Mode of action of trifluorothymidine (TFT) against DNA replication and repair enzymes.

Trifluorothymidine (TFT) is well known to be converted to TFT-monophosphate by thymidine kinase and to inhibit thymidylate synthase. In addition, TFT-triphosphate (TFT-TP) is also incorporated into DNA, resulting in cytocidal effects. However, the precise mechanism of TFT-induced DNA damage is still unclear. Therefore, we investigated the modes of action of TFT against DNA replication and repair enzymes, as compared with those of 5FU and FdUrd. When HeLa cells were treated with TFT at a concentration of 1 µM (IC50 value), the concentration of TFT in the DNA was calculated as 62.2±0.9 pmol/1x106 cells for 4 h. On the other hand, following treatment of the cells with FdUrd (0.5 µM) and 5FU (10 µM) at their IC50 doses, the drug concentrations in the DNA were 7.53, and 0.17 pmol/1 x 106 cells for 4 h, respectively. These results show the markedly greater degree of incorporation of TFT into the DNA of the HeLa cells compared with that of 5FU (approximately more than 300-fold for 4 h) or FdUrd (approximately more than 8-fold for 4 h). The primer extension assay demonstrated that TFT-TP was also incorporated into the T-sites of the growing DNA strand, however, it competed only weakly with thymidine triphosphate. The DNA glycosylase assay was performed using commercially available DNA glycosylase and fractionated HeLa cell extracts obtained by gel filtration. There was no detectable excision of the TFT pairing to adenine by uracil DNA glycosylase (UDG), thymine DNA glycosylase (TDG), methyl-CpG binding domain 4 (MBD4) or the fractionated HeLa cell extracts, however, TDG and MBD4 were able to excise the TFT pairing to guanine. Additional data indicate that small-interfering RNA-mediated knockdown of TDG or MBD4 significantly increased the resistance to the cytotoxic effects of FdUrd, but not to that of TFT. These studies show the greater degree of incorporation of TFT into the DNA than that of 5FU or FdUrd, and that such a high degree of incorporation of TFT residues into the DNA might be related to exhibit potent cytotoxic activity to be refractory to cleavage by these DNA glycosylases; thus, the DNA-directed cytotoxic effect of the compound is quite different from that of 5FU.

Therapeutic potential of the dual-targeted TAS-102 formulation in the treatment of gastrointestinal malignancies.

Current treatment modalities for cancer combine cytotoxic drugs against DNA and novel targeted drugs affecting signal transduction pathways, which are required for growth progression and metastasizing tumors. Classical chemotherapeutic regimens for gastro-intestinal tumors include antimetabolites based on 5-fluorouracil (5FU), the platinum analog oxaliplatin and the topoisomerase inhibitor irinotecan. The thymidine analog trifluorothymidine (TFT) has been shown to bypass resistance pathways for 5FU derivatives (S-1, UFT, Xeloda) in model systems, while concurrent application with a thymidine phosphorylase inhibitor (TPI) increases the bioavailability of TFT, thereby potentiating the in vivo efficacy of TFT. The formulation TAS-102 is given orally in a 1.0:0.5 molar ratio (TFT:TPI). The formulation is dual-targeted due to the cytotoxic effect of TFT, which is enhanced by TPI, while TPI also exerts antiangiogenic effects by inhibiting thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor. Evidence is accumulating from in vitro and in vivo preclinical studies that these properties favor further combinations with other cytotoxic agents currently being used in the treatment of gastro-intestinal tumors. Also treatment with targeted agents will synergistically down-regulate signal transduction pathways responsible for growth and progression of tumors. In this review, we summarize the available information on (clinical) pharmacology, mechanisms of action, pharmacodynamic and pharmacokinetic properties, early clinical trials and future directions of the new potent combination drug TAS-102.

Potentiation of the antitumor activity of alpha, alpha, alpha-trifluorothymidine by the co-administration of an inhibitor of thymidine phosphorylase at a suitable molar ratio in vivo.

Trifluorothymidine (FTD) is a thymidine analog that exhibits an antitumor activity through its inhibition of thymidylate synthase and its incorporation into DNA. However, FTD is rapidly hydrolyzed to an inactive form by thymidine phosphorylase (TP). We attempted to augment the antitumor activity of FTD by combining it with a potent and reversible inhibitor of TP, 5-chloro-6-(2-imino-propyrrolidin-1-yl) methyl-2, 4 (1H, 3H)-pyrimidinedione hydrochloride (TPI) in human tumor xenografts with a low sensitivity to 5-fluorouracil. The optimum ratio of TPI to FTD was determined by measuring the maximum plasma level of FTD after oral administration and the antitumor effect of FTD on human tumor xenografts in mice. When > 0.5 M of TPI and 1 M of FTD (10 mg/kg) were co-administered, the plasma FTD levels in mice and monkeys were elevated, almost reaching a maximal and constant value of 20-30 microg/ml and 15 microg/ml, respectively. When human gastrointestinal cancer cell lines (DLD-1, CO-3 and AZ521) were xenografted into nude mice, the antitumor activity of FTD was augmented by the co-administration of TPI, compared to that of FTD alone, and the ED50 value, used to indicate the antitumor effect, reached a maximum value (about 25, 20, and 10 mg/kg in the DLD-1, CO-3, and AZ521 tumors, respectively) when > 0.5 M of TPI was combined with 1 M of FTD. The oral administration of TPI markedly improved the FTD-induced toxicity, as evaluated by the decrease in the body weight of the mice. These results suggested that the optimum ratio of FTD to TPI was 1:0.5 M, enabling a high antitumor activity and a low toxicity. We further evaluated whether TPI inhibits TP-induced angiogenesis in a gelatin-sponge mouse model, based on the finding that TP is identical to platelet-derived endothelial cell growth factor. Ten and 30 mg/kg administration of TPI significantly inhibited TP-induced neovascularization in a dose-dependent manner in a mouse model. The above results suggest that the combination of TPI and an antitumor nucleoside, FTD, not only enhances the antitumor efficacy and decreases the toxicity of FTD, but also suppresses TP-induced angiogenesis.

A novel combination antimetabolite, TAS-102, exhibits antitumor activity in FU-resistant human cancer cells through a mechanism involving FTD incorporation in DNA.

TAS-102 is a new antimetabolite agent composed of a alpha, alpha, alpha-trifluorothymidine (FTD; 1 M) and thymidine phosphorylase inhibitor (TPI; 0.5 M). Here, we investigated the antitumor effect and mechanism of TAS-102 against 5-FU, or FdUrd, resistant human cancer cell lines. The respective tumor growth inhibition rate of orally administered FTD against 5-FU-resistant NUGC-3 was about 70% at a dose level of 200 mg/kg/day; this value was comparable to that against the parental NUGC-3. On the other hand, the tumor inhibition rates of 5-FU, FdUrd, and TS-1 against 5-FU-resistant NUGC-3 were lower than those against parental NUGC-3. Similar observations were made in an FdUrd-resistant human colorectal cancer cell line (DLD-1). TAS-102 was also effective in 5-FU-less sensitive human pancreatic cancer cell lines (PAN-12 and BxPC-3) and human esophagus cancer (T.T.) when compared with 5-FU or UFT. Our hypothesis was that a relatively short and high dosage of TAS-102 results in an additional mechanism of FTD incorporation into DNA other than thymidylate synthase (TS) inhibition. We then examined the effects of FTD on DNA at the cellular level. After treatment with FTD or FdUrd, the DNA fragmentation pattern was examined using filter elution and in situ nick translation. Treatment with FTD for 2 h resulted in marked DNA fragmentation. When the tumor cells were treated with FTD for 72 h or with FdUrd for 2 or 72 h, only a small amount of DNA fragmentation was observed, and the appearance of the tumor cells did not differ markedly from that of untreated cells. Moreover, the DNA fragmentation rate in the TAS-102 treatment group was significantly higher than that in the control group in vivo. These results suggest that when tumor cells are exposed to high concentrations of FTD for short periods of time, FTD manifests its antitumor activity primarily through the induction of DNA fragmentation after FTD incorporation into the DNA. We conclude that TAS-102 is expected to manifest antitumor effects against 5-FU-resistant tumors that are similar to those exerted in 5-FU-sensitive tumors.

Synthesis and evaluation of 6-methylene-bridged uracil derivatives. Part 1: discovery of novel orally active inhibitors of human thymidine phosphorylase.

A series of novel 6-methylene-bridged uracil derivatives have been prepared as inhibitors of human thymidine phosphorylase (TP). To enhance the in vivo antitumor activity of fluorinated pyrimidine 2'-deoxyribonucleosides such as 2'-deoxy-5-(trifluoromethyl)uridine (F(3)dThd), a potent TP inhibitor preventing their degradation to an inactive compound, has become a target of medicinal chemistry. We present here the synthesis and evaluation of novel human TP inhibitors. Introduction of an N-substituted aminomethyl side chain at the 6-position of 5-chlorouracil has improved water solubility and enhanced inhibitory activity compared with the known TP inhibitor, 6-amino-5-chlorouracil. Compound 42 was reasonably well absorbed in mice after oral administration. When combined with F(3)dThd, compound 42 exerted its TP inhibitory potency by increasing the maximum plasma concentrations of the former as evidenced in experiments with monkeys. Positive changes in pharmacokinetic profile were accompanied by the enhanced in vivo antitumor activity of this combination when compared to F(3)dThd alone, in mice bearing human tumor xenografts. Both biochemical and pharmacological effects appeared to fit the concept as anticipated.

Synthesis and evaluation of 6-methylene-bridged uracil derivatives. Part 2: optimization of inhibitors of human thymidine phosphorylase and their selectivity with uridine phosphorylase.

A series of novel 6-methylene-bridged uracil derivatives have been optimized for clinical use as the inhibitors of human thymidine phosphorylase (TP). We describe their synthesis and evaluation. Introduction of a guanidino or an amidino group enhanced the in vitro inhibitory activity of TP comparing with formerly reported inhibitor 1. Their selectivity for TP based on uridine phosphorylase inhibitory activity was also evaluated. Compound 2 (TPI) has been selected for clinical evaluation based on its strong TP inhibition and excellent modulation of 2'-deoxy-5-(trifluoromethyl)uridine (F(3)dThd) pharmacokinetics. As a result, TAS-102 (a combination of F(3)dThd and TPI) is currently in phase 1 clinical studies.

A novel antimetabolite, TAS-102 retains its effect on FU-related resistant cancer cells.

TAS-102 is a new oral anti-tumor drug preparation, composed of a 1:0.5 mixture (on a molar basis) of alpha,alpha,alpha-tri-fluorothymidine (FTD) and thymidine phosphorylase inhibitor (TPI). TAS-102 is currently undergoing clinical trials, and has been demonstrated to have at least 2 mechanisms; inhibition of thymidylate synthase (TS) and incorporation into DNA. 5-FU is widely used in the treatment of solid tumor, but the inherent or acquired resistance of certain tumors to 5-FU therapy is a major clinical problem. In the present study, we investigated FTD in vitro and in vivo comparing with 5-FU and using FU-resistant cells. There was no relationship between FTD and 5-FU growth inhibition effect in vitro. A different sensitivity pattern was observed by the log-mean graph. We next investigated the anti-tumor activity of TAS-102 in a FU-resistant xenograft model. Comparative efficacy was observed between FU-resistant cell and its parent cell. We also studied the influence of TAS-102 on liver metastasis in a mouse model of human colorectal cancer, because liver metastasis of colorectal cancer is associated with patient survival. Human cancer DNA was detected by PCR, and TAS-102 markedly inhibited the number of liver metastasis. A novel angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF), was shown to be identical to a previously characterized intracellular enzyme, thymidine phosphorylase, TAS-102 can be expected to have not only anti-tumor cytocidal effects but also antiangiogenesis activity and may inhibit liver metastasis. Our findings suggested that TAS-102 is a promising candidate for clinical use and can be expected to decrease minimal residual disease.

Thymidine kinase and thymidine phosphorylase level as the main predictive parameter for sensitivity to TAS-102 in a mouse model.

TAS-102 is a new oral anti-cancer drug preparation, composed of a 1:0.5 mixture (on a molar basis) of alpha,alpha,alpha-trifluorothymidine (FTD) and 5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]-2,4(1H,3H)-pyrimidinedione hydrochloride (TPI). TAS-102 currently undergoing clinical trials, has been demonstrated to have at least two mechanisms, inhibition of TS and incorporation into DNA. We hypothesized that the thymidine metabolism enzyme may be a crucial factor that affects the antitumor activity of TAS-102. In the present study, we measured the enzyme activity of thymidine kinase (TK), thymidine phosphorylase (TP) and thymidilate synthase (TS) in human cancer xenografts to investigate the contribution of these enzymes to the sensitivity of TAS-102. Antitumor activity of TAS-102 appears to be associated with TK, tumor growth and TS. However, the most related factors in this study were the TK and TP ratio. There was a significant correlation (p=0.04) between tumor growth inhibition and this ratio. These results suggested that the activation and degradation pattern of FTD plays an important role in the efficacy of TAS-102 and that it is possible to use the TK/TP ratio to predict response to TAS-102 therapy. We also studied the influence of TPI on the capacity of exogenous dThd to reverse FTD-dependent growth inhibition. Thymidine (dThd) levels rescued the effect of FTD in vitro and significantly increased in serum after administration of TAS-102 or TPI alone but not FTD alone. This may suggest the possibility of a decrease in antitumor effect. However, our study indicated that the therapeutic index was clearly increased by FTD combined with TPI, compared with FTD alone, suggesting FTD-induced toxicity to sensitive host tissue can be selectively reversed with dThd. In conclusion, TK and TPI effects on TP play important roles in the cytotoxic action of TAS-102, and it is possible to use the TK/TP ratio to predict more precisely individual resistance or sensitivity.

An optimal dosing schedule for a novel combination antimetabolite, TAS-102, based on its intracellular metabolism and its incorporation into DNA.

TAS-102 is a combination drug consisting of alpha,alpha,alpha-trifluorothymidine (FTD) and thymidine phosphorylase inhibitor (TPI). FTD is converted to F3TMP by thymidine kinase and inhibits the thymidylate synthetase (TS) activity by binding to TS. In addition, FTD triphosphate form, F3TTP is incorporated into DNA, which leads to cytocidal effects. Therefore, the incorporation of FTD into DNA is expected to be an important factor, discriminating it from 5-FU showing TS inhibitory activity as their main mechanism of action. To assess a clinically more effective regimen protocol, the intracellular metabolism and the incorporation of FTD into DNA were investigated using human cancer cell lines in vitro and in vivo. FTD was incorporated into DNA in a time-dependent manner, but not in a concentration-dependent manner. FTD was the most efficiently incorporated into DNA after treatment with a several-micro molar level of FTD for around 8 h. The intracellular F3TTP was rapidly eliminated from tumor cells, as soon as FTD was washed out from the culture medium, whereas the FTD incorporated into DNA was retained by 80% or more even at 24 h after a washing-out procedure. When TAS-102 was administered into tumor-bearing mice once daily or three times daily at 3-h intervals at a dose of 150 mg/kg/day for one or 3 consecutive days, incorporation of FTD into tumor DNA by divided dosing significantly higher than that of single dosing. Based on our findings, the antitumor effects of TAS-102 against 3 different human cancer cell xenografts into mice were examined. The divided daily dosing resulted in enhancement of the antitumor effects of TAS-102 without any additional side effects. It is concluded that multiple daily dosing may result in better clinical benefits of TAS-102, when compared with single daily dosing and TAS-102 is a promising candidate for not only FU-sensitive but also FU-resistant cancer patients.