Differential effects of gemcitabine on ribonucleotide pools of twenty-one solid tumour and leukaemia cell lines.

To gain a more detailed insight into the metabolism of 2', 2'-difluoro-2'-deoxycytidine (dFdC, gemcitabine, Gemzar) and its effect on normal ribonucleotide (NTP) metabolism in relation to sensitivity, we studied the accumulation of dFdCTP and the changes in NTP pools after dFdC exposure in a panel of 21 solid tumour and leukaemia cell lines. Both sensitivity to dFdC and accumulation of dFdCTP were clearly cell line-dependent: in this panel of cell lines, the head and neck cancer (HNSCC) cell line 22B appeared to be the most sensitive, whereas the small cell lung cancer (SCLC) cell lines were the least sensitive to dFdC. The human leukaemia cell line CCRF-CEM accumulated the highest concentration of dFdCTP, whereas the non-SCLC cell lines accumulated the least. Not only the amount of dFdCTP accumulation was clearly related to the sensitivity for dFdC (R=-0.61), but also the intrinsic CTP/UTP ratio (R=0.97). NTP pools were affected considerably by dFdC treatment: in seven cell lines dFdC resulted in a 1.7-fold depletion of CTP pools, in two cell lines CTP pools were unaffected, but in 12 cell lines CTP pools increased about 2-fold. Furthermore, a 1.6-1.9-fold rise in ATP, UTP and GTP pools was shown in 20, 19 and 20 out of 21 cell lines, respectively. Only the UTP levels after treatment with dFdC were clearly related to the amount of dFdCTP accumulating in the cell (R=0.64 (P<0.01)), but not to the sensitivity to dFdC treatment. In conclusion, we demonstrate that besides the accumulation of dFdCTP, the CTP/UTP ratio was clearly related to the sensitivity to dFdC. Furthermore, the UTP levels and the CTP/UTP ratio after treatment were related to dFdCTP accumulation. Therefore, both the CTP and UTP pools appear to play an important role in the sensitivity to dFdC.

Gemcitabine and paclitaxel: pharmacokinetic and pharmacodynamic interactions in patients with non-small-cell lung cancer.

PURPOSE: To assess possible pharmacokinetic and pharmacodynamic interactions between gemcitabine and paclitaxel in a phase I/II study in non-small-cell lung cancer (NSCLC) patients. PATIENTS AND METHODS: Eighteen patients with advanced NSCLC received the following in a 3-week schedule: gemcitabine 1,000 mg/m(2) (30 minutes, days 1 and 8) and paclitaxel 150 (n = 9) or 200 mg/m(2) (n = 9) before gemcitabine (3 hours, day 1). Plasma pharmacokinetics and pharmacodynamics in mononuclear cells were studied. RESULTS: Gemcitabine did not influence paclitaxel pharmacokinetics at 150 and 200 mg/m(2) (area under the concentration-time curve [AUC], 7.7 and 8.8 micromol/ L. h, respectively; maximum plasma concentration [C(max)], 3.2 and 4.0 micromol/L, respectively), and paclitaxel did not influence that of gemcitabine (C(max), 30 +/- 3 micromol/L) and 2',2'-difluorodeoxyuridine. Paclitaxel, however, dose-dependently increased the C(max) of gemcitabine triphosphate (dFdCTP), the active metabolite of gemcitabine, from 55 +/- 10 to 106 +/- 16 pmol/10(6) cells.( )No significant difference in the AUC of dFdCTP was observed. Moreover, the gemcitabine-paclitaxel combination significantly increased ribonucleotide levels, most pronounced for adenosine triphosphate (six- to seven-fold). Postinfusion paclitaxel AUC was related to pretreatment hepatic function (bilirubin: r = 0. 79; P <.001) and to the percentage decrease in platelets (r = 0.61; P =.009). The latter was also related to the duration of paclitaxel concentration above 0.1 micromol/L (r = 0.62; P =.007). Gemcitabine C(max )was related to the percentage decrease in platelets (r = 0. 58; P =.01), pretreatment hepatic function (bilirubin: r = 0.77; P <. 001), and to plasma creatinine (r = 0.5; P =.03). The pharmacokinetics and pharmacodynamics were not related to response or survival. CONCLUSION: Gemcitabine and paclitaxel pharmacokinetics were related to the percentage decrease in platelets. Paclitaxel did not affect the pharmacokinetics of gemcitabine, nor did gemcitabine affect the pharmacokinetics of paclitaxel, but paclitaxel increased dFdCTP accumulation. This might enhance the antitumor activity of gemcitabine.

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.

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.

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.

Schedule-dependent pharmacodynamic effects of gemcitabine and cisplatin in mice bearing Lewis lung murine non-small cell lung tumours.

The combination of 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) and cis-diammine-dichloroplatinum(II) (cisplatin, CDDP) is increasingly applied in clinical oncology. We studied the underlying mechanisms of the in vivo schedule dependency and supraadditive interaction between dFdC and CDDP in C57/B16 mice bearing Lewis lung (LL) tumours. Mice were treated with CDDP (6 mg/kg) and dFdC (60 mg/kg) either simultaneously or in a 4 or 24 h interval with dFdC preceding CDDP or vice versa. Four, 8 (in some cases 12) and 24 h after treatment mice were sacrificed and tumours, kidneys, blood and bone marrow (BM) were collected. Since CDDP acts by formation of Platinum (Pt)-DNA adducts and dFdC by incorporation of its triphosphate (dFdCTP) into DNA, we measured total Pt levels, dFdCTP accumulation and Pt-DNA adducts by atomic absorption spectrometry (AAS), high performance liquid chromatography (HPLC) and 2P-postlabelling, respectively. These levels were related to the previously determined antitumour efficacy and toxicity of the dFdC/CDDP combination. Peak dFdCTP accumulation in tumours (11 pmol/mg) was found 4 h after dFdC treatment, while CDDP tended to reduce this in a time-dependent way. Peak levels of total Pt in tumours were found 4 h after CDDP treatment (581 fmol/mg) and dropped 1.8-fold after simultaneous treatment with dFdC (P = 0.04). Treatment with dFdC 4 h after or simultaneously with CDDP increased Pt retention (level 24 h after CDDP treatment) 1.4- and 1.6-fold (P = 0.04 and P = 0.03, respectively). Peak Pt-DNA adduct levels in tumours were also found 4 h after CDDP treatment (7 fmol/microg DNA) and were decreased 3-fold by dFdC treatment 24 h prior to CDDP (P = 0.04). Pt-DNA adduct retention was only decreased when dFdC was given 4 h before CDDP (8-fold (P < 0.01)). The retention and the area-under the concentration time curve of Pt-DNA adducts were related to decreased tumour doubling time (linear regression coefficient (R) = 0.95; P < 0.05, 0.96 P = 0.04 and 0.90; P = 0.04. Pt-DNA adduct levels in the BM cells reached a plateau level 4-24 h after CDDP treatment (approximately 10 fmol/microg DNA), which was increased by dFdC when given either simultaneously with, 4 h before or 4 h after CDDP (6-, 3- and 5-fold at 28 h, 8 h and 28 h, respectively (P < or = 0.04)). Peak Pt-DNA adduct formation (24 h: 8 fmol/microg DNA) in kidneys was enhanced by dFdC when given simultaneously with or 4 h before CDDP (4 h timepoint) (P < 0.01). However, retention was 4- and 6-fold decreased when dFdC was given 4 or 24 h after CDDP, respectively (P < or = 0.01). dFdC given 24 h before CDDP decreased all Pt-DNA adduct levels in kidneys 3-fold or more (P < or = 0.03). Pt-DNA adduct levels were inversely related to kidney toxicity when the most toxic schedule was excluded from the analysis. Peak levels of total Pt in kidneys were reached 24 h after CDDP treatment (4.3 fmol/mg) and the 8 h levels were increased 2-fold by dFdC when given 4 h after CDDP (P = 0.07).

Combination chemotherapy studies with gemcitabine.

Gemcitabine (2',2'-difluorodeoxycytidine) is an antineoplastic agent with clinical activity against ovarian carcinoma, small cell and non-small cell lung cancers, head and neck cancer, bladder cancer, breast cancer, and pancreatic cancer. Cisplatin (CDDP), etoposide (VP-16), and mitomycin C (MMC) are well-known anticancer agents that are also active against many of these types of cancer. Because of the low toxicity profile of gemcitabine and the differences in mechanism of cytotoxicity, combinations of these drugs with gemcitabine were studied in vitro and in vivo. Cells were exposed in vitro for 1, 4, 24, or 72 hours to gemcitabine in combination with these drugs, either simultaneously or sequentially in a constant ratio. Another approach consisted of exposure to a combination of the approximate IC25 of one drug and varying concentrations of the other drug. Synergism for several of these combinations was found in the human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP, its gemcitabine-resistant variant AG6000, and in the non-small cell lung cancer cell lines H322 and Lewis lung (LL) after a 72-hour drug treatment. Studies of the possible mechanisms of action initially focused on the major metabolic features of each drug. CDDP did not enhance the accumulation of gemcitabine triphosphate and caused only marginal changes in the extent of DNA double-strand breaks (DSBs) induced by gemcitabine in these cell lines. Gemcitabine increased platinum accumulation only in the ADDP cell line, but the DNA platination was enhanced in the A2780, ADDP, AG6000, and LL cell lines. MMC did not influence the formation of DSBs by gemcitabine in the LL cell line. The combination of VP-16 and gemcitabine, however, resulted in the formation of more DSBs in this cell line than each drug alone. This effect was even more pronounced when cells were exposed to VP-16 4 hours before gemcitabine. In vivo, the antitumor activity of a combination of 50 mg/kg gemcitabine and 6 mg/kg CDDP was more effective against LL tumors than each compound alone. In conclusion, gemcitabine is an attractive drug to combine with a wide range of anticancer drugs; synergism is often schedule dependent.

Preclinical combination therapy with gemcitabine and mechanisms of resistance.

Cisplatin and gemcitabine both have activity in solid tumors, such as non-small cell lung, ovarian, and head and neck cancers. These drugs have the desired features needed to obtain synergistic activity, different side effect profiles, and mechanisms of action. Cisplatin acts by forming DNA-DNA cross-links (both intrastrand and interstrand) and DNA-protein cross-links; resistance to cisplatin is thought to be due to excision repair of the affected DNA. Gemcitabine acts by its incorporation into nucleic acids, leading to masked chain termination. By combining gemcitabine with cisplatin, it might be possible to achieve a better therapeutic effect than either drug alone and to bypass resistance to one or both drugs. Acquired resistance to gemcitabine was associated with a deoxycytidine kinase deficiency in vitro, but this was difficult to achieve in vivo. Proper scheduling may overcome intrinsic and transient resistance due to physiologic circumstances or aberrant biochemical properties. Preclinical in vitro and in vivo combination studies with cisplatin showed schedule- and model-dependent synergistic and additive effects between cisplatin and gemcitabine. Incorporation of gemcitabine into DNA might facilitate cisplatin-DNA adduct formation. Combining gemcitabine and cisplatin inhibited the DNA excision-repair process more than gemcitabine alone. This implies that if gemcitabine nucleotide is incorporated into the DNA strand, the action of the proofreading exonucleases is less efficient. In addition, both deoxyribonucleotide and ribonucleotide pools, essential for good functioning of DNA repair, are seriously depleted by gemcitabine. It is concluded that combining gemcitabine with cisplatin can be at least additive providing the right schedule is chosen, giving the best balance between acceptable toxicity and an enhanced antitumor activity.

Vitamin A and beta-carotene influence the level of benzo[a]pyrene-induced DNA adducts and DNA-repair activities in hamster tracheal epithelium in organ culture.

Although most studies concerning the effect of vitamin A and beta-carotene on chemical carcinogenesis are focused on tumour promotion and progression, these compounds may affect initiation as well. In this study the influence of vitamin A and beta-carotene on unscheduled DNA synthesis (UDS) was investigated in hamster tracheal epithelium in organ culture exposed to benzo[a]pyrene (B[a]P). DNA-repair activities were compared with the level of B[a]P-DNA adducts as measured both by 32P-postlabeling and by immunocytochemical detection. In hamster tracheal epithelial cells, both vitamin A and beta-carotene significantly increased B[a]P-induced UDS, with 40% and 45%, respectively. At the same time, vitamin A and beta-carotene decreased the level of B[a]P-DNA adducts in these cells with 18% and 40%, respectively as measured by 32P-postlabeling and with 12% and 35%, respectively as measured by immunocytochemistry. The effect of vitamin A on B[a]P-induced UDS and DNA-adduct levels in hamster tracheal epithelium appeared to depend on the dose of B[a]P vis-à-vis the concentration of vitamin A. The results of the present study show that both vitamin A and beta-carotene cause a decrease in B[a]P-DNA adduct levels by enhancing DNA-repair activities. Because the formation of B[a]P-DNA adducts is considered to be an early step in respiratory tract carcinogenesis, it is suggested that enhancement of DNA-repair activities by vitamin A and the subsequent removal of DNA adducts may be one of the mechanisms involved in vitamin A-mediated protection against cancer.

Combination chemotherapy studies with gemcitabine and etoposide in non-small cell lung and ovarian cancer cell lines.

Gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and etoposide (4'-demethylepipodo-phyllo-toxin-9-4,6-O-ethylidene-beta-D-g lucopyranoside, VP-16) are antineoplastic agents with clinical activity against various types of solid tumors. Because of the low toxicity profile of dFdC and the differences in mechanisms of cytotoxicity, combinations of both drugs were studied in vitro. For this purpose, we used the human ovarian cancer cell line A2780, its cis-diammine-dichloroplatinum-resistant and VP-16 cross-resistant variant ADDP, and two non-small cell lung cancer cell lines, Lewis Lung (LL, murine) and H322 (human). The interaction between the drugs was determined with the multiple drug effect analysis (fixed molar ratio) and with a variable drug ratio. In the LL cell line, the combination of dFdC and VP-16 at a constant molar ratio (dFdC:VP-16 = 1:4 or 1:0.125 after 4- or 24-hr exposure, respectively) was synergistic (combination index [CI], calculated at 50% growth inhibition = 0.7 and 0.8, respectively; CI <1 indicating synergism). After 24- and 72-hr exposure to both drugs at a constant ratio, additivity was found in the A2780, ADDP, and H322 cell lines (dFdC:VP-16 = 1:500 for both exposure times in these cell lines). When cells were exposed to a combination of dFdC and VP-16 for 24 or 72 hr, with VP-16 at its IC25 and dFdC in a concentration range, additivity was found in both the LL and H322 cells; synergism was observed in the A2780 and ADDP cells, which are the least sensitive to VP-16. Schedule dependency was found in the LL cell line; when cells were exposed to dFdC 4 hr prior to VP-16 (constant molar ratio, total exposure 24 hr), synergism was found (CI = 0.5), whereas additivity was found when cells were exposed to VP-16 prior to dFdC (CI = 1.6). The mechanism of interaction between the drugs was studied in more detail in the LL cell line; dFdCTP accumulation was 1.2-fold enhanced by co-incubation with VP-16, and was even more pronounced (1.4-fold) when cells were exposed to VP-16 prior to dFdC. dCTP levels were decreased by VP-16 alone as well as by the combination of both compounds, which may favor phosphorylation of dFdC, thereby increasing dFdCTP accumulation. DNA strand break (DSB) formation was increased for exposure to both compounds together compared to exposure to each compound separately, this effect being most pronounced when cells were exposed to VP-16 prior to dFdC (38% and 0% DSB for dFdC and VP-16 alone, respectively and 97% DSB for the combination). The potentiation in DSB formation might be a result of the inhibition of DNA repair by dFdC. Provided the right schedule is used, VP-16 is certainly a compound eligible for combination with dFdC.

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.

Effect of gemcitabine and cis-platinum combinations on ribonucleotide and deoxyribonucleotide pools in ovarian cancer cell lines.

Gemcitabine (dFdC) and cisplatin (CDDP) act synergistically by an increase in platinum-DNA adduct formation. Since ribonucleotide (NTP) and deoxyribonucleotide (dNTP) levels are essential for DNA-synthesis and repair of DNA damage, we investigated whether disturbances might account for differences in effects between sensitive and resistant cell lines. The human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP and its dFdC-resistant variant AG6000 were exposed for 24 h to dFdC or CDDP alone, or a combination causing moderate to strong growth inhibition. In AG6000 cells UTP levels were 2-fold lower and in ADDP cells almost 2-fold higher than in A2780 cells. Levels of dTTP, dATP and dCTP were 2-5-fold lower in the resistant cell lines. Drug treatment affected NTP and dNTP levels most pronounced in A2780 cells. dFdC alone, at 1.5 nM to 1 micro M increased ATP, GTP and CTP pools 1.2 to 2.0-fold, while 10 micro M dFdC increased UTP 2.5-fold. Combination of dFdC and CDDP increased all NTP levels at low dFdC and CDDP concentrations more than 1.2-fold, but at 20 micro M CDDP only CTP increased 2.4-fold. Only 1.5 nM dFdC increased all dNTP pools more than 1.6-fold, but at 0.1 and 1 micro M dFdC, dATP and dGTP decreased down to 10-fold, while dTTP increased 3-5-fold. CDDP and the combination increased all dNTP pools over 1.4 and 1.9-fold, respectively. In AG6000 cells dFdC and CDDP hardly affected the NTP and dNTP status, except at the high concentrations, which decreased ATP, GTP and UTP levels 1.2-1.8-fold. Both CDDP alone and the combination increased dTTP, dCTP and dATP pools up to 1.6-fold. In ADDP cells NTP and most dNTP levels were hardly affected, except dGTP levels which decreased to non-detectable levels. In conclusion, both dFdC and CDDP induce concentration and combination dependent changes in NTP and dNTP pools.

Selective cell kill of the combination of gemcitabine and cisplatin in multilayered postconfluent tumor cell cultures.

Both gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and cisplatin (cis-diammine-dichloroplatinum) have significant anticancer activity against ovarian, head and neck, and non-small cell lung cancer (NSCLC). dFdC can be incorporated into DNA and RNA, and inhibit DNA repair, while cisplatin can form Pt-DNA adducts. We previously observed schedule-dependent synergism of the combination of dFdC and cisplatin in monolayer cell cultures. We now evaluated whether the combination would also enable selective cell kill in multilayered postconfluent cell cultures, since each compound showed variable activity in multilayered cells. The combination was tested in multilayered cultures from cell lines with a different histological origin: the human head and neck squamous cell carcinoma cell line UMSCC-22B (22B), the human NSCLC cell line H322, and ADDP, a cisplatin-resistant variant of the human ovarian cancer cell line A2780. Sensitivity of the multilayered cells was dependent on exposure duration and sequence of the drug combinations, which were added in a constant molar ratio (dFdC:cisplatin 1:100), with a total exposure time of 96 h. The type of interaction was related to the degree of resistance of the cell lines to either dFdC or cisplatin. Thus, the very sensitive 22B cells only showed an additive effect when cells were preincubated for 24 h with dFdC prior to exposure to the combination. In contrast, in the resistant ADDP and H322 cells, synergism was the most common profile (three out of four schedules tested). This is of special relevance when we take into account that these drugs only show cytostatic effects when administered alone, whereas the combination produced cytotoxic cell killing. In conclusion, combining dFdC with cisplatin can be at least additive, but synergistic in multilayered postconfluent cells resistant to dFdC and cisplatin.

Gemcitabine-cisplatin: a schedule finding study.

PURPOSE: To evaluate the tolerability of four alternating cisplatin-gemcitabine schedules. A secondary aim was to evaluate the clinical efficacy of this combination. PATIENTS AND METHODS: Forty-one patients with advanced solid tumors received alternating sequences with a 4- and 24-hour interval of cisplatin and gemcitabine. Gemcitabine 800 mg/m2 was administered as a 30-min infusion on day 1, 8 and 15, and cisplatin 50 mg/m2 over 1 hour on day 1 and 8; in case of the 24-hour time interval the second drug was administered one day later. Four cisplatin-gemcitabine schedules were studied: gemcitabine four hour before cisplatin (10 patients), or vice versa (14 patients) and gemcitabine twenty-four hours before cisplatin (9 patients) or vice versa (8 patients). The sequence of drug administration was reversed in the second cycle of therapy in each individual patient, enabling the evaluation of sequence-dependent side effects. Twenty-six patients had received prior chemotherapy, of which twenty-one platinum-based. RESULTS: The main toxicity was myelosuppression. Overall, grade 3 and 4 thrombocytopenia was observed in 27 out of 41 patients (66%) and was not schedule dependent. No serious bleeding occurred. Leukopenia was significantly different between the 4 alternating schedules (P = 0.01); gemcitabine 24 hours before cisplatin was significantly less toxic compared to both cisplatin 4 hours and 24 hours before gemcitabine (P = 0.01 and P = 0.003, respectively). Furthermore, paired analysis of the 4-hour and 24-hour data sets showed that leukopenia was significantly more serious when cisplatin preceded gemcitabine (P = 0.005). Although most patients received prior treatment, both prior chemotherapy and radiotherapy were not related to toxicity. Overall, grade 3 and 4 leukopenia occurred in 19 out of 41 patients (46%). Anemia (Hb < or = 6.0 mmol/l) was not sequence dependent and was observed in 63% of patients. Myelotoxicity was cumulative between cycles and caused frequent omission of gemcitabine on day 15. Overall, in 51% of administered cycles there was no omission of gemcitabine. A mean of 3.5 therapy cycles was administered. Non-hematological toxicity was moderate, consisting mainly of grade 1 and 2 nausea/vomiting and fatigue, and was not schedule dependent. Recently, we described that the schedule in which cisplatin was administered 24 hours before gemcitabine produced the best pharmacological profile. Based on this and because toxicity was manageable, the schedule cisplatin 24 hours prior to gemcitabine was chosen for phase II evaluation. Nine out of thirty-six evaluable patients had an objective response. These responses were observed in head and neck squamous-cell carcinoma (HNSCC), non-small-cell lung cancer (NSCLC), melanoma, adenocarcinoma of unknown origin, ovarian and esophageal carcinoma. CONCLUSIONS: Myelosuppression was the most important toxicity. Leukopenia was schedule dependent: gemcitabine before cisplatin was less toxic than the reversed sequence, in this respect. Some encouraging responses were seen in patients with esophageal cancer. Currently, a phase II study with cisplatin 24 hours before gemcitabine is ongoing in patients with advanced upper gastro-intestinal tumors.

An integrated physical map of 210 markers assigned to the short arm of human chromosome 11.

Using a panel of patient cell lines with chromosomal breakpoints, we constructed a physical map for the short arm of human chromosome 11. We focused on 11p15, a chromosome band harboring at least 25 known genes and associated with the Beckwith-Wiedemann syndrome, several childhood tumors, and genomic imprinting. This underlines the need for a physical map for this region. We divided the short arm of chromosome 11 into 18 breakpoint regions, and a large series of new and previously described genes and markers was mapped within these intervals using fluorescence in situ hybridization. Cosmid fingerprint analysis showed that 19 of these markers were included in cosmid contigs. A detailed 10-Mb pulsed-field physical map of the region 11p15.3-pter was constructed. These three different approaches enabled the high-resolution mapping of 210 markers, including 22 known genes.

Pharmacokinetic schedule finding study of the combination of gemcitabine and cisplatin in patients with solid tumors.

PURPOSE: To determine possible schedule dependent pharmacokinetic and pharmacodynamic interactions between gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and cisplatin (cis-diammine-dichloroplatinum, CDDP) in patients with advanced stage solid tumors in a phase I trial. PATIENTS AND METHODS: A total of 33 patients with advanced stage solid tumors were treated with gemcitabine (30-min infusion, 800 mg/m2) and cisplatin (one-hour infusion, 50 mg/m2). Sixteen patients had a four-hour interval between gemcitabine (days 1, 8, 15) and cisplatin (days 1 and 8), followed by the reverse schedule and seventeen patients had a 24-hour interval between gemcitabine (days 1, 8, 15) and cisplatin (days 2 and 9), followed by the reverse schedule. Gemcitabine and cisplatin pharmacokinetics were measured in plasma and white blood cells (WBC), isolated from blood samples taken at several time points after the start of treatment. RESULTS: A four-hour time interval between both agents did not reveal major differences in plasma pharmacokinetics of gemcitabine, dFdU (deaminated gemcitabine) and platinum (Pt), and of gemcitabine-triphosphate (dFdCTP) accumulation and Pt-DNA adduct formation in WBC between the two different sequences of gemcitabine and cisplatin. In the patients treated with the 24-hour interval, cisplatin before gemcitabine did not significantly change peak gemcitabine levels and the AUC of plasma dFdU, but tended to increase dFdCTP AUC in WBC 1.5-fold (P < 0.06). Gemcitabine before cisplatin decreased the plasma AUC of Pt 2.1-fold (P = 0.03). No significant differences in Pt-DNA adduct levels in WBC were found, although gemcitabine before cisplatin tended to increase the 24-hour retention of Pt-DNA adducts. Creatinine clearance on day 28 was related to the peak plasma levels of total Pt (linear regression coefficient (r) = 0.47, P = 0.02, n = 26). Furthermore, the increase in the Pt-GG to Pt-AG ratio 24 hours after cisplatin treatment was related to the overall response of patients (r = 0.89, P < 0.01, n = 8). CONCLUSIONS: Of all schedules the treatment of patients with cisplatin 24 hours before gemcitabine led to the highest dFdCTP accumulation in WBC and total Pt levels in plasma. These characteristics formed the basis for further investigation of this schedule in a phase II clinical study.