Dihydropyrimidine dehydrogenase

Dihydropyrimidine dehydrogenase catalyzes the first step in the degradation of uracil and thymine. Inherited deficiencies of the enzyme are known, and the enzyme can play a critical role in cancer chemotherapy, for example, in the metabolism of 5 -fluorouracil. 

In the assay by Klein and Haas (1990), the substrate, 5-bromouracil, is separated from the reaction mixture by chromatography on Hypersil ODS 2. The mobile phase was 0.02 M potassium phosphate buffer (pH 5.6)-methanol (94 6, v/v). The column effluent was monitored at 275 nm. The rate of disappearance of substrate was obtained from the slope of the line obtained by plotting the concentrations of 5-bromouracil against the incubation times. 

The standard reaction mixture contained 0.20 mL of the enzyme source, 0.25 mL of NADPH (2.5 mg/mL), and 2.30 mL of Sorensen buffer (pH 7.4). The reaction was started by adding 0.05 mL of 5 bromouracil (0.9 mg/mL). At intervals, 50 /xL portions of the assay mixture were mixed with 150 /xL of 6% trichloroacetic acid. After centrifugation, the supemate was shaken with 200 /xL of 0.5 M trioctylamine solution in Freon and centrifuged again. Subsequently, 25 /xL of the upper phase was injected. When weak enzymatic activities were being measured, the volume of the reaction mixture was reduced to 1.0 mL. 

In the assay by Lu et al. (1992), the catabolites of uracil, thymine, or 5-fluorouracil were separated from the respective parent compounds by chromatography on two 5 /xm Hypersil columns (from Jones Chromatography, Littleton, CO) used in tandem. The mobile phase contained 1.5 mM potassium phosphate (pH 8.0 for 5-fluorouracil, pH 8.4 for thymine and uracil) and 5 mM tetrabutylammonium hydrogen sulfate. The column effluent was monitored at an appropriate wavelength in the UV region. 

The reaction mixture used to monitor enzyme purification contained 35 mM potassium phosphate (pH 7.4), 2.5 mM magnesium chloride, 10 mM 2-mercaptoethanol, 200 iM NADPH, 20 /xM 5-fluorouracil, and enzyme solution. The final volume was 2.0 mL. At various reaction times, 350 fiL of reaction sample was taken and added into an equal volume of ethanol. After filtering, an aliquot was analyzed by HPLC. 

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Capecitabine is used for the treatment of colorectal and breast cancers. It is contraindicated in patients with known hypersensitivity to capecitabine or any of its components or to 5-fluorouracil and in patients with known dihydropyrimidine dehydrogenase (DPD) deficiency. The use of capecitabine is restricted in patients with severe renal impairment. The drag can induce diarrhea, sometimes severe. Other side effects include anemia, hand-foot syndrome, hyperbilirubinemia, nausea, stomatitis, pyrexia, edema, constipation, dyspnea, neutropenia, back pain, and headache. Cardiotoxicity has been observed with capecitabine. A clinically important drag interaction between capecitabine and warfarin has been demonstrated. Care should be exercised when the drag is co-administered with CYP2X9 substrates. 

The aerobic degradation of several azaarenes involves reduction of the rings at some stage, and are discussed in Chapter 10, Part 1. Illustrative examples include the degradation of pyridines (3-alkyl-pyridine, pyridoxal) and pyrimidines (catalyzed by dihydropyrimidine dehydrogenases). Reductions are involved in both the aerobic and the anaerobic degradation of uracil and orotic acid. 

Lu Z-H, R Zhang, RB Diasio (1992) Purification and characterization of dihydropyrimidine dehydrogenase from human liver. J Biol Chem 267 17102-17109A. 

An additional determinate of 5-FU toxicity, regardless of the method of administration, is related to its catabolism and phar-macogenomic factors. Dihydropyrimidine dehydrogenase (DPD) is the main enzyme responsible for the catabolism of 5-FU to inactive metabolites.37 A pharmacogenetic disorder has been identified in which patients have a complete or near-complete... 

After intravenous administration, about 80-90% of the dose is catabolized in the liver by dihydropyrimidine dehydrogenase (DPD) [38] (Figure 14.3). The formation of the inactive 5-fluoro-5,6-dihydrouracil (5-FUH2) by DPD is the rate-limiting step of 5-FU catabolism [39]. DPD is widely distributed among tissues, with the highest levels found in the liver. Once 5-FU entered tumor cells, its antitumor effect is mainly dependent on the extent of 5-FU anabolism. After two sequential anabolic steps involving thymidine phosphorylase (TP) and thymidine kinase... 

Harris BE, Carpenter JT, Diasio RB. Severe 5-fluorouracil toxicity secondary to dihydropyrimidine dehydrogenase deficiency. A potentially more common phar-macogenetic syndrome. Cancer 1991 68 499-501. 

Lu Z, Zhang R, Diasio RB. Dihydropyrimidine dehydrogenase activity in human peripheral blood mononuclear cells and liver population characteristics, newly identified deficient patients, and clinical implication in 5-fluorouracil chemotherapy. Cancer Res 1993 53 5433-5438. 

Stephan F, Etienne MC, Wallays C et al. Depressed hepatic dihydropyrimidine dehydrogenase activity and fluorouracil-related toxicities. Am J Med 1995 99 685-688. 

Milano G, Etienne MC, Pierrefite V et al. Dihydropyrimidine dehydrogenase deficiency and fluorouracil-related toxicity. Br J Cancer 1999 79 627-630. 

Fleming RA, Milano GA, Gaspard MH, et al. Dihydropyrimidine dehydrogenase activity in cancer patients. Eur J Cancer 1993 29A 740-744. 

Wei X, McLeod HL, McMurrough J et al. Molecular basis of the human dihydropyrimidine dehydrogenase deficiency and 5-fluorouracil toxicity. J Clin Invest 1996 98 610-615. 

Van Kuilenburg AB, Vreken P, Beex LV et al. Severe 5-fluorouracil toxicity caused by reduced dihydropyrimidine dehydrogenase activity due to heterozygosity for a G ->A point mutation. J Inherit Metab Dis 1998 21 280-284. 

Johnson MR, Hageboutros A, Wang K et al. life-threatening toxicity in a dihydropyrimidine dehydrogenase-deficient patient after treatment with topical 5-fluorouracil. Clin Cancer Res 1999 5 2006-2011. 

Identification of novel mutations in the dihydropyrimidine dehydrogenase gene in a Japanese patient with 5-fluorouracil toxicity. Clin Cancer Res 1998 4 2999-3004. 

O et al. Population study of dihydropyrimidine dehydrogenase in cancer patients. J Clin Oncol 1994 12 2248-2253. 

Ridge SA, Sludden J, Wei X et al. Dihydropyrimidine dehydrogenase pharmacogenetics in patients with colorectal cancer. Br J Cancer 1998 77 497-500. 

McMurrough J, McLeod HL. Analysis of the dihydropyrimidine dehydrogenase polymorphism in a British population. 

Grem JL, Yee LK, Venzon DJ et al. Inter-and intraindividual variation in dihydropyrimidine dehydrogenase activity in pe-... 

Chazal M, Etienne MC, Renee N et al. Link between dihydropyrimidine dehydrogenase activity in peripheral blood mononuclear cells and liver. Clin Cancer Res 1996 2 507-510. 

Figure 11.3 shows spectra from a cubane pair in an enzyme called DPD, dihydropyrimidine dehydrogenase (Hagen et al. 2000). It catalyzes the first step in the breakdown of pyrimidine bases. The two cubanes have unusually low reduction potentials, and so they are reduced to the [4Fe-4S]1+ form with S = 1/2 ground state by means of... 

FIGURE 11.5 Half-held spectrum of two interacting cubanes. The signal from dihydropyrimidine dehydrogenase is of low intensity due to the relative weakness of the dipolar interaction. The g = 4.3 signal is a dirty iron contamination. 

Hagen, W.R., Vanoni, M.A., Rosenbaum, K., and Schnackerz, K.D. 2000. On the iron-sulfur clusters in the complex redox enzyme dihydropyrimidine dehydrogenase. European Journal of Biochemistry 267 3640-3646. 

In rare cases, patients deficient in dihydropyrimidine dehydrogenase, responsible for the catabolism of 5-FU, develop severe toxicity, including death, after 5-FU administration. 

AO is also effective in metabolizing a wide range of nitrogen-containing heterocycles such as purines, pyrimidines, pteridines, quinolines, and diazanaphthalenes (95). For example, phthalazine is rapidly converted to 1-phthalazinone by AO and the prodrug, 5-ethynyl-2-(l//)-pyrimidone, is oxidized to the dihydropyrimidine dehydrogenase mechanism-based inhibitor, 5-ethynyluracil, by AO (Fig. 4.40) (96). 

Dihydropyrimidine dehydrogenase is the first and the rate-limiting enzyme in the three-step metabolic pathway involved in the degradation of the pyrimidine bases uracil and thymine. In addition, this catabolic pathway is the only route for the synthesis of p-alanine in mammals. 

Takimoto, C.H., Lu, Z.H., Zhang, R., et al. (1996) Severe neurotoxicity following 5-fluorour-acil-based chemotherapy in a patient with dihydropyrimidine dehydrogenase deficiency. Clin. Cancer Res. 2, 477 81. 

Harris, B.E., Song, R., Soong, S.J., et al. (1990) Relationship between dihydropyrimidine dehydrogenase activity and plasma 5-fluorouracil levels with evidence for circadian variation of enzyme activity and plasma drug levels in cancer patients receiving 5-fluorouracil by protracted continuous infusion. Cancer Res. 50,197-201. 

Fleming, R.A., Milano, G., Thyss, A., et al. (1992) Correlation between dihydropyrimidine dehydrogenase activity in peripheral mononuclear cells and systemic clearance of fluorouracU in cancer patients. Cancer Res. 52, 2899-2902. 

Raida, M., Schwabe, W., Hausler, P., et al. (2001) Prevalence of a common fwint mutation in the dihydropyrimidine dehydrogenase (DPD) gene within the 5 -splice donor site of intron 14 in patients with severe 5-fluorouracil (5-FU)-related toxicity compared with controls. Clin. Cancer Res. 7, 2832-2839. 

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