Dihydropyrimidine Dehydrogenase Activity in Human Peripheral Blood Mononuclear Cells and Liver: Population Characteristics, Newly Identified Deficient Patients, and Clinical Implication in 5-Fluorouracil Chemotherapy1

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (FUra), one of the most widely used anticancer drugs. Previous studies from our laboratory demon strated the clinical importance of DPD in cancer patients (G. D. Heggie, J-P. Sommadossi, D. S. Cross, W. J. Muster, and R. B. Diasio. Cancer Res., 47: 2203-2206, 1987; B. E. Harris, R. Song, S-j. Soong, and R. B. Diasio. Cancer Res., 50: 197-201, 1990), particularly in those with DPD deficiency who experience severe FUra toxicity (including death) following FUra treatment [R. B. Diasio, T. L. Beavers, and J. T. Carpenter. J. Clin. Invest., 81: 47-51, 1988; B. E. Harris, J. T. Carpenter, and R. B. Diasio. Cancer (Phila.), 68: 499-501, 1991]. We now suggest that measurement of DPD activity may be useful in routine screening of cancer patients prior to FUra treatment. In this paper, we describe the following serial studies: (a) we developed a sensitive, accurate, and precise DPD assay and a storage method to stabilize DPD activity, permitting large scale DPD screening in cancer patients; 11»we demonstrated a normal distribution (Gaussian distribution) of human DPD activity from peripheral blood mononuclear cells (PBM-DPD) in a population study. Baselines for PBM-DPD with fresh and frozen samples were 0.425 ±0.124 (SD) and 0.189 ±0.064 nmol/min/mg protein, respectively. The 95% and 99% distribution ranges for both fresh and frozen samples were also determined, providing criteria for detection of DPD-deficient patients; (c) we identified nine new patients with profound or partial DPD deficiency; (d\ we determined a baseline for human liver DPD activity, which was shown to be 0.360 ±0.182 nmol/ min/mg protein (frozen samples); in we did a preliminary evaluation of liver DPD from deficient patients. Low liver DPD activity in two deficient patients correlated with low PBM-DPD activity. Using a polyclonal anti body raised against human liver DPD in our laboratory (Z. Lu, R. Zhang, and R. B. Diasio. J. Biol. Chem., 267: 17102-17109, 1992), Western blot analysis demonstrated decreased DPD protein in the liver cytosol from DPD-deficient patients compared to normal subjects. These results may be useful in improving the effectiveness and/or lessening the toxicity of FUra chemotherapy. INTRODUCTION DPD' (EC 1.3.1.2) is the initial and rate-limiting enzyme in FUra catabolism (1-4). Although it has been thought that the cytotoxic effects of FUra are directly mediated by the anabolic pathway (1-4), studies from our laboratory have demonstrated the importance of the catabolic pathway with more than 80% of administered FUra being catabolized by DPD (5). It has also been shown that the anticancer Received 6/9/93; accepled 9/8/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by USPHS Grant CA-62164 and Clinical Research Center Grant RR-00032. This study was presented in part at the 29th annual meeting of the American Society of Clinical Oncology (ASCO), Orlando. FL, May 16-18, 1993. Z. L. is the recipient of the 1993 ASCO Travel Awards. 2 To whom requests for reprints should be addressed, at Department of Pharmacology, University of Alabama at Birmingham. UAB Station, Box 600, Volker Hall 101, Birming ham. AL 35294-0019. 1 The abbreviations used are: DPD, dihydropyrimidine dehydrogenase; FUra, 5-fluo rouracil; PBM cells, peripheral blood mononuclear cells; SDS-PAGE. sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPLC. high-performance liquid chromatography. efficacy of FUra is related to DPD activity in an experimental animal tumor model (6). We (7) and others (8, 9) have demonstrated that DPD activity is correlated with FUra pharmacokinetics, suggesting an im portant role in the regulation of FUra metabolism and thus determin ing the amount of FUra available for anabolism. DPD activity in humans (7, 10) and experimental animals (11-14) follows a circadian pattern, which is inversely correlated with FUra plasma level in cancer patients treated with FUra by continuous infusion (7). Additional studies with competitive DPD inhibitors (15-18) have also shown the importance of DPD in FUra chemotherapy. More importantly, the role of DPD in FUra chemotherapy has been shown in cancer patients with confirmed (19-21) or suspected (22, 23) DPD deficiency. These patients experienced severe FUra toxicity (in cluding death) following FUra treatment. A clinical pharmacological study of one of these deficient patients (19) demonstrated minimal catabolism of FUra with a 10-fold longer FUra half-life compared to patients with normal DPD activity. Familial studies (19-21) have demonstrated that this pharmacogenetic syndrome follows an autosomal recessive pattern of inheritance. Although extensive studies in DPD biochemistry have been con ducted with experimental animals and crude human tissue prepara tions (24-27), little has been known about human DPD until recently. In our laboratory, human liver DPD has recently been purified and characterized with preparation of a polyclonal antibody to DPD (28, 29), permitting further investigation of DPD in cancer patients. Given the frequent use of FUra in cancer chemotherapy, the im portance of DPD in FUra pharmacokinetics, and the clinical signifi cance of DPD deficiency, we suggest the potential value of DPD screening in cancer patients prior to and during FUra treatment. In order to provide the basis for large scale DPD screening in cancer patients, the following studies were undertaken: (a) development of a sensitive, accurate, precise, and clinically useful DPD assay; (b) es tablishment of a baseline for DPD activity and criteria for diagnosis of partial and profound DPD deficiency; (c) identification of additional patients with DPD deficiency; (d) determination of liver DPD activity in the general population and in cancer patients with DPD deficiency initially identified by PBM-DPD assay. In addition, using a polyclonal antibody raised from purified human liver DPD (28), we also con ducted Western blot analyses to determine if DPD protein in the liver cytosol from DPDdeficient patients was decreased compared to that of normal subjects. The serial studies reported in this paper demon strate the potential value of measuring DPD activity. We believe that determination of DPD activity in cancer patients prior to and/or during FUra treatment may be beneficial in improving the clinical effective ness of FUra. MATERIALS AND METHODS Chemicals and Radiochemicals FUra, bovine serum albumin. NADPH, and Histopaque were purchased from the Sigma Chemical Co. (St. Louis, MO). ['H]FUra (25 Ci/mmol) was obtained from New England Nuclear Corp. (Boston. MA). The purity of