Radiol Oncol 2000; 34(4): 337-47.
Bone marrow protection by amifostine in Re-186-HEDP treatment: first results obtained in a rabbit animal model
Susanne Klutmann, Karl H. Bohuslavizki, Sabine Kroger, Lars Jenicke, Ralph Buchert, Janos Mester, Malte Clausen
Department of Nuclear Medicine, University Hospital Eppendorf, Hamburg, Germany
Background. In the last few years various reports dealt with radioprotective effects ofamifostine (Ethyol®, USB, Philadelphia, PA). Since amifostine is markedly accumulated in bone marrow it looks worthwhile to study the radioprotective effects ofamifostine on bone marrow in patients treated with Re-186-HEDP. As a first step animal shidies were performed using New Zealand Wute rabbits.
Materials and methods. A tatal of 18 rabbits received 300 MBq Tc-99m-HDP far whole-body scintigraphy. Thereafter, 9 animals were h-eated with 200 mg/kg body weight amifostine, and 9 rabbits served as controls receiving physiological saline solution. Then, these 18 rabbits received 400 MBq Re-186-HEDP i.v. Two rabbits served as untreated confrols and received neither Tc-99m-HDP nor Re-186-HEDP. Blood samples were taken at the beginning and in huo-week-intervals far the duration ofhuo months in all 20 animals. Laboratory findings were determined far white and red blood cells, far platelet count and far hemoglobin. Two months after therapy all animals were sacrificed, and both femora were removed surgically far histopathological examination ofbone marrow. Results. In contiols as well as in amifostine-treated animals, the red blood cell count and the hemoglobin were almost constant at all times ofobservation. In 9 control rabbits the mean platelet count was 265.22±127.41 Mrd/l prior to Re-186-HEDP-therapy. Two weeks after therapy the mean platelet count was reduced to 211.22±52.8 Mrd/l. Prior to Re-186-HEDP-therapy the mean platelet count of amifostine-treated rabbits was not significantly different (p>0.05) from control rabbits. Two weeks after injection of the radionuclide the mean platelet count decreased to 180.67±37.43 Mrd/l in the amifostine group. There was no significant difference behueen rabbits of the control goup and amifostine-h-eated animals (p>0.05). Tims, amifostine was not able to prevent a transient thrombocytopenia. Tivo weeks after therapy only a slight decrease ofthe white blood cell count was recognized in controls. In contrast, amifostine-treated rabbits showed a considerable decrease of the white blood cell count huo weeks after therapy with a mean value of3.39±0.91 Mrd/l. This differencewas statistically significant (p<0.0002). Discussion. The insufficient radioprotection ofamifostine concerning the platelet count was probably due to phar-macocinetics of amifostine. Since the generation offree radicals in the bone marrow caused by Re-186-HEDP lasts several times longer than the radioprotective effect ofamifostine given as one single dose prior to the application of Re-186-HEDP. However, the observed decline of white blood cells due to amifostine application is yet unknown. Conclusion. In order to use amifostine as a suitable agent far radioprotection multiple doses ofamifostine might be applied, i.e. huo shots per day far the duration of3 to 4 days after the application of Re-186-HEDP. The observed leucopenia should be studied infrirther animal studies.
Key words: Radiation-protection agents; bone marrow; amifostine; erythrocyte count; platelet count, hemoglobin; Re-186-HEDP; bone metastases
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Introduction
Patients with prostate cancer will develop bone metastases in nearly 70 %,1 which may result in pathological fractures as well as in severe bone pain.2-3 The application of p--emitting osteotropic radionuclides, i.e. Sr-89-chloride, Sm-153-EDTMP, and Re-186-HEDP is one therapeutic approach in palliative treatment of painful, multilocular bone metas-tases.4-10 Since bone metastases show a preferential uptake of bone-seeking radionuclides, i.e. Re-186-HEDP, this therapy is rather specific, while non-affected tissue is spared from the effects of the PMrradiation.11 In 75% of the patients pain relief occurs within one to two weeks after the application of Re-186-HEDP and lasts for about 1-6 months.3-10-12 However, Re-186-HEDP delivers a substantial dose to the bone marrow, thus, a potential bone marrow suppression is still the most important dose-limiting factor.3-10-13 This radiobiological side-effect is mainly confined to a decline of the platelet count, called thrombocytopenia. 14-15 Therefore, one inclusion criteria for patients undergoing rhenium-therapy is a platelet count of at least 150 Mrd/1 prior to therapy, and in clinical routine a blood count is performed directly prior to the application of the bone-seeking radionu-clide.16 However, some patients do not fulfill this inclusion criteria at the day of admission and therefore, rhenium-therapy can not be performed as planned. Thus, the reduction of radiobiological side-effects is of major interest in patients treated for painful bone metastases. On the other hand, therapy with stan-
Received 8 May 2000 Accepted 23 May 2000
Correspondence to: Karl H. Bohuslavizki, MD, PhD, Department of Nuclear Medicine, University Hospital Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany. Phone: +49 40 42803 4047; Fax: +49 40 42803 6775; E-mail: bohu@uke.uni-ham-burg.de
dard activities of 1200 MBq Re-186-HEDP is still a palliative treatment which does not influence the prognosis of the underlying disease at all. However, a reduction of side-effects of Re-186-HEDP might improve tolera-bility of rhenium-therapy. Thus, in the future, Re-186-HEDP might be applied not only as palliative but also as curative means in patients with multilocular bone metastases.
In the last few years various reports dealt with radioprotective effects of the phosphory-lated aminothiol amifostine (Ethyol®, USB, Philadelphia, PA).1729 Since amifostine markedly accumulates in salivary glands the application of amifostine has been successfully used both in external radiotherapy in patients with head and neck tumors30 and high-dose radioiodine therapy in patients with differentiated thyroid cancer.26-29-31-34 Since amifostine is also markedly accumulated in bone marrow25-35-38 it looks worthwhile to study the radioprotection of bone marrow in order to avoid/reduce bone marrow toxicity in patients treated with Re-186-HEDP, and thus, to increase the tolerability of rhenium-therapy. As a first step animal studies were performed.
Material and method
Animal studies
In order to investigate the radioprotective effects of amifostine an animal model was established using New Zealand White rabbits. A total of 20 animals aged between two and three months and weighing 2.1 to 3.1 kg with a mean weight of 2.86±0.11 kg were used. As a first step, 18 rabbits received 300 MBq Tc-99m-HDP as intravenous injection by a vein flow placed in the ear. For bone scintig-raphy 2 hrs p.i., all rabbits were positioned in prone position directly onto a low-energy high-resolution collimator of a large field of view gamma camera (Diacam, Siemens, Erlangen, Germany). For anesthesia, 50 mg/kg Ketanest® (Ketaminhydrochlorid,
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Parke-Davis, Berlin, Germany) and 4 mg/kg Rompun® (Xylazinhydrochlorid, BayerVital, Leverkusen, Germany) were administered in a mixed syringe as intramuscular injection in the upper leg directly prior to image acquisition. Images were acquired over 20 min each and stored digitally in a 256x256 matrix. Directly after bone scintigraphy 9 animals were treated with 200 mg/kg Amifostine® (Amifostine, USB, Philadelphia,PA) as slow intravenous infusion over 5 min. Nine rabbits served as controls receiving a corresponding volume of physiological saline solution. Then, these 18 rabbits received 400 MBq Re-186-HEDP dissolved in less than 0.6 ml. Whole-body scintigraphy was performed at 24 hrs or 48 hrs p.i. in order to evaluate the distribution of the Re-186-HEDP applied. Again, 50 mg/kg Ketanest® (Ketaminhydrochlorid, ParkeDavis, Berlin, Germany) and 4 mg/kg Rompun® (Xylazinhydrochlorid, BayerVital, Leverkusen, Germany) were administered for anesthesia in a mixed syringe as intramuscular injection in the upper leg. Rabbits were positioned as described above directly onto a low-energy high-resolution collimator of a large field of view gamma camera (Diacam, Siemens, Erlangen, Germany). Images were acquired over 20 min each and were stored digitally in a 256x256 matrix.
Two rabbits served as untreated controls and received neither Tc-99m-HDP nor Re-186-HEDP. Blood samples were taken at the beginning and in two-week-intervals for the duration of two months in all 20 animals. Laboratory findings were determined for white and red blood cells, for platelet count and for hemoglobin. Two months after therapy all animals were sacrificed, and both femora were removed surgically for histopatholog-ical examination of bone marrow.
Histopathological examination
The bone marrow was stained in conventional manner with Hematoxilin/Eosin, Giemsa,
and Berlin blue. An experienced pathologist performed histopathological examinations in a blind manner. Evaluation criteria were the cellularity of the bone marrow, the number and differentiation of the erthropoesis, gran-ulopoesis and thrombocytopoesis, the evaluation of bone marrow vessels, and the quantity of bone marrow's iron content.
Evaluation
Blood samples were directly transferred to the central laboratory and determined the same day in order to avoid an artificial decline of platelets. Hemoglobin was measured in g/dl. White blood cell count, red blood cell count, and platelet count were measured in Mrd/1, respectively.
Whole-body scintigramms acquired after injection of Tc-99m-HDP or Re-186-HEDP were.evaluated by visual inspection.
Statistics
Data are given as mean ± one standard deviation. Two-tailed Student's t-test for paired data was used to evaluate statistical differences between animal subsets. P<0.05 was considered to be significant.39
Results
Animal model
The animal model chosen was easy to handle. Due to the size of the ear veins both the injection of the radiopharmaceuticals and the drawing of blood samples were easy to perform. Moreover, the chosen anesthesia consisting of a mixture of Ketanest® and Rompun® was safe and allowed image acquisition without any movement artifacts.
Red blood cells
Details of the red blood cell count for all groups of rabbits are given in Table l. In con-
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trols, the red blood cell count was almost constant at all times of observation. Prior to therapy a mean red blood cell count of 6.07(0.84 Mrd/1 was measured in this group. At two, four, six and eight weeks after therapy the red blood cell count was 6.07±0.39, 6.61±0.42, 6.59±0.63 and 6.18±0.58 Mrd/1, respectively. Corresponding results were observed for animals of the amifostine-treated group. Prior to therapy a mean red blood cell count of 6.29±0.51 Mrd/1 was measured in these animals. Two, four, six and eight weeks after therapy the mean red blood cell count amounted to 6.25±0.46, 6.12±0.41, 5.82±0.34 and 6.19±0.4 Mrd/1, respectively. Thus, ami-fostine-treated animals showed almost unchanged red blood cell counts during Re-186-HEDP-therapy.
Two completely untreated animals showed an almost constant red blood cell count during all times of observation. In these two animals a mean red blood cell count of 6.0±0.13 Mrd/1 was measured at first time of observation and amounted to 6.25±0.13, 6.7±0.04, 6.07±0.86 and 5.98±0.23 in two-week-intervals up to eight weeks after beginning of the study.
Hemoglobin
Details of values for hemoglobin are given in Table l. In animals of the control group mean hemoglobin was 12.53±1.14 g/dl prior to therapy and amounted to 12.84±0.9, 14.32±0.75, 14.09±1.01 and 13.71±0.72 g/dl two, four, six and eight weeks after therapy, respectively. Thus, animals pretreated with physiological saline solution only showed near unchanged hemoglobin at all times of observation. Laboratory findings of hemoglobin determined prior and in two-week intervals showed corresponding results for amifostine-treated animals. In these rabbits mean hemoglobin amounted to 13.41±0.96 g/dl prior to therapy and was 13.23±0.94, 13.64±0.52, 12.74±0.54 and 13.87±0.86 g/dl at two, four, six and eight weeks after therapy, respectively.
Moreover, unchanged values for hemoglobin were observed in both untreated animals with hemoglobin amounting to 12.75±0.49 g/dl at the beginning and amounting to 13.4±0.99,14.6±0.99, 13.25±0.92 and 13.2±0.57 g/dl at two-week-intervals.
Table 1. Mean values and standard deviation of the red and white blood celi count, the platelet count and the hemoglobin prior to and 2, 4, 6 and 8 weeks after Re-186-HEDP treatment in the control group, the amifostine-treated group and in the untreated rabbits
Time with respect to Re-186-HEDP i.v
Before	2 weeks after	4 weeks after	6 weeks after	8 weeks after
Controls	6.07±0.53	6.07±0.39	6.61±0.42	6.59±0.63	6.18±0.6
RBC [Mrd/1] Amifostine	6.29±0.51	6.25±0.46	6.12±0.41	5.82±0.34	6.19±0.4
Untreated	6.00±0.13	6.25±0.13	6.70±0.04	6.07±0.86	5.98±0.2
Controls	12.50±1.11	12.84±0.90	14.32±0.75	14.09±1.01	13.71±0.72
Hgb [g/dl] Amifostine	13.40±1.12	13.23±0.94	13.64±0.52	12.74±0.54	13.87±0.86
Untreated	12.80±0.54	13.40±0.99	14.60±0.99	13.25±0.92	13.20±0.57
Controls	265.2±127.4	211.2±52.8	359.7±93.3	338.8±90.5	359.0±86.7
Platelets [Mrd/1] Amifostine	286.2±73.2	180.7±37.43	214.8±122.0	267±89.6	251.4±102.9
Untreated	378.0±161.2	315.5±129.4	379.5±112.4	275.5±105.4	327.0±70.7
Controls	6.64±1.27	5.43±0.88	6.96±1.41	8.1±1.46	7.79±0.84
WBC [Mrd/1] Amifostine	5.73±2.1	3.39±0.91	4.61±1.48	5.29±1.24	5.76±1.34
Untreated	6.6±1.41	6.15±0.21	6.75±1.63	9.2±4.53	9±2.55
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Platelet count
Details of platelet count are given for all rabbits in Table l. In 9 control rabbits mean platelet count was 265.22±127.41 Mrd/1 prior to Re-186-HEDP-therapy. Two weeks after injection of the radionuclide mean platelet count was reduced to 211.22±52.8 Mrd/1. Four and six weeks after therapy mean platelet count increased to 359.67±93.26 and 338.78±90.46 Mrd/1, respectively. Prior to the sacrification mean platelet count of 359±86.72 Mrd/1 was measured for all animals pre-treat-ed with physiological saline solution only.
Prior to Re-186-HEDP-therapy the mean platelet count of amifostine-treated rabbits was not significantly different (p>0.05) from control rabbits amounting to 286.22±73.2 Mrd/1. Two weeks after injection of the radionuclide the mean platelet count of the amifostine group decreased to 180.67±37.43
Mrd/1. There was no significant difference between rabbits of the control group and ami-fostine-treated animals (p>0.05). Four, six and eight weeks after Re-186-HEDP-therapy the platelet count increased to 214.78±121.97, 267.67±89.6 and 251.44±102.89 Mrd/1, respectively. Moreover, there was no significant difference between animals treated with either physiological saline solution or amifostine.
Laboratory findings of two untreated animals revealed platelet count of 378±161.22 Mrd/1 at the beginning and 315.5±129.4, 379.5±112.43, 275.5±105.36 and 237±70.71 Mrd/1 at two-week-intervals follow-up.
White blood cells
Values of the white blood cell count are given in detail in Table l. Prior to Re-186-HEDP-therapy mean white blood cell count of
Figure l. Whole body scintigraphy after injection of 300 MBq Tc-99m-HDP prior to (left column) and 2 months after Re-186-HEDP-therapy (right column) in a rabbit of the control group (lower row) and in an amifostine-treat-ed animal (upper row). Distribution of Re-186-HEDP 48 hrs after i.v. application is displayed in the middle column.
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6.64±1.27 Mrd/1 was measured for rabbits of the control group. Two weeks after therapy a slight decrease of white blood cell count was recognized in this animal group with a mean value of 5.43±0.88 Mrd/1. Four, six, and eight weeks after therapy white blood cell count increased to mean values of 6.96±1.41, 8.1±1.46 and 7.97±0.84 Mrd/1, respectively. Amifostine-treated animals revealed mean white blood cell count of 5.73±2.1 Mrd/1 prior to the injection of the radionuclide. In contrast to the control group, amifostine-treated rabbits showed a considerable decrease of white blood celi count two weeks after therapy with a mean value of 3.39±0.91 Mrd/1. This difference was statistically significant (p<0.0002). In follow-up studies the white blood celi count removed to 4.61±1.48, 5.29±1.24 and 5.76±1.34 Mrd/1 at four, six and eight weeks after therapy, respectively.
In two completely untreated animals white blood celi count remained almost unchanged with values of 6.6±1.41, 6.15±0.21, 6.75±1.63, 9.2±4.53 and 9±2.55 Mrd/1 at the beginning and in two-week intervals, respectively.
ution of Tc-99m-HDP in either control animals or amifostine-treated animals. Moreover, there was no visual difference between the distribution of the Tc-99m-HDP prior to and eight weeks after therapy with Re-186-HEDP.
Histopathological findings
Eight weeks after Re-186-HEDP-therapy ali 18 rabbits showed hyperemic bone marrow vessels as compared to untreated animals (Figure 2).
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Scintigraphic findings
Left column of Figure 1 shows examples of whole-body scintigraphy 2 hrs after injection of Tc-99m-HDP of one animal of the control group (lower row) and one animal of the amifostine-treated group (upper row). Prior to Re-186-HEDP-therapy there was no difference between the control animals and the amifos-tine-treated animals concerning the distribution of Tc-99m-HDP. Examples of whole-body scintigraphy 48 hrs after injection of Re-186-HEDP are displayed in the middle column of Figure 1. Hie visual evaluation of the scinti-gramms showed a distribution of Re-186-HEDP comparable to bone scans in both groups of rabbits. Examples of bone scintig-raphy at 8 weeks after Re-186-HEDP-therapy are shown in the right column of Figure 1. There was no visual difference of the distrib-
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Figure 2. Histological examination of bone marrow in conventional Giernsa staining, magnification: 200fold. A: animal which received neither Tc-99m-HEDP nor Re-186-HEDP. B: animal of the control group. C: animal of the amifostine group. Note the comparable cellularity and differentiation of the different cell lines of B and C as compared to the bone marrow of the completely untreated animal (A). However, in contrast to the untreated animal the blood vessels of B and C are filled with red blood cells, and thus, appear hyperernic.
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There was no difference between animals treated with or without amifostine concerning the cellularity of bone marrow, the number and differentiation of erythropoesis, gran-ulopoesis and thrombocytopoesis, and the quantity of iron. Moreover, histopathological examination revealed no difference between animals treated with amifostine and those rabbits, which received physiological saline solution only.
Discussion
Prostate cancer is the second most common malignancy in men in Western Europe.1 The incidence is 15-16 per 100.000 habitants per year with increasing tendency. As much as 80% of patients with prostate cancer will develop bone metastases.1 In about one third of all patients osseous metastases are detected at primary staging. Moreover, the skeleton is the only single site of metastases in a reasonable amount of patients.3 In case of multi-locular, osseous metastases a complete remission of prostate cancer is nearly impossible.
Since osseous metastases are often associated with bone pain, effective pain relief is the primary goal when caring for patients with prostate cancer and multiple osseous metastases. Traditional therapeutic approach is the application of central or peripheral analgesics in combination with neuroleptics.6 Moreover, a steroid medication, diphospho-nates and hormonal drugs may complete analgesic effects. However, the therapy with opioids is limited in many patients due to side-effects, i.e. nausea, vomits and gastrointestinal symptoms and thus, often associated with a loss of patient's quality of life.7
Skeletal pain confined to a single site metastasis usually responds to external beam radiotherapy in 70-80 %.6A0,4i in case of mul-tilocular, osseous metastases external beam radiation is helpful to avoid pathologic fractures or compression of the spinal cord.42
However, hemibody or whole-body irradiation for pain relief is often limited by bone marrow suppression, gastrointestinal symptoms and a radiation pneumonitis.14'43 Therefore, an effective relief of pain with low side-effects and an improvement in patient's quality of life is warranted in these patients.
The application of ^--emitting osteotropic radionuclides is a promising method to selectively irradiate osseous metastases by sparing normal tissues from short-range irradia-tion.11'13 Due to the osteoblastic character of osseous metastases the radiopharmaceutical is predominantly accumulated in malignant transformed cells, which leads to a rather selective irradiation of bone metastases. Re-186-hydroxyethylidendiphosphonate (Re-186-HEDP) has recently been developed for palliative treatment of painful osseous metas-tases.5 Re-186 has a therapeutic ^--emission of 1,07 MeV associated with a y-emission of 137 keV. Moreover, Re-186-HEDP and Tc-99m-HDP, which is commonly used for diagnostic bone scintigraphy, have an almost exactly similar bone distribution since both sorts of diphosphonates bridge to the hydrox-yapatite of bone substance. Therefore, pretherapeutic and posttherapeutic scinti-graphic imaging is possible which allows a control of Re-186-HEDP distribution as shown in Figure 1 (middle column). Re-186 has a short physical half-life of 3,8 days that allows a single application of activities of 1110 to 1850 MBq with high tumor doses as well as an easy handling of radioactive waste, i.e. urine. About 50 % and 70 % of the activity injected are excreted via the kidneys into the urine within the first 6 hours and 24 hours after application, respectively. Apart from the distribution in osseous structures Re-186-HEDP is not accumulated in any other structures of the body.
Pain relief is attained within two weeks after application of Re-186-HEDP and lasts for about 1-6 months. Response rates of Re-186-HEDP-therapy of 70-80 % have been
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reported.3-10-12 Especially in patients with oral medication of non-opioid analgesics, Re-186-HEDP-therapy led either to a reduction or to a stop of oral drug medication. Due to the short physical half-life, Re-186-HEDP treatment can be repeated after 4-6 months.
The main radiobiological side-effect of bone seeking radiotherapeuticals is their potential bone marrow toxicity.11-13-15 Thrombocytopenia plays the major role in this bone marrow suppression. The decline of platelets presents with a nadir about two to three weeks after its application. Since patients with prostate cancer often show a bleeding tendency caused by the additional use of non-steroid anti-inflammatory drugs and by a tumor infiltration of the bladder frequent controls of platelet count are necessary in posttherapeutic follow-up. In clinical practice platelet counts are regularly defined in two-week intervals for the duration of two months after Re-186-HEDP-therapy. Since thrombocy-topenia was proven to be the main side-effect a reduction of the platelet counts' decline would improve tolerability of Re-186-therapy. On the other hand, thrombocytopenia is the dose-limiting factor. Thus, by reducing the bone marrow toxicity of Re-186-HEDP higher activities of more than 1200 MBq might be injected as one single dose in the future. Thus, Re-186-HEDP might be applied not only as a palliative but also as curative means in patients with prostate cancer.
The pro-drug amifostine emerged as the most promising radioprotector synthesized and tested in a large study funded by the US army. In clinical trials amifostine was shown to protect salivary glands from irradiation damage in patients with thyroid cancer under high-dose radioiodine therapy.26-29'31-32'44'45 Moreover, amifostine was proven helpful in patients with cancer of the head and neck treated with chemo-irradiation.30'45'46 In these patients amifostine was able to significantly reduce side-effects, i.e. mucositis, xerostomia and thrombocytopenia as compared to a non-
amifostine treated patient group. Moreover, either in patients with thyroid cancer nor in patients with head and neck cancer amifos-tine was shown not to protect tumor tissue, which is a prerequisite for its potential use in tumor therapy.25-33'47
When administered intravenously, amifos-tine is rapidly cleared from the plasma with an alpha half-life of as less than one minute and a beta half-life of less than 10 minutes.48 In contrast to its brief systemic half-life, there is a prolonged retention of the drug and its metabolites in normal tissues.36 In the first 30 minutes after amifostine administration, the drug uptake into normal tissues such as salivary glands, liver, kidney, heart and bone-marrow demonstrated an up to 100-fold greater difference as compared to tumor tissue.36
In this preclinical study the radioprotective effect of amifostine on bone marrow suppression under Re-186-HEDP-therapy was studied using a rabbit bone marrow model. Therefore, in a total of 18 rabbits, 400 MBq Re-186-HEDP were applied intravenously in order to evaluate the bone marrow suppression in rabbits pretreated either with amifostine or with physiological saline solution only. Neither red blood cell count nor hemoglobin was changed by Re-186-HEDP in controls and in rabbits pretreated with amifostine. Moreover, by histopathological examination there was no difference concerning the red blood cell line in all three groups of animals. This is in accordance with the observations of other investigators who reported no influence of Re-186-HEDP on hemoglobin and red blood cells.16
In contrast, a marked decline of the platelet count of about 20% was observed two weeks after application of Re-186-HEDP in controls. This is again in accordance with the observation of other investigators.7-11-13-16 Following whole-body exposure by external radiation, thrombocytopenia develops slowly over a period of approximately 30 days after doses of 200-400 cGy. After the application of a dose of 600-1000 cGy the production of
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345
platelets is completely stopped, which leads to a decrease of the platelet count reflecting the life range of the platelets of approximately 9 days.15 It was estimated that standard activities of 1200 MBq Re-186-HEDP deliver a radiation dose of about 75 cGy to the bone marrow leading to the marked platelet suppression.8 In contrast to the investigations of de Klerk15 who reported a nadir of decline of platelet count at week 4 after therapy in this rabbit animal model a marked reversible decline of platelets occurred as early as two weeks after therapy. Thus, in amifostine-treat-ed animals peripheral platelet count showed a comparable decrease of about 37.1 %. Thus, amifostine could not reduce the transient thrombocytopenia in animals treated with Re-186-HEDP. This is probably due to the phar-macocinetic properties of amifostine. First, amifostine was applied as a single dose prior to Re-186-HEDP. Second, while Re-186-HEDP has both a biological and a physical half-life of about three days, the biological half-life of amifostine within the bone marrow is probably less than 24 hrs. Thus, the generation of free radicals within the bone marrow caused by Re-186-HEDP lasts several times longer than the radioprotective effect of amifostine. This leads to the conclusion that in order to use amifostine as a suitable agent for radio-protection, multiple doses of amifostine might be applied, e.g. two single shots per day for the duration of 3 to 4 days after the application of Re-186-HEDP. However, concerning the thrombocytopoesis 8 weeks after therapy the pathologist described no difference between animals treated with Re-186-HEDP and totally untreated animals. Thus, in order to investigate the myelotoxic effect of Re-186-HEDP the sacrification of the animals might be performed earlier than 8 weeks after therapy, e.g. 2 weeks after therapy while the platelet count decrease exhibits its nadir.
As far as white blood cells are concerned amifostine-treated animals showed a significant reduction of leucocytes as compared to
animals of the control group. There was no explanation for this side-effect of amifostine while nausea, vomiting and potentially hypotension are well-described side-effects after the administration of amifostine. However, a reduction of white blood cells is yet unknown. Moreover, there was no difference in the granulopoesis between all three groups of rabbits investigated as demonstrated by histopathology. Thus, in order to evaluate cytotoxic effects of amifostine or Re-186-HEDP on the genesis of white blood cells the sacrification of the rabbits might be performed earlier. However, the hyperemic blood vessels of all animals treated with RE-186-HEDP might be interpreted as a late reactive sign of bone marrow damage.
Conclusions
In this animal model amifostine given in one single dose was not able to avoid the transient thrombocytopenia in rabbits treated with Re-186-HEDP. However, in order to use amifos-tine as a suitable agent for radioprotection multiple doses of amifostine might be applied, e.g. two single shots per day for the duration of 3 to 4 days after the application of Re-186-HEDP. The observed leucopenia should be studied in further animal studies.
References
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