Partial protein binding of uracil and thymine affects accurate dihydropyrimidine dehydrogenase (DPD) phenotyping.

Fluorouracil is among the most used antimetabolite drugs for the chemotherapeutic treatment of various types of gastrointestinal malignancies. Dihydropyrimidine dehydrogenase (DPYD) genotyping prior to fluorouracil treatment is considered standard practice in most European countries. Yet, current pre-therapeutic DPYD genotyping procedures do not identify all dihydropyrimidine dehydrogenase (DPD)-deficient patients. Alternatively, DPD activity can be estimated by determining the DPD phenotype by quantification of plasma concentrations of the endogenous uracil and thymine concentrations and their respective metabolites dihydrouracil (DHU) and dihydrothymine (DHT). Liquid chromatography - mass spectrometry (LC-MS) detection is currently considered as the most adequate method for quantification of low-molecular weight molecules, although the sample preparation method is highly critical for analytical outcome. It was hypothesized that during protein precipitation, the recovery of the molecule of interest highly depends on the choice of precipitation agent and the extent of protein binding in plasma. In this work, the effect of protein precipitation using acetonitrile (ACN) compared to strong acid perchloric acid (PCA) on the recovery of uracil, thymine, DHU and DHT is demonstrated. Upon the analysis of plasma samples, PCA precipitation showed higher concentrations of uracil and thymine as compared to ACN precipitation. Using ultrafiltration, it was shown that uracil and thymine are significantly (60-65 %) bound to proteins compared to DHU and DHT. This shows that before harmonized cut-off levels of DPD phenotyping can be applied in clinical practice, the analytical methodology requires extensive further optimization.

A Simple and Rapid UPLC-UV Method for Detecting DPD Deficiency in Patients With Cancer.

Detecting patients with dihydropyrimidine dehydrogenase (DPD) deficiency is becoming a major concern in clinical oncology. Monitoring physiologic plasma uracil and/or plasma uracil-to-dihydrouracil metabolic ratio is a common surrogate frequently used to determine DPD phenotype without direct measurement of the enzymatic activity. With respect to the increasing number of patients rquiring analysis, it is critical to develop simple, rapid, and affordable methods suitable for routine screening. We have developed and validated a simple and robust ultraperformance liquid chromatography-ultraviolet (UPLC-UV) method with shortened (i.e., 12 minutes) analytical run-times, compatible with the requirements of large-scale upfront screening. The method enables detection of uracil (U) over a range of 5-500 ng/ml (265 nm) and of dihydrouracil (UH2) over a range of 40-500 ng/ml (210 nm) in plasma with no chromatographic interference. When used as part of routine screening for DPD deficiency, this method was fully able to discriminate nondeficient patients (i.e., with U levels < 16 ng/ml) from deficient patients at risk of severe toxicity (i.e., U > 16 ng/ml). Results from 1 month of routine testing are presented and, although no complete deficits were detected, 10.7% of the screened patients presented DPD deficiency and would thus require s decresed dose. Overall, this new method, using a simple preanalytical solid-phase extraction procedure, and based on use of a standard UPLC apparatus, is both cost- and time-effective and can be easily implemented in any laboratory aiming to begin routine DPD testing.

Lethal toxicities after capecitabine intake in a previously 5-FU-treated patient: why dose matters with dihydropryimidine dehydrogenase deficiency.

Dihydropryimidine dehydrogenase (DPD) deficiency is a pharmacogenetic syndrome associated with severe or lethal toxicities with oral capecitabine. Usually, patients with history of 5-FU-based therapy with no signs for life-threatening toxicities are considered as not DPD-deficient individuals who can be safely treated next with capecitabine if required. Here we describe the case of a woman originally treated with standard FEC100 protocol for metastatic breast cancer with little severe toxicities but grade-3 mucosities that were quickly resolved by symptomatic treatment. When switched to capecitabine + vinorelbine combo, extremely severe toxicities with fatal outcome were unexpectedly observed. Pharmacogenetic investigations were performed on cytidine deaminase and DPYD, and showed that this patient was heterozygous for the 2846A>T mutation on the DPYD gene. DPD phenotyping (i.e., uracil plasma levels >250 ng/ml, dihydrouracil/uracil ratio <0.5) confirmed that this patient was profoundly DPD deficient. Differences in fluoropyrimidine dosing between FEC100 (i.e., 500 mg/m2 5-FU) and capecitabine (i.e., 2250 mg daily) could explain why initial 5-FU-based protocol did not lead to life-threatening toxicities, whereas capecitabine rapidly triggered toxic death. Overall, this case report suggests that any toxicity, even when not life threatening, should be considered as a warning signal for possible underlying profound DPD deficiency syndrome, especially with low-dose protocols.

Prompt treatment with uridine triacetate improves survival and reduces toxicity due to fluorouracil and capecitabine overdose or dihydropyrimidine dehydrogenase deficiency.

Uridine triacetate has been shown to be an effective antidote against mortality and toxicity caused by either overdoses or exaggerated susceptibility to the widely used anticancer agents 5-fluorouracil (5-FU) and capecitabine. However, a direct assessment of efficacy based on when emergency treatment was initiated was not clinically feasible. In this study we used mouse models of 5-FU overdose and of dihydropyrimidine dehydrogenase (DPD) deficiency to compare the efficacy of uridine triacetate in reducing toxicity and mortality when treatment was initiated at time points from 4 to 144 h after administration of 5-FU. We found that uridine triacetate was effective both in the 5-FU overdose and DPD deficiency models. Starting treatment within 24 h was most effective at reducing toxicity and mortality in both models, while treatment starting more than 96 to 120 h after 5-FU was far less effective. Uridine triacetate also reduced mortality in the DPD deficiency model when mice were treated with the 5-FU prodrug capecitabine. The results of this study are supportive of clinical observations and practice, indicating that efficacy declined progressively with later and later treatment initiation. Prompt treatment with uridine triacetate, within 24 h, conferred the greatest protection against 5-FU overexposure.

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.

Beating the odds: efficacy and toxicity of dihydropyrimidine dehydrogenase-driven adaptive dosing of 5-FU in patients with digestive cancer.

AIMS: 5-FU is the backbone of most regimens in digestive oncology. Administration of standard 5-FU leads to 15-30% of severe side effects, and lethal toxicities are regularly reported with fluoropyrimidine drugs. Dihydropyrimidine dehydrogenase (DPD) deficiency is a pharmacogenetic syndrome responsible for most cases of life-threatening toxicities upon 5-FU intake, and pre-treatment checking for DPD status should help to reduce both incidence and severity of side effects through adaptive dosing strategies. METHODS: We have used a simple method for rapidly establishing the DPD phenotype of patients with cancer and used it prospectively in 59 routine patients treated with 5-FU-based therapy for digestive cancers. No patient with total DPD deficiency was found but 23% of patients exhibited poor metabolizer phenotype, and one patient was phenotyped as profoundly deficient. Consequently, 5-FU doses in poor metabolizer patients were cut by an average 35% as compared with non deficient patients (2390 ± 1225 mg vs. 3653 ± 1371 mg, P < 0.003, t-test). RESULTS: Despite this marked reduction in 5-FU dosing, similar efficacy was achieved in the two subsets (clinical benefit: 40 vs. 43%, stable disease: 40 vs. 37%, progressive disease: 20% in both subsets, P = 0.893, Pearson's chi-square). No difference in toxicities was observed (P = 0.104, Fisher's exact test). Overall, only 3% of early severe toxicities were recorded, a value markedly lower than the 15-30% ones usually reported with 5-FU. CONCLUSIONS: This feasibility study shows how simplified DPD-based adaptive dosing of 5-FU can reduce sharply the incidence of treatment-related severe toxicities while maintaining efficacy as part of routine clinical practice in digestive oncology.

Translating DPYD genotype into DPD phenotype: using the DPYD gene activity score.

The dihydropyrimidine dehydrogenase enzyme (DPD, encoded by the gene DPYD) plays a key role in the metabolism of fluoropyrimidines. DPD deficiency occurs in 4-5% of the population and is associated with severe fluoropyrimidine-related toxicity. Several SNPs in DPYD have been described that lead to absent or reduced enzyme activity, including DPYD*2A, DPYD*13, c.2846A>T and c.1236G>A/haplotype B3. Since these SNPs differ in their effect on DPD enzyme activity, a differentiated dose adaption is recommended. We propose the gene activity score for translating DPYD genotype into phenotype, accounting for differences in functionality of SNPs. This method can be used to standardize individualized fluoropyrimidine dose adjustments, resulting in optimal safety and effectiveness.

microRNAs miR-27a and miR-27b directly regulate liver dihydropyrimidine dehydrogenase expression through two conserved binding sites.

Dihydropyrimidine dehydrogenase (DPD, encoded by DPYD) is the rate-limiting enzyme in the uracil catabolic pathway and has a pivotal role in the pharmacokinetics of the commonly prescribed anticancer drug 5-fluorouracil (5-FU). Deficiency of DPD, whether due to inadequate expression or deleterious variants in DPYD, has been linked to severe toxic responses to 5-FU. Little is known about the mechanisms governing DPD expression in the liver. In this report, we show increased accumulation of RNA-induced silencing complex (RISC) proteins on DPYD mRNA in cells overexpressing the highly homologous microRNAs (miRNA) miR-27a and miR-27b. These miRNAs were shown to repress DPD expression through two conserved recognition sites in DPYD. The IC50 of 5-FU for HCT116 cells overexpressing miR-27a or miR-27b was 4.4 µmol/L (both), significantly lower than that for cells expressing a nontargeting (scramble) control miRNA (14.3 µmol/L; P = 3.3 × 10(-5) and P = 1.5 × 10(-7), respectively). Mouse liver DPD enzyme activity was inversely correlated with expression levels of miR-27a (R(2) = 0.49; P = 0.0012) and miR-27b (R(2) = 0.29; P = 0.022). A common variant in the hairpin loop region of hsa-mir-27a (rs895819) was also shown to be associated with elevated expression of the miR-27a in a panel of cell lines (P = 0.029) and in a transgenic overexpression model (P = 0.0011). Furthermore, rs895819 was associated with reduced DPD enzyme activity (P = 0.028) in a cohort of 40 healthy volunteers. Taken together, these results suggest that miR-27a and miR-27b expression may be pharmacologically relevant modulators of DPD enzyme function in the liver. Furthermore, our data suggest that rs895819 may be a potential risk allele for 5-FU sensitivity.

Evaluation of 5-fluorouracil pharmacokinetic models and therapeutic drug monitoring in cancer patients.

5-fluorouracil (5-FU) remains the cornerstone of all currently applied regimens for the treatment of patients with cancers of the gastrointestinal tract, breast, and head and neck. Unfortunately, a large variation in the clearance of 5-FU has been observed between patients, suggesting that some patients might receive nonoptimal 5-FU doses. However, therapeutic drug monitoring of 5-FU has been shown to result in reduced intra- and inter-individual variability in 5-FU plasma levels and pharmacokinetically guided dose adjustments of 5-FU-containing therapy results in a significantly improved efficacy and tolerability. To date, compartmental Michaelis-Menten elimination-based modeling has proven to be a sensitive and accurate tool for analyzing the pharmacokinetics of 5-FU and to identify patients with a dihydropyrimidine dehydrogenase deficiency. These Michaelis-Menten models also allow the use of a limited sampling strategy and offer the opportunity to predict a priori the 5-FU plasma concentrations in patients receiving adapted doses of 5-FU.

Pharmacogenomic biomarkers in dermatologic drugs.

Biomarkers are becoming increasingly important when considering the efficacy, toxicology, mechanism of action, and risk of adverse events in certain drugs. As availability of bio-genomic information increases, more treatments can be tailored to specific individuals, with a net effect of improved health outcomes. Many dermatology drugs have pharmacogenomic information on their labels. Knowing the risks and benefits associated with genomic biomarkers can aid physicians to make more knowledgeable decisions when identifying treatments for their patients.

Pharmacokinetics of orally administered uracil in healthy volunteers and in DPD-deficient patients, a possible tool for screening of DPD deficiency.

PURPOSE: Dihydropyrimidine dehydrogenase (DPD) deficiency can lead to severe toxicity in patients treated with standard doses of 5-fluorouracil (5-FU). Oral uracil administration and subsequent measurement of uracil and dihydrouracil (DHU) plasma concentrations might detect patients with DPD deficiency. This study compares the pharmacokinetics of uracil and DHU after oral uracil administration in subjects with normal and deficient DPD status. METHODS: Five hundred milligrams of uracil per metre square was administered orally to 11 subjects with normal DPD status and to 10 subjects with reduced DPD activity. Repeated administration (n = 3) of this dose was performed in 4 subjects, and 1,000 mg uracil/m(2) was administered to 4 subjects to assess intra-individual variation and linearity of pharmacokinetics. RESULTS: In subjects with normal DPD status, 500 mg/m(2) uracil resulted in uracil C (max) levels of 14.4 ± 4.7 mg/L at T (max) = 30.0 ± 11.6 min, and in DPD-deficient subjects, 20.0 ± 4.5 mg/L at 31.5 ± 1.1 min. The uracil AUC(0>180) was 31.2 ± 5.1 mg L/h in DPD-deficient subjects, which was significantly higher (P < 0.05) than in the subjects with normal DPD status (13.8 ± 3.9 mg L/h). Repeated uracil dosing showed reproducible uracil PK in subjects with normal DPD status, and dose elevation of uracil suggested linear pharmacokinetics. CONCLUSION: The pharmacokinetics of uracil differs significantly between subjects with a normal DPD activity and those with a deficient DPD status. The AUC and C (max) of uracil can be useful as a diagnostic tool to differentiate patients with regard to DPD status.

Children's toxicology from bench to bed--Liver injury (1): Drug-induced metabolic disturbance--toxicity of 5-FU for pyrimidine metabolic disorders and pivalic acid for carnitine metabolism.

Congenital disorders of metabolism show a wide spectrum of symptoms as a consequence of impairment of a certain metabolic pathway by mutated enzymes resulting in abnormal accumulation of enzyme substrates, deficiency of expected products, and abnormal burden to collateral metabolic pathways, etc. However, in some occasions, depending on which pathway up to what degree of disturbance, it can be asymptomatic until a certain kind of burden is placed on to the patient. Enzyme deficiency involved in pyrimidine degradation, such as Dihydropyrimidine dehydrogenase (DPD) and Dihydropyrimidinase (DHP), has been reported with convulsion or autism as symptoms, but many asymptomatic cases are also reported. However, when the patients are treated with 5-fluorouracil, a pyrimidine analogue anticancer drug, lethal side-effects can be seen even in asymptomatic patients. Some oral cephem antibiotics have pivalic acid side chain to increase absorption rate at intestine. These antibiotics degrade into active antibiotics and pivalic acid at the intestinal wall. This pivalic acid is carnitine-conjugated and excreted into urine. Carnitine acts as a carrier of long chain fatty acid to mitochondria and to beta-oxidation, thus an important molecule for energy production by beta-oxidation and maintenance of mitochondrial function. Because of this, long term administration of such antibiotics could induce depletion of carnitine from the body and lead to low ketotic hypoglycemia, convulsion and consciousness disturbance. This paper reports some possible serious side effects closely linked to drug metabolism.

Adjuvant therapy with raltitrexed in patients with colorectal cancer intolerant of 5-fluorouracil: British Columbia Cancer Agency experience.

BACKGROUND: Severe 5-FU toxicity in adjuvant therapy of colorectal cancer may require change of therapy. We retrospectively explored the safety and efficacy of adjuvant raltitrexed in patients intolerant of 5-FU. METHODS: Over a 5 year period, patients who received 5-FU and subsequent raltitrexed therapy were identified. RESULTS: There were 44 patients, (39 stage III). Median number of prior 5-FU cycles was 2. Three year relapse free and overall survival proportions for stage III patients were 70.8% and 83.6%, respectively. CONCLUSIONS: Raltitrexed adjuvant therapy can be given safely and effectively in patients where further 5-FU is contraindicated.