Interaction of iododeoxyuridine and its primary metabolite, iodouracil on radiation response

ht. J. Radarion Oncology Bid. P/y.. Vol. 12, pp. 1519-1522 Printed in the U.S.A. All rights reserved

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036&3016/86 1986 Pergamon

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0 Session VIII INTERACTION OF IODODEOXYURIDINE AND ITS PRIMARY METABOLITE, IODOURACIL ON RADIATION RESPONSE TIMOTHY

J. KINSELLA,

M.D.,

PATRICIA P. DOBSON, B.S. AND JAMES B. MITCHELL,

PH.D.

Radiobiology Section, Radiation Oncology Branch, National Cancer Institute, Bethesda, MD 20892 An in vitro model using Chinese hamster V79 cells was designed to test the interaction of iododeoxyuridine (IdUrd) and its primary metabolite, iodouracil (IU) on growth and radiation response. A recent clinical pharmacology study documented that while steady-state arterial plasma levels of IdUrd remained linear over the dose range of 2501200 mg/m2/12 hour intravenous infusion, plasma levels of IU rose to >l log higher (approaching 10e4 M) at the completion of the 1Zhour infusion. Using these clinically relevant doses of IdUrd and IU, we report no apparent effect on radiosensitization of IU alone, or in combination with IdUrd using exponentially growing V79 cells. Similarly, IU does not result in any growth delay. Thus, unlike the recent reports of 54uorouracil beii htcorporated into DNA following phosphorylation to FdUMP, iodouracil does not appear to follow similar metabolic pathways and is unlikely to contribute to the clinical radiosensitizing potential of iododeoxyuridine. Iododeoxyuridine, Iodouracil, Radiation response. been demonstrated using IdUrd in both in vivo and clinical

INTRODUCTION

studies.‘,5*12Recent studies, of the structurally similar fluoropyrimidines, have documented that while fluorodeoxyuridine (FdUrd) primarily affects DNA synthesis by inhibiting thymidylate synthetase and 54luorouracil (FU) is primarily incorporated into RNA, there is some conversion of FU to FdUMP, the active metabolite of FdUrd.7,g,” Using this metabolic pathway, 5-fluorouracil can be incorporated into DNA. Additionally, uracil can enhance FdUrd incorporation into DNA at least in MCF7 human breast cancer cells.8 Based on these data, we designed an in vitro study using Chinese hamster V79 cells to study possible interactions of IdUrd and IU on the growth characteristics and radiation response of these mammalian cells.

In a recent Phase I study of 5-iodo-2’deoxyuridine

(IdUrd) given by constant intravenous infusion (12 hrs/day X 2 weeks), linear pharmacokinetics for IdUrd were demonstrated over the dose range of 250- 1200 mg/m2/12 hour infusion.6 Steady-state plasma concentrations of IdUrd were achieved within 1 hr and ranged from l-8 X lop6 M. Total body clearance of IdUrd remained constant at .75 1/min/m2 and at the end of the infusion, IdUrd levels were no longer measurable (< 1Op7M) within 30 min (Fig. 1). However, the primary metabolite, 5-iodouracil (IU), did not achieve steady state during the 12 hr infusion and reached plasma levels 2 1 log higher (approaching low4 M) at the completion of the infusion. A 50- to lOO-fold increase in uracil and thymine were also measured during the infusion. The disappearance of IU, uracil, and thymine occurred slowly over the ensuing 12 hr following completion of the IdUrd infusion (Fig. 1). Thymidine and deoxyuridine plasma levels did not change during or following the infusion. These alterations in endogenous pyrimidine pools are consistent with competitive inhibition of the enzyme dihydrouracil dehydrogenase by IU. These pharmacological observations during the continuous intravenous infusion of IdUrd as a clinical radiosensitizer raised questions of whether IU might contribute to the cytotoxicity of normal tissues such as bone marrow or to the radiosensitization of tumors which have

Growth studies Exponentially growing Chinese hamster V79 cells were plated in F12 medium supplemented with 10% heat inactivated fetal bovine serum and pen/strep, and incubated at 37°C in 5% C02-95% air. Under these conditions, the doubling time is 9- 10 hours, and the plating efficiency is 85-95%. Media was removed and replaced with warmed medium containing the test conditions: lop4 and 1O-’ M

Presented at the Chemical Modifiers of Cancer Treatment Conference, Clearwater, Florida, 20-24 October 1985. Reprint requests to: Timothy J. Kinsella, M.D., Radiation

Oncology Branch, National Cancer Institute, Building 10, Room B3B69, Bethesda, MD 20892. Accepted for publication 25 February 1986.

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of IdUrd(O), IU(O), Uracil(A), and thymine during a la-hour continuous intravenous infusion of IUdR and for 12 hr following completion of the IUdR

Fig. 1. Plasma concentrations

infusion (adapted from Reference 7).

IU; or 10e5 and lop6 M IdUrd; and combinations of the two. Plates were returned to the incubator and duplicate plates of each condition were harvested at appropriate intervals to be compared against control plates. Harvesting was accomplished by removing the medium, rinsing one time with 5 ml PBS, and a known amount of trypsinVersene (0.03% trypsin, 0.01% EDTA) was added (usually 2 to 5 ml) and cells were allowed to detach. Cells were then dispersed with a transfer pipet and 0.1 ml was taken to count using an Elzone particle counter. Growth curves were constructed based on serial measurements corrected for plating efficiency over the 50-hour exposure to the various treatments and compared to controls.

Survival studies V79 cells were plated as for growth curves (above) and allowed to incubate with the test conditions (1 0m4M IU, 10m5M IdUrd, or 10m4M IU plus 10e5 M IdUrd) for 17 hours (two population doublings). Plates were kept in conditions of subdued yellow light at all times and given a dose of 0, 5, 11, or 16 Gy for control or IU cells; or 0, 3, 6, and 9 Gy for samples containing IdUrd and combinations of IdUrd and IU. Cell samples were irradiated using a 15 MeV linear accelerator with a dose rate of 5 Gy/min. Full electron equilibrium was provided by adequate lucite build-up. Immediately after irradiation, cells were removed with trypsin, counted and plated in triplicate at low density for colony formation. Plates were incubated for 6-7 days, fixed with methanol:acetic acid (3:1), stained with crystal violet, and scored for colony formation with any colony containing over 50 cells counted as a positive. Each survival point was plated in triplicate and experiments for the various test conditions were repeated twice. The mean value of each set of conditions, the plating efficiency of the control, and surviving fraction of the test conditions were calculated along with the standard error of the mean.

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Fig. 2. Growth curves for exponentially growing Chinese hamster V79 cells during a 50-hour exposure to IdUrd alone, IU alone or combined IdUrd/IU. The drug concentrations are shown above.

RESULTS Growth curves for control and treated V79 cells are presented in Figure 2. There appears to be a modest growth delay using IdUrd at IO-’ M, but no further change with the addition of IU at lo-’ and 10e5 M. Treatment with IdUrd at 10m6M; IU at lop4 M, or combinations of the two did not affect the growth characteristics of V79 cells over a 50-hour period. In Figure 3, radiation survival curves for exponentially growing V79 cells treated with IdUrd, IU, or both, are illustrated. Following a 17-hour exposure of IdUrd at 10e5 M, there is a marked change in both ii and Do compared to control cells (Panel A) (ii, = .75, iic = 2.25; DoI = 0.6 Gy, Dot = 1.5 Gy). Previous incorporation studies using 3H-IdUrd note that 16% of thymidine in DNA is replaced using this treatment protocol. (Kinsella, T.J., Dobson, P.P., Mitchell, J.B., Fornace, A-J., Jr., unpublished data, 1985). Treatment with IU for 17 hrat both 10e4 and 10s5 M resulted in no change in survival compared to controls (Panel B). Finally, co-treatment with IdUrd and IU in the clinically relevant range did not result in any increase in radiosensitization compared to IdUrd alone (Panel C). Thymidine replacement studies were not performed fol-

Interaction of iododeoxyuridine 0 T. J.

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Fig. 3. Radiation survival curves for exponentially growing V79 cells following a 17-hour exposure (2 population doublings) to IdUrd at lo-’ M, IU at 10m4and lo-’ M and combinations of IdUrd/IU.

IdUrd/IU treatment since no increased radiation effect on survival was observed. lowing

the combined

DISCUSSION

Use of the halogenated pyrimidine analogs as potential clinical radiosensitizers is an area of renewed experimental and clinical research at the National Cancer Institute and elsewhere.4 Early clinical trials using direct intra-arterial infusions of BrdUrd and bolus intravenous infusions of IdUrd suggested a therapeutic gain compared to radiation alone in poorly radioresponsive tumors. 1*3More recently, Phase I/II studies of both BrdUrd and IdUrd, given as continuous intravenous infusions, have documented steady state arterial (tumor bed) levels of lop6 - low5 M which can be maintained for up to 2 weeks.6,10Preliminary treatment results in poorly radioresponsive tumors, such as high grade gliomas and sarcomas, are encouraging.4,5 Cellular effects of these exogenous thymidine analogs (nucleosides) are mediated through conversion to nucleotides, followed by incorporation as nucleic acids.2 Nucleotides do not cross the cell membrane and cannot be administered directly. With the exception of Sfluorouracil, pyrimidine bases have not been administered clinically. While the exact mechanism of radiosensitization by these thymidine analogs is not clearly understood, it appears that phosphorylation and subsequent incorporation into DNA is necessary.2 A recent pharmacology study using 12-hour infusions of IdUrd documented significant

plasma elevations of the primary metabolite, 5-iodouracil (Fig. 1),6which had not been previously reported. In light of the recent literature reporting DNA incorporation of Sfluorouracil via conversion to FdUMP, we designed an in vitro study to address whether IU might contribute to either the cytotoxicity or the radiosensitizing potential of IdUrd. We report no apparent effect on exponential growth of V79 cells over a 50-hour period with the addition of IU to IdUrd using clinically relevant doses. In a previous study of cytotoxicity using human bone marrow CFU-c, we found that while a 24-hour exposure of IdUrd at 5 X 10e6 M resulted in a 50% decrease in colony formation, IU at the same concentration and at concentrations l-2 logs higher showed no cytotoxicity.6 We also report no enhancement of radiosensitization with cotreatment using IU and IdUrd compared to IdUrd alone. Based on these in vitro data, it appears that IU is unlikely to contribute to the clinical tumor radiosensitization or systemic normal tissue toxicity found in our Phase I/II study,5 in spite of significant elevations of IU during the continuous infusion of IdUrd for up to 2 weeks. However, it is recognized that other modulations of halogenated pyrimidine incorporation into DNA, such as co-administration of FdUrd, can potentially result in increased DNA incorporation and increased radiosensitization. l2 At present, we are determining the most optimal sequence of blocking endogenous thymidine synthesis using FdUrd and assaying IdUrd incorporation and radiosensitization in this in vitro V79 cell system prior to initiating clinical trials.

REFERENCES 1. Calabresi, P., Creasey, W.A., Prusoff, W.H., Welch, A.D.:

Clinical and pharmacological studies with 5-iodo-2’-deoxyuridine. Cancer Rex 23: 583-592, 1963. 2. Goz, B.: The effects of incorporation of S-halogenated deoxyuridines into the DNA of eukaryotic cells. Pharmacol. Rev. 29: 249-272, 1978.

3. Hoshino, T., Sano, K.: Radiosensitization of malignant brain tumors with bromouridine (thymidine analog). Acta Radiof. Ther. Phys. Biol. 8: 15-26, 1969. 4. Kit&la, T.J., Mitchell, J.B., Russo, A., Morstyn, G., Glatstein, E.: The use of halogenated pyrimidine analogs as clinical radiosensitizers: Rationale, current status, and future

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prospects. Int. J. Radiat. 0~01. Biol. Phys. 10: 1399- 1406, 1984. Kinsella, T.J., Russo, A., Mitchell, J.B., Collins, J.R., Rowland, J., Wright, D., Glatstein, E.: Phase I study of intravenous iododeoxyuridine as a clinical radiosensitizer. Int. J. Radiat. Oncol. Biol. Phys. 11: 1941-1946, 1985.

9.

10.

Klecker, R.W., Jenkins, J.F., Kinsella, T.J., Fine, R.L., Strong, J.M., Collins, J.M.: Clinical pharmacology of S-iodo2’-deoxyuridine and 54odouracil and endogenous pyrimidine modulation. Clin. Pharmacol. Ther. 38: 45-5 1, 1985. Kufe, D., Major, P.: 5-fluorouracil incorporation into human breast carcinoma RNA correlates with cytotoxicity. J. Biol. Chem. 256: 9802-9805, 1981. Kufe, D.W., Herrick, D., Gunner, L.: Uracil enhancement of 54uorodeoxyuridine incorporation into human breast

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carcinoma deoxyribonucleic acid. Biochem. Pharmacol. 33: 2329-2331, 1984. Roobol, C., DeDobbeleer, G.B.E., Bemheim, J.L.: 54uorouracil and 5-fluoro-2’deoxyuridine follow different metabolic pathways in the induction of cell lethality in L1210 leukemia. Brit. J. Cancer 49: 739-744, 1984. Russo, A., Gianni, L., Kin&la, T.J., Klecker, R.W., Jenkins, J., Rowland, J., Glatstein, E., Mitchell, J.B., Collins, J., Myers, C.: A pharmacologic evaluation of intravenous delivery of BUdR to patients with brain tumors. Cancer Res. 44: 1702-1705, 1984. Spears, C.P., Shahinian, A.H., Moran, R.G., Heidelberger, C., Corbett, T.H.: In vivokinetics of thymidylate synthetase inhibition in 5-fluorouracil sensitive and resistant murine colon adenocarcinomas. Cancer Res. 42: 450-455, 1982. Szybalski, W.: X ray sensitization by halo-pyrimidines. Cancer Chemother. Rep. 58: 539-557, 1974.