High-Dose Treatment with ¹⁸⁶Re-HEDP or ¹⁵³Sm-EDTMP Combined with Amifostine in a Rabbit Model

MATERIALS AND METHODS

Scintigraphic Skeletal Uptake Measurement

Whole-body images from anterior and posterior views were acquired simultaneously at 3 min, 3 h, and 24 h after a bolus injection of the respective radiopharmaceutical. The acquisition time was 1 min during the blood-pool phase and 10 min for the later images. Images were obtained under the same conditions for each rabbit, using a double-head gamma camera (Bodyscan; Siemens, Erlangen, Germany) equipped with high-resolution low-energy collimators. The energy window used was 137 keV ± 15% for ¹⁸⁶Re and 103 keV ± 15% for ¹⁵³Sm.

For each image, activity in the whole body, urinary bladder, and soft tissue of the flank was measured by region-of-interest (ROI) technique using both anterior and posterior views. Additionally, a rectangular ROI adjacent to the body was used to measure the bremsstrahlung of the β-particles of the radionuclide. An example of ROI positions is shown in Figure 1. From these data, the geometric mean for each ROI was calculated after correction for different acquisition times, radioactive decay, and bremsstrahlung. The initial whole-body activity of each rabbit was used as the reference value for further calculations of activity data, which are all given as a percentage of the whole-body activity. According to a simplified 3-compartment model, soft-tissue activity compared with initial whole-body activity equals activity in muscles compared with initial muscle activity at 3 min after injection. Urinary excretion was determined by the difference in whole-body activity between the reference image at 3 min and the image of interest plus urinary bladder activity. From these data, bone uptake at 24 h after injection was calculated as 100% of initial whole-body activity minus both urinary excretion and soft-tissue retention, as has been described in detail elsewhere (8).

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FIGURE 1.

ROI positions on whole-body scintigram of rabbit from posterior view 3 h after injection of 1,000 MBq ¹⁵³Sm-EDTMP.

RESULTS

DISCUSSION

The cytoprotective potential of thiol-containing compounds against radiation damage has been known for many years (19). Amifostine was selected from >4,000 of such compounds as the agent with the best clinical profile. In animal studies, amifostine protected normal tissues such as bone marrow, salivary glands, skin, and oral mucosa from the toxic effects of lethal doses of ionizing radiation, with no impact on malignant transformed cells (20,21). The mechanism of this selective radioprotection of amifostine has been shown to be related to its preferential rapid uptake into normal tissues and no or only slow uptake into tumor cells. This selectivity results mainly from differences in the presence of alkaline phosphatase at capillary endothelial cells and the pH of normal tissues compared with the pH of tumor tissue. These conditions favor the dephosphorylation of amifostine by the membrane-bound alkaline phosphatase to its active metabolite, the free thiol WR-1065 (Fig. 2), which immediately enters normal tissues (22). Once inside the cell, WR-1065 acts as a scavenger of oxygen free radicals by detoxifying these radicals before they can damage the DNA or RNA (23). After intravenous injection, amifostine is rapidly cleared from the plasma, with half-lives of <1 min and <8 min (23). In contrast to the brief systemic half-life, retention of amifostine and its metabolites in normal tissue is prolonged. During the first 30 min after administration, uptake into normal tissues such as salivary glands, kidneys, liver, and bone marrow has been shown to be >100-fold higher than uptake into tumor tissue, with intracellular retention up to 24 h after injection (24). As a result of these experimental findings, numerous clinical studies have been performed and have shown amifostine to be a potent cyto- and myeloprotective agent in patients undergoing chemotherapy or radiation therapy (16,25,26). Consequently, amifostine has gained drug approval in the United States and in Europe (13,27).

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FIGURE 2.

Chemical structure of amifostine and its active metabolite, the free thiol WR-1065.

In this rabbit animal model, amifostine was found to have a significant cytoprotective effect on platelets both in rabbits treated with ¹⁸⁶Re-HEDP and in rabbits treated with ¹⁵³Sm-EDTMP, and this finding is in line with the well-documented myeloprotective effects in radiation therapy (10,16,20,25,27). Of special interest is the finding that a single administration of amifostine 10–15 min before the radionuclide treatment could clearly reduce cytotoxic effects on platelets despite the somewhat long radiation exposure of bone marrow cells caused by the radiopharmaceuticals affixed to the bone matrix, with physical half-lives of 90.6 and 46.7 h for ¹⁸⁶Re-HEDP and ¹⁵³Sm-EDTMP, respectively. This observation, however, is in line with reports that single amifostine administrations have a cytoprotective effect on salivary glands during high-dose radioiodine treatment (12,28).

Completely different findings were obtained for leukocytes: Whereas for ¹⁸⁶Re-HEDP no significant differences could be found between amifostine-treated animals and control animals, the rabbits treated with SmA showed a significantly greater decrease in leukocytes than did animals treated with ¹⁵³Sm-EDTMP only. On the other hand, treatment with amifostine alone, without any radionuclide administration, showed no changes in leukocyte counts. These data indicate increased cytotoxic effects on leukocytes for the combination of ¹⁵³Sm-EDTMP and amifostine compared with the other subgroups in this study. Klutmann et al. (29) found similar leukotoxic effects in 9 rabbits after administration of 200 mg amifostine per kilogram of body weight in combination with 400 MBq ¹⁸⁶Re-HEDP: in amifostine-treated animals, leukocyte counts decreased from 5.7 ± 2.1/nL to 3.4 ± 0.9/nL, corresponding to a mean decrease of approximately 40%, whereas the control animals showed somewhat constant values. The difference between the 2 groups was reported to be highly significant, with P < 0.002. The authors therefore concluded that a selective cytotoxic effect on leukocytes has to be discussed for amifostine in combination with ¹⁸⁶Re-HEDP. Although we could not document a significant effect for amifostine in combination with ¹⁸⁶Re-HEDP, we did show similar results for amifostine in combination with ¹⁵³Sm-EDTMP. The myelotoxicity of high-dose amifostine is known, and lymphopenia and a corresponding decrease in total white blood cells caused by amifostine in both rats and dogs has been reported for the dosage administered in this study, that is, 200 mg/kg of body weight (30). For platelets, only a moderate decrease has been observed (30). Nevertheless, in this study we found neither leukotoxic nor platelet-toxic effects in rabbits when we administered amifostine alone in a dosage of 200 mg/kg of body weight. The same dosage had been used successfully in rabbits undergoing high-dose radioiodine therapy in previous studies (17,28). Thus, the myelotoxic effects of high-dose amifostine, although not detectable through measurement of platelet and leukocyte counts in blood, seemed to potentiate the myelotoxic effects of β-emitter–labeled bisphosphonates. However, in contrast to Klutmann et al., we found such an additive leukotoxic effect only for ¹⁵³Sm-EDTMP, not for ¹⁸⁶Re-HEDP, possibly because of a statistical problem caused by the small number of animals. Summarizing all these observations, we conclude that in rabbits, a dosage of 200 mg amifostine per kilogram of body weight is close to but below the threshold of measurable leukotoxic effects, which may become detectable during combined treatment with amifostine and myelotoxic drugs such as ¹⁵³Sm-EDTMP or ¹⁸⁶Re-HEDP. Therefore, for further studies in rabbits, the dosage of amifostine should be reduced if white blood cell counts are part of the study design. To our knowledge, however, there are no other reports on the use of amifostine in radionuclide bone therapy that might support our conclusions or explain the underlying mechanisms in more detail.

Our study used a recently introduced quantification method based on whole-body bone scanning to measure skeletal uptake, soft-tissue retention, and urinary excretion of radiolabeled bisphosphonates (8,31) and, thus, document drug interactions with amifostine. This step seemed necessary because of the known possibility that amifostine may alter renal drug clearance (14) and bone metabolism (15,16). For ¹⁸⁶Re-HEDP, bone uptake at 24 h after injection was significantly lower in animals treated with amifostine than in control animals. The remainder soft-tissue activity at 24 h after injection was the same in amifostine-treated rabbits as in control animals, suggesting that the higher amount of nonskeletal bound activity was completely excreted by the kidneys. For ¹⁵³Sm-EDTMP also, significantly reduced bone uptake was found in amifostine-treated animals. However, in these rabbits the main observation was a shift in activity toward the soft-tissue compartment, whereas urinary excretion was nearly the same for both amifostine-treated animals and untreated animals. Because amifostine can alter bone metabolism through both parathyroid hormone–independent and parathyroid hormone–dependent mechanisms, resulting in hypocalcemia and an inhibition of osteoclastic bone resorption (15,16,32), the reduced bone uptake of ¹⁸⁶Re-HEDP and ¹⁵³Sm-EDTMP after administration of amifostine might be explained by subsequent reduction of osteoblastic activity in response to transient osteoclastic inhibition. However, this hypothesis would not explain the increased remainder soft-tissue activity of ¹⁵³Sm-EDTMP, because excretion of nonskeletal-bound ¹⁵³Sm-EDTMP by the kidneys is expected, as shown in patients with bone metastases: the lower the number and extent of bone metastases, the lower was bone uptake and the higher was urinary excretion, whereas constant soft-tissue retention values proved to be independent of the number of metastases (8,33). However, because of the chemical structure of amifostine, it is a potentially chelating agent that presents thiol moieties containing electron-rich coordinating atoms for rhenium or samarium. Thus, amifostine might be able to displace bisphosphonates from their rhenium– and samarium–bisphosphonate complexes, building up new rhenium–amifostine or samarium–amifostine complexes. This hypothesis would explain both the reduced bone uptake of ¹⁵³Sm and the reduced bone uptake of ¹⁸⁶Re, because rhenium– or samarium–amifostine complexes are not expected to have bone affinity, and different biodistribution pattern are caused by the different biochemical characteristics of amifostine–samarium and amifostine–rhenium complexes. Because the plasma half-life of amifostine is only about 8 min, administration of the radiolabeled bisphosphonates as late as 30 min after the infusion of amifostine might be helpful to ensure almost complete intracellular uptake of amifostine and its metabolites and to reduce the possible formation of rhenium– or samarium–amifostine complexes. Furthermore, amifostine cannot, at this point in its investigation, be recommended for general use in patients undergoing radionuclide therapy of painful bony metastases, because the lower uptake in the skeleton may reduce the efficacy of this treatment modality.

CONCLUSION

In rabbits undergoing high-dose treatment with 1,000 MBq of either ¹⁸⁶Re-HEDP or ¹⁵³Sm-EDTMP, we found amifostine to have a highly significant cytoprotective effect on platelets. However, no leukoprotection was found, and even leukotoxicity was shown for the combination of amifostine and ¹⁵³Sm-EDTMP. This effect most probably relies on the intrinsic myelotoxicity of high-dose amifostine, which seemed to potentiate radiopharmaceutical-induced myelodepression. These findings suggest the use of lower doses of amifostine in rabbits. Furthermore, significantly reduced bone uptake of ¹⁸⁶Re-HEDP and ¹⁵³Sm-EDTMP was found in amifostine-treated animals. Amifostine, because of its potential to form complexes, may be able to displace bisphosphonates from the rhenium– and samarium–bisphosphonate complexes, resulting in reduced bone uptake and an altered biodistribution pattern.