Highly efficient, processive and multifunctional recombinant endoglucanase RfGH5_4 from Ruminococcus flavefaciens FD-1 v3 for recycling lignocellulosic plant biomasses.

Gene encoding endoglucanase, RfGH5_4 from R. flavefaciens FD-1 v3 was cloned, expressed in Escherichia coli BL21(DE3) cells and purified. RfGH5_4 showed molecular size 41 kDa and maximum activity at pH 5.5 and 55 °C. It was stable between pH 5.0-8.0, retaining 85% activity and between 5 °C-45 °C, retaining 75% activity, after 60 min. RfGH5_4 displayed maximum activity (U/mg) against barley ß-D-glucan (665) followed by carboxymethyl cellulose (450), xyloglucan (343), konjac glucomannan (285), phosphoric acid swollen cellulose (86), beechwood xylan (21.7) and carob galactomannan (16), thereby displaying the multi-functionality. Catalytic efficiency (mL.mg-1 s-1) of RfGH5_4 against carboxymethyl cellulose (146) and konjac glucomannan (529) was significantly high. TLC and MALDI-TOF-MS analyses of RfGH5_4 treated hydrolysates of cellulosic and hemicellulosic polysaccharides displayed oligosaccharides of degree of polymerization (DP) between DP2-DP11. TLC, HPLC and Processivity-Index analyses revealed RfGH5_4 to be a processive endoglucanase as initially, for 30 min it hydrolysed cellulose to cellotetraose followed by persistent release of cellotriose and cellobiose. RfGH5_4 yielded sufficiently high Total Reducing Sugar (TRS, mg/g) from saccharification of alkali pre-treated sorghum (72), finger millet (62), sugarcane bagasse (38) and cotton (27) in a 48 h saccharification reaction. Thus, RfGH5_4 can be considered as a potential endoglucanase for renewable energy applications.

Circadian variations in the pharmacokinetics of the oral anticancer agent tegafur-uracil (UFT) and its metabolites in rats.

Uracil-tegafur (UFT) is an oral anticancer drug containing uracil and 5­fluorouracil prodrug tegafur and is widely used for adjuvant chemotherapy of colorectal cancer. Although clinical data show circadian variations in plasma 5­fluorouracil concentrations during its long-term infusion, and feasibility studies of chronomodulated administration have been previously reported, the circadian pattern in plasma 5­fluorouracil concentration after UFT administrations remains unclear. The aim of this study was to identify factors causing circadian variations in UFT pharmacokinetics and estimate circadian patterns of plasma 5­fluorouracil concentration corresponding to UFT dosing time in rats. Rats were orally administered UFT (15 mg/kg as tegafur) at three different times of the day: 07:00 (23 h after light onset, HALO), 13:00 (5 HALO), or 19:00 (11 HALO), and then plasma concentrations of tegafur, 5­fluorouracil, and uracil were measured after UFT administration. We found that the area under the plasma concentration-time curves (AUC0-∞) of 5­fluorouracil depended on the UFT dosing time of day with a 2.4-fold difference between the peak (at 19:00: 13.7 ±â€¯1.4 µmol·h/L) and trough (at 13:00: 5.6 ±â€¯1.3 µmol·h/L). The simulated population mean clearance of 5­fluorouracil followed a 24-h cosine circadian curve, with the highest value in the early light phase being 2.2-fold higher than the lowest value in the early dark phase, which was an inverse circadian pattern compared to the plasma 5­fluorouracil concentration. The plasma tegafur levels suggested that circadian variation in tegafur absorption and conversion to 5­fluorouracil are factors causing variations in plasma 5­fluorouracil levels following UFT administration. In conclusion, the circadian pattern of 5­fluorouracil clearance and circadian variations in tegafur pharmacokinetics are important determinants of plasma 5­fluorouracil concentrations following UFT administration. This knowledge could help in developing a chronomodulated administration strategy of UFT for improving clinical outcomes.

Novel prodrugs of tegafur that display improved anticancer activity and antiangiogenic properties.

New and more potent prodrugs of the 5-fluorouracyl family derived by hydroxymethylation or acyloxymethylation of 5-fluoro-1-(tetrahydro-2-furanyl)-2,4(1H,3H)-pyrimidinedione (tegafur, 1) are described. The anticancer activity of the butyroyloxymethyl-tegafur derivative 3 and not that of tegafur was attenuated by the antioxidant N-acetylcysteine, suggesting that the increased activity of the prodrug is in part mediated by an increase of reactive oxygen species. Compound 3 in an in vitro matrigel assay was found to be a more potent antiangiogenic agent than tegafur. In vivo 3 was significantly more potent than tegafur in inhibiting 4T1 breast carcinoma lung metastases and growth of HT-29 human colon carcinoma tumors in a mouse xenograft. In summary, the multifunctional prodrugs of tegafur display selectivity toward cancer cells, antiangiogenic activity, and anticancer activities in vitro and in vivo, superior to those of tegafur. 5-fluoro-1-(tetrahydro-2-furanyl)-2,4(1 H,3 H)-pyrimidinedione (tegafur, 1), the oral prodrug of 5-FU, has been widely used for treatment of gastrointestinal malignancies with modest efficacy. The aim of this study was to develop and characterize new and more potent prodrugs of the 5-FU family derived by hydroxymethylation or acyloxymethylation of tegafur. Comparison between the effect of tegafur and the new prodrugs on the viability of a variety of cancer cell lines showed that the IC50 and IC90 values of the novel prodrugs were 5-10-fold lower than those of tegafur. While significant differences between the IC50 values of tegafur were observed between the sensitive HT-29 and the resistant LS-1034 colon cancer cell lines, the prodrugs affected them to a similar degree, suggesting that they overcame drug resistance. The increased potency of the prodrugs could be attributed to the antiproliferative contribution imparted by formaldehyde and butyric acid, released upon metabolic degradation. The anticancer activity of the butyroyloxymethyl-tegafur derivative 3 and not that of tegafur was attenuated by the antioxidant N-acetylcysteine, suggesting that the increased activity of the prodrug is in part mediated by an increase of reactive oxygen species. Compound 3 in an in vitro matrigel assay was found to be a more potent antiangiogenic agent than tegafur. In vivo 3 was significantly more potent than tegafur in inhibiting 4T1 breast carcinoma lung metastases and growth of HT-29 human colon carcinoma tumors in a mouse xenograft. In summary, the multifunctional prodrugs of tegafur display selectivity toward cancer cells, antiangiogenic activity and anticancer activities in vitro and in vivo, superior to those of tegafur.

Synthesis and cytotoxicity of novel fatty acid-nucleoside conjugates.

N(3)-Hydroxyethyltegafur (3) was synthesized in 91.0% yield under a new condition. A series of novel fatty acid esters of 3 were synthesized. These fatty acid-nucleoside conjugates have shown cytotoxicities against Ec9706 cells and A549 cells, and the structure activity relationship was discussed.

Synthesis, structure, and conformation of anti-tumor agents in the solid and solution states: hydroxyl derivatives of Ftorafur.

The pyrimidine antimetabolite Ftorafur [FT; 5-fluoro-1-(tetrahydro-2-furyl)uracil] has shown significant antitumor activity in several adenocarcinomas with a spectrum of activity similar to, but less toxic than, 5-fluorouracil (5-FU). It is considered as a prodrug that acts as a depot form of 5-FU, and hence the two drugs exhibit a similar spectrum of chemotherapeutic activity. Ftorafur is metabolized in animals and humans when hydroxyl groups are introduced into the tetrahydrofuran moiety. These metabolites are also thought to be as active as ftorafur but less toxic than 5-FU. Hydroxyl derivatives: 2'-hydroxyftorafur (III), 3'-hydroxyftorafur (IV) and 2',3'-dihydroxyftorafur (II) were synthesized and X-ray and NMR studies of these hydroxyl derivatives were undertaken in our laboratories to study the structural and conformational features of Ftorafur and its metabolites in the solid and solution states. X-ray crystallographic investigations were carried out with data collected on a CAD-4 diffractometer. The structures were solved and refined using the SDP crystallographic package of Enraf-Nonius on PDP 11/34 and Microvax computers. All of the compounds studied had the base in the anti conformation. The glycosidic torsion angles varied from -20 to 60 degrees. There is an inverse correlation between the glycosyl bond distances and the chi angle. Molecules with a lower chi angle have a larger bond distance and vice versa. The sugar rings show a wide variation of conformations ranging from C2'-endo through C3'-endo to C4'-exo. The crystal structures are stabilized by hydrogen bonds involving the base nitrogen atom N3 and the hydroxyl oxygen atoms of the sugar rings as donors and the keto oxygens O2 and O4 of the base and the hydroxyl oxygen atoms O2' and O3' as acceptors. The NMR studies were carried out on Brüker 400 and 600 MHz instruments. Simulated proton spectra were obtained through Laocoon, and pseudorotational parameters were solved by Pseurot. Presence of syn or anti forms was demonstrated with the use of NOE experiments. The glycosyl conformations in solution vary more widely than in the solid state. The conformations of the sugar molecules are in agreement with the values obtained in the solid state. The studies of the structure and conformation in the solid and solution states give a model for the Ftorafur molecule that could be used in structure, function and biological activity correlation studies.

Relevance of the pharmacology of oral tegafur to its use as a 5-FU pro-drug.

We have studied the pharmacology of iv and oral tegafur (FT) and compared the results with similar studies using continuously infused 5-FU. All patients received daily abdominal irradiation as well as FT. In eight patients receiving 1.0 g/m2 of iv FT, serum FT levels were essentially the same as those in five patients receiving the same dose of oral FT. Oral FT appeared well-absorbed, even with abdominal irradiation. The mean serum FT achieved on a daily basis was a linear function of the oral FT dose between 1.0 and 2.5 g/m2 and was consistently about 1000-fold higher than the resultant 5-FU level. The major FT metabolite, dehydro-FT (DHFT), was persistently present at about ten times the 5-FU level. Because of their long half-lives, both FT and DHFT accumulate during continuous therapy. When the mean serum 5-FU levels with oral FT were compared to those found during continuous 5-FU infusions, we found that oral FT was the equivalent of low-level 5-FU infusion. Oral FT at 1.0 g/m2 was the equivalent of about 11.5 mg/kg/24 hours of 5-FU, increasing to about 17.5 mg/kg of 5-FU for oral FT at 2.5 g/m2. The pharmacologic properties of FT appear to dictate its most useful schedule (continuous oral dosing in multiple doses) and explain why FT alone is not ideal as a 5-FU pro-drug. In addition, insight into the pharmacokinetic limitations of FT also suggests means by which its usage may be improved, including its potential application as a radiosensitizer.

Synthesis and antitumor activity of a series of ftorafur analogues: the effect of varying electronegativity at the 1'-position.

To test the effect of changes in electronegativity within the alicyclic N-1 substituent 5-fluorouracil analogues on cytotoxic activity, a series of derivatives of ftorafur, 1-(2'-tetrahydrofuranyl)-5-fluorouracil, was synthesized and tested for antitumor activity in the P388 lymphocytic leukemia screen and cytotoxic activity in the L1210 cell culture screen. Two compounds of N-1 substituent with high electronegativity, the 2'-tetrahydrothiophene 1'-oxide and the 2'-tetrahydrothiophene 1',1'-dioxide derivatives, demonstrated the highest in vitro L1210 cell inhibition (84.5% and 92.0%, respectively). Furthermore, against P388 lymphocytic leukemia in vivo, the 2'-tetrahydrothiophene 1'-oxide derivative showed significant activity (T/C = 143). Other compounds of similar or lower electronegativity within the N-1 cyclic substituent were inactive against P388 lymphocytic leukemia and less active against L1210 cells.

[Cerebrospinal fluid distributions of systemically administered fluorinated pyrimidines].

Transfer of systemically administered fluorinated pyrimidines (Tegafur, TAC-278, HCFU and FD-1) to cerebrospinal fluid was studied in 7 patients primary brain tumors. Seven patients had had irradiation and also had V-P shunt operation for hydrocephalus 5-FU concentration in CSF was extremely high in FD-1 and TAC-278 administration, but not in Tegafur and HCFU administration. In addition, Tegafur and HCFU did not reveal any cumulative effects of 5-FU in CSF by continuous prolonged systemic administration. The facts suggest strongly the usefullness of the agents in the treatment of intracranial neoplasms, which have high CSF concentrations. However, intermediate metabolites of 5-FU in CSF are different from those in systemic pathway, and FD-1 and TAC-278 produce CNS toxicities. Therefore, further extensive studies are necessary to utilize these agents for the treatment of intracranial neoplasms.

[Clinical experiences with UFD-1].

FD-1, 1,3-bis(tetrahydro-2-furanyl)-5-fluorouracil, is an anticancer agent newly developed in Japan and is a kind of marked compound of 5-fluorouracil. FD-1 changes to 3-FT and tegafur and is then converted to 5-fluorouracil. From our clinical observations, FD-1 showed excellent clinical effects in a daily dose of 600 mg. However, in some instances toxicities of central nervous system were developed. On the other hand, there is evidence that uracil enhances antitumor activity of FD-1 in the treatment of sarcoma 180 bearing mice and AH130 bearing rats. On oral administration of FD-1 plus uracil in various combination ratios, the high T/B value (ratio of the concentration of 5-fluorouracil in the tumor and blood) is obtained at a ratio of uracil to tegafur of 20 to 50. Fifteen cases with advanced cancer were treated with UFD-1 (mixture of FD-1 and uracil under molar ratio of 1: 20) in a daily dose of 300 mg of FD-1. However, no tumor regression was observed in any of our cases. On the contrary, toxic manifestations were experienced in five of fifteen cases. They mainly consisted of mild G.I. toxicities. Furthermore, in one case, ataxia developed. Our clinical studies revealed no usefulness of UFD-1 in the treatment of advanced adenocarcinoma cases.

Effects of 1,3-bis(tetrahydro-2-furanyl)-5-fluoro-2,4-pyrimidinedione (FD 1) on the central nervous system: (2) Effects on nigro-striatal dopaminergic neurons.

A single high oral dose of 1,3-bis(tetrahydro-2-furanyl)-5-fluoro-2, 4-pyrimidinedione (FD-1) (486.0-729.0 mg/kg) followed by daily administration of lower doses of FD-1 (121.5 mg/kg) for 2-4 weeks increased the concentration of striatal dopamine (DA) in rats. The decrease in striatal DA following an injection of alpha-methyl-p-tyrosine methyl ester (alpha-MT) was lessened by a dose of FD-1 (364.8 mg/kg, p.o.) given immediately before the alpha-MT. This may indicate a decrease in striatal DA utilization by FD-1. The increase in striatal DA after FD-1 was inhibited by two antagonists of gamma-aminobutyric acid (GABA) which act on postsynaptic receptors: bicuculline (0.5-2.0 mg/kg, i.p.) and picrotoxin (2.0 mg/kg, i.p.). The increase of DA induced by the GABA transaminase inhibitor aminooxyacetic acid (AOAA) was enhanced by FD-1. These results suggest that the action of FD-1 is mediated by GABA. FD-1 probably causes inhibition of DA release from nerve terminals resulting in an accumulation of DA in the corpus striatum.

Serum concentrations of 5-FU, ftorafur, and a major serum metabolite following ftorafur chemotherapy.

Daily 24-hour serum levels of ftorafur (FT), 5-FU, and a major FT metabolite, dehydroftorafur (DFT), were monitored by high-performance liquid chromatography as part of a phase I study designed to evaluate FT as a radiosensitizing 5-FU pro-drug in patients with advanced gastrointestinal cancers. At a daily iv bolus FT dose of 1.0 g/m2, 5-FU was not detected in serum concentrations above the reliable assay limits of approximately 25 ng/ml; FT did not generate the extracellular (serum) 5-FU concentrations required for sensitization by 5-FU per se. DFT was present in every patient serum tested and was confirmed to be a metabolite of FT by in vitro conversion to 5-FU. Chemical ionization solid-probe mass spectrometry of the DFT metabolite indicates the dehydro FT structure proposed by other researchers. In six of eight patients monitored, a consistent relationship was noted between serum levels of FT and DFT.

Differential effects of 5-fluorouracil analogs on humoral and cell-mediated responses in mice: augmentation of delayed hypersensitivity response against picryl chloride by 1,3-bis(tetrahydro-2-furanyl)-5-fluoro-2,4-pyrimidinedione (FD-1), a new anti-tumor drug.

The inhibitory effects of three fluorinated pyrimidines, i.e., 5-fluorouracil (FU), N1-(2-tetrahydrofuryl)-5-fluorouracil (ftorafur, FT), and 1,3-bis(tetrahydro-2-furanyl)-5-fluoro-2,4-pyrimidinedione (FD-1), on humoral and cell-mediated responses were compared. Both primary and secondary humoral responses against sheep red blood cells were strongly suppressed, and the suppressive activities of FD-1, FT and FU were directly proportional to the plasma level of active substance, FU. However, allogeneic skin graft rejection was not suppressed by FD-1 and FT. Furthermore, the delayed hypersensitivity response (DHR) against picryl chloride (PCl) was augmented by FD-1 but not by FT and FU if they were administrated for 3 days after PCl immunization. Spleen cells from mice immunized 7 days previously with PCl were able to suppress the occurrence of the DHR when they were transfused intravenously to normal syngeneic mice simultaneously immunized with PCl. The suppressor activity of spleen cells was due to B cells since the activity was abrogated by treatment with anti-IgG serum plus complement, but not by anti-T cell serum plus complement. However, the suppressor activity disappeared when donor mice were administrated with FD-1 and FU. It is suggested that FD-1 has a great inhibitory effect on suppressor cells than effector cells induced by PC1, and then the immunosuppressive potency of these drugs is not always reflected in the level of FU.

Effects on 1,3-bis(tetrahydro-2-furanyl)-5-fluoro-2,4-pyrimidinedione(FD-1) on the central nervous system: 1. Effects on monoamines in the brain.

The effect of 1,3-bis(tetrahydro-2-furanyl)-5-fluoro-2,4-pyrimidinodione(FD-1) on the central nervous system was examined by measuring regional changes in the concentrations of its metabolites and of monoamines in the brain of rats after single or repeated doses of FD-1. After single doses of FD-1, its metabolites were found to be evenly distributed in the brain. Chronic administration of FD-1 increased the concentration of two metabolites, namely 5-fluoro-2,4-pyrimidinedione(5-FU) and gamma-hydroxybutyrate (GHB) in all parts of the brain. Single treatment with FD-1 and GHB-Na increased the concentration of dopamine (DA) in the corpus striatum; treatment with FD-1, 5-fluoro-1-(tetrahydro-2-furanyl)-2,4-pyrimidinedione(FT) or GHB-Na decreased the level of acetylcholine (ACh) in the corpus striatum; treatment with 5-FU decreased the level of 5-hydroxytryptamine in the corpus striatum and hypothalamus; treatment with FD-1, FT, 5-FU or GHB-Na decreased the level of ACh in the brain stem. It is concluded that the effects of FD-1 on the central nervous system may be mediated by GHB and/or 5-FU and are probably due to an effect on the dopaminergic neurons.

Metabolism, antitumor activity, and acute toxicity of 5-fluoro-1,3-bis(tetrahydro-2-furanyl)-2,4-pyrimidinedione by oral administration to animals.

The metabolism, antitumor activity, and acute toxicity of 5-fluoro-1,3-bis-(tetrahydro-2-furanyl)-2,4-pyrimidinedione (FD-1) were investigated in animals, compared with 5-fluoro-1-(tetrahydro-2-furanyl)-2,4-pyrimidinedione (FT). It was found that after oral administration of FD-1, the level of 5-fluorouracil (5-FU) was maintained higher and longer than after administration of FT, and that a large amount of 5-FU was released from FD-1 by liver microsomal drug-metabolizing enzymes or spontaneous hydrolysis via 5-fluoro-3-(tetrahydro-2-furanyl)-2,4-pyrimidinedione (3-FT) and FT. FD-1 had a significant activity against the solid form of Ehrlich carcinoma, sarcoma-180, hepatoma AH130, Yoshida sarcoma, Walker carcinosarcoma-256, and leukemia L1210 and P388, but not the ascitic forms, and it produced greater inhibition of tumor growth than FT. The acute toxicity of FD-1 was less than that of FT.

Review of a new antimetabolic agent, 1,3-bis(tetrahydro-2-furanyl)-5-fluoro-2,4-pyrimidinedione (FD-1).

FD-1, a new anticancer drug, is a fluorinated pyrimidine derivative that has shown less acute toxicity than 5-FU and FT-207, and higher antitumor activity than FT-207 in Ehrlich carcinoma, S-180, AH-130, Yoshida sarcoma, and Walker 256. A principal feature of FD-1 is that, in comparison with FT-207, in oral administration it can maintain a higher concentration of 5-FU both in plasma and in tumor tissues. FD-1 is considered to be activated into 5-FU by a drug-metabolizing system in liver microsomes. In the plase I study of FD-1, the maximum tolerated dose was greater than 20 mg/kg in a single administration. The dose-limiting factor in FD-1 administration is gastrointestinal toxicity that causes side effects such as nausea and vomiting. The recommended dosage for daily oral administration of FD-1 is 6-12 mg/kg/day. In the phase I study of the sustained released form of FD-1 (FD-1-S), frequency of nausea and vomiting could be definitely reduced by the oral administration of FD-1-S, which showed higher tolerability. FD-1-S can be continuously administered at a dose ranging from 600 mg to 1200 mg/body/day for for 4 weeks and can be expected to have higher efficacy. In further studies on the long-term administration of FD-1-S, CNS toxicity has been reported in some cases.

Synthesis and biological activities of ftorafur metabolites. 3'- and 4'-hydroxyftorafur.

Four isomers of ftorafur were synthesized as authentic samples of possible ftorafur (FT) metabolites. 2,3-Dihydrofuran was treated with perbenzoic acid in MeOH to give 2-methoxy-3-hydroxytetrahydrofuran, which upon treatment with Ac2O/pyridine yielded the key intermediate 2-methoxy-3-acetoxytetrahydrofuran. The other intermediate, 2-ethoxy-4-acetoxytetrahydrofuran, was prepared by acid hydrolysis (HCl/50% EtOH) of 1,1-diethoxy-3,4-dihydroxybutane, followed by acetylation (Ac2O/pyridine). Treatment of 2,4-bis(trimethylsilyl)-5-fluorouracil with either 2-methoxy-3-acetoxytetrahydrofuran or 2-ethoxy-4-acetoxytetrahydrofuran in 1,2-dichloroethane at room temperature using SnCl4 as catalyst afforded cis- and trans-3'-OAc-FT or 4'-OAc-FT, respectively. However, trans-3'-OAc-FT and cis-4'-OAc-FT were the major condensation products. In each case, separation of these cis and trans isomers was achieved by silica gel column chromatography. Treatment of 3'- or 4'-OAc-FT with NH3/CH3OH at 5 degrees C overnight yielded the described hydroxylated FT. Both trans-3'-OH-FT and cis-4'-OH-FT showed no significant activity against L1210 up to 100 mg/kg. These two agents produced an inhibitory effect on HeLa cell growth equal to that of ftorafur, with ID50 = 200 MICROGRAMS/KG.