Cytochrome P-450-Dependent Oxidative Cleavage of 1-(Tetra hydro-2-furanyl)-5-fluorouracil

Transcription

1 Cytochrome P-450-Dependent Oxidative Cleavage of 1-(Tetra hydro-2-furanyl)-5-fluorouracil to 5-Fluorouracil Sumio KAWATA, Yuzo MINAMI, Seiichiro TARUI, Teruyoshi MARUNAKA*, Mitsuhiro OKAMOTO** and Toshio YAMANO**, *** Second Department of Internal Medicine, and **Department of Biochemistry, Osaka University Medical School, Nakanoshima, Kita-ku, Osaka 530, Japan *Taiho Pharmaceutical Co. Ltd., Tokushima , Japan Accepted June 1, 1984 Abstract-Cytochrome P-450-dependent oxidative cleavage of 1 -(tetrahydro-2 furanyl)-5-fluorouracil (FT) was investigated in a reconstituted system containing purified phenobarbital-inducible cytochrome P-450 (P-4501) or 3-methylcholan threne inducible cytochrome P-450 (P-4481). FT was converted into 5-fluorouracil (5-FU) in the reconstituted system, and its rate was 71 pmol 5-FU formed/min/ nmol P-4501 and 45 pmol 5-FU/min/nmol P Cytochrome P-450, NADPH cytochrome P-450 reductase and NADPH were required for 5-FU production. Inhibitors of cytochrome P-450 such as carbon monoxide and metyrapone markedly decreased the rate. FT was found to interact with the purified cytochrome P-450, causing a reverse type I spectral change. From these observations, we concluded that the hepatic microsomal cytochrome P-450-dependent mixed function oxidase system participates in the oxidative cleavage of FT. A pyrimidine antimetabolite, 5-fluorouracil (5-FU), is known to have a significant therapeutic effect on several adenocarci nomas. Its masked form, 1 -(tetrahydro-2 furanyl)-5-fluorouracil (FT), is also used as an anti-cancer agent, and it has lower toxicity than 5-FU (1). FT has been con sidered to be slowly converted into 5-FU by two main pathways in the liver: one occurring in the microsomal fraction and the other in the 100,000xg supernatant fraction (2-4). Involvement of the microsomal cytochrome P-450-dependent mixed function oxidase system in the conversion of FT into 5-FU has been suggested by the observations that FT was decomposed at a higher rate in liver microsomes from phenobarbital-pretreated mice than in those from untreated mice and that addition of FT to rat liver microsomal preparation caused a substrate binding spectral change (5). However, unequivocal evidence for the involvement of cytochrome P-450 has not been reported yet. We studied the conversion of FT into 5-FU in a reconstituted system containing purified cytochrome P-450 obtained from pheno barbital or 3-methylcholanthrene-pretreated rabbits, and we confirmed that the cytochrome P-450-dependent mixed function oxidase system participates in the oxidative cleavage of FT to form 5-FU. Materials and Methods Preparation of microsomes: Male New Zealand rabbits (weighing 2 to 2.5 kg) were injected intraperitoneally with 80 mg of phenobarbital or 40 mg of 3-methylchol anthrene per kg body weight once daily for 4 days. The animals were deprived of food for 20 hr before sacrifice. The livers were perfused with 0.25 M sucrose solution and homogenized in eight volumes of the same solution. After centrifugation at 10,000 x g for 20 min, the microsomal pellet was obtained by further centrifugation at 105,000 x g for 90 min. After washing once with 1.15% KCI solution, the pellet was frozen and stored at -80 C until further use for purification of

2 cytochrome P-450. Analytical methods: The content of cyto chrome P-450 was determined as described by Omura and Sato (6), using an extinction coefficient of 91 mm-1 cm-1. Protein concen tration was measured by the method of Lowry et al. (7), using bovine serum albumin as a standard. FT was quantified with a Shimadzu Model LC-2 high pressure liquid chromatograph (HPLC) equipped with a UV detector operating at 254 nm as previously described (8). 5-FU was measured as its trimethylsilylated derivative with a JEOL Model JMS-D 300 mass spectrometer with an electron-impact ion source connected to a JEOL MODEL JGC-20KP gas chromato graph as previously described (8, 9). Enzyme assay in a reconstituted system: The activity of purified NADPH-cytochrome P-450 reductase was assayed by measuring NADPH-cytochrome c reductase activity by the method of Mihara and Sato (10). One unit of the enzyme was defined as the amount catalyzing the reduction of 1 Pmol of the acceptor per min at 25'C. The reaction mixture for the assay of mixed function oxidase activity contained 2.5 /N cytochrome P-450, 1.0 unit of NADPH-cytochrome P 450 reductase, Triton X-100 (0.05%, w/v), 1 mm NADPH, 0.1 M potassium phosphate buffer (ph 7.4) and 1.6 mm FT as a substrate in a final volume of 1.0 ml as previously described (11-13). The reaction mixture was incubated at 37'C for 20 min. The conversion of FT to 5-FU proceeded linearly during this incubation time. The rate of the conversion was determined by subtracting the amount of 5-FU formed per min in the buffer alone from that in the system, since a small amount (about 0.05%) of FT is nonenzymatically converted into 5-FU in the buffer at 37'C during this incubation time. Spectral change caused by addition of FT to purified cytochrome P-450 preparation: Equal amounts of cytochrome P-4501 (4.3,W) or cytochrome P-4481 (2.2 W) in 0.1 M potassium phosphate buffer (ph 7.4) were placed in sample and reference cuvettes, and difference spectra were measured by successively adding grains of FT to the sample cuvette. After each difference spectrum had been taken, 0.1 ml of the solution from the sample cuvette was used to determine the FT concentration. The extent of the spectral change was determined as the difference in absorbance at two fixed wavelengths with a Hitachi 557 dual wavelength spectrometer. The dissociation constant (Ks) was determined from reciprocal plots of the absorbance changes as described by Schenkman et al. (14). Preparation of microsomal enzymes: Cytochrome P-4501 and cytochrome P-4481 were purified from liver microsomes of phenobarbital and methylcholanthrene treated rabbits using (-aminooctyl Sepharose, hydroxyapatite and CM-Sepharose chromato graphies by a modification of the procedure reported by Imai et al. (15) to purities of 17.2 and 16.0 nmols/mg of protein, re spectively. Detergent solubiiized NADPH cytochrome P-450 reductase was purified by the method of Yasukochi and Masters (16) using 2',5'-ADP-Sepharose affinity chroma tography. Reagents and biochemicals: FT was syn thesized and purified as described by Yasumoto et al. (17). NADPH was obtained from Oriental Yeast Co. Triton X-100 was purchased from Wako Pure Chemical Industries Ltd. Cytochrome c (Type III, from horse heart) was purchased from Sigma Chemical Co., and 2',5'-ADP-Sepharose was from Pharmacia Fine Chemicals. Metyrapone was a generous gift from Ciba-Geigy (Japan), Ltd. Phenyl isocyanide was synthesized by the method of Schmidt and Stern (18). Catalase from bovine liver and superoxide dismutase from bovine erythrocytes were purified according to Sumner and Dounce (19) and McCord and Fridovich (20), respectively. Results Interaction of FT with purified cytochrome P-450: The addition of FT to 0.1 M potassium phosphate buffer (ph 7.4) containing 4.3 /LM cytochrome P-4501 caused a reverse type I spectral change (21) characterized by the appearance of a trough at 382 nm and an absorption peak at 416 nm (Fig. 1A). The dissociation constant (KS) based on the difference spectra was calculated as 13.7 mm from reciprocal plots of the absorbance

3 changes at 416 nm relative to 382 nm (Fig. 1 B). Addition of 5-FU to the buffer con taining cytochrome P-4501 could not cause the spectral change. This observation indicates that the tetrahydrofuran moiety is required for binding of FT to cytochrome P-450. The addition of FT to the buffer containing 2.2 /cm cytochrome P-4481 also caused the reverse type I spectral change characterized by the appearance of a trough at 380 nm and an absorption peak at 414 nm (Fig. 2A). K; was calculated to be 28.6 mm from reciprocal plots of the absorbance changes at 414 nm relative to 380 nm (Fig. 2B). Conversion of FT into 5-FU in a recon stituted system containing purified cyto chrome P-450: Conversion of FT into 5-FU was studied in a reconstituted system con taining purified cytochrome P-450 and NADPH-cytochrome P-450 reductase. Sig nificant production of 5-FU was observed in the reconstituted system, and its rate was 71 pmols/min/nmol P-450 (Table 1). Cytochrome P-450, NADPH-cytochrome P 450 reductase and NADPH were required for the production of 5-FU in the recon stituted system. Neither superoxide dismutase Fig. 1. Interaction of FT with purified cytochrome P-450,. A, spectral change obtained by successive addition of grains of FT to 0.1 M potassium phosphate buffer (ph 7.4) containing 4.3 fim cytochrome P-4501 at 37 C; B, reciprocal plot of changes in absorbance at 416 nm relative to 382 nm. Fig. 2. Interaction of FT with purified cytochrome P A, spectral change obtained by successive addition of grains of FT to 0.1 M potassium phosphate buffer (ph 7.4) containing 2.2 im cytochrome P-4481 at 37'C; B, reciprocal plot of changes in absorbance at 414 nm relative to 380 nm.

4 Table 1. Rate of 5-FU production in a reconstituted system containing cytochrome P-450, Table 2. Effect of inhibitors of cytochrome P-450 on the rate of 5-FU production in a reconstituted system containing cytochrome P-4501 nor catalase had a significant effect on 5-FU production in the reaction mixture. Therefore, superoxide anion and hydrogen peroxide may not be directly involved in the conversion of FT. To further evaluate the participation of cytochrome P-450 in the reaction, the effects of various inhibitors of cytochrome P-450 on the rate of the conversion were examined in the reconstituted system (Table 2). Marked inhibition was caused by 0.1 mm metyrapone or 20 /em phenyl isocyanide. Under an atmosphere of 90% carbon monoxide and 10% oxygen, the rate of 5-FU production decreased to 30% of that under an atmosphere of 90% nitrogen and 10% oxygen. These observations indicated that conversion of FT into 5-FU is catalyzed by the hepatic microsomal cytochrome P-450 dependent mixed function oxidase system. The kinetics of 5-FU production from FT Fig. 3. A Lineweaver-Burk plot of 5-FU production in the reaction mixture containing 2.5 pm cyto chrome P-4501, 1.0 unit of NADPH-cytochrome P-450 reductase, Triton X-100 (0.05%, w/v), 1 mm NADPH, 0.1 M potassium phosphate buffer (ph 7.4) and 1.6 mm FT as the substrate in a final volume of 1.0 ml. were studied in the reconstituted system. The apparent Km calculated from Lineweaver

5 Table 3. Rate of 5-FU production in a reconstituted system containing cytochrome P-4481 Burk plots was 1.2 mm, while Vmax was 125 pmol 5-FU formed/min/nmol cytochrome P-450 (Fig. 3). The conversion of FT into 5-FU was also examined in a reconstitued system containing purified cytochrome P The rate of 5-FU production was 45 pmol 5-FU/min/nmol cytochrome P-448 (Table 3). This indicated that the phenobarbital-inducible species of cytochrome P-450 has a higher catalytic activity for 5-FU production than the 3 methylcholanthrene-inducible one. Discussion The conversion of FT into 5-FU was observed in a reconstituted system containing purified cytochrome P-4501 or P Cytochrome P-450, NADPH-cytochrome P 450 reductase and NADPH were required for 5-FU production in the reconstituted system. Neither superoxide anion nor hydrogen peroxide was directly involved in the reaction. Inhibitors of cytochrome P-450 such as carbon monoxide and metyrapone markedly decreased the 5-FU production rate in the reconstituted system. Moreover, FT interacted with cytochrome P-4501 or P-4481, causing a reverse type I spectral change. From these observations, we concluded that the hepatic microsomal cytochrome P-450 dependent mixed function oxidase system participates in the oxidative cleavage of FT. FT interacted with cytochrome P-450, causing the reverse type I spectral change. Alcohols, glucocorticoids and phenacetin are known to induce the spectral change of cytochrome P-450, and hydroxyl groups may play an important role in the reverse type I interaction (14, 21, 22). From our observation that 5-FU could not induce the spectral change, the tetrahydrofuran moiety seems to be required for the reverse type I interaction of FT with cytochrome P-450. The dissociation constant (K5) of FT to cytochrome P-4501 was 13.7 mm, and it was found to be much larger than the Michaelis constant (Km), 1.2 mm, of the drug to the cytochrome. The reason for the discrepancy between the values of KS and Km remains to be elucidated. However, there are several examples of this kind of discrepancy reported for the type II substrates and the reverse type I substrates. For instance, Schenkman et al. (14) have reported a similar discrepancy between KS and Km for a type II substrate, aniline. After they conducted a detailed study on the reverse type I spectral change, they (21) concluded that the reverse type I spectral change does not appear to be related to substrate metabolism like the type I spectral change. Since FT was converted into 5-FU in the reconstituted system containing purified cytochrome P-450 and other enzyme systems were not required for the reaction, FT seems to be first oxidized at one of the C-2', -3', -4' and -5' positions of the tetra hydrofuran moiety by the mixed function oxidase system, resulting in formation of a chemically labile intermediate which spon taneously cleaves to form 5-FU. Oxidation at the C-3' or C-4' position of the tetra hydrofuran moiety is known to yield stable products, 3'-hydroxy-FT or 4'-hydroxy-FT, as described by Marunaka et al. (23), and therefore, it could not be the mechanism for 5-FU production by the mixed function oxidase system. The remaining molecular

6 sites of the possible oxidation by the system are the C-2' or C-5' position of FT; 2' hydroxy-ft or 5'-hydroxy-FT seems to be a chemically unstable intermediate. Hypo thetically, 2'-hydroxy-FT has been con sidered to cleave spontaneously to form 5-FU and r-butyrolactone, and 5'-hydroxy FT to form 5-FU and succinaldehyde (3, 4). Sayed and Sadee (4) recently suggested that FT is converted into 5-FU through oxidation at the C-5' position by liver microsomes since succinaldehyde, but not T-butyrolactone, was found in the reaction mixture containing rat liver microsomes and NADPH. Therefore, FT may be oxidized at the C-5' position by the cytochrome P-450 dependent mixed function oxidase system. However, further investigation is required to elucidate the mechanism for oxidative cleavage of FT. In conclusion, using the reconstituted system, we confirmed that the hepatic microsomal cytochrome P-450-dependent mixed function oxidase system catalyzes the oxidative cleavage of FT. Phenobarbital inducible cytochrome P-450 showed higher catalytic activity than 3-methylcholanthrene inducible P-448. Cytochrome P-450-dependent drug hydro xylase activity in liver microsomes often seems to be impaired in tumor-bearing animals with liver disorders (24). Induction of phenobarbital-inducible cytochrome P-450 may increase the activity of the conversion of FT into 5-FU in the liver and thereby enhance the effectiveness of FT treatment in tumor-bearing patients with impaired drug metabolism. Acknowledgments: A part of this work was supported by research grants from the Ministry of Education, Science and Culture and the Science and Technology Agency of Japan. References 1 Blokhina, N.G., Vozny, E.K. and Grain. A.M.: Results of treatment of malignant tumors with ftorafur. Cancer 30, (1972) 2 Toide, H., Akiyoshi, H., Minato, Y., Okuda, H. and Fujii, S.: Comparative studies on the me tabolism of 2-(tetrahydrofuranyl)-5-fluorouracil and 5-fluorouracil. Gann 68, (1977) 3 Lai-Sim, A. and Sadee, W.: Activation of ftorafur [R,S-1 -(tetra hydro-2-furanyl)-5-fluoro uracil] to 5-fluorouracil and r-butyrolactone. Cancer Res. 40, (1980) 4 Sayed, E.I. and Sadee, W.: Metabolic activation of ftorafur [R,S-1 -(tetra hydro-2-furanyl)-5 fluorouracil]: The microsomal oxidative pathway. Biochem. Pharmacol. 31, (1982) 5 Cohen, A.M.: The disposition of ftorafur in rats after intravenous administration. Drug Metab. Dispos. 3, (1975) 6 Omura, T. and Sato, R.: The carbon monoxide binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239, (1964) 7 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, (1951) 8 Marunaka, T., Umeno, Y., Yoshida, K., Naga machi, M., Minami, Y. and Fujii, S.: High pressure liquid chromatographic determination of ftorafur [1-(tetrahydro-2-furanyl)-5-fluoro uracil] and Gic-mass spectrometric determi nation of 5-fluorouracil and uracil in biological materials after oral administration of uracil plus ftorafur. J. Pharm. Sci. 69, (1980) 9 Marunaka, T., Umeno, Y. and Minami, Y.: Analysis of pyrimidine bases in biological materials by gas chromatography-mass spectro metry. J. Chromatogr. 190, (1980) 10 Mihara, K. and Sato, R.: Partial purification of NADPH-cytochrome b5 reductase from rabbit 11 Sugiyama, T., Miki, N. and Yamano, T.: NADH and NADPH-dependent reconstituted p-nitro anisole 0-demethylation system containing cyto chrome P-450 with high affinity for cytochrome b5. J. Biochem. 87, (1980) 12 Sugiyama, T., Kawata, S., Yamano T., Miki, N., Miura, R. and Miyake, Y.: Interaction between cytochrome b5 and cytochrome P-450 in the reconstituted mixed function oxidase system. In Microsomes, Drug Oxidations, and Drug Toxicity, Edited by Sato, R. and Kato, R., p , Japan Scientific Societies Press, Tokyo (1980) 13 Kawata, S., Sugiyama, T.. Seki, K., Tarui, S., Okamoto, M. and Yamano, T.: Stimulatory effect of cytochrome b5 induced by p-nitroanisole and diisopropyl 1,3-dithiol-2-y!idenemalonate on rat 14 Schenkman, J.B., Remmer, H. and Estabrook, R.W.: Spectral studies of drug interaction with

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