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Targeted protein degradation (TPD) has emerged as an important strategy to target disease-relevant proteins that were previously considered difficult to drug or even undruggable. Cereblon (CRBN) plays an outsized role in TPD as a preferred degradation-inducing effector protein for several reasons, including its anticipated broad protein substrate scope and its ligandability with drug-like small molecules. Notably, CRBN-based molecular glue degraders (MGDs) and proteolysis targeting chimeras (PROTACs) have shown success in clinical trials and, in some cases, as approved drugs. Thus, the interest in CRBN ligands within the pharmaceutical industry and academia has increased dramatically in recent years, highlighting the need for robust synthetic approaches towards them. This short review summarizes tactics and strategies to synthesize CRBN ligands, including the most recent developments in the field. Particular emphasis is put on the construction and direct functionalization of key CRBN binding motifs such as glutarimides and dihydrouracils. 1 Introduction 2 Cereblon Ligands with Glutarimide Binding Motif 3 Cereblon Ligands with Dihydrouracil Binding Motif 4 Cereblon Ligands with Other Binding Motifs 5 Conclusions and Outlook [ABSTRACT FROM AUTHOR]

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. 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). 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. 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. [ABSTRACT FROM AUTHOR]

This document is a reply to a letter discussing the effectiveness of uracil for pre-emptive DPD testing. The authors acknowledge the limitations of their study, including the omission of testing for haplotype B3 and the lack of information on renal and liver functions. They disagree with the assumption that a non-detectable UH2 is necessary for a true DPD deficient status. The authors suggest that a combination of indirect phenotyping and extended genotyping may improve the identification of patients at risk for severe fluoropyrimidine toxicity. [Extracted from the article]

This article discusses the impact of hemolysis on uracilemia in the context of dihydropyrimidine dehydrogenase (DPD) deficiency testing. DPD deficiency is a known cause of severe toxicities in patients receiving fluoropyrimidine anticancer drugs. The French Health Authorities recommend screening for DPD deficiency by measuring plasma uracil concentration before administering these drugs. The study found that sample hemolysis can significantly affect uracilemia levels, potentially leading to misinterpretation of DPD activity. The authors recommend careful evaluation of sample hemolysis and requesting a new blood sample if uracilemia is above the threshold value in hemolyzed samples. [Extracted from the article]

A letter to the editor of Clinical Chemistry & Laboratory Medicine discusses the limitations and concerns regarding the use of uracil testing for pre-emptive dihydropyrimidine dehydrogenase (DPD) deficiency testing in fluoropyrimidine treatment. The authors argue that the exclusion of certain genetic variants in the genotyping panel used in the study hampers adequate comparison between genotyping and uracil testing. They also highlight the importance of proper identification of DPYD variant carriers and suggest that both genotyping and phenotyping should be used in combination for accurate testing. The authors urge medical societies and regulatory organizations to refrain from recommending fluoropyrimidine dosing strategies based on uracil levels alone. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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%). [ABSTRACT FROM AUTHOR]

Keywords: dihydropyrimidine dehydrogenase (DPD) deficiency; dihydrouracil; pre-analytical; uracil EN dihydropyrimidine dehydrogenase (DPD) deficiency dihydrouracil pre-analytical uracil e112 e115 4 04/05/22 20220401 NES 220401 To the Editor, Fluoropyrimidines (5-fluorouracil and its prodrug capecitabine) are chemotherapeutics frequently used to treat solid tumors. In the fasting and non-fasting group, respectively, the CIs were (4.1-13.4) vs. (3.8-15.1) for uracil, (43.9-103.2) vs. (31.8-75.5) for UH SB 2 sb , and (3.2-13.8) vs. (2.8-8.3) for the UH SB 2 sb /uracil ratio. Therefore, in this study, uracil and UH SB 2 sb concentrations, as well as UH SB 2 sb /uracil ratios were compared between fasting and non-fasting individuals. Boxplots representing data distribution for uracil (A), dihydrouracil or UH2 (B), and UH2/uracil ratios (C) in both fasting healthy volunteers sampled between 8 and 10 a.m. (n=96) and non-fasting healthy volunteers sampled randomly throughout the day (n=42). [Extracted from the article]

Objective　To establish an UHPLC-MS/MS method for the determination of uracil (U) and dihydrouracil (UH2) in human plasma. Methods　A positive ion detection mode was adopted on the Agilent 6460A mass spectrometer. Chlorouracil was used as the internal standard. 3% bovine serum albumin was used as surrogate plasma matrix. The pretreatment of plasma sample was completed based on liquid-liquid extraction with ethyl acetate. The chromatographic separation was achieved on an Agilent Poroshell 120 SB-Aq (2.1 mm×100 mm, 2.7 μm) column with gradient elution. The mobile phase was 5 mmol/L ammonium acetate aqueous solution and acetonitrile solution. The flow rate was 0.3 ml/min. The column temperature was 30°C. The injection volume was 5 μl. Results　The linear range of uracil and dihydrouracil was 10.0-1 500.0 ng/ml. Both of uracil and dihydrouracil had good linear relationship with correlation coefficient (r)>0.990. Both of inter- and intra-day precision was <15%. Conclusion　The established method is simple, selective, and suitable for the determination of U and UH2 in human plasma. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: To establish an analytical method for simultaneous determination of the concentrations of fluorouracil, uracil and dihydrouracil in human plasma, and to apply it clinically. Methods: The LC-MS/MS method was established with bromouracil as the internal standard. Chromatographic separation was performed on Waters Atlantis T 3 column (100 mm x 3.0 mm, 3 µm). 0.1% formic acid aqueous solution (A) and methanol (B) were used as the mobile phase by gradient elution (0-2 min, 5% B, 2.0-2.1 min, 5% -40% B, 2.1-5 min 40% B, 5.0-5.1 min, 40%-5% B, 5.1-7.0 min, 5% B), and flow rate was 0.35 ml /min. Electrospray ion source and multiple reaction monitoring mode (MRM) were used for MS analysis. The detected ion pairs were m/z 129→42 (fluorouracil), m/z 113→70 (uracil),m/z 115→55 (dihydrouracil) and m/z 189→42 (bromouracil). Ethyl acetate isopropanol (85 : 15, V/V) was used as the liquid-liquid extraction solvent. The established method was used for therapeutic drug monitoring of the patients with gastrointestinal cancer. Results: Good linearity was shown when fluorouracil and dihydrouracil were within the range of 10-1000 ng /ml and when uracil was within the range of 5-500 ng /ml. The intra-day and inter-day accuracy were in the range of 88.3%-111. 9%, and relative standad deviation (RSD) were less than 8%. The matrix effect was in the range of 100%-106.9%. The steady state blood concentration of fluorouracil in the 9 patients was 174.66-634.95 ng /ml, and the ratio of plasma concentration of dihydrouracil to uracil after administration was significantly lower than that prior to administration. Conclusion: The established method had high specificity and high sensitivity, and could quickly and accurately determine the concentrations of fluorouracil, uracil and dihydrouracil in human plasma. For this reason, it could meet the requirements of drug concentration monitoring and help to provide good evidence for the optimization of clinical chemotherapy regimens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (r s  = 0.960; r s  = 0.828; r s  = 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, r s  = −0.373, r s  = 0.377) and toxicity ( r  = −0.363, r s  = −0.523, r s  = 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 tomeasure plasma concentrations of uracil and dihydrouracil in colorectal cancer patients scheduled to receive fluoropyrimidinebased chemotherapy. [ABSTRACT FROM AUTHOR]

Objective The aim of this study was to evaluate the use of plasma and saliva uracil (U) to dihydrouracil (UH 2 ) metabolic ratio and DPYD genotyping, as a means to identify patients with dihydropyrimidine dehydrogenase (DPD) deficiency and fluoropyrimidine toxicity. Methods Paired plasma and saliva samples were obtained from 60 patients with gastrointestinal cancer, before fluoropyrimidine treatment. U and UH 2 concentrations were measured by LC–MS/MS. DPYD was genotyped for alleles * 7 , * 2A , * 13 and Y186C . Data on toxicity included grade 1 to 4 neutropenia, mucositis, diarrhea, nausea/vomiting and cutaneous rash. Results 35% of the patients had severe toxicity. There was no variant allele carrier for DPYD . The [UH 2 ]/[U] metabolic ratios were 0.09–26.73 in plasma and 0.08–24.0 in saliva, with higher correlation with toxicity grade in saliva compared to plasma (r s = − 0.515 vs r s = − 0.282). Median metabolic ratios were lower in patients with severe toxicity as compared to those with absence of toxicity (0.59 vs 2.83 saliva; 1.62 vs 6.75 plasma, P < 0.01). A cut-off of 1.16 for salivary ratio was set (AUC 0.842), with 86% sensitivity and 77% specificity for the identification of patients with severe toxicity. Similarly, a plasma cut-off of 4.0 (AUC 0.746), revealed a 71% sensitivity and 76% specificity. Conclusions DPYD genotyping for alleles 7 , * 2A , * 13 and Y186C was not helpful in the identification of patients with severe DPD deficiency in this series of patients. The [UH 2 ]/[U] metabolic ratios, however, proved to be a promising functional test to identify the majority of cases of severe DPD activity, with saliva performing better than plasma. [ABSTRACT FROM AUTHOR]

Quantification of the endogenous dihydropyrimidine dehydrogenase (DPD) substrate uracil (U) and the reaction product dihydrouracil (UH 2 ) in plasma might be suitable for identification of patients at risk of fluoropyrimidine-induced toxicity as a result of DPD deficiency. In this paper, we describe the development and validation of a rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) assay for quantification of U and UH 2 in human plasma. Analytes were extracted by protein precipitation, chromatographically separated on an Acquity UPLC ® HSS T3 column with gradient elution and analyzed with a tandem mass spectrometer equipped with an electrospray ionization source. U was quantified in the negative ion mode and UH 2 in the positive ion mode. Stable isotopes for U and UH 2 were used as internal standards. Total chromatographic run time was 5 min. Validated concentration ranges for U and UH 2 were from 1 to 100 ng/mL and 10 to 1000 ng/mL, respectively. Inter-assay bias and inter-assay precision for U were within ±2.8% and ≤12.4%. For UH 2 , inter-assay bias and inter-assay precision were within ±2.9% and ≤7.2%. Adequate stability of U and UH 2 in dry extract, final extract, stock solution and plasma was demonstrated. Stability of U and UH 2 in whole blood was only satisfactory when stored up to 4 hours at 2–8 °C, but not at ambient temperatures. An accurate, precise and sensitive UPLC-MS/MS assay for quantification of U and UH 2 in plasma was developed. This assay is now applied to support clinical studies with fluoropyrimidine drugs. [ABSTRACT FROM AUTHOR]

A novel substrate, 6-(4-nitrophenyl)dihydropyrimidine-2,4(1 H,3 H)-dione ( pNOPheDU), was chemically synthesized and analytically verified for the potential biocatalytic synthesis of enantiopure β-amino acids. The hydantoinase (EC 3.5.2.2) from Arthrobacter crystallopoietes DSM20117 was chosen to prove the enzymatic hydrolysis of this substrate, since previous investigations showed activities of this enzyme toward 6-monosubstituted dihydrouracils. Whole cell biotransformations with recombinant Escherichia coli expressing the hydantoinase showed degradation of pNOPheDU. Additionally, the corresponding N-carbamoyl- β-amino acid ( NCarb pNO βPhe) was chemically synthesized, an HPLC-method with chiral stationary phases for detection of this product was established and thus (S)-enantioselectivity toward pNOPheDU has been shown. Consequently this novel substrate is a potential precursor for the enantiopure β-amino acid para-nitro- β-phenylalanine ( pNO βPhe). [ABSTRACT FROM AUTHOR]

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. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The synthesis of hydantoins and dihydrouracils from ureidoacetamides has been carried out at high temperature in glycol solvents. A series of substrates were prepared and examined to determine the effect of substrate structure, N -acyl substitution (X), and solvent on the course of the reaction. A dramatic effect was observed when using ureidoacetamides (e.g., X= N -methyl- N -phenyl), which led to higher yields, faster reaction times, and lower racemization of chiral substrates. The rate of racemization of a chiral hydantoin in the presence of dibenzylamine and N -methyl aniline has also been determined. The thermal cyclization methodology has been applied to the preparation of a complex hydantoin. [ABSTRACT FROM AUTHOR]

Purpose: 5-fluorouracil (5-FU) competes with uracil (Ura) as a substrate for dihydropyrimidine dehydrogenase (DPD). Low DPD activity impairs breakdown of Ura to dihydrouracil (UH) and is associated with toxicity during 5-FU-based chemotherapy. Calculation of the 5-FU dose is based on body surface area, and new tools are needed to individualize treatment. The aim of study was to measure Ura and UH in saliva of patients with colorectal cancer and relate levels to treatment-induced toxicity. Methods: Saliva was collected from 73 patients with stage III colorectal cancer prior to adjuvant 5-FU-based treatment. Ura and UH were analyzed by a column-switching HPLC method. Toxicity was evaluated before each treatment cycle and the highest grade was noted at end of treatment. Results: Toxicity was more common and severe among women compared with men. The Ura and UH concentrations in saliva were 5.0 ± 6.8 and 5.0 ± 4.0 nmol/ml, respectively. The UH/Ura ratio was lower in women compared with men (1.2 ± 1.0 and 2.2 ± 2.5, respectively, p = 0.0026). Patients who needed to reduce the drug dose during treatment (or terminate treatment) due to toxicity had a lower ratio (1.3 ± 0.85) compared to patients who completed treatment without dose reduction (4.1 ± 4.3, p < 0.0001). Conclusion: Sampling of saliva is a quick, noninvasive, safe and painless process that gives information about patients Ura and UH levels prior to chemotherapeutical treatment. This information may be useful in order to predict and prevent occurrence of treatment-related toxicities which otherwise may limit drug administration. [ABSTRACT FROM AUTHOR]

Aim: Decreased DPD activity is a major cause of 5-fluorouracil (5-FU) toxicity, but known reduced-function variants in the DPD gene ( DPYD) explain only a part of DPD-related 5-FU toxicities. Here, we evaluated the baseline (pretherapeutic) plasma 5,6-dihydrouracil:uracil (UH2:U) ratio as a marker of DPD activity in the context of DPYD genotypes. Materials & methods: DPYD variants were genotyped and plasma U, UH2 and 5-FU concentrations were determined by liquid chromatography-tandem mass spectrometry in 320 healthy blood donors and 28 cancer patients receiving 5-FU-based chemotherapy. Results: Baseline UH2:U ratios were strongly correlated with generally low and highly variable U concentrations. Reduced-function DPYD variants were only weakly associated with lower baseline UH2:U ratios. However, the interindividual variability in the UH2:U ratio was reduced and a stronger correlation between ratios and 5-FU exposure was observed in cancer patients during 5-FU administration. Conclusion: These results suggest that the baseline UH2:U plasma ratio in most individuals reflects the nonsaturated state of DPD and is not predictive of decreased DPD activity. It may, however, be highly predictive at increased substrate concentrations, as observed during 5-FU administration. Original submitted 9 May 2014; Revision submitted 20 August 2014 [ABSTRACT FROM AUTHOR]

Highlights: [•] The DNA adduct 7-(2′-carboxyethyl)guanine is present in human liver. [•] Nitrosation of dihydrouracil in vivo leads to elevated 7-CEGua in rat hepatic DNA. [•] The hepatocarcinogen N-nitrosodihydrouracil can be formed in vivo in rats. [•] The normal human pyrimidine metabolite dihydrouracil may be a carcinogen precursor. [•] Mass spectrometry detects 7-carboxyethylguanine in human leukocyte DNA. [Copyright &y& Elsevier]

Abstract: The N,N′-disubstituted urea derivatives such as amino acid hydantoins and dihydrouracil derivatives were prepared starting from natural and unnatural amino acid esters using dibutylphosphate (DBP). During the attempted synthesis of N-heterocycles with larger than six-membered rings containing the N,N′-disubstituted urea functionalities, three unexpected products namely squamolone, N-methyl pyrrolidine-2-one, and diketopiperazine were isolated. [Copyright &y& Elsevier]

A new strategy for the synthesis of 1,3-disubstituted uracils has been developed. The 1,3-disubstituted uracils were synthesized with good yields by amino-selenenylation of α,β-unsaturated esters and cyclization with isocyanates, followed by oxidation-elimination of the phenylseleno group. [ABSTRACT FROM AUTHOR]

Abstract: We examined the metabolic fate of [2-14C]uridine, [2-14C]cytidine, [2-14C]uracil and [2-14C]dihydrouracil in leaf disks of Bruguiera sexangula in 0mM or 250mM NaCl. Uridine was readily converted to uracil nucleotides, and cytidine was converted to cytosine nucleotides and also uracil nucleotides, possibly after conversion to uridine. These nucleotides were utilised for synthesis of RNA and UDP-glucose 3h after administration of labelled precursors. Degradation of uridine and cytidine was extremely limited. In contrast, salvage of [2-14C]uracil was low, and up to 45% of uracil was degraded to CO2. [2-14C]Dihydrouracil, which is a possible intermediate in the reductive pathway of pyrimidine catabolism, was converted to β-ureidopropionic acid and CO2. No inhibitory effect of 250mM NaCl was apparent on pyrimidine metabolism of B. sexangula, although RNA synthesis was reduced slightly. The metabolic fate of [2-14C]uridine was surveyed in seven other mangrove species, Bruguiera gymnorrhiza, Kandelia candel, Rhizophora stylosa, Sonneratia alba, Avicennia marina, Lumnitzera racemosa and Pemphis acidula. The general profile of [2-14C]uridine in these mangrove leaves was similar to that in B. sexangula; almost all uridine was salvaged to nucleotides, UDP-glucose and RNA, and catabolism was very low. No or very little inhibitory effect of 250mM NaCl on uridine salvage was found in leaf disks of mangrove species, except L. racemosa. A stimulatory effect of 250mM NaCl on uridine salvage was found in P. acidula. In contrast, uptake and metabolism of [2-14C]uridine, especially a salvage pathway, in the leaves of the non-halophyte plant Populus alba, were drastically impaired by 250mM NaCl. [Copyright &y& Elsevier]

Abstract: Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and it catalyses the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine to N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid, respectively. To date, only nine individuals have been reported suffering from a complete DHP deficiency. We report two siblings presenting with strongly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in plasma, cerebrospinal fluid and urine. One of the siblings had a severe delay in speech development and white matter abnormalities, whereas the other one was free of symptoms. Analysis of the DHP gene (DPYS) showed that both patients were compound heterozygous for the missense mutation 1078T>C (W360R) in exon 6 and a novel missense mutation 1235G>T (R412M) in exon 7. Heterologous expression of the mutant enzymes in Escherichia coli showed that both missense mutations resulted in a mutant DHP enzyme without residual activity. Analysis of the crystal structure of eukaryotic DHP from the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum suggests that the W360R and R412M mutations lead to structural instability of the enzyme which could potentially impair the assembly of the tetramer. [Copyright &y& Elsevier]

Background: This study was designed to measure the dihydrouracil (UH2)/uracil (U) ratio in plasma as a surrogate marker for dihydropyrimidine dehydrogenase (DPD) activity and to investigate the relationships of the UH2/U ratios in plasma with the toxicities of 5-fluorouracil (5-FU)-based adjuvant chemotherapy and 5-FU plasma concentrations in colorectal cancer patients. Methods: Thirty colorectal cancer patients received adjuvant chemotherapy of leucovorin plus 5-FU after operations. The concentrations of UH2, U and 5-FU were assayed by the high-performance liquid chromatography method. The relationships of the UH2/U ratios with the 5-FU toxicities and 5-FU plasma concentrations were analyzed.Results: There was a negative relationship between the UH2/U ratios and 5-FU plasma concentrations (p <0.001). 5-FU toxicities had a negative correlation with the UH2/U ratios and a positive correlation with 5-FU plasma concentrations (p < 0.05). Conclusion: The UH2/U ratio in plasma has close correlations with the 5-FU plasma concentration and 5-FU toxicity during chemotherapy, which may highlight the theoretical base of individual therapy for patients with colorectal cancer. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]