Radiotherapy and Oncology, 21 (1991) 149-156

~ 1991 Elsevier Science Publishers B.V. 0167-8140/91/$03.50

149

RADION 00850

Cyclophosphamide 24 hours before or after total body irradiation: effects on lung and bone marrow Rendi Y a n i, Lester J. Peters 2 and Elizabeth L. Travis 1 JDepartment of Experimental Radiotherapy, and 2Division of Radiotherapy, The University of Texas M. D. Anderson Cancer Center Houston, Texas, U.S.A.

(Received 8 January 1990, revision received 12 February 1991, accepted 20 March 1991)

Key words: Cyclophosphamide; Total body irradiation; Lung; Bone marrow transplantation

Summary Preparative regimens for bone marrow transplantation (BMT) use a sequence of drugs, such as cyclophosphamide, in combination with radiation. However, the optimum sequencing of the two agents that will maximize tumor cell kill and minimize normal tissue damage is unknown and controversial. The studies presented here were done in order to determine the effect of cyclophosphamide on bone marrow and lung damage in mice when given 24 h before or after total body irradiation (TBI). A range of single doses of TBI was given before or after a single sublethal dose of 180 mg/kg of cyclophosphamide. The bone marrow of all mice intended for lung damage assessment was reconstituted with 5 x 106 syngeneic bone marrow cells. Lung damage was assessed by breathing rate and lethality; bone marrow damage by lethality at 30 days. LDso values for pneumonitis were obtained between 30 and 84 days after cyclophosphamide and radiation and between 80 and 180 days after radiation alone. Dose modifying factors were obtained as the ratio of LDsos for mice given only TBI compared to those for mice given cyclophosphamide and TBI. Cyclophosphamide enhanced radiation pneumonitis when given before or after TBI, giving DMFs of 1.4 and 1.2 (1.1-1.4, 95 ~ c.1.) respectively. The effect ofcyclophosphamide on radiation pneumonitis was drug dose-dependent. The LDso for death from bone marrow damage was reduced when cyclophosphamide was given either before or after TBI but the effect was greater, i.e. the LDso was lower when cyclophosphamide was given after TBI. These data show that cyclophosphamide given 24 h after TBI causes less lung damage but more bone marrow damage in this mouse model.

Introduction Drugs are routinely used in conjunction with total body irradiation (TBI) as a standard conditioning regimen for bone marrow transplantation (BMT) in the treatment of hematologic malignancies as well as other malignancies. One of the major treatment-related toxicities is idiopathic pneumonitis. However, there is no consensus concerning the role of the radiation or the drugs in the pulmonary morbidity. Clinically, the drugs are most often given 1 or 2 days before TBI with few exceptions. However, whether this is the optimum sequencing or time interval separating the two modalities to achieve minimum lung damage and maximum tumor

cell kill is unknown. Many studies in mice have shown that both sequencing and time between drugs and radiation are critical to the effect in both malignant and normal cells and tissues [2,3,9,10,28,29]. Cyclophosphamide is one of the drugs commonly used in conditioning regimens for BMT. The enhancement of radiation-induced lung damage by cyclophosphamide in experimental animals is well established but a wide range of dose enhancement factors, from 1.07 to 2.35, has been reported [2,14,15,18,19,27,29]. This variability is at least partially due to differences in time intervals between the drug and radiation in the various studies as well as order of administration. Variations in the dose rate of the radiation and the dose of cyclophos-

Address for correspondence: E. L. Travis, Department of Experimental Radiotherapy, Box 66, The University of Texas M. D. Anderson Cancer

Center, 1515 Holcombe Blvd., Houston, TX 77030, U.S.A.

150 phamide used also contribute to the difficulty in comparing results from different laboratories. Perhaps most critical to the BMT setting is that in all of these studies of lung damage only the whole thorax was irradiated, not the whole body, In contrast to its universal enhancement of radiation-induced lung damage, the effects of cyclophosphamide on radiation damage in bone marrow are less clear, some investigators report enhancement while others report protection [ 9-11,28 ]. However, what may be critical to the transplant setting is that in all studies there were more deaths from bone marrow damage when cyclophosphamide was given 24 h after TBI than at anytime before TBI. The aim of the studies presented here was to compare the effect of cyclophosphamide on lung damage and bone marrow damage when given at a fixed time, 24 h, before or after TBI. These times were chosen to simulate those times used in clinical conditioning regimens for bone marrow transplantation.

and the dose rate was 1.19 Gy" min-~. All doses are quoted at midline of the thorax or whole body as measured by TLDs in a suitable mouse-size phantom of tissue equivalent material or by ion chamber measurements in jigs in the treatment set-up. These radiation measurements indicate a variation of dose on the order of + 12~o from the nominal midline dose.

Cyclophosphamide Cyclophosphamide was dissolved in sterile distilled water. The dose of 180 mg/kg, which is 2/3 of the LDso for cyclophosphamide in our mice was injected intraperitoneally as a single dose at a constant volume of 0.01 ml/g animal weight. Cyclophosphamide was given only at 2 times, 24 h before or 24 h after, irradiation.

Assays of damage Lung. Breathing rate and lethality were used to

Materials and methods

Animals All experiments were performed in specific pathogenfree male C3Hf/Kam mice, 10-12 weeks of age, weighing 26-30 g. Mice were housed five to seven mice per cage and allowed access to sterilized pellet food and sterilized acidified water ad libitum. All mice were bred and maintained in our specific pathogen-free colony for the duration of the experiments.

Irradiations Radiation was delivered by a Philips 250 kVp X-ray unit operated at 15 mA with a total filtration of 0.5 mm Cu plus an aluminum compensator yielding a HVL of 1.68 mm Cu. For the TBI, groups of five mice were held in a Lucite box measuring 14 x 14 x 2.8 cm which was placed on a platform fixed to the head of the X-ray machine. The target-to-skin distance was 30.10 cm and the dose rate was 1.69 Gy" min-~, as described previously [20,23]. A flattening filter limited the dose variation across the field to ~t 12 Gy TBI but after only a dose of 6 Gy TBI in mice given cyclophosphamide. Tooth damage was observed also in mice given cyclophosphamide alone. Approximately 50~o of all mice in this study exhibited broken teeth. Tooth deformities appeared at 6 weeks after treatment but were normal by 12 weeks. Powdered food was provided to the animals during this time.

Bone marrow damage Discussion

Figure 6 shows survival at 30 days plotted as a function of single doses of total body radiation alone or TBI given 24 h before or after 180 mg/kg cyclophosphamide. LDso values with 95 ~o confidence limits and DMFs are given in Table I. When cyclophosphamide was given 24 h after TBI, there was a clear shift of the doseresponse curve to the left, and the LDso/3o days was reduced by a factor of 1.2. The dose-response curve for mice given cyclophosphamide 24 h before TBI is not well defined because the percent of mice dying did not exceed 4 0 ~ , even after the highest radiation dose (see Fig. 6). This curve has been drawn by eye and an LDso is not reported. These data do show however that cyclophosphamide given 24 h before TBI does enhance bone I

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Dose (Gy) Fig. 6. Number of animals dead from bone marrow damage 30 days after single doses of total body irradiation (TBI) alone (A), 24 h before (O), or 24 h after ( 0 ) 180 mg/kg eyclophosphamide given intraperitoneally. The curve for cyclophosphamide 24 h before TBI (O) is fitted by eye, the other two curves were fitted by logit analysis. The error bars at the LDso are 95~ confidence limits.

All of the experimental studies reported in the literature on the effect of cyclophosphamide on lung damage irradiated only the whole thorax. To more closely approximate clinical bone marrow transplant conditioning regimens, we irradiated the whole body 24 h before or after one dose of cyclophosphamide and compared these data with irradiation of the whole thorax. The data presented here show that cyclophosphamide enhances radiation-induced lung damage, as demonstrated by a significant decrease in the radiation LDs0 for lung damage in mice given both cyclophosphamide and radiation compared to that in mice given radiation alone, in agreement with previous reports [2,6,14,15, 18,19,27,29,30]. In addition, the latent time to expression of the damage was shorter in mice given both treatments than in those given only the radiation, as was the entire time course of the damage. Lung damage was observed as soon as one month after combined treatment and persisted up to 3 months, with few deaths occurring after this time, which is also in agreement with published data [2,6,29]. Despite this difference in the time the lung damage was expressed, the histologic changes were similar regardless of the treatment given and were characteristic of radiation pneumonitis as reported previously [2,6,12,17,25]. Thus, data obtained between 1 and 3 months for the mice given drug and radiation were compared with those obtained between 3 and 7 months for mice given radiation alone, the latter being the standard assay time for radiation pneumonitis. The DMFs we obtained for lung damage, which ranged from 1.2 and 1.4 after total body irradiation to 1.7 after whole thorax irradiation, were in agreement

155 with previously reported values but seem to suggest that cyclophosphamide has more effect on the lung when only the thorax is irradiated than when the whole body is irradiated. However, in these studies, considering only the DMFs is misleading since the variability in these values are not dependent solely on whether cyclophosphamide was given, but also are dependent on whether the whole lung or total body was irradiated. In our studies, the LDso for radiation pneumonitis after whole body irradiation was significantly less than after irradiation of only the thorax, 10.77 Gy and 12.5 Gy, respectively. Although it has been reported that deaths between one and 3 months after irradiation techniques that encompass the head, such as TBI, may be attributed erroneously to lung damage when, in fact, damage to the incisors during this time contributes to a reduction in the LDso [4] ; in the studies reported here deaths in the TBI-treated mice occurred later than 3 months after irradiation and at doses lower than those reported by Down et al. [4] and Pearson and Phelps [13] as causing tooth damage. Nonetheless, all mice including those given only TBI were monitored for tooth damage and fed soft food, thus preventing any deaths from starvation. Thus it is most likely that the lower LDs0 after TBI than after whole thorax irradiation (WTI) in the present studies does reflect lung damage. If one considers only the dose for isoeffective lung damage then as a more accurate indicator of the effect of cyclophosphamide on radiation-induced lung damage, it is clear that cyclophosphamide had significantly less effect on lung damage when given 24 h after TBI. The isoeffective dose was significantly higher (by about 2 Gy with no overlapping confidence limits) in the mice given cyclophosphamide 24 h after TBI than 24 h before TBI. The isoeffect dose for lung damage was not only lower in the mice given cyclophosphamide 24 h before TBI but was remarkably similar to that of mice given cyclophosphamide before or after whole thorax irradiation (see Table I). Although we have no explanation why cyclophosphamide has less effect in the lung when given 24 h after TBI, these data suggest that it would be prudent to give the drug after the completion of TBI at least from the standpoint of lung damage. The bigger enhancement of bone marrow damage when cyclophosphamide was given after but not before TBI is in agreement with the data of von der Maase et al. [28] who obtained DEFs of 1.75 or 1.15 for bone marrow deaths when cyclophosphamide was given after or before TBI, respectively. Although the dose of cyclophosphamide used in their studies was higher (250 mg/kg) than that used by us, both sets of data indicate that the effects of cyclophosphamide on normal bone marrow are more pronounced when given

after TBI. Although the mechanism of this effect is unknown, it is well established that CFUs survival decreases within the first day after irradiation, reaching a nadir between 12 and 24 h (post-irradiation dip) [5,8]. Cyclophosphamide given 24 h after TBI may be more effective simply because there are fewer cells present resulting in a lower LDso. In addition, it has been shown [9-11 ] that cyclophosphamide given 1 to 3 days before TBI actually protects mice against death from bone marrow damage. Clearly, studies are needed using experimental leukemia models to determine whether these findings in normal murine marrow are relevant. Interstitial pneumonitis is the single most devastating complication of allogeneic BMT in leukemia patients. Radiation therapy is often implicated in 50 To of these patients but the experimental data, as well as the clinical data, suggest that at the total radiation doses, dose rates, and doses per fraction used, radiation exerts a minimal influence on this treatment complication. The data presented here and in other published studies show that the drugs used in these preparative regimens enhance the radiation response in the lung, causing significant morbidity from what otherwise are radiation doses well below accepted tolerance. Differences do exist in the actual enhancement factors obtained, ranging from 1.3 to 2.3 ; however, it is clear from all of these studies that cyclophosphamide augments radiation-induced lung damage. Only two studies in rats suggest that idiopathic pneumonitis is due mainly to radiation with little, if any, contribution from cyclophosphamide [27,30]. The objective of the studies presented here was simple: to compare the effect on bone marrow and lung of cyclophosphamide given 24 h before or after TBI as a preparative regimen for BMT. The data from these studies show that: (1) Less enhancement of radiation pneumonitis was observed when cyclophosphamide was given 24 h after than 24 h before TBI. (2) Cyclophosphamide given 24 h after TBI causes more damage in normal bone marrow than when given 24 h before TBI. Thus, if there are no compelling biological reasons to give the drugs pre TBI, it might be prudent to give them after TBI.

Acknowledgements We kindly thank Mrs. Lori Verhalen and Ms. Shelia Buckner for preparation of this manuscript. This investigation was supported by grant number CA-06294, awarded by the National Cancer Institute.

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Cyclophosphamide 24 hours before or after total body irradiation: effects on lung and bone marrow.

Preparative regimens for bone marrow transplantation (BMT) use a sequence of drugs, such as cyclophosphamide, in combination with radiation. However, ...
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