C.J.A. van Moorsel

Basis for effective combination cancer chemotherapy with antimetabolites.

Most current chemotherapy regimens for cancer consist of empirically designed combinations, based on efficacy and lack of overlapping toxicity. In the development of combinations, several aspects are often overlooked: (1) possible metabolic and biological interactions between drugs, (2) scheduling, and (3) different pharmacokinetic profiles. Antimetabolites are used widely in chemotherapy combinations for treatment of various leukemias and solid tumors. Ideally, the combination of two or more agents should be more effective than each agent separately (synergism), although additive and even antagonistic combinations may result in a higher therapeutic efficacy in the clinic. The median-drug effect analysis method is one of the most widely used methods for in vitro evaluation of combinations. Several examples of classical effective antimetabolite-(anti)metabolite combinations are discussed, such as that of methotrexate with 6-mercaptopurine or leucovorin in (childhood) leukemia and 5-fluorouracil (5FU) with leucovorin in colon cancer. More recent combinations include treatment of acute-myeloid leukemia with fludarabine and arabinosylcytosine. Other combinations, currently frequently used in the treatment of solid malignancies, include an antimetabolite with a DNA-damaging agent, such as gemcitabine with cisplatin and 5FU with the cisplatin analog oxaliplatin. The combination of 5FU and the topoisomerase inhibitor irinotecan is based on decreased repair of irinotecan-induced DNA damage. These combinations may increase induction of apoptosis. The latter combinations have dramatically changed the treatment of incurable cancers, such as lung and colon cancer, and have demonstrated that rationally designed drug combinations offer new possibilities to treat solid malignancies.

Mechanisms of synergism between cisplatin and gemcitabine in ovarian and non-small-cell lung cancer cell lines

Summary2′,2′-Difluorodeoxycytidine (gemcitabine, dFdC) and cis-diammine-dichloroplatinum (cisplatin, CDDP) are active agents against ovarian cancer and non-small-cell lung cancer (NSCLC). CDDP acts by formation of platinum (Pt)–DNA adducts; dFdC by dFdCTP incorporation into DNA, subsequently leading to inhibition of exonuclease and DNA repair. Previously, synergism between both compounds was found in several human and murine cancer cell lines when cells were treated with these drugs in a constant ratio. In the present study we used different combinations of both drugs (one drug at its IC25 and the other in a concentration range) in the human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP, its dFdC-resistant variant AG6000 and two NSCLC cell lines, H322 (human) and Lewis lung (LL) (murine). Cells were exposed for 4, 24 and 72 h with a total culture time of 96 h, and possible synergism was evaluated by median drug effect analysis by calculating a combination index (CI; CI < 1 indicates synergism). With CDDP at its IC25, the average CIs calculated at the IC50, IC75 IC90 and IC95 after 4, 24 and 72 h of exposure were < 1 for all cell lines, indicating synergism, except for the CI after 4 h exposure in the LL cell line which showed an additive effect. With dFdC at its IC25, the CIs for the combination with CDDP after 24 h were < 1 in all cell lines, except for the Cls after 4 h exposure in the LL and H322 cell lines which showed an additive effect. At 72 h exposure all Cls were < 1. CDDP did not significantly affect dFdCTP accumulation in all cell lines. CDDP increased dFdC incorporation into both DNA and RNA of the A2780 cell lines 33- and 79-fold (P < 0.01) respectively, and tended to increase the dFdC incorporation into RNA in all cell lines. In the AG6000 and LL cell lines, CDDP and dFdC induced > 25% more DNA strand breaks (DSB) than each drug alone; however, in the other cell lines no effect, or even a decrease in DSB, was observed. dFdC increased the cellular Pt accumulation after 24 h incubation only in the ADDP cell line. However, dFdC did enhance the Pt–DNA adduct formation in the A2780, AG6000, ADDP and LL cell lines (1.6-, 1.4-, 2.9- and 1.6-fold respectively). This increase in Pt–DNA adduct formation seems to be related to the incorporation of dFdC into DNA (r = 0.91). No increase in DNA platination was found in the H322 cell line. dFdC only increased Pt–DNA adduct retention in the A2780 and LL cell lines, but decreased the Pt–DNA adduct retention in the AG6000 cell line. In conclusion, the synergism between dFdC and CDDP appears to be mainly due to an increase in Pt–DNA adduct formation possibly related to changes in DNA due to dFdC incorporation into DNA.

Scheduling of gemcitabine and cisplatin in Lewis Lung tumour bearing mice

We used the gemcitabine (dFdC) and cisplatin (cis-diamine dichloroplatinum CDDP) resistant murine NSCLC tumour Lewis Lung (LL) in C57/B16 mice to optimise scheduling of both drugs, since in previous in vivo studies no effective combination schedule of both compounds was found to overcome resistance to either drug. dFdC could not be combined at the previously determined maximum tolerated dose (MTD) (120 mg/kg, q3dx4) with CDDP at its MTD (9 mg/kg, q6dx2) (mean weight loss < 15% and < 15% toxic deaths), because of additive toxicity. Therefore, we lowered the dose of dFdC to 60 mg/kg (q3dx4) and of CDDP to 3 mg/kg (q6dx2), which caused an increase in antitumour effect compared with the activity of each compound alone at its MTD (growth delay factor (GDF) = 0.55, 0.13 and 2.56 for dFdC and CDDP alone and the combination, respectively). Changing the CDDP treatment schedule giving the total dose (6 mg/kg) only at day 0 caused unacceptable toxicity. This effect was not seen when mice were treated with the total dose of CDDP on day 9, but, the anti-tumour effect was not enhanced. To decrease toxicity, the dosage of dFdC was lowered to 50 mg/kg and combined with the total dose of CDDP on day 0, which caused a better antitumour effect than the combination of 60 mg/kg dFdC and 3 mg/kg CDDP (q6dx2) with acceptable toxicity. Schedule dependency was found for the combination: dFdC preceding CDDP by 4 h was the best treatment schedule in the LL tumours (GDF: 2.1) with acceptable toxicity. However, when the interval was increased to 24 h, toxicity became unacceptable (> 30% weight loss). The reverse schedule, in which CDDP preceded dFdC, did not lead to an increased antitumour effect or to increased toxicity. Adding amifostine, a selective chemoprotector, to the treatment decreased toxicity of the combination without affecting the antitumour effect. Increasing the CDDP dose to 9 mg/kg (day 0) under amifostine protection led to an improved therapeutic index.

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.

Induction of in vivo resistance against gemcitabine (dFdC, 2',2'-difluoro-deoxycytidine).

Gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) is a cytidine analog with major activity against several solid tumors (1,2). Until now acquired resistance has only been associated with deoxycytidine kinase (dCK) deficiency, after continuous exposure to dFdC in vitro (3). For l-β-D-arabinofuranosylcytosine (ara-C) the main in vitro resistance mechanism also is dCK deficiency (4,5). In patients however, the mechanism for ara-C resistance is less clear (5). Since dFdCis now widely used in the treatment of non-small cell lung and pancreatic cancer, it is very likely that resistance to gemcitabine will develop. Therefore it is important to determine mechanisms of resistance in an in vivo tumor model. For this purpose we used a tumor with moderate in vivo sensitivity to dFdC (6), Colon 26-A. We induced resistance by repeated dFdC treatment. To facilitate mechanism studies, cell lines were derived form the parental and resistant tumors. The questions we sought to answer were: 1. Is in vivo resistance to dFdC associated with dCK deficiency? And 2. If not, what mechanism is responsible for this resistance?