 0.70 32.4 0.40 2.1 0.21 1.08 0.23 1.37 0.40
1.38 3.32 0.31 6.23 0.05 0.42 0.08 0.24 2.34 0.07 0.015
(0.54) (0.09) (0.39) (5.16) (0.10) (0.6) (0.09) (0.19) (0.11) (0.681) (0.23)
(0.88) (0.83) (0.11) (0.75) (0.02) (0.29) (0.01 ) (0.03) (2.03) (0.06) (0.015)
%ID/g
%/ID
1.11 (0.65) 1.32 (0.315) 0.41 (0.22) 23.04 (2.30) 0.42 (0.04) 2.45 (1.70) 0.19 (0.01) 0.52 (0.17) 0.24 (0.21) 0.05 (0.05) 0.15 (0.1)
1.88 3.74 0.38 9.27 0.01 0.17 0.027 0.14 1.38 0.26 0.809
%ID/g (0.19) (0.35) (0.29) (1.50) (0.01) (0.04) (0.014) (0.02) (0.84) (0.18) (0.21)
1.49 1.54 1.53 35.2 0.3 1.44 0.25 0.26 0.15 0.15 0.32
(0.15) (0.23) (1.23) (6.42) (0.01) (0.27) (0.10) (0.09) (0.11) (0.11) (0.2)
Values in parentheses represent standard deviations.
(F0.09) %/g at 3 h pi. Optimum tumor uptake was 0.52 (F0.17) %/g at 1 h pi. 4. Discussion Annexin V has been labeled with 99mTc via the HYNIC conjugation method for clinical studies to date, and 99mTcHYNIC-annexin V has been the main 99mTc-annexin V radiopharmaceutical [26,38,39]. However, the product has shown high uptake in liver and kidney, and, hence, a need to develop 99mTc-labeled annexin V with better biodistribution profile has been felt [26,40]. The N terminal domain of annexin is considered as the regulatory region of the protein as it contains the major sites for phosphorylation, proteolysis or interactions with other proteins [28]. Annexin V has very high affinity for phospholipids (nanomolar range) and it is attributed to the N terminal domain constituting 13 amino acid sequence [28]. Being a peptide, the N terminal domain constituting 13 amino acid sequence can be easily synthesized and functionalized with a chelating group for labeling with 99mTc, rather than with whole annexin V molecule. Hence, 99mTc-labeled annexin 13 is attractive for exploration as a tool for apoptosis imaging. Hence, the N terminal domain constituting 13 amino acid residue of annexin V was functionalized with cysteine, dicysteine and histidine, and the functionalized annexin 13 fragments were labeled with 99mTc using [99mTc-(N)]2+ and [99mTc-(CO)3(H2O)3]+ precursor to investigate their potential for imaging apoptosis. CC-Anx13 was synthesized for the preparation of asymmetric nitrido complex in the absence of PNP ligand. C-Anx13 is expected to interact with 99mTc-nitrido intermediate to form a symmetric complex with two annexin 13 fragments. Introduction of two biologically active bidentate fragments in a symmetrical manner into a 99mTc-nitrido core is not preferable as this could lead to poor bioavailability. It would be preferable to design a complex with two different bidentate ligands to avoid the contemporary insertion of two biofragments on the same core. 99mTc-(N)-(PNP)-C-Anx13 was hence
prepared with the intention of making such an asymmetrical heterocomplex [41]. Of four different 99mTc-Anx13 complexes studied, although all were formed in high yields of N 95%, it was observed that 99mTc-(N)-CC-Anx13 and 99mTc-(CO)3-HAnx13 were formed as single species, while 99mTc-(N)-CAnx13 and 99mTc-(N)-PNP-C-Anx13 were formed with more than one species. Since complexes with multiple species are generally not acceptable as radiopharmaceuticals, owing to the possibility of differences in their pharmacokinetics [42,43], these complexes were not evaluated for their biological behavior. Hence, in vitro studies were carried out only with 99mTc-(N)-CC-Anx13 and 99mTc(CO)3-H-Anx13 in apoptotic HL60 cells. Among these, 99m Tc-(N)-CC-Anx13 exhibited specific uptake in apoptotic HL60 cells, which could be inferred from poor uptake in untreated HL60 cells as well as in treated cells in the presence of excess cold peptide Anx13. In the case of 99m Tc-(CO)3-H-Anx13, the uptake was higher but not found to be specific. Earlier studies reported on 99mTc carbonyl complex of annexin V mutant have shown high retention of biological activity [44] However, our studies with 99mTc(CO)3-H-Anx13 in apoptotic HL60 cells did not substantiate this finding. One reason could be the loss of biological activity of H-Anx13 fragment after labeling with 99mTc via a carbonyl precursor. Being a peptide, H-Anx may have been more sensitive to loss of activity than high-molecularweight annexin V mutant protein. Imaging apoptosis has many applications with new ones emerging with time. The most widely used application is in cancer treatment [45] for assessing tumor response to novel therapies [46] as tumor often respond to radiation as well as to chemotherapy by direct induction of apoptosis. Again, in the evaluation of the extent of damage to the heart muscle in a patient with myocardial infarction, clinicians welcome the use of an agent to image apoptotic regions. Thus, it is expected that the utility of an apoptosis imaging radiopharmaceutical would be considered important and would grow. In this context, a small molecule that can be easily
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synthesized, labeled and quality controlled would be preferable to a large protein such as annexin V. Economic viability would also be favorable for smaller molecules, as annexin V is expensive. Hence, in our studies the Anx13 peptides were evaluated as possible molecules for imaging apoptosis. Among the four tested complexes,99mTc-(N)-CC-Anx13, which showed the best characteristics, was further evaluated in vivo in tumor-bearing Swiss mice to study its suitability to assess response to cancer therapy. 99mTc-(N)-CC-Anx13 has shown reasonably good tumor uptake. Apart from the above, large molecules do not clear from the body quickly, often accompanied by soft tissue uptake of the radiopharmaceuticals. In the case of 99mTc-HYNIC-annexin V too, earlier studies in normal Balb/c mice have shown high soft tissue retention (spleen, 17 %ID/g; liver, 15.2 %ID/g; stomach, 5.4 %ID/g; and kidney, 187 %ID/g) at 1 h pi [25]. 99m Tc-HYNIC-annexin V has also shown high retention of activity in the abdominal region in murine tumor model, making the product not suitable for evaluation of apoptosis in abdominal regions [11]. In our studies with 99mTc-(N)CC-Anx13 in tumor-bearing Swiss mice, the retention in soft tissues is relatively low in comparison to 99mTcHYNIC-annexin V [25]. Thus, 99mTc-(N)-CC-Anx13 could be a potential agent for apoptosis imaging. 5. Conclusion The N terminal chain of annexin V consisting of 13 amino acids, which is considered to bind to phosphatidylserine exposed on apoptotic cells, could be labeled with 99m Tc using novel chemical approaches in 99mTc radiopharmaceutical chemistry. This study is the first one to report on the use of 99mTc-labeled annexin 13 fragment for apoptosis detection. Among the four different complexes tested, 99m Tc-(N)-CC-Anx13 has shown specific uptake in apoptotic human leukemia HL-60 cells in the in vitro studies. Biodistribution studies of 99mTc-(N)-CC-Anx13 in tumorbearing Swiss mice also yielded encouraging results in tumor uptake and revealed superiority in soft tissue clearance in comparison with 99mTc-HYNIC annexin V. Being a small peptide that can be synthesized in large quantities, 99mTc-(N)-CC-Anx13 warrants further evaluation for imaging apoptosis. Acknowledgments This work was carried out as part of the IAEA CRP project on bDevelopment of 99mTc-based small biomolecules using novel 99mTc cores.Q The work on peptide synthesis was supported by a grant (RET08/2004-DNT) from the National Bureau of Research and Technology in Hungary. The authors are thankful to Dr. Duatti for the supply of the nitrido kit. The authors are also grateful to Dr. V. Venugopal (Director, Radiochemistry and Isotope Group,
Bhabha Atomic Research Centre) and Dr. M.R.A. Pillai (former head, Radiopharmaceuticals Division) for their encouragement and support.
References [1] Boris Z, Bertrand J, Orrenius S. Tumor radiosensitivity and apoptosis. Expt Cell Res 1999;248:10 – 7. [2] Kabelitz D. Apoptosis, graft rejection and transplantation tolerance. Transplantation 1998;65:869 – 75. [3] Mattson MP, Culmsee C, Yu ZF. Apoptosis and antiapoptotic mechanisms in stroke. Cell Tissue Res 2000;301:173 – 87. [4] Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995;267:1456 – 62. [5] Zucker RM, Hunter III ES, Rogers JM. Apoptosis and morphology in mouse embryos by confocal laser scanning microscopy. Methods 1999;18:473 – 80. [6] Hakumaki JM, Poptani H, Sandmair AM, Yia-Herttuala S, Kauppinen RA. 1H MRS detects polyunsaturated fatty acid accumulation during gene therapy of glioma: implications for the in vivo detection of apoptosis. Nat Med 1999;5:1323 – 7. [7] Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 1992;148:2207 – 16. [8] Funakoshi T, Heimark RL, Hendrickson LE, McMullen BA, Fujikawa K. Human placental anticoagulant protein: isolation and characterization. Biochemistry 1987;26:5572 – 8. [9] Reutelingsperger CPM, Hornstra G, Hemker HC. Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord. Eur J Biochem 1985;151:625 – 9. [10] Maurer-Fogy I, Reutelingsperger CPM, Pieters J, Bodo G, Stratowa C, Hauptmann R. Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+-dependent phospholipid-binding protein. Eur J Biochem 1988;174:585 – 92. [11] Subbarayan M, Hafeli UO, Feyes DK, Unnithan J, Emancipator SN, Mukhtar H. A simplified method for preparation of 99mTc-annexin V and its biologic evaluation for in vivo imaging of apoptosis after photodynamic therapy. J Nucl Med 2003;44:650 – 6. [12] Steinmetz ND, Green Am. Chemotherapy induced change in 99mTc HYNIC-annexin V uptake as an early predictor of response to platinum therapy in advanced non-small cell lung cancer. J Nucl Med 2004;45:36P. [13] Post AM, Katsikis PD, Tait JF, Geaghan SM, Strauss HW, Blankenberg FG. Imaging cell death with radiolabeled annexin V in an experimental model of rheumatoid arthritis. J Nucl Med 2002;43:1359 – 65. [14] Blankenberg FG, Naumovski L, Tait JF, Post AM, Strauss HW. Imaging cyclophosphamide-induced intramedullary apoptosis in rats using 99mTc radiolabeled annexin V. J Nucl Med 2001;42:309 – 16. [15] Zijlstra S, Gunawan J, Burchert W. Synthesis and evaluation of a 18F labeled recombinant annexin-V derivative for identification and quantification of apoptotic cells with PET. Appl Radiat Isot 2003;58:201 – 7. [16] Toretsky J, Levenson A, Weinberg IN, Tait JF, Uren A, Mease RC. Preparation of F-18 labeled annexin V: a potential PET radiopharmaceutical for imaging cell death. Nucl Med Biol 2004;31:747 – 52. [17] McQuade P, Jones LA, Vanderheyden JL, Welch MJ. 94mTc and 64Cu labeled annexin V positron-emitting radiopharmaceuticals. J Label Compd Radiopharm 2003;46:S1 – S4. [18] Dekker B, Keen H, Shaw D, Disley L, Hastings D, Hadfield J, et al. Functional comparison of annexin V analogues labeled indirectly and directly with iodine 124. Nucl Med Biol 2005;32:403 – 13. [19] Glaser M, Collingridge DR, Aboagye EO, Hayes LB, Hutchinson OC, Martin SJ, et al. Iodine-124 labeled annexin-V as a potential
A. Mukherjee et al. / Nuclear Medicine and Biology 33 (2006) 635 – 643
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30] [31]
[32]
radiotracer to study apoptosis using positron emission tomography. Appl Radiat Isot 2003;58:55 – 62. Lahorte C, Slegers G, Philippe J, van de Wiele C, Dierckx RA. Synthesis and in vitro evaluation of 123I-labeled human recombinant annexin V. Biomol Eng 2001;17:51 – 3. Russell J, O’Donoghue JA, Finn R, Koziorowski J, Ruan S, Humm JL, et al. Iodination of annexin V for imaging apoptosis. J Nucl Med 2002;43:671 – 7. Blankenberg FG, Katsikis PD, Tait JF, Davis RE, Naumovski L, Ohtsuki K, et al. In vivo detection and imaging of phophatidylserine expression during programmed cell death. Proc Natl Acad Sci U S A 1998;95:6349 – 54. Hoftstra L, Liem IH, Dumont EA, Boersma HH, van Heerde WL, Doevendans PA, et al. Visualization of cell death in vivo in patients with acute myocardial infarction. Lancet 2000;356:209 – 12. Keen HG, Dekker BA, Disley L, Hastings D, Lyons S, Reader AJ, et al. Imaging apoptosis in vivo using 124I annexin V and PET. Nucl Med Biol 2005;32:395 – 402. Blankenberg FG, Katsikis PD, Tait JF, Davis RE, Naumovski L, Ohtsuki K, et al. Imaging of apoptosis (programmed cell death) with 99m Tc annexin V. J Nucl Med 1999;40:184 – 91. Boersma HH, Bennaghmouch A, Heidenal GAK, Kietselaer BLJH, Hofstra L, Stolk LML, et al. Past present and future of annexin A5: from protein discovery to clinical application. J Nucl Med 2005;46: 2035 – 50. Mearly T, Meers P. Calcium dependent annexin binding to phospholipids: stoichiometry, specificity and role of negative charge. Biochemistry 1993;32:11711 – 21. Raynal P, Pollard HB. Annexins: the problem of assessing the biologica role for gene family multifunctional calcium- and phospholipid-binding proteins. Biochim Biophys Acta 1994;1197: 63 – 93. Hayashi H, Owada MK, Sonobe S, Kakunaga T. Characterization of two distinct Ca2+ dependent phospholipid binding proteins of 68 kDa isolation from placenta. J Biol Chem 1989;264:17222 – 30. Pepinsky RB, Sinclair LK, Chow EP, Obrinegreco B. A dimeric form of lipocortin-1 in human placenta. Biochem J 1989;263:97 – 103. Signore A, Annovazzi A, Chianelli M, Corsetti F, Van de Wiele C, Watherhouse RN. Scopinaro, peptide radiopharmaceuticals for diagnosis and therapy. Eur J Nucl Med 2001;28:1555 – 65. Kornyei J, Toth G, Szemenyei E, Duatti A. Development of annexin V fragments for labeling with 99mTc. Report 2nd research coordination meeting on bDevelopment of 99mTc based small biomolecules using novel 99mTc coresQ. Vienna7 IAEA; 2004.
643
[33] Kothari K, Pillai MRA. International symposium on radiochemistry and radiation chemistry, AR-19, 1-2, Bhabha Atomic Research Center, Bombay, India; 2000. [34] Stewart JM, Young JD. Solid phase peptide synthesis. 2nd ed Rockford (IL)7 Pierce Chemical Company; 1984. [35] Tfmbfly C, Kfve´r K, Pe´ter A, Tourwe´ D, Biyeshev D, Benyhe S, et al. Structure–activity on the Phe side chain arrangement of endomorphins using conformationally constrained analogues. J Med Chem 2004;447:735 – 43. [36] Gisin BF. The preparation of Merrifield-resins through total esterification with cesium salts. Helv Chim Acta 1973;56:1476 – 82. [37] Alberto R, Schibli R, Egli A, Schubiger AP, Abram U, Kaden TA. A novel organometallic aqua complex of technetium for the labeling of biomolecules: synthesis of [Tc-99m (OH2)3(CO)3]+ from [(TcO4)-Tc99m] in aqueous solution and its reaction with a bifunctional ligand. J Am Chem Soc 1998;120:7987 – 8. [38] Boersma HH, Stolk LM, Kenis H, Deckers NN, Vanderheyden JL, Guido LH, et al. The ApoCorrect assay: a novel, rapid method to determine the biological functionality of radiolabeled and fluorescent annexin A5. Anal Biochem 2004;327:126 – 34. [39] Kemerink GJ, Liu X, Kieffer D, Kieffer D, Ceyssens S, Mortelmans L, et al. Safety, biodistribution, and dosimetry of 99mTc-HYNICannexin V, a novel human recombinant annexin V for human application. J Nucl Med 2003;44:947 – 52. [40] Glasier M, Collingridge DR, Aboagye EO, Hayes LB, Hutchinson OC, Martin SJ, et al. Iodine-124 labelled annexin V as a potential radiotracer to study apoptosis using positron emission tomography. Appl Radiat Isot 2003;58:55 – 62. [41] Boschi A, Bolzati C, Benini E, Malago E, Uccelli L, Duatti A, et al. A novel approach to the high specific activity labeling of small peptides with the technetium-99m fragment [99mTc(N)(PXP)]2+ (PXP= diphosphine ligand). Bioconjug Chem 2001;12:1035 – 42. [42] Hom R, Katzenellenbogen J. Technetium-99m labeled receptorspecific small molecule radiopharmaceuticals: recent developments and encouraging results. Nucl Med Biol 1997;24:485 – 98. [43] Dilworth JR, Parrot S. The biomedical chemistry of technetium and rhenium. Chem Soc Rev 1998;27(Suppl):43 – 55. [44] Tait JF, Smith C, Gibson DF. Development of annexin V mutants suitable for labeling with Tc (i)-carbonyl complex. Bioconjug Chem 2002;13:1119 – 23. [45] Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995;267:1456 – 62. [46] Blankenberg FG, Strauss HW. Nuclear medicine applications in molecular imaging. J Magn Reson Imaging 2002;16:352 – 61.