1~. J. Radiation Oncology Ho/. Phys Vol. Printed in the U.S.A. All rights reserved.

16, pp.

0360-3016/90 53.00 + $4 copyright 0 1990 Pergamon Press plc

1377-1385

??Original Contribution

ENHANCED IUdR RADIOSENSITIZATION BY 241Am PHOTONS RELATIVE TO **‘jRaAND “‘1 PHOTONS AT 0.72 Gy/hr RAVINDER NATH, PH.D., PAUL BONGIORNI, M.S., PETER I. ROSSI, B.S. AND SARA ROCKWELL, PH.D. Department

of Therapeutic Radiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 065 10

The dependence of IUdR radiosensitization on photon energy was investigated by irradiating Chinese hamster cells in vitro under aerobic conditions at a dose rate of 0.72 Gy/hr which is typical of temporary brachytherapy implants. It had been observed previously that the IUdR radiosensitization with the 60 keV photons from 241Am is about 1.5 times greater than that with 830 keV (average) photons from ‘%a. It was hypothesized that the enhanced IUdR radiosensitlzation for 60 keV photons was a result of a larger production of Auger electron cascades from the filling of K-shell vacancies in the iodine atoms, which have a K-shell binding energy of 33.2 keV. Since most of the photons from a “‘1 source have energies below 33.2 keV, it would be expected that IUdR radiosensitization with 28 keV (average) photons from “‘1 and 830 keV (average) photons from *%Ra would both be smaller than the radiosensitization with the 60 keV photons from %lArn. To test this hypothesis we compared IUdR radiosensitization for *%t, 241Am, and 125Iat 0.72 Gy/hr, using Chinese hamster lung cells in vitro. The measured survival curves led to RBEs of 1.20 + 0.10 and 1.30 _+0.11 for “‘Am and ‘*‘I photons relative to *=Ra; to IUdR radiosensitization factors at a lo-’ M concentration of 1.35 + 0.1 1, 1.67+ 0.09, and 1.47 f 0.08 for *%Ra,u’Am, and ‘=I, respectively; and to radiosensitization factors at a 10T4 M concentration of 1.89 + 0.16,3.04 f 0.13, and 2.48 + 0.17 for *%a, “‘Am and 1251,respectively. These results indicate that IUdR produces significant radiosensitization with all three isotopes (*%a, =‘Arn, and ‘*’I) for continuous low dose rate irradiations at 0.72 Gy/hr. Also, we observed greater radiosensitization with “‘Am photons compared to **‘%aon the higher energy side and to ‘*‘I on the lower energy side. These findings support the concept that photon-induced Auger electrons produce a significant increase in IUdR radiosensitization when photons with energies just above the K-edge of the iodine atom are employed for continuous low dose rate irradiations. These findings suggest that regimens combining IUdR infusion with temporary brachytherapy implants using low energy photons in relatively quiescent sites such as brain tumors may have clinical potential, and indicate the need for rigorous preclinical evaluation of this approach. Iododeoxyurdine electrons.

(IUdR),

Americium-241,

Iodine-125,

Radium-226,

Brachytherapy,

Radiosensitization,

Auger

cause of excessive radiosensitization in dose-limiting, rapidly proliferating, normal tissues (1, 14). In recent years, there has been renewed interest in the use of IUdR in the treatment of selected, rapidly growing tumors, such as brain tumors and osteogenic sarcomas, located in sites with relatively quiescent dose-limiting normal tissue ( 15, 20-22,26, 36, 38). All of these studies have used external beam radiotherapy. Because brachytherapy offers much greater localization of the dose to tumor volume than does external beam therapy, use of brachytherapy with IUdR might decrease normal tissue radiation doses, and therefore improve the therapeutic gain when compared with external beam therapy. It is therefore interesting to investigate the effects

INTRODUCTION

Halogenated thymidine analogs such as IUdR and BUdR have been long known to be potent radiosensitizers of rapidly proliferating cells and tissues (2, 8, 19, 27). These halogenated pyrimidines are incorporated into the DNA of cells at the time of DNA synthesis, and are therefore incorporated almost exclusively into actively proliferating cells. It was postulated that in the case of a growing neoplasm located in a region of non-proliferating normal tissues, selective IUdR incorporation, and thus differential radiosensitization, could be used to improve the therapeutic ratio. Early clinical trials using IUdR or BUdR to produce selective radiosensitization failed, primarily be-

periments from the 3 M company, Medical Products Division. Also, we thank Ms. Deanna Jacobs for preparing and editing this manuscript. Accepted for publication 20 December 1989.

Reprint requests to: Ravinder Nath. Supported in part by USPHS grants CA-39044 and CA-44537 awarded by the National Cancer Institute. Acknowledgements-The authors gratefully acknowledge the generous gift of high activity iodine-125 sources for these ex1377

1378

1. J. Radiation Oncology 0 Biology 0 Physics

of halogenated pyrimidines at the dose rates of interest to brachytherapists. In a recent review on the use of nonhypoxic radiosensitizers by Mitchell et al. (29) it is noted that BUdR has been found to produce similar radiosensitization at high (2 Gy/min) and low (.028 Gy/min) dose rates. We had recently observed that IUdR at concentrations of 10e5 M and 10m4M produces significant radiosensitization in Chinese hamster cells in vitro under continuous low dose rate irradiation (CLDRI) (30). More interestingly, it was observed that IUdR radiosensitization with 60 keV photons from 241Am at a dose rate of 0.57 Gy/hr was about 1.5 times greater than the radiosensitization with higher energy photons from 226Ra. This enhancement of IUdR radiosensitization by 60 keV photons, relative to higher energy photons, may be the result of a greater production of photon-induced Auger electrons from the iodine atom of the IUdR molecule at 60 keV than at higher energies, because these 60 keV photons have energies just above the K-edge of the photon absorption in the iodine atom (33.2 keV). Therefore, it would be reasonable to expect that the radiosensitization produced with ‘25l photons (which have energies below 33.2 keV) would be smaller than that with 24’Am photons. To test this hypothesis, the present experiments were performed with 226Ra,241Am,and ‘25lsources which provide one photon energy below the iodine K-absorption edge (1251),one just above the K-edge (24’Am), and one much greater than the K-edge (226Ra). METHODS

AND MATERIALS

June 1990, Volume 18, Number 6

growth, the growth medium was removed and replaced by growth medium containing 10e5 or 1Op4M IUdR. The medium on the cultures was replaced by fresh medium containing IUdR just before the beginning of the irradiation, to ensure that IUdR was available in the medium throughout the irradiations, which lasted 1 to 48 hr. During irradiation, cells were maintained in a humidified 95% air-5% CO2 environment at 37°C. Unirradiated controls were maintained analogously. After irradiation, the cells were washed with Hanks’ balanced salt solution, trypsinized, and counted using a cell counter,+ equipped with a Channelizer to allow assessment of and correction for any changes in cell size. Cells were plated for colony formation in at least four dishes per data point and were allowed to grow in a humidified 95% sir/5% CO2 environment at 37°C for 10 days. The colonies were then fixed, stained, and counted. Experiments were planned to obtain approximately 120 colonies in each dish, by adjusting the number of cells plated per dish appropriately. Controls receiving IUdR treatment but no irradiation were also examined to assess the toxicity of the drug alone. Plating efficiencies for untreated cells were approximately 90% in the experiments reported here. Cell survivals are usually calculated as the ratios of the plating efficiencies in the treated cells relative to those of untreated cells under control conditions. In these experiments, however, the number of cells in cultures treated with CLDRI were significantly lower than the number of cells in control cultures. This could reflect either the death and lysis of some cells during the prolonged radiation and drug treatments, or a decreased rate of cell proliferation in the treated cultures. Few cells were observed floating in the cell culture medium. The surviving fractions were therefore calculated to account for this cell loss during CLDRI, using the formula:

Cells Experiments were performed using Chinese hamster lung cells.* This system has been in place in our laboratory for several years; the characteristics of the cell system and some of the details of the methodology have been described previously (30, 34). Chinese hamster cells are grown as monolayers in 75 cm* Falcon tissue culture flasks, in a humidified 95% air-5% CO2 atmosphere at 37°C. These cells are maintained in basal medium with Eagle’s salts, supplemented with fetal calf serum ( 15% V/ V), antibiotics ( 1% V/V), MEM vitamins ( 1% V/V), and L-glutamine ( 1% V/V). Under these conditions, the population doubling time during exponential growth is approximately 12 hr. Stock cultures are subcultured at 3-to-4 day intervals.

are the numbers of colonies obNexptl and Ncontrol served per 120 cells plated for experimental and control samples, respectively; and Cexptland Ccontrolare the numbers of cells per ml in the experimental and control samples, respectively. The “cell loss” in the following sections of this paper has been calculated as (1 - Cexpt&,ntrol).

Cytotoxicity studies Monolayers for experiments were prepared by plating cells, suspended from exponentially growing stock cultures, into 60-mm Falcon tissue culture dishes. The cells were incubated for 18 hr prior to IUdR treatment or irradiation, to allow them to attach and to progress into exponential growth. Once the cells were in exponential

Measurement of IUdR incorporation Average incorporation of IUdR into the DNA of the cells was measured using “‘IUdR tracer. Initially, lop5 cells were plated into petri dishes. After 18 hr had elapsed (ensuring exponential growth) the growth medium (Eagle’s Basal Medium) with Earle’s salts supplemented with fetal bovine serum ( 15% V/V), antibiotics, MEM vitamins, and

* DON LINE,

Rockville, MD.

American Type Culture Collection CCL 16,

S-

N0 N control

C exptl Gntro~

where

+ Coulter

Corp., Model ZBI, Hialeah, FL.

1379

Enhanced IUdR radiosensitization 0 R. NATH etal. L&&mine ( 1% V/V) was replaced with 5 ml of lz51UdR medium (hot) plus 5 ml of IUdR medium (cold). Concentrations of the hot plus cold IUdR used were lop5 M or 10m4M. The IUdR medium was replaced 3 times with fresh IUdR medium during the initial 24 hr exposure to IUdR medium. IUdR medium was left on the cells for tag times up to 96 hr. The tagging procedure was done at 37°C with a 95% sir/5% CO2 atmosphere. After the tag time was completed, the IUdR medium was removed and the attached cells were washed gently 3 times with phosphate buffered salines to remove any non-specifically attached ‘251UdR. The IUdR medium, removed this way, was counted for activity using a 3 X 3” NaI scintillation detector system. After the decanted IUdR medium was counted, the dish with the radiolabelled cells was counted. The sum of the activity in the decanted medium plus the activity in the cells should equal the amount of activity used to tag the cells.

26 ‘25Isource& with an activity of 40 mCi each. Twentyfour of the 1251sources are arranged in a circle with a diameter of 6 cm, and 2 sources are placed next to each other at the center of the circle. The 1251sources are contained in a 1.25 cm thick, 10 cm diameter polystyrene disk, which is placed in a larger polystyrene irradiator with outside dimensions of 20 X 20 X 10 cm. Different dose rates spanning the range from 0.1 to 1.O Gy/hr were obtained by varying the thickness of the polystyrene spacers. The 24’Am and ‘25I irradiators were placed in a 37°C water-jacketed incubator and surrounded in lead foil of 1 mm thickness to shield the gamma rays. Adequate shielding of the 226Ra irradiator within a commercial incubator would not be easily accomplished because of the thickness and weight of lead necessary. Shielding the whole incubator would have required a tremendous amount of lead. Therefore, our radium irradiator was placed in a specially designed small incubator which was surrounded by lead and kept in a well-shielded, restricted area.

A total= Acellsf Amedium The initial tag amount in every dish was 0.0 1 &i. Cold dishes and a reference dish containing the same tag, 0.0 1 &I, were used to calibrate the counter for background and sensitivity. The cell activity counts were adjusted for background and for counter sensitivity, and the amount of activity per cell (pCi/cell) was calculated. Using the specific activity of ‘25I(2 17 1 Ci/mmole), it can be shown that 1 pCi of ‘251contains 2.774 X lo5 atoms of ‘251.The number of IUdR molecules per cell was then calculated from the measured activity per cell by using the dilution factor of hot versus cold IUdR. To determine percent thymidine replaced, we assumed the number of thymidine moieties in the DNA of Chinese hamster cells to be 4.30 X lo9 bases per DNA molecule (23). Irradiation techniques Cells in petri dishes were irradiated with specially designed ‘251,24’Am and 226Ra source irradiators made of polystyrene with a central hole for placing the petri dishes on top of the sources, with or without some polystyrene spacers in between the sources and the tissue culture dish. The 24’Am irradiator contains a disc source of 241Amwith an activity of 9.7 Ci (31). The americium is in the form of americium oxide, which is bonded to an aluminum substrate and then double encapsulated in 1 mm of titanium. The 226Ra irradiator consists of 137 mg of 226Ra spread around in a circular pattern to provide a uniform dose distribution at the petri dish. The 226Rasources and the petri dish are contained in a larger polystyrene cylindrical phantom with outside dimensions of 20 cm diameter and 10 cm height. The ‘25I irradiator consists of * Dulbecco’s formulation, Gibco Laboratories, Grand Island, NY. BModel 6702, 3M Medical Products, St. Paul, MN.

Dosimetry The dose to cells in the monolayers was measured using an FeS04 Fricke dosimeter with standard formulation (1 .O mM ferrous sulfate, 1.0 mM sodium chloride, 0.8 N sulfuric acid). The ferric ion yields, or G-values, were taken to be 14.4 for ‘251, 14.9 for 24’Am and 15.7 for 226Ra. Optical absorbance measurements were taken at 224 nm and 304 nm. Five ml of dosimeter solution was placed in 60 mm petri dishes,** and the dishes were then placed in the identical locations used for petri dishes containing experimental monolayers. Petri dishes were used for Fricke dosimetry because the inner surfaces of these dishes have been found to have a negligible effect on the optical absorbance of the unirradiated dosimeter solution. The dose to the monolayer of cells adhering to the petri dish was calculated from the dose to the FeS04 solution, by multiplying by the ratio of the mass energy absorption coefficients of muscle and FeS04 solution and applying a correction factor for the interface effect ( 16). Further details of the dosimetry will be presented elsewhere; suffice it to note that for a determination of radiosensitization factors for different isotopes, it is not necessary to determine doses to the cells with extreme precision, because the dosimetric factors are identical for cultures irradiated in the presence and absence of IUdR, and the radiosensitization factor is therefore a relative measurement. RESULTS The variation in IUdR incorporation as a function of the incubation time is shown in Figure 1. For both 10m5 M and lop4 M concentrations, the IUdR incorporation ** Falcon petri dishes, Becton Dickinson Labware, Lincoln Park, NJ.

1380

1. J. Radiation Oncology 0 Biology 0 Physics

June 1990, Volume 18, Number 6 Radlatlon Alone

./

. :i

0

5

10

15

M

I

25

Time (hr)

Fig. 1. The percent IUdR incorporation as a function of incubation time for 10m5 and 10m4 M concentrations. The lines through the data points are smooth lines to guide the eye.

10 5 M IUdR Alone

10 5 M IUdR & Radlatlon

ibi

0

08

increased rapidly with time, reaching plateau values after 24 and 12 hr for 10e5 M and 10m4M concentrations, respectively. In all of the radiation sensitization experiments, cells were incubated with IUdR for 24 hr prior to irradiation; the percentages of the thymidines replaced by IUdR were 22% and 45% for 10m5M and 1O-4 M concentrations, respectively. Cell numbers in cultures treated with IUdR or radiation were lower than those in untreated control cultures. As described above the cell deficit or “cell loss” was calculated from the experimental and control cell counts as (1 - CexptKcontrol ). As shown in Figure 2, cell loss increased linearly with treatment time; the rates of cell loss were different for different treatments. The cell loss per hour was observed to be 0.018 + 0.001, 0.016 + 0.002 and 0.022 f 0.004 for radiation alone, 10m5M IUdR alone, and 10m4M IUdR alone, respectively. For irradiation in the presence of IUdR at concentrations of lop5 M and lop4 M, the cell loss per hour increased to 0.026 + 0.002 and 0.036 f 0.004, respectively. All cytotoxicity data were corrected for the cell loss during treatment, as described in Methods and Materials. Results of the drug toxicity experiments are shown in Figure 3. This figure shows the surviving fraction as a function of IUdR treatment time on a semilogarithmic scale identical to those used below to show the effects of radiation plus drug. Using a statistical package,++ these drug toxicity data were fitted by regression analysis with straight lines, which had slopes of 0.025 and 0.060 per hour for drug concentrations of 10P5 M and lop4 M, respectively. Figures 4-6 show the surviving fractions as a function of radiation dose, with and without IUdR, for **‘jRa, 24’Am and 1251,respectively. These figures also show the surviving fractions for the regimens combining IUdR and radiation, corrected for drug toxicity: All of these curves ++PRODAS, Professional Database Analysis System, Conceptual Software, Inc., Houston, TX.

. .. .i-:-:_l

3

st

05

.

0

02

I

.;III8.. 5

10 4 M IUdR Alone

’

0

08

D 305 % 0

10 4 M IUdR & Radiation

’

1

(e)

.

??

.

?? . .

5

’

.

=,

.

i

.

.

??

1. . .

??

. 0

’

.

1%’

.

02

I

IO

I5

Time (hr)

2-2

25

0

5

10

15

20

25

Time (hr)

Fig. 2. Cell loss as a function of treatment time in hours for: (a) radiation treatment at 0.72 Gy/hr alone, (b) lo-’ M IUdR treatment alone, (c) 10e5 M IUdR treatment combined with CLDRI, (d) 10m4M IUdR treatment alone, (e) 10m4M IUdR treatment with CLDRI. The straight lines through the data points are the result of regression analysis using a linear model with no intercept.

were fitted using a linear model, In S = -aD, because the inclusion of a quadratic term did not improve the goodness of the fit significantly. The values of the coefficient (Yfor these data are shown in Tables 1 and 2 for concentrations of 10m5M and 10m4M, respectively. For radiation alone, the values of (Yincreased as the photon energy decreased; these values were 0.37 1 + 0.029,0.444 * 0.014,

1381

Enhanced IUdR radiosensitization 0 R. NATH et al.

studies provide a basis for considering other potential clinical applications of IUdR in radiotherapy. Regimens combining IUdR with brachytherapy coulci theoretically offer certain advantages over regimens combining IUdR with fractionated external beam radiotherapy. The greater localization of the radiation dose obtainable with brachytherapy (especially with low energy photons) will minimize normal tissue reactions. In ad-

DRUG TOXICITY 1Ci5M IUdR

RADlUW226 Time (hr)

Time (hr)

Radiation Alone

1

Fig. 3. Surviving fraction as a function of IUdR treatment time for 10e5 and 10m4M concentrations. The straight lines through the data points represent the results of regression analysis using a linear model with no intercept.

and 0.48 1 + 0.010 Gy-’ for 226Ra, 241Am, and ‘*‘I, re-

spectively. This is consistent with the change in the LETS for these photon radiations, which increase with decreasing photon energy. RBEs were calculated as the ratios of the coefficients cy.The RBEs of 24’Am and “‘1 relative to 226Ra were found to be 1.20 + 0.10 and 1.30 + 0.11, respectively (Table 3). Radiosensitization factors (RSF), calculated as ratios of the coefficients (Y for the combined radiation/drug treatments relative to radiation treatments alone, are shown in Table 3. The RSF for 241Am was higher than the RSFs for j2’I and 226Ra. For 10e5 M IUdR, the RSFs were 1.35 + 0.11, 1.67 + 0.09, and 1.47 + 0.08 for 226Ra, 24’Am and ‘251,respectively. For 10m4M IUdR, the RSFs were 1.89 * 0.16, 3.04 f 0.13, and 2.48 + 0.17 for 226Ra, 24’Am, and ‘251,respectively. These RSF data, illustrated in Figure 7, show that 60 keV photons produce greater radiosensitization than either lower energy photons (28 keV) or higher energy photons (830 keV). To evaluate the combined effects of drug and radiation, a cytotoxicity factor (CF) was also calculated, as the ratio of the coefficient (Yfor treatment with radiation plus drug relative to that for treatment with radiation alone. The CFs are shown in Table 3. The CFs are generally larger than the RSFs because the CF measures the total toxic and radiosensitizing effect of the drug, while the RSF excludes the effect of the toxicity of the drug alone.

(a) , 0

5

,

,

,

IO IS Dose (GY)

1 o-5 M

P

10’5MNJdR&Radiakm Corrected for Drug Toxicity

IWR 8 Radiin Measured Data

6)

10.4 M IUdR & Radiation Corrected for Drug Toxicity

lr.? M IUdR 8 Radiation Measured Data

DISCUSSION IUdR has recently received renewed attention as a radiosensitizer for use in the treatment of rapidly proliferating tumors located in sites with relatively quiescent doselimiting normal tissues (15, 20-22, 26, 36, 38). Clinical trials suggest that IUdR augments the response of certain tumors to external beam radiotherapy. Ongoing clinical trials are optimizing the regimens of delivery of IUdR, and are defining the pharmacokinetics, patterns of incorporation, and toxicity of IUdR. These ongoing clinical

(e) 0

Dose(GY)

5

IO

15

20

25

Do= (GY)

Fig. 4. Surviving fraction as a function of radiation dose for: (a) radiation alone using 226Ra at 0.72 Gy/hr, (b) treatment with 10e5 M IUdR in combination with CLDRI, (c) treatment with lOA M IUdR in combination with CLDRI, corrected for drug toxicity, (d) treatment with 10e4 M IUdR in combination with CLDRI, (e) treatment with 10m4M IUdR in combination with CLDRI, corrected for drug toxicity.

1382

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June 1990, Volume 18, Number 6

allow utilization of the “extra” radiosensitization resulting from the photon-induced Auger cascades. Examination of the use of halogenated pyrimidines with continuous low dose rate irradiation therefore appears warranted. Data defining the effect of halogenated pyrimidines on the efficacy of CLDRI are sparse. The only report, other than ours, known to the authors is the one by Mitchell and Phillips, noted briefly in a review by Mitchell et al. (29). This study shows that the BUdR radiosensitization factors for a high dose rate (2 Gy/min) and a low dose

AMERICIUM-241 Radntlon Alone

IODINE-125 Dose (Gy)

Radlatlon Alone 10 ’ M IUdR & Radiation Corrected for Drug Toxicity

io.5 M IUdR 8 Radlatlon Measured Data

0

5

10

15

20

25

Dose (Gy)

10 ’ M IUdR 8 Radiation Corrected for Drug Toxicity

10SMIUdRBAbne Measured Data 10 4 M IUdR 8 Radlatlon Measured Data

10 ’ M IUdR & Radlatlon

. . . \

.

J

.d),

,

,

,

5

10

15

20

Dose (Gy)

L

1O-4M IUdR 8 Alone Measured Data

25

Dose (Gy)

Fig. 5. Surviving fraction as a function of radiation dose for: (a) radiation alone using 24’Am at 0.72 Gy/hr, (b) treatment with 10m5 M IUdR in combination with CLDRI, (c) treatment with IO-’ M IUdR in combination with CLDRI, corrected for drug

toxicity, (d) treatment with 10e4 M IUdR in combination CLDRI, (e) treatment with 10m4M IUdR in combination CLDRI, corrected for drug toxicity.

[CC)

with with

dition, many brachytherapy regimens deliver large radiation doses over relatively short overall treatment times (a few days); this would allow intensive radiation to be delivered during the period of maximum IUdR incorporation. IUdR levels would have to be maintained at a high level for a shorter time period for brachytherapy than for a typical external beam treatment. Moreover, the use of isotopes emitting appropriate low energy photons would

,

,

lo.4 M IUdR 8 Radiation Corrected for Drug Toxicity

F . . . .

6

(d) !III!I

(a) 5

10 1s Dose (Gy)

20

2s

0

5

10 15 Dose (Gy)

20

5

Fig. 6. Surviving fraction as a function of radiation dose for: (a) radiation alone using “‘1 at 0.72 Gy/hr, (b) treatment with 10m5 M IUdR in combination with CLDRI, (c) treatment with 10m5 M IUdR in combination with CLDRI, corrected for drug toxicity, (d) treatment with 10m4 M IUdR in combination with CLDRI, (e) treatment with 10e4 M IUdR in combination with CLDRI, corrected for drug toxicity.

1383

Enhanced IUdR radiosensitization 0 R. NATH et al.

Table 1. Coefficient (Y’S for the effects of 10m5M IUdR and CLDRI Coefficient a(Gy-‘)

A: radiation alone B: radiation and drug C: radiation and drug corrected for drug toxicity

1251

24’Am

226Ra

Treatment

0.37 1 f 0.029

0.444 * 0.014

0.545 + 0.011

0.786 f 0.033

0.481 + 0.010 0.754 + 0.038

0.502 f 0.010

0.743 + 0.031

0.7 11 + 0.035

rate (0.028 Gy/min) were similar for V79 cells exposed to X rays. We had observed previously (30) that IUdR at a concentration of 1O-5 M produces radiosensitization factors of about 1.8 and 2.5 in Chinese hamster cells in vitro irradiated at acute dose rates by 4 MV and 250 kV (unfiltered, 0.5 mm Cu HVL) X rays, respectively. In the IUdR radiosensitization studies reported here, we observe that IUdR at a concentration of 10e5 M produces radiosensitization factors of 1.4 and 1.7 in Chinese hamster cells in vitro irradiated continuously at a low dose rate of 0.72 Gy/hr by 60 keV photons from 24’Am and 830 keV (average) photons from 226Ra, respectively. Thus, we observe lower IUdR radiosensitization by CLDRI than acute dose rate irradiation. However, these two studies were performed with slightly different protocols of IUdR infusion and data analysis; for example, cell loss and IUdR toxicity corrections were not made in the acute dose rate study (30). These technical differences between the two studies may have caused the observed differences in RSF. On the other hand, it is possible that IUdR radiosensitization dose depends upon the dose rate. Cell proliferation effects play a major role in the response of mammalian cells to CLDRI in the brachytherapy dose rate range of 0.1 to 1 Gy/hr; (25) a number of

studies have demonstrated a dose rate dependence for RBE’s. Time-dose relationships will be complex for regimens combining IUdR with CLDRI because they will depend upon the kinetics of IUdR incorporation and removal, as well as upon cell proliferation and the repair of radiation damage. The time-dose-effect relationship may also depend upon the photon energy, because induced Auger cascades behave like high LET radiations, which are relatively unrepairable and have elevated RBE’s for both cell killing and perturbation of cell proliferation. For these reasons, further radiobiological studies with IUdR and CLDRI at different dose rates of interest to radiation therapy are warranted. In the IUdR radiosensitization studies reported here, potent radiosensitization by 10e5 M IUdR (RSF’s 1.351.67) is observed for CLDRI with 226Ra,24’Am, and ‘251. Therefore, we would expect radiosensitization with any of the commercially available brachytherapy sources (‘Q, 19*Ir ‘251,‘03Pd,or 19*Au),all of which emit photons in the ene&y range 20 keV to 1 MeV represented by the three radioisotopes studied in this work. From the point of view of radiation protection, the lower energy photons have a distinct advantage over the higher energy photon emitters. Although CLDRI with all of the three radioisotopes studied here produced significant radiosensitization,

Table 2. Coefficient cy’sfor the effects of 10e4 M IUdR and CLDRI Coefficient a(Gy-‘) Treatment

226Ra

24’Am

1251

A: radiation alone B: radiation and drug C: radiation and drug corrected for drug toxicity

0.37 1 + 0.029 0.775 f 0.026

0.444 f 0.014 1.460 f 0.043

0.481 k 0.010 1.320 k 0.089

0.700 + 0.022

1.349 f 0.04 1

1.193 f 0.081

Table 3. Effect of the photon energy on the RBE and on the radiosensitization Parameter RBE relative to 226Ra Radiosensitization factor* = C/A Cytotoxicity factor* = B/A

IUdR concentration None 1O-5 M 1O-4 M 1O-5 M 1O-4 M

* Symbols A, B and C refer to the rows shown in Tables 1 and 2.

226Ra 1.oo 1.35 + 0.11 1.89 k 0.16 1.47 k 0.12 2.08 + 0. I8

obtained with IUdR 1251

24’Am 1.20 1.67 3.04 1.77 3.29

+ + k k +

0.10 0.09 0.13 0.09 0.14

1.30 1.47 2.48 1.57 2.74

-+ 0.11 + 0.08 + 0.17 + 0.09 -t 0.19

1. J. Radiation Oncology 0 Biology 0 Physics

0 l-125 I Am-241 pzzd Ro-226

IUdR

CONCENTRATION

(M)

Fig. 7. Radiosensitization factors for ‘251,24’Am and 226Ra for IUdR concentrations of 10m5and 10m4M.

24’Am photons produced greater radiosensitization than either 226Ra and ‘251;for example, using 10e4 M IUdR, 60 keV photons from 24’Am results in an RSF of 3.04 whereas 830 keV photons from 226Raproduce an RSF of 1.89, and 1251,an RSF of 2.48. This observation of higher radiosensitization for 60 keV photons compared to both lower and higher energy photons confirms our earlier conclusion that Auger electron cascades induced by photons just above the binding energy of the electrons in the K-shell of the iodine atom in the IUdR molecule can make a significant contribution to IUdR radiosensitization. Enhancement of IUdR radiosensitization by Auger electron cascades induced by low energy photons has been observed in chemical systems (7), microorganisms (17, 24), and mammalian cell tissue cultures (9-l 1, 13, 28, 30, 39). The production of Auger electron cascades by photons with energies slightly above 33.2 keV binding energy of the K shell not only amplifies the usual radio-

June 1990, Volume 18, Number 6

sensitization mechanism, but also results in the production of high LET radiations, with extremely short (subcellular) ranges, in the DNA molecule holding the IUdR. These high LET lesions are similar to those produced by ‘25I decay in ‘251-labelled IUdR, which have been studied extensively (3). The exact mechanisms for the production of radiosensitization by photon-induced Auger electrons remain to be elucidated. Use of track structure models, such as those developed by Charleton et al., (4, 5, 6, 18, 37) would be helpful in elucidating these effects by predicting quantitatively the yield of Auger electrons from the K-shell and other shells of halogenated pyrimidines as a function of photon energy. These physical data would be useful in examining the complex time-dose-response relationships for CLDRI combined with halogenated radiosensitizers. The studies reported here show that the radiosensitization obtained with IUdR at the low photon energies characteristic of certain new brachytherapy sources (12, 32, 33, 35) (24’Am and I45Sm) is actually greater than that obtained with higher, more conventional photon energies. The studies reported here used a very artificial model system of Chinese hamster cells in rapid exponential growth in vitro, exposed to relatively high IUdR concentrations and incorporating relatively large amounts of IUdR to assess whether radiosensitization and the extra radiosensitization resulting from induced Auger cascades occurred with IUdR and CLDRI. More clinically relevent in vivo radiobiology studies with animal models, using IUdR with low energy photons, such as those from 14’Srn,24’Am and ‘251,are warranted. These should examine further the incorporation of IUdR into the cells of tumors and normal tissues, the heterogeneity of the IUdR incorporation, the toxicity of the drug, and the effect of IUdR on the response of tumors and on the therapeutic ratio.

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