The Uracil Breath Test in theAssessment of Dihydropyrimidine Dehydrogenase Activity: Pharmacokinetic Relationship between Expired

Purpose:Dihydropyrimidine dehydrogenase (DPD) deficiency is critical in the predisposition to 5-fluorouracil dose-related toxicity. We recently characterized the phenotypic [2-C]uracil breath test (UraBT) with 96% specificity and100% sensitivity for identification of DPD deficiency. In the present study, we characterize the relationships among UraBT-associated breath CO2 metabolite formation, plasma [2-C]dihydrouracil formation, [2-C]uracil clearance, and DPD activity. Experimental Design:An aqueous solutionof [2-C]uracil (6mg/kg)was orally administered to 23 healthy volunteers and 8 cancer patients. Subsequently, breath CO2 concentrations and plasma [2-C]dihydrouracil and [2-C]uracil concentrations were determined over180 minutes using IR spectroscopy and liquid chromatography-tandemmass spectrometry, respectively.Pharmacokinetic variables were determined using noncompartmental methods. Peripheral blood mononuclear cell (PBMC) DPDactivity was measured using the DPD radioassay. Results: The UraBT identified 19 subjects with normal activity, 11 subjects with partial DPD deficiency, and 1 subject with profound DPD deficiency with PBMC DPD activity within the corresponding previously established ranges. UraBT breath CO2 DOB50 significantly correlated with PBMC DPD activity (rp = 0.78), plasma [2C]uracil area under the curve (rp = 0.73), [2-C]dihydrouracil appearance rate (rp = 0.76), and proportion of [2-C]uracil metabolized to [2-C]dihydrouracil (rp = 0.77; all Ps < 0.05). Conclusions: UraBT breath CO2 pharmacokinetics parallel plasma [2C]uracil and [2-C]dihydrouracil pharmacokinetics and are an accurate measure of interindividual variation in DPDactivity.These pharmacokinetic data further support the future use of the UraBTas a screening test to identify DPD deficiency before 5-fluorouracil-based therapy. Dihydropyrimidine dehydrogenase (EC 1.3.1.2, DPD) is the rate-limiting enzyme in uracil and 5-fluorouracil (5-FU) catabolism, converting >80% of an administered dose of 5-FU to inactive metabolites (1, 2). The initial step of catabolism is mediated by DPD converting 5-FU to 5-dihydrofluorouracil, with subsequent catabolism by dihydropyrimidinase and h-ureidopropionase enzymes to ultimately produce fluoroh-alanine, ammonia, and CO2. The latter final metabolic endproducts are excreted in the urine and breath (3). The pharmacogenetic syndrome of complete and partial DPD deficiency is prevalent in f0.1% and 3% to 5% of the general population, respectively (4). DPD deficiency is a significant pharmacogenetic factor in the predisposition of cancer patients to increased risk of altered 5-FU pharmacokinetics and associated toxicity. Specifically, 60% of patients presenting with severe 5-FU-related hematologic toxicity showed reduced DPD activity (5). Recent studies have investigated the predictive value of the ratio of plasma dihydrouracil area under the curve (AUC) to uracil AUC (DUUR) for the assessment of DPD activity and potential individualization of 5-FU therapy. Specifically, 5-FU dose optimization may be based on the plasma DUUR observed before 5-FU administration (6). Jiang et al. have also showed that the pre-5-FU treatment DUUR may be a good index of DPD activity (7, 8). Our laboratory recently reported the rapid noninvasive phenotypic [2-C]uracil breath test (UraBT) for assessment of DPD activity with 96% specificity and 100% sensitivity (9). Application of the UraBT to a large population of cancer-free subjects (n = 255) showed an observed 86% sensitivity (with 12 of 14 DPD-deficient subjects identified as DPD deficient) and Cancer Therapy: Clinical Authors’Affiliations: Divisions of Clinical Pharmacology and Toxicology and Biostatistics, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama and Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan Received 9/15/05; revised10/18/05; accepted11/7/05. Grant support: NIH grant CA62164 (R.B. Diasio) and National Center for Research Resources grant M01RR-00032 (General Clinical Research Center, University of Alabama at Birmingham). 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 with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Robert B. Diasio, Division of Clinical Pharmacology and Toxicology, Comprehensive Cancer Center, University of Alabama at Birmingham, 1824 6th Avenue South,Wallace Tumor Institute, Room 620, Birmingham, AL 35294-3300. Fax: 205-975-5650; E-mail: [email protected]. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-2020 www.aacrjournals.org Clin Cancer Res 2006;12(2) January15, 2006 549 Research. on June 8, 2017. © 2006 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 99% specificity (with 239 of 241 subjects with DPD activity in the reference range identified as normal; ref. 10). To date, however, the clinical relationship between pharmacokinetics of the [2-C]uracil probe substrate and its metabolites in plasma and breath remains to be elucidated. Based on our initial characterization of the UraBT, we hypothesize that (a) [2-C]uracil metabolite pharmacokinetic variables in the breath and plasma are reflective of DPD activity and (b) breath CO2 concentrations as measured through the UraBT correlate with plasma [2-C]uracil metabolite pharmacokinetics. In the present study, we provide a detailed characterization of the UraBT showing the relationship among breath CO2 metabolite formation, plasma [2C]dihydrouracil formation, [2-C]uracil clearance, and DPD activity. Materials andMethods Subjects. Thirty-one subjects (16 men and 15 women; ages 19-70 years) participated in this institutional review board – approved pharmacokinetic examination that was conducted at the General Clinical Research Center at the University of Alabama at Birmingham. Eight subjects were cancer patients who were referred by their oncologist due to known or suspected DPD deficiency. Twenty-three subjects were participants from the University of Alabama at Birmingham campus who volunteered for examination after reading an institutional review board– approved advertisement placed in the campus newspaper. Due to the rarity of DPD deficiency in the general population, we included six DPD-deficient individuals previously phenotyped [UraBT and DPD peripheral blood mononuclear cell (PBMC) radioassay] and genotyped (denaturing high-performance liquid chromatography analysis of the DPYD gene) in the current pharmacokinetic examination (9). Subjects with a history of gastric (i.e., dyspepsia) or respiratory (i.e., asthma) disease were excluded from