Lethal drug interactions of the new antiviral, sorivudine, with anticancer prodrugs of 5-fluorouracil

In 1993 eighteen Japanese patients with cancer and herpes zoster, a viral disease, died from interactions of the new oral antiviral drug, sorivudine (SRV: 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil), with oral anticancer prodrugs of 5-fluorouracil (5-FU) within 40 d after SRV was approved by the Japanese government and began to be used clinically. Before the death, most of these patients had severe symptoms of toxicity, including diarrhea with bloody flux and marked decreases in white blood cell and platelet counts. All of these patients received SRV daily for several days while being administered long-term anticancer chemotherapy with one of the oral 5-FU prodrugs. There was no acute toxic symptom in patients who received SRV alone or SRV and the other types of anticancer drugs. A toxicokinetic study was carried out using rats to investigate the mechanism of the acute death in the patients caused by drug interactions between SRV and 5-FU prodrugs. Rats were orally coadministered SRV with tegafur (FT: 1-(2-tetrahydrofuryl)-5-fluorouracil), a 5-FU prodrug that most of the patients were considered to receive before the death. All the rats receiving SRV and FT once daily showed extremely elevated levels of 5-FU in the plasma and tissues, including bone marrow and small intestines, and died within 10 d, while the animals given the same repeated dose of SRV or FT alone were still alive over 20 d without any appreciable toxic symptom. Before their death, there were a marked damage of bone marrow, a marked atrophy of intestinal membrane mucosa, marked decreases in white blood cells and platelets, diarrhea with bloody flux, and severe anorexia as reported for the patients. Data obtained by in vivo and in vitro studies indicated that (E)-5-(2-bromovinyl)uracil (BVU), generated from SRV by the gut flora and absorbed through the intestinal membrane, was reduced in the presence of NADPH to a reactive form by hepatic dihydropyrimidine dehydrogenase (DPD), a key enzyme regulating the tissue 5-FU levels from FT, bound covalently to DPD as a suicide inhibitor, and markedly retarded the catabolism of 5-FU. An irreversible inactivation by BVU of rat and human DPDs, expressed in E. coli for the latter, was observed in the presence of NADPH with their purified preparations in a manner reciprocal to radiolabelling of the enzyme proteins with [14C]BVU. SRV showed no inhibitory effect on the rat and human DPDs in the presence of NADPH.     

A possible mechanism of eighteen patient deaths caused by interactions of sorivudine, a new antiviral drug, with oral 5-fluorouracil prodrugs

A toxicokinetic study was performed using rats to investigate the possible mechanism of 18 acute deaths in Japanese patients with cancer and herpes zoster by interactions of the new oral antiviral drug, sorivudine (SRV), with one of the oral 5-fluorouracil (5-FU) prodrugs within 40 days after approval of the use of SRV. Tegafur, an anticancer 5-FU prodrug suggested to be used by most of the patients who died, and SRV were orally administered to rats simultaneously once daily. All of these rats died within 10 days, whereas rats given SRV or tegafur alone under the same dosage conditions showed no appreciable change over 20 days compared with controls. In the rats given both drugs, bone marrow and intestinal membrane mucosa were greatly damaged at an early stage of the coadministration, and before death, the animals showed marked decreases in white blood cell and platelet counts, diarrhea with bloody flux, and severe anorexia, as was also manifested by the patients who subsequently died. In the rats given both drugs for 6 days, extremely enhanced 5-FU levels were observed from the first day of administration in plasma and in all tissues examined, including bone marrow and intestines. The extreme enhancement of the tissue 5-FU levels was attributable to the facile inactivation by (E)-5-(2-bromovinyl)uracil (BVU) of hepatic dihydropyrimidine dehydrogenase (DPD), a key enzyme regulating the systemic 5-FU level in the rat and human. BVU, a major metabolite formed from SRV by gut flora, was found at considerable levels in the liver of rats orally administered SRV alone or SRV and tegafur, and there was a marked decrease in hepatic DPD activity. In the presence of NADPH, DPD purified from rat liver cytosol was rapidly and irreversibly inactivated by [14C]BVU as a suicide inhibitor with concomitant incorporation of the radioactivity into the enzyme protein, although SRV showed no inhibitory effect on DPD under the same conditions. Human liver DPD was recently demonstrated by us to be inactivated with BVU in a manner very similar to rat DPD.     

Late phase II study of novel oral fluoropyrimidine anticancer drug S-1 (1 M tegafur-0.4 M gimestat-1 M otastat potassium) in advanced gastric cancer patients

S-1 is a novel oral anticancer drug, composed of tegafur (FT), gimestat (CDHP) and otastat potassium (Oxo) in a molar ratio of 1:0.4:1, based on the biochemical modulation of 5-fluorouracil (5-FU). CDHP inhibits dihydropyrimidine dehydrogenase (DPD), an enzyme which degrades 5-FU, and maintains prolonged 5-FU concentrations in the blood and tumours. Oxo is distributed in the gastrointestinal tract at a high concentration after oral administration and alleviates gastrointestinal toxicity due to 5-FU. S-1 improves the tumour-selective toxicity of 5-FU by the actions of two modulators, CDHP and Oxo. We conducted a late phase II clinical trial of S-1 as an open trial in patients with advanced gastric cancer, to confirm its antitumour effect and adverse reactions. 51 patients with advanced gastric cancer were enrolled in the trial. S-1 was administered orally twice daily after meals, at a standard dose of 80 mg/m2/day. One course consisted of consecutive administration for 28 days and 14 days' rest. Administration was repeated over four courses. A complete response was obtained in 1 patient and partial responses in 24 patients, producing a response rate of 49% (25/51) (95% confidence interval (CI) 35.9-62.3%). The incidence of adverse reactions was 78% (40/51) and that of adverse reactions of grades 3 and 4 was 20%. Adverse reactions of grades 3 and 4 included a decrease in the haematocrit, leucopenia, granulocytopenia, diarrhoea, malaise and proteinuria. No serious unexpected adverse reactions were observed. In conclusion, S-1 was effective and well tolerated in patients with advanced gastric cancer.     

Outpatient utilization patterns of integrated and split psychotherapy and pharmacotherapy for depression

Dihydropyrimidine dehydrogenase (DPD) deficiency with a defect of the pyrimidine catabolic pathway has recently become the focus of considerable attention, due to the severe 5-fluorouracil (5-FU) toxicities occurring in DPD deficiency patients. Studies also suggest that 5-FU toxicities could occur in another pyrimidine metabolic disorder, dihydropyrimidinuria (DHPuria). This study shows that urinary dihydrothymine (DHT) and thymine (THY) are useful indexes for detection of DPD deficiency and DHPuria. We measured urinary DHT and THY in 276 Japanese adults to establish reference ranges. When males and females were compared, both DHT and THY levels were found to be significantly higher in females. The reference ranges (mean +/- SD with logarithmic values) for males were found to be 1.56-5.70 micromol/g of creatinine for DHT and 0.40-1.47 micromol/g of creatinine for THY. The reference ranges for females were found to be 1.89-8.33 micromol/g of creatinine for DHT and 0.58-2.30 micromol/g of creatinine for THY. In addition to this study we analyzed a DPD deficiency case and a DHPuria case. In the DPD deficiency case, the THY concentrations of all urine samples were out of the reference range. However, uracil levels in most of the samples were within the normal range. The DHPuria case excreted large amounts of DHT and dihydrouracil, both out of the normal range.     

Severe neurotoxicity following 5-fluorouracil-based chemotherapy in a patient with dihydropyrimidine dehydrogenase deficiency

Patients with decreased dihydropyrimidine dehydrogenase (DPD) activity are at increased risk for experiencing serious adverse reactions following 5-fluorouracil (5-FU)-based chemotherapy. Symptoms include severe and potentially life-threatening gastrointestinal toxicity, myelosuppression, and neurological toxicity. In the present study, we describe a 50-year-old Caucasian man who developed severe encephalopathy during his second cycle of 5-FU chemotherapy. The patient remained in a comatose state for 4 days but then showed dramatic improvement in his neurological status following continuous i.v. infusion of thymidine at 8 g/m2/day. Laboratory studies revealed the patient to be severely DPD deficient, as demonstrated by DPD enzyme activity from peripheral blood mononuclear cells being below the lower limit of the 95th percentile of a control population and by Western immunoblot analysis showing undetectable levels of DPD protein. Additional studies revealed a significant defect in pyrimidine catabolism with a 3.3- and 365-fold increase in the levels of uracil in plasma and urine, respectively, compared to normal subjects. Family studies suggest that the inheritance pattern of this syndrome is complex and most consistent with an autosomal recessive trait. This study demonstrates that cancer patients with DPD deficiency are at increased risk for developing severe neurological toxicity secondary to 5-FU chemotherapy, and that infusional thymidine should be considered as a potential rescue agent against this particular host toxicity.     

Dihydropyrimidine dehydrogenase activity in cancer patients

Dihydropyrimidine dehydrogenase (DPD) is the major catabolic enzyme of pyrimidines and fluoropyrimidines. The clinical course of 2 patients with suspected DPD deficiency is described. Both patients had significantly delayed clearance of fluorouracil (5-FU), elevated plasma uracil concentrations, and subsequent lethal toxicity. The prevalence of DPD deficiency in the general population is unknown, but given the large number of cancer patients treated with 5-FU, it may be of great clinical significance. Lymphocytes have been previously shown to be a useful marker of systemic DPD activity. Because DPD activity has not been previously reported in a large population of cancer patients using 5-FU as the substrate, we determined DPD activity in lymphocytes from 66 patients with cancer. DPD activity was determined by a sensitive high performance liquid chromatography method. The mean DPD activity (S.D.) in 66 patients with head and neck cancer was 0.189 (0.071) nomol/min/mg protein with wide interpatient variability (range 0.058-0.357). DPD activity was not correlated to age (r = -0.164, P = 0.188). The mean DPD activity in men [0.192 (0.074)] was not significantly different from that in women [0.172 (0.057); t-test P = 0.418]. Likewise, there was no statistical difference in DPD activity in patients who had not received prior chemotherapy [0.195 (0.066)] to patients receiving one or more cycles of chemotherapy [0.186 (0.074); t-test P = 0.638].     

Identification of novel mutations in the dihydropyrimidine dehydrogenase gene in a Japanese patient with 5-fluorouracil toxicity

5-Fluorouracil (5-FU) is used widely in the treatment of several common neoplasms. Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-FU. Several recent studies have described a pharmacogenetic disorder in which cancer patients with decreased DPD activity develop life-threatening toxicity following exposure to 5-FU. We reported recently the first Japanese case of decreased DPD activity accompanied by severe 5-FU toxicity. The present study describes the results of molecular analysis of this patient and her family, in which three novel mutations (Arg21Gln, Val335Leu, and Glu386Ter) of the gene coding for DPD were identified. We also revealed that Arg21Gln and Glu386Ter are on the same allele and that Val335Leu is on the other allele, on the basis of analysis of the family genome. Expression analysis in Escherichia coli showed that Val335Leu and Glu386Ter led to mutant DPD protein with significant loss of enzymatic activity and no activity, respectively. The Arg21Gln mutation, however, resulted in no decrease in enzymatic activity compared with the wild type. The present data represent the first molecular genetic analysis of DPD deficiency accompanied by severe 5-FU toxicity in a Japanese patient.     

Invention of a tumor-selective 5-fluorouracil derivative named S-1 by biochemical modulation of 5-fluorouracil

A new oral type of 5-fluorouracil (5-FU) derivative possessed of both potent antitumor activity and less gastrointestinal (GI) toxicity was investigated and developed in the form of a combination of tegafur (FT), a masked form of 5-FU, and its two peculiar biochemical modulators. One is 5-chloro-2,4-dihydroxypyridine (CDHP), a new potent inhibitor of 5-FU degradation in vivo, and another is potassium oxonate (Oxo), a characteristic inhibitor of 5-FU phosphorylation, which distributes much higher in GI tract after p.o. administration. 5-FU levels in blood of rats following administration of FT, were markedly elevated and persisted for a long-time by co-oral CDHP corresponding to over 0.4 molar ratio to FT, like the case in continuous infusion of 5-FU, which resulted in an augmentation of antitumor efficacy in Yoshida sarcoma-bearing rats, although severe GI toxicity simultaneously occurred. To reduce 5-FU-induced toxicities such as diarrhea and body weight loss and to maintain the augmented antitumor activity, 0.5 to 2 molar Oxo was orally given to rats with one molar FT plus 0.4 molar CDHP. As a result, both severe GI injury and body weight loss were markedly inhibited by coadministration of 0.5 to 1.0 molar Oxo while high antitumor efficacy (about 90% inhibition of tumor growth) was maintained. However, such almost complete antitumor effect was reduced to about 50% inhibition of tumor growth by over 2 molar Oxo combined with one molar FT plus 0.4 molar CDHP. Based on these results, a novel 5-FU derivative, named S-1, was composed of one molar FT, 0.4 molar CDHP and one molar Oxo. S-1 showed an antitumor activity over 3-fold stronger than UFT (one molar FT plus 4 molar uracil) against Yoshida sarcoma and Sato lung carcinoma in rats and human colon carcinoma (KM12C) xenografted in nude rats when its minimum toxic dose was administered. Co-oral Oxo also significantly reduced the incidence of diarrhea and stomatitis induced by administration of FT-CDHP in beagle dogs. These results suggest that high antitumor activity and less GI toxicity of S-1 was brought about by the elevation in blood and tumor tissues and by selective decrease of 5-fluoronucleotides, an active metabolite of 5-FU, in GI tract.     

Effect of gastrectomy on the pharmacokinetics of tegafur, uracil, and 5-fluorouracil after oral administration of a 1:4 tegafur and uracil combination

The effects of gastrectomy on the pharmacokinetics of UFT, a combined oral preparation of 1-(2-tetrahydrofuryl)-5-fluorouracil (tegafur) and uracil at a molar ratio of 1:4, were examined in 26 patients with macroscopic Stage I gastric cancer. In all, 200 mg UFT (in terms of tegafur) was given to 17 patients who underwent partial gastrectomy (9 cases of Billroth I reconstruction, 8 cases of Billroth II reconstruction) and to 9 patients who underwent total gastrectomy with modified Roux-en-Y reconstruction. Before the operation, the area under the curve (AUC) for tegafur, uracil, and 5-fluorouracil (5-FU) was 79.28 +/- 26.88, 4.41 +/- 1.78, and 0.51 +/- 0.20 micrograms h ml-1, respectively. Partial (Billroth I and II) and total gastrectomy did not alter the AUC of tegafur, and partial gastrectomy using the Billroth I and II methods decreased the AUCs of uracil and 5-FU during the first 2 weeks postoperation. However, plasma levels of uracil and 5-FU reverted to preoperative values at 3 months postsurgery. Our findings show that when UFT is prescribed for patients treated in the early postoperative period following partial gastrectomy for cancer, dose increases and the timing of administration should be given close attention.     

Adenovirus-mediated transduction of Escherichia coli uracil phosphoribosyltransferase gene sensitizes cancer cells to low concentrations of 5-fluorouracil

5-fluorouracil (5-FU), although a widely used chemotherapeutic agent, has a limited effect in the treatment of human solid tumors due to their resistance to the cytotoxic effects of 5-FU. Escherichia coli uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that catalyzes the synthesis of UMP from uracil and 5-phosphoribosyl-alpha-1-diphosphate. The present study demonstrates that adenovirus-mediated transduction of E. coli UPRT gene results in marked sensitization of colon, gastric, liver, and pancreas cancer cell lines to low concentration of 5-FU in vitro. The in vitro bystander effect was observed when only 10% of the hepatoma Hep3B cells were infected with UPRT-expressing adenovirus. In addition, 5-FU treatment of human hepatoma or gastric cancer xenografts in nude mice transduced with UPRT was demonstrated to result in significant in vivo antitumor effects. The adenovirus vector transduction of the UPRT gene followed by 5-FU administration is representative of a new chemosensitization strategy for cancer gene therapy.     

Metabolism of 5'-ether prodrugs of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil in rats

1-beta-D-Arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) is a selective antiherpesviral agent that has been shown to be metabolically stable in mice. However, E-5-(2-bromovinyl)uracil (BVU) is the major metabolite found after oral dosing in animals other than mice. When BV-araU was given orally to germ-free rats, only small amounts of BVU were found in the plasma, suggesting an important role of enterobacteria in the formation of BVU. Then, the metabolism of BV-araU prodrugs was studied in specific-pathogen free rats to select oral prodrugs of BV-araU with enhanced metabolic stability. 5'-O-Ethyl BV-araU (Et-BV-araU) gave about a 2-fold higher BV-araU blood concentration 3 and 6 hr after administration than after oral dosing of BV-araU, while the level of BVU was lower. Other aliphatic alkyl prodrugs also gave a lower level of BVU, but did not give the same elevation in blood concentration of BV-araU as did Et-BV-araU. Dosing of 5'-O-acetyl BV-araU resulted in blood concentrations of BV-araU and BVU similar to those after oral administration of BV-araU. 5'-O-Aromatic alkyl prodrugs showed poor bioavailability. A nearly 2-fold higher urinary recovery rate was seen for Et-BV-araU than for BV-araU or 5'-O-acetyl BV-araU. The conversion of Et-BV-araU to BV-araU was demonstrated in vitro using rat liver extract in the presence of co-factors, although the reaction was slow. The 5'-O-aliphatic alkyl prodrugs were completely resistant to degradation by enterobacteria, whereas the esters were partially degraded to BVU. Et-BV-araU may be a useful oral prodrug of BV-araU due to its increased metabolic stability and bioavailability.     

Putative role of dihydropyrimidine dehydrogenase in the toxic side effect of 5-fluorouracil in colorectal cancer patients

Dihydropyrimidine dehydrogenase (DPD) is the first and rate limiting enzyme in the catabolism of 5-fluorouracil (5-FU). It has been reported from various laboratories that the plasma concentration of 5-FU was influenced by DPD activities in various normal human organs (e.g. liver or lymphocytes). Since the congenital deficiency in DPD caused severe, in some cases lethal, FU-related toxicity, it was decided to collect data about the DPD activity in colorectal cancer patients in order to investigate the possible correlation between the enzyme activity and appearance of the side effects of 5-FU. Assuming that DPD activity in lymphocytes represents the 5-FU catabolic capacity of the organism, DPD activity was determined in the lymphocytes of 48 patients with colorectal cancer after surgery during the therapeutic course with 5-FU and folinic acid. On the basis of the enzyme activity, patients were divided into three categories: low (DPD <5.03 pmol/min/10(6) lymphocytes); medium (DPD = 5.04-13.25 pmol/min/10(6) lymphocytes), and high (DPD > 13.26 pmol/min/10(6) lymphocytes) activity groups. By evaluating the toxic side effects during the 5-FU + folinic acid treatment, the following results were obtained. In the low DPD activity group, 9 of 11 patients had 5-FU-related side effects (mucositis, diarrhea, myelotoxicity, angina pectoris, hypertension). In 3 patients, no change of the therapy was needed, in 3 patients symptoms could be reversed by dose reduction of 5-FU while in 3 patients interruption of 5-FU therapy was needed. In the medium DPD activity group, mild toxicity (diarrhea, transitory hypertension) occurred in 5 of 29 and in the high activity group (diarrhea) in 1 of 8 patients, respectively. In these last two groups, no dose reduction of 5-FU was necessary. The present study furnished further evidence for the possible correlation between the 5-FU side effects and DPD function. Consequently, it is recommended to measure DPD activity prior to 5-FU based chemotherapy, which might be helpful in avoiding drug-related toxicity by adjusting the dose of 5-FU individually.     

Hematopoietic stem cells found in lineage-positive subsets in the bone marrow of 5-fluorouracil-treated mice

It is known that treatment of mice with 5-fluorouracil (5-FU, 150 mg/kg) confers radioprotection. To investigate this effect, we performed bone marrow transplantation (BMT) using C57BL/6-Ly5 congenic mice treated with 5-FU five days prior to experiments. The mononuclear cells (MNC) in 5-FU-treated bone marrow (BM) were 10 times more radioprotective than those in untreated BM. Moreover, the number of BM MNC expressing c-kit on their surface from 5-FU-treated mice was markedly decreased relative to those from untreated controls. These results showed that the surface characteristics of cells that contributed to this radio-protective effect differ from those of stem cells as reported recently. BM MNC of mice treated with 5-FU were separated on the basis of expression of the lineage-specific antigens (Lin), c-kit, and Ly6A/E. When injected into lethally irradiated mice, 1,000 Lin+ and Lin-c-kit+Ly6A/E+ cells showed radioprotective effects such that 100% and 60% survived, respectively. Flow cytometric analysis 165 days after BMT showed that 88.8% and 65.1% of peripheral blood (PB) in mice transplanted with Lin+ and Lin-c-kit+Ly6A/E+ was derived from donor mice, respectively. After six months, donor-derived Lin-c-kit+Ly6A/E+ cells which showed radioprotective effects on a secondary irradiated host were detected from mice transplanted with Lin+ cells from 5-FU-treated mice. Taken together, these findings demonstrated that stem cells expressing Lin+ present in the BM of mice treated with 5-FU other than Lin-c-kit+Ly6A/E+ cells and these Lin+ cells play an important role in the recovery of myeloablative mice.     

First-pass metabolism of 5-fluorouracil in the perfused rat small intestine

The first-pass intestinal metabolism of 5-fluorouracil (5-FU) was investigated by single-pass perfusion of the rat small intestine. At the low concentration of 0.06 mg/ml, the fraction of 5-FU absorbed into (i.e., appeared in) the mesenteric venous blood (Fa,b) was about 50% smaller than the fraction absorbed (disappeared) from the intestinal lumen (Fa), indicating the first-pass extraction of 5-FU in the intestinal mucosa. By addition of uracil (6 mg/ml), the Fa of 5-FU was reduced presumably by competition for the pyrimidine carrier at the process of intestinal uptake (entry into the mucosa). The Fa,b was also reduced, but to a lesser extent, resulting in insignificant first-pass extraction. These results suggest that the extraction of 5-FU in the absence of uracil is caused by metabolism and that uracil is a competitor for this pathway. When 5-FU concentration was raised from 0.06 to 0.6 mg/ml in the absence of uracil, the Fa was reduced by about 50%, consistent with the suggestion of the involvement of saturable uptake by the pyrimidine carrier, and thereafter remained unchanged at 6 mg/ml. However, since Fa,b was also reduced by a similar extent, the intestinal availability (FI=Fa,b/Fa) was unchanged at about 0.5, indicating that the intestinal first-pass extraction of 5-FU is independent of concentration with the extraction ratio (difference between unity and FI) of about 0.5 over the wide range of concentration from 0.06 to 6 mg/ml. Thus, the present study demonstrates that the significant first-pass metabolic extraction of 5-FU occurs in the mucosa of the small intestine, supporting our previous suggestion that 5-FU undergoes first-pass metabolism not only in the liver but also in the small intestine after oral administration. Considering that the oral bioavailability of 5-FU in the human (28%) is reportedly comparable with that in the rat (28%), it is likely that intestinal first-pass metabolism may be significant also in the human. Intestinal first-pass metabolism should be taken into account to explore more efficient and controlled oral 5-FU therapy.     

Dihydropyrimidine dehydrogenase but not thymidylate synthase expression is associated with resistance to 5-fluorouracil in colorectal cancer

BACKGROUND/AIMS: In planning adjuvant treatment of colorectal cancer, it is of critical importance to optimize the treatment by identifying subsets of patients that will respond or not to chemotherapy. Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are key enzymes involved in the biochemical functions of the antimetabolite 5-fluorouracil (5-FU). In searching for the factors determining the 5-FU sensitivity of colorectal cancer, TS and DPD were analyzed in relation to the inhibitory effect of 5-FU on cell proliferation in a series of human colorectal cancer cell lines. METHODOLOGY: TS and DPD protein expressions were quantified in 5 human colorectal cancer cell lines, using TS binding assay and Western blotting, respectively. Cellular growth inhibition was assessed by MTT assay after 48 hours of continuous exposure to 5-FU or cisplatin (CDDP). RESULTS: TS protein expression was detected in all but one of the cell lines studied and varied within a 17-fold range, while DPD protein expression was detectable in only one cell line (CaR1). CaR1, which expressed the highest level of DPD and no detectable TS, showed remarkable resistance to 5-FU. The other colorectal cancer cell lines with undetectable DPD expression were sensitive to 5-FU. There was no correlation between TS expression and 5-FU sensitivity. All of the cell lines studied showed similar sensitivity to CDDP. CONCLUSIONS: These data suggest that DPD, but not TS, expression predicts 5-FU sensitivity in colorectal cancer cell lines.     

In vitro diffusion of 5-fluorourcil and tegafur through excized pig cornea

Permeability kinetics of 5-fluorouracil (5-FU), tegafur (TGF) and ftorafur (FT) through synthetic, lipophilic membranes and the pig excised cornea were studied. During the dialysis process, the drugs were dissolved in Ringer's solutions of either pH 6.89 or 7.98 and the drug concentrations were measured vs time by UV spectrophotometry at 37 degrees C. The diffusion processes of 5-FU, TGF and FT through the synthetic membranes were pH dependent and could be interpreted according to pseudo-first-order kinetics. However, the drug diffusion through the pig excised cornea was a zero-order process. The permeability rate of FT (TGF) across the pig cornea was greater if compared to that of 5-FU, whose partition coefficient (ko/w) was higher than those of the former drugs.     

Heterozygosity for a point mutation in an invariant splice donor site of dihydropyrimidine dehydrogenase and severe 5-fluorouracil related toxicity

Dihydropyrimidine dehydrogenase (DPD) is responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5-FU), thereby limiting the efficacy of the therapy. It has been suggested that patients suffering from 5-FU toxicities due to a low activity of DPD are genotypically heterozygous for a mutant allele of the gene encoding DPD. In this study we investigated the cDNA and a genomic region of the DPD gene of a cancer patient experiencing severe toxicity following 5-FU treatment for the presence of mutations. Although normal activity of DPD was observed in fibroblasts, the DPD activity in leucocytes of the cancer patient proved to be in the heterozygous range. Analysis of the DPD cDNA showed heterozygosity for a 165bp deletion that results from exon skipping. Sequence analysis of the genomic region encompassing the skipped exon showed that the tumour patient was heterozygous for a G-->A point mutation in the invariant GT splice donor sequence in the intron downstream of the skipped exon. So far, the G-->A point mutation has also been found in 8 out of 11 patients suffering from a complete deficiency of DPD. Considering the frequent use of 5-FU in the treatment of cancer patients, the severe 5-FU-related toxicities in patients with a low activity of DPD and the high frequency of the G-->A mutation in DPD deficient patients, analysis of the DPD activity and screening for the G-->A mutation should be routinely carried out prior to the start of the treatment with 5-FU.     

5-Fluorouracil prodrug: role of anabolic and catabolic pathway modulation in therapy of colorectal cancer

Following p.o. administration to rats bearing advanced colorectal carcinoma, Ftorafur (FT) is converted to 5-fluorouracil (FUra) by microsomal P450 in the liver. To optimize the therapeutic selectivity of the FUra generated from FT, three approaches were utilized: (a) inhibition of FUra degradation to dihydrofluorouracil by uracil as an alternative substrate for uracil reductase in the molar ratio of 4 uracil:1 FT (UFT); (b) modulation of drug inhibition of thymidylate synthase by leucovorin (LV); and (c) by increasing the level of FUra incorporation into cellular RNA by N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbomylase. The maximum tolerated dose (MTD) of FT and UFT, administered 3 times a day for 28 days, was 150 mg/kg/day and 60 mg/kg/day, respectively. The MTDs were not significantly modified by LV (150 or 600 mg/kg/day), administered by the p.o. route with the drugs, or by PALA (100 mg/kg) administered weekly by the i.v. route. The dose-limiting toxicity of FT alone and in combination with the modulators was stomatitis. The severe alopecia observed with FT alone was reduced significantly by uracil. At the MTD, the antitumor activity of UFT was superior to those of FT and FUra alone and in combination with LV and/or PALA. The 3-month sustained complete tumor regression for UFT, FT, and FUra was 38%, 0%, and 13% (for the weekly schedule), respectively. Although uracil, LV, and PALA individually increased the antitumor activity of FT at its MTD, the combination of the three modulators produced the highest therapeutic efficacy in rats bearing advanced colorectal carcinoma, in which 100% of the treated animals achieved complete and sustained tumor regression. The therapeutic efficacy observed with FT modulation could not be achieved with FUra administered by different schedules, each at its MTD alone or in combination with either LV or PALA. In brief, modulation of FT produced greater therapeutic efficacy and selectivity than FUra. Furthermore, the combined use of modulators capable of inhibiting the degradation pathway of FUra and potentiating the effects of the anabolic metabolites action appears to offer the greatest therapeutic potential.     

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.     

Phase I clinical and pharmacological studies of benzylacyclouridine, a uridine phosphorylase inhibitor

Benzylacyclouridine (BAU, IND 039655) is a potent and specific inhibitor of uridine phosphorylase (UrdPase; EC 2.4.2.3). This enzyme plays a major role in regulating uridine homeostasis and also catalyzes the conversion of fluoropyrimidine nucleosides to their respective bases. Inhibition of UrdPase enzyme activity 18-24 h after 5-fluorouracil (5-FU) administration increased plasma levels of uridine and enhanced the therapeutic index of 5-FU by rescuing normal tissues. Moreover, in vitro preclinical studies have also shown that inhibiting UrdPase enzyme activity by BAU prior to administration of 5-FU increased cytotoxicity in a number of human cancer cell lines. A series of preclinical studies was performed in dogs and pigs to evaluate the pharmacological and pharmacodynamic properties of BAU. These data showed a sustained elevation in plasma uridine concentration in both animal models. The rapid degradation of a tracer dose of uridine into uracil was virtually arrested by BAU administered both p.o. or i.v. The t1/2 of BAU was 1.8-3.6 h in dogs, with bioavailability levels of 85% (30 mg/kg) and 42.5% (120 mg/kg). In pigs, the half-life varied from 1.6 to 2.3 h, with a bioavailability of 40% at 120 mg/kg. The drug was distributed into most tissues with a tissue: plasma ratio of approximately 0.7. On the basis of these preclinical studies, we performed a Phase I clinical trial of BAU in patients with advanced cancer. Patients received 200, 400, 800, and 1600 mg/m2 BAU as a single oral dose. Toxicities included grade 2 anemia, grade 1 fever, grade 1 fatigue, grade 1 constipation, and grade 1 elevation in alkaline phosphatase; none of these toxicities were observed to be dose dependent. The maximum tolerated dose and dose-limiting toxicity were not reached at the doses given. BAU plasma concentrations and area under the curve correlated linearly with the oral dose level. The pharmacokinetics of BAU were consistent with a first-order clearance, with average peak concentrations ranging from 19 microM (200 mg/m2) to 99 microM (1600 mg/m2) and tbeta1/2 ranging from 3.0 to 3.9 h at the four dose levels. Compared with baseline plasma uridine, treatment of patients with 200, 400, 800, and 1600 mg/m2 BAU increased peak uridine concentrations by 120, 150, 250, and 175%, respectively. On the basis of this clinical study, the suggested Phase II starting dose of BAU in combination with 5-FU is 800 mg/m2. Studies combining BAU with 5-FU and incorporating appropriate molecular and biochemical end points to assess the effects of this drug combination on tumor and/or surrogate tumor tissue are under way.