Background

[PubMed]

Apoptosis, or programmed cell death, plays an important role in the pathophysiology of many diseases, such as cancer, neurodegenerative disorders, vascular disorders, and chronic hepatitis, as well as in the biology of normal cells, such as epithelial cells and immune cells (1). Apoptosis is gene regulated (2) and involves the proteolysis of intracellular components by activation of a series of proteolytic enzymes, called caspases, and changes of plasma membrane structure by translocase, floppase, and scramblase (3-5). As a result, there is rapid redistribution of phosphatidylserine (PS) from the inner membrane leaflet to the outer membrane leaflet of apoptotic cells, exposing the anionic head group of PS. On the other hand, PS is also accessible for annexin V binding in necrotic cells because of disruption of the plasma membrane.

Annexin V is a 36-kDa endogenous human protein that is produced by the epithelial cells of many tissues, such as placenta, umbilical vessels, liver, spleen, kidney, heart, uterus, and skeletal muscle, as well as by erythrocytes, leukocytes, endothelial cells, and platelets (6). Annexin V binds to PS with high affinity (K_d = 7 nM) with 8 annexin V molecules per PS molecule (3, 7, 8). Apoptosis can be induced by chemicals, radiation, cytokines, hormones, and various pathologic conditions (5); therefore, the ability to monitor apoptosis in association with disease progression or regression should provide important information for clinical applications. Annexin V has been radiolabeled with ¹²³I, ¹²⁵I, and ^99mTc for single-photon emission computed tomography (SPECT) imaging (9, 10), and hydrazinonicotinamide (HYNIC) has been conjugated to human recombinant annexin V to form HYNIC-annexin V (11). ^99mTc-HYNIC-annexin V is being developed as an imaging agent to study apoptotic and necrotic cells in humans.

Synthesis

[PubMed]

In the method described by Blankenberg et al. (11), HYNIC was conjugated to human recombinant annexin V to form HYNIC-annexin V by use of succinimidyl 6-HYNIC. HYNIC-annexin V was purified by dialysis with a yield of >94%, and the ratio of HYNIC to annexin V was 0.9:1. A mixture of HYNIC-annexin V (100 μg) and [^99mTc]pertechnetate (148-740 MBq (4-20 mCi)) was reacted in a solution containing tricine/SnCl₂ at room temperature for 60 min. ^99mTc-HYNIC-annexin V was purified by column chromatography to give a yield of 56%, with a radiochemical purity >97% and a specific activity >1.95 MBq/μg of protein (50 μCi/μg of protein). Verbeke et al. (12) reported optimized preparation of ^99mTc-HYNIC-annexin V with 95% labeling yields by reaction of 60-90 μg of HYNIC-annexin V, 0.37-1.11 GBq (10-30 mCi) of ^99mTcO₄^-, 10-20 μg of SnCl₂·2 H₂O, and 1.5 mg of tricine.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

^99mTc-HYNIC-annexin V has been shown to selectively bind to apoptotic Jurkat T cells (induced by serum deprivation, doxorubicin, and anti-FAS antibody) and thymocytes (induced by dexamethasone) (13). The increases in ^99mTc-HYNIC-annexin V radioactivity were correlated with fluorescein isothiocyanate (FITC)-annexin V fluorescence intensities as measured by flow cytometry. The IC₅₀ values of annexin V, HYNIC-annexin V, and decayed ^99mTc-HYNIC-annexin V for inhibition of FITC-annexin V binding to PS were 8, 10.5, and 12.3 nM, respectively. There thus was only a minimal loss of binding affinity by the modified annexin V.

Animal Studies

Human Studies

[PubMed]

Various SPECT studies using ^99mTc-HYNIC-annexin V have been performed in patients with head-and-neck carcinoma and showed that absolute uptake values in the primary tumors correlate well with the number of apoptotic cells as measured by the TUNEL assay or other histologic analyses (16-19). However, SPECT failed to identify most of the lymph node lesions detected by computed tomography, mainly because the lymph nodes are smaller in size than the primary tumors and contain mostly focal metastases.

In a study of 33 patients with lymphoma (n = 26) and other tumors, Kartachova et al. (20) concluded that partial or complete tumor response to therapy was associated with a marked increase in ^99mTc-HYNIC-annexin V in the tumors. In contrast, patients with stable or progressive disease showed low ^99mTc-HYNIC-annexin V accumulation before treatment and no increase after treatment. Therefore, ^99mTc-HYNIC-annexin V SPECT may be used to identify patients who would respond to therapies and to assess the effectiveness of therapies.

Kemerink et al. (21) reported on SPECT studies in 6 normal volunteers after injection of 250 MBq (6.8 mCi) of ^99mTc-HYNIC-annexin V. The kidneys received the highest dose of radioactivity (49.7% ID) at 3 h after injection, followed by the liver (13.1% ID), the red marrow (9.2% ID), and the spleen (4.6% ID). More than 90% of the blood activity was cleared rapidly with a half-life of 24 min. The radioactivity was excreted almost exclusively through the urine (22.5% ID at 24 h), and hardly any radioactivity was seen in the feces. Absorbed doses were 196 μGy/MBq (0.73 rad/mCi) for the kidneys, 41 μGy/MBq (0.15 rad/mCi) for the spleen, 16.9 μGy/MBq (0.063 rad/mCi) for the liver, and 8.4 μGy/MBq (0.031 rad/mCi) for the red marrow. The effective dose was 11.0 ± 0.8 μSv/MBq (40.7 ± 3.0 mrem/mCi).

Human Studies

HL-47151, HL-61717

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