Biology Contributions
Maximizing therapeutic gain with gemcitabine and fractionated radiation

Presented at the 40th Annual Meeting of ASTRO, October 28, 1998, Phoenix, AZ.
https://doi.org/10.1016/S0360-3016(99)00134-0Get rights and content

Abstract

Purpose/Objective: The nucleoside analogue gemcitabine inhibits cellular repair and repopulation, induces apoptosis, causes tumor growth delay, and enhances radiation-induced growth delay. After single doses of drug and radiation, maximum enhancement of tumor response was obtained when gemcitabine preceded radiation by at least 24 h. Conversely, the cellular radioresponse of the normal gastrointestinal epithelium was slightly protected when gemcitabine and radiation were separated by 24 h. This differential response created a time frame within which therapeutic gain could be maximized. In our present investigation, we sought to define the most therapeutically beneficial scheme of gemcitabine administration when combined with fractionated radiotherapy.

Methods and Materials: C3Hf/Kam mice were given identical drug and radiation schedules of administration, and both normal tissue (jejunal mucosa) and tumor (Sa-NH) responses were measured. Irradiation was given once per day for 5 days in normal tissue and tumor growth delay studies and twice per day for the tumor cure endpoint. A total dose of 25 mg/kg gemcitabine was given i.p. in 1 of 3 schedules: a single dose of 25 mg/kg 24 h before the start of fractionated irradiation, 12.5 mg/kg 24 h before the first and third radiation doses, or 24 h before each of 5 radiation doses. Groups of mice bearing 7- or 8-mm diameter tumors were treated with gemcitabine alone or in combination with fractionated irradiation under ambient or hypoxic conditions. The survival response of the jejunal mucosa was quantified by the microcolony assay and histologically by quantifying apoptosis, mitosis, S-phase fraction, and crypt cellularity.

Results: For tumor growth delay, dose-modifying factors (DMFs) were similar (1.34–1.46) for all 3 schedules of drug administration. In contrast, the response of the jejunum was strongly dependent on the schedule of gemcitabine administration. A single dose of gemcitabine before the start of fractionated radiotherapy resulted in slight radioprotection (DMF 0.96). Two doses and 5 daily doses of gemcitabine enhanced radiation response by factors of 1.09 and 1.23, respectively. Major factors affecting the response of the jejunal mucosa were apoptotic death of S-phase cells exposed to gemcitabine and cell cycle synchrony of surviving cells. Tumor reoxygenation was found to be a major mechanism for tumor radioenhancement, in addition to those reported earlier.

Conclusion: All 3 schedules of drug administration produced therapeutic gain; however, when gemcitabine was given more than once in a 5-fraction radiation treatment schedule, normal tissue toxicity increased. The highest therapeutic gain (1.4) was achieved by giving a single dose of gemcitabine (25 mg/kg) 24 h before the start of fractionated radiotherapy.

Introduction

Several biological factors are known to limit the efficacy of radiotherapy when given as a single treatment modality to patients with locally or regionally advanced cancer. They include the large number of tumor cell clonogens, the radioresistance of hypoxic tumor cells within the tumor mass, efficient DNA repair mechanisms, and recovery from injury in the period between radiation fractions. Also, distant metastases may develop from occult tumor cells that exist outside the irradiated field. For these reasons, increased attention is being given to combining radiotherapy with other treatment modalities, especially chemotherapy 1, 2, 3. Chemotherapeutic drugs act on both the primary tumor and disseminated cancer cells. In the primary tumor they destroy tumor cells by their own cytotoxic action, and may enhance the effects of radiotherapy by overcoming resistance mechanisms associated with this treatment modality. The results of the combined use of radiotherapy with chemotherapy have been encouraging, both in terms of increased local tumor control and decreased rates of distant failures 4, 5, 6, 7, 8. The combined treatments are, however, often associated with increased toxicity to normal tissue.

The use of newer cytotoxic drugs to further improve the combined efficacy of radiotherapy plus chemotherapy, and particularly, to increase their ability to overcome radiation resistance mechanisms has recently been explored 9, 10, 11, 12. Among these drugs are nucleoside analogues such as fludarabine and gemcitabine (2′2′-difluoro-2′-deoxycytidine, or dFdC) 11, 12. These drugs were investigated in combination with radiation because they are inhibitors of DNA replication and are DNA chain terminators 13, 14, 15, 16; consequently, they might inhibit DNA repair in radioresistant tumor cells and slow tumor regrowth during a fractionated radiation schedule.

Our own earlier studies were focused mainly on fludarabine 17, 18, 19, 20, 21. Using preclinical mouse tumor models, we demonstrated that fludarabine could enhance tumor radioresponse by factors of 1.24 to 2.14, depending on the timing of fludarabine administration relative to radiation, the dose of fludarabine, tumor type, and schedule of radiation 17, 18, 19, 21. Radioenhancement was associated with preferential loss of S-phase cells through fludarabine-induced apoptosis, delayed cell cycle progression, and subsequent parasynchronization of tumor cells into the radiosensitive G2/M phases of the cell cycle (21). Because fludarabine did not appreciably modify the radioresponse of a number of normal tissues 17, 19, 20, it improved the therapeutic ratio of radiotherapy.

Fludarabine and gemcitabine have similar mechanisms of action; however, the latter has better membrane permeability, and its active metabolites have a longer cellular half-life (22). Also, while fludarabine has shown little single-agent activity in solid tumors 18, 23, gemcitabine exerts antitumor activity in a number of murine solid tumors and human tumor xenografts 24, 25, 26, and has shown significant clinical activity in several solid tumor types traditionally treated with radiotherapy, including pancreatic, head and neck, and lung carcinomas (27). These properties of gemcitabine make this nucleoside analogue an attractive candidate for combining with radiation.

In cells, gemcitabine undergoes phosphorylation by deoxycytidine kinase to the active metabolites gemcitabine diphosphate (dFdCDP) and triphosphate (dFdCTP) (28). dFdCDP interferes with DNA synthesis by inhibiting ribonucleotide reductase; hence, reducing the deoxynucleotide pools 29, 30. dFdCTP competes with deoxycytidine triphosphate for incorporation into elongating DNA strands; only one additional deoxynucleotide can be incorporated after the insertion of dFdCTP, thus halting DNA polymerization 29, 30.

Several in vitro studies demonstrated that gemcitabine can radiosensitize tumor cells when administered before radiation, by enhancement factors (EF) of up to 1.8 12, 31, 32, 33. In general, higher concentrations of gemcitabine and longer presence of the drug in cell cultures before irradiation were found to be more effective. Studies from our laboratory, and those of others, have shown that gemcitabine inhibited chromosome repair following irradiation, thus increasing the frequency of residual chromosome breaks 34, 35, 36. Information on the ability of gemcitabine to enhance tumor response to radiation and improve the therapeutic ratio, however, are limited. A recent report showed that gemcitabine, when given once or twice weekly, significantly enhanced the fractionated radioresponse of a human squamous cell carcinoma grown in nude mice (37). Using a murine sarcoma, designated SA-NH, and tumor growth delay and tumor cure rate as treatment endpoints, we recently showed that gemcitabine has strong radioenhancing properties when combined with single-dose local tumor irradiation (1). Depending on experimental conditions, the enhancement factors ranged from 1.1 to 2.0, with the greatest effect observed when gemcitabine administration preceded irradiation by 24–72 h. In addition, gemcitabine was highly effective in eliminating microscopic metastases in the lungs of mice whose primary tumors were controlled by irradiation.

The experiments described in the present study explored the optimal dose and schedule with which to combine gemcitabine and fractionated irradiation to achieve the most favorable therapeutic ratio. SA-NH sarcoma was used to investigate the effect of the combination on tumor responses, and mouse jejunum was selected to assess the effect of gemcitabine on normal tissue damage.

Section snippets

Mice and tumors

C3Hf/Kam mice, bred and maintained in our specific-pathogen-free mouse colony, were 3–4-months old at the beginning of experiments. The tumor used, the sarcoma SA-NH, is syngeneic to and nonimmunogenic in this strain of mice. Solitary tumors were produced in the muscles of the right leg by the inoculation of 5 × 105 cells. Tumor cell suspensions were prepared by mechanical disruption and enzymatic digestion of nonnecrotic tumor tissue (38).

Gemcitabine

Gemcitabine (2′,2′-difluoro-2′-deoxycytidine) was

Antitumor activity of gemcitabine alone

Before assessing the potential of gemcitabine to enhance tumor response to fractionated radiation, it was important to establish a dose of gemcitabine that could be given to mice safely every day for several days, and that would exert an appreciable antitumor effect. In our earlier study, doses of gemcitabine ranging from 50 to 400 mg/kg were strongly effective against SA-NH tumor, and were well tolerated when given as a single bolus. To determine the effect of multiple injections, mice bearing

Discussion

The data presented here show that gemcitabine can significantly enhance the response of the murine sarcoma SA-NH to fractionated irradiation, as well as enhance or reduce radiation response of jejunal mucosa. These effects depended on gemcitabine’s dose and schedule relationship with irradiation; however, all schedules tested were able to increase the therapeutic ratio of radiotherapy. Tumor radioresponse was enhanced by factors of 1.34–1.46 (using the isoeffective radiation dose assessments of

Acknowledgements

We are grateful to Lane Watkins and his staff for the supply and care of the mice used in these studies. Animals used in this study were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the United States Department of Agriculture and Department of Health and Human Services. We also thank Mr. Kuriakose Abraham for preparation of histology slides, Ms. Nalini Patel for BrdUrd

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