Can we identify patients carrying targeted deleterious DPYD variants with plasma uracil and dihydrouracil? A GPCO-RNPGx retrospective analysis.

OBJECTIVES: Dihydropyrimidine dehydrogenase (DPD) deficiency is the main cause of severe fluoropyrimidine-related toxicities. The best strategy for identifying DPD-deficient patients is still not defined. The EMA recommends targeted DPYD genotyping or uracilemia (U) testing. We analyzed the concordance between both approaches. METHODS: This study included 19,376 consecutive French patients with pre-treatment plasma U, UH2 and targeted DPYD genotyping (*2A, *13, D949V, *7) analyzed at Eurofins Biomnis (2015-2022). RESULTS: Mean U was 9.9 ± 10.1 ng/mL (median 8.7, range 1.6-856). According to French recommendations, 7.3 % of patients were partially deficient (U 16-150 ng/mL) and 0.02 % completely deficient (U≥150 ng/mL). DPYD variant frequencies were *2A: 0.83 %, *13: 0.17 %, D949V: 1.16 %, *7: 0.05 % (2 homozygous patients with U at 22 and 856 ng/mL). Variant carriers exhibited higher U (median 13.8 vs. 8.6 ng/mL), and lower UH2/U (median 7.2 vs. 11.8) and UH2/U2 (median 0.54 vs. 1.37) relative to wild-type patients (p<0.00001). Sixty-six% of variant carriers exhibited uracilemia <16 ng/mL, challenging correct identification of DPD deficiency based on U. The sensitivity (% patients with a deficient phenotype among variant carriers) of U threshold at 16 ng/mL was 34 %. The best discriminant marker for identifying variant carriers was UH2/U2. UH2/U2<0.942 (29.7 % of patients) showed enhanced sensitivity (81 %) in identifying deleterious genotypes across different variants compared to 16 ng/mL U. CONCLUSIONS: These results reaffirm the poor concordance between DPD phenotyping and genotyping, suggesting that both approaches may be complementary and that targeted DPYD genotyping is not sufficiently reliable to identify all patients with complete deficiency.

Quantitative impact of pre-analytical process on plasma uracil when testing for dihydropyrimidine dehydrogenase deficiency.

AIMS: Determining dihydropyrimidine dehydrogenase (DPD) activity by measuring patient's uracil (U) plasma concentration is mandatory before fluoropyrimidine (FP) administration in France. In this study, we aimed to refine the pre-analytical recommendations for determining U and dihydrouracil (UH2 ) concentrations, as they are essential in reliable DPD-deficiency testing. METHODS: U and UH2 concentrations were collected from 14 hospital laboratories. Stability in whole blood and plasma after centrifugation, the type of anticoagulant and long-term plasma storage were evaluated. The variation induced by time and temperature was calculated and compared to an acceptability range of ±20%. Inter-occasion variability (IOV) of U and UH2 was assessed in 573 patients double sampled for DPD-deficiency testing. RESULTS: Storage of blood samples before centrifugation at room temperature (RT) should not exceed 1 h, whereas cold (+4°C) storage maintains the stability of uracil after 5 hours. For patients correctly double sampled, IOV of U reached 22.4% for U (SD = 17.9%, range = 0-99%). Notably, 17% of them were assigned with a different phenotype (normal or DPD-deficient) based on the analysis of their two samples. For those having at least one non-compliant sample, this percentage increased up to 33.8%. The moment of blood collection did not affect the DPD phenotyping result. CONCLUSION: Caution should be taken when interpreting U concentrations if the time before centrifugation exceeds 1 hour at RT, since it rises significantly afterwards. Not respecting the pre-analytical conditions for DPD phenotyping increases the risk of DPD status misclassification.

Synthesis and Characterization of Dihydrouracil Analogs Utilizing Biginelli Hybrids.

Dihydrouracil presents a crucial intermediate in the catabolism of uracil. The vital importance of uracil and its nucleoside, uridine, encourages scientists to synthesize novel dihydrouracils. In this paper, we present an innovative, fast, and effective method for the synthesis of dihydrouracils. Hence, under mild conditions, 3-chloroperbenzoic acid was used to cleave the carbon-sulfur bond of the Biginelli hybrids 5,6-dihydropyrimidin-4(3H)-ones. This approach led to thirteen novel dihydrouracils synthesized in moderate-to-high yields (32-99%).

Food-effect study on uracil and dihydrouracil plasma levels as marker for dihydropyrimidine dehydrogenase activity in human volunteers.

AIMS: This study aimed to determine the effect of food intake on uracil and dihydrouracil plasma levels. These levels are a promising marker for dihydropyrimidine dehydrogenase activity and for individualizing fluoropyrimidine anticancer therapy. METHODS: A randomized, cross-over study in 16 healthy volunteers was performed, in which subjects were examined in fasted and fed state on two separate days. In fed condition, a high-fat, high-caloric breakfast was consumed between 8:00 h and 8:30 h. Whole blood for determination of uracil, dihydrouracil and uridine plasma levels was drawn on both test days at predefined time points between 8:00 h and 13:00 h. RESULTS: Uracil levels were statistically significantly different between fasting and fed state. At 13:00 h, the mean uracil level in fasting state was 12.6 ± 3.7 ng ml-1 and after a test meal 9.4 ± 2.6 ng ml-1 (P < 0.001). Dihydrouracil levels were influenced by food intake as well (mean dihydrouracil level at 13:00 h in fasting state 147.0 ± 36.4 ng ml-1 and in fed state 85.7 ± 22.1 ng ml-1 , P < 0.001). Uridine plasma levels showed curves with similar patterns as for uracil. CONCLUSIONS: It was shown that both uracil and dihydrouracil levels were higher in fasting state than in fed state. This is hypothesized to be an direct effect of uridine plasma levels, which were previously shown to be elevated in fasting state and reduced after intake of food. These findings show that, when assessing plasma uracil and dihydrouracil levels for adaptive fluoropyrimidine dosing in clinical practice, sampling should be done between 8:00 h and 9:00 h after overnight fasting to avoid bias caused by circadian rhythm and food effects.

The impact of liver resection on the dihydrouracil:uracil plasma ratio in patients with colorectal liver metastases.

PURPOSE: The dihydrouracil (DHU):uracil (U) plasma ratio is a promising marker for identification of dihydropyrimidine dehydrogenase (DPD)-deficient patients. The objective of this study was to determine the effect of liver resection on the DHU:U plasma ratio in patients with colorectal liver metastases (CRLM). METHODS: An observational study was performed in which DHU:U plasma ratios in patients with CRLM were analyzed prior to and 1 day after liver resection. In addition, the DHU:U plasma ratio was quantified in six additional patients 4-8 weeks after liver resection to explore long-term effects on the DHU:U plasma ratio. Quantification of U and DHU plasma levels was performed using a validated ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay. RESULTS: The median (range) DHU:U plasma ratio in 15 patients prior to liver resection was 10.7 (2.6-14.4) and was significantly reduced to 5.5 (< quantification limit (LLOQ-10.5) 1 day after resection (p = 0.0026). This reduction was caused by a decrease in DHU plasma levels from 112.0 (79.8-153) ng/mL to 41.2 (< LLOQ-160) ng/mL 1 day after resection (p = 0.0004). Recovery of the DHU:U plasma ratio occurred 4-8 weeks after liver resection, which was shown by a median (range) DHU:U plasma ratio in six patients of 9.1 (6.9-14.5). CONCLUSION: Liver resection leads to very low DHU:U plasma ratios 1 day after liver resection, which is possibly caused by a reduction in DPD activity. Quantification of the DHU:U plasma ratios directly after liver resection could lead to false-positive identification of DPD deficiency and is therefore not advised.

A new sample preparation and separation combination for the precise, accurate, and simultaneous determination of uracil and dihydrouracil in human plasma by reversed-phase HPLC.

We have developed an efficient procedure and detection method using reversed-phase high-performance liquid chromatography for the simultaneous measurement of uracil and dihydrouracil in human plasma. The procedure, including chromatographic conditions and sample preparation, was optimized and validated. Optimization of the sample preparation included deproteinization, extraction, and cleanup. A new sample preparation method which resulted in an improved extraction yield of analytes and significantly reduced interference at low-wavelength UV detection was developed. The developed method was validated for specificity, linearity, limits of detection and quantitation, precision, and accuracy. All calibration curves showed excellent linear regression (R2  > 0.9990) within the testing range. The limit of detection for uracil and dihydrouracil was 2.5 and 5.0 ng/mL, respectively. The extraction yields were >94% for uracil and 91% for dihydrouracil. Intra- and interassay precision and accuracy for uracil and dihydrouracil were lower than 8% at all tested concentrations. The proposed method was successfully applied to measure plasma concentrations of uracil and dihydrouracil in colorectal cancer patients scheduled to receive fluoropyrimidine-based chemotherapy.

The Herb-Drug Pharmacokinetic Interaction of 5-Fluorouracil and Its Metabolite 5-Fluoro-5,6-Dihydrouracil with a Traditional Chinese Medicine in Rats.

BACKGROUND: Xiang-Sha-Liu-Jun-Zi-Tang (XSLJZT) is the most common traditional formula given to colorectal and breast cancer patients in Taiwan, according to a statistical study of the National Health Insurance Research Database. 5-Fluorouracil (5-FU) is widely used as the first line of treatment for colorectal cancer. Thus, the aim of study is to investigate the pharmacokinetic interaction of XSLJZT and 5-FU. METHODS: To investigate the herb-drug interaction of XSLJZT with 5-FU as well as its metabolite 5-fluoro-5,6-dihydrouracil (5-FDHU) using pharmacokinetics, a high-performance liquid chromatography (HPLC) system coupled with a photodiode array detector was developed to monitor 5-FU and 5-FDHU levels in rat blood. Rats were divided into three cohorts, one of which was administered 5-FU (100 mg/kg, iv-intravenous) alone, while the other two groups were pretreated with low and high doses of XSLJZT (600 mg/kg/day or 2400 mg/kg/day for 5 consecutive days) in combination with 5-FU. RESULTS: The results demonstrated that 5-FU level was not significantly different between the group treated with only 5-FU and the group pretreated with a normal dose of XSLJZT (600 mg/kg/day). However, pharmacokinetic analysis revealed that pretreatment with a high dose of XSLJZT (2400 mg/kg/day) extended the residence time and increased the volume of distribution of 5-FU. No significant distinctions were found in 5-FDHU pharmacokinetic parameters at three doses of XSLJZT. CONCLUSIONS: Overall, the pharmacokinetic results confirm the safety of coadministering 5-FU with XSLJZT, and provide practical dosage information for clinical practice.

Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III.

Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3'-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln(41) and Leu(81) as DNA lesion sensors.

Shotgun metabolomic profiles in maternal urine identify potential mass spectral markers of abnormal fetal biochemistry - dihydrouracil and progesterone in the metabolism of Down syndrome.

In Down syndrome (DS) in particular, the precise cellular mechanisms linking genotype to phenotype is not straightforward despite a clear mapping of the genetic cause. Metabolomic profiling might be more revealing in understanding molecular-cellular mechanisms of inborn errors of metabolism/syndromes than genomics alone and also result in new prenatal screening approaches. The urinary metabolome of 122 maternal urine from women with and without an aneuploid pregnancy (predominantly Down syndrome) were compared by both zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) and reversed-phase liquid chromatography (RPLC) coupled to hybrid ion trap time of flight mass spectral analysis. ZIC-HILIC mass spectrometry resolved 10-fold more unique molecular ions than RPLC mass spectrometry, of which molecules corresponding to ions of m/z 114.07 and m/z 314.20 showed maternal urinary level changes that significantly coincided with the presence of a DS fetus. The ion of m/z 314.20 was identified as progesterone and m/z 114.07 as dihydrouracil. A metabolomics profiling-based maternal urinary screening test modelled from this separation data would detect approximately 87 and 60.87% (using HILIC-MS and RPLC-MS, respectively) of all DS pregnancies between 9 and 23 weeks of gestation with no false positives.

Improved synthesis of N1-substituted orotic acid derivatives.

An improved method for the synthesis of N1-substituted orotic acid derivatives is reported. The method involves sequential incorporation of nitrogen atoms to the pyrimidine structure from simple starting materials and thus allows the synthesis of N1-substituted orotic acid derivatives with single 15N label at either N-1 or N-3.

Identification of DPYD variants and estimation of uracil and dihydrouracil in a healthy Indian population.

Aim: This study aimed to identify DPYD variants and the related but previously unexplored phenotype (plasma uracil, dihydrouracil [DHU], and the DHU-to-uracil ratio) in a healthy adult Indian population. Methods: Healthy adult volunteers (n = 100) had their uracil and DHU levels measured and were genotyped for selected variants. Results: Among the nine variants studied, c.1906-14763G>A and c.85T>C were the most prevalent. Participants with any of the variants except for c.85T>C and c.1627A>G had a significantly lower DHU-to-uracil ratio and those with c.1905+1G>A variant had significantly increased uracil concentration compared with wild-type. Conclusion: Participants with five variants were identified as having altered phenotypic measures, and 40% of the intermediate metabolizers had their phenotype in the terminal population percentiles.

Background: 5-fluorouracil (5-FU) is a medicine used in cancer treatment. It is eliminated from body by the enzyme DPD. Identifying deficiency in DPD before initiating 5-FU can save patients from oral, intestinal, and bone marrow toxic effects. Methods: The uracil and dihydrouracil (DHU, produced by DPD enzyme action) levels were measured and DPD gene (for identifying defects) was sequenced in 100 healthy adults. Results: Participants with DPD gene sequence that is known to be defective had higher plasma uracil levels and a low DHU-to-uracil ratio compared with those who did not have a defective gene. Conclusion: Measuring plasma uracil and DHU-to-uracil ratio can help identify people with defective DPD genes.

Fast, Direct Dihydrouracil Quantitation in Human Saliva: Method Development, Validation, and Application.

Background. Salivary metabolomics is garnering increasing attention in the health field because of easy, minimally invasive saliva sampling. Dihydrouracil (DHU) is a metabolite of pyrimidine metabolism present in urine, plasma, and saliva and of fluoropyrimidines-based chemotherapeutics. Its fast quantification would help in the identification of patients with higher risk of fluoropyrimidine-induced toxicity and inborn errors of pyrimidine metabolism. Few studies consider DHU as the main salivary metabolite, but reports of its concentration levels in saliva are scarce. We propose the direct determination of DHU in saliva by reversed-phase high-performance liquid chromatography (RP-HPLC-UV detector) as a simple, rapid procedure for non-invasive screening. Methods. The method used was validated and applied to 176 saliva samples collected from 21 nominally healthy volunteers and 4 saliva samples from metastatic colorectal cancer patients before and after receiving 5-fluorouracil chemotherapy. Results. DHU levels in all samples analyzed were in the µmol L-1 range or below proving that DHU is not the main metabolite in saliva and confirming the results found in the literature with LC-MS/MS instrumentation. Any increase of DHU due to metabolism dysfunctions can be suggestive of disease and easily monitored in saliva using common, low-cost instrumentation available also for population screening.

Comparison of a thymine challenge test and endogenous uracil-dihydrouracil levels for assessment of fluoropyrimidine toxicity risk.

PURPOSE: Standard dosages of fluoropyrimidine chemotherapy result in severe toxicity in a substantial proportion of patients, however, routine pre-therapeutic toxicity prediction remains uncommon. A thymine (THY) challenge test can discriminate risk of severe gastrointestinal toxicity in patients receiving fluoropyrimidine monotherapy. We aimed to measure endogenous plasma uracil (U) and its ratio to dihydrouracil (DHU), and assess the performance of these parameters compared with the THY challenge test to evaluate risk of severe toxicity. METHODS: Plasma samples, previously collected from 37 patients receiving 5-fluorouracil (5-FU) or capecitabine monotherapy for a THY challenge test (ACTRN12615000586516; retrospectively registered), were assessed for endogenous plasma concentrations of U and DHU using a validated LC-MS/MS method. Renal function was estimated from blood creatinine, and patients with ≥ grade 3 toxicity (CTCAE v4.0) were classified as cases. RESULTS: There were no differences in median endogenous U plasma concentrations or U/DHU ratios between severe toxicity cases and non-cases. Significant differences between cases and non-cases were noted when these measures were normalised to the estimated renal function (CrCL), Unorm p = 0.0004; U/DHUnorm p = 0.0083. These two parameters had a sensitivity of 29%, compared with 57% for the THY challenge test in the same patients. Genotyping for clinically relevant DPYD variants was inferior to either of these pyrimidine phenotyping tests (sensitivity of 14%). CONCLUSIONS: The endogenous uracil-based parameters, adjusted to CrCL, were more predictive of increased risk of severe fluoropyrimidine toxicity than DPYD genotyping. However, endogenous U measurement detected fewer cases of severe toxicity than the THY challenge test.

Local Irradiation Modulates the Pharmacokinetics of Metabolites in 5-Fluorouracil-Radiotherapy-Pharmacokinetics Phenomenon.

BACKGROUND: The effects of radiotherapy (RT) on the pharmacokinetics (PK) of 5-FU and 5-fluoro-5,6-dihydro-uracil (5-FDHU) were investigated by animal experiments. METHODS: Whole-pelvis RT with 0.5 and 2 Gy was delivered to Sprague-Dawley rats. 5-FU at 100 mg/kg was intravenously infused 24 h after radiation. The pharmacokinetics of 5-FU and 5-FDHU in the plasma and bile system were calculated. RESULTS: The areas under the concentration versus time curve (AUC) of 5-FU in the plasma were reduced by local irradiation by 23.7% at 0.5 Gy (P < 0.001) and 35.3% at 2 Gy (P < 0.001). The AUCs of 5-FDHU were also reduced by 21.4% at 0.5 Gy (P < 0.001) and 51.5% at 2 Gy (P < 0.001). Irradiation significantly increased the clearance values (CLs) of 5-FU by 30.6% at 0.5 Gy and 50.1% at 2 Gy, respectively. The CLs of 5-FDHU were increased by 27.2% at 0.5 Gy and 106% at 2 Gy. The AUCs of 5-FU in the bile were increased by 36.7% at 0.5 Gy (P < 0.001) and 68.6% at 2 Gy (P = 0.005). The AUCs of 5-FDHU in the bile were increased by 40.3% at 0.5 Gy (P < 0.001) and 248.1% at 2 Gy (P < 0.001). The CLs of 5-FU in the bile were increased by 31.8% at 0.5 Gy and 11.2% at 2 Gy. However, the CLs of 5-FDHU in the bile were decreased by 29.1% at 0.5 Gy and 71.0% at 2 Gy. CONCLUSION: Both conventional and low-dose irradiation can affect the pharmacokinetics of 5-FU and its metabolite, 5-FDHU. RT plus 5-FU could cause more adverse events than 5-FU alone by increasing the AUC ratio of 5-FU/5-FDHU. Irradiation decreases the AUC of 5-FU in the plasma, which may cause poor clinical outcomes.

DPD functional tests in plasma, fresh saliva and dried saliva samples as predictors of 5-fluorouracil exposure and occurrence of drug-related severe toxicity.

OBJECTIVE: to evaluate plasma and salivary uracil (U) to dihydrouracil (UH2) ratios as tools for predicting 5-fluorouracil systemic exposure and drug-related severe toxicity, and clinically validate the use of dried saliva spots (DSS) as an alternative sampling strategy for dihydropyrimidine dehydrogenase (DPD) deficiency assessment. METHODS: Pre-chemotherapy plasma, fresh saliva and DSS samples were obtained from gastrointestinal patients (N = 40) for measurement of endogenous U and UH2 concentrations by LC-MS/MS. A second plasma sample collected during 5FU infusion was used for 5FU area under the curve (AUC) determination by HPLC-DAD. Data on toxicity was reported according to CTCAE. RESULTS: 15% of the patients developed severe 5FU-related toxicity, with neutropenia accounting for 67% of the cases. U, UH2 and [UH2,]/[U] were highly correlated between fresh and dried saliva samples (rs = 0.960; rs = 0.828; rs = 0.910, respectively). 5FU AUC ranged from 11.3 to 37.31 mg h L-1, with 46.2% of under-dosed and 10.3% over-dosed patients. The [UH2]/[U] ratios in plasma, fresh saliva and dried saliva samples were moderately correlated with 5FU AUC and adverse events grade, indicating a partial contribution of the variables to drug exposure (r = -0.412, rs = -0.373, rs = 0.377) and toxicity (r = -0.363, rs = -0.523, rs = 0.542). Metabolic ratios were lower in patients with severe toxicity (P < .01 salivary ratios, and P < .5 plasma ratios), and 5FU AUC were in average 47% higher in this group than in moderate toxicity. The diagnostic performance of [UH2]/[U] ratios in fresh saliva and DSS for the identification of patients with severe toxicity were comparable. CONCLUSIONS: The [UH2]/[U] metabolic ratios in plasma, fresh saliva and DSS were significantly associated with 5FU systemic exposure and toxicity degree. This study also demonstrated the applicability of DSS as alternative sampling for evaluating DPD activity.

Kinetics and Thermodynamics of DNA Processing by Wild Type DNA-Glycosylase Endo III and Its Catalytically Inactive Mutant Forms.

Endonuclease III (Endo III or Nth) is one of the key enzymes responsible for initiating the base excision repair of oxidized or reduced pyrimidine bases in DNA. In this study, a thermodynamic analysis of structural rearrangements of the specific and nonspecific DNA-duplexes during their interaction with Endo III is performed based on stopped-flow kinetic data. 1,3-diaza-2-oxophenoxazine (tCO), a fluorescent analog of the natural nucleobase cytosine, is used to record multistep DNA binding and lesion recognition within a temperature range (5-37 °C). Standard Gibbs energy, enthalpy, and entropy of the specific steps are derived from kinetic data using Van't Hoff plots. The data suggest that enthalpy-driven exothermic 5,6-dihydrouracil (DHU) recognition and desolvation-accompanied entropy-driven adjustment of the enzyme-substrate complex into a catalytically active state play equally important parts in the overall process. The roles of catalytically significant amino acids Lys120 and Asp138 in the DNA lesion recognition and catalysis are identified. Lys120 participates not only in the catalytic steps but also in the processes of local duplex distortion, whereas substitution Asp138Ala leads to a complete loss of the ability of Endo III to distort a DNA double chain during enzyme-DNA complex formation.