5-Fluorouracil incorporation into RNA and DNA in relation to thymidylate synthase inhibition of human colorectal cancers.

BACKGROUND: The mechanism of action of 5-fluorouracil (5-FU) has been associated with inhibition of thymidylate synthase (TS) and incorporation of 5-FU into RNA and DNA, but limited data are available in human tumor tissue for the latter. We therefore measured incorporation in human tumor biopsy specimens after administration of a test dose of 5-FU alone or with leucovorin. PATIENTS AND METHODS: Patients received 5-FU (500 mg/m(2)) with or without high-dose leucovorin, low-dose leucovorin or l-leucovorin, and biopsy specimens were taken after approximately 2, 24 or 48 h. Tissues were pulverized and extracted for nucleic acids. 5-FU incorporation was measured using gas chromatography/mass spectrometry after complete degradation to bases of isolated RNA and DNA. RESULTS: Maximal incorporation into RNA (1.0 pmol/micrograms RNA) and DNA (127 fmol/micrograms DNA) of 59 and 46 biopsy specimens, respectively, was found at 24 h after 5-FU administration. Incorporation into RNA but not DNA was significantly correlated with intratumoral 5-FU levels. However, DNA incorporation was significantly correlated with the RNA incorporation. Primary tumor tissue, liver metastasis and normal mucosa did not show significant differences, while leucovorin had no effect. Neither for RNA (30 patients) nor DNA (24 patients) incorporation was a significant correlation with response to 5-FU therapy found. However, in the same group of patients, response was significantly correlated to TS inhibition (mean TS in responding and non-responding groups 45 and 231 pmol/h/mg protein, respectively; P=0.001). CONCLUSIONS: 5-FU is incorporated at detectable levels into RNA and DNA of human tumor tissue, but no relation between the efficacy of 5-FU treatment and incorporation was found, in contrast to TS.

Induction of thymidylate synthase as a 5-fluorouracil resistance mechanism.

Thymidylate synthase (TS) is a key enzyme in the de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP) from 2'-deoxyuridine-5'-monophosphate (dUMP), for which 5,10-methylene-tetrahydrofolate (CH(2)-THF) is the methyl donor. TS is an important target for chemotherapy; it is inhibited by folate and nucleotide analogs, such as by 5-fluoro-dUMP (FdUMP), the active metabolite of 5-fluorouracil (5FU). FdUMP forms a relatively stable ternary complex with TS and CH(2)THF, which is further stabilized by leucovorin (LV). 5FU treatment can induce TS expression, which might bypass dTMP depletion. An improved efficacy of 5FU might be achieved by increasing and prolonging TS inhibition, a prevention of dissociation of the ternary complex, and prevention of TS induction. In a panel of 17 colon cancer cells, including several variants with acquired resistance to 5FU, sensitivity was related to TS levels, but exclusion of the resistant variants abolished this relation. For antifolates, polyglutamylation was more important than the intrinsic TS level. Cells with low p53 levels were more sensitive to 5FU and the antifolate raltitrexed (RTX) than cells with high, mutated p53. Free TS protein down-regulates its own translation, but its transcription is regulated by E2F, a cell cycle checkpoint regulator. Together, this results in low TS levels in stationary phase cells. Although cells with a low TS might theoretically be more sensitive to 5FU, the low proliferation rate prevents induction of DNA damage and 5FU toxicity. TS levels were not related to polymorphisms of the TS promoter. Treatment with 5FU or RTX rapidly induced TS levels two- to five-fold. In animal models, 5FU treatment resulted in TS inhibition followed by a two- to three-fold TS induction. Both LV and a high dose of 5FU not only enhanced TS inhibition, but also prevented TS induction and increased the antitumor effect. In patients, TS levels as determined by enzyme activity assays, immunohistochemistry and mRNA expression, were related to a response to 5FU. 5FU treatment initially decreased TS levels, but this was followed by an induction, as seen with an increased ratio of TS protein over TS-mRNA. The clear retrospective relation between TS levels and response now forms the basis for a prospective study, in which TS levels are measured before treatment in order to determine the treatment protocol.

A phase II study of active specific immunotherapy and 5-FU/Leucovorin as adjuvant therapy for stage III colon carcinoma.

Active specific immunotherapy, using vaccines with autologous tumour cells and BCG, significantly reduces the rate of tumour recurrence in stage II colon cancer patients, while no clinical benefit has yet been observed in stage III patients. Adjuvant treatment with 5-Fluorouracil/Leucovorin is now considered standard therapy for stage III colon carcinoma and results in an absolute survival benefit of approximately 10%. Yet, the 5-year overall survival rate of stage III colon cancer patients is only 40-50%. Combining chemotherapy and immunotherapy might improve prognosis for stage III patients, especially when considering that active specific immunotherapy and chemotherapy have shown synergistic effects in pre-clinical tumour models. We performed a phase II study with 56 patients, using the combination of active specific immunotherapy and chemotherapy as an adjuvant therapy in stage III colon cancer patients to assess the influence of 5-Fluorouracil/Leucovorin on anti-tumour immunity induced by autologous tumour cell vaccinations. Anti-tumour immunity was measured before and after chemotherapy by means of delayed type hypersensitivity reactions, taken 48 h after the third and the fourth vaccination. We also investigated the toxicity of this combined immuno-chemotherapy treatment. Delayed type hypersensitivity reactions before chemotherapy had a median size of 20.3 mm, while after chemotherapy delayed type hypersensitivity size was 18.4 mm (P=0.01), indicating that chemotherapy hardly affected anti-tumour immunity. The severity of ulcers at the BCG vaccination sites was comparable to previous studies. In 30% of the patients grade III or grade IV chemotherapy related toxicity was seen; this is comparable to what is normally observed after adjuvant chemotherapy alone. This study shows that the active specific immunotherapy-induced anti-tumour immune response is only minimally impaired by consecutive chemotherapy and that the combined treatment of stage III colon cancer patients with active specific immunotherapy and 5-Fluorouracil/Leucovorin does not cause unexpected toxicity.

Downstream molecular determinants of response to 5-fluorouracil and antifolate thymidylate synthase inhibitors.

Thymidylate synthase (TS) is an essential enzyme for the de novo synthesis of thymidylate and subsequently DNA synthesis. TS has been used as a target for cancer chemotherapy in the development of fluoropyrimidines such as 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine and of novel folate-based TS inhibitors such as ZD1694 (Tomudex, Raltitrexed), ZD9331, LY231514 (ALIMTA, Pemetrexed), AG337 (Thymitaq, Nolatrexed) and AG331. Although TS has been considered as a target for chemotherapy, the precise mechanism by which TS inhibition leads to cell death is still not completely resolved. TS inhibition results in depletion of dTTP, an essential precursor for DNA, and an increase in dUTP. This results in the so-called thymine-less death due to misincorporation of dUTP into DNA; its excision, catalysed by uracil-DNA glycosylase, results in DNA damage. Both this imbalance in dTTP/dUTP and DNA damage can result in induction of downstream events, leading to apoptosis. On the other hand a specific interaction exists between oncogenes and TS, by binding of TS protein to the p53 and c-myc RNA, while wt p53 can also inhibit TS promotor activity. TS inhibition by either 5-FU or antifolates can also result in a depression of TS protein mediated inhibition of TS mRNA translation leading to induction of more TS protein synthesis, and p53 protein may further deregulate this process. These complex indirect and direct interactions between oncogenes and TS may have as yet unclear clinical implications, since most data are based on in vitro or in vivo studies and some results are contradictive. In some preliminary clinical studies evidence was postulated for a combined prognostic role for TS and p53. This knowledge should be used to design clinical studies with the aim to deliver effective treatment to potentially sensitive patients both in the adjuvant setting and in advanced stage disease.

Thymidylate synthase expression in patients with colorectal carcinoma using a polyclonal thymidylate synthase antibody in comparison to the TS 106 monoclonal antibody.

Colorectal cancer is one of the most common human cancers, for which 5-fluorouracil (5FU) is usually part of the treatment. Thymidylate synthase (TS), the target enzyme for 5FU, can be predictive for the outcome of 5FU-based therapy. TS levels in tumor samples can be determined with radiochemical enzyme assays, RT-PCR, and immunohistochemical staining. We validated TS immunohistochemistry with a polyclonal rabbit anti-human TS antibody using the avidin-biotin method. This antibody can be used on paraffin-embedded, formalin-fixed material using an antigen retrieval method with citrate buffer and microwave treatment. The antibody shows a granular cytosolic staining pattern. The reproducibility in cross-sections from colorectal tumors from 50 patients was 90% and the interobserver variability was acceptable with a kappa of 0.45. On Western blotting it detects purified TS at 36 kD, while in 5FU-treated cells the ternary complex between FdUMP, TS, and 5, 10-methylene-tetrahydrofolate is clearly visible at 38 kD, with no other interfering bands. In a separate set of tumors, immunostaining was compared with enzyme levels; Western blots correlated with enzyme levels. Because both this polyclonal antibody and the monoclonal antibody TS-106 are being used for large-scale studies, we also determined whether they could be used interchangeably. No differences were observed. This polyclonal antibody is specific and gives reproducible results. A study on a larger scale is ongoing to determine the role of TS as a predictive parameter in patients with colorectal cancer treated either with postoperative adjuvant 5FU/levamisole or with surgery only.

Development of a panel of 15 human ovarian cancer xenografts for drug screening and determination of the role of the glutathione detoxification system.

OBJECTIVES: We have established a panel of 15 human ovarian cancer xenografts grown subcutaneously in the flank of the nude mouse. Similar to the clinic, the xenografts show differences in histological subtype and volume doubling time. We determined whether the panel is useful for drug screening by testing the sensitivity to six conventional anticancer agents. In addition, we investigated whether the glutathione detoxification system affects sensitivity to cisplatin and cyclophosphamide, major drugs in the treatment of ovarian cancer. METHODS: Mice bearing well-established tumors were treated at maximum tolerated doses as defined by a reversible weight loss up to 15% of their initial weight: cisplatin 5 mg/kg iv weekly x2, cyclophosphamide 150 mg/kg ip 2-weekly x2, doxorubicin 8 mg/kg iv weekly x2, hexamethylmelamine ip 150 mg/kg every other day x4, methotrexate ip 150 mg/kg weekly x2, and 5-fluorouracil 60 mg/kg ip weekly x4. Glutathione levels and the activities of three different glutathione-dependent enzymes were measured in untreated xenograft tissues. RESULTS: Growth inhibition >75% was reached for cisplatin in 40%, for cyclophosphamide in 27%, and for doxorubicin in 20% of the xenografts. Methotrexate and 5-fluorouracil did not induce growth inhibition of importance. Hexamethylmelamine showed >75% growth inhibition in 53% of the xenografts, which may have been caused by the favorable metabolism of the drug in mice when compared with that in patients. Glutathione levels varied 3.6-fold in the xenografts and did not show a relation with sensitivity to cisplatin, cyclophosphamide, or doxorubicin. No relation was found between the activities of glutathione S-transferase and glutathione peroxidase and the sensitivities to the three anticancer agents. Glutathione reductase activity, however, showed a weak, inverse relation with the efficacy of cisplatin and cyclophosphamide (r values of -0.55 and -0.58, respectively). CONCLUSIONS: The sensitivity to the six anticancer agents of our panel of 15 human ovarian cancer xenografts reflects the response rates known for similar drugs in ovarian cancer patients. In that respect, the panel may be useful for drug screening as well as studies on the relevance of drug resistance features in vivo. The various components of the glutathione detoxification system did not predict for primary drug resistance which confirms clinical data in ovarian cancer.

The phenotypic profile of CD34-positive peripheral blood stem cells in different mobilization regimens.

The type of regimen used might result in mobilization of phenotypically and functionally different CD34(+) cells. We compared the phenotype of CD34(+) cells in leukapheresis products of three homogeneous groups: I, healthy individuals treated with granulocyte colony-stimulating factor (G-CSF) alone (n = 13); II, patients mobilized with G-CSF following chemotherapy (n = 16); and III, patients mobilized with G-CSF after high-dose chemotherapeutic pretreatment (n = 24). Multiparameter flow cytometry was performed for CD34(+) subpopulation analysis and focused on adhesion molecules, differentiation markers and megakaryocytic markers relevant for stem cell homing, with special reference to the importance of L-selectin expression. Regimens I and II led to higher numbers of mobilized CD34(+) cells (mean 468 x 10(6) and 491 x 10(6) CD34(+) cells per leukapheresis procedure respectively) than regimen III (mean 41 x 10(6) CD34(+) cells per leukapheresis procedure). Both the expression of L-selectin and CD54 on CD34(+) cells was significantly lower in group III, as was the percentage of megakaryocytic (CD41(+)) progenitors. A higher percentage of primitive (CD38(-) and/or HLA(-)DR(-)) CD34(+) cells was found in group III, correlating with a higher clonogenicity of the CD34(+) cells. However, when comparing the CD34(+)_ subpopulations that were also positive for L-selectin, there was no significant difference between the three regimens. A similar approach for the megakaryocytic CD34+ population resulted in an even worse quality of regimen III: 5.1% of CD34(+) being CD41(+)/L-selectin(+) compared with 9.2% and 8.9% in regimens I and II respectively. We concluded that the phenotypes of the CD34(+) cells in the G-CSF (group I) and G-CSF-chemotherapy (group II) regimens are similar, whereas the phenotype of the CD34(+) cells mobilized in the high-dose regimen (group III) displayed features that might negatively influence homing of the cells. Future studies will be directed towards regimens that will lead to the mobilization of a higher amount of CD34(+) cells with a phenotypically favourable phenotype.

Current chemotherapeutic possibilities in the treatment of colorectal cancer.

To date, the best treatment modality for colorectal cancer is a surgical excision of the primary tumour. Adjuvant therapy can be added to the surgical treatment and can consist of adjuvant chemo-, immuno- or radiotherapy. In the U.S.A., adjuvant chemotherapy with 5-fluorouracil (5FU) and levamisole is advocated as standard treatment for patients with localised poor risk (Dukes stage C) colon cancer. Not every clinician is convinced of the usefulness of adjuvant chemotherapy. Therefore, confirmatory clinical trials are still ongoing to compare no adjuvant treatment with 5FU/levamisole adjuvant treatment. Treatment with 5FU/leucovorin has been shown to be effective as adjuvant therapy. In rectal cancer, radiotherapy can be added to the primary surgical treatment. It is still unproven whether radiotherapy should be given pre-, peri, or postoperatively, and whether chemotherapy should be added to this multimodality regimen. If chemotherapy is applied as a radio-sensitiser, a continuous infusion is preferable to daily bolus injection. Much effort has been put into the improvement of the response rate of 10-15% 5FU, used as a single agent in the treatment of advanced colorectal cancer. Biochemical modulation of 5FU with leucovorin and interferon, different schedules of 5FU administration and hepatic arterial therapy have all been attempted. Higher response rates have been reported with these treatment modalities, unfortunately without improvement of survival, except for the intra-arterial approach. Recently, two new drugs have shown efficacy in the treatment of advanced colorectal cancer. A phase II trial with Tomudex (ZD1694), a new antifolate thymidiylate synthase inhibitor, produced a response rate of 25% in patients with advanced colorectal cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Thymidylate synthase and drug resistance.

Thymidylate synthase is an important target for both fluorinated pyrimidines and for new folate analogues. Resistance to 5-fluorouracil (5FU) can be related to insufficient inhibition of thymidylate synthase. The 5FU-nucleotide FdUMP induces inhibition of thymidylate synthase which is enhanced and retained for longer in the presence of increased folate pools, for which leucovorin is a precursor. In a murine model system, 5FU treatment caused a 4-fold induction of thymidylate synthase levels which may have contributed to resistance. Addition of leucovorin to this treatment prevented this induction and increased the antitumour effect 2-3-fold. In the clinical setting, 5FU administration to patients resulted in approximately 50% inhibition of TS after 48 h. The combination with leucovorin resulted in a more pronounced inhibition after 48 h (approximately 70%). A significant relationship was observed with outcome of treatment; when thymidylate synthase levels were high and inhibition was low, no response was observed. A separate study showed that low thymidylate synthase levels appeared to be an independent prognostic factor for adjuvant therapy.

Thymidylate synthase inhibition after administration of fluorouracil with or without leucovorin in colon cancer patients: implications for treatment with fluorouracil.

PURPOSE: To determine the time-dependence of fluorouracil (5FU)-induced thymidylate synthase (TS) inhibition in colon cancer patients, the effect of leucovorin (LV), and the relation to response. PATIENTS AND METHODS: A 5FU injection (500 mg/m2) was given to 47 patients with advanced colorectal cancer; tumor biopsy specimens were obtained 1 to 72 hours after laparotomy. Eleven patients received LV (2-hour infusion of 500 mg/m2) with 5FU midinfusion; biopsies were obtained after 45 hours. TS inhibition was evaluated by comparing the number of total and free 5-fluoro-2'-deoxy-uridine-5'- monophosphate (UMP) (FdUMP) binding sites and the total and residual catalytic activity of TS. RESULTS: The total catalytic TS activity varied from 0 to 621 pmol/h/mg protein and the total number of FdUMP binding sites varied from 0 to 976 fmol/mg protein. The residual catalytic TS activity after 2, 23, and 45 hours was 41%, 65%, and 74% of the total catalytic activity; the number of free FdUMP binding sites was 12%, 27%, and 49% of the total number, respectively. LV enhanced TS inhibition after 45 hours; the residual catalytic activity decreased from 74% to 49%, and the number of free FdUMP binding sites from 49% to 24%. Eleven of 19 patients treated with hepatic arterial infusion of 5FU had a partial response (PR). In the nonresponding patients, total TS activity was significantly higher (P < .05) than in responding patients. A high TS activity with a poor inhibition correlated with no response. CONCLUSION: Residual and total TS activity are predictive for response to 5FU. The findings may be applicable for treatment of patients with advanced disease and TS should be evaluated as a prognostic factor in adjuvant chemotherapy studies.

Response of peritoneal solid tumours after intraperitoneal chemohyperthermia treatment with cisplatin or carboplatin.

The combination of heat and chemotherapy was studied in an intraperitoneal tumour model. Rats bearing peritoneal CC531 tumours (2-6 mm) were treated i.p. with cDDP or CBDCA [maximal tolerated dose (MTD)] in combination with regional hyperthermia (41.5 degrees C, 1 h) of the peritoneal cavity. The addition of hyperthermia to the i.p. treatment led to a decrease in the MTD of cDDP by 33.3% at 41.5 degrees C. This was due to increased nephrotoxicity. The MTD of CBDCA did not change as a result of hyperthermia treatment. The chemo-hyperthermia treatment resulted in more cDDP or CBDCA DNA adducts in peritoneal tumours after the combined treatment than after chemotherapy alone. The increased tumour platinum concentrations, rising from 1.3 micrograms Pt g-1 tumour at 37 degrees C to 5.4 micrograms Pt g-1 tumour at 41.5 degrees C for cDDP and from 0.2 microgram Pt g-1 tumor to 0.7 microgram Pt g-1 tumour at 41.5 degrees C for CBDCA, contributed considerably to the enhanced numbers of cDDP or CBDCA DNA adducts. As a result of the latter, i.p. chemotherapy combined with regional hyperthermia led to an increase in tumour growth delay (TGD) after increasing the temperature to 41.5 degrees C for cDDP and CBDCA (by 40 days for cDDP, 22 days for CBDCA). These data were in agreement with the in vitro findings, i.e. that higher temperatures led to increased cytotoxicity.

Pharmacokinetics of paclitaxel and metabolites in a randomized comparative study in platinum-pretreated ovarian cancer patients.

PURPOSE: To investigate the pharmacokinetics and pharmacodynamics of paclitaxel in a randomized comparative study with four different treatment arms in patients with platinum-pretreated ovarian carcinoma. PATIENTS AND METHODS: Eighteen patients were entered onto this study in which paclitaxel was administered at a high dose of 175 mg/m2 versus a low dose of 135 mg/m2 on a 3- or 24-hour infusion schedule. A solid-phase extraction technique for sample pretreatment followed by a reverse-phase high-performance liquid chromatographic (HPLC) assay was used for analysis of plasma. RESULTS: Grade 3 neutropenia occurred in all four treatment arms. However, it was more severe on the 24-hour infusion schedule. Paclitaxel concentrations as low as 0.012 mumol/L were measured with the HPLC assay. With this low quantitation threshold, we found the plasma disappearance of paclitaxel to be triphasic, with half-lives t1/2(alpha), t1/2(beta), and t1/2(gamma) mean values for the different treatment arms of 0.19 hours (range, 0.01 to 0.4), 1.9 hours (range, 0.5 to 2.8), and 20.7 hours (range, 4 to 65), respectively. Eleven possible metabolites were found, of which three were identified as taxanes by on-line HPLC-photodiode array (PDA) detection. Investigation of pharmacodynamics shows no clear relationship between the pharmacokinetic parameters area under the plasma concentration time curve (AUC), area under the plasma concentration moment curve (AUMC), maximal plasma concentration (Cmax), clearance, and toxicity. However, a relationship was found between the duration of plasma concentrations above a threshold of 0.1 mumol/L with absolute neutrophil count (ANC) and white blood cell count (WBC). CONCLUSION: Paclitaxel is metabolized, and putative metabolic products can be found in plasma of patients treated with the drug. Our results indicate that myelosuppression can be predicted by the measurement of the duration of plasma concentrations above the threshold of 0.1 mumol/L.

Modulation of fluorouracil toxicity with uridine.

The antineoplastic agent 5-fluorouracil (5-FU) is used in the treatment of various tumor types. However, its antitumor activity is limited. To be active, 5-FU has to be metabolized. Its mechanisms of action have been largely elucidated but are complex. Combining 5-FU with biochemical modulating agents that interfere with 5-FU metabolism may enhance its therapeutic index. Uridine is one of these biochemical modulating agents. The aim of combining 5-FU with uridine is that the latter will reduce the toxicity of 5-FU while its antitumor activity is retained. This will enable the use of higher 5-FU doses with a potential increased antitumor effect. The combination 5-FU/uridine has shown clear activity in preclinical models. However, application in the clinic is limited. From the preclinical experience, it seemed that high doses of uridine giving rise to prolonged exposure of uridine to the tissues would be required to achieve the biochemical effect. Thus, initial clinical studies investigated tolerance and toxicities of high-dose uridine in humans. Dose-limiting toxicity was fever. High-dose uridine given as intermittent intravenous infusions was feasible and reversed 5-FU-induced leukopenia. High-dose uridine led to millimolar plasma concentrations of uridine. However, its half life was short due to rapid catabolism. Oral administration of uridine has also been studied, but bioavailability was low. Further studies are required to define the role of uridine in the biochemical modulation of 5-FU activity.

A rationale for carboplatin treatment and abdominal hyperthermia in cancers restricted to the peritoneal cavity.

The purpose of this study was to optimize the treatment of cancers restricted to the peritoneal cavity by combining i.p. chemotherapy with abdominal hyperthermia. In vitro experiments demonstrated that the uptake of carboplatin into CC531 tumor cells was increased at temperatures higher than 41.5 degrees C at dose levels of 5 and 50% cell kill. Carboplatin-DNA adduct formation and cytotoxicity, however, were already increased at temperatures of about 40 degrees C, indicating that carboplatin-DNA adduct formation and consequently cytotoxicity could be enhanced by mild hyperthermia (temperatures in the range of 39-41.5 degrees C). CC531 tumor bearing rats were treated i.v. and i.p. with carboplatin (6.15 mg/kg) in combination with regional hyperthermia of the abdomen (41.5 degrees C for 1 h). The mean temperature was 41.5 +/- 0.3 degrees C (SD) in the peritoneal cavity and 40.5 +/- 0.3 degrees C in the esophagus. Enhanced platinum concentrations were found in peritoneal tumors (factor 3) and in kidney, liver, spleen, and lung (a factor 2 average), after the combined i.v. or i.p. carboplatin-hyperthermia treatment. Pharmacokinetic data of i.p. CBDCA combined with hyperthermia demonstrated an increased tumor exposure for total and ultrafiltered platinum in plasma. The areas under the concentration x time curve for total platinum at 37 degrees C and 41.5 degrees C were 69 and 210 microM/h, respectively; for ultrafiltered platinum these values were 47 and 173 microM/h. This may have been due to a slower elimination of platinum from the blood at the higher temperature (t1/2 beta for total platinum 99 and 156 min at 37 and 41.5 degrees C, respectively). The direct exposure of the tumor via the peritoneal fluid appeared to diminish, since the area under the curve for total platinum was lower at 41.5 degrees C than at 37 degrees C (576 microM/h versus 1255 microM/h, respectively). Our results indicate that the advantage of adding hyperthermia is caused by an increased drug exposure of the tumor via the circulation. This was supported by the fact that platinum concentrations in peritoneal tumors after carboplatin treatment at elevated temperatures were similar for the i.p. and i.v. routes.

Reversal of 5-fluorouracil-induced myelosuppression by prolonged administration of high-dose uridine.

The effect of high-dose uridine on 5-fluorouracil (5-FU)-induced toxicity was investigated. Nine patients were treated weekly with 5-FU at increasing dosages. Five patients developed dose-limiting leukopenia, and four patients developed thrombocytopenia. At dose-limiting toxicity, 5-FU treatment was repeated and followed after 3 hours by intermittent iv infusion of uridine (2 g/m2 per hr) during 72 hours. Leukopenia was reversed for several weeks but thrombocytopenia was not. Side effects consisted of mild rises in body temperature. The pharmacokinetics of uridine were similar to those observed with single-agent uridine. Our data indicate that high-dose uridine can reduce the severity of 5-FU-induced myelosuppression.

The main steps in the development of anticancer agents.

The development of new anticancer agents is a long-term process, which involves the acquisition of new compounds, screening for antitumor activity, production and formulation, animal toxicology and finally, evaluation of toxicity and antitumor activity of the compound in man (Table I). In this paper, the main steps in the early development of new cytotoxic agents up to phase II clinical studies will be discussed. However, clinical phase III-IV trails, where the efficacy of the drug has been proven, should be dealt with separately.

In vitro biochemical and in vivo biological studies of the uridine 'rescue' of 5-fluorouracil.

The effect of delayed uridine administration on the in vitro growth inhibitory effects of 5-fluorouracil (5FU) and on the in vivo antitumour activity and toxicity was studied. In vitro growth inhibition of the human intestinal cell lines WiDr and Intestine 407 by 3 microM 5FU could be reversed by 1.0 mM uridine; the effect was more pronounced with WiDr cells. At 0.1 mM uridine an intermediate effect was observed. Inhibition of colony formation in both cell lines could also be reversed by delayed administration of uridine at 0.1 and 1 mM. Incorporation of 5FU into RNA of WiDr cells did not proceed after addition of uridine, in contrast to Intestine 407 cells. In these cells only a partial inhibition was observed. In vivo we studied the effect of uridine on two colon carcinoma tumour lines, the 5FU sensitive Colon 38 and the relatively resistant Colon 26. 5FU was administered i.p. in a weekly schedule. With Colon 26 delayed administration of uridine (3500 mg kg-1) at 2 and 20 h after 5FU enabled us to increase the 5FU dose from 100 to 250 300mg kg-1. The combination of high-dose 5FU and uridine resulted both in a superior antitumour effect and an increase in life span. In the 5FU sensitive Colon 38 we determined whether the sensitivity to 5FU was affected by uridine. Mice were treated at the non-lethal dose of 100 mg kg-1 which inhibited tumour growth almost completely. Delayed administration of uridine did not significantly affect the antitumour effect. In non-tumour bearing mice we studied the time course of the reversal of the haematological toxicity of 5FU. The effective dose of 100 mg kg-1 induced a significant decrease in leukocytes; in combination with delayed uridine the leukopenia was less severe and recovered more rapidly. 5FU also induced a decrease in haematocrit, which could be prevented by delayed administration of uridine. In conclusion, in cell culture the reversal of 5FU cytotoxicity could be achieved at a low concentration of 0.1 mM uridine, the extent of the reversal might be related to the 5FU incorporation into RNA. In vivo the relatively resistant tumour Colon 26 could be treated with a higher dose of 5FU in the presence of uridine. The sensitivity to 5FU of the sensitive Colon 38 was not affected by delayed administration of uridine, while the haematological toxicity of 5FU was less. So, delayed administration of uridine after 5FU resulted in an improved therapeutic effect in both a relatively resistant and sensitive tumour.

Effect of pyrimidine nucleosides on body temperatures of man and rabbit in relation to pharmacokinetic data.

The effect of high-dose uridine on body temperatures of rabbits and man has been studied in relation to plasma concentrations of uridine and its catabolite uracil. Uridine induced fever in both rabbits and man. High-dose cytidine had no influence on body temperature in rabbits. Plasma concentrations of uridine were between 1 and 1.5 mM at 30 min after an iv bolus injection of 400 mg uridine/kg in rabbits and reached peak levels of 2 mM after a 1-hr infusion of 12 g uridine/m2 in man. The plasma concentration of cytidine in rabbits was about 0.5 mM and that of uridine was 0.30 mM at 30 min after an iv bolus injection of 400 mg cytidine/kg. The mean residence time for uridine in patients and rabbits varied between 80 and 195 min. The area under the plasma concentration-time curve (AUC) for uridine in rabbits was 2.0 mmol.hr/liter, and that for cytidine was 0.6 mmol.hr/liter. A large AUC for uridine indicates a prolonged exposure of tissues to uridine, which might lead to extensive formation of degradation products. The administration of some of these catabolites, dihydrouracil (at 20-40 mg/kg), carbamyl-beta-alanine (at 60 mg/kg), and beta-alanine (at 300-400 mg/kg), resulted in a significant increase in body temperature. It is concluded that the change in body temperature associated with uridine administration was not due to bacterial pyrogens but that one of the degradation products might be involved in thermoregulation.

Human tumor xenografts in the nude mouse and their value as test models in anticancer drug development (review).

In an attempt to increase the predictability of preclinical antitumor testing, the value of human tumor lines in immune-deficient nude mice is assessed by reviewing the relevant literature. This test model is rather elaborate due to the nature of the animal as well as test and evaluation procedure. However, it represents a realistic simulation of clinical drug treatment. This is demonstrated by (a) a good correlation of drug effects in the nude mouse with clinical results in the donating patient's tumor and (b) by a good predictability of a panel of human tumor lines for clinically effective drugs. In order to avoid clinical trials with inactive drugs and no therapeutic benefit for a large number of patients, the application of human tumor xenografts in anticancer drug development is warranted.

Clinical and pharmacokinetic studies of prolonged administration of high-dose uridine intended for rescue from 5-FU toxicity.

A clinical and pharmacokinetic investigation of prolonged administration of high-dose uridine was performed in seven patients with advanced-stage cancer. Uridine administration was examined as a continuous infusion at 1 and 2.5 g/m2/hr (two patients) and as a series of intermittent infusions during 72 hrs at doses of 1-3 g/m2/hr, whereby 3-hr uridine administration was alternated with a 3-hr treatment-free interval (six patients). Continuous infusions of uridine resulted in plasma uridine concentrations of 0.5-1 mM, but was discontinued due to rapid increase in body temperature. Further studies focused on the intermittent schedule in an attempt to avoid the development of fever. Intermittent uridine infusion resulted in markedly elevated plasma uridine levels in the millimolar range. However, during the treatment-free period, rapid elimination of uridine was observed, resulting in plasma levels of 138-335 microM for 3 g/m2/hr. Plasma uracil concentrations also increased markedly, but smaller fluctuations compared to uridine were seen. Total urinary excretion of uridine was 15%-40% of the dose, while uracil excretion in urine was 2%-17%. Intermittent uridine infusion resulted in little or no rise in body temperature (less than or equal to 1.0 degrees C) in ten of 12 courses, and fever of greater than 39 degrees C in two courses. Both intermittent and continuous infusion of uridine gave rise to phlebitis, which necessitated central venous administration. These data show that using an intermittent infusion schedule, long-term administration of uridine is tolerable, with fever being dose-limiting. Intermittent infusion provides for the maintenance of markedly elevated plasma uridine levels and long-term uridine exposure to the tissues, and may be useful in further studies aimed at testing the potential of uridine to rescue patients from 5-FU toxicity.