Tipiracil Research Articles

P2-26-7 - Efficacy of Trifluridine for 5-Fluorouracil-Resistant Human Gastric Cancer Cell Lines and Their Mechanisms

Background: TAS-102 consisting of trifluridine (FTD) and tipiracil hydrochloride (TPI) was found to significantly improve the overall survival of patients with metastatic colorectal cancer who were refractory to treatment with a fluoropyrimidine, irinotecan, and oxaliplatin in a phase II study. Although significant survival benefits from 5-fluorouracil (5-FU)-based chemotherapy have also been reported in patients with gastric cancer, many patients experience recurrences after 5-FU-based chemotherapy. In this study, the efficacy of FTD against 5-FU-resistant human gastric cancer cell lines was investigated.Method: 5-FU-resistant cell lines were established by continuously exposing the parent cell lines (MKN-45, MKN-74, and KATOIII) to 5-FU over a 1-year. The cytotoxicity of FTD was evaluated by a crystal violet staining assay. To elucidate the mechanism by which resistance is overcome, the mRNA levels of TK1, which converts FTD into an active form, and hENT1, which is involved in the cellular uptake of FTD, were determined by RT-PCR.Results: KATOIII/5FU, MKN74/5FU, and MKN45/5FU exhibited a 2.0-, 4.8-, and 14.2-fold resistance to 5-FU, compared with their parent cell lines, respectively. MKN-45/5FU also showed a 3.7-fold resistance to FTD, whereas MKN-74/5FU and KATOIII/5FU showed no cross-resistance to FTD. The TK1 mRNA level was decreased by 40% in the MKN-45/5FU, compared with the parent cell line. Furthermore, this cell line showed a 60% decrease in the mRNA level for hENT1. In contrast, the hENT1 mRNA level increased by 1.6-fold in the KATOIII/5FU cells, with a 50% decrease in the TK1 mRNA level. The TK1 and hENT1 mRNA levels in MKN-74/5FU were increased by 1.9- and 1.7-fold, respectively.Conclusion: FTD was able to overcome the resistance to 5-FU in 2 out of 3 resistant cell lines, with MKN45/5FU exhibiting a partial cross-resistance. Therefore, TAS-102 might be a promising agent for gastric cancer relapses after 5-FU-based treatment.

IS7-5 - New Drug Development for Refractory Metastatic Colorectal Cancer in Japan

Cytotoxic agents, fluoropyrimidine, irinotecan and oxaliplatin, and monoclonal antibodies, bevacizumab, cetuximab and panitumumab have been shown to significantly improve overall survival (OS) for metastatic colorectal cancer (mCRC). Recently, two other agents targeting vascular endothelial growth factor (VEGF) have shown promise. Regorafenib is an orally active inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases, which showed statistically significant improvement in OS for mCRC who had progressed after standard therapies. Ziv-aflibercept is a fusion protein consisting of key domains of VEGF receptor 1 and 2, which showed statistically significant improvement in OS for previously treated mCRC. However, no standard salvage therapy available, although many patients retain good performance status.TAS-102 is a novel oral nucleoside antitumor agent consisting of 5-trifluorothymidine (FTD) and a thymidine phosphorylase inhibitor, tipiracil-hydrochloride (TPI). FTD is the active antitumor component of TAS-102 while TPI prevents degradation of FTD, thereby enabling sustained and effective cytotoxic levels of this novel anti-metabolite. TAS-102 is effective against human colorectal tumor cell lines with innate and acquired resistance to 5FU, both in-vitro as well as in-vivo models. A prior randomized phase 2 study demonstrated a significant survival improvement of TAS-102 over placebo in refractory mCRC (HR = 0.56 p = 0.0011, Yoshino T., et al., Lancet Oncology 2012). Based on these results, TAS-102 has been approved in Japan before the rest of the world. An international phase III trial, called the RECOURSE, was conducted to evaluate the efficacy and safety of TAS-102 in patients with mCRC refractory to standard therapies (ClinicalTrials.gov NCT01607957). We have started the phase 1/2 trial to investigate the efficacy and safety of TAS-102 plus bevacizumab in refractory mCRC (UMIN000012883). Update on promising new agents will be presented.

Abstract 781: Efficacy of trifluridine for 5-fluorouracil-resistant human gastric cancer cell lines and their mechanisms

Abstract Background: TAS-102 is a novel oral nucleoside antitumor agent consisting of trifluridine (FTD) and tipiracil hydrochloride (TPI). TAS-102 was found to significantly improve the overall survival of patients with metastatic colorectal cancer who were refractory to treatment with a fluoropyrimidine, irinotecan, and oxaliplatin in a double-blind randomized phase II study. Although significant survival benefits from 5-fluorouracil (5-FU)-based chemotherapy have also been reported in patients with gastric cancer, many patients experience recurrences after several courses of 5-FU-based chemotherapy. The resistance of gastric tumors to 5-FU therapy is thus a major clinical problem. In this study, the efficacy of FTD against 5-FU-resistant human gastric cancer cell lines was investigated. Method: 5-FU-resistant cell lines established by continuously exposing the parent cell lines (MKN-45, MKN-74, and KATOIII) to escalating concentrations (1-5 μM) of 5-FU over a 1-year period were used. The sensitivities of the cell lines to FTD were evaluated using a crystal violet staining assay. To elucidate the mechanism by which resistance is overcome, the mRNA levels of TK1, which converts FTD into an active monophosphate form, and hENT1, which is involved in the cellular uptake of FTD, were determined using RT-PCR. Results: The resistant cell lines KATOIII/5FU, MKN74/5FU, and MKN45/5FU exhibited a 2.0-fold, 4.8-fold, and 14.2-fold resistance to 5-FU, compared with their parent cell lines, respectively. MKN-45/5FU also showed a 3.7-fold resistance to FTD, whereas MKN-74/5FU (1.0-fold) and KATOIII/5FU (1.2-fold) showed no cross-resistance to FTD. The TK1 mRNA level was decreased by 40% in the MKN-45/5FU cells, compared with the parent cell line. Furthermore, this cell line showed a 60% decrease in the mRNA level for hENT1. In contrast, the hENT1 mRNA level increased by 1.6-fold in the KATOIII/5FU cells, with a 50% decrease in the TK1 mRNA level. The TK1 and hENT1 mRNA levels in MKN-74/5FU were increased by 1.9-fold and 1.7-fold, respectively. Conclusion: FTD was able to overcome the resistance to 5-FU in 2 out of 3 resistant cell lines in vitro, with MKN45/5FU exhibiting a partial cross-resistance to FTD. These results suggest that TAS-102 might be a promising chemotherapeutic agent for the treatment of gastric cancer relapses after 5-FU-based treatment. Furthermore, TK1 and hENT1 might be involved in FTD-related cytotoxicity. Citation Format: Kazuaki Matsuoka, Takashi Kobunai, Teiji Takechi. Efficacy of trifluridine for 5-fluorouracil-resistant human gastric cancer cell lines and their mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 781. doi:10.1158/1538-7445.AM2014-781

Repeated oral dosing of TAS-102 confers high trifluridine incorporation into DNA and sustained antitumor activity in mouse models.

TAS-102 is a novel oral nucleoside antitumor agent containing trifluridine (FTD) and tipiracil hydrochloride (TPI). The compound improves overall survival of colorectal cancer (CRC) patients who are insensitive to standard chemotherapies. FTD possesses direct antitumor activity since it inhibits thymidylate synthase (TS) and is itself incorporated into DNA. However, the precise mechanisms underlying the incorporation into DNA and the inhibition of TS remain unclear. We found that FTD-dependent inhibition of TS was similar to that elicited by fluorodeoxyuridine (FdUrd), another clinically used nucleoside analog. However, washout experiments revealed that FTD-dependent inhibition of TS declined rapidly, whereas FdUrd activity persisted. The incorporation of FTD into DNA was significantly higher than that of other antitumor nucleosides. Additionally, orally administered FTD had increased antitumor activity and was incorporated into DNA more effectively than continuously infused FTD. When TAS-102 was administered, FTD gradually accumulated in tumor cell DNA, in a TPI-independent manner, and significantly delayed tumor growth and prolonged survival, compared to treatment with 5-FU derivatives. TAS-102 reduced the Ki-67-positive cell fraction, and swollen nuclei were observed in treated tumor tissue. The amount of FTD incorporation in DNA and the antitumor activity of TAS-102 in xenograft models were positively and significantly correlated. These results suggest that TAS-102 exerts its antitumor activity predominantly due to its DNA incorporation, rather than as a result of TS inhibition. The persistence of FTD in the DNA of tumor cells treated with TAS-102 may underlie its ability to prolong survival in cancer patients.

Phase III Recourse Trial of Tas-102 Vs. Placebo, with Best Supportive Care (Bsc), in Patients (Pts) with Metastatic Colorectal Cancer (Mcrc) Refractory to Standard Therapies

ABSTRACT Aim: TAS-102 is a combination of a novel oral nucleoside, trifluridine (FTD) with the thymidine phosphorylase inhibitor, tipiracil hydrochloride (TPI), which prevents the degradation of FTD, enabling sustained and effective FTD levels. RECOURSE (Sponsor: Taiho Oncology Inc. / Taiho Pharmaceutical Co. Ltd.) was conducted to evaluate the efficacy and safety of TAS-102 in pts with mCRC refractory to standard therapies. Methods: mCRC-patients (ECOG PS 0-1) who failed fluoropyrimidines, irinotecan, oxaliplatin, bevacizumab and cetuximab/panitumumab (if KRAS wild-type) were eligible. The primary endpoint (overall survival, OS) and the key secondary efficacy endpoint (progression-free survival, PFS) were evaluated using univariate and multivariate analyses for prospectively defined subgroups and a retrospectively defined subgroup with prior regorafenib. Results: 800 pts were randomized to TAS-102 (534 pts) or placebo (266 pts). The hazard ratios for OS and PFS were 0.68 (95% CI: 0.58 - 0.81; p Subgroups OS N HR 95% CI KRAS status Wild-type 393 0.58 0.45-0.74 Mutant 407 0.80 0.63-1.02 Region Western 534 0.64 0.52-0.80 Asia 266 0.75 0.57-1.00 PS 0 448 0.73 0.58-0.93 1 352 0.61 0.48-0.79 Prior Use of regorafenib Yes 144 0.69 0.45-1.05 No 656 0.69 0.57-0.83 Conclusions: OS and PFS benefit was observed in all prospectively defined subgroup analyses, including prior regorafenib therapy. Disclosure: K. Yamazaki: Other substantive relationships (speakers bureaus) from Taiho Pharmaceutical Co., Ltd; Y. Shimada: Membership on an advisory board, Corporate-sponsored research finding, and Other substantive relationships (speakers bureaus) from Taiho Pharmaceutical Co., Ltd; M. Aivado: Sponsor's employee; L. Makris: Consulting engagement for Taiho Pharmaceutical Co. Ltd. and Taiho Oncology Inc. All other authors have declared no conflicts of interest.

Thymidine phosphorylase influences [18F]fluorothymidine uptake in cancer cells and patients with non-small cell lung cancer

Thymidine phosphorylase (TP), a key enzyme in the pyrimidine nucleoside salvage pathway, catalyses the reversible phosphorylation of thymidine, thereby generating thymine and 2-deoxy-D-ribose-1-phosphate. By regulating the levels of endogenous thymidine, TP may influence [(18)F]fluorothymidine ([(18)F]FLT) uptake. We investigated the effect of TP activity on [(18)F]FLT uptake by tumours. Uptake of [(3)H]FLT and [(3)H]thymidine ([(3)H]Thd) and the activities of TP, thymidine kinase 1 (TK1), and equilibrative nucleoside transporter 1 (ENT1) were determined in exponentially growing A431, A549, HT29, HOP92, ACHN, and SKOV3 cells in the presence or absence of tipiracil hydrochloride, a TP inhibitor. Eighty-five non-small cell lung cancer tissues from a patient cohort that was previously studied with [(18)F]FLT positron emission tomography (PET) were retrieved and subjected to immunohistochemical analysis of TP expression. Factors that affected the maximum standardised uptake value (SUVmax) of [(18)F]FLT-PET were identified by multiple linear regression analysis. A431 cells had the highest TP activity; A549 and HT29 cells had moderate TP activity; and ACHN, SKOV3, and HOP92 cells had little detectable TP activity. Cell lines with high TP activity took up more [(3)H]FLT than [(3)H]Thd, whereas cells with little TP activity took up more [(3)H]Thd than [(3)H]FLT. In cells with high TP activity, TP inhibition decreased [(3)H]FLT uptake and increased [(3)H]Thd uptake. However, TP inhibition had no effect on ACHN, SKOV3, and HOP92 cells. TP inhibition did not change TK1 or ENT1 activity, but did increase the intracellular level of thymidine. The SUVmax of [(18)F]FLT was affected by three independent factors: Ki-67 expression (P < 0.001), immunohistochemical TP score (P < 0.001), and tumour size (P = 0.015). TP activity influences [(18)F]FLT uptake, and may explain preferential uptake of [(18)F]FLT over [(3)H]Thd. These results provide important insights into the biology of [(18)F]FLT as a proliferation marker.