The potential superiority of bromodeoxyuridine to iododeoxyuridine as a radiation sensitizer in the treatment of colorectal cancer.

Although the thymidine analogues 5-bromo-2'-deoxyuridine (BrdUrd) and 5-iodo-2'-deoxyuridine (IdUrd) have been used successfully as radiation sensitizers in clinical trials, it is not clear which of these agents is the more promising to pursue. To begin to assess this question with regard to colorectal cancer metastatic to the liver, a study was carried out using HT29 human colon cancer cells in culture and implanted in nude mice as xenografts. Cells and animals were treated with BrdUrd +/- the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (FdUrd), and the results compared to our previous studies with IdUrd +/- FdUrd (T. S. Lawrence, M. A. Davis, P. E. McKeever, J. Maybaum, P. L. Stetson, D. P. Normolle, and W. D. Ensminger. Cancer Res., 51: 3900-3905, 1991). Using cultured cells, it was found that FdUrd (at concentrations of greater than 10 nM) increased: (a) the incorporation of BrdUrd into the DNA of cultured tumor cells; (b) BrdUrd-mediated radiosensitization; (c) BrdUrd-mediated increase in radiation-induced DNA damage; and (d) BrdUrd-mediated decrease in the repair of radiation-induced damage. The incorporation of BrdUrd was greater than or equal to the incorporation of IdUrd previously determined under the same exposure conditions. Studies using nude mice bearing HT29 xenografts showed that FdUrd increased BrdUrd incorporation more into tumors than into the normal liver. Most tumor cells incorporated BrdUrd (labeling index after a 4-day infusion = 87 +/- 2%; SE); in the liver, labeling was confined chiefly to nonparenchymal cells. In both the presence and absence of FdUrd, the incorporation of BrdUrd into tumors was significantly and consistently greater than the incorporation of IdUrd measured under the same conditions of drug administration (by a factor of 1.2-3.6). Furthermore, the administration of BrdUrd +/- FdUrd tended to produce less weight loss and hematological toxicity than IdUrd +/- FdUrd. These findings suggest that BrdUrd may be superior to IdUrd as a radiation sensitizer in the treatment of colorectal cancer metastatic to the liver.     

Fluorodeoxyuridine improves imaging of human glioblastoma xenografts with radiolabeled iododeoxyuridine.

Use of radiolabeled nucleotides for tumor imaging is hampered by rapid in vivo degradation and low DNA-incorporation rates. We evaluated whether blocking of thymidine (dThd) synthesis by 5-fluoro-2'-deoxyuridine (FdUrd) could improve scintigraphy with radio-dThd analogues, such as 5-iodo-2'-deoxyuridine (IdUrd). We first show in vitro that coincubation with FdUrd substantially increased incorporation of [125I]IdUrd and [3H]dThd in the three tested human glioblastoma lines. Flow cytometry analysis showed that a short coincubation with FdUrd (1 h) produces a signal increase per labeled cell. We then measured biodistribution 24 h after i.v. injection of [125I]IdUrd in nude mice s.c. xenografted with the three glioblastoma lines. Compared with animals given [125I]IdUrd alone, i.v. preadministration for 1 h of 10 mg/kg FdUrd increased the uptake of [125I]IdUrd in the three tumors 4.8-6.8-fold. Compatible with previous reports, there were no side effects in mice observed for 2 months after receiving such a treatment. The tumor uptake of [125I]IdUrd was increased < or =13.6-fold when FdUrd preadministration was stepwise reduced to 1.1 mg/kg. Uptake increases remained lower (between 1.7- and 5.8-fold) in normal proliferating tissues (i.e., bone marrow, spleen, and intestine) and negligible in quiescent tissues. DNA extraction showed that 72-80% of radioactivity in tumor and intestine was bound to DNA. Scintigraphy of xenografted mice was performed at different times after i.v. injection of 3.7 MBq [125I]IdUrd. Tumor detection was significantly improved after FdUrd preadministration while still equivocal after 24 h in mice given [125I]IdUrd alone. Furthermore, background activity could be greatly reduced by p.o. administration of KClO4 in addition to potassium iodide. We conclude that FdUrd preadministration may improve positron or single photon emission tomography with cell division tracers, such as radio-IdUrd and possibly other dThd analogues.     

Fluorodeoxyuridine modulation of the incorporation of iododeoxyuridine into DNA of granulocytes: a phase I and clinical pharmacological study

The amount of iododeoxyuridine (IdUrd) incorporated into DNA determines the degree of radiosensitization. Fluorodeoxyuridine (FdUrd) has been shown to biochemically modulate IdUrd incorporation into DNA in vitro and in vivo. In this Phase I study, these drugs were coadministered to patients during 14-day continuous i.v. infusion periods in order to investigate whether the incorporation of IdUrd into DNA in vivo could be increased without increasing the dose of IdUrd. IdUrd plasma concentrations and incorporation of IdUrd into DNA of granulocytes were measured by high-performance liquid chromatography. Up to 8.8% substitution of thymidine by IdUrd was observed. Even at 3.5 mg/m2/day FdUrd for 14 days (78% of the maximum-tolerated dose as a single agent), no clinically relevant enhancement of incorporation of IdUrd into DNA of granulocytes was observed. Also, no changes in plasma levels of IdUrd were observed with escalating doses of FdUrd. Toxicity patterns (stomatitis, diarrhea, and bone marrow depression) and isobologram analysis suggested that IdUrd and FdUrd had additive, rather than synergistic, effects.     

Modulation of iododeoxyuridine-mediated radiosensitization by 5-fluorouracil in human colon cancer cells.

The incorporation of 5-iodo-2'-deoxyuridine (IdUrd), a thymidine analog radiosensitizer, can be increased by the use of modulators such as 5-fluorouracil (FUra). FUra is a particularly attractive potential modulator to use against colorectal cancer, as it is the most active single agent in the treatment of this disease. To begin to define the conditions for the optimal combination of IdUrd and FUra in the treatment of patients with colorectal cancer, a study was conducted of the effect of FUra on IdUrd-mediated radiosensitization in cultured HT29 human colon cancer cells. It was found that when cells were exposed to concentrations of IdUrd typical of those obtained through intravenous exposure (1-3 microM), FUra (1 microM) increased radiosensitization beyond that which would be predicted for the same extent of incorporation produced by incubation with IdUrd alone. This increase appeared to result from a combination of at least two effects: FUra-mediated cell cycle redistribution and increased IdUrd incorporation. When a higher concentration of IdUrd (10 microM) was used with FUra (1 microM), cell cycle distribution returned to nearly normal, and radiosensitization was equal to that predicted by the extent of incorporation of IdUrd. These data demonstrate that the combination of FUra and IdUrd can produce radiosensitization both through increased IdUrd incorporation and cell cycle redistribution. Furthermore, they suggest that, in the presence of a modulator, it may not be necessary to achieve high levels of IdUrd incorporation to produce significant tumor radiosensitization.     

Preclinical toxicity and efficacy study of a 14-day schedule of oral 5-iodo-2-pyrimidinone-2'-deoxyribose as a prodrug for 5-iodo-2'-deoxyuridine radiosensitization in U251 human glioblastoma xenografts.

In anticipation of an initial clinical Phase I trial in patients with high-grade gliomas of p.o. administered 5-iodo2-pyrimidinone-2'-deoxyribose (IPdR) given daily for 14 days as a prodrug for 5-iodo-2'-deoxyuridine (IUdR)-mediated tumor radiosensitization, we determined the systemic toxicities and the percentage IUdR-DNA incorporation in normal athymic mouse tissues and a human glioblastoma xenograft (U251) after this dosing schedule of IPdR. Using a tumor regrowth assay of s.c. U251 xenografts, we also compared radiosensitization with this IPdR-dosing schedule to radiation therapy (XRT) alone (2 Gy/day for 4 days) or to XRT after continuous infusion IUdR for 14 days at the maximum tolerated dose in mice (100 mg/kg/day). Athymic mice with and without U251 s.c. xenografts tolerated 750 or 1500 mg/kg/day of p.o. IPdR (using gastric lavage) for 14 days without weight loss or activity level changes during treatment and for a 28-day posttreatment observation period. The percentage IUdR-DNA incorporation in U251 tumor cells was significantly higher after p.o. IPdR (750 and 1500 mg/kg/day) for 14 days (3.1 +/- 0.2% and 3.7 +/- 0.3%, respectively) than continuous infusion IUdR for 14 days (1.4 +/- 0.1%). Compared to XRT alone, a significant sensitizer enhancement ratio (SER) was found with the combination of p.o. IPdR (1500 mg/kg/d) + XRT (SER = 1.31; P = 0.05) but not for the combination of continuous infusion IUdR + XRT (SER = 1.07; P = 0.57) in the U251 xenografts. The percentage IUdR-DNA incorporation after IPdR at 1500 mg/kg/day for 14 days in normal bone marrow, normal small intestine, and normal liver were 1.2 +/-0.2%, 3.3 +/- 0.3%, and 0.2 +/- 0.1%, respectively. We conclude that a 14-day p.o. schedule of IPdR at up to 1500 mg/kg/day results in no significant systemic toxicity in athymic mice and is associated with significant radiosensitization using this human glioblastoma multiforme xenograft model. Based on these data and our previously published data using shorter IPdR dosing schedules, which also demonstrate an improved therapeutic index for IPdR compared to IUdR, an initial clinical Phase I and pharmacokinetic study of p.o. IPdR daily for 14 days is being designed.     

Variability of 5-bromo-2'-deoxyuridine incorporation into DNA of human glioma cell lines and modulation with fluoropyrimidines.

Human glioma-derived cell lines were found to vary in their ability to incorporate the radiosensitizer 5-bromo-2'-deoxyuridine (BrdUrd) into DNA after one cell doubling. The U-251 cell line was the best incorporator of BrdUrd, whereas U-118 and D-54 demonstrated poor incorporation with respective C50 (BrdUrd concentration required for 50% of the maximum amount of BrdUrd incorporation into DNA) values of 2.8- and 6-fold greater than that of U-251 (P less than 0.001). Modulation of radiosensitizer uptake into DNA could be achieved using the thymidylate synthase inhibitors 5-fluorouracil or 5-fluoro-2'-deoxyuridine (FdUrd). Incorporation into U-251 cells increased only slightly in the presence of the fluoropyrimidines. The BrdUrd concentration required for 50% of the maximum amount of BrdUrd incorporation into DNA changed (P less than 0.001) from 1.8 +/- 0.11 microM (SD) in the absence of a modulator to 1.1 +/- 0.09 or 1.1 +/- 0.16 microM in the presence of 10 microM 5-fluorouracil or 5 nM FdUrd, respectively. The D-54 cell line, which was the worst incorporator of BrdUrd, was found to have an extensive amount of BrdUrd into DNA following biomodulation. The C50 in the absence of modulation was 7.3 +/- 1.3 microM, which was reduced (P less than 0.001) to 0.62 +/- 0.04 and 0.32 +/- 0.13 microM, respectively, in the presence of 10 microM 5-fluorouracil and 5 nM FdUrd. This represents a 12- to 22-fold reduction in the concentration of radiosensitizer required to achieve the same level of BrdUrd incorporation into DNA. Furthermore, this enhancement of BrdUrd DNA incorporation seen in the presence of the fluoropyrimidines is observed at clinically achievable concentrations. The degree of radiosensitization was solely dependent upon the amount of BrdUrd incorporated into DNA. D-54 cells grown in the presence of 0.18 microM BrdUrd plus 5 nM FdUrd or 2.8 microM BrdUrd alone yielded a similar level of BrdUrd incorporation into DNA and radiosensitization, though a 15-fold lower BrdUrd concentration was used in the presence of FdUrd. The combined use of a radiosensitizer with a fluoropyrimidine may overcome poor incorporation of BrdUrd into DNA that may exist among resistant subpopulations of cells within malignant glioma.     

Biochemical modulation of 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine incorporation into DNA in VX2 tumor-bearing rabbits.

The thymidine analogues 5-bromo-2'-deoxyuridine (Brd-Urd) and 5-iodo-2'-deoxyuridine (IdUrd) compete with thymidine for incorporation into the DNA of replicating cells. This incorporation results in radiosensitizing effects which are directly related to the degree of analogue substitution. In vitro and in vivo evidence suggests that preadministration or coadministration of the thymidylate synthetase inhibitors fluorouracil and 5-fluoro-2'-deoxyuridine (FdUrd) can modulate analogue incorporation into DNA. We have evaluated in the rabbit VX2 tumor model the effects of thymidylate synthetase inhibitor (fluorouracil or FdUrd) coadministration (as 24-hour, intravenous infusions) on the incorporation of BrdUrd or IdUrd into the DNA of relevant normal tissues (bone marrow, gut mucosa) and intrahepatic VX2 tumor. Tissues were harvested and processed for gas chromatography-mass spectrometry analysis of the thymine, 5-bromouracil, and 5-iodouracil contents in hydrolyzed DNA. Coadministration of FdUrd resulted in statistically significant (P less than .01) enhancement of IdUrd incorporation into the DNA of intrahepatic VX2 tumor and normal (bone marrow and duodenal mucosa) rabbit tissues. Coadministered fluorouracil, on the other hand, significantly enhanced IdUrd incorporation only into DNA of intrahepatic VX2 tumor. Statistically significant enhancement of BrdUrd incorporation was achieved only with FdUrd coadministration and then only into the DNA of intrahepatic VX2 tumor. The percent of thymine replaced by analogue (I) is related to the steady-state arterial plasma drug concentration (C) by the Michaelis-Menten equation: I = I(MAX.) C/(C50 + C). The primary effect of FdUrd coadministration on BrdUrd incorporation into VX2 tumor DNA was a reduction of the C50 parameter (plasma BrdUrd concentration eliciting I = I(MAX)/2) from 8.17 microM to 1.78 microM. On the other hand, the I(MAX) parameter (I as C approaches infinity) was only slightly affected (29.7% to 25.2%). Thus, the degree to which the modulator enhanced analogue incorporation varied inversely with the analogue's steady-state plasma concentration. These results, which describe potential tissue specificity of modulator efficacy and characterize the effects of thymidylate synthetase inhibitor modulation on thymidine analogue incorporation pharmacodynamics, should provide guidance as to dose scheduling of BrdUrd and IdUrd in clinical trials for improved tumor specificity of uptake.     

Clinical pharmacological studies of concurrent infusion of 5-fluorouracil and thymidine in treatment of colorectal carcinomas

The effects of thymidine (dThd) coadministration on the pharmacokinetics and metabolism of 5-fluorouracil (FUra) were investigated in 29 colorectal cancer patients. Five patients received 5-day i.v. infusion of FUra at 15 mg/kg/day and 24 patients received infusion of FUra (7.5 mg/kg/day, 5 days) and dThd (216 mg/kg/day, 6 days) preceded by a bolus dose of dThd (405 mg/kg). Plasma and urine concentrations of FUra, 5-fluorodeoxyuridine (FdUrd), thymine, and dThd were quantitated by a high-pressure liquid chromatographic assay. Concurrent dThd administration reduced the plasma clearance of FUra at steady state from 389.1 +/- 153.5 (S.D.) to 56.0 +/- 36.4 liters/kg/day. The mean steady-state plasma concentration of FUra in patients receiving FUra alone was 0.38 microM and was significantly lower than the 1.30 microM in patients receiving FUra-dThd. Plasma concentrations of FUra were linearly dependent on those of thymine. Furthermore, the metabolic and renal clearances of FUra decreased inversely with thymine concentrations indicating that the elimination of FUra was reduced by thymine. In contrast to the absence of FdUrd as a circulating metabolite in patients treated with FUra alone, microM concentrations of FdUrd were detected in plasma of most of the patients treated with FUra-dThd. This together with the linear correlation of FdUrd and dThd concentrations indicates that the interconversion of FUra to FdUrd was enhanced by dThd. The incidence of dose-limiting leukopenia in the FUra-dThd combination therapy was 40%. There is an inverse correlation between the plasma clearance of FUra at steady state and hematological toxicity. The plasma clearance of FUra in the toxic population was 32.0 +/- 16.8 liters/kg/day and was significantly lower than the clearance of 72.0 +/- 37.3 liters/kg/day in the nontoxic population (p less than 0.001). The corresponding critical toxic steady-state FUra plasma concentration was 1.5 microM. The biochemical effects of dThd on the incorporation of FUra and FdUrd into RNA and into acid-soluble 5-fluorodeoxyuridine monophosphate (FdUMP) in human colon tumor cells were studied in vitro. At 100 microM, dThd increased the incorporation of FUra into RNA up to 4-fold but diminished the acid-soluble FdUMP pool. Similarly, the incorporation of FdUrd into acid-soluble FdUMP was inhibited by dThd. The response rate of colorectal carcinoma to FUra was not improved by coadministration of dThd; only one of the 11 patients who had no prior FUra therapy achieved partial remission. The lack of clinical response in these patients may be partly due to the inhibition of anabolism of FUra and FdUrd to FdUMP by dThd.     

Fluorodeoxyuridine-induced radiosensitization and inhibition of DNA double strand break repair in human colon cancer cells

The halogenated pyrimidine, fluorodeoxyuridine (FdUrd), has been used in combination with radiation for the treatment of human neoplasms. In an attempt to improve the clinical use of this combination, FdUrd-radiation interactions were studied in vitro using human HT29 colon cancer cells. It was found that FdUrd produced radiosensitization at clinically achievable (1-100 nM) concentrations. Sensitization depended critically on the timing of exposure. When cells were irradiated after a 12-hr exposure to 100 nM FdUrd, marked sensitization was produced (mean inactivation dose (MID) = 2.01 +/- 0.01, compared to control of 4.35 +/- 0.16, p less than .01). No radiosensitization occurred when cells were irradiated 4 hr prior to incubation (MID = 3.95 +/- 0.05, p greater than 0.4). Radiosensitization appeared to result from an inhibition of thymidylate synthase since concentrations of FdUrd which produced radiosensitization depleted intracellular TTP pools and blocked the incorporation of deoxyuridine into DNA. Furthermore, radiosensitization was completely inhibited by co-incubation with thymidine. FdUrd also decreased the repair, but not the formation, of radiation-induced DNA double strand breaks (DSB's). These data are consistent with the hypothesis that FdUrd produces radiosensitization by depleting thymidine pools which leads to a decreased rate of DNA DSB repair. Furthermore, they suggest that in clinical trials FdUrd should be infused at least 8 hr before irradiation.     

Inhibition of base excision repair potentiates iododeoxyuridine-induced cytotoxicity and radiosensitization

5-Iodo-2'-deoxyuridine (IdUrd) is a halogenated thymidine analogue recognized as an effective in vitro and in vivo radiosensitizer in human cancers. IdUrd-related cytotoxicity and/or radiosensitization are correlated with the extent of IdUrd-DNA incorporation replacing thymidine. IdUrd cytotoxicity and radiosensitization result, in part, from induction of DNA single-strand breaks (SSB) with subsequent enhanced DNA double-strand breaks leading to cell death. Because base excision repair (BER) is a major DNA repair pathway for SSB induced by chemical agents and ionizing radiation, we initially assessed the role of BER in modulating IdUrd cytotoxicity and radiosensitization using genetically matched Chinese hamster ovary cells, with (AA8 cells) and without (EM9 cells) XRCC1 expression. XRCC1 plays a central role in processing and repairing SSBs and double-strand breaks. We found that EM9 cells were significantly more sensitive than parental AA8 cells to IdUrd alone and to IdUrd + ionizing radiation. The EM9 cells also demonstrate increased DNA damage after IdUrd treatment as evaluated by pulse field gel electrophoresis and single cell gel electrophoresis (Comet Assay). BER-competent EM9 cells, which were stably transfected with a cosmid vector carrying the human XRCC1 gene, showed responses to IdUrd similar to AA8 cells. We also assessed the role of methoxyamine, a small molecule inhibitor of BER, in the response of human colon cancer cells (HCT116) to IdUrd cytotoxicity and radiosensitization. Methoxyamine not only was able to increase IdUrd cytotoxicity but also increased the incorporation of IdUrd into DNA of HCT116 human colon cancer cells leading to greater radiosensitization. Thus, a genetic or biochemical impairment of BER results in increased IdUrd-induced cytotoxicity and radiosensitization in mammalian cells.     

Phase I trial of hepatic artery infusion of 5-iodo-2'-deoxyuridine and 5-fluorouracil in patients with advanced hepatic malignancy: biochemically based combination chemotherapy

Eighteen patients with hepatic metastases primarily from colorectal carcinoma were treated on a phase I protocol employing hepatic artery infusion (HAI) of 5-fluorouracil (FUra) and 5-iodo-2'-deoxyuridine (IdUrd) via implantable infusion pump. Patients received a 14-day continuous HAI of 300 mg/day FUra. During days 8-14 of therapy, patients received IdUrd as a separate 3-h HAI daily x 7. Treatment cycles were repeated every 28 days. IdUrd was escalated from 0.1 to 2.86 mg/kg/day x 7. Myelosuppression and stomatitis were mild and not dose limiting. Hepatotoxicity was dose limiting and similar to that reported for 5-fluoro-2'deoxyuridine alone administered as a 14-day infusion every month. One patient developed a clinical picture consistent with sclerosing cholangitis and another had biopsy-proven cholestasis and triaditis. Catheter complications occurred in 7 of 18 patients. Plasma concentrations of FUra during the 7-day continuous HAI of FUra alone were consistently either undetectable or very low (less than or equal to 0.1 microM). At level 3 (1.0 mg/kg/day IdUrd) and beyond, measurable plasma concentrations of FUra, iodouracil, and IdUrd were found at the end of the daily 3-h infusion of IdUrd. The maximum tolerated dose of IdUrd as administered in this trial is 2.2 mg/kg/day x 7 and the recommended starting dose for further clinical investigation is 1.7 mg/kg/day x 7.     

Highly efficient DNA incorporation of intratumourally injected [125I]iododeoxyuridine under thymidine synthesis blocking in human glioblastoma xenografts

Intratumoural (i.t.) injection of radio-iododeoxyuridine (IdUrd), a thymidine (dThd) analogue, is envisaged for targeted Auger electron- or beta-radiation therapy of glioblastoma. Here, biodistribution of [(125)I]IdUrd was evaluated 5 hr after i.t. injection in subcutaneous human glioblastoma xenografts LN229 after different intravenous (i.v.) pretreatments with fluorodeoxyuridine (FdUrd). FdUrd is known to block de novo dThd synthesis, thus favouring DNA incorporation of radio-IdUrd. Results showed that pretreatment with 2 mg/kg FdUrd i.v. in 2 fractions 0.5 hr and 1 hr before injection of radio-IdUrd resulted in a mean tumour uptake of 19.8% of injected dose (% ID), representing 65.3% ID/g for tumours of approx. 0.35 g. Tumour uptake of radio-IdUrd in non-pretreated mice was only 4.1% ID. Very low uptake was observed in normal nondividing and dividing tissues with a maximum concentration of 2.9% ID/g measured in spleen. Pretreatment with a higher dose of FdUrd of 10 mg/kg prolonged the increased tumour uptake of radio-IdUrd up to 5 hr. A competition experiment was performed in FdUrd pretreated mice using i.t. co-injection of excess dThd that resulted in very low tumour retention of [(125)I]IdUrd. DNA isolation experiments showed that in the mean >95% of tumour (125)I activity was incorporated in DNA. In conclusion, these results show that close to 20% ID of radio-IdUrd injected i.t. was incorporated in tumour DNA after i.v. pretreatment with clinically relevant doses of FdUrd and that this approach may be further exploited for diffusion and therapy studies with Auger electron- and/or beta-radiation-emitting radio-IdUrd.     

Iododeoxyuridine (IdUrd) incorporation into DNA of human hematopoietic cells, normal liver and hepatic metastases in man: as a radiosensitizer and as a marker for cell kinetic studies

Iododeoxyuridine (IdUrd) was administered as a continuous infusion for 14 days to patients with glioblastoma and sarcoma, and for 3 days to patients with metastatic colorectal carcinoma. In the first group, the maximum incorporation of IdUrd into DNA was determined, taking granulocytes as parameter. In the second group, selective incorporation into DNA of normal liver and hepatic metastases of colorectal cancer was investigated. The highest dose of 675 mg/sq.m./day for 14 days produced IdUrd plasma concentrations of 1.8 +/- 0.3 microM, and a substitution of dThd by IdUrd in the range of 7.1-11.7%. Coadministration of fluorodeoxyuridine did not show significant enhancement of IdUrd-incorporation in granulocytes. Three-day intravenous infusions of IdUrd 1000 mg/sq.m./day produced 1.7-4.5% IdUrd-incorporation in hepatic metastases. Three-day intraarterial infusions (hepatic artery) produced 3.8-10.5% dThd-replacement, whereas, in 9/10 patients this was less than 1% in normal liver. In tumor tissue there was a trend towards FdUrd-modulated enhancement of IdUrd-incorporation, although there was considerable scatter. Cell kinetic studies revealed that IdUrd-incorporation in monocytes and granulocytes was very similar. In lymphocytes, a much lower fraction incorporated IdUrd. Liver tumor contained a considerably higher fraction of IdUrd-labeled cells, compared with normal liver. Potential doubling times for the tumors were estimated to be 10 days.     

Iododeoxyuridine incorporation and radiosensitization in three human tumor cell lines.

Iododeoxyuridine is a halogenated pyrimidine and non-hypoxic cell radiosensitizer currently being used in clinical trials. The amount of radiosensitization by IdUrd is related to the amount of incorporation of the drug into a cell's DNA. These experiments were carried out in three human tumor cell lines (lung, glioma, and melanoma) in monolayer culture exposed to concentrations of IdUrd from 0.1-10 microM for one and three cell cycles before irradiation to determine incorporation and sensitization as a function of drug exposure. Except for the lung cell line, which required greater than 1 microM IdUrd, these cells demonstrate radiosensitization when exposed to 0.1 microM or greater of IdUrd. Maximum sensitization occurred at 10 microM IdUrd for all the cell lines at three cell cycles. The percent thymidine replacement by IdUrd increased with increasing concentrations, but was cell line dependent. Maximum percent replacement occurred at 10 microM at three cell cycles for all the cell lines: lung = 22.4%, glioma = 32.0%, and melanoma = 39.1%. The relationships between percent thymidine replacement and sensitization are not identical across these human tumor cell lines. If IdUrd is going to be a successful radiosensitizer in clinical trials, sustained plasma levels of 10 microM or greater for at least three cell cycles should be achieved during irradiation. This may be best accomplished with repeated short exposures to IdUrd (three cell cycles or approximately 4 days in these cell lines) every 1-2 weeks during radiation. Measurements of thymidine replacement in a tumor biopsy should be attempted prior to radiation to develop a predictive assay for radiosensitization.     

Modulation of IdUrd-DNA incorporation and radiosensitization in human bladder carcinoma cells

5' Amino-5'-deoxythymidine (5'-AdThd) has been demonstrated previously to antagonize dTTP-mediated feedback inhibition of purified thymidine kinase from 647V, a human bladder cancer cell line. Low concentrations of 5'-AdThd (3-30 microM) have also been shown to stimulate cellular uptake of iododeoxyuridine (IdUrd) in 647V cells at clinically relevant IdUrd concentrations (2 microM). We report that the combination of 30 microM 5'-AdThd plus 2 microM IdUrd results in a significant increase of IdUrd replacement of thymidine (dThd) (18%) in the DNA of 647V cells over that obtained by exposure to 2 microM IdUrd alone (7.9%). However, increasing the 5'-AdThd concentration to 300 microM inhibited the incorporation of IdUrd into DNA (3%). IdUrd-induced radiosensitization of 647V cells, as measured by clonogenic survival, was enhanced by coincubation with 30 microM 5'-AdThd, while 300 microM 5'-AdThd reduced the IdUrd radiosensitization. Additionally, radiation-induced single strand break generation when IdUrd was incorporated into 647V DNA, as measured by rapid alkaline elution, was also enhanced by coincubation with 30 microM 5'-AdThd, while 300 microM 5'-AdThd resulted in a decrease in the number of single strand breaks produced. In T24, another bladder cancer cell line, and SV-HUC-TT1, a tumorigenic cell line derived from SV-HUC, 3-10 microM 5'-AdThd was also able to enhance IdUrd replacement of dThd in DNA. However, no stimulation of dThd replacement by 5'-AdThd occurred in SV-HUC, a prototypic "normal" bladder urothelial cell line. Since 5'-AdThd is not a substrate for mammalian thymidine kinase and has little or no cytotoxicity in vitro and in vivo, it may be a selective modulator of IdUrd radiosensitization of human bladder carcinoma and should be tested in vivo.     

Additivity of radiosensitization by the combination of SR 2508 (etanidazole) and Ro 03-8799 (pimonidazole) in a murine tumor system

The nitroimidazole radiosensitizers SR 2508 and Ro 03-8799 have different dose-limiting toxicities in man and hence can be used in combination. Such therapy will be beneficial only if their radiosensitizing properties are additive, which this study sought to determine using clinically relevant radiosensitizer concentrations in the EMT6 tumor in the flanks of BALB/c mice. 240 mg/kg of each drug gave tumor concentrations (+/- 2 se) 55 min after i.v. administration of the combination of 50.4 +/- 10.6 micrograms/g (236 +/- 50 nmol/g) for SR 2508 and 39.7 +/- 9.0 micrograms/g (137 +/- 31 nmol/g) for Ro 03-8799. The radiosensitization by both agents administered both singly and in combination at 240 mg/kg and singly at 480 mg/kg was measured, giving sensitizers 30 min before 20 Gy of 250 kV X rays. Tumor response was assayed by clonogenic cell survival. SER values (with 95% confidence limits) were 1.28 (1.20-1.37) for 240 mg/kg SR 2508, 1.20 (1.10-1.30) for 240 mg/kg Ro 03-8799, 1.46 (1.33-1.59) for 240 mg/kg of both drugs in combination, 1.46 (1.38-1.55) for 480 mg/kg SR 2508 and 1.46 (1.31-1.62) for 480 mg/kg Ro 03-8799. These data confirm the additivity of radiosensitization by the two drugs administered in combination.     

Schedule-dependent drug effects of oral 5-iodo-2-pyrimidinone-2'-deoxyribose as an in vivo radiosensitizer in U251 human glioblastoma xenografts.

PURPOSE: 5-Iodo-2-pyrimidinone-2'-deoxyribose (IPdR) is an oral prodrug of 5-iodo-2'-deoxyuridine (IUdR), an in vitro/in vivo radiosensitizer. IPdR can be rapidly converted to IUdR by a hepatic aldehyde oxidase. Previously, we found that the enzymatic conversion of IPdR to IUdR could be transiently reduced using a once daily (q.d.) treatment schedule and this may affect IPdR-mediated tumor radiosensitization. The purpose of this study is to measure the effect of different drug dosing schedules on tumor radiosensitization and therapeutic index in human glioblastoma xenografts. EXPERIMENTAL DESIGN: Three different IPdR treatment schedules (thrice a day, t.i.d.; every other day, q.o.d.; every 3rd day, q.3.d.), compared with a q.d. schedule, were analyzed using athymic nude mice with human glioblastoma (U251) s.c. xenografts. Plasma pharmacokinetics, IUdR-DNA incorporation in tumor and normal proliferating tissues, tumor growth delay following irradiation, and body weight loss were used as end points. RESULTS: The t.i.d. schedule with the same total daily doses as the q.d. schedule (250, 500, or 1,000 mg/kg/d) improved the efficiency of IPdR conversion to IUdR. As a result, the percentage of IUdR-DNA incorporation was higher using the t.i.d. schedule in the tumor xenografts as well as in normal small intestine and bone marrow. Using a fixed dose (500 mg/kg) per administration, the q.o.d. and q.3.d. schedules also showed greater IPdR conversion than the q.d. schedule, related to a greater recovery of hepatic aldehyde oxidase activity prior to the next drug dosing. In the tumor regrowth assay, all IPdR treatment schedules showed significant increases of regrowth delays compared with the control without IPdR (q.o.d., 29.4 days; q.d., 29.7 days; t.i.d., 34.7 days; radiotherapy alone, 15.7 days). The t.i.d. schedule also showed a significantly enhanced tumor growth delay compared with the q.d. schedule. Additionally, the q.o.d. schedule resulted in a significant reduction in systemic toxicity. CONCLUSIONS: The t.i.d. and q.o.d. dosing schedules improved the efficiency of enzymatic activation of IPdR to IUdR during treatment and changed the extent of tumor radiosensitization and/or systemic toxicity compared with a q.d. dosing schedule. These dosing schedules will be considered for future clinical trials of IPdR-mediated human tumor radiosensitization.     

The dependence of halogenated pyrimidine incorporation and radiosensitization on the duration of drug exposure

The influence of the duration of exposure to the halogenated pyrimidines iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) on incorporation into DNA and the resulting radiosensitization was studied in cultured human colon cancer cells. Cells were incubated with either 10 microM BrdUrd or IdUrd for periods up to 7 days. They were also assessed for up to 4 days after removal of drug from the medium. Replacement of thymidine by fraudulent bases was measured using a sensitive gas chromatographic, mass spectrometric (GC/MS) assay. Incorporation of BrdUrd and IdUrd plateaued at 35% and 30%, respectively, after 4 days of exposure. Prolonging the time of exposure to 7 days increased cytotoxicity without affecting either incorporation or radiosensitization. Incorporation remained constant for 1-2 days after removal of drug from the medium. Radiosensitization was linearly related to incorporation throughout the range of conditions assessed. These data suggest that it may be possible to develop a predictive assay for radiosensitization based on measurements of halogenated pyrimidine incorporation in a tumor biopsy specimen. They also suggest that a clinical approach based on repeated short exposures to halogenated pyrimidines may present certain advantages over the current practice of prolonged continuous exposure. A Phase I/II trial using IdUrd and external beam irradiation for the treatment of patients with poor prognosis soft tissue sarcomas has been initiated based on this concept.     

Differential radiosensitization in DNA mismatch repair-proficient and -deficient human colon cancer xenografts with 5-iodo-2-pyrimidinone-2'-deoxyribose.

PURPOSE: 5-iodo-2-pyrimidinone-2'-deoxyribose (IPdR) is a pyrimidinone nucleoside prodrug of 5-iododeoxyuridine (IUdR) under investigation as an orally administered radiosensitizer. We previously reported that the mismatch repair (MMR) proteins (both hMSH2 and hMLH1) impact on the extent (percentage) of IUdR-DNA incorporation and subsequent in vitro IUdR-mediated radiosensitization in human tumor cell lines. In this study, we used oral IPdR to assess in vivo radiosensitization in MMR-proficient (MMR+) and -deficient (MMR-) human colon cancer xenografts. EXPERIMENTAL DESIGN: We tested whether oral IPdR treatment (1 g/kg/d for 14 days) can result in differential IUdR incorporation in tumor cell DNA and subsequent radiosensitization after a short course (every day for 4 days) of fractionated radiation therapy, by using athymic nude mice with an isogenic pair of human colon cancer xenografts, HCT116 (MMR-, hMLH1-) and HCT116/3-6 (MMR+, hMLH1+). A tumor regrowth assay was used to assess radiosensitization. Systemic toxicity was assessed by daily body weights and by percentage of IUdR-DNA incorporation in normal bone marrow and intestine. RESULTS: After a 14-day once-daily IPdR treatment by gastric gavage, significantly higher IUdR-DNA incorporation was found in HCT116 (MMR-) tumor xenografts compared with HCT116/3-6 (MMR+) tumor xenografts. Using a tumor regrowth assay after the 14-day drug treatment and a 4-day radiation therapy course (days 11-14 of IPdR), we found substantial radiosensitization in both HCT116 and HCT116/3-6 tumor xenografts. However, the sensitizer enhancement ratio (SER) was substantially higher in HCT116 (MMR-) tumor xenografts (1.48 at 2 Gy per fraction, 1.41 at 4 Gy per fraction), compared with HCT116/3-6 (MMR+) tumor xenografts (1.21 at 2 Gy per fraction, 1.20 at 4 Gy per fraction). No substantial systemic toxicity was found in the treatment groups. CONCLUSIONS: These results suggest that IPdR-mediated radiosensitization can be an effective in vivo approach to treat "drug-resistant" MMR-deficient tumors as well as MMR-proficient tumors.     

Anti-stromal therapy with imatinib inhibits growth and metastasis of gastric carcinoma in an orthotopic nude mouse model

Recent studies have revealed that platelet-derived growth factor (PDGF) plays a role in promoting progressive tumor growth in several organs; however, whether PDGF plays such a role in gastric carcinoma is undetermined. We examined whether inhibition of PDGF receptor (PDGF-R) tyrosine kinase signaling by imatinib affects tumor growth and metastasis in an orthotopic nude mouse model of human gastric carcinoma. TMK-1 human gastric carcinoma cells were injected into the gastric wall of nude mice. Groups of mice (n = 10 each) received sterile water (control), low-dose imatinib (50 mg/kg/day), high-dose imatinib (200 mg/kg/day), cancer chemotherapeutic agent irinotecan (5 mg/kg/week), or imatinib (50 mg/kg/day or 200 mg/kg/day) and irinotecan (5 mg/kg/week) in combination for 28 days. Tumor growth and metastasis were assessed. Resected tumors were analyzed immunohistochemically. Carcinoma-associated fibroblasts, pericytes and lymphatic endothelial cells in stroma expressed high levels of PDGF-R; carcinoma cells did not. Treatment with imatinib alone did not inhibit tumor growth and metastasis; however, treatment with irinotecan alone or combined with imatinib significantly inhibited tumor growth. Only treatment with high-dose imatinib and irinotecan in combination inhibited lymph node and peritoneal metastases. Immunohistochemically, only imatinib alone or in combination with irinotecan was shown to significantly decrease the stromal reaction, microvessel area and pericyte coverage of tumor microvessels. These effects were marked with high-dose imatinib. In conclusion, administration of PDGF-R tyrosine kinase inhibitor in combination with irinotecan appears to impair the progressive growth of gastric carcinoma by blockade of PDGF-R signaling pathways in stromal cells.