Abstract
Background: NUC-3373 is the first Nucleotide Analogue able to bypass the key drug resistance mechanisms associated with 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (FUDR) that severely hinder their clinical activity. The key cellular mechanisms causing drug resistance in cancer cells are the limited expression of activating kinases and nucleoside transporters, and overexpression of catabolic enzymes. The anti-neoplastic activity of 5-FU is largely attributed to its active metabolite, 5-fluorodeoxyuridine monophosphate (FdUMP), which inhibits the enzyme thymidylate synthase. As it already bears the monophosphate moiety, NUC-3373 is a pre-activated form of the active anti-cancer agent FdUMP. Here, we report potent in vitro and in vivobiological activity of NUC-3373.
Methods: The cytotoxicity of 5-FU and the ProTide NUC-3373 was monitored with EC50 in vitro viability assays using 4 colorectal (Colo-205-luc; HT-29; HCT-116 and SW620); 3 lung (H1975; H1703 and SK-MES-1); 2 ovarian (OVCAR3 and A2780); 1 acute lymphoblastic leukemia (CCRF-CEM) and 1 cervix (HeLa) human tumour cell lines. These assays were also performed in conditions mimicking cancer resistance, where the activating enzyme thymidine kinase (TK) and the nucleoside transporter, hENT1, were mutated. Sensitivity of NUC-3373 and 5-FU to dihydropyrimidine dehydrogenase (DPD) degradation was assessed by absorption spectroscopy and UPLC-MS/MS. NUC-3373 anti-cancer activity was further evaluated in HT-29 nude mouse xenograft models.
Results: NUC-3373 was more cytotoxic than 5-FU, achieving 2 to 333 fold lower EC50 values in the majority of cancer cell lines tested. TK inhibition reduced the cytotoxicity of NUC-3373 and FUDR by 4- and 136-fold respectively, suggesting NUC-3373 is more independent of TK. Inhibition of nucleoside transport decreased cytotoxic activity of NUC-3373 only mildly compared to a 63-fold reduction in FUDR cytotoxicity. NUC-3373 and 5-FU sensitivity to DPD degradation was assessed in cell lysates with or without the DPD inhibitor, gimeracil. NUC-3373 concentrations remained unaffected in the cell lysate irrespective of gimeracil treatment. In contrast, when the experiment was conducted with 5-FU, gimeracil treatment significantly increased the 5-FU concentration (p = 0.039) suggesting that NUC-3373, unlike 5-FU, is not a substrate for DPD metabolism. In colorectal cancer xenografts, NUC-3373 demonstrated greater tumour growth inhibition (47%) than 5-FU (25%). A toxicology study in which NUC-3373 was administered daily for 5 days/week for 4 consecutive weeks, at doses greater than 4 mg/kg/day in beagle dogs, compared favourably with that described following a single bolus intravenous administration of 5-FU.
Conclusions: The novel ProTide, NUC-3373, overcomes the key cancer resistance mechanisms associated with 5-FU. NUC-3373 has efficacy in vitro and in vivo, and is resistant to DPD-mediated degradation. Results from formal toxicology assessments support the initiation of human clinical studies. A Phase I/II clinical study at Oxford University has been initiated to explore the safety, pharmacokinetic and clinical activity of NUC-3373 in participants with advanced solid tumours.
Citation Format: Essam A. Ghazaly, Magdalena Slusarczyk, Christopher McGuigan, David Harrison, Sarah P. Blagden. NUC-3373: A novel pyrimidine nucleotide analogue that overcomes key cancer drug resistance limiting patient survival. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B46.
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