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Feasibility of ^sup 99m^Tc-Annexin V for Repetitive Detection of Apoptotic Tumor Response to Chemotherapy: An Experimental Study Using a Rat Tumor Model

Posted on: Tuesday, 2 March 2004, 06:00 CST

Annexin V (annexin A5), a human protein with a high affinity for phosphatidylserine, labeled with ^sup 99m^Tc can detect apoptosis in vivo. In the repetitive detection of apoptosis with ^sup 99m^Tc- annexin V, however, the specific binding of annexin V to phosphatidylserine might affect the subsequent detection of apoptosis with this compound. To determine whether there is interference with repetitive doses of annexin V, we evaluated the effects of previous administration of cold annexin V on accumulation of ^sup 99m^Tc-annexin V in tumors in an experimental tumor model. Methods: Rats bearing hepatoma received cyclophosphamide (150 mg/ kg, intraperitoneally) 11 d after the tumor inoculation. Cold annexin V (20 g/kg, intravenously) was administered 24 h before or after the cyclophosphamide treatment (n = 7/group). ^sup 99m^Tc- Annexin V was injected intravenously (radioactive dose, 5-23 MBq/ kg; mass dose, 20 g/kg), and radioactivity in tissues was determined 6 h later. Results: Accumulation of ^sup 99m^Tc-annexin V in tumors was not significantly affected by previous treatment with cold annexin V before or after chemotherapy. Conclusion: These results demonstrate the feasibility of ^sup 99m^Tc-annexin V imaging for repetitive detection of apoptosis, which is highly required in the clinical setting.

Key Words: ^sup 99m^Tc-annexin V; apoptosis; tumor; chemotherapy; rat

Apoptosis plays an important role in both normal physiology and many disease processes (1). One of the earliest events in apoptosis is the externalization of phosphatidylserine, a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer. Annexin V (annexin A5), a human protein with a high affinity for membrane-bound phosphatidylserine (2), can be labeled with fluorescent markers for in vitro detection of apoptotic cells (3) and with radioactive agents, such as ^sup 99m^Tc, to detect apoptosis in vivo (4).

Successful chemotherapy or radiotherapy of tumors induces apoptosis in neoplastic cells (5). Previous studies indicated that radiolabeled annexin V imaging can detect this apoptotic tumor response in vivo in experimental models (4,6,7) and in patients (8). In a clinical setting, annexin V injection and imaging have been performed both immediately before and after an initial treatment. The pre- and posttreatment injections may be only a few days apart. Because annexin V binds to about 3 phosphatidylserine molecules on the cell surface with a nanomolar affinity, it is possible that the first injection of annexin V may still be resident on the cell surface, tying up phosphatidylserine and thereby compromising the ability of the second dose to localize. To determine whether there is interference with repetitive doses of annexin V, we evaluated the effects of previous administration of cold annexin V on accumulation of radiolabeled annexin V in tumors in an experimental tumor model.

MATERIALS AND METHODS

All procedures involving animals were performed in accordance with the institutional guidelines of Hokkaido University and the current laws in Japan.

Male Wistar King Aptekman/Hok rats (supplied by the Experimental Animal Institute, Graduate School of Medicine, Hokkaido University) were inoculated with a suspension of KDH-8 rat hepatoma cells (1 10^sup 6^ cells per rat) into the left calf muscle and divided into 6 groups (n = 6-7/group; Table 1). Rats in groups A, B, D, and E received a single dose of cyclophosphamide (150 mg/kg, intraperitoneally) 11 d after tumor inoculation (day 11). Rats in group A received unlabeled (cold) recombinant human annexin V (annexin A5; Alexis Corp.; 20 g/kg, intravenously) 24 h before the cyclophosphamide treatment (day 10), and rats in group C received cold annexin V (20 g/kg, intravenously) 24 h after the cyclophosphamide treatment (day 12) under light ether anesthesia. Rats in groups B and D served as controls for groups A and C, respectively, and rats in groups C and F served as untreated controls.

TABLE 1

Treatment of Each Group of Rats Inoculated with Hepatoma Cells

Annexin V-117, a mutant molecule of annexin V engineered to contain a binding site for ^sup 99m^Tc without reducing the affinity for phosphatidylserine, was produced by expression in Escherichia coll. The protein was labeled with ^sup 99m^Tc to produce ^sup 99m^Tc-annexin V as previously described (9). The specific activity of ^sup 99m^Tc-annexin V was 0.25-1.15 MBq/g. With the tumor- bearing rats (body weight, 170-230 g) under light anesthesia, ^sup 99m^Tc-annexin V (radioactive dose, 5-23 MBq/kg; mass dose, 20 g/ kg) was injected intravenously. Groups A-C were injected on day 12, and groups C-E on day 13. Six hours after ^sup 99m^Tc-annexin V injection, the animals were sacrificed and the tumors, blood, and samples of normal tissues were collected. The tissue samples were weighed, and radioactivity was determined with a well-type scintillation counter (1480 Wizard 3''; Wallac Co., Ltd.). The accumulation of ^sup 99m^Tc-annexin V in the tissues was expressed as percentage injected dose per gram of tissue after normalization to the animal's weight ((%ID/g) kg).

TABLE 2

Biodistribution of ^sup 99m^Tc-Annexin V in Rats Inoculated with Hepatoma Cells

All values are shown as mean SD. Statistical analysis was performed using the unpaired Student t test to evaluate the significance of differences in values between the 2 groups. A 2- tailed value of P < 0.05 was considered significant.

RESULTS

Tissue distribution of ^sup 99m^Tc-annexin V is shown in Table 2. In untreated rats (groups C and F), uptake of ^sup 99m^Tc-annexin V was highest in the kidneys, followed in decreasing order by the spleen, liver, and bone marrow. Cyclophosphamide treatment (groups B and E) significantly increased the accumulation of ^sup 99m^Tc- annexin V in several tissues, including the tumor, thymus, spleen, and bone marrow.

Cold annexin V injection before cyclophosphamide treatment did not significantly affect the accumulation of ^sup 99m^Tc-annexin V in tumors (group A, 0.021 0.002 [%ID/g] kg), compared with that in the corresponding control group (group B, 0.022 0.003 [%ID/g] kg) (Fig. 1; Table 2). No significant change in the accumulation of ^sup 99m^Tc-annexin V in tumors was detected in the rats administered cold annexin V after cyclophosphamide treatment (group D, 0.026 0.002 [%TD/g] kg), compared with that in the corresponding control group (group E, 0.026 0.0024 [%ID/g] kg) (Fig. 1; Table 2). There were no significant differences in ^sup 99m^Tc-annexin V accumulation in other tissues, including the thymus, spleen, and bone marrow, between groups A and B and between groups D and E.

DISCUSSION

The results of these experiments demonstrate that accumulation of ^sup 99m^Tc-annexin V in tumors is not significantly affected by treatment with cold annexin V before or after chemotherapy. Consequently, it appears that the ability of radiolabeled ^sup 99m^Tc-annexin V to concentrate in tumors remains unchanged in the presence of marked increases in the circulating level of annexin V.

In the clinical evaluation of tumor response to therapy using ^sup 99m^Tc-annexin V, imaging is usually performed at baseline, to determine the degree of apoptosis in the untreated state, and after 1 or 2 treatments, to determine whether the therapy is efficacious. In the repetitive detection of apoptosis with ^sup 99m^Tc-annexin V, however, the specific binding of annexin V to phosphatidylserine might affect the subsequent detection of apoptosis with this compound. Our preliminary imaging study with ^sup 99m^Tc-annexin V suggested prolonged retention of annexin V on phosphatidylserine expressed by tumor cells. Groups A and B were compared to elucidate the feasibility of ^sup 99m^Tc-annexin V for imaging of tumors in subjects before initiation of chemotherapy and immediately after a single dose of chemotherapy. On the other hand, groups D and E were compared to elucidate the feasibility of ^sup 99m^Tc-annexin V for imaging of tumors in subjects repetitively after chemotherapy. The present results indicate that repetitive detection of apoptosis with ^sup 99m^Tc-annexin V is feasible for both imaging protocols. Our study also showed that injection of cold annexin V did not significantly affect accumulation of ^sup 99m^Tc-annexin V in tumors. In addition, there was no change in tracer concentration in the thymus, spleen, or bone marrow, where apoptosis appeared to be induced by the cyclophosphamide treatment. These results further support the feasibility of repetitive detection of apoptosis using ^sup 99m^Tc-annexin V.

FIGURE 1. Uptake of ^sup 99m^Tc-annexin V in tumors in rats inoculated with hepatoma cells. NS = not statistically significant.

A dose of 20 g/kg was selected as the treatment dose of unlabeled (cold) and labeled annexin V, considering the clinical doses used in ^sup 99m^Tc-annexin V imaging (10). Accumulation of ^sup 99m^Tc- annexin V in tumors and in other tissues was not affected by previous administration of cold annexin V. The amounts of cold annexin V given (20 g/kg) were probably far below the amount needed to saturate available binding sites on the tumor. It is reported that doses of 300-1,000 g/kg were neede\d to produce measurable anticoagulation in vivo in rats (77). The fact that anticoagulation requires that a significant fraction of the membrane surface be occupied by annexin V provides some indication of the amount of annexin V that would be needed to saturate available binding sites on tumors. Higher doses of annexin V might affect the accumulation of ^sup 99m^Tc-annexin V in tumors and other tissues, although it is unlikely that such higher doses of annexin V are used in clinical imaging with ^sup 99m^Tc-annexin V. It is also important to consider the phosphatidylserine-expression kinetics in relation to the amount of annexin V administered to rats, since phosphatidylserine expression is regarded as a dynamic process. The evidence with annexin V suggested that there are at least 2, peaks of phosphatidylserine expression, one occurring early, within hours of the initiation of chemotherapy, and another probably 24-72 h after the completion of treatment (12). Thus, in our study, it is expected that phosphatidylserine is significantly expressed throughout the time studied (24-48 h after treatment). The kinetics of phosphatidylserine expression may not be a responsible factor in the present results.

In the present study, we used annexin V-117 labeled with ^sup 99m^Tc. Several chelation sites have been proposed for radiolabeling annexin V with ^sup 99m^Tc (4,6,9,10). Annexin V-117, a mutant molecule of annexin V with a high affinity for membrane phosphatidylserine, was produced by expression in E. coli and could be used for imaging of cyclophosphamide-induced apoptosis in vivo (9). In our untreated rats (groups C and F), the concentrations of ^sup 99m^Tc-annexin V-117 in the blood and tissues were relatively lower than those of ^sup 99m^Tc-hydrazinonicotinamide (HYNIC)- annexin V (7) and ^sup 99m^Tc-ethylenedicysteine (EC)-annexin V (6), although the biodistribution pattern of ^sup 99m^Tc-annexin V-117 was similar to those of ^sup 99m^Tc-HYNIC-annexin V and ^sup 99m^Tc- EC-annexin V. Clearance of ^sup 99m^Tc-annexin V-117 may be more rapid than that of ^sup 99m^Tc-HYNIC-annexin V (9). On the other hand, cyclophosphamide (groups B and E) treatment significantly increased the accumulation of ^sup 99m^Tc-annexin V in the tumor, thymus, spleen, and bone marrow, further confirming the ability of ^sup 99m^Tc-annexin V-117 to detect cyclophosphamide-induced apoptosis (9).

CONCLUSION

Our results demonstrate the feasibility of ^sup 99m^Tc-annexin V imaging for repetitive detection of apoptosis, which is highly required in clinical evaluation of tumor response to therapy.

ACKNOWLEDGMENTS

The authors thank Dr. Futoshi Okada of the Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, for generously providing tumor cells. The authors are grateful to Professors Shinzo Nishi, Kazuo Miyasaka, and Toshiyuki Ohnishi of the Central Institute of Isotope Science, Hokkaido University, for supporting this work. The authors thank Drs. Koutaro Suzuki, Hidenori Katsuura, Hidehiko Omote, and Hiroshi Arai for assistance. This work was supported in part by a grant from the Japanese Foundation for Multidisciplinary Treatment of Cancer and by a grant from the Association for Nuclear Technology in Medicine.

J Nucl Med 2004; 45:309-312

REFERENCES

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6. Yang DJ, Azhdarinia A, Wu P, et al. In vivo and in vitro measurement of apoptosis in breast cancer cells using ^sup 99m^Tc- EC-annexin V. Cancer Blather Radiopharm. 2001;16:73-83.

7. Mochizuki T, Kuge Y, Zhao S, et al. Detection of apoptotic tumor response in vivo after a single dose of chemotherapy with ^sup 99m^Tc-annexin V. J Nucl Med. 2003;44:92-97.

8. Belhocine T, Steinmetz N, Hustinx R, et al. Increased uptake of the apoptosisimaging agent ^sup 99m^Tc recombinant human annexin V in human tumors after one course of chemotherapy as a predictor of tumor response and patient prognosis. Clin Cancer Res. 2002;8:2766- 2774.

9. Tait JF, Brown DS, Gibson DF, Blankenberg FG, Strauss HW. Development and characterization of annexin V mutants with endogenous chelation sites for ^sup 99m^Tc. Bioconjug Chem. 2000;11:918-925.

10. Kemerink GJ, Boersma HH, Thimister PW, et al. Biodistribution and dosimetry of ^sup 99m^Tc-BTAP-annexin-V in humans. Eur J Nucl Med. 2001;28:1373-1378.

11. Romisch J, Seiffge D, Reiner G, Paques EP, Heimburger N. In- vivo antithrombotic potency of placenta protein 4 (annexin V). Thromb Res. 1991;61:93-104.

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Yuji Kuge, PhD1; Masayuki Sato, BS1-3; Songji Zhao, MD2; Toshiki Takei, MS2; Kunihiro Nakada, MD2; Koh-ich Seki, PhD1,3; H. William Strauss, MD4; Francis G. Blankenberg, MD5; Jonathan F. Tait, PhD6; and Nagara Tamaki, MD2

1 Department of Tracer Kinetics, Graduate School of Medicine, Hokkaido University, Sapporo, Japan; 2 Department of Nuclear Medicine, Graduate School of Medicine, Hokkaido University, Sapporo, Japan; 3 Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Japan; 4 Department of Nuclear Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York; 5 Pediatric Radiology, Stanford University School of Medicine, Palo Alto, California; and 6 Department of Laboratory Medicine, University of Washington, Seattle, Washington

Received Jul. 16, 2003; revision accepted Oct. 23, 2003.

For correspondence or reprints contact: Nagara Tamaki, MD, Department of Nuclear Medicine, Graduate School of Medicine, Hokkaido University, Kita15 Nishi7, Kita-ku, Sapporo 060-8638, Japan.

E-mail: natamaki@med.hokudai.ac.jp

Copyright Society of Nuclear Medicine Feb 2004

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