Cancer Letters

Cancer Letters

Volume 122, Issues 1–2, 9 January 1998, Pages 107-113
Cancer Letters

Suicidal inactivation of human dihydropyrimidine dehydrogenase by (E)-5-(2-bromovinyl)uracil derived from the antiviral, sorivudine

https://doi.org/10.1016/S0304-3835(97)00377-7Get rights and content

Abstract

An enzymatic study was performed to clarify the mechanism of 18 acute deaths in patients who had received the new oral antiviral drug, sorivudine (SRV), during anticancer chemotherapy with 5-fluorouracil (5-FU) prodrugs. Human dihydropyrimidine dehydrogenase (hDPD), playing a key role in the liver as the rate-limiting enzyme in catabolism of 5-FU, was expressed in E. coli, purified and incubated in the presence of NADPH with SRV or (E)-5-(2-bromovinyl)uracil (BVU), a metabolite of SRV produced by human gut flora. hDPD was rapidly and irreversibly inactivated by BVU, but not by SRV. Radioactivity of [14C]BVU was incorporated into hDPD in the presence of NADPH in a manner reciprocal to the enzyme inactivation. In the absence of NADPH, hDPD was not inactivated by BVU, nor radiolabeled with [14C]BVU. Thus, as we demonstrated previously with studies using the rat, the acute deaths were strongly suggested to be attributable to markedly elevated tissue 5-FU levels which were responsible for irreversible inhibition of hDPD by covalent binding of a reduced form of BVU as a suicide inactivator.

Introduction

In 1993, there were 15 acute deaths in Japan in patients orally administered the new antiviral drug, sorivudine (1-β-d-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil, SRV), for herpes zoster within 40 days after SRV was approved by the Japanese government for clinical use [1]. According to a report from the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, Japan [1], all of these patients, in addition to the three who died during the phase II clinical trial of SRV, were daily receiving one of the oral 5-fluorouracil (5-FU) prodrugs widely used in Japan for long-term anticancer chemotherapy while being orally administered SRV for a period of several days. Before death, there were severe toxic symptoms, such as markedly decreased white blood cell and platelet counts and diarrhea with bloody flux. However, there was no acute death following such severe symptoms of toxicity in patients who had herpes zoster and who received SRV alone or who were receiving both SRV and anticancer drugs other than 5-FU or its prodrugs.

Very recently we demonstrated with rats the mechanism of the acute deaths caused by the interaction between SRV and the oral 5-FU prodrug, tegafur (1-(2-tetrahydrofuryl)-5-fluorouracil, FT) [2], which most of the patients were receiving and which had been shown to generate 5-FU by hepatic cytochrome P-450 after being absorbed [3]. In the animals, orally received SRV was converted in part by gut flora into (E)-5-(2-bromovinyl)uracil (BVU) with no antiviral activity [4]. BVU, microbially generated, was absorbed through intestinal membrane and irreversibly inactivated hepatic dihydropyrimidine dehydrogenase (DPD), playing a key role as the rate-limiting enzyme in 5-FU catabolism to α-fluoro-β-alanine and markedly elevated the tissue levels of 5-FU formed from simultaneously administered FT [2]. The elevated 5-FU levels in bone marrow and intestines after repeated simultaneous administration of FT and SRV resulted in the acute death of all the animals used within 10 days, whereas no toxic symptom was observed on day 20 in animals given the same repeated dose of FT or SRV alone. Before death, the animals exhibited severe toxic symptoms very similar to those reported in the patients.

DPD purified from rat liver cytosol (rDPD) was completely inactivated by BVU as a suicide inactivator in the presence of NADPH, but not by SRV in the presence of NADPH or by BVU in the absence of the cofactor to any appreciable extent [2]. Furthermore, purified rDPD was radiolabeled with [14C]BVU in the presence of NADPH, but not in the absence of the cofactor [2].

Before our study, Desgranges et al. [5]had demonstrated that partially purified rDPD was inactivated by unlabeled BVU in the presence of NADPH and that the activity of the inactivated rDPD could not be restored by dialysis.

BVU has been demonstrated to be a major metabolite of SRV in humans [6]and to be readily formed from the antiviral by Bacteroides species, such as B. eggerthill and B. vulgatus, abundantly existing in human gut flora [7].

DPD is a soluble homodimeric protein with a molecular mass of 204–216 kDa containing FMN/FAD and an Fe/S cluster in each subunit 8, 9, 10, 11and is primarily distributed in the liver in rats [8]and humans [12]. The primary structure of human DPD (hDPD) has been elucidated by molecular cloning [13]. However, heterologous expression of hDPD has not been achieved.

Recently, it has been noted through studies on genetic polymorphism of hDPD that 5-FU should not be used for anticancer chemotherapy in patients who are DPD-deficient or have very low DPD activity in the liver or in mononuclear cells in peripheral blood, as they will suffer from severe toxic symptoms or die from markedly elevated tissue 5-FU levels 14, 15. Actually, a number of such DPD-deficient patients have been reported to have died when treated with clinical doses of 5-FU [14]. Our study on the lethal drug interaction in rats strongly suggested that the acute deaths might have been caused in patients who became artificially DPD-deficient by the irreversible inactivation of the enzyme with BVU formed from SRV. The present study was undertaken to obtain direct evidence for suicidal inactivation of hDPD by BVU. For this purpose, we used recombinant hDPD expressed in E. coli.

Section snippets

Materials

SRV and [14C]BVU were prepared as previously reported [2]. Restriction enzymes, T4 DNA ligase and Taq polymerase were purchased from Takara Shuzo (Kyoto, Japan), ampicillin (Na), NADPH, NADP+, Triton X-100 and uracil were purchased from Wako Pure Chemical Industries (Osaka, Japan), Xpress™ protein expression system (pTrcHis) and ProBond™ resin were purchased from Invitrogen (San Diego, CA), 2′,5′-ADP-Sepharose 4B and a PD-10 column were purchased from Pharmacia LKB Biotechnology (Uppsala,

Results and discussion

Potent 5-FU-reducing activity was observed in the dialyzed cytosolic fraction from E. coli transformed by the expression plasmid, pTrcHis(hDPD). However, dialyzed cytosol from E. coli transformed by the control vector, pTrcHisA, had no detectable 5-FU-reducing activity. The 5-FU-reducing activity of the bacterial cytosol (1.5–3.0 nmol/mg protein/min) was about five to 10 times higher than the highest such activity reported with human liver cytosols (0.054–0.29 nmol/mg protein/min) 10, 15.

References (18)

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