Development and evaluation of a novel (99m)tc-labeled annexin A5 for early detection of response to chemotherapy.

(99m)Tc-HYNIC-annexin A5 can be considered as a benchmark in the field of apoptosis imaging. However, (99m)Tc-HYNIC-annexin A5 has characteristics of high uptake and long retention in non-target tissues such as kidney and liver. To minimize this problem, we developed a novel (99m)Tc-labeled annexin A5 using a bis(hydroxamamide) derivative [C3(BHam)2] as a bifunctional chelating agent, and evaluated its usefulness as an imaging agent for detecting apoptosis. The amino group of C3(BHam)2 was converted to a maleimide group, and was coupled to thiol groups of annexin A5 pretreated with 2-iminothiolane. (99m)Tc labeling was performed by a ligand exchange reaction with (99m)Tc-glucoheptonate. Biodistribution experiments for both (99m)Tc-C3(BHam)2-annexin A5 and (99m)Tc-HYNIC-annexin A5 were performed in normal mice. In addition, in tumor-bearing mice, the relationship between the therapeutic effects of chemotherapy (5-FU) and the tumor accumulation of (99m)Tc-C3(BHam)2-annexin A5 just after the first treatment of 5-FU was evaluated. (99m)Tc-C3(BHam)2-annexin A5 was prepared with a radiochemical purity of over 95%. In biodistribution experiments, (99m)Tc-C3(BHam)2-annexin A5 had a much lower kidney accumulation of radioactivity than (99m)Tc-HYNIC-annexin A5. In the organs for metabolism, such as liver and kidney, radioactivity after the injection of (99m)Tc-HYNIC-annexin A5 was residual for a long time. On the other hand, radioactivity after the injection of (99m)Tc-C3(BHam)2-annexin A5 gradually decreased. In therapeutic experiments, tumor growth in the mice treated with 5-FU was significantly inhibited. Accumulation of (99m)Tc-C3(BHam)2-annexin A5 in tumors significantly increased after 5-FU treatment. The accumulation of radioactivity in tumor correlated positively with the counts of TUNEL-positive cells. These findings suggest that (99m)Tc-C3(BHam)2-annexin A5 may contribute to the efficient detection of apoptotic tumor response after chemotherapy.

Annexin A5-conjugated polymeric micelles for dual SPECT and optical detection of apoptosis.

UNLABELLED: Imaging of apoptosis can allow noninvasive assessment of disease states and response to therapeutic intervention for a variety of diseases. The purpose of this study was to develop and evaluate a multimodal nanoplatform for the detection of apoptosis. METHODS: To modulate the pharmacokinetics of annexin A5, a 36-kDa protein that binds specifically with phosphatidylserine, annexin A5 was conjugated to polyethylene glycol-coated, core-cross-linked polymeric micelles (CCPMs) dually labeled with near-infrared fluorescence fluorophores and a radioisotope ((111)In). To evaluate the specificity of the binding of annexin A5-CCPM to apoptotic cells, both fluorescence microscopy and cell-binding studies were performed in vitro. Pharmacokinetics, biodistribution, dual nuclear and optical imaging, and immunohistochemical studies were performed in 2 xenografted tumor models to evaluate the potential applications of annexin A5-CCPM. RESULTS: In cell-based studies, annexin A5-CCPM exhibited strongly specific binding to apoptotic tumor cells. This binding could be efficiently blocked by annexin A5. In mice, annexin A5-CCPM displayed a mean elimination half-life of 12.5 h. The mean initial concentration in blood was 22.4% of the injected dose/mL, and annexin A5-CCPM was mainly distributed in the central blood compartment. In mice bearing EL4 lymphoma treated with cyclophosphamide and etoposide and in mice bearing MDA-MB-468 breast tumors treated with poly(L-glutamic acid)-paclitaxel and cetuximab (IMC-C225) anti-epidermal growth factor receptor antibody, the tumor apoptosis was clearly visualized by both SPECT and fluorescence molecular tomography. In contrast, there was little accumulation of this nanoradiotracer in the tumors of untreated mice. The biodistribution data were consistent with the imaging data, with tumor-to-muscle and tumor-to-blood ratios of 38.8 and 4.1, respectively, in treated mice, and 14.8 and 2.2, respectively, in untreated mice bearing EL4 lymphoma. Moreover, further studies demonstrated that the conventional (99m)Tc-labeled hydrazinonicotinamide annexin A5 and the plain CCPM control exhibited significantly lower uptake in the tumors of the treated mice than annexin A5-CCPM. Immunohistochemistry staining study showed that radioactivity count correlated with fluorescence signal from the nanoparticles, and both signals colocalized with the region of tumor apoptosis. CONCLUSION: Annexin A5-CCPM allowed visualization of tumor apoptosis by both nuclear and optical techniques. The complementary information acquired with multiple imaging techniques should be advantageous in assessing and validating early response to therapy.

Imaging and dosimetry of 99mTc EC annexin V: preliminary clinical study targeting apoptosis in breast tumors.

BACKGROUND: Early detection of cellular events is important to predict the outcome of the patients. This study was aimed to use (99m)Tc EC-annexin V to image tumor cells undergoing apoptosis. METHODS: In 10 patients with breast cancer, scintigraphic images and dosimetric estimates were obtained after administering (99m)Tc EC-annexin V. RESULTS: Nine of the 10 cases showed detectable (99m)Tc EC-annexin V uptake in tumor. Higher values of T/N ratios are associated with patient after treatment. CONCLUSIONS: Apoptosis can be quantified using (99m)Tc EC-annexin V.

Preparation of F-18 labeled annexin V: a potential PET radiopharmaceutical for imaging cell death.

The clinical response to antitumor therapy is measured using imaging, such as CT or MRI, 6-12 weeks following chemotherapy treatment. The images at that time reflect both tumor cell death and new growth. Therefore, the amount of tumor cell death caused by chemotherapy cannot be efficiently quantified with current imaging modalities. A quantitative measurement of tumor cell death immediately following chemotherapy is needed to help validate both new agents and to optimize administration of existing therapies. Annexin V is a 36kD protein that binds to exposed phosphatidylserine (PS) on dying cells. In order to synthesize a probe that can detect cell death in vivo, the positron emitter F-18 was conjugated to annexin V via the compound N- succinimidyl-4-[18F]fluorobenzoate, [18F]SFB. The decay corrected radiochemical yield of F-18 labeled annexin V from 18F fluoride was 17.6 +/- 5.6% (n = 4) in three hours. The stepwise radiochemical yield of the conjugation step with annexin V was as high as 70% when a protein concentration of 5 mg/ml was used. Cancer cells treated with the chemotherapeutic agent, etoposide, showed an 88% increase in the binding of F-18 labeled annexin V compared to untreated cells. We conclude that [18F] labeled annexin V can be readily prepared by the conjugation of annexin V with [18F]SFB and that the positron-emitting compound is biologically active in detecting apoptosis.

Production of [F-18]fluoroannexin for imaging apoptosis with PET.

Recombinant human-annexin-V was conjugated with 4-[F-18]fluorobenzoic acid (FBA) via its reaction with the N-hydroxysuccinimidyl ester (FBA-OSu) at pH 8.5. A series of reactions using varying amounts of annexin-V, unlabeled FBA-OSu, and time produced products with different conjugation levels. Products were characterized by mass spectrometry and a cell-binding assay to assess the effect of conjugation. In each case, the conjugated protein was a mixture of proteins with a range of conjugation. Annexin-V could be conjugated with an average of two FBA mole equivalents without decreasing its affinity for red blood cells (K(d) 6-10 nM) with exposed phosphatidylserine. An average conjugation of 7.7 (range 3-13) diminished the binding 3-fold. Large-scale production and purification of [F-18]FBA-OSu from [F-18]fluoride was accomplished within 90 min and in 77% radiochemical yield (decay-corrected to the end of cyclotron bombardment). The conjugation reaction of annexin with [F-18]FBA-OSu was studied with respect to activity level, protein mass, and concentration. Under the most favorable conditions, >25 mCi [F-18]fluoroannexin (FAN) was isolated in 64% yield (decay-corrected for a 22 min conjugation process) from labeling 1.1 mg of annexin-V. A pilot PET imaging study of [F-18]fluoroannexin in normal rats showed high uptake in the renal excretory system and demonstrated sufficient clearance from most other internal organs within 1 h. [F-18]Fluoroannexin should prove useful in imaging targeted apoptosis.

Improved radiolabeling of PEGylated protein: PEGylated annexin V for noninvasive imaging of tumor apoptosis.

We have developed a one-step procedure to introduce both polyethylene glycol (PEG) and the metal chelator diethylenetriaminepentaacetic acid (DTPA) to proteins through a heterofunctional PEG precursor. The PEG precursor contains DTPA at one end and an amine-reactive isothiocyanate (SCN-) functional group at the other end. It was obtained as lyophilized powder and could be stored at 4 degrees C for several months. Protein conjugation was achieved by simply mixing the proteins and the PEG precursor SCN-PEG-DTPA in an aqueous solution. As exemplified by the PEGylation and radiolabeling of annexin V, the resulting conjugate 111In-DTPA-PEG-annexin V showed selective binding to apoptotic cells in vitro and increased blood half-life in vivo. The PEGylated, radiolabeled annexin V may be useful in the noninvasive imaging of apoptosis.

Optimization of the preparation of 99mTc-labeled Hynic-derivatized Annexin V for human use.

Hydrazino nicotinate (Hynic) is one of the most attractive bifunctional agents designed for the labeling of proteins with (99m)Tc. Recently, a (99m)Tc-labeled Hynic-Annexin V derivative has been described and successfully evaluated in animal models of apoptosis. Prior to a phase I human study, the preparation of (99m)Tc-Hynic-Annexin V has been optimized. The influence of the Hynic-load of Annexin V, amount of protein, nature and amount of reducing agent, activity and co-ligand on the labeling yield were evaluated using ITLC and size-exclusion FPLC. Optimal labeling yields were obtained when 60-90 microgram Hynic-Annexin V was labeled with up to 1.11 GBq (30 mCi) (99m)TcO(4)-using 10-20 microgram SnCl(2).2H(2)O as reducing agent and 1.5 mg tricine as the co-ligand. Biodistribution in normal mice was comparable to literature data.

Near-infrared fluorescent imaging of tumor apoptosis.

Noninvasive imaging using radioactive annexin V is an emerging strategy for the assessment of cell death in vivo (F. G. Blankenberg, and H. W. Strauss. Apoptosis, 6: 117-123, 2001.). Therefore, we investigated whether annexin V labeled with the fluorophore Cy5.5 (Cy) could serve as a probe for imaging of tumor apoptosis using near infrared fluorescence (NIRF). We prepared active Cy-annexin (an equimolar dye:protein ratio) that bound to apoptotic Jurkat T cells and an inactive Cy-annexin probe (>2 dyes/mol protein) that did not. Active Cy annexin was used to image a 9L gliosarcoma, constitutively expressing green fluorescent protein marker, and the CR8 variant of Lewis lung carcinoma, stably transfected to express DsRed2. The expression of transfected fluorescent protein provided an indication of tumor margins and a means of defining tumor-associated NIRF signal intensity with both tumor models. Tumors were imaged with and without cyclophosphamide treatment. In both tumor models active Cy-annexin V tumor NIRF signal increased two to three times after the treatment. Tumor NIRF signal developed by 75 min after active Cy-annexin injection and remained for a 20-h observation period. Inactive annexin V was used as a control in the CR8 carcinoma experiments and resulted in a low nonspecific signal. With the 9L gliomosacrcoma model, active Cy-annexin V bound to both tumor cells (Cy-annexin V staining only) and endothelial cells (costained with Cy-annexin V and antibody to the endothelial marker CD31). Our results demonstrate that active Cy-annexin can be used as a NIRF probe to image apoptosis from outside an intact living animal and may provide nonradioactive method of measuring the antiproliferative effects of cancer chemotherapeutic regimens.

Synthesis and in vitro evaluation of 123I-labelled human recombinant annexin V.

Annexin V was radiolabelled with iodine-123 in order to develop a SPECT-ligand for imaging atherosclerosis and apoptosis. Iodination by means of electrophylic substitution resulted in radiochemical yields up to 70% and specific activities of 7.4-92.5 MBq/microg protein. Binding experiments with blood platelets indicated that 123I-labelled annexin V remained its biological activity.