Albert Leyva

Purine nucleosides as cell-specific modulators of 5-fluorouracil metabolism and cytotoxicity.

Purine nucleosides and ribose-5-phosphate (Rib-5-P) were used to modulate the metabolism and cytotoxicity of 5-fluorouracil (5-FU) in order to get a better understanding of the mechanism of action of 5-FU. In extracts from five different cell lines both Rib-5-P and inosine were relatively good precursors for Rib-1-P, but deoxyinosine was a moderate to poor precursor for deoxyRib-1-P. In the human colon carcinoma WiDr and the human epithelial intestinal Intestine 407 inosine enhanced Rib-1-P concentrations 3-6-fold. Incubation with deoxyinosine resulted in the appearance of deoxyRib-1-P in both cell lines in levels comparable to those of Rib-1-P. dIMP had the same effect as deoxyinosine in Intestine 407 cells, but not in WiDr cells. Both inosine and deoxyinosine caused a depletion of phosphoribosyl-pyrophosphate. In WiDr cells deoxyinosine (0.1-1.0 mM) clearly potentiated the growth inhibition by 0.1-0.5 microM 5-FU after 24 h of culture, but growth between 24 and 48 h was normal. In Intestine 407 cells the potentiation of 5-FU cytotoxicity by deoxyinosine was even more pronounced at 48 h than at 24 h. In WiDr cells dIMP did not potentiate 5-FU cytotoxicity, but in Intestine 407 cells the effect was comparable to that of deoxyinosine. The lack of potentiation in WiDr was accompanied by a low metabolism of dIMP. Growth inhibition by 5-FU and deoxyinosine could be reversed by thymidine in Intestine 407 cells but not completely in WiDr cells. Since the predominant target of the deoxyinosine-5-FU combination was thymidylate synthase, we analyzed the inhibition of this enzyme by FdUMP and the retention of the inhibition in cell culture. In both cell lines FdUMP was a potent competitive inhibitor of thymidylate synthase with a Ki of between 0.5 and 2 nM. Culture of cells in the presence of 5-FU and deoxyinosine resulted in an almost complete inhibition of thymidylate synthase activity after 24 h but after 48 h the activity was partly recovered. In Intestine 407 cells replenishment of the culture medium at 24 h even enhanced the recovery. Analysis of 5-FU anabolism into nucleic acids demonstrated that deoxyinosine inhibited the incorporation of 5-FU into RNA. It is concluded that in Intestine 407 cells addition of deoxyinosine enhanced the effects of 5-FU on growth inhibition due to increased formation of FdUMP leading to enhanced inhibition of thymidylate synthase.(ABSTRACT TRUNCATED AT 400 WORDS)

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-β-alanine (at 60 mg/kg), and β-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.

Separation of several 5-fluorouracil metabolites in various melanoma cell lines. Evidence for the synthesis of 5-fluorouracil-nucleotide sugars.

5-Fluorouracil (5FU) metabolism was studied in intact cancer cells kept in suspension by incubation with tritiated 5FU. Metabolites were analyzed using various chromatographic procedures, including a one-directional separation on PEI-cellulose sheets, which separated 5FU from the mono-, di- and triphosphate forms and from nucleotide sugars. The monophosphate ester was present as FdUMP, as could be demonstrated with another chromatographic procedure. In the human melanoma cell lines IGR3 and M5 the main metabolite of 5FU was 5-fluorouridine, while in the murine B16 melanoma only a small amount of 5-fluorouridine was formed. In B16 cells more 5FU label was present as the triphosphate ester while in M5 cells more FdUMP was formed. With all three cell lines 5FU was incorporated into RNA; this incorporation was stimulated by 1 mM N-(phosphonacetyl)-L-aspartate (PALA). PALA did not significantly affect the conversion of 5FU into other metabolites, nor did it affect the incorporation of 5FU into DNA. A 5FU-nucleotide sugar, present as the diphosphate-glucose, was a predominant 5FU-metabolite in M5 cells but not in the other cell lines. Its identity was confirmed by thin-layer chromatography and high-performance liquid chromatography. Its possible function is discussed.

Inhibition of cell growth by N-(phosphonacetyl)-L-aspartate in human and murine cells in vitro☆

Two murine cell lines, L1210 leukemia (T-cell) and B16 melanoma, and 3 human cell lines, CCRF-CEM leukemia (T-cell), NC37 lymphoblasts (B-cell) and IPC-48 melanoma were compared with respect to sensitivity to N-(phosphonacetyl)-L-aspartate (PALA), growth rate and aspartate transcarbamylase activity. No correlation between drug sensitivity and growth rate was found. The melanoma cell lines were more sensitive to PALA than were the lymphocytic cell lines. The 2 T-cell leukemia lines had similar sensitivities to PALA while the B-lymphoblasts were more resistant at 10(-3) M PALA and less resistant at 10(-4) M PALA than were L1210 and CCRF-CEM cells. Aspartate transcarbamylase activity was similar among the 2 melanoma cell lines and among the 3 lymphocytic cell lines and was 2-fold higher in the latter.

The concentration of 5-phosphoribosyl 1-pyrophosphate in monolayer tumor cells and the effect of various pyrimidine antimetabolites

5-Phosphoribosyl 1-pyrophosphate (PRPP) was determined in several murine and human cancer cell lines grown in monolayer, and harvested by trypsinization. For all cell lines a large variation in the PRPP concentration (5-1300 pmol/ 10(6) cells was found. A 1-hr incubation in Dullbeccos medium reduced the variation in PRPP concentration. After this incubation the highest concentration was found in the murine B16 melanoma cell line (about 200 pmol/10(6) cells). The human melanoma cell lines IGR3 and M5 and the human colon carcinoma cell line WiDr contained about 100 pmol/10(6) cells. After this preincubation of 1-hr these cell suspensions were used to study the effect of several antimetabolites on PRPP concentration. A 2-hr incubation with 1mM N-(phosphonacetyl)-L-aspartate (PALA) increased the PRPP concentration only in M5 cells, whereas methotrexate caused an increase in all cell lines. When 5-fluorouracil (5FU) was added, no significant decrease was found in any cell line. Addition of 5FU after a 2-hr preincubation with PALA resulted in a lower concentration in B16, M5 and WiDr cells. The prodrug, 5-fluoro-5 deoxyuridine altered the PRPP concentration only in in WiDr cells when it was added after PALA. The activity of the 5FU metabolizing enzyme orotate phosphoribosyl transferase was comparable in B16, M5 and WiDr cells, but much lower in IGR3 cells.

Plasma concentrations of carminomycin and carminomycinol in man, measured by high pressure liquid chromatography

In 9 patients with advanced malignant disease who received carminomycin (CMM) in an i.v. bolus injection (dose 18 mg/m2), curves of plasma concentrations of CMM and carminomycinol (CMMOH), a metabolite, versus time were constructed. For determination of plasma concentrations, high pressure liquid chromatography was used. For CMM and CMMOH the median areas under the curves (AUCs) were 31 (range 4-57) X 10(-8) mol/Ql/hr (measured over 24 hr) and 100 (range 309-158) X 10(-8) mol/l/hr (measured over 48 hr) respectively. From the data an accumulation of CMMOH in patients receiving treatments separated by brief intervals ban be predicted (half-life time of plasma disappearance for CMMOH was 2 days). Clinical toxicity was lowest in those 3 patients showing the lowest AUC for both CMM and CMMOH.

Purine modulation of thymidine activity in L1210 leukemia cells in vitro

Alteration of purine metabolism using adenine was studied in mouse L1210 leukemia cells for its effect on dThd-mediated inhibition of growth and deoxyribonucleotide pool perturbations. Inhibition of cell growth caused by 10 to 50 microM dThd was enhanced more than additively by 100 microM adenine which was only slightly inhibitory when administered alone. Adenine at 100 microM affected ribonucleotide levels by expanding the ATP pool and causing slight decreases in the GTP, UTP and CTP pools, while dThd alone or in combination with adenine had no effect on ribonucleotide pools. dThd at 10 microM caused a more than 2-fold increase in the dTTP pool and a marked but transient decrease in the dCTP pool with lesser effects on purine deoxyribonucleotide levels. Adenine at 100 microM produced only slight, transient increase in the dATP pool. Exposure of cells to dThd plus adenine compared with individual agents produced more than additive increases in dTTP and dATP pools. The decrease in the dCTP level was more with combined agents than with dThd alone. The results showed that an alteration in adenine nucleotide pools modifies the activity of dThd through greater enhancement of dTTP levels leading to a greater suppression of the dCTP pool.

Toxicity and antitumor effect of 5-fluorouracil and its rescue by uridine.

The pyrimidine analog 5-fluorouracil (5FU) has been used in the treatment against several solid carcinomas, mainly of the colon, for several decades (1). However, overall response rate is only about 20%, while gastrointestinal and myeloid toxicity are dose-limiting (1). Research on 5FU has been directed towards enhancing 5FU anabolism by combination with e.g. methotrexate (2) or thymidine (3) and towards the development of several analogs of 5FU. One recent analog, Doxifluridine (5′-deoxy-5-fluorouridine, 5’dFUR) showed an improved therapeutic index against a broad range of murine and rat tumors (4,5) and therapeutic activity in human advanced rectosigmoid adenocarcinoma (6).

Clinical and biochemical studies of high-dose thymidine treatment in patients with solid tumors

SummaryIn a clinical study of high-dose thymidine (TdR) treatment, toxic effects, TdR metabolism, and the influence of TdR on pyrimidine and purine metabolism were examined. Ten patients with solid tumors were treated with continuous infusion of TdR at 34–75 g/m2/day for 3 to 5 days. Hematologic toxicity occurred with 5-day TdR infusion at 75 g/m2/day but not when plasma TdR concentration failed to reach millimolar levels. In three patients who received similar TdR doses, plasma TdR levels were related to elimination rates of TdR and its metabolites from plasma. In one patient in whom urinary excretion was studied, 100% of the TdR dose given was recovered in the form of TdR, thymine (Thy), β-aminoisobutyrate, and 5-hydroxymethyluracil (5-HMUra). The latter metabolite, which had not been previously described in high-dose TdR treatment, was also found in plasma at levels from 5% to 10% of those of TdR. No effects of high-dose TdR infusion on purine levels in plasma were observed, while a substantial increase in uracil levels was noted both in plasma and urine. These data provide further information on high-dose TdR treatment with regard to clinical, pharmacokinetic, and biochemical effects.

Determination of thioproline in plasma using high performance liquid chromatography

The determination of thioproline in plasma of cancer patients, using high performance liquid chromatography (HPLC) is reported. As column support, a silica-bonded cation exchanger was used. Detection was performed at 205 nm. The detection limit of the method was 5 X 10(-6) M and the linear dynamic range was over 500. No sample clean-up procedure was necessary other than deproteinization of the plasma. The method was applied to the measurement of plasma drug levels in 3 patients, part of a clinical trial testing the effectiveness of thioproline as an anti-cancer agent.