Thymidylate synthase (TS) is an essential enzyme in the de novo synthesis of thymidylate and because it is a critical enzyme for cell proliferation, it has been an important target for drug development in cancer therapy. Although 5-fluorouracil and methotrexate are active agents in the treatment of patients with squamous cell carcinoma (SCC) of the head and neck, lack of therapeutic selectivity and resistance remain major obstacles to curative therapy with these agents. Recently, antimetabolites which are folate-based specific TS inhibitors became available; D1694, AG331 and AG337. These agents are now at various stages of clinical evaluation with demonstrated clinical activity in colorectal cancer. Overall goal of this research is to develop basic knowledge that can be evaluated clinically concerning the biochemical and molecular mechanisms associated with in vivo therapeutic selectivity of antimetabolites TS inhibitors in SCC tumors.To achieve this goal, less-differentiated human SCC of the pharynx (FaDu), and well-differentiated human epidermoid carcinoma of the neck (A253) cell lines will be used to achieve the following specific aims:1)to establish that the therapeutic selectivity of TS inhibitors is schedule dependent Hypothesis: G.I. toxicity observed clinically and in model system with a single i.v. push of ZD1694 can be altered by fractionated doses or by continuous i.v. infusion. It is likely that peak plasma concentration of the drug contributes to the observed tissue specific toxicity, namely gastrointestinal as the dose limiting toxicity. With few cells in solid tumors which are in S-phase, a single drug dose may be less therapeutic. Thus SCC cell lines will be used in nude mice to define the optimal dose-schedule relationship associated with maximum therapeutic index. 2)to evaluate the role of plasma dThd and tissue dthyd salvage pathway in the therapeutic selectivity of TS inhibitors The role of dThyd phosphorylase, interferon and dipyridamole (DP) will be evaluated. Hypothesis: High plasma level dThyd in mice (approximately 1 micromole) with active tissue dThyd kinase salvage pathway reduce the therapeutic potential of de novo thymidylate synthesis inhibitors. The effects of reducing plasma dThyd by dThyd phosphorylase, inhibition of nucleoside transport by DP, and activation of dThyd phosphorylase by interferon on thymidylate pools in normal (G.I., bone marrow) and tumor tissues will be assessed. 3)to identify biochemical and molecular determinants associated with drug therapeutic selectivity. Hypothesis: high degree and sustained inhibition of TS (>95%, for at least 3 cell doubling time) are essential prerequisites for inducting irreversible G1/S block, upregulation of p53 (wild) and high level and sustained DNA damage in tumor tissue. 4)to determine if the lack of antitumor activity is associated with development of tumor drug resistance Hypothesis: Resistance to D1694 is associated with decreased folypolyglutamate synthase and to AG331 and AG337 with altered TS resulting in a decrease in degree and duration of TS inhibition. With the knowledge gained in model system with these new agents, selective treatment may be designed and verified in patients with head & neck cancer and other solid tumor malignancies.
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