Emile Laurensse

A comparison of 5-fluorouracil metabolism in human colorectal cancer and colon mucosa

The metabolism of 5‐fluorouracil (5‐FU) was studied in biopsy specimens of primary colorectal cancer and healthy colonic mucosa obtained from previously untreated patients immediately after surgical removal. The conversion of 5‐FU to anabolites was measured under saturating substrate (5‐FU) and cosubstrate concentrations. For all enzymes, the activity was about threefold higher in tumor tissue compared with healthy mucosa of the same patient. The activity of pyrimidine nucleoside phosphorylase with deoxyribose‐1‐phosphate (dRib‐1‐P) was about tenfold higher (about 130 and 1200 nmol/hr/mg protein in tumors) than with ribose‐1‐phosphate (Rib‐1‐P), both in tumor and mucosa. Synthesis of the active nucleotides (5‐fluoro‐uridine‐5′‐monophosphate [FUMP] and 5‐fluoro‐2′deoxyuridine‐5′‐monophosphate [FdUMP]) was studied by adding physiologic concentrations of adenosine triphosphate (ATP) to the reaction mixture; the rate of FdUMP synthesis was 50% of that of FUMP (about 4 and 7 nmol/hr/mg protein in tumors). Direct synthesis of FUMP from 5‐FU in the presence of 5‐phosphoribosyl‐1‐pyrophosphate (PRPP) was about 2 nmol/hr/mg protein. With the natural substrate for this reaction, orotic acid, the activity was about 14‐fold higher. To obtain insight into the recruitment of precursors for these cosubstrates, the authors also tested the enzyme activity of pyrimidine nucleoside phosphorylase with inosine and ribose‐5‐phosphate (Rib‐5‐P, as precursors for Rib‐1‐P) and deoxyinosine (as a precursor for dRib‐1‐P); enzyme activities were approximately 7%, 7%, and 3%, respectively, of that with the normal substrates, both in tumors and mucosa. However, when ATP and Rib‐5‐P were combined, the synthesis of FUMP was about 70% of that with PRPP, but only in tumors. In normal tissues no activity was detectable. These data suggest a preference of colon tumor over colon mucosa for the conversion of 5‐FU to active nucleotides by a direct pathway; a selective antitumor effect of 5‐FU may be related to this difference.

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)

Thymidylate synthase from untreated human colorectal cancer and colonic mucosa: Enzyme activity and inhibition by 5-fluoro-2′-deoxyuridine-5′-monophosphate

Inhibition of thymidylate synthase (TS) by the 5-fluorouracil (5-FU) metabolite FdUMP is considered to be the main mechanism of action of 5-FU. TS from colorectal tumours and normal colon mucosa from 10 untreated patients was studied. There was a large variation in the activity of tumour TS both at 1 and 10 mumol/l of its substrate dUMP; in normal mucosa this variation was less. Inhibition by 10 nmol/l FdUMP in tumours varied from 80 to 90% at 1 mumol/l dUMP; in normal mucosa, inhibition varied from 10 to 80%. The number of FdUMP binding sites ranged from 0.1 to 1 in tumours but such binding sites were not detectable in normal mucosa. The ratio between TS activity and FdUMP binding sites varied considerably in tumours but not in normal mucosa. The deviations from normal kinetics may represent a mutant TS form. Alterations in TS may partly account for differences in response to 5-FU.

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.

Retention of in vivo antipyrimidine effects of brequinar sodium (DUP-785; NSC 368390) in murine liver, bone marrow and colon cancer

Brequinar sodium (DUP-785) is a potent inhibitor of the pyrimidine de novo enzyme, dihydroorotic acid dehydrogenase (DHO-DH). In order to determine whether in vitro data could be extrapolated to the in vivo situation we investigated antipyrimidine effects of DUP-785 in mice bearing colon cancer. Two tumor models were used, Colon 26 and Colon 38, resistant and moderately sensitive to DUP-785, respectively. DUP-785 at 50 mg/kg caused a depletion of plasma uridine in mice, and depleted tissue uridine levels in Colon 38 down to 10%, which was retained for several days; in Colon 26 the decrease was less and tissue uridine levels recovered rapidly. In livers of these mice no significant effect on uridine was observed. DUP-785 depleted UTP in bone marrow cells within 2 hr to 25% of control levels, after 4 days normal levels were found. In livers of both Balb-c mice (bearing Colon 26) and C57Bl/6 mice (bearing Colon 38) a small decrease of uridine nucleotide pools was found. In Colon 26 DUP-785 increased uridine nucleotide pools to 170% after 2 hr, at 1 day normal levels were observed, but after 2 days again an increase was found. In Colon 38 DUP-785 decreased the uridine nucleotide pool by 50% after 1 and 2 days. DUP-785 did not affect cytidine nucleotide pools of livers and of Colon 26 and Colon 38. The ratio between uridine nucleotides and cytidine nucleotides decreased from 2.2 to 0.90 in Colon 38, in the other tissues the decrease was less. DHO-DH was measured in bone marrow cells and Colon 26 and 38 before and after treatment. Basal levels of DHO-DH were 3 times higher in Colon 26 than in Colon 38. In treated tumors DHO-DH was initially inhibited by more than 90%, after 7 days enzyme activity in Colon 26 was 50% and in Colon 38 about 200% of basal levels. In bone marrow cells DHO-DH was also rapidly inhibited but recovered within 4 days. It is concluded that the retention of antipyrimidine effects of DUP-785 in Colon 38 were more pronounced than in Colon 26, which is in agreement with the better antitumor effect of DUP-785 in Colon 38.

A sensitive non-radioactive assay for pyrimidine nucleoside phosphorylase using reversed-phase high performance liquid chromatography

A new sensitive assay for pyrimidine nucleoside phosphorylase using non-radioactive substrates is described. With the natural substrate uridine (UR) and the analog, 5-deoxy-5-fluorouridine (5dFUR) conditions have been optimized to measure the product formation with reversed-phase high-performance liquid chromatography. Using automated injection large series of samples may be analyzed. The assay for UR phosphorylase appeared to be comparable to existing methods with radiolabeled UR as substrate regarding sensitivity and linearity. The assay has been used to measure kinetic parameters for 5dFUR and UR in two cell lines from intestinal origin.

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.

Fluoropyrimidine metabolism in human head and neck cancer xenografts and murine colon tumors

Human head and neck xenografts (HNX) tumor lines represent an unique model to study the action of anticancer drugs. 1/4 HNX lines resistant to 5-fluorouracil (5FU) was sensitive to its analog 5′deoxy-5-fluorouridine (5′dFUR). To explain these differences we studied metabolism of 5FU and 5′dFUR in 4 HNX lines (DU, KE, E, G) and for comparison also in two murine colon carcinoma lines (Colon 26 and 38). Initial conversion of 5′dFUR to 5FU catalyzed by pyrimidine nucleoside phosphorylase (PNP), was highest in Colon 26, 15-20 times lower in DU, KE and Colon 38 and intermediate in both other tumors. The Km for 5′dFUR in all tumors was 1-2 mM. PNP also catalyzes further anabolism of 5FU to flu-orouridine (FUR) or 2′-deoxyfluorouridine (FUdR); the same pattern of activity was found as with 5′dFUR as substrate. In all HNX tumors 5FU conversion to FUdR was 5-10 fold than of 5FU to FUR; in the colon tumors this was 3 fold. The conversion of 5FU to the active nucleotides FUMP and FdUMP (via FUR and FUdR, resp) was measured by addition of ATP to the PNP assay; FUMP synthesis showed the following pattern: Colon 26 » DU > G > E > KE » Colon 38, and FdUMP synthesis: Colon 26 ≥ DU = KE>E > G = Colon 38. For the direct conversion of 5FU to FUMP the following pattern was observed: Colon 26 > Colon 38 > KE > E = DU = G. Colon 26, 38 and KE were sensitive to 5′dFUR. Conclusion: the anabolism of 5′dFUR to 5FU and subsequently to nucleotides (via 5FU-> FUMP) may be related to the differential sensitivity of the tumors.

113 THE RELATIONSHIP BETWEEN DIHYDROOROTIC ACID DEHYDROGENASE AND IN VITRO AND IN VIVO CYTOSTATIC EFFECTS OF BREQUINAR (DUF-785; NSC 368390)

DUP-785 (Brequinar sodium) is a novel potent inhibitor of the pyrimidine de novo enzyme dihydroorotic acid dehydrogenase (DHO-DH). We studied the relationship between DHO-DH activity and in vitro growth-inhibitory effects and in vivo antitumor effects of DUP-785. Seven cell lines from different histological origin were continuously exposed to DUP-785 for 48 hr. The human squamous carcinoma cell line 14C was most sensitive (IC50 0.19 μM), three other cell lines had a comparable IC50 of about 0.4 μM, while rat hepatoma H35 and murine leukemia L1210 had a IC50 of 2.6 and 5.9 μM, resp. DHO-DH activity varied between 15 and 20 μmol/hr.106 cells in the cell lines tested. Inhibition by 1.3 μM DUP-785 was 80-90% in the most sensitive lines, compared to 50% in H35 and 30% in L1210. The sensitivity of two murine colon tumors was investigated in vivo by injection of 50 mg/kg at day 0, 4, 8 and 12. The doubling time of Colon 26 for untreated and treated tumors was 2.7 and 2.8 days, resp, while for Colon 38 these values were 5.1 and 8.3 days, resp. DHO-DH activity was 230 and 167 nmol/hr per mg protein, resp. In both tumors 50 mg DUP-785/kg decreased DHO-DH activity to below 10% after 4 hr, which was partly recovered after 1 day. After 1 day the concentration of uridine nucleotides in Colon 26 decreased by 30% and in Colon 38 by 50%, followed by an increase of 130 and 180%, resp, after 4 days. In conclusion; in vitro the extent of inhibition of DUP-785 was related to the growth-inhibition, but in vivo this relation was less evident.

MODULATION OF CYTOTOXICITY AND METABOLISM OF 5-FLUOROURACIL (FU) IN TWO INTESTINE CELL LINES: 156

Initial FU metabolism is catalyzed by either pyrimidine phosphorylase (PNP) or orotate phosphoribosyltransferase (OPRT). The human colon carcinoma cell line WiDr is 3 times more sensitive to FU and 5 times more sensitive to 5deoxy-5-fluorouridine than the intestinal cell line Intestine 407. However, in both cell lines PNP activity with either ribose-1-P (Rib-1-P) or deoxyRib-1-P (dRib-1-P) is comparable with OPRT activity (1-2 nmol/hr per 106 cells). Modulation of the availability of co-substrates with purine nucleosides might elucidate the mechanism of action of FU. PNP activity with inosine as co-substrate was 30 and 86% in WiDr and Intestine 407, and with deoxyinosine 7 and 19%, respectively, of the activity with Rib-1-P and dRib-1-P. Inosine increased the Rib-1-P levels 2-4-fold, while deoxyinosine enhanced dRib-1-P levels several fold. Inosine did not show a synergism with FU in growth-inhibition. Deoxyinosine at 0.1-1 mM showed synergism with FU at non-toxic concentrations (0.1-0.5 uM). Deoxyinosine itself was not toxic. The synergism was greater with Intestine 407 cells than with WiDr. Examination of the medium at 24 hr showed that in both cell lines deoxyinosine was rapidly broken down to hypoxanthine (at 0.1 mM for 80% in Intestine 407 and 50% in WiDr). Growth inhibition could be reversed by 2-10 uM thymidine in Intestine 407, but only partly in WiDr cells. The results show that low non-toxic concentrations of FU can be made toxic by supplementary dRib-1-P. The effect of thymidine shows that inhibition of thymidylate synthase differs in the cell lines.