[CANCER RESEARCH 43, 4039-4044, September 1983]
Metabolic Activation of RS-1-(Tetrahydro-2-furanyl)-5-fluorouracil (Ftorafur) to 5-Fluorouracil by Soluble Enzymes1
Yousry M. El Sayed2 and Wolfgang Sadée3
Departments of Pharmacy and Pharmaceutical Chemistry, School at Pharmacy, University of California, San Francisco, California 94143
ABSTRACT the N-1—C-2' bond of ftorafur to yield 4-hydroxybutanal as the intermediate which is further metabolized to GBL or GHB. There are two major fl,S-1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur) activation pathways to 5-fluorouracil, one that is me diated by microsomal cytochrome P-450 oxidation at C-5' of the MATERIALS AND METHODS tetrahydrofuran moiety and one that is mediated by soluble All chemicals and reagents were of spectroquality or analytical grade. enzymes. This report demonstrates that the soluble enzyme Ftorafur was supplied by the Chemical and Drug Procurement Section, pathway proceeds via enzymatic cleavage (possibly hydrolytic) of the N-1—C-2' bond to yield 5-fluorouracil and 4-hydroxybu- Division of Cancer Treatment, National Cancer Institute, Bethesda, Md. NADPH and NADH were purchased from the Sigma Chemical Co. (St. tanal, which is immediately further metabolized to 7-butyrolac- Louis, Mo.). Alcohol dehydrogenase from horse liver (specific activity, tone or -y-hydroxybutyric acid. The soluble activation pathway about 2.7 units/mg) was purchased from Boehringer Mannheim, Indian was present in liver, small intestine, and brain. Because of the apolis, Ind. Because of their chemical instabilities, 4-hydroxybutanal and limited distribution of cytochrome P-450 in body tissues and succinaldehyde had to be synthesized immediately preceding their use. because of the lack of redistribution of 5-fluorouracil via the 4-Hydroxybutanal was generated from the precursor 2,3-dihydrofuran systemic circulation after ftorafur administration, we propose (Aidrich Chemical Co., Milwaukee, Wis.) under acidic conditions (21). Succinaldehyde was generated from 2,5-dimethoxytetrahydrofuran (Aid- that the soluble enzyme pathway is at least in part responsible rich Chemical Co.) under acidic conditions (17). for organ toxicity and possibly antitumor effect. Distinction of the microsomal (C-5') and the soluble enzyme (C-2') activation path Tissue Homogenate Preparations and in Vitro Incubations. Male Dutch rabbits were stunned and decapitated. The liver, brain, and small ways can be exploited in the design of more selective prodrug intestine were excised and rinsed in ice-cold isotonic (1.15%) KCI. Each analogues. organ tissue was homogenized in ice-cold 0.01 M potassiunrsodium phosphate buffer (pH 7.4):1.15% KCI (1:2, w/v) using a Potter-Elvehjem Teflon-pestle homogenizer. The homogenates were centrifuged at INTRODUCTION 10,000 g for 20 min at 0-4° to yield the 10,000 g supernatant fraction. The pyrimidine antimetabolite ftorafur4 has shown activity The supernatant fractions were recentrifuged at 100,000 g for 1 hr at 0- 4°. The 100,000 g supernatant fraction may be used immediately or against several adenocarcinomas with less myelotoxicity but frozen at -20° until use. more central nervous sytem toxicity than those of 5-fluorouracil (29). Ftorafur is considered a prodrug of 5-fluorouracil, and it is Several experiments were carried out with dialyzed 100,000 g liver homogenates. The dialysis tubes containing the homogenates were slowly metabolized to 5-fluorouracil by several metabolic path incubated in 200 volumes of 100 mM Tris buffer, pH 7.4, for 5 hr at 4°. ways (3, 4, 10). A high concentration of Tris buffer was chosen to accelerate solute Recently, we identified 2 major in vitro pathways of ftorafur exchange; moreover, the buffer was replaced every hr. activation to 5-fluorouracil that do not involve the known ftorafur All in vitro incubations were earned out at pH 7.4 at 37°for 1 hr. Final metabolites (that retain the tetrahydrofuran moiety), one occur concentration of all preparations was standardized to 1 g tissue (wet ring in the 100,000 g supernatant fraction (soluble enzymes) of weight) per 2 ml incubation mixture. Chemical reagents and cofactors liver homogenate and one in the 100,000 g microsomal pellet (3, were added at the concentrations indicated. The concentrations of 10). The microsomal pathway involving cytochrome P-450 pro NADPH and NADH were 1 mM each. When incubations were performed duces succinaldehyde and 5-fluorouracil via oxidation at the C- for more than 1 hr, additional amounts of NADPH and NADH were added 5' position of ftorafur (10). In contrast, GBL or its ring open every hr (yielding concentration increments of 1 mw each) in order to prevent cofactor depletion. Higher concentrations of NADPH and NADH congener GHB was isolated as a major metabolite in the super failed to increase the rate of product formation. The rate of GBL or GHB natant soluble enzyme fractions. Chart 1 shows the potential formation from ftorafur was linear over at least 4 hr. activation pathways that can occur at either position C-2' or C- GG Assay for GBL or GHB, 4-Hydroxybutanal, and 1,4-Butanediol. 5' of ftorafur with GBL or GHB as the possible common end GBL or GHB, 4-hydroxybutanal, and 1,4-butanediol were measured by product. the previously published GC assay of GBL or GHB (3) with the following Precise knowledge of the activation mechanisms of ftorafur modifications. The column, injector, and detector temperatures were may lead to the development of more selective 5-fluorouracil 145,190, and 210°,respectively. GC retention times for GBL, 4-hydroxy butanal, and 1,4-butanediol were 3.2, 1.5, and 9.5 min, respectively. prodrugs. In this report, we provide evidence that the soluble There was no detectable conversion of 4-hydroxybutanal and succinal- enzyme pathway of ftorafur activation proceeds via cleavage of dehyde to GBL or GHB under the assay conditions. Sensitivity for GBL 1This work was supported by National Cancer Institute Grant CA 27866. was approximately 0.5 ¿/g/mltissue homogenates. Standard curves were 2 Recipient of a scholarship from the Egyptian Government, in partial support of linear over the range of 0.5 to 200 / SEPTEMBER 1983 4039 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1983 American Association for Cancer Research. Y. M. El Sayed and W. Sadée Chart 2 shows the percentage of conversion of succinaldehyde (500 MM)to GBL or GHB in rabbit liver homogenates (100,000 g C-2' IIIMTMI supernatant fraction). The addition of NADPH or NADH greatly O°^ enhanced the conversion of succinaldehyde to GBL or GHB, en tu while incubation without any cofactor added to the soluble enzyme preparation generated only 12% GBL or GHB. In addi tion, incubations without enzyme and only with phosphate buffer did not generate any detectable GBL or GHB. Acid treatment of o— succinaldehyde which is part of the GC assay procedure did not 4-11 1,4-10 produce any detectable amounts of GBL:GHB, thereby ruling out any procedural artifacts. Incubation of lower concentrations of succinaldehyde (50 Õ/M)gave similar yields of GBL or GHB. Incubations of succinaldehyde (500 A/M)with the 100,000 g supernatants in the presence of NADPH and hydrazine (2 mw) O«/* COOH _HO RESULTS Metabolism of Succinaldehyde, 4-Hydroxybutanal, and GBL or GHB. Under a variety of homogenates conditions tested (10,000 g and 100,000 g supernatants), GBL or GHB was essentially stable over the incubation period. In contrast, both 4- hydroxybutanal and succinaldehyde were rapidly metabolized, with GBL or GHB as a major product. In order to understand the Chart 2. Conversion of succinaldehyde (500 »M)toGBL or GHB by the 100,000 intermediate role that either one of these 2 tetrahydrofuran g supernatants of rabbit liver homogenates under different incubation conditions. Incubation time was 1 hr. Control samples contained supernatant alone. The metabolites may play in the conversion of ftorafur to GBL or inhibition effect of hydrazine was tested in the presence of NADPH, which affords GHB, it was necessary to study their metabolism in more detail. an efficient conversion of succinaldehyde to GBL or GHB. Reagent concentrations 1,4-Butanediol was not measurable in detectable quantities in were 1 mw NADPH, 2 HIM NADH, and 2 mM hydrazine. The reducing cofactors NADPH and NADH were added, since the conversion of succinaldehyde to GBL or any of the incubations. GHB includes a reduction of one of the aldehydic functions to an alcohol. 4040 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1983 American Association for Cancer Research. Ftorafur Activation by Soluble Enzymes Chart4. Conversion of ftorafur (2 mM) to GBL or GHB by the 100,000 g Charts. Conversion of 4-hydroxybutanal to GBL or GHB by the 100,000 g supernatant of rabbit liver homogenates under different incubation conditions. supernatant of rabbit liver homogenates under different incubation conditions. The Incubation time was 1 hr. Control samples contained supernatant alone. Reagent incubation time was 1 hr. Hydrazine was tested in the absence of reducing concentrations were 1 mM NAOPH, 2 mM NADH, 2 mM hydrazine, and horse liver cofactors, since they are not required but indeed inhibit the conversion of 4- alcohol dehydrogenase {ADH) (4 mg/ml). hydroxybutanal to GBL or GHB. Reagent concentrations were 1 mM NADPH, 2 HIM NAOH, 2 mM hydrazlne, and horse liver alcohol dehydrogenase (ADH) (4 mg/ sion to GBL or GHB was affected only to a small extent (Table 1). Furthermore, the enzyme preparation was dialyzed as de icarbazide (2 mM),and pyrazole (2 mM)again had no significant scribed ("Materials and Methods") in order to remove phosphate effect on the yield of GBL or GHB, while incubations in the which is required for the phosphorylation reaction. Subsequent presence of EDTA (5 mw) produced only a slight inhibition incubation of ftorafur with the dialyzed enzyme preparation with (~20%). Preheating of the 100,000 g supernatant liver homog or without phosphate buffer (10 HIM)yielded similar amounts of enates at 100°for30 min produced 85 to 90% inhibition of GBL GBL or GHB. However, dialysis resulted in partial loss (~70%) or GHB yield, which suggested that a small portion of the reaction of enzymatic activity, which prevents any firm conclusions that may occur nonenzymatically in the homogenate incubations. may have been derived from this experiment. Omission of the liver homogenates from the incubation buffer Activation of Ftorafur by Different Tissue Homogenates. In eliminated GBL or GHB yield from 4-hydroxybutanal. vitro experiments were carried out using various organ tissue Metabolic Conversion of Ftorafur to GBL or GHB and 5- homogenates. Table 2 represents the results of in vitro incuba Fluorouracil.Chart 4 shows the conversionof ftorafurto GBL tions of ftorafur with rabbit liver, brain, and small intestine or GHB (percentage of control) under different 100,000 g liver 100,000 g supernatant enzyme fractions. The yield of 5-fluoro homogenate incubation conditions. Potential inhibitors selected uracil from ftorafur is lower than that of GBL or GHB. Therefore, for this experiment were those active as inhibitors of either 4- the rate of disappearance of 5-fluorouracil (77 /¿M)wasmeasured hydroxybutanal or succinaldehyde conversion to GBL or GHB. in 100,000 g liver supernatants (without the addition of any Ftorafur is converted to GBL or GHB in highest yield without cofactor). Within the first 20 min of incubation, 5-fluorouracil was cofactor addition. Hydrazine did not reduce GBL or GHB yield, rapidly metabolized by more than 80%, which was possibly a while addition of NADPH and, more strongly, NADH (±horse result of the presence of residual endogenous cofactor (e.g., liver alcohol dehydrogenase) inhibited GBL:GHB formation from NADPH, ribose 1-phosphate, 5-phosphoribosyl 1-pyrophos- ftorafur to a similar extent as that from 4-hydroxybutanal. Incu phate). After this initial rapid phase, 5-fluorouracil metabolism bation of ftorafur with phosphate buffer (pH 7.4) at 37°for 1 hr was much slower (f1/2~ 150 min). Although exact quantitative resulted in no detectable 4-hydroxybutanal or GBL or GHB. In calculations of the expected 5-fluorouracil yield from ftorafur addition, preheating of the liver homogenates (100°for 30 min) were not readily possible under these conditions, the data are eliminated the formation of GBLGHB from ftorafur, which sug roughly compatible with the hypothesis that 5-fluorouracil and gests that the reaction is enzymatic. GBL or GHB were generated in stoichiometric amounts. Liver In order to address the question of whether or not thymidine homogenates showed highest levels of ftorafur activation, fol or undine phosphorylase is responsible for metabolic activation lowed by small intestines and brain. Although 5-fluorouracil con of ftorafur to 5-fluorouracil, we carried out in vitro incubations centrations were rather small in the brain homogenates, they are with 10 mw thymidine and with 2'-deoxy-5-trifluoromethyluridine clearly above the nonenzymatic background (<0.08% conver at a concentration (1.6 HIM)that was shown to inhibit nucleoside sion). In order to estimate the Michaelis-Menten constant («„,) phosphorylase (7, 14). Under these conditions, ftorafur conver- for the overall reaction of ftorafur to GBL or GHB, we carried out SEPTEMBER 1983 4041 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1983 American Association for Cancer Research. Y. M. El Sayed and W. Sadée Tabte! to 4-hydroxybutanal followed by oxidation; or (b) oxidation to In vitro metabolism of ftorafur (2 mu) to GBL or GHB by rabbit liver 100,000g succinic semialdehyde followed by reversible reduction to GBL supernatant enzyme fraction or GHB. It has been previously reported that succinic semialde Condition % of conversion to GBL or GHB/hr hyde (an intermediate metabolite in 7-aminobutyric acid metab 100,000 g supernatant atone 1.4 ±0.2° (5)" 100,000 g supernatant + 2'-deoxy-5'-trifluo- 1.1" olism) is reversibly metabolized to GBH by soluble enzyme romethyluridine (1.6 mM) fractions of brain and other tissue homogenates (11). The con 100,000 g supernatant + thymidine (10 RIM) 1.0 ±0.1 (2) version of succinaldehyde to GBL or GHB requires NADPH as a 100,000 g dialyzed supernatant 0.4 ±0.03 (4)" cofactor, and this conversion was strongly inhibited by hydrazine 100,000 g dialyzed supernatant + phosphate 0.5 ±0.04 (2) (an aldehyde-trapping reagent which can undergo bifunctional buffer. pH 7.4 (10 mm) * Mean ±S.D., unless 2 determinations; then, value is the mean ±one-half of condensation with succinaldehyde to form a heterocyclic ring the difference between the 2 observations. structure) (Chart 2). Kaufman and Nelson (18) had previously '' Numbers in parentheses, number of determinations. c Single observation. used hydrazine to trap succinic semialdehyde produced from " The dialyzed enzyme preparations were prepared as described under "Mate oxidation of GHB in vitro. In contrast, hydrazine had very little rials and Methods" using 10 mM Tris buffer for homogenization and 100 mM Tris effect on the conversion of both 4-hydroxybutanal and ftorafur buffer for accelerated dialysis. to GBL or GHB (Charts 3 and 4). This result rules out succinal Table2 dehyde as an intermediate of the conversion of ftorafur to GBL In vitro metabolism of ftorafur (2 mu) to GBL or GHB and 5-fluorouracil by or GHB and thus rules out C-5' oxidation by the soluble enzymes 100,000 g supernatant fractions of rabbit liver, small intestine, and brain as the major activation pathway. % of converston/hr There are also several pathways of 4-hydroxybutanal metab Liver Small intestine Brain olism by the soluble enzymes of 100,000 g supernatants (Chart 1.4 ±0.2*(5)" GBLGHB 0.9 ±0.2 (2) 0.43 ±0.04 (2) 1), 4-Hydroxybutanal could be directly oxidized to GBL or GHB, 5-Fluorouracil 0.6 ±0.1 (2) 0.7 ±0.1 (2) 0.2 ±0.03(2) reversibly oxidized to succinaldehyde or reversibly reduced to * Mean ±S.D., unless 2 determinations; then, value is the mean ±one-half 1,4-butanediol. Each of these reactions could involve a different range. 6 Numbers in parentheses, number of determinations. enzymatic mechanism, requiring different cofactors. The meta bolic conversion of 1,4-butanediol to GBL or GHB as the major product has been demonstrated previously (12, 24). We have in vitro incubations with increasing ftorafur concentrations (2, 5, found that the conversion of 4-hydroxybutanal to GBL or GHB and 10 HIM)and determined the rate of formation of GBL or GHB in 100,000 g homogenates is extremely rapid. Therefore, it was per hr. Reaction rates were linear over at least 4 hr. The hourly not possible to directly demonstrate the presence of 4-hydroxy yields of GBL or GHB formed were 33.8, 51.4, and 64.8 fiM butanal in ftorafur incubations. Conversion of 4-hydroxybutanal (percentage of conversion, 1.7, 1.03, and 0.65, respectively). Using the double-reciprocal plot (Lineweaver-Burk) of these data, to GBL or GHB did not require any external cofactor but it was slightly reduced by NADPH and strongly inhibited (80%) by NADH the apparent Kmof the enzyme is approximately 3 ITIM.Since the reaction rate of 4-hydroxybutanal to GBL or GHB was much plus added horse liver alcohol dehydrogenase. Similarly, NADPH greater at rather high 4-hydroxybutanal levels (10 to 100 UM), it and, more strongly, NADH (±horse liver alcohol dehydrogenase) inhibited GBL or GHB formation from ftorafur to a similar extent is likely that this Kmvalue is associated with the direct activation as that from 4-hydroxybutanal. These results provide strong of ftorafur. evidence in support of the hypothesis that 4-hydroxybutanal is DISCUSSION an essential intermediate in the conversion of ftorafur to GBL or GHB. Moreover, the lack of any cofactor requirements argues The formation of GBL or GHB and 5-fluorouracil from ftorafur against an oxidation of ftorafur at C-2' to give 5-fluorouracil and in the soluble enzyme preparation of 100,000 g supernatant GBL or GHB directly. fractions may be mediated by either of the 3 pathways described It has been previously reported that horse liver thymidine in Chart 1. These include oxidation at C-2' and C-5' positions phosphorylase and rat liver undine phosphorylase were inactive which leads to chemically labile intermediates that spontaneously against ftorafur (5, 30). Recently, Kono ef al. (19) suggested that cleave to 5-fluorouracil and to GBL or GHB (C-2' oxidation) or the cleavage of ftorafur to 5-fluorouracil was catalyzed by a to succinaldehyde (C-5' oxidation). Another potential pathway of thymidine phosphorylase activity, which was enhanced in human ftorafur activation involves C-2' hydrolytic cleavage which pro tumor tissues. Moreover, ftorafur activation was suppressed in duces 5-fluorouracil and 4-hydroxybutanal. Furthermore, succin the presence of a 7-fold excess amount of thymidine (19). Lack aldehyde and 4-hydroxybutanal can undergo enzymatic conver of a rigorous quantitative analysis of the data makes it difficult sion to GBL or GHB. In fact, GBL or GHB was isolated as a to assess the significance of their results. In contrast, in our major metabolite of ftorafur in the soluble enzyme fraction of experiments, addition of thymidine had little effect on the yield 100,000 g supernatants (3). of GBL or GHB, and the presence of the nucleoside phospho Since GBL or GHB is a major product of several possible rylase inhibitor (2'-deoxy-5-trifluoromethyluridine) did not inhibit metabolic pathways of ftorafur activation, it is necessary to first the metabolic activation of ftorafur to GBL or GHB. These results study the metabolic activation of the postulated intermediates, argue against the hypothesis that activation occurs via phospho- 4-hydroxybutanal and succinaldehyde. The specific inhibition of rolysis of the N-1—C-2' bond of ftorafur, yielding 5-fluorouracil their conversion to GBL to GHB can then be applied to test GBL and 2-hydroxytetrahydrofuran 2-phosphate, which is then con or GHB formation pathways from ftorafur. verted to 4-hydroxybutanal by phosphatases. Rather, we pro The formation of GBL or GHB from succinaldehyde in the pose that a hydrolytic enzyme mechanism is responsible for soluble enzyme preparation could occur by either one of the 2 activation of ftorafur in the 100,000 g supernatants. reaction sequences described in Chart 1: (a) reversible reduction Although ftorafur is thought to act predominantly via 5-fluoro- 4042 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1983 American Association for Cancer Research. Ftorafur Activation by Soluble Enzymes uracil formation, plasma concentrations of 5-fluorouracil gener soluble enzyme pathway is responsible for part of the organ ated in vivo were shown to be negligible (5); this finding is toxicity as well as for the antitumor effects of ftorafur. Other consistent with the hypothesis that intracellulariy formed 5- previously reported activation pathways are unlikely to contribute fluorouracil is further metabolized without subsequent redistri significantly to the ftorafur effects. Further development of pro- bution via the systemic circulation (2, 5). Therefore, the mecha drugs related to ftorafur should therefore be directed towards nism of metabolic activation of ftorafur to 5-fluorouracil might be separating the microsomal (C-5') and soluble enzyme (C-2') a determinant of its selective tissue toxicity towards the gastroin activation pathways of the tetrahydrofuran ring moiety. More testinal tract and the central nervous system as well as of its over, assay of the soluble activation pathway of ftorafur may antitumor effects. We demonstrate here that these tissues are serve as a predictor of the antitumor efficacy of the drug. capable of enzymatically activating ftorafur to 5-fluorouracil by soluble enzyme catalysis. Although the extent of in vitro activa REFERENCES tion is rather small, it must be considered that ftorafur is present in rather large concentrations after therapeutic doses. Moreover, 1. Ansfieid. F., Kallas, G., and Singson, J. Clinical results with Tegafur (ftorafur) in cotorectal and breast cancer. Proc. Am. Assoc. Cancer Res., 22: 450,1981. because of the long elimination half-life (6 to 17 hr) of ftorafur (5, 2. Au, J. L, and Sadée,W. 5-Fluorouracil concentrations in human plasma 6,16), tissue exposure to 5-fluorouracil as a result of the soluble following fl,S-1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur) administration. Cancer Res.. 39; 4289-4290,1979. ftorafur activation pathway could be sufficient to account for the 3. Au, J. L., and Sadée,W. Activation of ftorafur [fl,S-1-(tetrahydro-2-furanyl)-5- observed organ toxicities of the drug. On the other hand, we fluorouracil] to 5-fluorouracil and -y-butyrolactone. Cancer Res., 40: 2814- cannot rule out the possibility that metabolites other than 5- 2819, 1980. 4. Au, J. L., and Sadée,W. The pharmacology of ftorafur (fl,S-1-(tetrahydro-2- fluorouracil contribute to organ toxicity (1, 13). For example, furanyO-5-fluorouracil). Recent Results Cancer Res., 76:100-114,1981. GHB occurs physiologically in brain and cerebrospinal fluids (9, 5. Au, J. L., Wu, A. T., Friedman. M. A., and Sadée,W. Pharmacokinetics and 23, 25, 28) and has been used as an anesthetic adjuvant (15). metabolism of ftorafur in man. Cancer Treat. Rep., 63: 343-350,1979. 6. Benvenuto, J. A., Lu, K., Hall, S. W., Benjamin, R. S., and Loo, T. L. Disposition Its effects on the central nervous system lead to behavioral and metabolism of 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur) in humans. depression and an induction of an epileptic-like stupor (lethargy) Cancer Res., 38: 3867-3870,1978. 7. Cooper, G. M., and Gréer,S. Irreversible inhibition of dehalogenation of 5- (6, 27). However, the plasma concentrations of GBL or GHB ¡odouracilby 5-diazouraoil and réversibleinhibition by 5-cyanouracil. Cancer required for such effects are far above those generated from Res., 30: 2937-2941,1970. ftorafur. 8. Culp, H. W., and McMahon, R. E. ReducÃase for aromatic aldehydes and ketones. J. Biol. Chem., 243: 848-852,1958. In conclusion, this report demonstrates that ftorafur is acti 9. Doherty, J. D., Hattox, S. E., Snead, O. C., and Roth, R. H. Identification of vated to 5-fluorouracil by the soluble enzymes of target tissues endogenous •y-hydroxybutyratein human and bovine brain and its regional via cleavage of the N-1—C-2' bond (Chart 5). The intermediate distribution in human, guinea pig and rhesus monkey brain. J. Pharmacol. Exp. Ther., 207:130-139,1978. product, 4-hydroxybutanal, is rapidly converted to GBL or GHB 10. El Sayed, Y. M., and Sadée,W. Metabolic activation of ftorafur [fl,S-1- which has been observed previously as a major ftorafur metab (tetrahydrc-2-furanyl)-5-fluorouracil]: the microsomal oxidative pathway Brachem. Pharmacol., 37: 3006-3008,1982. olite in circulating plasma. The other major metabolic activation pathway, i.e., ftorafur oxidation at C-5' by a cytochrome P-450- 11. Fishbein, W. N., and Bessman, S. P. -x-Hydroxybutyrate in mammalian brain. Reversible oxidation by lactic dehydrogenase. J. Bio). Chem.. 239: 357-361, mediated mechanism (10), leads to 5-fluorouracil and succinal- 1964. 12. Gessa, G. L., Spano, P. F., Vargin, L., Crabai, F., Tagliamonte, A., and Mameli. dehyde (Chart 5), the latter being also converted, at least par L. Effect of 1,4-butanediol and other butyric acid congeners on brain cate- tially, to GBL or GHB. Since cytochrome P-450 is mainly localized cholamines. Life Sci., 17: 289-298,1968. in the liver, with lower levels in the gastrointestinal tract and 13. Harrison, S. D., Jr., Donine, E. P., and Giles, H. D. Evidence for toxicologie activity of ftorafur independent of conversion to 5-fluorouracil. Cancer Treat. much lower levels in the brain, and since 5-fluorouracil is not Rep., 63: 1389-1391,1979. redistributed via the circulation (2, 5), it is possible that the 14. Heidelberger, C., Bimie, G. D., Boohar, J., and Wentland, D. Fluorinated pyrimidines XX. Inhibition of the nucleoside phosphorylase cleavage of 5- fluoro-2'-deoxyuridine by 5-trifluoromethyl-2'-deoxyuridine. Btochim. Biophys. Acta, 76:315-318,1963. 15. Helrich, M., McAslan, R. C.. and Skolnik, S. Correlation of blood levels of 4- hydroxybutyrate with stages of consciousness. Anesthesiology. 23:771-775, 1964. 16. Hills. E. B.. Godefroi, V. G., O'Leary, I. A., Burke, M., Amdrezejewski, D., Brukwinski, W., and Horwitz, J. P. GLC determination for ftorafur in biological ?//* "W fluids. J. Pharm. Sci., 66:1497-1499,1977. 17. Hjeds, H., and Larsen. I. K. A reinvestigation of the structure of the conden **/> V« sation products of phenylhydrazine and succinaldehyde. Acta Chem. Scand., 25:2394-2402,1971. 18. Kaufman, E. E., and Nelson, T. Kinetics of coupled -x-hydroxybutyrate oxidation and D-glucuronate reduction by an NADP*-dependent oxidoreductase. J. Bid. o + Chem., 256: 6890-6894,1981. 19. Kono, K., Hará,Y., and Matsushima, Y. Enzymatic formation of 5-fluorouracil from 1-{tetrahydro-2-furanyl)-5-fluorouracil (Tegafur) in human tumor tissues. Chem. Pharm. Bull.. 29: 1486-1488,1981. 20. Marunaka, T., Minami, Y., Umeno, Y., Yasudia, A., Sato, T., and Fujii, S. Identification of metabolites of 1-(tetrahydro-2-furanyl)-5-fluorouracil (FT-207) formed in vitro by rat liver microsomes. Chem. Pharm. Bull., 28: 1795-1803, 1980. 21. Paul, R., Fluchaire. M., and Collardeau, G. Transposition des dihydro-2.5 furannes en dihydro-2.3 furannes. Application a la préparationde l'hydroxy-4 butanal. Bull. Soc. Chim. Fr., 668-671,1950. 22. Raskin, N. H., and Sokoloff, L. Alcohol dehydrogenase activity in rat brain and Chart 5. The 2 major activation pathways of ftorafur (FT) to 5-fluorouracil (FUra). liver. J. Neurochem., 17: 1677-1687,1970. Note that the 2 pathways attack at different sites of the ftorafur moiety. While only 23. Roth, R. H., and Giarman, N. J. -y-Butyrolactone and yhydroxybutyric. i. a fraction of the generated succinaldehyde (SA) is converted to GBL or GHB. 4- Distribution and metabolism. Brachem. Pharmacol., 75:1333-1348,1966. hydroxybutanal (4-HB) quantitatively yields GBL or GHB. 24. Roth, R. H., and Giarman, N. J. Evidence that central nervous system depres- SEPTEMBER 1983 4043 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1983 American Association for Cancer Research. Y. M. El Sayed and W. Sadée sten by 1,4-butaned¡olismediated through a metabolite, gamma-hydroxybu- graphic and behavioraleffects. Neurology, 26: 51-56,1976. tyrate. Biochem. Pharmacol.. 17:735-739,1968. 28. Tabakoff, B., and Radulavacki,M. -,-Hydroxybutyrate in CSF during steepand 25. Roth, R. H., and Giarman, N. J. Natural occurrence of f-hydroxybutyrate in wakefulness. Res. Commun. Chem. Pathol. Pharmacol., 14: 587-590,1976. mammalianbrain. Biochem. Pharmacol., 19:1087-1093,1970. 29. Valdivieso, M., Bodey, G. P., Gottlieb, J. A., and Freireich, E. J. Clinical 26. Snead. O. C. Gamma-hydroxybutyrate in the monkey. I. Electroencephalo- evaluation of ftorafur (pynmidine-deoxyribose W-1^'-Ãuranidyl-S-fluorouracil). graphic, behavioral and pharmacokmetic studies. Neurology, 28: 636-648, Cancer Res., 36:1821-1824,1976. 1978. 30. Wu, A. T., Au, J. L., and Sadée,W. Hydroxylated metabolites of fl.S-1- 27. Snead, O. C., Yu, R. K., and Hutttentocher, P. R. Gamma-hydroxybutyrate, (tetrahydro-2-furanyl)-5-fluorouracil(ftorafur) in rats and rabbits. Cancer Res., correlation of serum and cerebrospinal fluid levels with electroencephalo- 38:210-214,1978. 4044 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1983 American Association for Cancer Research. Metabolic Activation of R, S -1-(Tetrahydro-2-furanyl)-5-fluorouracil (Ftorafur) to 5-Fluorouracil by Soluble Enzymes Yousry M. El Sayed and Wolfgang Sadée Cancer Res 1983;43:4039-4044. Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/43/9/4039 E-mail alerts Sign up to receive free email-alerts related to this article or journal. Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected]. Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/43/9/4039. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site. Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1983 American Association for Cancer Research.