Clinical phase I and pharmacology study of gemcitabine (2', 2'-difluorodeoxycytidine) administered in a two-weekly schedule.

Gemcitabine (dFdC) was tested in a Phase I trial at 14 doses (40-5700 mg/m(2)), administered every 2 weeks as a (1/2) -h infusion to 52 patients with refractory solid cancer. Gemcitabine and its deaminated metabolite difluorodeoxyuridine (dFdU), measured with HPLC, reached plasma peak levels of 2-3 microM at 40 mg/m(2) which increased to 512 microM at 5700 mg/m(2). Gemcitabine was eliminated rapidly with a t(1/2) beta of 2.3-15.8 min in the 40-5700 mg/m(2) dose range, with one exception of 38 min at 4500 mg/m(2) . dFdU was still present at a plateau of +/- 20 microM from 4-24 h at doses >960 mg/m(2). Up to 3650 mg/m(2) linear pharmacokinetics were observed for gemcitabine, while those for dFdU were linear over the whole range. Gemcitabine clearance varied between 1.5-12.6 l/min and was 1.5-fold higher in males than in females (p= 0.024); its volume of distribution was 45.2-248 l. In lymphocytes peak levels of the active metabolite dFdCTP were 100-380 pmol/10( 6 )cells in the first course. Apparently a plateau was reached which was confirmed by incubation of white blood cells with increasing gemcitabine concentrations up to 500 microM, reaching a plateau of about 350 pmol/10(6 )cells; in contrast in cancer cells this concentration dependence did not exist and accumulation reached about 1590 pmol/10( 6 )cells. In tumors isolated from patients treated with gemcitabine dFdCTP reached about 70 pmol/g wet weight. Gemcitabine itself was eliminated only to a limited extent in the urine, but dFdU was eliminated almost completely in the urine in the first 24 h (51-92%). In conclusion, dFdC was rapidly eliminated in contrast to dFdU, which was present for at least 18 h, as well as dFdCTP in lymphocytes.

The role of thymidine phosphorylase and uridine phosphorylase in (fluoro)pyrimidine metabolism in peripheral blood mononuclear cells.

Thymidine phosphorylase (TP) and uridine phosphorylase (UP) catalyze the (in)activation of several fluoropyrimidines, depending on their catalytic activity and substrate specificity. Blood cells are the first compartment exposed to most anticancer agents. The role of white blood cells in causing toxic side effects and catalyzing drug metabolism is generally underestimated. Therefore we determined the contribution of the white blood cell compartment to drug metabolism, and we investigated the activity and substrate specificity of TP and UP for the (fluoro)pyrimidines thymidine (dThd), uridine (Urd), 5'-deoxy-5-fluorouridine (5' dFUrd) and 5-fluorouracil (5FU) in peripheral blood mononuclear cells (PBMC) and undifferentiated monocytes and differentiated monocytes: macrophages and dendritic cells. PBMC had an IC50 of 742 microM exposed to 5'dFUrd, increasing to > 2000 microM when both TP and UP activities were inhibited. Total phosphorolytic activity was higher with dThd than with Urd, 5'dFUrd or 5FU. Using a specific TP inhibitor (TPI) and UP inhibitor (BAU) we concluded that dThd and Urd were preferentially converted by TP and UP, respectively, while 5'dFUrd and 5FU were mainly converted by TP (about 80%) into 5FU and FUrd, respectively. 5FU was effectively incorporated into RNA. dThd conversion into thymine was highest in dendritic cells (52.6 nmol thymine/h/10(6) cells), followed by macrophages (two-fold) and undifferentiated monocytes (eight-fold). TPI prevented dThd conversion almost completely. In conclusion, PBMC were relatively insensitive to 5'dFUrd, and the natural substrates dThd and Urd were preferentially converted by TP and UP, respectively. TP and UP were both responsible for converting 5'dFUrd/5FU into 5FU/FUrd, respectively.

Antifolate resistance mediated by the multidrug resistance proteins MRP1 and MRP2.

Transfection of multidrug resistance proteins (MRPs) MRP1 and MRP2 in human ovarian carcinoma 2008 cells conferred a marked level of resistance to short-term (1-4 h) exposure to the polyglutamatable antifolates methotrexate (MTX; 21-74-fold), ZD1694 (4-138-fold), and GW1843 (101-156-fold). Evidence for MRP-mediated antifolate efflux relies upon the following findings: (a) a 2-3.3-fold lower accumulation of [3H]MTX and subsequent reduced formation of long-chain polyglutamate forms of MTX; (b) reversal of MTX resistance by probenecid in both transfectants, and (c) ATP-dependent uptake of [3H]MTX in inside-out vesicles of MRP1 and MRP2 transfectants. This report provides a mechanistic basis for resistance to polyglutamatable antifolates through an MRP-mediated drug extrusion.

Phase I clinical and pharmacokinetic study of oral S-1 in patients with advanced solid tumors.

PURPOSE: To investigate the side effects, determine the maximum-tolerated dose (MTD), and study the pharmacokinetics of S-1, an oral fluoropyrimidine-based antineoplastic agent consisting of the fluorouracil (5-FU) prodrug tegafur combined with two modulators, 5-chloro-2,4-dihydroxypyridine and potassium oxonate. PATIENTS AND METHODS: Patients with advanced solid tumors received S-1 bid for 28 days, followed by 1 week of rest. 5-FU pharmacokinetics were investigated after a single initial dose of S-1 during the first 24 hours and weekly thereafter. RESULTS: Twenty-eight patients received S-1 at the four consecutive dose levels of 25, 45, 35, and 40 mg/m(2). The MTD was initially found at 45 mg/m(2), with diarrhea as the dose-limiting toxicity (DLT). Diarrhea was also the DLT at the dose of 40 mg/m(2), which was the MTD for patients exposed to extensive prior chemotherapy. Other toxicities were generally mild. Two patients had a reduction of more than 50% in tumor dimension. Plasma pharmacokinetics of 5-FU were linear; at the highest S-1 dose level, 5-FU plasma peak concentrations reached 1 to 2 micromol/L, and the half-life of 5-FU was 3 to 4 hours. A statistically significant relationship was observed between the severity of diarrhea and pharmacokinetic parameters of 5-FU. CONCLUSION: The recommended dose of S-1 in chemotherapy-naive or minimally chemotherapy-exposed patients is 40 mg/m(2) bid on 28 consecutive days, every 5 weeks. In heavily pretreated patients, the recommended dose is 35 mg/m(2) bid. Phase II trials are warranted in tumors known to be responsive to 5-FU treatment.

mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method.

Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of beta-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three- and four-fold higher, respectively; P < 0.001), while FPGS expression was lower (three-fold, P = 0.006) compared to common/preB-ALL. The ratio of (DHFR x FPGH)/(RFC x FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P < 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P = 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (four-fold higher; P = 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r = 0.80; P < 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance.

Modulation of 5-fluorouracil in mice using uridine diphosphoglucose.

Uridine diphosphoglucose (UDPG) is a precursor of uridine that can be used as a rescuing agent from 5-fluorouracil (5FU) toxicity. Four doses of UDPG (2000 mg/kg i.p. or p.o. at 2, 6, 24, and 30 h after 5FU bolus) allowed the escalation of a weekly bolus of 5FU from 100 mg/kg (5FU100) to 150 mg/kg (5FU150) in healthy and tumor-bearing BALB/c, C57/BI, and CD8F1 (BALB/c x DBA/8) mice. 5FU150 without rescuing agents is not tolerated by the animals. When followed by UDPG, on the contrary, it is possible to increase the dose of 5FU even when it is modulated by leucovorin. Toxicity was the same for 5FU100 and 5FU150 + UDPG, and the nadir values (expressed as a percentage of pretreatment values) were 83 and 85% for weight, 45 and 45% for hematocrit, and 45 and 61% for leukocytes, respectively. Platelets were not affected by treatment. A protective effect was also shown for the gastrointestinal tract. The enzymes thymidine kinase, maltase, and sucrase were measured in the intestinal mucosa at different times after 5FU treatment with or without UDPG rescue. Even if the nadir values in enzyme activities were similar in mice receiving or not receiving UDPG, the pattern of recovery showed that cell repopulation was more rapid in the group treated with UDPG. 5FU150 + UDPG had enhanced antitumor activity against CD8F1 mammary carcinoma and against the resistant tumor Colon 26 (tumor doubling time 1.9 days for controls, 8.5 days for 5FU100, 13.7 days for 5FU150 + UDPG, and 15.9 days for 5FU150 + leucovorin + UDPG). We demonstrated that UDPG administered at 2, 24, and 30 h after 5FU100 does not reduce the antitumor activity of 5FU in two sensitive tumors (Colon 38 and Colon 26-10). In conclusion, UDPG is a promising rescuing agent for 5FU; it reduces the toxic side effects and increases the therapeutic index.

Therapeutic efficacy of fluoropyrimidines depends on the duration of thymidylate synthase inhibition in the murine colon 26-B carcinoma tumor model.

5-Fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) are common chemotherapeutic drugs for the treatment of advanced colorectal cancer. Two recognized mechanisms of action of these agents are inhibition of thymidylate synthase (TS) and incorporation of fluorinated UTP into cellular RNA. In previous studies on drug scheduling of both fluoropyrimidines, we observed the highest therapeutic efficacy by using a weekly i.v. push schedule. Furthermore, weekly 400-mg/kg FdUrd is superior to equitoxic weekly 80-mg/kg FUra in murine Colon 26-B carcinoma. We evaluated the most important pharmacokinetic and pharmacodynamic effects of both fluoropyrimidines to delineate the biochemical mechanisms underlying their differences in therapeutic activity in this tumor model. FUra concentrations and elimination in tumors after FdUrd or FUra administration were comparable, and the level of FUra incorporation into cellular RNA following treatment with FUra or FdUrd was similar. Free tumoral 5-fluoro-dUMP levels were initially 3-fold higher after FdUrd but diminished rapidly thereafter. The number of free [3H]5-fluoro-dUMP-binding sites decreased to about 25 and 15% of control values within 2 h after treatment with equitoxic doses of FUra and FdUrd and remained low for 72 h. The duration of TS inhibition was significantly longer following treatment with FdUrd compared with FUra, 168 and 72 h, respectively. The superiority of the antitumor activity of an i.v. push of FdUrd over FUra in the treatment of Colon 26-B tumors correlates with maintenance of TS inhibition and repeated drug administration when TS remains low, whereas FUra incorporation into RNA does not appear to distinguish the antitumor response of FdUrd from that of FUra in this tumor model.

Thymidylate synthase level as the main predictive parameter for sensitivity to 5-fluorouracil, but not for folate-based thymidylate synthase inhibitors, in 13 nonselected colon cancer cell lines.

Thymidylate synthase (TS), a critical enzyme in the de novo synthesis of thymidylate, is an important target for fluoropyrimidines and folate-based TS inhibitors. In a panel of 13 nonselected human colon cancer cell lines, we evaluated the role of TS levels in sensitivity to 5-fluorouracil (5FU) and four folate-based TS inhibitors that have been introduced recently into the clinic: ZD1694 (Tomudex, Raltitrexed, TDX), GW1843U89 (GW), LY231514 (LY), and AG337 (Thymitaq, AG). Because the latter compounds have different transport and polyglutamylation characteristics, we also related these parameters with drug sensitivity, measured by the sulforhodamine B assay after 72 h of drug exposure. For 5FU, the IC50s varied from 0.8 to 43.0 microM. Leucovorin (LV) potentiated the activity of 5FU in only 4 of 13 cell lines. Sensitivity to folate-based TS inhibitors was variable; IC50s were in the range of: 5.3-59.0 nM TDX; 11.0-1570 nM LY; and 0.5-8.9 nM GW. Eleven of 13 cell lines had an IC50 for AG between 1.3 and 5.3 microM. Two cell lines were resistant to AG, Colo201 and SW1116, with IC50s of 27 and 29 microM, respectively. TS catalytic activity (conversion of dUMP to dTMP) varied from 62 to 777 pmol/h/10(6) cells. The number of FdUMP binding sites varied from 32 to 231 fmol/10(6) cells. Regression analysis showed a significant relation between TS catalytic activity and IC50s for 5FU and 5FU/LV. Kis for FdUMP showed a significant Spearman rank correlation with the IC50s of AG and GW. The role of antifolate transport, accumulation, and polyglutamylation was determined with [3H]methotrexate (MTX) as a reference compound. [3H]MTX influx via the reduced folate carrier varied from 18.6 to 150 fmol/10(6) cells/min. Folylpolyglutamate synthetase (FPGS) activity showed a range from 47 to 429 pmol/10(6) cells/h. A total of 24 h of [3H]MTX accumulation showed a 20-fold variation, from 1.2 to 21.8 pmol/10(6) cells. FPGS levels showed a Spearman rank positive correlation with cytotoxicity to TDX. In conclusion, in a heterogeneous nonselected human colon cancer cell line panel, the best predictor for sensitivity to 5FU and 5FU/LV was TS activity. Multiple sensitivity determinants were of importance for antifolate TS inhibitors, including FPGS activity and TS enzyme kinetics.

Biochemical mechanisms of interferon modulation of 5-fluorouracil activity in colon cancer cells.

The antiproliferative effect of 5-fluorouracil (5-FU) in colon cancer can be enhanced by interferons (IFN-alpha and IFN-gamma). The mechanisms by which IFNs modulate 5-FU activity are not completely elucidated. IFN-alpha may elevate the levels of the active 5-FU metabolite 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) in the cell, possibly leading to increased inhibition of the target enzyme thymidylate synthase (TS), which might enhance DNA damage. It has been shown that IFN-gamma can prevent 5-FU induced overexpression of TS. We studied IFN modulation in three colon cancer cell lines (SW948, WiDr, human; C26-10, murine) and the sublines WiDr/F and C26-10/F, which were adapted to low folate levels. A 1.5-fold increase in 5-FU sensitivity was observed in C26-10 and C26-10/F (by murine IFN-alpha, beta); in SW948, WiDr and WiDr/F (by human IFN-gamma) and in SW948 and WiDr/ F (by human IFN-alpha). In none of the cell lines did human IFN-alpha, IFN-gamma or murine IFN-alpha, beta increase FdUMP levels after exposure to 5-FU. TS activity, indirectly measured by incorporation of [6-3H]-deoxyuridine into DNA, was inhibited by 5-FU, but the IFNs did not enhance inhibition. DNA damage was measured as a drug-induced decrease of double-stranded (dss) DNA compared to control cells. After 5-FU exposure, dss DNA decreased to 60-75% in WiDr, WiDr/F and SW948 cells. Human IFN-alpha alone caused minimal DNA damage (95% dss DNA), but increased 5-FU-induced effects to 35-50% dss DNA. IFN-gamma did not cause DNA damage and did not enhance 5-FU-mediated DNA damage. Expression of TS protein, analysed by ELISA, was increased after 5-FU exposure of SW948 cells, but this increase was not affected by addition of either IFN-alpha or IFN-gamma. It is concluded that one of the mechanisms involved in modulation of 5-FU activity is the effect of IFN-alpha on 5-FU-mediated DNA damage, but for IFN-gamma no mechanism of action was found.

Cross-resistance in the 2',2'-difluorodeoxycytidine (gemcitabine)-resistant human ovarian cancer cell line AG6000 to standard and investigational drugs.

Gemcitabine (2'-2'-difluorodeoxycytidine; dFdC) is a deoxycytidine analogue which is effective against solid tumours, including lung cancer and ovarian cancer. dFdC requires phosphorylation by deoxycytidine kinase (dCK) for activation. In the human ovarian cancer cell line A2780 and its 30,000-fold dFdC-resistant variant AG6000 (P<0.001), we investigated the cross-resistance profile to several drugs. AG6000, which has a complete dCK deficiency, was approximately 1000-10,000-fold resistant to other deoxynucleoside analogues such as 1-beta-D-arabinofuranosyl cytosine, 2-chloro-deoxyadenosine, aza-deoxycytidine and 2', 2'-difluorodeoxyguanosine (dFdG) (P<0.001). dFdG can be activated by dCK and deoxyguanosine kinase (dGK), but the latter enzyme was not altered in AG6000 cells. Thus dFdG resistance was only due to dCK deficiency. AG6000 was 1.6- and 46.7-fold resistant to 5-fluorouracil (5-FU) and ZD1694, respectively (the latter was significant; P<0.01), which may be due to the 1.7-fold higher thymidylate synthase (TS) activity, but AG6000 cells were also 2. 7-fold resistant to the lipophilic TS inhibitor AG337 (P<0.05). Remarkably, AG6000 cells were 2.5-fold more sensitive to methotrexate (MTX) (P<0.01) than A2780 cells, but 1.6-fold more resistant to trimetrexate (TMQ) (P<0.10). However, no differences in reduced folate carrier activity, folylpolyglutamate synthetase (FPGS) activity and polyglutamation of MTX were found between the cell lines. AG6000 cells were approximately 2 to 7.5-fold more resistant to doxorubicin (DOX), daunorubicin (DAU), epirubicin and vincristine (VCR) (the latter was significant; P<0.02) and approximately 4-fold more resistant to the microtubule inhibitors paclitaxel and docetaxel (P<0.001). Fluorescent activated cell sorter (FACS) analysis revealed no P-glycoprotein (Pgp) or multidrug resistance-associated protein (MRP) expression, but less fluorescence of intercalated DAU in AG6000 cells. An approximately 2-fold resistance to the topoisomerase I and II inhibitors etoposide, CPT-11 and SN38 was found in AG6000 cells. Topoisomerase I and IIalpha RNA expression was decreased in AG6000 cells. AG6000 was 2.4, 2.4, 2.3 and 3.7-fold more resistant to EO9 (P<0.02), mitomycin-C (MMC) (P<0.05), cisplatin (CDDP) (P<0.10) and maphosphamide (MAPH), respectively. DT-diaphorase (DTD), which activates EO9, was 2.2-fold lower in AG6000 cells. CDDP resistance might be related to a reduced retention of DNA adducts in AG6000. However, glutathione levels were equal in A2780 and AG6000 cells. A 24 h exposure to DOX, VCR and paclitaxel at equimolar and equitoxic concentrations, resulted in more double-strand breaks (1.5- to 2-fold) in A2780 than in AG6000 cells. MAPH at 1120 nM and 17 nM of EO9 did not cause DNA damage in either cell line. In conclusion, AG6000 is a cell line highly cross-resistant to a wide variety of drugs. This cross-resistance might be related to altered enzyme activities and/or increased DNA repair.

Antitumour activity, toxicity and inhibition of thymidylate synthase of prolonged administration of 5-fluorouracil in mice.

Continuous infusions of 5-fluorouracil (5-FU) are increasingly used in the treatment of cancer. Their optimal use, however, has still to be determined since the availability of suitable animal models is limited. We studied continuous infusions in mice using subcutaneously implanted pellets that release 5-FU over a period of 3 weeks. At the maximum tolerated dose (MTD) (based on the systemic toxicity in healthy animals) we assessed the antitumour activity, haematological toxicity, inhibition of thymidylate synthase (TS) in tumours and the concentration of 5-FU in plasma during the 3-week period. We also studied the addition of leucovorin in different schedules. The dose-limiting toxicity was weight loss, and at the MTD of 10 mg of 5-FU released in 21 days per mouse myelosuppression was tolerable (nadir for leucocytes and thrombocytes was approximately 40% of pretreatment levels). In several independent experiments using the 5-FU-resistant Colon 26 tumour, a good antitumour activity was observed during the first part of the infusion, but thereafter the growth of the tumours resumed; the overall effect of continuous infusions was thus comparable to that of bolus injections. Coadministration of leucovorin did not enhance the therapeutic results; depending on the schedule used, it proved ineffective or only increased toxicity. Similar results were obtained with head and neck squamous cell carcinomas and with the 5-FU-sensitive tumour Colon 38. In Colon 26 tumours the TS activity (FdUMP-binding assay) initially decreased to 20-30% of controls and returned to normal after 11 days. In the catalytic TS assay a slight inhibition was observed for the continuous infusion, followed after 11 days by a marked (4-fold) increase in activity. 5-FU plasma levels varied from 0.1 to 1 microM following a circadian rhythm (with a peak at 6 h after light onset), and were maintained during the entire period. Subcutaneously implanted pellets represent a suitable model to study prolonged administration of 5-FU in mice and to evaluate the effect of modulating agents in laboratory animals before transferring data obtained in vitro to the clinic.

Intermittent continuous infusion of 5-fluorouracil and low dose oral leucovorin in patients with gastrointestinal cancer: relationship between plasma concentrations and clinical parameters.

Modulation of 5-fluorouracil (5-FU) by leucovorin and continuous infusion of 5-FU can both result in enhanced therapeutic efficacy. The main objective of this study was to determine the maximum tolerated dose (MTD) of oral leucovorin in combination with continuous infusion of 5-FU for 14 days every 4 weeks at a dose of 300 mg/m2/day in 30 patients with gastrointestinal cancer. The MTD of oral leucovorin was established at 10 mg/day. Dose-limiting toxicities were mucositis, diarrhoea and hand-foot syndrome. Plasma leucovorin concentrations were below the detection limit of the assay (< 0.5 microM). Plasma 5-FU concentrations varied considerably from 0.06 to 11.3 microM. A relation between toxicity, response and plasma concentration of 5-FU could not be established. Our data may indicate that even very low plasma concentrations of leucovorin are able to modulate 5-FU. In 17 patients with colorectal cancer the response rate was 24% (95% CI: 7-50%), which is comparable to other treatment schedules with leucovorin or to continuous infusion of 5-FU alone.

A possible role for methotrexate in the treatment of childhood acute myeloid leukaemia, in particular for acute monocytic leukaemia.

Acute myeloid leukaemia (AML) is thought to be methotrexate (MTX)-resistant. However, a small study suggested that acute monocytic leukemia (AML-M5) is sensitive to MTX. We measured MTX accumulation/polyglutamylation in 20 AML-nonM5, 37 AML-M5 and 83 common/preB-acute lymphoblastic leukaemia (c/preB-ALL) samples. Membrane transport was determined in 11 childhood AMLs (including 3 AML-M5) and in 25 c/preB-ALL samples. MTX sensitivity was determined in 23 AML-nonM5, 15 AML-M5 and 63 common/preB-ALL samples using the thymidylate synthase (TS) inhibition assay. MTX transport was higher in AML samples compared with c/preB-ALL precluding a transport defect in AML. Accumulation of long-chain polyglutamates MTX-Glu(4-6) was 3-fold lower for AML-nonM5 compared with c/preB-ALL cells (median 268 versus 889 pmol MTX-Glu(4-6)/10(9) cells; P < or = 0.001); for AML-M5 samples, median accumulation of MTX-Glu(4-6) was 0 pmol/10(9) cells (P < or = 0.001). After short-term MTX exposure, AML-nonM5 was 6-fold more resistant to MTX compared with c/preB-ALL cells (2.16 versus 0.39 microM; P < 0.001), while AML-M5 was 2-fold more resistant (P = 0.02). In both AML-nonM5 and AML-M5 cells, MTX resistance was circumvented by continuous MTX exposure (median TSI(50) values: 0.052 and 0.041 microM, respectively) compared with a c/preB-ALL value of 0.066 microM. In conclusion, AML-M5 is relatively sensitive to MTX compared with other AML-subtypes even though polyglutamylation of MTX is poor. Using continuous exposure, AML-nonM5 and AML-M5 cells were at least as sensitive to MTX as c/preB-ALL cells. This report suggests that MTX might be an overlooked drug in the treatment of childhood AML.

Effect of folinic acid on fluorouracil activity and expression of thymidylate synthase.

Folinic acid (leucovorin, [LV]) can potentiate the growth inhibitory effects of fluorouracil (5-FU) in vitro and in vivo. LV is a precursor for 5,10-methylene-tetrahydrofolate (CH2-THF). Sufficient levels of CH2-THF enhance the inhibition of the enzyme thymidylate synthase by the 5-FU metabolite FdUMP. This study describes the effects of 5-FU and LV in two murine (C26-10, C38-1) and two human (WiDr, HT-29) colon carcinoma cell lines and in two murine tumors (Colon 26 and Colon 38). In vitro, only C38-1 was more sensitive for the combination of LV/5-FU compared with 5-FU alone. This effect was not dose or schedule dependent. l-LV, a purified stereo-isomere of LV, is thought to be the biological active form. Tests with this compound in vitro did not show a better effect than the mixture of d- and l-LV. In vivo, dl-LV could potentiate 5-FU antitumor effect in two murine colon tumors (Colon 26, Colon 38). This effect was clearly schedule dependent. dl-LV administered 1 hour before and together with 5-FU was much better than only simultaneous or posttreatment, but there was no dose dependency, while like in vitro l-LV effect was comparable to dl-LV. TdR was used to study the role of TS inhibition in the growth inhibitory effect of 5-FU with and without LV. TdR can reverse growth inhibition caused by TS inhibition due to 5-FU. In vitro, a partial reversal of growth inhibition of 5-FU and 5-FU/LV was observed, but in vivo there was no reversal. In vivo, TdR combinations led to high toxicity. Measurements of TS amounts in cells and tumors showed that those of human origin had much lower TS than the murine. C38-1 and Colon 38 with low TS were more sensitive to 5-FU than Colon 26 with higher TS amounts. TS inhibition was studied in the two murine colon tumors at several time points after weekly 5-FU or LV and 5-FU administration. LV did not increase the extent or retention of TS inhibition due to 5-FU during the first week. After three courses of treatment a fourfold increase of TS levels was seen in Colon 26 after 5-FU therapy. This resulted in a less effective TS inhibition after this treatment. Tumors treated with 5-FU and LV also showed an increase of TS, but to a lower extent, while the effect on TS inhibition remained the same.(ABSTRACT TRUNCATED AT 400 WORDS)

Importance of pharmacodynamics in the in vitro antiproliferative activity of the antifolates methotrexate and 10-ethyl-10-deazaaminopterin against human head and neck squamous cell carcinoma.

The pharmacodynamic profiles of methotrexate (MTX) and 10-ethyl-10-deazaaminopterin (10-EdAM) were determined in three head and neck squamous cell carcinoma (HNSCC) cell lines. Cell growth inhibition was tested using a semi-automated 96-well based proliferation assay, the sulforhodamine B (SRB) assay. Drug concentrations ranged from 10(-5) to 10(-9) M, with exposure periods of 4, 24, 48, 72 and 96 hr. The SRB-test was performed after each of these periods of continuous exposure and after an additional period of 24 and 48 hr in drug-free medium. Without a drug-free period the IC50 values strongly depended on the time of exposure. For example, with respect to MTX, IC50 values at 24 hr ranged from 2.9 (UM-SCC-14C) to over 10 microM (UM-SCC-22B and -11B), but when exposed continuously for 96 hr, IC50 values varied between 0.039 and 0.1 microM. 10-EdAM followed a similar sensitivity pattern with 5-20-fold lower IC50 values. The minimal time to achieve significant growth inhibition varied between the cell lines, < 24 hr for UM-SCC-14C, > 24 and > 48 hr for UM-SCC-11B and -22B, respectively. The cell lines also varied with respect to growth behaviour when placed in drug-free medium for an additional period. Growth of UM-SCC-14C cells was recovered significantly after removing the drug, whereas UM-SCC-22B showed a different pattern: when cultured for over 48 hr, cell growth was strongly inhibited, independent of the drug being removed. This variable pattern of sensitivity could be correlated with the capacity of the cells to form polyglutamate derivatives. After 24 hr, drug accumulation was at least three times lower in UM-SCC-14C than in both other cell lines. The low level of antifolate accumulation in UM-SCC-14C is in line with the recovery from growth inhibition at culture in drug-free medium, while the persistent growth inhibition observed in UM-SCC-22B agrees with the intracellular accumulation of higher polyglutamates. In conclusion, these experiments show that the pharmacodynamic profile varies between HNSCC cell lines and plays an important role in the growth inhibition by antifolates. Both exposure time and the intrinsic capacity to synthesize polyglutamates are important factors in the sensitivity of HNSCC to antifolate drugs.

Schedule dependence of sensitivity to 2',2'-difluorodeoxycytidine (Gemcitabine) in relation to accumulation and retention of its triphosphate in solid tumour cell lines and solid tumours.

2',2'-Difluorodeoxycytidine (Gemcitabine, dFdC) is a relatively new deoxycytidine antimetabolite, with established activity against ovarian cancer and non-small-cell lung cancer. dFdC is assumed to exert its antitumour effect mainly by incorporation of the triphosphate dFdCTP into DNA. We determined the sensitivity to dFdC of six cell lines derived from solid tumours; two ovarian carcinoma (A2780 and OVCAR-3), two colon carcinoma (WiDr and C26-10) and two squamous cell carcinoma cell lines (UM-SCC-14C and UM-SCC-22B). In vitro sensitivity to dFdC was strongly time dependent. Under all conditions A2780 was the most sensitive cell line with an IC50 (the concentration of dFdC causing 50% growth inhibition) of 31 and 0.6 nM at 1 and 48 hr exposure, respectively. WiDr and C26-10 cells were relatively insensitive, with IC50s of 468 and 1133 nM, respectively, at 1 hr exposure, but of 11 and 6 nM at 48 hr exposure. Accumulation of the triphosphate dFdCTP was also time dependent. After 4 hr exposure to 10 microM dFdC, A2780, WiDr and C26-10 cells accumulated 223, 136 and 267 pmol/10(6) cells, respectively; after 24 hr exposure they accumulated 1045, 619 and 617 pmol/10(6) cells, respectively. A2780 cells retained the high dFdCTP concentration longer than 24 hr. For comparison purposes we also studied dFdCTP kinetics in the corresponding solid tumours, showing the same sensitivity pattern as the cell lines. In general, sensitivity to dFdC in vitro related with dFdCTP accumulation and retention, but in vivo this relation was less clear. Unexpectedly, remarkable in vitro and in vivo changes were observed in the ribonucleotide pools. The most predominant in vitro cell line dependent changes were a decrease in CTP concentrations, accompanied by an increase in UTP and GTP concentrations. In vivo CTP, UTP and GTP pools increased in all tumours. In conclusion, in this study we demonstrate that dFdCTP is accumulated and retained in solid tumours and cell lines. dFdCTP is not only important as a DNA precursor, but also appears to interfere with normal ribonucleotide metabolism.

Modulation of metabolism and cytotoxicity of cytosine arabinoside with N-(phosphon)-acetyl-L-aspartate in human leukemic blast cells and cell lines.

Cytosine arabinoside (Ara-C) activation to cytosine arabinoside triphosphate (Ara-CTP) and subsequent incorporation into DNA is regulated by the pyrimidine nucleotides UTP, CTP and dCTP. Inhibition of the de novo synthesis of these pyrimidine nucleotides by N-(phosphon)-acetyl-L-aspartate (PALA) may enhance the cytotoxicity of Ara-C. We therefore studied the effect of PALA on Ara-C cytotoxicity and on Ara-CTP accumulation and incorporation into DNA on cell lines and patient samples. Fifty micromolar PALA increased the growth inhibitory effect of Ara-C in U937 cells several fold both with pre- and coincubation. Ara-C cytotoxicity was not potentiated by PALA in Hl60 cells. However, coincubation with PALA did not enhance Ara-CTP accumulation both in HL60 and U937 cells, nor affect Ara-C incorporation into DNA. Ara-C cytotoxicity to leukemic blast cells from 11 untreated patients with different types of leukemia was only modulated to a small extent by high PALA concentrations in only two cases. Ara-CTP accumulation in leukemic blast cells varied from non-detectable levels to 200 pmol/10(6) cells. Fifty micromolar PALA enhanced the accumulation of Ara-CTP significantly in only one patient with no apparent effect on UTP and CTP levels. Raising PALA to 500 microM decreased UTP and CTP levels to 50% but had no effect on Ara-CTP levels. In conclusion, modulation by PALA of Ara-C cytotoxicity and metabolism is limited in leukemic cells, both in culture and from patients. This suggests the possibility for selective modulation of other agents by PALA on non-hematological cells.

Modulation of resistance to ara-C by bryostatin in fresh blast cells from patients with AML.

Bryostatin has shown promise both as a cytotoxic agent and more recently as a modulator of 1-beta-D-arabinofuranosylcytosine (ara-C) resistance. This compound is currently in phase I and II trials as a single agent. We have used the 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay as a means of investigating the direct effects of bryostatin and the effects of co-incubating this agent with ara-C on fresh blast cells from 53 patients with acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). Additional studies evaluated the levels of accumulation and retention of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) in cells exposed to ara-C with and without bryostatin. Cells were exposed to bryostatin at a range of concentrations (0.1-100 nM) for 48 h and at 1 nM for both modulation studies and assessment of ara-CTP production. We found bryostatin to be cytotoxic in 18/58 (31%) tests whilst potentiation of formazan production in the MTT assay was seen in 21/58 (36%) patients. On co-incubation with bryostatin, 16/58 (27%) tests showed increased cytotoxicity to ara-C. Furthermore, there was a significant increase in the accumulation of ara-CTP on co-incubation with bryostatin (p = 0.0401). We found patients with in vitro resistance were more likely to become sensitised following exposure to bryostatin (p < 0.01). This study has emphasised the need to optimise treatment regimens for individual patients using this approach.

Interference of gemcitabine triphosphate with the measurements of deoxynucleotides using an optimized DNA polymerase elongation assay.

The main mechanism of action of the anticancer drug gemcitabine is assumed to be incorporation of its triphosphate (dFdCTP) into DNA, resulting in inhibition of DNA polymerization, inhibition of DNA synthesis and repair. Another mechanism is inhibition of ribonucleotide reductase leading to imbalance in the deoxyribonucleotide (dNTP) pools. One assay to measure dNTP pools is based on oligonucleotide elongation mediated by DNA polymerase. Since the latter may be affected by dFdCTP, we studied the effect of 0.1-600 pmol dFdCTP on this assay; 10 pmol and more dFdCTP significantly increased the average dpm of the blank (absence of other dNTP) and that of the calibration line of dATP (1.4-1.6-fold); 0.1 pmol and more increased that of the standard dGTP curve significantly (1.1-1.8-fold); 10-75 pmol decreased that of dCTP while 75 and 100 pmol significantly increased that of dCTP (1.3-fold); 50 pmol significantly increased that of dTTP (1.3-1.5-fold). For dATP, dGTP and dTTP, a saturation was reached at 100 pmol dFdCTP, but not yet for dCTP. To minimize these effects, we added an excess of 200 pmol dFdCTP to all samples and calibration lines when measuring dNTP levels of gemcitabine treated samples. In this way the effects of gemcitabine on dNTP levels were studied in human A2780 ovarian, HT29 colon, K562 myelogenous leukemia, H322 non-small cell lung cancer cell lines and the murine lung cancer cell line Lewis Lung. In all cell lines, intrinsic dTTP pools (3-77 pmol/106 cells) were the highest, followed by dATP (1.5-31), dCTP (0.7-27) and (nd-14) dGTP. Exposure to 1 and 10 microM gemcitabine for 4-h concentration dependently decreased dATP 3-10-fold and dGTP to undetectable levels, but dCTP at most 3-fold, while dTTP increased. In conclusion, dFdCTP affects dNTP measurements with the DNA polymerase elongation assay, but its effect could be controlled by addition of similar amounts of dFdCTP to each assay.

5-ethyl-2'-deoxyuridine, a modulator of both antitumour action and pharmacokinetics of 5-fluorouracil.

The aim of the present studies was to elucidate the effects and optimal modulatory conditions of 5-ethyl-2'-deoxyuridine (EtdUrd) on the antitumour efficacy, pharmacokinetics and catabolism of 5-fluorouracil (5-FU) on Colon-26 and Colon-38 murine tumours. HPLC and GC-MS techniques were used to measure the concentrations of 5-FU, dihydro-5-fluorouracil, EtdUrd, 5-ethyluracil and uridine in the plasma and that of 5-FU and 5-fluoro-2'-deoxyuridine monophosphate (FdUMP) in the tumours. It was shown that EtdUrd, given 1 h before 5-FU, selectively enhanced the antitumour action of 5-FU, without significantly increasing its toxic side-effects, thus resulting in an approximately three times higher therapeutic index. Pharmacokinetic studies revealed that 1 h after 400 mg/kg EtdUrd administration - i.e. at the time of 5-FU treatment - the plasma concentration of EtdUrd was 269 microM, and that of 5-ethyluracil, as the major metabolite of EtdUrd, was 421 microM. It is of interest that EtdUrd pretreatment did not change the maximal plasma concentration of 5-FU; however, the half-life of the terminal elimination increased from 114.5 min to 171.2 min and thus the mean residence time of 5-FU rose significantly (P < 0.05). After the combined treatment, the maximal concentration of dihydro-5-fluorouracil in the plasma decreased from 61.06 microM to 29.70 microM (P < 0.01). The intratumoral concentrations of 5-FU were 34%-158% higher 6-96 h after the combined treatment than after the single 5-FU treatment. EtdUrd also caused a moderate increase in the intratumoral level of FdUMP. It is noteworthy, that EtdUrd increased the endogenous uridine concentration in the plasma from 18 microM to a maximum of 249 microM, and the level remained high for longer than 6 h. The present studies indicate that EtdUrd enhances the therapeutic index of 5-FU by reducing the catabolism, prolonging the plasma and intratumoral concentrations of 5-FU and, at the same time, offering protection to normal organs by increasing the endogenous uridine level.