[Antitumor Molecular Mechanism of Trifluridine and Tipiracil Hydrochloride (TAS-102: TFTD)].

Treatment options for patients with metastatic colorectal cancer (mCRC), who are refractory to standard chemotherapy, are limited. In a global multicenter randomized double-blind phase III study (RECOURSE study), TAS-102 (TFTD) administration significantly improved overall survival rate with favorable safety profile in mCRC patients refractory to standard chemotherapy (HR=0.68, p<0.001). TFTD was approved initially in Japan in March 2014 and is currently under review by health authorities in the United States and Europe. TFTD is expected to play an important role in salvage-line treatment for patients with mCRC. In this review, we present the history of its clinical development and the experimental data that elucidate the underlying molecular mechanism of action of TFTD and its key component, trifluridine.

Antiviral activities of two nucleos(t)ide analogs against vaccinia, mpox, and cowpox viruses in primary human fibroblasts.

Many poxviruses are significant human and animal pathogens, including viruses that cause smallpox and mpox (formerly monkeypox). Identifying novel and potent antiviral compounds is critical to successful drug development targeting poxviruses. Here we tested two compounds, nucleoside trifluridine, and nucleotide adefovir dipivoxil, for antiviral activities against vaccinia virus (VACV), mpox virus (MPXV), and cowpox virus (CPXV) in physiologically relevant primary human fibroblasts. Both compounds potently inhibited the replication of VACV, CPXV, and MPXV (MA001 2022 isolate) in plaque assays. In our recently developed assay based on a recombinant VACV expressing secreted Gaussia luciferase, they both exhibited high potency in inhibiting VACV replication with EC50s in the low nanomolar range. In addition, both trifluridine and adefovir dipivoxil inhibited VACV DNA replication and downstream viral gene expression. Our results characterized trifluridine and adefovir dipivoxil as strong poxvirus antiviral compounds and further validate the VACV Gaussia luciferase assay as a highly efficient and reliable reporter tool for identifying poxvirus inhibitors. Given that both compounds are FDA-approved drugs, and trifluridine is already used to treat ocular vaccinia, further development of trifluridine and adefovir dipivoxil holds great promise in treating poxvirus infections, including mpox.

Involvement of concentrative nucleoside transporter 1 in intestinal absorption of trifluorothymidine, a novel antitumor nucleoside, in rats.

ααα-Trifluorothymidine (TFT), an anticancer nucleoside analog, is a potent thymidylate synthase inhibitor. TFT exerts its antitumor activity primarily by inducing DNA fragmentation after incorporation of the triphosphate form of TFT into the DNA. Although an oral combination of TFT and a thymidine phosphorylase inhibitor has been clinically developed, there is little information regarding TFT absorption. Therefore, we investigated TFT absorption in the rat small intestine. After oral administration of TFT in rats, more than 75% of the TFT was absorbed. To identify the uptake transport system, uptake studies were conducted by using everted sacs prepared from rat small intestines. TFT uptake was saturable, significantly reduced under Na(+)-free conditions, and strongly inhibited by the addition of an endogenous pyrimidine nucleoside. From these results, we suggested the involvement of concentrative nucleoside transporters (CNTs) in TFT absorption into rat small intestine. In rat small intestines, the mRNAs coding for rat CNT1 (rCNT1) and rCNT2, but not for rCNT3, were predominantly expressed. To investigate the roles of rCNT1 and rCNT2 in TFT uptake, we conducted uptake assays by using Xenopus laevis oocytes injected with rCNT1 complementary RNA (cRNA) and rCNT2 cRNA. TFT uptake by X. laevis oocytes injected with rCNT1 cRNA, and not rCNT2 cRNA, was significantly greater than that by water-injected oocytes. In addition, in situ single-pass perfusion experiments performed using rat jejunum regions showed that thymidine, a substrate for CNT1, strongly inhibited TFT uptake. In conclusion, TFT is absorbed via rCNT1 in the intestinal lumen in rats.

Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs.

Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly 15N-labeled T2M4SOD1 was prepared from minimal media using 15NH4Cl as the 15N source. The T2M4SOD1 monomer (both 15N labeled and unlabeled) was correctly folded as confirmed by 1H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their 1H NMR and/or 19F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, 1H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the 15N-NMR spectra of 15N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR.

Collision induced dissociation studies of alkali metal adducts of tetracyclines and antiviral agents by electrospray ionization, hydrogen/deuterium exchange and multiple stage mass spectrometry.

The collision induced dissociation (CID) mass spectra were obtained for the X(+)-adducts (X=Na(+) or Li(+)) of five tetracyclines, four pyrimidine and three purine derivatives and their fully D-exchanged species in which the labile hydrogens were replaced by deuterium by either gas phase or liquid phase exchange. The CID spectra were obtained for [M + Na](+) and [M + Li](+) and the exchanged analogs, [M(D) + Na](+) and [M(D) + Li](+), and compositions of product ions and mechanisms of decomposition were determined by comparison of the MS(n) spectra of the undeuterated and deuterated species. Metal ions are bound to the base of purine and pyrimidine antiviral agents and dissociate primarily to give the metal complexes of the base [B + X](+). For vidarabine monophosphate, however, the metal ions are bound to the phosphate group, resulting in unique and characteristic cleavage reactions not observed in the uncomplexed system, and dissociate through the loss of phosphate and/or phosphate metal ion complex. The [B + X](+) of these antiviral agents are relatively stable and show no or little fragmentation compared to [B + H](+). The CID of [B + X](+) of guanine derivative occurs mainly through elimination of NH(3) and that of trifluoromethyl uracil dissociates primarily through the loss of HF. For tetracyclines, metal ions are bound to ring A at the tricarbonylmethyl group and dissociate initially by the loss of NH(3)/ND(3) from [M(H) + X](+) and [M(D) + X](+). The CID spectra of [M + X](+) of tetracyclines are somewhat similar to those of [M + H](+). The dominant fragments from the metal complexes of these compounds are charge remote decompositions involving molecular rearrangements and the loss of small stable molecules. Additionally, tetracyclines and the antiviral agents show more selectivity towards Li+ ion than the corresponding complexes with Na(+) or K(+).

Use of 5-trifluoromethyldeoxycytidine and tetrahydrouridine to circumvent catabolism and exploit high levels of cytidine deaminase in tumors to achieve DNA- and target-directed therapies.

5-Trifluoromethyl-2'-deoxycytidine (F3methyl-dCyd), when coadministered with tetrahydrouridine (H4Urd), surpasses the efficacy of 5-trifluorothymidine and 5-trifluoromethyl-2'-deoxycytidine when administered alone as demonstrated with adenocarcinoma 755 and Lewis lung carcinoma as solid tumors implanted in C57BL X DBA/2 F1 mice. It appears that the reason for the heightened efficacy of F3methyl-dCyd, when coadministered with low concentrations of H4Urd, is decreased systemic deamination and subsequent systemic catabolism by pyrimidine nucleoside phosphorylases, which do not act on deoxycytidine and its analogues. Furthermore, the elevated levels of cytidine deaminase in these mouse tumors may result in selective conversion of F3methyl-dCyd to 5-trifluorothymidine at the tumor site. This suggests an approach to the treatment of human tumors possessing elevated levels of cytidine deaminase such as certain leukemias, bronchogenic carcinoma of the lung, adenocarcinomas of the colon and rectum, astrocytomas, and certain tumors which are refractory to chemotherapy with 1-beta-D-arabinofuranosylcytosine. In contrast to fluorinated pyrimidines in current use, F3methyl-dCyd + H4Urd potentially allows an exclusive DNA-, rather than both a DNA- and RNA-, directed approach. The major mechanism of the antitumor activity of F3methyl-dCyd appears to be via inhibition by 5-trifluorothymidine-5'-monophosphate of thymidylate synthetase, the target enzyme of fluoropyrimidine analogues in current use. However, the established and potential differences in the mode of action, anabolism, nature of incorporation into DNA, repair and cofactor requirements of F3methyl-dCyd and its anabolites, compared to that of the commonly utilized fluorinated pyrimidines, indicate that F3methyl-dCyd + H4Urd is a novel combination of agents. In comparative studies with Lewis lung carcinoma, F3methyl-dCyd (+ H4Urd) was shown to surpass the efficacies of 5-fluorouracil and 5-fluorodeoxyuridine and to be essentially equal in efficacy to 5-fluorodeoxycytidine (+ H4Urd). The optimum established protocol against Lewis lung carcinoma is F3methyl-dCyd, 175 mg/kg, + H4Urd, 25 mg/kg, once per day for 7 days. Studies utilizing high concentrations of H4Urd coadministered with F3methyl-dCyd indicate that the major pathway of tumor inhibition is via conversion of F3methyl-dCyd to 5-trifluorothymidine in view of the fact that tumor inhibition diminishes at doses of H4Urd which result in extensive (93%) inhibition of tumor cytidine deaminase.(ABSTRACT TRUNCATED AT 400 WORDS)

Trifluridine Induces p53-Dependent Sustained G2 Phase Arrest with Its Massive Misincorporation into DNA and Few DNA Strand Breaks.

Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102, which consists of FTD and a thymidine phosphorylase inhibitor. Like 5-fluoro-2'-deoxyuridine (FdUrd), a deoxynucleoside form of 5-fluorouracil metabolite, FTD is sequentially phosphorylated and not only inhibits thymidylate synthase activity, but is also incorporated into DNA. Although TAS-102 was effective for the treatment of refractory metastatic colorectal cancer in clinical trials, the mechanism of FTD-induced cytotoxicity is not completely understood. Here, we show that FTD as well as FdUrd induce transient phosphorylation of Chk1 at Ser345, and that this is followed by accumulation of p53 and p21 proteins in p53-proficient human cancer cell lines. In particular, FTD induced p53-dependent sustained arrest at G2 phase, which was associated with a proteasome-dependent decrease in the Cyclin B1 protein level and the suppression of CCNB1 and CDK1 gene expression. In addition, a p53-dependent increase in p21 protein was associated with an FTD-induced decrease in Cyclin B1 protein. Although numerous ssDNA and dsDNA breaks were induced by FdUrd, few DNA strand breaks were detected in FTD-treated HCT-116 cells despite massive FTD misincorporation into genomic DNA, suggesting that the antiproliferative effect of FTD is not due to the induction of DNA strand breaks. These distinctive effects of FTD provide insights into the cellular mechanism underlying its antitumor effect and may explain the clinical efficacy of TAS-102.

Involvement of Concentrative Nucleoside Transporter 1 in Intestinal Absorption of Trifluridine Using Human Small Intestinal Epithelial Cells.

TAS-102, which is effective for refractory metastatic colorectal cancer, is a combination drug of anticancer trifluridine (FTD; which is derived from pyrimidine nucleoside) and FTD-metabolizing enzyme inhibitor tipiracil hydrochloride (TPI) at a molecular ratio of 1:0.5. To evaluate the intestinal absorption mechanism of FTD, the uptake and transcellular transport of FTD by human small intestinal epithelial cell (HIEC) monolayer as a model of human intestinal epithelial cells was investigated. The uptake and membrane permeability of FTD by HIEC monolayers were saturable, Na(+) -dependent, and inhibited by nucleosides. These transport characteristics are mostly comparable with those of concentrative nucleoside transporters (CNTs). Moreover, the uptake of FTD by CNT1-expressing Xenopus oocytes was the highest among human CNT transporters. The obtained Km and Vmax values of FTD by CNT1 were 69.0 µM and 516 pmol/oocyte/30 min, respectively. The transcellular transport of FTD by Caco-2 cells, where CNT1 is heterologously expressed, from apical to basolateral side was greater than that by Mock cells. In conclusion, these results demonstrated that FTD exhibits high oral absorption by the contribution of human CNT1.

The corneal penetration of trifluorothymidine, adenine arabinoside, and idoxuridine: a comparative study.

Trifluorothymidine (F3TdR) and idoxuridine (IDU) were observed to penetrate through the cornea from the epithelial side at a greater rate than adenine arabinoside (ARA-A) during in vitro corneal perfusions. Removal of the epithelium increased the rate of penetration of F3TdR and IDU by about twofold and the rate of ARA-A penetration by fivefold. The kinetics of antiviral penetration did not display saturation points at high antiviral concentrations, thus indicating that these three antiviral drugs penetrate the cornea by nonfacilitated diffusion. The sole breakdown product detected following F3TdR penetration in vitro, in situ, and in controls was 5 carboxy-2'-deoxyuridine (5-COOH-2'-dUd). The sole breakdown product isolated during ARA-A penetration experiments was hypoxanthine arabinoside (ARA-HX), and control experiments indicated that ARA-A was stable at pH 7.6. IDU was degraded to 2'-deoxyuridine (dUd) in control experiments, but during corneal penetration experiments IDU was degraded to a mixture of dUd and iodouracil (IU).

Trifluridine: a review of its antiviral activity and therapeutic use in the topical treatment of viral eye infections.

Trifluridine (trifluorothymidine) is an antiviral agent for topical use in the eye, and is structurally related to idoxuridine. In vitro studies have shown that it effectively inhibits the replication of herpes simplex virus type 1, which causes primary keratoconjunctivitis and recurrent epithelial keratitis in man. In masked comparative studies, predominantly in patients with dendritic ulcers, trifluridine 1% solution was effective in over 90% of patients; in such studies it was comparable with vidarabine in treating dendritic ulcers, and was at least as effective as, and in some studies more effective than, idoxuridine. The drug was also effective in treating a small number of patients with geographic ulcers (sometimes associated with the usage of topical corticosteroids), and this could be an important advantage if confirmed in further well-designed studies. However, experience at present is too limited to reliably determine the usual response rate in this difficult therapeutic area. In open studies the drug proved to be particularly useful in treating ulcers previously unresponsive to idoxuridine or vidarabine, and in treating patients intolerant of idoxuridine, with a high success rate and minimal side effects being reported. The role of trifluridine in treating deep stromal disease, uveitis, or adenovirus kerato-conjunctivitis has not been established. The drug is well tolerated and cross-hypersensitivity and cross-toxicity between trifluridine, idoxuridine and vidarabine are rare. Thus, trifluridine is an effective alternative to the drugs available for treating herpetic keratitis, and seems especially useful in 'difficult' cases.

Detection of new metabolites of trifluridine (F3TdR) using 19F NMR spectroscopy.

The metabolism of 5-trifluoromethyl-2'-deoxyuridine (trifluridine, F3TdR) in male BALB/C mice has been studied by 19F NMR spectroscopy. Contrary to previous reports, a number of fluorinated metabolites were observed in urine, whole livers and blood samples taken from mice after i.p. injection of F3TdR. The present study describes the identification of two new metabolites in mouse urine using the 19F NMR technique. The NMR of crude urine showed the presence of F3TdR 5-trifluorothymine (F3T), the newly-identified metabolites, 5-trifluoromethyl-5,6-dihydrouracil (DHF3T) and 5-trifluoromethyl-5,6-dihydroxyracil (DOHF3T), and several new, as yet unidentified fluorinate metabolites. These two new metabolites were characterized by comparison to authentic compounds prepared synthetically from F3T.

alpha-Trifluoromethyl-beta-alanyl glycine (F3MBAG): a novel mammalian metabolite of trifluridine (F3TdR).

19F NMR spectroscopy has been used to study further the metabolism of 5-trifluoromethyl-2'-deoxyuridine (trifluridine; F3TdR). The synthesis and characterization of alpha-trifluoromethyl-beta-alanyl glycine (F3MBAG), a putative new metabolite of F3TdR, are now reported. This study describes ex vivon and in vivo detection of F3MBAG and other previously reported metabolites of trifluridine, using 19F NMR spectroscopy, in male BALB/C mice bearing EMT-6 tumors. A parallel 19F NMR spectroscopic study was also performed on rats dosed with F3TdR, to observe the qualitative pattern of F3TdR metabolism in another species. Unexpectedly, 5-trifluoromethyl-5,6-dihydroxyuracil (DOHF3T), alpha-trifluoromethyl-beta-ureidopropionic acid (F3MUPA) and fluoride, which result from the metabolic degradation of F3TdR and which were detected in various biological samples from mice dosed with F3TdR, could not be identified in rat urine or in homogenized tissue extracts. The presence of these metabolites in intact tissues is uncertain since in this study 19F NMR spectroscopy of these samples always displayed a broad resonance "hump" across the range of chemical shifts that would encompass these metabolites. No clear explanation for the loss of spectroscopic resolution in this region has been rationalized. N-Carboxy-alpha-trifluoromethyl-beta-alanine (F3MBA-CO2), alpha-trifluoromethyl-beta-alanyl alanine (F3MBAA) and N-acetyl-alpha-trifluoromethyl-beta-alanine (Ac-F3MBA) were synthesized and characterized, but were not detected as metabolites in any of the biological specimens examined.

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.

Intraocular antiviral penetration.

Aqueous levels of topically applied vidarabine monophosphate, trifluridine, and acyclovir were determined following topical administration in patients with normal corneas prior to cataract extraction. Meager levels in aqueous were found for vidarabine monophosphate, and none for trifluridine. Substantial levels of acyclovir were detected that were well within the therapeutic range of herpes simplex susceptibility.

Development of a novel mouse tk+/- embryonic stem cell line for use in mutagenicity studies.

A tk+/- mouse embryonic stem (ES) cell line, designated 1G2, has been created in which one allele of the thymidine kinase (tk) gene was inactivated by targeted homologous recombination. This line is an analog of the mouse lymphoma tk+/- L5178Y cell line, which is used widely to assess the mutagenicity of chemical agents. Treatment of 1G2 cells with the alkylating agent N-ethyl-N-nitrosourea (ENU) resulted in a dose-related increase in trifluorothymidine-resistant colonies. Mutant frequencies of 152 and 296 per 10(6) cells were determined for 0.1 and 0.3 mg/ml doses of ENU, compared with a spontaneous mutant frequency of 15 per 10(6) cells. The data indicate that tk+/- 1G2 ES cells may be useful for the creation of a transgenic mouse model for assessing in vivo mutation using an endogenous autosomal gene.

Intraocular penetration of trifluridine.

We studied intraocular penetration of topically applied trifluridine in five patients with herpetic keratitis undergoing penetrating keratoplasty. We compared the concentration of trifluridine and its metabolite 5-carboxy 2'-deoxyuridine in the aqueous humor to those of normal control patients undergoing routine cataract extraction without preoperative antiviral therapy. Significant concentrations of intact trifluridine were achieved in the acqueous humor after topical application of 1% trifluridine ophthalmic drops. The metabolite, 5-carboxy 2'-deoxyuridine, was not found in the aqueous humor. Unlike idoxuridine and vidarabine, it is possible to achieve therapeutic levels of trifluridine at intraocular sites that would be advantageous in the treatment of deep herpetic disease involving the stroma and iris.

Dermal penetration enhancement profile of hexamethylenelauramide and its homologues: in vitro versus in vivo behavior of enhancers in the penetration of hydrocortisone.

Several amides of cyclic amines were prepared and tested as penetration enhancers in the diffusion of various drugs through hairless mouse skin in vitro. Hexamethylenelauramide (hexahydro-1-lauroyl-1H-azepine) was selected as a broad spectrum penetration enhancer worthy of further study. Later, the duration of the effect of various enhancers on the penetration barrier in vivo was determined by evaluating the in vitro diffusion of hydrocortisone through skins that had been pretreated in vivo. We found that the longer the pretreatment, the smaller the amount of penetrated hydrocortisone. Furthermore, our results suggested that differences exist in the retention of various enhancers in living mouse skin. The in vitro pretreatment experiments revealed that the penetration through dead skin is slow compared with the penetration through living skin. Neither the nature of the receptor phase, nor the increased temperature of the in vitro experiments, explain the striking differences between the in vivo and the in vitro experiments. Finally, the penetration of hydrocortisone through the stratum corneum in the presence of enhancers, as well as the penetration of 1-dodecylhexahydro-2H-azepin-2-one (laurocapram), hexamethylenelauramide, and oleic acid, were determined using a stratum corneum stripping technique. More hydrocortisone penetrated through the stratum corneum during the first 3 h in the presence of hexamethylenelauramide than in the presence of laurocapram or oleic acid.

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.

Can the L5178Y Tk+/- mouse lymphoma assay detect epigenetic silencing?

The mouse lymphoma L5178Y Tk(+/-) assay is broadly used in toxicology to assess genotoxicity because of its known sensitivity to genotoxicants that act through a variety of mechanisms, which may include epigenetic DNA methylation. This brief article highlights the studies that have contributed to this conjecture and suggests an addition to the experimental design that could identify if the test substance is a potential epimutagen acting via hypermethylation.

Highly regioselective synthesis of 3'-O-acyl-trifluridines catalyzed by Pseudomonas cepacia lipase.

3'-O-Acyl-trifluridines were prepared successfully through an enzymatic approach for the first time. Among the ten commercially available lipases tested, Pseudomonas cepacia lipase displayed the highest regioselectivity towards the acylation of 3'-hydroxyl of trifluridine. Furthermore, the effects of some crucial factors on the enzymatic myristoylation of trifluridine were examined. The optimal reaction medium, molar ratio of trifluridine to vinyl myristate and reaction temperature were found to be anhydrous THF, 1:7 and 50 °C, under which the reaction rate, substrate conversion, and 3'-regioselectivity were 63.9 mM/h, >99.0%, and 99%, respectively. Additionally, the enzyme recognition of the chain length of the acyl donors was investigated. The results showed that 3'-regioselectivity of the enzyme maintained 99% with the increment of acyl chain length (C6, C10, and C14). The reason might derive from the strong hydrophobic interaction between 5-CF(3) group of the base moiety and Leu 287 located in the medium-sized pocket of the active site.