In Vitro Assessment of Fluoropyrimidine-Metabolizing Enzymes: Dihydropyrimidine Dehydrogenase, Dihydropyrimidinase, and β-Ureidopropionase

Abstract

Fluoropyrimidine drugs (FPs), including 5-fluorouracil, tegafur, capecitabine, and doxifluridine, are among the most widely used anticancer agents in the treatment of solid tumors. However, severe toxicity occurs in approximately 30% of patients following FP administration, emphasizing the importance of predicting the risk of acute toxicity before treatment. Three metabolic enzymes, dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP), and β-ureidopropionase (β-UP), degrade FPs; hence, deficiencies in these enzymes, arising from genetic polymorphisms, are involved in severe FP-related toxicity, although the effect of these polymorphisms on in vivo enzymatic activity has not been clarified. Furthermore, the clinical usefulness of current methods for predicting in vivo activity, such as pyrimidine concentrations in blood or urine, is unknown. In vitro tests have been established as advantageous for predicting the in vivo activity of enzyme variants. This is due to several studies that evaluated FP activities after enzyme metabolism using transient expression systems in Escherichia coli or mammalian cells; however, there are no comparative reports of these results. Thus, in this review, we summarized the results of in vitro analyses involving DPD, DHP, and β-UP in an attempt to encourage further comparative studies using these drug types and to aid in the elucidation of their underlying mechanisms.

DPYDgenetic polymorphisms in non-European patients with severe fluoropyrimidine-related toxicity: A systematic review

“…Up to 77% of the in silico prediction tools we utilised predicted this variant to be deleterious or probably damaging and this variant was classified as deleterious by APF (Table 3). In vitro functional analysis containing the Tyr186Cys amino acid substitution showed a ∼15% reduction in DPD enzyme activity relative to the wild-type (Table 3) 57,65 . Maharjan and colleagues (2019) did not include c.557A>G genetic testing in their cohort of African American patients 63 .…”

“…Up to 92% of the in silico prediction tools we utilised predicted c.1774C>T to be deleterious and the APF classified this variant as deleterious (Table 3). Previously published in vitro functional characterization of c.1774C>T reported a reduction in DPD catalytic activity compared to the wild-type (Table 3) 45,57,59,65 .…”

“…Heterozygous carriers of the intron 14 splice donor variant c.1905+1G>A were reported in one Thai cohort patient 77 and 14 Chinese cohort patients in which significantly higher incidences of grade 3-4 myelosuppression, hand-foot syndrome, diarrhoea, gastrointestinal reactions and mucositis were observed (OR = unreported; p < 0.001 for each severe side effect) compared to wild-type carriers 69 . 100% of the in silico prediction tools we utilised predicted this variant to be deleterious and published in vitro expression analysis reported c.1905+1G>A to be catalytically inactive (Table 3) 56,65 .…”

Importance of Rare DPYD Genetic Polymorphisms for 5-Fluorouracil Therapy in the Japanese Population

“…To date, more than 450 genetic variants have been identified in the DPYD gene ( Sistonen et al, 2012 ; Thomas et al, 2016 ; Hishinuma et al, 2020 ). In Caucasians, four DPYD risk variants, including c.1905+1G>A (IVS14+1G>A, DPYD*2A ), c.1129-5923C>G/hapB3, c.1679T>G ( DPYD*13 , p.I560S), and c.2846A>T (p.D949V), have been reported ( Froehlich et al, 2015 ; Meulendijks et al, 2015 ).…”

Functional Characterization of 12 Dihydropyrimidinase Allelic Variants in Japanese Individuals for the Prediction of 5-Fluorouracil Treatment-Related Toxicity

“…Thus, DPYS polymorphisms could become essential in predicting 5-FU-related toxicity in patients, including those from Asian populations. We previously described the functional characterization of 21 DHPase variants that have been identified in various ethnic groups (Hishinuma et al, 2017;Hishinuma et al, 2020). Most of these variants may contribute to the large interindividual variability in the pharmacokinetics and pharmacodynamics of 5-FU.…”