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Biomedical subjects

E Laurensse

Publications and source records attributed to E Laurensse.

At least 19 recordsLinked to original sources
PubMed1992

Time course of inhibition of thymidylate synthase in patients treated with fluorouracil and leucovorin.

The inhibition of thymidylate synthase (TS) by the fluorouracil (5-FU) metabolite 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) is considered to be one of the main mechanisms of action of 5-FU. The inhibition of TS is mediated by the formation of a ternary complex between TS, FdUMP, and the folate cofactor 5,10-methylene tetrahydrofolate. The activity of TS, its inhibition by FdUMP, and the binding of FdUMP to TS have been determined in biopsy specimens of colorectal tumors, liver metastases, normal colon mucosa, and liver obtained from patients who never had received chemotherapy, and patients treated with 5-FU or 5-FU with leucovorin (LV). In nontreated patients we observed a large variation in the activity of TS both at 1 microM and 10 microM dUMP (40- to 80-fold difference). In contrast, in normal colonic mucosa this variation was less than 10-fold. FdUMP binding in tumors also varied considerably but was not detectable in normal mucosa. The deviations from normal (ie, as found in mucosa) kinetic patterns of TS may represent a mutant TS form. Thirty-five patients with advanced colorectal cancer received 5-FU (500 mg/m2) at 1 to 48 hours prior to surgery. In biopsy specimens of tumor and normal tissues the residual catalytic activity of TS and the percentage of free-binding sites for FdUMP (TS-free) were determined. After dissociation of FdUMP, total catalytic activity of TS- and total FdUMP-binding sites (TS-tot) were determined. Total and residual catalytic TS activity in primary tumors and metastases showed a large variation. TS-tot and TS-free in tumors also varied considerably. At least eight patients with an undetectable TS-free showed response to subsequent intraarterial treatment with 5-FU. To several patients leucovorin (2-hour infusion of 500 mg/m2) was administered with a 5-FU bolus (500 mg/m2) in the middle of the infusion. Biopsy specimens were obtained about 48 hours after treatment. In these patients inhibition of TS was markedly enhanced compared with patients who did not receive LV. The large variation in TS may be related to the observed variation in clinical response to 5-FU treatment.

Adult↗
PubMed1991

Clinical and pharmacologic study of orally administered uridine.

Effects of oral administrations of uridine were investigated in a study of six healthy volunteer control subjects and nine patients with metastatic colorectal cancer. Oral uridine was studied as single-dose administrations at doses escalating from 0.3 to 12 g/m2 and as multiple-dose administrations every 6 hours for 3 days at doses from 5 to 10 g/m2. The maximum tolerated dose (MTD) was 10 to 12 g/m2 for a single dose of uridine and 5 g/m2 for the multiple-dose regimen. Diarrhea was the dose-limiting toxic effect. Single-dose oral uridine resulted in an increase in plasma uridine concentrations in the range of 60 to 80 microM after doses of 8 to 12 g/m2. At these doses, bioavailability of oral uridine ranged from 5.8% to 9.9%. At the MTD of 5 g/m2 in the multiple-dose uridine schedule, steady-state plasma uridine levels of approximately 50 microM were achieved. Further studies should explore the role of oral uridine in the modulation of the toxicity of fluorouracil.

Administration, Oral↗
PubMed1989

The relation between inhibition of cell growth and of dihydroorotic acid dehydrogenase by brequinar sodium.

The growth inhibitory effects of Brequinar Sodium (DUP-785; NSC 368390) in 7 different cell lines were related to growth rates and to the inhibition of dihydroorotic acid dehydrogenase (DHO-DH) activity. IC50 values were between 0.2 and 5.8 microM; the fastest growing cell line was least sensitive. Despite a large variation in sensitivity, basal activity of DHO-DH showed little variation (only 2-fold) between the different cell lines. Residual activity of DHO-DH in the presence of Brequinar Sodium varied 30-fold. Drug sensitivity correlated with this residual DHO-DH activity; DHO-DH activity was only slightly inhibited by Brequinar Sodium in the most resistant lines, and almost completely in the most sensitive.

Animals↗
PubMed1989

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.

Aged↗
PubMed1988

DUP 785 (NSC 368390): schedule-dependency of growth-inhibitory and antipyrimidine effects.

DUP 785 (NSC 368390; Brequinar sodium) is a new inhibitor of pyrimidine de novo biosynthesis with antitumor activity against several experimental tumors. DUP 785 inhibits the mitochondrial enzyme dihydroorotate dehydrogenase, blocking the conversion of dihydroorotate to orotate. We examined the influence of exposure time to DUP 785 on its growth-inhibitory effects in L1210 murine leukemia and WiDR human adenocarcinoma cells and the effects of pyrimidine (deoxy) nucleosides on reversal of growth-inhibition. The results were correlated with changes in intracellular pyrimidine nucleotide pools and cell cycle distribution. In L1210 cells, a continuous exposure to 25 microM DUP 785 up to 96 hr caused complete growth inhibition. A 2 hr exposure of cells to the drug did not affect growth. In WiDR cells, exposure to the drug for 1-24 hr, followed by cultivation in drug-free medium resulted in recovery of growth. However, cells exposed to the drug for 48 hr or longer were not able to resume growth when recultured in drug-free medium. Reversal studies were performed to know whether selective depletion of one of the pyrimidine (deoxy) nucleotides might be related to the growth-inhibitory effects of DUP 785. Neither thymidine, deoxycytidine alone, deoxycytidine plus tetrahydrouridine; nor cytidine plus tetrahydrouridine added after 24 hr were able to reverse cell growth inhibition induced by 25 microM DUP 785. However, uridine and cytidine alone reversed growth inhibition. UTP and CTP pools in L1210 cells decreased to about 30-40% of control levels after 4 hr of drug exposure, while dTTP and dCTP pools decreased to about 30% of control levels. There were no significant changes in purine nucleotide pools. In WiDR cells, UTP and CTP pools decreased rapidly after drug exposure and were substantially depleted after 24 hr. Reculture of cells in drug-free medium resulted in a significant recovery of UTP and CTP levels only for cells exposed to DUP 785 for 1-24 hr. For cells exposed to the drug for 48 and 72 hr recovery of nucleotide pools was minimal. In L1210 cells, a 12-hr exposure to the drug caused an accumulation of cells in the early S-phase. In WiDR cells, there was a clear accumulation of cells in the S-phase of the cell cycle after 24 hr drug exposure.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

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.

Animals↗
PubMed1987

A sensitive, nonradiometric assay for dihydroorotic acid dehydrogenase using anion-exchange high-performance liquid chromatography.

A new method to assay the mitochondrial pyrimidine de novo enzyme, dihydroorotate (DHO) dehydrogenase, which catalyzes the dehydrogenation of DHO, with orotic acid as the product was developed. The assay was optimized using a rat liver mitochondrial preparation. Orotic acid was quantified with high-performance liquid chromatography using an anion-exchange column (Partisil-SAX) with uv detection at 280 nm. Isocratic elution with low phosphate buffer at pH 4.0 was used. The detection limit was 20 pmol per injection, which is comparable to previously described radiometric assays. The HPLC assay was compared with a spectrophotometric assay measuring orotic acid formation in a deproteinized reaction mixture. Absorbance was measured at the optimal wavelength for orotic acid, 278.5 nm. This assay is less sensitive and less specific than the HPLC assay, which can also detect UMP which might be formed from orotic acid in whole homogenates. With both assays kinetic parameters of the enzyme were determined. In the high concentration range (80-1000 microM) both Km and Vmax values were comparable. With the HPLC assay the concentration range was extended down to 12 microM and initial rates could be determined. The apparent Km was about 12 microM. The HPLC assay is also suitable for use in the study of inhibition of DHO dehydrogenase.

Animals↗
PubMed1987

Inhibition of pyrimidine de novo synthesis by DUP-785 (NSC 368390).

The mechanism of action of NSC 368390 (DUP-785, 6-fluoro-2-(2'-fluoro-1, 1'-biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid sodium salt) was studied using three different approaches. First, we studied growth inhibition by DUP-785 in L1210 leukemia cells and M5 melanoma cells. The concentrations causing 50% growth inhibition after 48 hr of culture were 5.8 and 0.6 microM, respectively. DUP-785 had to be present continuously throughout culture. Growth inhibition by 25 microM DUP-785 could be prevented by addition of 1 mM uridine or orotic acid to cultures of these cell lines; in M5 cells cytidine was also able to prevent growth inhibition. Dihydro-orotic acid (DHO) and carbamyl-aspartate were not able to prevent growth inhibition by DUP-785. Second, we studied accumulation of orotic acid and of orotidine induced by incubation with 1 microM pyrazofurin, an inhibitor of the orotate phosphoribosyl-transferase-orotidine-monophosphate decarboxylase complex. Addition of DUP-785 to the culture medium prevented the orotic acid accumulation. Furthermore, DUP-785 prevented accumulation of H14CO3- into orotic acid of pyrazofurin-treated L1210 cells. Third, we measured the effect of DUP-785 on DHO-dehydrogenase (DHO-DH), since the results indicated that this enzyme was affected by DUP-785. DHO-DH was assayed in isolated rat liver mitochondria. The Km for L-DHO was about 12 microM. DUP-785 appeared to be a potent inhibitor of DHO-DH with an apparent Ki of about 0.1 microM and an apparent Ki' of about 0.8 microM. The mode of inhibition appeared to be linear mixed type. After exposure of L1210 cells to 25 microM DUP-785 for 2 hr DHO-DH was almost completely inhibited. After suspension in fresh medium without drug, DHO-DH activity was recovered to about 60% after 24 hr. In conclusion, DUP-785 is a potent inhibitor of pyrimidine de novo biosynthesis, by inhibition of the mitochondrial enzyme DHO-DH.

Amides↗
PubMed1987

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)

Animals↗
PubMed1987

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.

Animals↗
PubMed1986

Sensitivity of human, murine, and rat cells to 5-fluorouracil and 5'-deoxy-5-fluorouridine in relation to drug-metabolizing enzymes.

Six cell lines differing in histological origin were studied regarding the growth inhibitory effect of fluoropyrimidines in relation to their metabolism. The human colon carcinoma cell line WiDr was most sensitive to 5-fluorouracil (FUra) (50% growth inhibitory concentration, 0.7 microM) and to its analogue 5'deoxy-5-fluorouridine (5'dFUR) (50% growth inhibitory concentration, 18 microM). The murine B16 melanoma cell line was moderately sensitive to FUra but least sensitive to 5'dFUR. The 50% growth inhibitory concentration values in the human melanoma cell lines IGR3 and M5, the transformed human intestine cell line intestine 407 and the rat hepatoma cell line H35 varied for FUra between 1.7 and 5.0 microM, and for 5'dFUR between 54 and 160 microM. Several enzymes from pyrimidine metabolism responsible for FUra metabolism were measured with FUra as a substrate. The activity of uridine phosphorylase, which catalyzes the conversion of 5'dFUR to FUra, was lowest in B16 cells correlating with the low sensitivity to 5'dFUR. When adenosine 5'-triphosphate was included in the reaction mixture for uridine phosphorylase, FUra was rapidly channeled into FUra nucleotides via its nucleoside. The rate of channeling appeared to correlate with the nucleoside phosphorylase activity in the various cell lines. In several cell lines activities of nucleotide-degrading enzymes were rather high and interfered with the measurement of orotate phosphoribosyl transferase (OPRT) with FUra as substrate. Addition of the phosphatase inhibitor glycerol-2-phosphate partly prevented breakdown of the newly formed 5-fluorouridine 5'-monophosphate and enabled measurement of OPRT. The WiDr cell line had a relatively high OPRT activity which could explain its sensitivity to FUra. The activity of thymidylate synthase was measured at a suboptimal concentration of 1 microM and at the optimal concentration of 10 microM deoxyuridine 5'-phosphate. With all cell lines the ratio between the activities at 10 and 1 microM was between 2.3 and 3.6. The activity of thymidylate synthase was lowest in WiDr and IGR3 cells and 3-4 times higher in M5 and Intestine 407 cells. The inhibition of 0.01 microM 5-fluorodeoxyuridine 5'-monophosphate was 80-90% at 1 microM deoxyuridine 5'-phosphate and 50-70% at 10 microM deoxyuridine 5'-phosphate with all cell lines. At 0.1 microM 5-fluorodeoxyuridine 5'-monophosphate enzyme activity was inhibited by 95-100%. The incorporation of FUra into RNA was relatively low in IGR3 cells and 3-5 times higher in all other cell lines.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

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 Dullbecco's 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.

Animals↗
PubMed1984

Fluctuations in phosphoribosyl pyrophosphate levels in monolayer tumor cell lines. Effects of drugs.

The concentration of PRPP (phosphoribosyl pyrophosphate) measured in tumor cells grown in monolayer showed a large variation with the various harvesting methods examined, including trypsinization. This variation could be reduced by a 1-h incubation of trypsinized cells as a suspension in Dulbecco's medium. After this preincubation these cell suspensions were suitable for the study of modulation of PRPP. One microM methotrexate caused a 2-3-fold increase and 1 mM N- phosphonoacetyl -L-aspartate a slight increase, but inosine and deoxyinosine drastically reduced PRPP concentrations. 5-Fluorouracil had no effect. This study demonstrates that metabolic parameters such as PRPP concentrations can be studied conveniently in suspensions of cells which are commonly cultivated as monolayers.

Animals↗
PubMed1984

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.

Animals↗