Biology contribution
Interaction of pemetrexed disodium (ALIMTA, multitargeted antifolate) and irradiation in vitro

https://doi.org/10.1016/S0360-3016(01)02794-8Get rights and content

Abstract

Purpose: Pemetrexed disodium (Alimta, multitargeted antifolate, LY231514; Eli Lilly and Co., Indianapolis, Indiana) (“pemetrexed”) is a new folate antimetabolite with significant antitumor activity. Different from classic antifolates, pemetrexed inhibits several key enzymes of thymidylate and purine synthesis, but a radiosensitizing potential may also be presumed. Therefore, the interaction of pemetrexed and ionizing radiation was studied for in vitro clonogenic survival of different human tumor cell lines.

Methods and materials: Human colon (Widr), breast (MCF-7), cervix (Hela), and lung (LXI) carcinoma cells from log-phase cultures were exposed to pemetrexed (2 h) in combination with different radiation doses given 1 h before pemetrexed washout (all cell lines) or at different points of time before or after pemetrexed addition (Widr). Survival curves were analyzed according to the linear-quadratic (LQ) model, and mean inactivation doses (MID) and radiation enhancement ratios were calculated from the survival curve parameters. Cell-cycle progression of serum-stimulated and pemetrexed- or mock-treated Widr cells was monitored by flow cytometry.

Results: Radiosensitization was found for all cell lines at moderately toxic pemetrexed exposures (0.05–0.3 μg/ml [106–636 nM]), but this was cell-type dependent and was most pronounced at roughly isotoxic concentrations, for the least pemetrexed-sensitive Widr cells. Enhancement ratios ranged from about 1.2 (MCF-7 and Hela) to 1.8 (Widr), with a tendency to increase with pemetrexed concentration. Little, if any, change of radiosensitization was observed (Widr) when the time of irradiation was varied from 4 h before to 10 h after the beginning of pemetrexed treatment. Cell-cycle progression of serum-stimulated Widr cells was only marginally affected by pemetrexed.

Conclusions: Pemetrexed enhances radiation-induced cell inactivation at moderately toxic exposures and over many hours after drug removal. This effect is not due to disturbed cell-cycle progression, but likely involves an interaction of pemetrexed with long-lived (>4 h) cellular radiation damage and needs to be considered when introducing a combined clinical application.

Introduction

Alimta (Pemetrexed disodium, LY231514: Eli Lilly and Co., Indianapolis, Indiana) is a new pyrrolopyrimidine-based folate antimetabolite. Different from classic antifolates, pemetrexed inhibits several key enzymes of thymidylate and purine synthesis 1, 2. Pemetrexed is brought into cells by a reduced folate carrier system. Intracellularly, pemetrexed is modified to a negatively charged polyglutamated form by the enzyme folypolyglutamate synthetase (FPGS). This prevents efflux from cells and aids binding to target enzymes. Cytotoxicity and cytostasis result from the inhibition of thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyl transferase (GARFT), as well as of other folate-requiring enzymes. Cytoprotection can only be achieved by addition of thymidine and hypoxanthine. Thus, the reversal pattern differs from other antifolates 3, 4, 5. Pemetrexed shows a significant antitumor activity for a broad spectrum of cancers, including pancreatic, colorectal, gastrointestinal, lung, head and neck, breast, and cervix cancers. Various Phase II studies are ongoing or completed (6).

Combined modality treatment with radiation and cytotoxic agents that might improve clinical outcome have gained increasing interest. The potential of antifolates to change radiation effects has been known for a long time (7). The classic antifolate, 5-fluorouracil (5-FU), is used in simultaneous combination with radiotherapy of different tumors such as anal and rectal carcinomas 8, 9. Raltitrexed, a thymidylate synthase inhibitor, increases ionizing radiation-induced cell killing in vitro (10). Although the activity of pemetrexed can be distinguished from that of common antifolates, a potential to modify radiation effects may be presumed. Therefore, the interaction of pemetrexed and ionizing radiation was studied in different human tumor cell lines by measuring clonogenic survival.

Section snippets

Tumor cell lines

Four different human carcinoma cell lines were used. Widr (colon), MCF-7 (breast), Hela (cervix uteri), and LXI (lung) were provided by the Tumorbank of the German Cancer Research Center (dkfz Heidelberg). Cells were grown as monolayers in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum in 6% CO2 at 37°C and were subcultured as log-phase cells every 3 days. The population doubling times were approximately 28 h for Widr and MCF-7, 32 h for Hela, and 24 h for LXI cells, respectively.

Pemetrexed toxicity

The toxicity of a 2-h pemetrexed exposure for the different cell lines is depicted in Fig. 1. LXI was the most sensitive cell line with a decrease of clonogenic survival to 10% at concentrations of >0.1 μg/ml (>212 nM). In contrast, a 20-fold higher dose was needed for MCF-7 to reduce survival to a comparable level. Widr and Hela cells showed a response below 0.5 μg/ml (1.06 μM) that was similar to the MCF-7 cells, but also a marked threshold behavior around 1 μg/ml (2.12 μM). As it was

Discussion

Pemetrexed is a promising new antifolate/antimetabolite. Considering the possibility of its use in combined modality cancer treatment together with radiotherapy, preclinical in vitro investigations on potential interaction phenomena appeared desirable. The present results with four different human tumor cell lines clearly demonstrate that a concomitant exposure to pemetrexed and ionizing radiation inhibited clonogenic survival in excess of independent toxicities (Fig. 2, Fig. 3, Fig. 4, Fig. 5,

Acknowledgements

The authors gratefully acknowledge the excellent technical assistance of Ute Haner and Thuy Trinh.

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