Pharmacokinetics of 5-Fluorouracil in Patients Heterozygous for the IVS14+1G > A Mutation in the Dihydropyrimidine Dehydrogenase Gene

5-Fluorouracil (5FU) and capecitabine are two of the most frequently prescribed chemotherapeutic drugs for the treatment of patients with cancer. Administration of test doses of 5FU to eight patients heterozygous for the IVS14+1G > A mutation and five control patients showed that the AUC and clearance were weak parameters with respect to the identification of patients with a DPD deficiency. However, highly significant differences were observed for the terminal half life of 5FU between DPD patients and controls. Thus, a DPD deficiency could be predicted from 5FU blood concentrations measured after the administration of a test dose of 5FU.     

High-performance liquid chromatographic assay with ultraviolet detection for quantification of dihydrofluorouracil in human lymphocytes: application to measurement of dihydropyrimidine dehydrogenase activity

The anticancer drug 5-fluorouracil (5FU) undergoes extensive biotransformation to 5-dihydrofluorouracil (5FUH2) by the enzyme dihydropyrimidine deshydrogenase (DPD). A new HPLC method with direct UV detection for the determination of 5FUH2 in peripheral lymphocytes has been developed to detect DPD deficiency in patients treated with 5FU-based therapy. The method has been shown to be valid over the 5FUH2 concentration range of 1.14–37.88 nmol/ml. Optimal enzymatic conditions for DPD activity measurement were studied: incubation time, protein and 5FU concentrations. The assay was successfully cross-validated with the existing method using HPLC with radiochemical detection.     

Altered Dihydropyrimidine Dehydrogenase Activity Associated with Mild Toxicity in Patients Treated with 5-Fluorouracil Containing Chemotherapy

Dihydropyrimidine dehydrogenase (DPD) plays a pivotal role in the metabolism of 5-fluorouracil (5FU). In patients treated with capecitabine or 5FU combined with other chemotherapeutic drugs, DPD activity in peripheral blood mononuclear cells was increased in patients experiencing grade I/II neutropenia. In contrast, decreased DPD activity proved to be associated with grade I/II dermatological toxicity, including hand-foot syndrome. Thus, patients with a low-normal or high-normal DPD activity proved to be at risk of developing mild toxicity upon treatment with 5FU-based chemotherapy, demonstrating the important role of DPD in the etiology of toxicity associated with 5FU and the catabolites of 5FU.     

Altered dihydropyrimidine dehydrogenase activity associated with mild toxicity in patients treated with 5-fluorouracil containing chemotherapy

Dihydropyrimidine dehydrogenase (DPD) plays a pivotal role in the metabolism of 5-fluorouracil (5FU). In patients treated with capecitabine or 5FU combined with other chemotherapeutic drugs, DPD activity in peripheral blood mononuclear cells was increased in patients experiencing grade I/II neutropenia. In contrast, decreased DPD activity proved to be associated with grade I/II dermatological toxicity, including hand-foot syndrome. Thus, patients with a low-normal or high-normal DPD activity proved to be at risk of developing mild toxicity upon treatment with 5FU-based chemotherapy, demonstrating the important role of DPD in the etiology of toxicity associated with 5FU and the catabolites of 5FU.     

Intragenic deletions and a deep intronic mutation affecting pre-mRNA splicing in the dihydropyrimidine dehydrogenase gene as novel mechanisms causing 5-fluorouracil toxicity

Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme acting in the catabolism of the widely used antineoplastic agent 5-fluorouracil (5FU). DPD deficiency is known to cause a potentially lethal toxicity following administration of 5FU. Here, we report novel genetic mechanisms underlying DPD deficiency in patients presenting with grade III/IV 5FU-associated toxicity. In one patient a genomic DPYD deletion of exons 21–23 was observed. In five patients a deep intronic mutation c.1129–5923C>G was identified creating a cryptic splice donor site. As a consequence, a 44 bp fragment corresponding to nucleotides c.1129–5967 to c.1129–5924 of intron 10 was inserted in the mature DPD mRNA. The deleterious c.1129–5923C>G mutation proved to be in cis with three intronic polymorphisms (c.483 + 18G>A, c.959–51T>G, c.680 + 139G>A) and the synonymous mutation c.1236G>A of a previously identified haplotype. Retrospective analysis of 203 cancer patients showed that the c.1129–5923C>G mutation was significantly enriched in patients with severe 5FU-associated toxicity (9.1%) compared to patients without toxicity (2.2%). In addition, a high prevalence was observed for the c.1129–5923C>G mutation in the normal Dutch (2.6%) and German (3.3%) population. Our study demonstrates that a genomic deletion affecting DPYD and a deep intronic mutation affecting pre-mRNA splicing can cause severe 5FU-associated toxicity. We conclude that screening for DPD deficiency should include a search for genomic rearrangements and aberrant splicing.     

Identification of three novel mutations in the dihydropyrimidine dehydrogenase gene associated with altered pre-mRNA splicing or protein function

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil and thymine, as well as of the widely used chemotherapeutic drug 5-fluorouracil (5FU). Analysis of the DPD gene ( DPYD ) in two patients presenting with complete DPD deficiency and the parents of an affected child showed the presence of three novel mutations, including one splice site mutation IVS11 + 1G-->T and the missense mutations 731A-->C (E244V) and 1651G-->A (A551T). The G-->T mutation in the invariant GT splice donor site flanking exon 11 (IVS11 + 1G-->T) created a cryptic splice site within exon 11. As a consequence, a 141-bp fragment encoding the aminoacid residues 400-446 of the primary sequence of the DPD protein was missing in the mature DPD mRNA. Analysis of the crystal structure of pig DPD suggested that the E244V mutation might interfere with the electron flow between NADPH and the pyrimidine binding site of DPD. The A551T point mutation might prevent binding of the prosthetic group FMN and affect folding of the DPD protein. The identification of these novel mutations in DPYD will allow the identification of patients with an increased risk of developing severe 5FU-associated toxicity.     

Circadian Rhythm in Dihydropyrimidine Dehydrogenase Activity and Reduced Glutathione Content in Peripheral Blood of Nasopharyngeal Carcinoma Patients

Dihydropyrimidine dehydrogenase (DPD) is a rate‐limiting enzyme of 5‐fluorouracil (5‐FU) catabolism. Glutathione (GSH) is a tripeptide involved in platinum complex detoxification. This study explored the circadian rhythms of DPD activity and GSH concentration in the peripheral blood of 16 patients with histologically proven nasopharyngeal carcinoma (NPC) in order to guide the establishment of chronotherapeutic schedules for this cancer. DPD activity and GSH concentration were determined by high performance liquid chromatography (HPLC). Both variables displayed significant circadian rhythms (Cosinor analysis: p=0.009 and 0.012, respectively). Peak DPD activity occurred at about 02:30 h; whereas, peak GSH concentration occurred around 12:40 h. The differences between the peak and nadir mean values were 25.5% and 38.7%, respectively. The study showed that the circadian rhythms in DPD activity and GSH concentration in Chinese NPC are similar to those reported for western patients with colorectal cancer, despite the differences in race and kinds of cancer. These findings imply that the chronotherapeutic schedule of 5‐FU and platinum used to treat European colorectal cancer patients probably is applicable to Chinese NPC patients.     

Clinical pharmacokinetics of 5-fluorouracil with consideration of chronopharmacokinetics

Even though 5-fluorouracil (FU) is one of the oldest anticancer drugs, its use in cancer chemotherapy continues to increase. Fluorouracil is a pro-drug that requires intracellular activation to exert its effects. This makes it difficult to associate blood drug concentration with cell toxicity directly, although data from the literature show the existence of such a relationship. The relationship between FU pharmacokinetics and patient response has been explored extensively and reports attest a link between systemic drug exposure and response and survival. This has led to the concept of maximal tolerated exposure, and strategies to achieve this rely on pharmacokinetic follow-up and individual dose adjustment. More than 80% of the administered FU dose is eliminated by catabolism through dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme. Dihydropyrimidine dehydrogenase activity is found in most tissues but is highest in the liver. Peripheral blood mononuclear cells (PBMC) are used to monitor clinically DPD activity. A significant, but weak correlation between PBMC and liver DPD activity has been observed. The relationship between PBMC–DPD activity and FU systemic clearance is weak (r²=0.10); thus, simply determining PBMC–DPD is not sufficient to predict accurately FU clearance. Population pharmacokinetic analysis identified patient co-variables that influence FU clearance; drug kinetics is significantly reduced by increased age, high serum alkaline phosphatase, length of drug infusion, and low PBMC–DPD. Autoregulation of FU metabolism also is suggested; inhibition of DPD activity was observed after FU administration in both colorectal cancer patients and an animal model. Circadian rhythmicity in DPD activity is suggested from both human and animal investigations. In patients receiving protracted low dose 5-FU infusion, the circadian rhythm in FU plasma concentration peaks at 11:00h and is lowest at 23:00h, on average. The inverse relationship observed between the circadian profile of FU plasma concentration and PBMC–DP activity in these same patients suggests a link between DPD activity and FU pharmacokinetics. The impact of the biological time of drug administration was also studied with short venous infusions; clearance was 70% greater at 13:00h than at 01:00h. Similarly, peak drug concentration occurred in the first half of the night in patients receiving constant rate 5-FU infusion for 2–5 d. Several studies describe wide interindividual variation in the timing of the peak and trough of the 24h rhythm in DPD activity. The rational for FU chronomodulated therapy has been the circadian rhythm in host drug tolerance, which is greatest during the night time when the proliferation of normal target tissue is least. A randomized study of chronomodulated FU therapy with maximal delivery rate at 04:00h was shown clearly to be significantly more effective and less toxic than control flat FU therapy. Future research must focus on easy-to-obtain markers of specific rhythms to individualize the chronomodulated FU delivery.     

Clinical pharmacokinetics of 5-fluorouracil with consideration of chronopharmacokinetics

Even though 5-fluorouracil (FU) is one of the oldest anticancer drugs, its use in cancer chemotherapy continues to increase. Fluorouracil is a pro-drug that requires intracellular activation to exert its effects. This makes it difficult to associate blood drug concentration with cell toxicity directly, although data from the literature show the existence of such a relationship. The relationship between FU pharmacokinetics and patient response has been explored extensively and reports attest a link between systemic drug exposure and response and survival. This has led to the concept of maximal tolerated exposure, and strategies to achieve this rely on pharmacokinetic follow-up and individual dose adjustment. More than 80% of the administered FU dose is eliminated by catabolism through dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme. Dihydropyrimidine dehydrogenase activity is found in most tissues but is highest in the liver. Peripheral blood mononuclear cells (PBMC) are used to monitor clinically DPD activity. A significant, but weak correlation between PBMC and liver DPD activity has been observed. The relationship between PBMC-DPD activity and FU systemic clearance is weak (r²=0.10); thus, simply determining PBMC-DPD is not sufficient to predict accurately FU clearance. Population pharmacokinetic analysis identified patient co-variables that influence FU clearance; drug kinetics is significantly reduced by increased age, high serum alkaline phosphatase, length of drug infusion, and low PBMC-DPD. Autoregulation of FU metabolism also is suggested; inhibition of DPD activity was observed after FU administration in both colorectal cancer patients and an animal model. Circadian rhythmicity in DPD activity is suggested from both human and animal investigations. In patients receiving protracted low dose 5-FU infusion, the circadian rhythm in FU plasma concentration peaks at 11:00h and is lowest at 23:00h, on average. The inverse relationship observed between the circadian profile of FU plasma concentration and PBMC-DP activity in these same patients suggests a link between DPD activity and FU pharmacokinetics. The impact of the biological time of drug administration was also studied with short venous infusions; clearance was 70% greater at 13:00h than at 01:00h. Similarly, peak drug concentration occurred in the first half of the night in patients receiving constant rate 5-FU infusion for 2-5 d. Several studies describe wide interindividual variation in the timing of the peak and trough of the 24h rhythm in DPD activity. The rational for FU chronomodulated therapy has been the circadian rhythm in host drug tolerance, which is greatest during the night time when the proliferation of normal target tissue is least. A randomized study of chronomodulated FU therapy with maximal delivery rate at 04:00h was shown clearly to be significantly more effective and less toxic than control flat FU therapy. Future research must focus on easy-to-obtain markers of specific rhythms to individualize the chronomodulated FU delivery.     

Pharmacogenetic studies on the prediction of efficacy and toxicity of fluoropyrimidine-based adjuvant therapy in colorectal cancer

The cytotoxic effect of 5-fluorouracil (5-FU) is mediated by the inhibition of thymidylate synthase (TS), however, at the same time 5-FU is catabolized by dihydropyrimidine dehydrogenase (DPD). Efficacy of 5-FU may therefore depend on the TS and DPD activity and on pharmacogenetic factors influencing these enzymes. Our aims were (1) to determine the distribution of DPD activity, the frequency of DPD deficiency and the DPD (IVS14+1G>A) mutation in the peripheral blood mononuclear cells of colorectal cancer (CRC) patients, and study the relationship between DPD deficiency and toxicity of 5-FU; (2) to investigate the influence of TS polymorphisms and DPD activity on the survival of CRC patients receiving 5-FU-based adjuvant therapy. The frequency of DPD deficiency was determined by radiochemical methods in the peripheral blood mononuclear cells (PBMCs) of 764 CRC patients treated with 5-FU. The relationship between the TS polymorphisms, DPD activity and the disease-free and overall survival was studied in 166 CRC patients receiving 5-FU-based adjuvant therapy. TS polymorphisms were determined in the DNA samples separated from the PBMCs, by PCR-PAGE and PCR-RFLP-PAGE (restriction fragment length polymorphism) methods. Low DPD values (<10 pmol/min/106 PBMCs) were demonstrated in 160/764 patients (20.9%), and of those DPD deficiency (<5 pmol/min/106 PBMCs) was verified in 38 patients (4.9%). In the latter group severe (>Gr 3) toxicity was found in 87%. The prevalence of the DPD IVS14+1G>A mutation among the 38 DPD-deficient patients was 7.8% (3/38) and was accompanied by severe Gr 4 toxic symptoms (neutropenia, mucositis, diarrhea). TS polymorphisms showed a relationship with the survival of CRC patients. It is important to mention that by combining the 3-3 genotypes of 5'-TSER and 3'-TSUTR polymorphisms the obtained 8 genotype combinations showed significantly different Kaplan-Meier survival curves. The evaluation of these curves with Cox regression analysis resulted in two prognostically different groups: "A" good prognosis (RR<1) and "B" bad prognosis (RR>1). The disease-free- and overall survival of these two groups were significantly different. DPD activity also showed correlation with the survival; patients with DPD activity <10 pmol/min/106 PBMCs showed significantly longer disease-free and overall survival. The determination of DPD activity proved to be a more valuable parameter in the evaluation of serious 5-FU-related toxicity compared to the IVS14+1G>A mutation analysis. According to the Cox multivariate analysis the combination of germline TS polymorphisms and DPD activity is/an independent prognostic marker of survival in CRC patients treated with adjuvant 5-FU therapy.     

Dihydropyrimidine dehydrogenase (DPD) deficiency: identification and expression of missense mutations C29R, R886H and R235W

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 ΔC1897 frameshift 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. Received: 20 June 1997 / Accepted: 26 August 1997     

A pivotal role for beta-aminoisobutyric acid and oxidative stress in dihydropyrimidine dehydrogenase deficiency?

Dihydropyrimidine dehydrogenase (DPD) constitutes the first step of the pyrimidine degradation pathway in which the pyrimidine bases uracil and thymine are catabolised to beta-alanine and beta-aminoisobutyric acid (beta-AIB), respectively. The mean concentration of beta-AIB was approximately 5- to 8-fold lower in urine of patients with a DPD deficiency, when compared to age-matched controls. Comparable levels of 8-hydroxydeoxyguanosine (8-OHdG) were present in urine from controls and DPD patients at the age <2 year. In contrast, slightly elevated levels of 8-OHdG were detected in urine from DPD patients with an age >2 year, suggesting the presence of increased oxidative stress.     

Pyrimidine Degradation Defects and Severe 5‐Fluorouracil Toxicity

5‐Fluorouracil (5FU) remains one of the most frequently prescribed chemotherapeutic drugs for the treatment of cancer. Recently, the pivotal role of the catabolic pathway of 5FU in the determination of toxicity towards 5FU has been highlighted. Patients with a (partial) dihydropyrimidine dehydrogenase deficiency proved to be at risk of developing severe toxicity after the administration of 5FU. A partial dihydropyrimidinase deficiency proved to be a novel pharmacogenetic disorder associated with severe 5FU toxicity.     

Pyrimidine degradation defects and severe 5-fluorouracil toxicity

5-Fluorouracil (5FU) remains one of the most frequently prescribed chemotherapeutic drugs for the treatment of cancer. Recently, the pivotal role of the catabolic pathway of 5FU in the determination of toxicity towards 5FU has been highlighted. Patients with a (partial) dihydropyrimidine dehydrogenase deficiency proved to be at risk of developing severe toxicity after the administration of 5FU. A partial dihydropyrimidinase deficiency proved to be a novel pharmacogenetic disorder associated with severe 5FU toxicity.     

A Pivotal Role for β-Aminoisobutyric Acid and Oxidative Stress in Dihydropyrimidine Dehydrogenase Deficiency?

Dihydropyrimidine dehydrogenase (DPD) constitutes the first step of the pyrimidine degradation pathway in which the pyrimidine bases uracil and thymine are catabolised to β-alanine and β-aminoisobutyric acid (β-AIB), respectively. The mean concentration of β-AIB was approximately 5- to 8-fold lower in urine of patients with a DPD deficiency, when compared to age-matched controls. Comparable levels of 8-hydroxydeoxyguanosine (8-OHdG) were present in urine from controls and DPD patients at the age <2 year. In contrast, slightly elevated levels of 8-OHdG were detected in urine from DPD patients with an age >2 year, suggesting the presence of increased oxidative stress.     

A high-throughput denaturing high-performance liquid chromatography method for the identification of variant alleles associated with dihydropyrimidine dehydrogenase deficiency

Dihydropyrimidine dehydrogenase (DPD) is the initial, rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU). A pharmacogenetic syndrome has been described in which DPD-deficient patients are at risk for toxicity following administration of 5-FU. To date, there are at least 21 previously described mutations and/or polymorphisms that have been associated with DPD deficiency. In this study we describe the development of a highly specific, sensitive, inexpensive, and robust denaturing HPLC (DHPLC) method for rapidly identifying sequence variations (mutations and/or polymorphisms) in the gene (DPYD) that codes for the DPD enzyme. DHPLC conditions were optimized at three temperatures for analysis of the 23 exons of the DPYD gene using 25 amplicons representing the entire coding sequence, including all intron/exon boundaries (splice sites). Resolution of all 25 amplicons at the optimized temperature can be performed in 4.2 h. All 21 previously described sequence variations (mutations and/or polymorphisms) were prepared using site-directed mutagenesis from the wild-type DPYD gene, confirmed by sequence analysis, and subsequently resolved by DHPLC using the optimized conditions. These analyses generated reference chromatogram patterns for all known sequence variations previously encountered in DPD-deficient patients. In order to examine the utility and sensitivity of this approach, samples from patients with known sequence variations in the DPYD gene were analyzed. This DHPLC technique resolved 100% of the known DPYD sequence variations and differentiated between homozygous and heterozygous genotypes. We conclude that this DHPLC method is a highly specific and sensitive technique for rapidly detecting known sequence variations in the DPYD gene. In addition, this approach can be used to identify currently unrecognized unknown sequence variations in the DPYD gene and should be useful in future pharmacogenetic studies examining DPD deficiency.     

Simple gas chromatographic–mass spectrometric procedure for diagnosing pyrimidine degradation defects for prevention of severe anticancer side effects

Inborn errors of pyrimidine degradation, dihydropyrimidine dehydrogenase deficiency and dihydropyrimidinase deficiency, are less rare than has generally been assumed. Many asymptomatic cases have been reported, and in patients with symptoms, the clinical abnormalities are variable and nonspecific. Withdrawal of pyrimidine analogues such as 5-fluorouracil (5FU), a commonly used anticancer drug, from the cancer chemotherapy regimens of patients with pyrimidine degradation deficiencies, however, is critical because 5FU is degraded in vivo by pyrimidine-degradative enzymes. Patients with these deficiencies suffer from severe neurotoxicity, sometimes leading to death, following administration of 5FU, and even otherwise asymptomatic homozygotes or heterozygotes may develop severe clinical symptoms upon administration of such medication. Therefore, a rapid and specific method for identifying cancer patients with these enzyme deficiencies prior to treatment with 5FU is critical. To address this problem, we established methods for highly sensitive yet specific determinations of thymine, uracil, dihydrothymine, dihydrouracil, orotate and creatinine simultaneously in 0.1-ml liquid urine or filter-paper urine. This method involves stable isotope dilution, a simplified urease treatment previously described and gas chromatography–mass spectrometry without prior fractionation. The high recovery and low C.V. values were obtained and healthy control values were also determined for these metabolites. Using artificially prepared urine specimens simulating these disorders, the chemical diagnosis can be made clearly, and no further analysis appears to be required for differential chemical diagnosis.     

Analysis of severely affected patients with dihydropyrimidine dehydrogenase deficiency reveals large intragenic rearrangements of DPYD and a de novo interstitial deletion del(1)(p13.3p21.3)

Dihydropyrimidine dehydrogenase (DPD) deficiency is an infrequently described autosomal recessive disorder of the pyrimidine degradation pathway and can lead to mental and motor retardation and convulsions. DPD deficiency is also known to cause a potentially lethal toxicity following administration of the antineoplastic agent 5-fluorouracil. In an ongoing study of 72 DPD deficient patients, we analysed the molecular background of 5 patients in more detail in whom initial sequence analysis did not reveal pathogenic mutations. In three patients, a 13.8 kb deletion of exon 12 was found and in one patient a 122 kb deletion of exon 14–16 of DPYD. In the fifth patient, a c.299_302delTCAT mutation in exon 4 was found and also loss of heterozygosity of the entire DPD gene. Further analysis demonstrated a de novo deletion of approximately 14 Mb of chromosome 1p13.3–1p21.3, which includes DPYD. Haploinsufficiency of NTNG1, LPPR4, GPSM2, COL11A1 and VAV3 might have contributed to the severe psychomotor retardation and unusual craniofacial features in this patient. Our study showed for the first time the presence of genomic deletions affecting DPYD in 7% (5/72) of all DPD deficient patients. Therefore, screening of DPD deficient patients for genomic deletions should be considered.