Uridine triacetate for prevention of 5FU toxicity due to dihydropyrimidine dehydrogenase (DPD) deficiency.

To the editor, With interest we read the article by Dr Cubero and colleagues, in which they evaluated the safety of tegafur-uracil (UFT®) in five cases with partial dihydropyrimidine dehydrogenase (DPD) deficiency [Cubero et al. 2012]. Based on our previous experience [Deenen et al. 2010], however, we would like to express our concern about their conclusion that UFT is a safe alternative for the treatment of patients with partial DPD deficiency. Cubero and colleagues make the erroneous and unproven statement that the presence of uracil in UFT creates an artificial DPD deficiency, and that the DPD activity in patients with normal DPD activity would then be similarly as low as in DPD-deficient patients. This assumption, however, is incorrect. As uracil is a competitive inhibitor of DPD, it competes with 5-fluorouracil (5-FU) for DPD-mediated metabolism. This does not mean that the activity of DPD is depleted, as suggested by Cubero and colleagues, in contrast, its activity is fully utilized, as well as for the metabolism of uracil, as for the metabolism of 5-FU. We would like to caution that treating patients with partial DPD deficiency with the standard dose of UFT may unnecessarily lead to severe, potentially lethal toxicity. Unlike the cases described by Cubero and colleagues, we could previously describe four cases presenting with comparable severe toxicity profiles upon treatment with UFT as had previously occurred during treatment with capecitabine or 5-FU. In all subjects an underlying partial DPD deficiency was identified by genotype and phenotype analyses [Deenen et al. 2010]. Furthermore, there are several pharmacological lines of argument that support our clinical observation, i.e. that the standard dose of UFT is not safe in (partial) DPD-deficient patients. First, pharmacokinetic studies have shown that DPD remains essential for the metabolism of UFT, with significantly longer half-lives of 5-FU after administration of UFT compared with 5-FU administered intravenously [Ho et al. 1998]. This is due to the presence of uracil in UFT. Since DPD-deficient patients already have longer half-lives of 5-FU than other patients [Mattison et al. 2006], presence of uracil increases its half-life even further. This in turn leads to prolonged and elevated circulating levels of 5-FU, with a subsequently increased risk of 5-FU-induced severe toxicity. Another argument underscoring the importance of normal DPD function in the safe application of UFT, is the experience with S-1. S-1 is another drug combination of tegafur, consisting of tegafur, 5-chloro-2,4-dihydroxypyridine (CDHP) and potassium oxonate in a molar ratio of 1:0.4:1. CDHP inhibits DPD 200-fold more potently than does uracil [Shirasaka et al. 1996a, 1996b]. Even after administration of S-1, the primary 5-FU metabolite formed by DPD is observed in significant concentrations in plasma [Kim et al. 2007]. Thus, DPD remains an essential detoxification enzyme of 5-FU, even when its activity is strongly inhibited. The ultimate proof of theory is the occurrence of 18 treatment-related deaths in patients with cancer and herpes zoster given UFT plus the antiviral drug sorivudine [Pharmaceutical Affairs Bureau, 1994]. Subsequent studies in rats showed that a metabolite of sorivudine, (E)-5-(2-bromovinyl)uracil, instantly and irreversibly inactivates DPD by covalent binding, which has been identified as the underlying mechanism of these toxic deaths [Ogura et al. 1998; Okuda et al. 1998]. It is for these arguments that the Summary of Product Characteristics of UFT notes a known DPD deficiency as a contra-indication [Merck Serono, 2011]. The fact that the patients described by Cubero and colleagues did not develop significant toxicity might be due to patient selection, the slightly decreased dose intensity of 90%, or despite their DPYD*2A genotype a DPD enzyme activity within the (lower) range of normal. We are not aware of this, because DPD enzyme activity was not determined in these patients. In summary, we would like to state that standard-dose UFT is not a safe treatment in (partial) DPD-deficient patients. Instead, dose reductions of on average 50% of either capecitabine, 5-FU or UFT with careful monitoring of safety and further dose titration are proposed as the standard of care [Deenen et al. 2011].

e13505 Background: The enzyme dihydropyrimidine dehydrogenase (DPD) catalyzes the first step in degradation of 5-fluorouracil (5FU). Patients with complete or partial DPD deficiency (about 0.1% and 3% of the population, respectively) can experience severe or lethal toxicities after receiving standard doses of 5FU, and impaired clearance may underlie a large fraction of the ∼1,300 deaths per year due to 5FU toxicity in the US. Orally-administered uridine triacetate (vistonuridine) prevents or diminishes toxicities when administered after 5FU overexposure, and has been used successfully as an antidote in more than 28 patients to date who had received accidental overdoses of 5FU. Because DPD deficiency may alter 5FU clearance kinetics (versus 5FU overdoses in the setting of normal DPD activity), studies were conducted on reversal of 5FU toxicity in mice pretreated with the potent DPD inhibitor ethynyluracil (eniluracil; EU) to model DPD deficiency. Methods: Balb/C mice received 2 mg/kg EU i.p., followed by 100 mg/kg 5FU (weekly bolus MTD in mice). Groups of 10 mice each then received either vehicle or oral uridine triacetate (2,000 mg/kg t.id. × 5) beginning at a range of times after 5FU. Survival was monitored. Results: 100 mg/kg 5FU was lethal in mice pretreated with EU. Uridine triacetate administration beginning within 24 hours after EU + 5FU resulted in survival of all the mice in the treatment groups. Fewer mice survived if treated with uridine triacetate at later time points after EU plus 5FU. Conclusions: Timely treatment with uridine triacetate reduced 5FU toxicity and mortality in DPD-inhibited mice. Its benefit has previously been demonstrated in patients (and mice) overdosed with 5FU. Therefore, DPD-deficient patients who have received 5FU should also benefit from treatment with uridine triacetate if the deficiency is identified soon enough after 5FU dosing. Therapeutic monitoring of 5FU during or after infusions could permit rapid detection of 5FU overexposure due to DPD-deficiency, enabling the use of uridine triacetate as an antidote in DPD-deficient patients. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Wellstat Therapeutics Corporation

e20592 Background: 5-Fluorouracil (5-FU) is broadly used to treat solid tumors. It is typically administered by IV infusion, at or near its maximum tolerated dose, over several days. The use of infusion pumps increases the possibility of life-threatening or lethal toxicity due to errors in pump programming, infusion reservoir errors and dosage miscalculations. Partial or total dihydropyrimidine dehydrogenase (DPD) deficiency, which predisposes patients (up to 3% of the population) to impaired 5-FU elimination, can also result in serious or lethal toxicity, Uridine triacetate is an orally bioavailable direct biochemical antagonist of 5-FU toxicity that has been used successfully to treat patients in emergency overdose situations as well as patients with known or suspected overexposure to 5-FU due to DPD deficiency or other causes. Approximately 100 patients at excess risk of 5-FU toxicity due to 5-FU overdose, accidental Xeloda (capecitabine) ingestion, or possible DPD deficiency (rapid onset of severe toxicities) have been treated with uridine triacetate using a common treatment regimen and protocol. More than 80 of these cases have occurred since uridine triacetate data were presented at ASCO in 2009. Methods: Uridine triacetate was provided under emergency IND provisions or an expanded access protocol when requested by qualified clinical sites following 5-FU overexposures, most due to infusion pump errors. Patients received uridine triacetate (10g q6h for 20 doses) as soon as possible after recognition of the overdose or possible clearance defect. Clinical outcomes, including safety and resumption of chemotherapy, were monitored. Results: To date, 98 patients overexposed to 5-FU have been treated with uridine triacetate. 96 of these 98 patients recovered fully. Reductions in or absence of GI, hematologic, and other toxicities associated with 5-FU poisoning were observed. Adverse events attributable to urdine triacetate were infrequent and mild in severity. Conclusions: Uridine triacetate appears to be a safe and effective life-saving antidote for 5-FU overexposure in emergency situations.

BackgroundPretherapeutic screening for dihydropyrimidine dehydrogenase (DPD) deficiency is recommended or required prior to the administration of fluoropyrimidine-based chemotherapy. However, the best strategy to identify DPD-deficient patients remains elusive.MethodsAmong a nationwide cohort of 5886 phenotyped patients with cancer who were screened for DPD deficiency over a 3 years period, we assessed the characteristics of both DPD phenotypes and DPYD genotypes in a subgroup of 3680 patients who had completed the two tests. The extent to which defective allelic variants of DPYD predict DPD activity as estimated by the plasma concentrations of uracil [U] and its product dihydrouracil [UH2] was evaluated.ResultsWhen [U] was used to monitor DPD activity, 6.8% of the patients were classified as having DPD deficiency ([U] > 16 ng/ml), while the [UH2]:[U] ratio identified 11.5% of the patients as having DPD deficiency (UH2]:[U] < 10). [U] classified two patients (0.05%) with complete DPD deficiency (> 150 ng/ml), and [UH2]:[U] < 1 identified three patients (0.08%) with a complete DPD deficiency. A defective DPYD variant was present in 4.5% of the patients, and two patients (0.05%) carrying 2 defective variants of DPYD were predicted to have low metabolism. The mutation status of DPYD displayed a very low positive predictive value in identifying individuals with DPD deficiency, although a higher predictive value was observed when [UH2]:[U] was used to measure DPD activity. Whole exon sequencing of the DPYD gene in 111 patients with DPD deficiency and a “wild-type” genotype (based on the four most common variants) identified seven heterozygous carriers of a defective allelic variant.ConclusionsFrequent genetic DPYD variants have low performances in predicting partial DPD deficiency when evaluated by [U] alone, and [UH2]:[U] might better reflect the impact of genetic variants on DPD activity. A clinical trial comparing toxicity rates after dose adjustment according to the results of genotyping or phenotyping testing to detect DPD deficiency will provide critical information on the best strategy to identify DPD deficiency.

Patients with partial or complete DPD deficiency have decreased capacity to degrade fluorouracil and are at risk of developing toxicity, which can be even life-threatening. A 43-year-old man with moderately differentiated rectal adenocarcinoma on capecitabine presented to the emergency department with complaints of nausea, vomiting, diarrhea, weakness, and lower abdominal pain for several days. Laboratory findings include grade 4 neutropenia (ANC 10) and thrombocytopenia (platelets 36,000). Capecitabine is used as a component of first-line adjuvant therapy by approximately 2million patients worldwide each year. Capecitabine is metabolized to fluorouracil via the enzyme dihydropyrimidine dehydrogenase (DPD). With worsening pancytopenia and diarrhea, genetic testing for DPD deficiency was sent. Prompt treatment with uridine triacetate was initiated for presumed DPD deficiency. Unfortunately, he passed away from an infectious complication and was later confirmed to have a heterozygous DPYD*2A mutation. Our case demonstrates uneven testing guidelines for DPD prior to initiating 5-FU chemotherapy, appropriateness of treating with uridine triacetate, and analysis of next-generation sequencing (NGS) on tumor samples and co-incidentally obtaining germline DPD deficiency status. Our case also highlights the severe clinical impact of having DPD deficiency even with early uridine triacetate therapy. It is our recommendation to perform DPD deficiency in curative intent cancer treatment and this information can increasingly be obtained in standard tumor NGS profiling, a growing norm in medical oncology.

Dihydropyrimidine dehydrogenase (DPD) deficiency is prevalent in 3-5% of the Caucasian population; however, the frequency of this pharmacogenetic syndrome in the Indian population and other racial and ethnic groups remains to be elucidated. We describe an Indian patient who presented to clinic for the treatment of gastric adenocarcinoma with 5-flurouracil (5-FU) therapy who subsequently was diagnosed with DPD deficiency by using the peripheral blood mononuclear cell (PBMC) DPD radioassay. This observation prompted us to examine the data generated from healthy (cancer-free) Indian subjects who were enrolled in a large population study to determine the sensitivity and specificity of the uracil breath test (UraBT) in the detection of DPD deficiency. Thirteen Indian subjects performed the UraBT. UraBT results were confirmed by PBMC DPD radioassay. The Indian cancer patient demonstrated reduced DPD activity (0.11 nmol/min/mg protein) and severe 5-FU toxicities commonly associated with DPD deficiency. Of the 13 Indian subjects [ten men and three women; mean age, 26 years (range: 21-31 years)] enrolled in the UraBT, 12 Indian subjects demonstrated UraBT breath profiles and PBMC DPD activity within the normal range; one Indian subject demonstrated a reduced breath profile and partial DPD deficiency. DPD deficiency is a pharmacogenetic syndrome which is also present in the Indian population. If undiagnosed, the DPD deficiency can lead to death. Future epidemiological studies would be helpful to determine the prevalence of DPD deficiency among racial and ethnic groups, allowing for the optimization of 5-FU chemotherapy.

Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme in the catabolism of 5-fluorouracil (5FU) and DPD deficiency is an important pharmacogenetic syndrome. So far, only very limited information is available regarding the pharmacokinetics of 5FU in patients with a (partial) DPD deficiency and no limited sampling models have been developed taking into account the non-linear pharmacokinetic behaviour of 5FU. The aim of this study was to evaluate the pharmacokinetics of 5FU and to develop a limited sampling strategy to detect decreased 5FU elimination in patients with a c.1905+1G>A-related DPD deficiency. Thirty patients, heterozygous for the c.1905+1G>A mutation in DPYD, and 18 control patients received a dose of 5FU 300 mg/m2 and/or 5FU 450 mg/m2, followed by pharmacokinetic analysis of the 5FU plasma levels. A population pharmacokinetic analysis was performed in order to develop a compartmental pharmacokinetic model suitable for a limited sampling strategy. Clinical aspects of treating DPD-deficient patients with 5FU-based chemotherapy were assessed from the retrospectively collected clinical data. In a two-compartment model with Michaelis-Menten elimination, the mean maximum enzymatic conversion capacity (V(max)) value was 40% lower in DPD-deficient patients compared with controls (p < 0.001). Using a limited sampling strategy, with V(max) values calculated from 5FU concentrations at 30 or 60 minutes, significant differences were observed between DPD-deficient patients and controls at both dose levels (p < 0.001). The positive predictive value and negative predictive value for V(max), calculated from 5FU levels at 60 minutes, were 96% and 88%, respectively, in patients treated with a single dose of 5FU 300 mg/m2. All seven DPD-deficient patients (two males and five females) who had been genotyped prior to initiation of standard 5FU-containing chemotherapy developed grade 3-4 toxicity, with one case of lethal toxicity in a female patient. No grade 4 toxicity or lethal outcome was observed in 13 DPD-deficient patients treated with reduced doses of 5FU. The average dose of 5FU in DPD-deficient patients with mild toxicity (grade ≤2) was 61 ± 16% of the normal 5FU dose (n = 10). Profound differences in the elimination of 5FU could be detected between DPD-deficient patients and control patients. Pharmacokinetic 5FU profiling, using a single 5FU concentration at 60 minutes, may be useful for identification of DPD-deficient patients in order to reduce severe toxicity. Furthermore, treatment of DPD-deficient patients with standard 5FU-containing chemotherapy was associated with severe (lethal) toxicity.

Artificial increase of uracilemia during fluoropyrimidine treatment can lead to DPD deficiency misinterpretation

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

Dihydropyrimidine dehydrogenase (DPD) deficiency is critical in the predisposition to 5-fluorouracil dose-related toxicity. We recently characterized the phenotypic [2-(13)C]uracil breath test (UraBT) with 96% specificity and 100% sensitivity for identification of DPD deficiency. In the present study, we characterize the relationships among UraBT-associated breath (13)CO(2) metabolite formation, plasma [2-(13)C]dihydrouracil formation, [2-(13)C]uracil clearance, and DPD activity. An aqueous solution of [2-(13)C]uracil (6 mg/kg) was orally administered to 23 healthy volunteers and 8 cancer patients. Subsequently, breath (13)CO(2) concentrations and plasma [2-(13)C]dihydrouracil and [2-(13)C]uracil concentrations were determined over 180 minutes using IR spectroscopy and liquid chromatography-tandem mass spectrometry, respectively. Pharmacokinetic variables were determined using noncompartmental methods. Peripheral blood mononuclear cell (PBMC) DPD activity was measured using the DPD radioassay. The UraBT identified 19 subjects with normal activity, 11 subjects with partial DPD deficiency, and 1 subject with profound DPD deficiency with PBMC DPD activity within the corresponding previously established ranges. UraBT breath (13)CO(2) DOB(50) significantly correlated with PBMC DPD activity (r(p) = 0.78), plasma [2-(13)C]uracil area under the curve (r(p) = -0.73), [2-(13)C]dihydrouracil appearance rate (r(p) = 0.76), and proportion of [2-(13)C]uracil metabolized to [2-(13)C]dihydrouracil (r(p) = 0.77; all Ps < 0.05). UraBT breath (13)CO(2) pharmacokinetics parallel plasma [2-(13)C]uracil and [2-(13)C]dihydrouracil pharmacokinetics and are an accurate measure of interindividual variation in DPD activity. These pharmacokinetic data further support the future use of the UraBT as a screening test to identify DPD deficiency before 5-fluorouracil-based therapy.

Dihydropyrimidine dehydrogenase (DPD) deficiency (McKusick 274270) is an autosomal recessive disease characterized by thymine-uraciluria in homozygous-deficient patients and associated with a variable clinical phenotype. Cancer patients with this defect should not be treated with the usual dose of 5-fluorouracil because of the expected lethal toxicity. In addition, heterozygosity for mutations in the DPD gene increases the risk of toxicity in cancer patients treated with this drug. Sequence analysis in a patient with complete DPD deficiency, previously shown to be heterozygous for the delta C1897 frame-shift mutation, revealed the presence of a novel missense mutation, R235W. Expression of this novel mutation and previously identified missense mutations C29R and R886H in Escherichia coli showed that both C29R and R235W lead to a mutant DPD protein without significant residual enzymatic activity. The R886H mutation, however, resulted in about 25% residual enzymatic activity and is unlikely to be responsible for the DPD-deficient phenotype. We show that the E. coli expression system is a valuable tool for examining DPD enzymatic variants. In addition, two new patients who were both heterozygous for the C29R mutation and the common splice donor site mutation were identified. Only one of these patients showed convulsive disorders during childhood, whereas the other showed no clinical phenotype, further illustrating the lack of correlation between genotype and phenotype in DPD deficiency.

Lethal 5-fluorouracil toxicity associated with a novel mutation in the dihydropyrimidine dehydrogenase gene

Is Capecitabine Safe in Patients with Gastrointestinal Cancer and Dihydropyrimidine Dehydrogenase Deficiency?

Dihydropyrimidine dehydrogenase (DPD) deficiency, a known pharmacogenetic syndrome associated with 5-fluorouracil (5-FU) toxicity, has been detected in 3% to 5% of the population. Genotypic studies have identified >32 sequence variants in the DPYD gene; however, in a number of cases, sequence variants could not explain the molecular basis of DPD deficiency. Recent studies in cell lines indicate that hypermethylation of the DPYD promoter might down-regulate DPD expression. The current study investigates the role of methylation in cancer patients with an unexplained molecular basis of DPD deficiency. DPD deficiency was identified phenotypically by both enzyme assay and uracil breath test, and genotypically by denaturing high-performance liquid chromatography. The methylation status was evaluated in PCR products (209 bp) of bisulfite-modified DPYD promoter, using a novel denaturing high-performance liquid chromatography method that distinguishes between methylated and unmethylated alleles. Clinical samples included five volunteers with normal DPD enzyme activity, five DPD-deficient volunteers, and five DPD-deficient cancer patients with a history of 5-FU toxicity. No evidence of methylation was detected in samples from volunteers with normal DPD. Methylation was detected in five of five DPD-deficient volunteers and in three of five of the DPD-deficient cancer patient samples. Of note, one of the two samples from patients with DPD-deficient cancer with no evidence of methylation had the mutation DPYD*2A, whereas the other had DPYD*13. Methylation of the DPYD promoter region is associated with down-regulation of DPD activity in clinical samples and should be considered as a potentially important regulatory mechanism of DPD activity and basis for 5-FU toxicity in cancer patients.

Prevention of 5-fluorouracil-induced early severe toxicity by pre-therapeutic dihydropyrimidine dehydrogenase deficiency screening: Assessment of a multiparametric approach