Characterization of Dihydropyrimidine Dehydrogenase Catabolism of 5-Fluorouracil in Human Liver

Abstract

5-fluorouracil (5-FU) is a cytotoxic drug used in the treatment of colorectal and breast cancers. Interpatient variability in 5-FU response is a significant clinical problem with ~30% of patients experiencing severe, dose-limiting toxicity. The activity of the enzyme dihydropyrimidine dehydrogenase (DPD) is an important factor in the risk and severity of toxicity of 5-FU. DPD catalyses the rate-limiting step in the pathway responsible for >80% of 5-FU metabolic clearance and the production of FBAL, a suspected cardiotoxic agent. A large number of DPYD gene variants have been reported, with loss-of-function variants associated with severe toxicity. Few studies have examined in depth DPD activity in human liver which is the primary site of 5-FU metabolism. The aims of this project were to measure the range of activity and expression of DPD in human liver tissue, to assess the effect of DPYD variants on activity, and to produce an analytical HPLC method to determine the relationship between DPD activity and FBAL formation. A wide range of DPD activity was found within 13 livers (k=0.0-12.7 % loss.min-1), including 4 null activity and 1 very high activity liver. Genotyping of these livers revealed the presence of a known severe null function variant (IVS14+1G>A), as well as several livers with gain-of-function variants (1 M166V and 6 C29R carriers). No correlation between DPD activity and DPYD variants, mRNA, protein expression, miRNA, was found. However, an inverse relationship between endogenous uracil concentration and DPD activity was observed (P <0.05). Development of an HPLC assay for FBAL was partially successful, however chromatographic resolution of FBAL was poor in the liver samples due to interference by endogenous amino acids. Further work is required confirm the existence of what appears to be an ultra-rapid 5-FU metaboliser phenotype and to establish the prevalence and consequences of increased DPD activity. However, since uracil is an endogenous substrate of DPD it may act as a competitive inhibitor of the reaction. This may influence the apparent null phenotype observed in the three livers which were not loss-of-function variant carriers. Additional work to establish the clinical relevance of this biochemical mechanism is required.