Daphne A. Voorn

Common resistance mechanisms to deoxynucleoside analogues in variants of the human erythroleukaemic line K562

Resistant variants of the human leukaemic line K562 were developed using selection with the deoxynucleoside analogues cytosine arabinoside, 2‐chlorodeoxyadenosine, fludarabine and gemcitabine. The resistant lines displayed a high degree of cross resistance to all deoxynucleoside analogues, with little or no cross resistance to other agents. There was a profound accumulation defect of all nucleoside analogues in the resistant variants but no significant defect in nucleoside transport in any of the variants. 5′ nucleotidase activity was strongly increased and deoxycytidine kinase activity was moderately reduced in all of the resistant variants, resulting in reduced accumulation of triphosphate analogues. In addition a deletion in one of the alleles of the deoxycytidine kinase was detected in the fludarabine‐resistant line. Ribonucleotide reductase activity was found to be strongly increased in the gemcitabine‐selected line and purine nucleoside phosphorylase was increased in the 2‐chlorodeoxyadenosine‐selected line. Free nucleotide pools were increased in the 2‐chlorodeoxyadenosine‐selected line. There was no expression of the mdr1 gene by the resistant lines. Karyotypic analysis and FISH experiments using a 6q21 specific probe showed alterations in the 6(q16‐q22) region which contains the 5′‐nucleotidase gene. Early events in the activation and degradation of deoxynucleoside analogues appear to constitute common mechanisms of resistance to these compounds.

Sequence dependent effect of paclitaxel on gemcitabine metabolism in relation to cell cycle and cytotoxicity in non-small-cell lung cancer cell lines

Gemcitabine and paclitaxel are active agents in the treatment of non-small-cell lung cancer (NSCLC). To optimize treatment drug combinations, simultaneously and 4 and 24 h intervals, were studied using DNA flow cytometry and multiple drug effect analysis in the NSCLC cell lines H460, H322 and Lewis Lung. All combinations resulted in comparable cytotoxicity, varying from additivity to antagonism (combination index: 1.0–2.6). Gemcitabine caused a S (48%) and G1 (64%) arrest at IC-50 and 10 × IC-50 concentrations, respectively. Paclitaxel induced G2/M arrest (70%) which was maximal within 24 h at 10 × IC-50. Simultaneous treatment increased S-phase arrest, while at the 24 h interval after 72 h the first drug seemed to dominate the effect. Apoptosis was more pronounced when paclitaxel preceded gemcitabine (20% for both intervals) as compared to the reverse sequence (8%, P = 0.173 for the 4 h and 12%, P = 0.051 for the 24 h time interval). In H460 cells, paclitaxel increased 2-fold the accumulation of dFdCTP, the active metabolite of gemcitabine, in contrast to H322 cells. Paclitaxel did not affect deoxycytidine kinase levels, but ribonucleotide levels increased possibly explaining the increase in dFdCTP. Paclitaxel did not affect gemcitabine incorporation into DNA, but seemed to increase incorporation into RNA. Gemcitabine almost completely inhibited DNA synthesis in both cell lines (70–89%), while paclitaxel had a minor effect and did not increase that of gemcitabine. In conclusion, various gemcitabine–paclitaxel combinations did not show sequence dependent cytotoxic effects; all combinations were not more than additive. However, since paclitaxel increased dFdCTP accumulation, gemcitabine incorporation into RNA and the apoptotic index, the administration of paclitaxel prior to gemcitabine might be favourable as compared to reversed sequences.

Role of deoxycytidine kinase (dCK), thymidine kinase 2 (TK2), and deoxycytidine deaminase (dCDA) in the antitumor activity of gemcitabine (dFdC).

Deoxycytidine kinase (dCK) and deaminase (dCDA) are as activating and inactivating enzymes, respectively, in the metabolism of several chemotherapeutically important deoxynucleoside analogues [1]. 2′2′-Difluorodeoxycytidine (dFdC; gemcitabine) has considerable antitumor activity against solid tumors, such as against the chemoresistant non-small cell lung cancer (NSCLC) and pancreatic cancer [2]. dCK catalyses the rate-limiting phosphorylation of CdR and its analogues to their corresponding monophos-phates [1,3]. To avoid an overestimation of the dCK activity by thymidine kinase 2 (TK2), which can also efficiently phosphorylate CdR [3], dCK activity was measured in the presence of thymidine (TdR) to inhibit TK2 [4]. dCDA inactivates cytidine (CR), CdR and its analogues to their deaminated products [5,6]. Previously we could not establish a relationship between antitumor activity and the dCK and dCDA activities [6], while in a cell line study more precise measurements of dCK showed a relation between sensitivity to dFdC and efficiency of dCK [7]. We now reevaluated the role of dCK, TK2 and dCDA in the antitumor effect of dFdC against different solid tumors.

Mechanisms of synergism between gemcitabine and cisplatin.

2′,2′-difluorodeoxycytidine (Gemcitabine, dFdC) is an antineoplastic agent with clinical activity against several cancer types.1 cis-Diamminedichloroplatinum (cisplatin, CDDP) is a drug with long established anticancer activity, which acts by Platinum (Pt)-DNA adduct formation.2,3 Because of the low toxicity profile of dFdC and the differences in mechanism of cytotoxicity, preclinical studies were performed that demonstrated synergism between dFdC and CDDP in several cancer cell lines and in vivo,4–8 which is likely to be related to increased formation of Pt-DNA adducts.8 Pre-treatment with dFdC gave the best results both in vitro and in vivo.6,7,8 Several potential mechanisms underlying the synergism were studied in vitro, based on these results several schedules were studied in patients.

The Effect of Food on the Pharmacokinetics of S-1 after Single Oral Administration to Patients with Solid Tumors

Purpose: The purpose is to determine the effect of food on the bioavailability of S-1, an oral formulation of the 5-fluorouracil (5FU) prodrug Ftorafur (FT), 5-chloro-2,4-dihydroxypyridine (CDHP), a dihydropyrimidine dehydrogenase inhibitor, and oxonic acid (an inhibitor of 5FU phosphoribosylation in normal gut mucosa) in a molar ratio of 1:0.4:1. Experimental Design: Eighteen patients received a single dose of S-1 of 35 mg/m2 with (535–885 kcal) or without food in a crossover study design: in arm A without breakfast on day −7 and with breakfast on day 0 and in arm B the reversed sequence. Blood samples were taken before and after S-1 administration. This food effect was evaluated according to the Food and Drug Administration guidelines using log-transformed data. Results: Pharmacokinetic parameters for 5FU without breakfast were as follows: Tmax, 107 min; Cmax, 1.60 μm; area under the plasma concentration-time curve (AUC) 441 μm × min; and T1/2, 104 min. Fasting decreased Tmax of FT, 5FU, CDHP, and oxonic acid significantly (P < 0.006) and increased the Cmax (P < 0.013). The food/fast ratio for the AUC of FT was not different, which for 5FU was 0.84 (P = 0.041), for CDHP was 0.89 (P = 0.191), for oxonic acid was 0.48 (P < 0.0005), and for cyanuric acid, the breakdown product of oxonic acid, was 5.1 (P = 0.019). Accumulation of uracil, indicative for dihydropyrimidine dehydrogenase inhibition, was not affected, as well as the T1/2 of FT, 5FU, CDHP, and oxonic acid. Evaluation of the log-transformed data demonstrated that the 90% confidence interval for the food/fast ratio for the Cmax and AUC of FT, 5FU, CDHP, and uracil were within 70–143% and 80–125%, respectively, indicating no food effect. Only for oxonic acid and cyanuric acid were these values outside this interval. Conclusions: Food intake affected only the pharmacokinetics of the S-1 constituent oxonic acid but not of FT, CDHP, and 5FU. Because oxonic acid is included to protect against gastrointestinal toxicity, this observation might affect the gastrointestinal toxicity and thus the efficacy of S-1.

High-Dose 5-Fluorouracil with Uridine-Diphosphoglucose Rescue Increases Thymidylate Synthase Inhibition but Not 5-Fluorouracil Incorporation into RNA in Murine Tumors

5-Fluorouracil (5FU) shows a steep dose response curve in several experimental systems, but the clinical use of high doses is hampered by the toxic side effects of this drug. Uridine diphosphoglucose (UDPG) rescue allows an increase in the maximum tolerated dose of 5FU in mice from 100 (FU100) to 150 mg/kg (5FU150+UDPG) and the higher dose is more effective than the standard treatment against several tumors. In the present paper we report on the effect of high-dose 5FU on thymidylate synthase (TS) levels and on 5FU incorporation into RNA. In the resistant murine tumor (Colon 26A) high-dose 5FU inhibited TS catalytic activity 8 h after treatment (4-fold; p = 0.00041) and the inhibition persisted until day 3 (p < 10–4). Standard-dose 5FU did not significantly inhibit TS activity. In a relatively sensitive tumor (Colon 26-10), there was no difference in the initial extent of TS inhibition by the two 5FU doses, but TS was still inhibited (2-fold) on day 3 after (5FU150+UDPG) while it was within the normal range after 5FU100. In both tumor types TS activity showed an impressive rebound (3-fold) on days 3–7, and this occurred after both 5FU doses. In Colon 26A, however, a new 5FU injection on day 7 was still able to inhibit TS but not as effectively as the first dose. 5FU incorporation into RNA reached similar peak values (8 pmol/µg RNA) after the two 5FU doses, but the clearance was faster in mice receiving UDPG rescue. We conclude that UDPG does not interfere with the extent of TS inhibition by 5FU, but UDPG allows the use of a higher dose of 5FU resulting in enhanced TS inhibition. UDPG, however, increases 5FU clearance from RNA. In this experimental system the inhibition of TS seems essential in order to obtain a good antitumor activity, while 5FU incorporation into RNA does not seem to play a role in the antitumor activity of 5FU. Since preliminary results indicate that UDPG is well tolerated by patients, the use of higher 5FU doses may improve the response rate of human tumors.