5-[123i]iodo-2′-deoxyuridine in the radiotherapy of an early ascites tumor model

Int. J. Radiation Oncology Bid. Phys., Vol. 21. pp. 1541-1551 03603016/91 $3.00 + .oo

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??Biology Original Contribution

5-[1231]IODO-2’-DEOXYURIDINE IN THE RADIOTHERAPY OF AN EARLY ASCITES TUMOR MODEL

JANINA BARANOWSKA-KORTYLEWICZ, PH.D., G. MIKE MAKRIGIORGOS, PH.D., ANNICK D. VAN DEN ABBEELE, M.D., ROBERT M. BERMAN, B.A., S. JAMES ADELSTEIN, M.D., PH.D.

AND AMIN I. KASSIS, PH.D.

Department of Radiology (Nuclear Medicine), Shields Warren Radiation Laboratory, Harvard Medical School, 50 Binney Street, Boston, MA 02115

The extreme biological toxicity of Auger emitters is caused by the decay-associated, highly localized deposition of energy. The antineoplastic capability of an Auger-electron emitter, iodine-123, incorporated into the thymidine analog, 5-iodo-2’-deoxyuride (IUdR) was evaluated in an intraperitoneal (Lg) murine ovarian tumor (MOT) ln female C3HeB/FeJ mice. Total doses of 0.37 to 8.88 MBq (lo-240 uCi) IUdR were administered i.p. in five equally divided fractions at 24, 28, 32, 36, and 40 hr after the i.p. inoculation of 0.5 to 1.6 x lo6 tumor cells per mouse. Control tumor-bearing animals were injected with identical volumes of saline at 4-hr intervals. Biodistrlbutlon studies demonstrated a distinct and localized uptake of lUIUdR in the MOT cells (1 % of the injected dose was associated with MOT cells 24 hr after the last injection), whereas in animals without tumor there was no radioactivity associated with the peritoneal cells. Analogous results were obtained from scintigraphic images where the focal area of abdominal activity persisted only in MOT-bearing mice while it cleared from the abdomen of the controls. The 50% survival (median survival) of the control group was 19 days for an inoculum of 1.6 x lo6 MOT cells per animal, whereas the median survival of MOT-bearing animals treated with ?UdR increased by 11 days for the highest administered dose (8.88 MBq, 240 l&i) and resulted in a 20% absolute survival at 7 weeks. Statistically significant absolute survival prolongation was found with all of the total administered doses. The prolongation of both median and absolute survival time of the tumor-bearing animals treated with ‘=IUdR conclusively indicates the substantial antineoplastic activity of the Auger-electron emitter iodine-123.

5-Iodo-2’-deoxyuridine, Auger-electron emitters, Iodine radioisotopes, Radiotherapy, Murine ovarian tumor, Biodistribution, Scintigraphy, Dosimetry.

INTRODUCTION

The Auger-electron-emitting radioisotope, iodine- 125, pos- sesses significant cytotoxic activity when located in the cell nucleus as 5-[ ‘251]iodo-2’-deoxyuridine [ ‘251UdR] (5-7, 22). The radiotoxicity of 1251UdR incorporated into DNA has been demonstrated in cell-survival studies, by chromo- somal aberration and DNA strand-break measurements in mammalian cells in vitro, and by diminished survival of 1251UdR prelabeled cells assayed in vivo (e.g., 8, 23, 24). Furthermore, the radiotherapeutic effects of 1251UdR using increased life span as a criterion have been established. The site-specific delivery of this radiopharmaceutical has resulted in remarkable tumor cell killing and total eradica- tion of an ascitic tumor (5-7).

Unfortunately, the sensitivity of normal cell renewal systems to DNA-bound Auger-electron emitters is a major

disadvantage of 1251UdR in cancer therapy. 5-[ 1251]Iodo- 2’-deoxyuridine is incorporated into the DNA of all rapidly proliferating tissues, including intestinal epithelium and bone marrow (10, 13, 21, 37). Two months after 1251UdR administration (ll), its presence in cells with a long life span, such as liver and kidney, has been detected by auto- radiography. When given to mice less than 1 week old, an appreciable amount of the label is retained over a year (11). Although a blood-brain barrier to ‘251UdR has been reported (35), radiolabeled microglial cells were found in the central nervous system of animals injected subcutaneously with 1251UdR after peripheral nerve injury (40). While uptake of 1251UdR for DNA synthesis is approximately 5 to 10 times less than that of tritiated thymidine (26, 34), up to 10% of 1251UdR liberated by dead tumor cells is report- edly reutilized by dividing cells (9, 19, 20, 36), further escalating the risk of normal tissue exposure. Thus, the

This work was supported by NIH Grant ROl CA 15523-15. Presented in part at the Society of Nuclear Medicine 36* An-

School, Shields Warren Radiation Laboratory, 50 Binney Street, Boston, MA 02115.

nual Meeting, St. Louis, MO, 13-16, June 1989. Accepted for publication 27 March 199 1. Reprints requests to: Amin I. Kassis, Ph.D., Harvard Medical

1541

1542 I. J. Radiation Oncology 0 Biology 0 Physics November 1991, Volume 21, Number 6

relatively long (60-day) half-life of 1251 poses problems when considered in the context of radiation/radionuclide therapy. 1251UdR therapy in humans has been reported in which chemical modulation of DNA synthesis was applied with some success to decrease incorporation of the drug into normal tissue (2).

Although treatment of cancer by any type of radiotherapy is necessarily a compromise between causing maximum tumor damage and minimizing normal tissue exposure, in the case of Auger-electron emitters the odds can be improved by selecting an appropriate short-lived isotope. Iodine-123, with its half-life of 13.2 hr, also emits Auger electrons; approximately 11 are expected per decay (39) compared with 20 for 1251. 1231UdR, like 1251UdR, is selective in labeling only cells synthesizing DNA, and its in vitro radiotoxicity to mammalian cells has been demonstrated (31). Although there are some disadvantages to working with a short half-life isotope like iodine-123 (i.e., the synthesis and purification of the radiopharmaceutical must be simple and rapid), there is a good probability that the isotope will decay while still incorporated in the DNA. Moreover, with its considerably shorter half-life compared with 1251UdR, the period of normal tissue exposure to 1231UdR could be substantially reduced, and the fraction of 1231UdR available for reutilization could be significantly less. Thus, 1231UdR presents several advantages for cancer endoradiotherapy with unsealed, internally emitting radionuclides .

taneously in C3H female mice (18). It was maintained by serial intraperitoneal (i.p.) transplantation in its ascitic form in female C3HeB/FeJ mice. Cells were routinely har- vested by peritoneal lavage with physiological saline containing 4 U/mL heparin. Mice showing marked abdominal distention were selected at random. Their ascitic fluid was combined and centrifuged at 650 X g. The cell pellet was suspended in physiological saline and lo5 to lo6 MOT cells were injected i.p. into experimental animals (0.2 to 0.6 ml/mouse). The viability of injected cells as determined by a trypan blue exclusion assay was routinely > 95%.

Therapy studies

The objective of this study was to determine the antineoplastic potential of 1231UdR in an experimental murine ascites tumor model following intraperitoneal administration of this radiopharmaceutical using a system in which the tumor cells are directly exposed to the radiohaloge- nated pyrimidine deoxynucleoside (5).

METHODS AND MATERIALS

Chemistry No-carrier-added Na1231* and Na1251t. were purchased.

5-[ 1231]- and 5-[ 1251]Iodo-2’-deoxyuridine were synthesized as described previously (3) and used after purification on a C,, reverse phase hplc column.

1231UdR is effective in killing cells only when it is incorporated into the newly formed DNA molecules either during the S period or at any time that the DNA molecules of a cell are being replicated. Therefore, it was important to synchronize the dose fractionation with the cell cycle. Since the growth fraction of a l-day-old ascites tumor approaches unity (7), all cells can theoretically be labeled by selecting an appropriate dose regimen. The total cell cycle of the MOT cells is 14.3 hr with a DNA synthetic phase of 5.8 hr (6). Accordingly, various doses of carrier-free ‘231UdR (0.37-8.88 MBq/mouse; lo-240 @i/mouse) in physiological saline were administered i.p. in five fractionated 0.5 mL injections 24 hr after inoculation of 0.5 to 1.6 x lo6 MOT cells (10 animals per group). The interval

between doses was 4 hr. Control animals, inoculated with an identical number of MOT cells, were injected i.p. every 4 hr with a volume of saline equal to that of the 1231UdR injection. Survival time and development of ascites were recorded for each mouse by daily inspection up to 7 weeks. All deaths were associated with ascites forma- tion (only one animal in a group of over 120 developed a solid, subcutaneous tumor at the site of inoculation 6 weeks after the MOT cell injection). Median and absolute survival of animals was determined and the surviving fractions of tumor cells were calculated as described previously (7). Therapeutic response was evaluated by the prolongation of the absolute survival using the Wilcoxon signed ranks test.

Biodistribution studies

Animals Female 5-week-old C3HeB/FeJ mice+ were given food

and water ad libitum. Starting 2 days before injection of 1231UdR and continuing through the first 10 days after treatment, their drinking water was supplemented with a 0.1% solution of potassium iodide to prevent thyroidal accumulation of radioiodide. All animals were of the same age and sex, and of similar weight (about 22 g).

Tumor model The ascites cell line studied, an ovarian embryonal cell

carcinoma (murine ovarian tumor, MOT), originated spon-

Iodine- 125-labeled-IUdR biodistribution studies were conducted on mice inoculated with 5 X lo5 MOT cells. Animals were treated i.p. every 4 hr with five equal doses of 1251UdR (total dose 0.30-0.37 MBq; 8-10 $i>. Two to 120 hr after the injection of the final dose, mice were anesthetized with ether, bled through the periorbital plexus, and then sacrificed by ether asphyxiation. The abdominal skin was removed and heparinized saline (4 U/mL; 3 mL) was injected i.p., dispersed throughout the abdominal cavity by gently massaging the flanks of the mouse, and aspirated with the same syringe to remove MOT cells. The volume of MOT suspension from each animal was measured. At

*Nordion International Incorporated, Kanata, Canada. tNew England Nuclear, North Billerica, MA.

*Jackson Laboratories, Bar Harbor, ME.

‘231UdR in cancer radiotherapy 0 J. BARANOWSKA-KORTYLEWICZ et al. 1543

Table 1. Biodistribution studies of ‘?UdR in MOT-bearing micea

Percent injected dose per gram wet tissueb: time following last injection of ‘?UdR T 112

2hr 4hr 8hr 24 hr 120 hr (hr)E

Blood 1.28 (0.11) 0.57 (0.17) 0.23 (0.09) 0.04 (0.01) 0.00 4.8 (0.9) Bone 0.67 (0.07) 0.49 (0.08) 0.28 (0.09) 0.08 (0.02) 0.02 (0.01) 7.6 (0.8) Brain 0.04 (0.02) 0.03 (0.01) 0.01 (0.01) 0.00 0.00 5.6 (0.6) Heart 0.44 (0.07) 0.19 (0.04) 0.10 (0.04) 0.02 (0.01) 0.00 5.1 (0.8) Intestine (large) 1.54 (0.71) 1.51 (0.83) 0.98 (0.53) 0.89 (0.48) 0.16 (0.05) 38.2 (3.5)d Intestine (small) 2.22 (0.15) 1.74 (0.78) 2.10 (0.60) 1.54 (1.41) 0.13 (0.07) 28.9 (1.9)d Kidney 0.83 (0.25) 0.47 (0.22) 0.20 (0.05) 0.07 (0.01) 0.03 (0.01) 6.5 (1.5) Liver 0.42 (0.07) 0.20 (0.06) 0.11 (0.03) 0.04 (0.01) 0.01 (0.01) 7.3 (1.9) Lung 0.80 (0.07) 0.46 (0.10) 0.18 (0.06) 0.06 (0.01) 0.02 (0.00) 6.4 (1.5) Muscle 0.25 (0.04) 0.16 (0.04) 0.10 (0.03) 0.03 (0.02) 0.01 (0.00) 8.1 (1.2) Skin 0.84 (0.12) 0.80 (0.08) 0.54 (0.23) 0.34 (0.21) 0.08 (0.04) 17.4 (3.3) Spleen 1.60 (0.19) 1.53 (0.21) 1.03 (0.18) 0.43 (0.07) 0.11 (0.01) 11.5 (0.8) Stomach’ 8.14 (2.18) 6.22 (1.82) 2.24 (1.12) 0.18 (0.05) 0.07 (0.02) 4.0 (0.4) Thyroid’ 1.56 (0.37) 0.85 (0.14) 0.69 (0.16) 0.05 (0.01) 0.02 (0.01) 4.5 (0.3) Uterus 0.52 (0.08) 0.43 (0.12) 0.30 (0.11) 0.20 (0.07) 0.11 (0.08) 17.7 (4.7)

“Each animal received i.p. five fractionated doses of 0.06 MBq (1.6’* pCi) ‘251UdR every 4 hr; biodistribution studies were initiated 2 hr following last injection.

bMean (standard deviation), SD values smaller than 0.005% were omitted, SD values between 0.005% and 0.009% were rounded to 0.01%.

‘Mean (standard deviation) of the fast component unless otherwise indicated: mean values of half-lives were obtained from the initial loss rate constants; n=5 for each time point.

dMonophasic clearance rate. ‘Including its content. fIncluding surrounding tissue.

least 98% of the saline injectate and abdominal fluid was recovered. The cell number was determined in an elec- tronic particle counter,* and the cell-size distribution was analyzed using a multi-channel particle size analyzer.* During these procedures, the cells were kept on ice. Ali- quots of the MOT cell suspensions were centrifuged at 650 x g, the cell pellets were washed twice with saline and the tumor-cell-associated radioactivity measured in a gamma counter.? A group of healthy animals (n= 10) was treated in the same manner to determine radioactivity associated with normal peritoneal cells. Dissected tissues (listed in Table 1) were rinsed three times in physiological saline, blotted dry, weighed, and their radioactivity was determined in a gamma a counter.

Scintigraphy Gamma camera imaging was performed at 1 or 2 hr and

16 to 24 hr after a single i.p. injection of 1231UdR (11.1 MBq/mouse; 300 pCi/mouse) using a standard-field-of- view scintillation cameraS equipped with a medium energy, parallel hole collimator. Two groups of animals were imaged, mice bearing MOT tumor cells (n= 3) injected 24 hr earlier and control animals (n=2) injected with a volume of sahne equivalent to that of the MOT cell inoculum. No attempt was made to block the thyroid up-

take of 1231. Animals were anesthetized (pentobarbital, 1 mg/mouse, i.p.) and placed prone (anterior view) on the collimator. For the early time points (1 or 2 hr), static images were acquired for a preset total of 300,000 counts while the later time points (16 or 24 hr) were recorded for a fixed time of 10 min. A 128 x 128 matrix and a mag- nification of four were used. Regions of interest (ROI) were drawn over the abdomen (target) and the entire animal (total body counts) to measure serial changes. An identical area was analyzed for each animal at the two time points. Background activity was assessed in the shoulder area and subtracted from the ROI. A mean activity per pixel was derived -for each ROI, and the percentage of residual activity remaining in the target calculated using the following equation: residual activity = (total counts in the target - background)/(total body counts - background).

Dosimetry in mice and extrapolation to humans Organ dosimetry. Approximate radiation doses in hu-

mans for ‘231UdR/‘251UdR administered i.p. (five injections at 4-hr intervals) were derived by extrapolation of the mouse biodistribution data and by using the standard MIRD tables (41). The dose was calculated as follows:

1. The average radioactivity levels in the mouse organs

*Coulter Electronics Incorporated, Hialeah, FL. tPackard Instrument Company, Sterling, VA.

.+Starcam, General Electric Company, Milwaukee, WI.

I. J. Radiation Oncology 0 Biology 0 Physics November 199 1, Volume 21, Number 6 1544

z 100

6 8 a 60

a .$ ? 60

r3 0 2 40

2 2 20

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3 4 5 6 7

Weeks After Last ‘a31UdR Injection

Fig. 1. Absolute survival of host animals as measure of response of ascitic murine ovarian tumor in viva to therapeutic doses of 1231UdR; 10 mice per group were inoculated with 1.6 x lo6 MOT cells per animal 24 hr prior to first ‘231UdR injection; ‘231UdR was administered in five equal doses every 4 hr. Con- trol mice were injected every 4 hr with volume of saline equal to that of ‘231UdR injection. For clarity only curves for four doses are shown.

2.

were expressed as a fraction of the injected radioactivity per gram of tissue of interest. The mouse organ cumulated activity (Bq X hr) was calculated by integrating time-activity curves derived from the biodistribution data 20 to 96 hr following the first injection of radioactivity. Cumulated radioactivity in the period 0 to 20 hr was estimated in the absence of biodistribution data using the following assumptions: (a) after each injection of 1251UdR, the radionuclide local-

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18 0 50 100 150 200 250

Total Injected Dose (pCi)

Fig. 2. Median survival curves for mice treated with therapeutic doses of 1231UdR 24 hr after i.p. injection of 1.6 X lo6 MOT cells; 10 mice per group; k231UdR administered i.p. in five fractionated doses every 4 hr.

3.

4.

izes immediately in the organ of interest (this approximation is adequate for small molecules like IUdR that diffuse rapidly); and (b) the activity in each organ in the 4-hr period between injections follows the clearance pattern observed during the 20-to-24-m time period for which the experimental data are available. In other words, the biological half-life of the radionuclide in each organ is constant throughout the experiment. The extrapolation of accumulated radioactivity in human organs for a total injected dose of 37 MBq (1 mCi) was based on a commonly used assumption (12) that the relative uptake per gram of tissue is proportional to the total body weight in both humans and mice. Calculation of the dose (Gy) to human organs from activity outside each organ was based on the assumption of uniform distribution of the remaining radioactivity in the whole body. Summation of the activity associated with each organ (corrected for the body mass not collected during dissection) gave the approximate total remaining activity. From this, absorbed doses for each human organ were calculated using standard MIRD tables (41). Calculation of the corresponding doses from 1231UdR was based on the ‘*‘IUdR biodistribution studies since the biological behavior of both compounds is identical, the only difference being the physical half- life of the radionuclide.

Tumor dosimetry. Radiation dose estimates to the MOT cells in mice were derived from the activity measured in the collected ascites using the following assumptions:

The only cells taking up ‘251UdR/1231UdR in the peritoneum are MOT cells (this assumption is based on the experimental data obtained from the biodistribution studies conducted on MOT-bearing mice and mice without tumor; see Results). For the first 24 hr following inoculation with MOT, the cells do not divide; after this time all MOT cells divide continuously with a doubling time of 14.3 hr (6). This may be valid only for a total dose of 0.30 MBq (8 pCi) used in this study where it was established that the number of cells recovered from the peritoneal cavity at various time points correlated well with numbers expected for a logarithmically growing cell population. This assumption may be an underestimate for other total doses since it is known that in the presence of 1251UdR the cell doubling time increases (31), and 1251 decays in the DNA cause massive G, arrest even after relatively low doses (25 and references cited therein). The effect of this assumption on the dosimetry is as follows: if the doubling time of MOT cells were to increase to 28.6 hr (twice that without radiation), the effective half-life would be about 6.5 hr, causing the total dose received by MOT cells to increase by 35%. All radioactivity associated with MOT cells is DNA bound.

lz31UdR in cancer radiotherapy 0 J. BARANOWSKA-KORTYLEWICZ ef al. 1545

The average cumulated radioactivity (i.e., the total number of decays) in MOT cells was derived by integration of the time-activity curves (see above). The methods of Kassis er al. (29,31) were then used to convert the number of 123Y 12’1 decays in the cell nucleus to radiation dose. An MOT cell diameter of 13 p,m, as measured with a multi-channel particle size analyzer, was used for these calculations. It was deduced that each 1231 and 1251 decay in the nucleus contributes 4.5 x 10e3 and 1.0 x 10e2 Gy per decay, respectively, to the total nuclear dose.

RESULTS

Following i.p. transplantation of MOT cells (1.6 X 106/ mouse; n = lo), all control animals died within 24 days. Typical absolute survival curves for treated animals are shown in Figure 1. Statistically significant absolute survival prolongation was found with all of the total administered lz31UdR doses. For example, the null hypothesis (no significant difference between two sets of paired measurements: treated versus control mice) was rejected at the 5% level for animals treated with total doses of 0.37 MBq (10 p,Ci) and 0.56 MBq (15 PCi) 1231UdR compared to control mice injected with saline (results not shown). With total doses greater than 0.56 MBq, the null hypothesis was rejected at the 0.01% level. No significant differences in the absolute survival prolongation for animals injected with 7.03 MBq (190 &i) and 8.88 MBq (240 pCi) doses were found at the 20% level.

Similar results were obtained from a comparison of the median survival (50% survival) of animals treated with ‘231UdR and of control groups (Fig. 2). The 50% survival

of untreated mice inoculated with 1.6 X lo6 MOT cells was 19 days, whereas with a total dose as low as 0.93 MBq (25 PCi), one-half of the treatment group was still alive after 24 days. Five doses of 1.48 MBq each (5 X 40 p,Ci) prolonged median survival to 30 days and resulted in 20% disease-free survival at 7 weeks.

The ascites in the control mice was visible 7 days after tumor cell inoculation. Animals treated with therapeutic doses of 1231UdR began to manifest tumor 4 to 42 days later depending on the amount of injected radioactivity. All mice, regardless of the administered dose, appeared healthy and alert before development of ascites.

Since the biological behavior of ‘231UdR and ‘251UdR is identical, biodistribution studies were performed with ‘251UdR (the longer half-life of this isotope facilitates de- termination of the organ-accumulated radioactivity). Bio- distribution studies were initiated 2 hr following the last of five i.p. injections of 1251UdR. The results are shown in Table 1 (percent injected dose/gram wet tissue, %ID/g) and Table 2 (percent injected dose/organ). Maximum or near maximum incorporation of 1251UdR into all of the exam- ined tissues was observed within 2 hr after the last injection. Relatively high levels of radioactivity persisted in spleen, uterus, and intestine for 24 hr.The clearance of 1251 from bone, brain, heart, kidney, liver, lung, muscle, stomach, and thyroid was virtually parallel to the blood radioactivity levels. Blood clearance studies indicated that after 4.8 hr, 50% of the initial radioactivity was still in circulation but after 120 hr the radioisotope was not detectable in blood. Elimination of 1251UdR from these organs can be divided into fast (i.e., in equilibrium with the blood; 2 to 24 hr post-injection) and slow (later than 24 hr post-injection) components. Similar two-stage clearance rates were

Table 2. Distribution of iodine-125 in organs and tumor of MOT-bearing micea

Percent injected dose per organb: time following last injection of ‘*‘IUdR

2 hr 4hr 8hr 24 hr 120 hr

Brain 0.02 (0.01) 0.01 0.01 0.00 0.00 Heart 0.04 (0.01) 0.02 0.01 0.00 0.00 Kidney 0.25 (0.11) 0.13 (0.06) 0.05 (0.01) 0.02 0.01 Liver 0.50 (0.15) 0.25 (0.07) 0.12 (0.03) 0.04 (0.01) 0.02 Lung 0.11 (0.01) 0.06 (0.02) 0.04 (0.01) 0.01 0.00 Spleen 0.13 (0.02) 0.12 (0.02) 0.09 (0.02) 0.04 (0.01) 0.02 Stomach’ 2.40 (0.40) 1.61 (0.66) 0.75 (0.29) 0.04 0.02 Thyroidd 0.17 (0.05) 0.08 (0.04) 0.07 (0.03) 0.01 0.00 Uterus 0.06 (0.02) 0.05 (0.01) 0.03 (0.02) 0.02 0.01 Tumor” 0.94 (0.22) 1.15 (0.31) 1.06 (0.27) 0.88 (0.08) 0.31 (0.14)

“Each animal received i.p. five fractionated doses of 0.06 MBq (1.6 @i) lz51UdR every 4 hr, biodistribution studies were initiated 2 hr following last injection.

bMean (standard deviation), SD values smaller than 0.005% were. omitted, SD values between 0.005% and 0.009% were rounded to 0.01%.

‘Including its content. dIncluding surrounding tissue. ‘Percent injected dose per peritoneal cell content was estimated from radioactivity associated with cell pellets obtained as described in

the Experimental Section; means were calculated from percent injected dose values of all tumors in two independent groups of animals; n = 10 for each time point.

1546 I. J. Radiation Oncology 0 Biology 0 Physics November 199 1, Volume 21, Number 6

i ‘.,, o..

1 100

Hours Postinjection

Fig. 3. Radioactivity associated with MOT cells (0) and endog- enous murine macrophages (V) recovered from peritoneum of mice treated i.p. with five equal doses of ‘*?UdR every 4 hr (0.06 MBq [1.6 p,Ci] per injection); MOT-bearing animals (n = 10) were inoculated i.p. with tumor cells (106) 24 hr prior to the first ‘*‘IUdR injection; healthy mice (n = 5) were given i.p. equal volumes of saline prior to the first ‘*‘IUdR injection.

observed for skin and spleen, although the loss rate constants (k) of the fast component were notably lower, 0.040 hr-’ (SD 0.0075) and 0.060 hr-i (SD 0.0044), respectively. The kinetics of the distribution of radioactivity in the liver also had a two-stage character; however, the first rapid phase appeared to last only about 8 hr corresponding to k = 0.220 hr-’ (SD 0.050) with the half-life (T,,,) 3.2 hr (SD 0.60). The slow component had a loss rate constant of 0.015 hr-’ (SD 0.0061) and T,,, 44.9 hr (SD 12.0). This suggests that labeling involved reticuloendothelial

rather than hepatic cells (42). Similarly, a fast initial phase existed for the 1251 clearance from the uterus (T,,* 7.3 hr, SD 0.14; and k = 0.0955 m-i, SD 0.0019), followed by a slow decline of radioactivity levels (T,,, 86.1 hr, SD 17.0; and k = 0.0081 hi-‘, SD 0.0018). Larger variations observed for uterus were probably related to the estrus cycle, and those for skin were possibly due to errors result- ing from urine contamination. Small and large intestine seemed to eliminate the tracer in a monophasic fashion over the entire period of analysis, with a loss rate constant of 0.024 hr-’ (SD 0.0015) for the small intestine and 0.018 hr-’ (SD 0.0017) for the large intestine. This might represent rapid, initial labeling of the DNA of mucosal cells and their slow migration to the tips of the villi where they are sloughed into the lumen (25).The ‘251UdR half- lives for all organs are listed in Table 1.

There was a marked accumulation of radioactivity in tumor cells; for example, at 8 hr after the last of five administered doses, 1.06% ID (SD 0.27) was still associated with tumor cells (Fig. 3, Table 3), whereas the cell-associated radioactivity in the control animals treated with identical amounts of 1251UdR was detectable only at 2 hr (0.16% ID, SD 0.08). Measurements at 2 hr showed that the MOT-cell-associated radioactivity comprised about 37% of the total peritoneal 1251. Later time points reflected most likely the radioactivity already incorporated into the DNA since the total and cell-bound tracer levels were equal within the limits of experimental error. Elimination of “‘IUdR and/or its degradation products from the tumor, analyzed using the concentration of the label within the cells (Bq/cell), presumably reflected the fate of the radio- actively labeled tumor DNA. It was a slow, single-step process with a half-life of 20.3 hr (SD 0.7) and an activity loss rate constant of 0.034 bt- ’ (SD 0.0010). On the ba- sis of these data the cellular dosimetric calculation gave a total of 0.158 Gy (15.8 decays) and 0.037 Gy (8.2 decays) in MOT cells for 1251UdR and ‘231UdR, respectively, for the injected dose of 3700 Bq (0.1 $i).

Table 3. Distribution of iodine-125 in the peritoneum of healthy and MOT-bearing mice after intraperitoneal injections of ‘251UdR”

Time post iniection (6

Percent of injected ‘*‘I activity associated with cell pelletb

MOT Healthy

Percent of total peritoneal “‘1 activity associated with

cell pelletb

MOT Healthy

pCi/cell

MOT

2 0.94 (0.22) 0.16 (0.08) 37 (18) 39 (21) 0.020 (0.0085) 4 1.15 (0.31) 0.01 (0.01) 94 (32) 3 (3) 0.024 (0.0072) 8 1.06 (0.27) 0.05 (0.02) 83 (24) 31 (20) 0.018 (0.0035) 24 0.88 (0.08) 0.04 (0.03) 100 (17) 0 0.011 (0.0021) 120 0.31 (0.14) 0 97 (18) 0 0.0004 (0.0001)

“Each animal received i.p. five fractionated doses of 0.06 MBq (1.6 PCi) ‘251UdR every 4 hr; biodistribution studies were initiated 2 hr following last injection.

bMean (standard deviation), SD values smaller than 0.005% were omitted, SD values between 0.005% and 0.009% were rounded to 0.01%; percent injected dose per peritoneal cell content was estimated from radioactivity associated with cell pellets obtained as described in the Experimental Section; means were calculated from percent injected dose values of all tumors in two independent groups of animals; n = 10 for each time point.

‘231UclR in cancer radiotherapy 0 J. BARANOWSKA-KORTYLEWICZ et al. 1547

Fig. 4. Scintigraphic images obtained in normal mice and mice bearing MOT tumor cells i.p. 1 hr (A) and 24 hr (B) after i.p. injection of 1231UdR. Peritoneal tumor uptake is indicated by TU. Thyroid (TH) uptake is observed since mice were not placed on KI supplement.

The dosimetry for human organs obtained by extrapolation of the mice biodistribution data is shown in Table 4 for lz31UdR and 1251UdR for an injected activity of 37 MBq (1 mCi). There is a reduction in dose with 1231 relative to 125I because of its short half-life.The small intestine appears to receive the highest cumulative dose (1.35 X lop4 and 5.39 X 10T4Gy for 1231UdR and ‘251UdR, respectively). None of these doses exceeds currently acceptable limits.

Tumor-to-normal-tissue ratios derived from the biodistribution results were very favorable, in particular 24 hr after the last injection, ranging from 20 for organs with actively proliferating cells (uterus, intestine, stomach) to

0 50 100 150 200 250

Total Injected Dose (@Ii)

Fig. 5. Dose-response function of MOT cells in viva to therapeutic doses of ‘231UdR. Solid circles and solid line repesent mice (n= 10 per data point) inoculated with 1.6 x 10 MOT cells treated with five fractionated doses of ‘231UdR every 4 hr; dashed line taken from Bloomer & Adelstein (6) is shown for comparison and contains ‘%klR therapy results (seven fractionated doses at 4-hr intervals; lo5 tumor cells/animal).

over 400 for those with few dividing cells (e.g., brain, heart).

Analogous results were obtained from the scintigraphic images acquired 1, 2, 16, and 24 hr following a single injection of 11.1 MBq (300 l&i) ‘231UdR. At 1 hr (Fig. 4A) the focal localization of radioactivity was observed in the abdomen of both tumor-bearing mice (15.77% residual activity [RA] in the target, SD 3.39) and control animals (Fig. 4B; 14.60% RA, SD 1.36). At later time points (Fig. 4B), the focal area of abdominal activity persisted only in MOT-bearing mice (15.77% RA, SD 3.08) while it cleared

Table 4. Calculated average doses to human organs following five I.P. injections of ‘231UdR/L2sIUdR (total 37 MBq) at 4-hour intervals

Target organ Self

‘251UdR doses

Total

lz31UdR doses

[Gy] X 1O-6 Self Total

Stomach 325 336 119 129 Thyroid 64 74 39 46 Spleen 189 201 66 76 Lung 47 59 24 33 Liver 33 41 20 29 Kidney 58 69 27 36 Bone 25 41 14 25 Large intestine 248 260 95 105 Small intestine 527 539 124 135

1548 I. .J. Radiation Oncology 0 Biology 0 Physics November 199 1, Volume 2 1, Number 6

from the abdomen of animals without tumor (3.69% RA, SD, 0.75), confirming the biodistribution data.

DISCUSSION

The decay of the Auger-electron emitter iodine-123, in the form of ‘231UdR, produces lethal effects that surpass expectations based on classical dose calculations, when the radionuclide is incorporated into the DNA of mammalian cells. Previous work from our laboratory (31) showed that ‘231UdR is seven to eight times more effective in causing clonogenic death than 280-kVp X rays per unit of energy absorbed in the nucleus, and its mean lethal dose to the nucleus is about the same as that obtained with ‘251UdR. These studies suggest that ‘231UdR might present a prom- ising new approach to the treatment of malignant disease.

Using the peritoneal cavity as a model tumor site, the therapeutic usefulness of 1231UdR has been evaluated in vivo. The magnitude of the therapeutic response to 1231UdR treatment greatly depends on the extent of thymidine replacement by this analog, which in turn is related to the distribution of cell generation times within the tumor cell population and to the administered dose. Studies of cell proliferation kinetics indicated that, in general, the time for 99% of viable cells to pass through at least one S phase is 1.5 or more times the mean cell cycle, but a definition of the distribution of cell cycle times could not be made (33). There appear to be no data showing the relative time for 99.9% or more of the cells of any mammalian line (in vitro or in vivo) to complete one cell cycle. Nevertheless, it was expected that in early ascites tumor where the growth fraction approaches unity (7), labeling of a large portion of the tumor cell population could be accomplished by matching dose fractionation to the cell cycle. Intraperitoneal administration of ‘231UdR allowed direct access of the agent to ascites cells before absorption into the systemic circulation and hepatic degradation, further increasing the probability of reaching a large fraction of tumor cells. From the prac- tical standpoint, five divided doses administered every 4 hr (1.5 cell cycles) presented a rational approach to extending treatment to the majority of actively cycling cells without producing overt signs of normal tissue toxicity.

Calculated surviving-cell-fraction values are probably affected by the cell division delay (25, 31). However for the sake of comparison with previously published data (5- 7), the dose-response curve was determined from the standard median survival curve as described in reference 7. This hypothetical dose-response curve (Fig.5) derived from the median survival data indicates that although the opti- mum time-dose fractionation was not achieved, five ‘231UdR injections (7.03 MBq or 8.88 MBq) eradicate over 99.9% of the tumor cell population (it is assumed that the treatment did not alter those tumor cells destined to survive.) It is plausible that surviving cells were not in their DNA synthetic phase (i.e., generation times exceeded the time course of therapy) and most likely did not reach the G, phase either, since, as noted by Iliakis (28), the repair of sublethal damage by cells in the G, phase involves the

synthesis of DNA. From the doubling time of MOT cells (14.3 hr), one would expect only 7 to 8 days prolongation of the generation time when 99.9% cells were killed. Yet, the 50% survival time was increased by 11 to 12 days for the highest administered doses. The difference may be ex- plained by the cell division delay following the treatment or by a 2-day error observed for several values on the standard median survival curve. Augmentation of the observed therapeutic effect by host defenses is rather unlikely considering the increase of median survival of the treated animals by 5 days even with doses as low as 0.93 MBq (25 pCi), whereas a tenfold decrease in the cell inoculum of untreated animals led to only 24- to-48 hr prolongation of the median survival time (7). It is also apparent that the therapeutic effects due to the chemical toxicity of IUdR play a role in either tumor development or survival of tumor-bearing hosts, because even with the highest delivered dose, the amount of IUdR (high specific activity > 8510 TBq/mmol, [230,000 Ci/mmol], “no-carrier added” prep- aration) is at least 10,000 times lower than the quantity expected to exhibit minimal chemotoxic effects (4, 16).

As stated earlier, the therapeutic response to DNA pre- cursors is also dose-dependent. In a study of the therapeutic potential of 1251UdR, Bloomer and Adelstein (6) have shown that the logarithm of the surviving-cell fraction is directly proportional to dose from about 0.74 MBq (20 l&i) to 5.18 MBq (140 FCi) 1251UdR, reaching a plateau at higher doses (surviving fraction of about 10P5; seven treatments at 4-hr intervals; Fig. 5, dashed line). On the other hand, the region of proportionality for 1231UdR appears to extend between total doses of 0.37 MBq (10 l&i) and 7.03 MBq (190 pCi; five treatments every 4 hr; Fig. 5, solid line). The existence of proportionality reflects an increased number of cells being lethally irradiated as a result of increasing ‘231UdR dosage (only a few ‘23I decays are necessary to cause cell death in vitro (31)). The Wilcoxon signed ranks test performed on the absolute and median survival data did not demonstrate statistically significant differences between the two highest administered doses indicating that for ‘231UdR a plateau may have been reached with 7.03 MBq (190 l&i) and higher doses. The plateau in the dose-response curve is most likely an indi- cation of the heterogeneity of cell cycle times (i.e., the presence of “nonproliferative” cells, those that did not en- ter the S phase during the course of treatment). It is possible that similar to the case with ‘251UdR (6), an increase in the number of doses might well have depressed the plateau.

Although the primary objective of this study was to demonstrate the antineoplastic effect of ‘231UdR, biodistribution studies were conducted to evaluate the degree of preferential uptake of this radiopharmaceutical by tumor cells. After intraperitoneal injection of 5 X 0.06 MBq (5 x I .6 l&i) 1251UdR per mouse, about 1% of the administered radioactivity was incorporated and remained associated with tumor cells for at least 24 hr. The essential assumption in the estimation of cell-bound radioactivity

‘231UdR in cancer radiotherapy 0 J. BARAN~W~KA-K~RTYLE~I~~Z et al. 1549

was that the cell number was not decreased at the time of measurement due to the radiotoxicity of nuclear lz51. The number of cells recovered from the peritoneal cavity of experimental mice at various times correlated well with values predicted for a logarithmically growing cell population with the exception of 26 hr after MOT inoculation, when the number of resident peritoneal cells (2-3 X 106; our observation and ref. 1) was nearly equal to the number of tumor cells. It then appears that the tumor growth pattern was not changed following administration of the total dose (0.30 MBq (8 pCi) 1251UdR. Furthermore, earlier studies with the same cell line did not show any detectable cell killing with doses lower than 0.63 MBq (17 kCi> (5- 7). Since the radioactivity of dead tumor cells is not com- pletely and instantaneously excreted from tumor-bearing mice (24), the degree of 1251UdR incorporation estimated using the number of recovered cells can be regarded as a fair approximation of the actual cellular uptake.

Retention of 1231UdR in tumor-bearing mice and the fast elimination of the tracer from healthy animals was also seen in the scintigraphic images following a single i.p. injection of Il. 1 MBq (300 l&i) of ‘231UdR (Fig. 4). In fact, these scintigraphic images indicated clearly the diagnostic potential of IUdR when radiolabeled with the gamma emitter 1231. These results agree well with values obtained from biodistribution studies. For example, at 2 hr, about 70% of the unbound radioactivity was recovered from the peritoneal fluid and the cell pellet. At this time point the tracer distribution was similar in control and tumor-bearing mice. The pool of available 1251UdR in the peritoneal cavity decreased dramatically within 4 hr after the last injection. The control mice retained only traces of the injected dose; in MOT-bearing animals the total peritoneal radioactivity was associated with tumor cells, indicating that the incorporation of 1251UdR took place during the initial 4 hr after each injection. Since the amount of radioactivity recovered from the lipid, RNA, and protein residue after injection of ‘*‘IUdR in mice was insignificant compared with the amount in the DNA (17), it is reasonable to assume that the cell-associated radioactivity was DNA-bound and the 1251UdR uptake was complete in about 4 hr. Therefore, the supplementation of the 1231UdR pool every 4 hr in the therapy studies seems warranted. Accumulation and retention of radioactivity were higher in tumor than in any other tissue after 24 and 120 hr. At earlier times several organs (stomach, intestine, spleen) had more 1251 than the tumor when expressed in terms of %ID/gram wet tissue. How- ever, it is generally accepted that during the first few hours after injection, catabolic products significantly contribute to organ radioactivity. Consequently, the initial clearance must include activity as iodide, and only when this is elim- inated are the residual tissue counts a fair measure of the labeled DNA. Since most of the adult tissues are non- proliferating, there is only a small chance of damaging their DNA when the main concentration of the Auger- electron emitter is located outside the cell or in the cyto- plasm (30).

Although the duration of clearance varied for each organ, the radioactivity declined considerably after 24 hr and only intestine, spleen, and stomach maintained higher amounts. For 1231UdR, rapid clearance and 1231 decay take place concurrently; therefore, even actively proliferating tissues retain lower levels of radioactivity. For example, 0.7% ID 1251 remained in the large intestine at Ti,*; corrected for 1231 decay, this value becomes 0.1% ID 1231. Similarly, the small intestine had 1.2% ID 1251 at T,,,, with decay only 0.25% ID 1231 is expected to persist. The values are still rather high from the point of view of potential tissue damage, but for diagnostic purposes they decline adequately to allow clear delineation of tumor from background radioactivity. Dosimetric calculations (Table 4) for human organs obtained by standard extrapolation of the mouse biodistribution data and by use of the MIRD approximation, assuming uniform distribution of radioactivity throughout the organ, show that in principle very high doses from 1231UdR could be tolerated by the patient. This may not be entirely accurate: IUdR will be partially deha- logenated after passing from the peritoneum into the circulation. However, a certain proportion may still be in the form of IUdR and may be taken up by dividing cells. In these cases MIRD dosimetry has been shown to underestimate the dose delivered to the labeled cells (32). There- fore, although dosimetric results indicate that doses received by normal tissue are relatively low, in reality the possible underestimation from using the MIRD approximation should be taken into account. According to data on biodistribution and excretion of IUdR available from this and other studies (10, 14, 34, 38), the largest doses would be received by intestine, stomach, spleen, and kidney, pro- viding that the thyroid uptake of 1231 is blocked. The average doses are lower than those calculated for 1251UdR. This observation, combined with a much shorter overall tissue half-life of 1231UdR due to isotope decay, also reduces the concern of possible additional damage of the normal tissue renewal systems because of reutilization of 1231UdR released from dying tumor cells. Most incorporation of label from these cells involves hydrolysis of liberated DNA to the nucleoside levels and subsequent reutilization of these products in processes requiring two or more days (25, 34) following cell death, which exceeds the 13.2~hr half- life of 1231. Conversely, it might appear that the residence time of 1231UdR in DNA of tumor cells is not sufficient to produce lethal damage and might limit the therapeutic value of 1231UdR. However, even with a cell uptake as low as 7.4 X 10m4 Bq/cell (0.02 pCi/cell) observed with the total dose of 0.30 MBq (8 pCi) 1231UdR, 5.5 x lo3 molecules of thymidine are replaced by 1231UdR (1.7 x 10d4%), a quantity sufficient to generate in the first hr alone about 280 decays of 1231, theoretically equivalent to approximately 200 double-strand breaks (0.73 dsb/decay; (27)). Even if only 10% of 1231, about 30 decays, were to generate double-strand breaks, it might still be sufficient to produce lethal effects. For the same degree of thymidine replacement by lz51UdR, fewer than three decays and the-

1550 I. J. Radiation Oncology 0 Biology 0 Physics November 1991, Volume 21, Number 6

oretically three double-strand breaks are expected within 1 hr (1.1 dsb/decay; (27)). This simple calculation reaffirm- sthe therapeutic potential of 1231UdR. Many compounds used in cancer chemotherapy express their activity only through direct modification of DNA, but the number of interacting molecules per nucleus required to elicit cytotoxic response are staggering in comparison to that for radiolabeled IUdR. For example, at least 1% replacement of thymidine (3.2 x 10’ molecules) by nonradioactive IUdR is necessary to produce discemable chemotoxicity (4); about IO’ molecules of daunomycin must intercalate with DNA for 50% cytotoxicity (15). In contrast, the number of Auger-electron-emitter-containing molecules needed to produce the same tumoricidal effect is very low. From the survival curve in our studies, it can be estimated that 50% cytotoxicity seen with about 0.93 MBq (25 I&i) of ‘231UdR (specific activity > 8510 TBq/mmol) corresponds to a displacement of less than 1 X lo3 molecules of thy-

midine (2 x 10P5% replacement), using our value of about 1% ID uptake by the tumor.

The results substantiate the antineoplastic potential of 5-[1231]iodo-2’-deoxyuridine and at the same time intro- duce an important qualification. The propensity of tumor cells to survive can be influenced by many determinants, including phase of the cell cycle and access of the radiopharmaceutical to the target. These factors affect neoplastic cells in an arbitrary manner regardless of their growth potential. To achieve therapeutic ratio, it is critical to op- timize the time-dose fractionation schedule and to develop an efficient IUdR delivery system for each type of tumor. The ovarian tumor used in these experiments had a high growth factor and was exposed directly to the radiopharmaceutical before the latter entered the circulation. For the treatment of solid tumors where the growth fraction is considerably less than unity and access is less immediate, many challenges are yet to be met.

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5-[123i]iodo-2′-deoxyuridine in the radiotherapy of an early ascites tumor model

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