Abstract
Annexin V (annexin A5), a human protein with a high affinity for phosphatidylserine, labeled with 99mTc can detect apoptosis in vivo. In the repetitive detection of apoptosis with 99mTc-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 99mTc-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 μg/kg, intravenously) was administered 24 h before or after the cyclophosphamide treatment (n = 7/group). 99mTc-Annexin V was injected intravenously (radioactive dose, 5–23 MBq/kg; mass dose, 20 μg/kg), and radioactivity in tissues was determined 6 h later. Results: Accumulation of 99mTc-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 99mTc-annexin V imaging for repetitive detection of apoptosis, which is highly required in the clinical setting.
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 99mTc, 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 × 106 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 μg/kg, intravenously) 24 h before the cyclophosphamide treatment (day 10), and rats in group C received cold annexin V (20 μg/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.
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 99mTc without reducing the affinity for phosphatidylserine, was produced by expression in Escherichia coli. The protein was labeled with 99mTc to produce 99mTc-annexin V as previously described (9). The specific activity of 99mTc-annexin V was 0.25–1.15 MBq/μg. With the tumor-bearing rats (body weight, 170–230 g) under light anesthesia, 99mTc-annexin V (radioactive dose, 5–23 MBq/kg; mass dose, 20 μg/kg) was injected intravenously. Groups A–C were injected on day 12, and groups C–E on day 13. Six hours after 99mTc-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 99mTc-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).
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 99mTc-annexin V is shown in Table 2. In untreated rats (groups C and F), uptake of 99mTc-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 99mTc-annexin V in several tissues, including the tumor, thymus, spleen, and bone marrow.
Biodistribution of 99mTc-Annexin V in Rats Inoculated with Hepatoma Cells
Cold annexin V injection before cyclophosphamide treatment did not significantly affect the accumulation of 99mTc-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 99mTc-annexin V in tumors was detected in the rats administered cold annexin V after cyclophosphamide treatment (group D, 0.026 ± 0.002 [%ID/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 99mTc-annexin V accumulation in other tissues, including the thymus, spleen, and bone marrow, between groups A and B and between groups D and E.
Uptake of 99mTc-annexin V in tumors in rats inoculated with hepatoma cells. NS = not statistically significant.
DISCUSSION
The results of these experiments demonstrate that accumulation of 99mTc-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 99mTc-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 99mTc-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 99mTc-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 99mTc-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 99mTc-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 99mTc-annexin V for imaging of tumors in subjects repetitively after chemotherapy. The present results indicate that repetitive detection of apoptosis with 99mTc-annexin V is feasible for both imaging protocols. Our study also showed that injection of cold annexin V did not significantly affect accumulation of 99mTc-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 99mTc-annexin V.
A dose of 20 μg/kg was selected as the treatment dose of unlabeled (cold) and labeled annexin V, considering the clinical doses used in 99mTc-annexin V imaging (10). Accumulation of 99mTc-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 μg/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 μg/kg were needed to produce measurable anticoagulation in vivo in rats (11). 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 99mTc-annexin V in tumors and other tissues, although it is unlikely that such higher doses of annexin V are used in clinical imaging with 99mTc-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 99mTc. Several chelation sites have been proposed for radiolabeling annexin V with 99mTc (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 99mTc-annexin V-117 in the blood and tissues were relatively lower than those of 99mTc-hydrazinonicotinamide (HYNIC)-annexin V (7) and 99mTc-ethylenedicysteine (EC)-annexin V (6), although the biodistribution pattern of 99mTc-annexin V-117 was similar to those of 99mTc-HYNIC-annexin V and 99mTc-EC-annexin V. Clearance of 99mTc-annexin V-117 may be more rapid than that of 99mTc-HYNIC-annexin V (9). On the other hand, cyclophosphamide (groups B and E) treatment significantly increased the accumulation of 99mTc-annexin V in the tumor, thymus, spleen, and bone marrow, further confirming the ability of 99mTc-annexin V-117 to detect cyclophosphamide-induced apoptosis (9).
CONCLUSION
Our results demonstrate the feasibility of 99mTc-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.
Footnotes
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{at}med.hokudai.ac.jp
