Development and molecular characterization of a 2',2'-difluorodeoxycytidine-resistant variant of the human ovarian carcinoma cell line A2780.

2',2'-Difluorodeoxycytidine (gemcitabine, dFdCyd) is a deoxycytidine analogue with promising antitumor activity. In order to be active it must be phosphorylated by deoxycytidine kinase (dCK). We induced resistance to dFCyd in the human ovarian carcinoma cell line A2780 by exposure to increasing concentrations of dFdCyd. The IC50, defined as the concentration of dFdCyd causing 50% growth inhibition, at 72 h exposure increased from 0.6 nM dFdCyd in A2780 to 92 microM in the resistant variant, named AG6000. Although the resistant cell line is routinely cultured in 6 microM dFdCyd, the resistant phenotype can be maintained for at least 10 passages without dFdCyd. AG6000 is cross-resistant to other drug which require activation by dCK, such as 1-beta-D-arabinofuranosylcytosine, 5-aza-2'-deoxycytidine, and 2-chlorodeoxyadenosine. There was no specific dCK activity in extracts from AG600 cells. Western blot analysis using a polyclonal anti-dCK antibody did not reveal any dCK protein in AG6000 cell extracts. Reverse-transcribed and PCR-amplified mRNA, using specific dCK primers, demonstrated that AG6000 expressed a normal length amplicon of 701 base pairs, besides an aberrant amplicon of 500 base pairs. Chromosome spreads from the cell lines showed no major differences between A2780 and AG6000. The latter cell line was also cross-resistant to 2',2'-difluorodeoxyurdine, the deamination product of dFdCyd. Additionally, cross-resistance to the multidrug resistant drugs doxorubicin and vincristine was observed. This was not associated with the induction of P-glycoprotein, as determined by the RNase protection assay. Injection of AG6000 cells s.c. into nude mice demonstrated that the cell line had retained its tumorigenicity; AG6000 xenografts were not sensitive to dFdCyd treatment, in contrast to the parental A2780 tumors. No dFdCyd triphosphate accumulation was found in the resistant tumors, in contrast to the parental A2780 tumors. These results indicate that the dFdCyd resistance phenotype is stable, and mainly due to dCK deficiency.

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.

Synergistic interaction between cisplatin and gemcitabine in vitro.

2',2'-Difluorodeoxycytidine (dFdC; gemcitabine) is a new antineoplastic agent that is active against ovarian carcinoma, non-small-cell lung carcinoma, and head and neck squamous cell carcinoma. cis-diamminedichloroplatinum (CDDP; cisplatin) is used commonly for the treatment of these tumors. Because the two drugs have mechanisms of action that might be complementary, we investigated a possible synergism between dFdC and CDDP on growth inhibition. The combination was tested in the human ovarian carcinoma cell line A2780, its CDDP-resistant variant ADDP and its dFdC-resistant variant AG6000, the human head and neck squamous cell carcinoma cell line UMSCC-22B, and the murine colon carcinoma cell line C26-10. The cells were exposed to dFdC and CDDP as single agents and to combinations in a molar ratio of 1:500 for 1, 4, 24, and 72 h with a total culture time of 72 h. Synergy was evaluated using the multiple drug effect analysis. In A2780 and ADDP cells, simultaneous exposure to the drugs for 24 and 72 h resulted in synergism, but shorter exposure times were antagonistic. No synergism was found in the UMSCC-22B and C26-10 cell lines at prolonged simultaneous exposure. However, a preincubation with CDDP for 4 h followed by a dFdC incubation for 1, 4, 24, and 72 h was synergistic in all cell lines except C26-10 cells. A 4-h preincubation with dFdC followed by an incubation with the combination for 20 and 68 h was synergistic in all cell lines. Initial studies of the mechanism of interaction concentrated on the effect of CDDP on dFdCTP accumulation and DNA strand break formation. In all cell lines, CDDP failed to increase dFdCTP accumulation at 4- or 24-h exposure to dFdC; in two cell lines, CDDP even tended to decrease dFdCTP accumulation. Neither dFdC nor CDDP caused more than 25% double strand break formation, whereas in the combination, CDDP even tended to decrease this type of DNA damage. The synergistic interaction between the two drugs is possibly the result of dFdC incorporation into DNA and/or CDDP-DNA adduct formation, which may be affected by each other.

Schedule-dependent therapeutic efficacy of the combination of gemcitabine and cisplatin in head and neck cancer xenografts.

Gemcitabine and cisplatin are both drugs with proven clinical activity in various tumour types, have no overlapping toxic side-effects and are different with respect to cellular metabolism. We, therefore, performed an in vivo study to determine the efficacy of the combination of these two drugs using two human head and neck squamous cell carcinoma xenograft lines, subcutaneously growing in athymic nude mice. 100 mg/kg gemcitabine was given intraperitoneally on days 0, 3, 6 and 9 and 4 mg/kg cisplatin intravenously on days 0 and 6. In one tumour line, the combination treatment resulted in better effects than those observed when the drugs were administered individually. In the other cell line, addition of cisplatin did not increase the moderate effect of gemcitabine. Experiments with single dose injections of both drugs showed adverse effects when the interval was extended to 24 h. These data are of potential interest for clinical application, and suggest that the drugs should be administered either simultaneously or with a short time interval in which cisplatin should precede gemcitabine.

Postconfluent multilayered cell line cultures for selective screening of gemcitabine.

The in vitro cytotoxicity of gemcitabine (dFdC) was tested in ovarian and colon cancer cell lines grown as monolayers and three-dimensional multilayered cell cultures. In our model, dFdC showed slight selectivity in cytotoxicity against ovarian over colon cancer cells, when cell lines were grown as monolayers. However, when cell lines were grown as multilayers, this selectivity was accentuated: A2780 multilayers were 14 times less sensitive than monolayers, but the colon cancer cell lines were more than 1000 times more resistant than their corresponding monolayers. The accumulation of the active metabolite, dFdCTP, after 24 h exposure to 1 microM dFdC varied between 1100 and 1900 pmol/10(6) cells in monolayers. This was 5 times lower in multilayers compared with monolayers of all four cell lines, which can, in part, explain the lower sensitivity of the multilayers. In addition, it appears that the amount of the active metabolite retained is more important than the amount accumulated initially, since the differences between the ovarian and the colon cancer cell lines were more evident in retention experiments. Exposure to dFdC caused a 2-3-fold increase in the levels of several nucleotides, except for the CTP pools in the colon cancer lines, which were reduced by 3-fold at the highest dFdC concentration (10 microM). The findings with the multilayer model are in better agreement with in vivo activity in ovarian cancer and colon cancer than those with the monolayer system. This indicates the potential of the multilayer system to be a better predictive model than the conventionally used monolayer cultures.

Deoxycytidine kinase and deoxycytidine deaminase activities in human tumour xenografts.

Deoxycytidine kinase (dCK) and deaminase (dCDA) are both key enzymes in the activation and inactivation, respectively, of several deoxycytidine antimetabolites. We determined the total dCK and dCDA activities using standard assays, in 28 human solid tumours grown as xenografts in nude mice, and four corresponding cell lines. dCK activities in colon tumours varied from 11 to 12 nmol/h/mg protein, in ovarian tumours from 3 to 10 nmol/h/mg protein, in soft tissue sarcomas from 2 to 7 nmol/h/mg protein and in squamous cell carcinomas of the head and neck about 45-fold, between 0.4 and 18 nmol/h/mg protein. The dCDA activities showed a larger variation, from 243 to 483, 14 to 1231, 3 to 7 and 1 to 222 nmol/h/mg protein, respectively. The ratios of dCK vs. dCDA activities in these tumours varied from 0.025 to 0.046, 0.004 to 0.240, 0.581 to 1.123 and from 0.012 to 4.227, respectively. In four cell lines (A2780, OVCAR-3, WiDr and UM-SCC-14C), sources for some of the above mentioned tumours, a different pattern in dCK and dCDA was observed than in the corresponding tumours. The variation in dCDA activities was in a smaller range (20-fold) than in the tumours (40-fold). In all cell lines dCK activity was higher than dCDA activity, in contrast to the corresponding tumours, in which the reverse pattern was observed. Previously, some of the tumours were tested for sensitivity to the deoxycytidine analogues 5-aza-deoxycytidine and 2',2'-difluorodeoxycytidine. In the sensitive tumours, both the highest and lowest dCK activity was observed, indicating that dCK activity in solid tumours is high enough to activate deoxycytidine analogues.

Induction of resistance to 2',2'-difluorodeoxycytidine in the human ovarian cancer cell line A2780.

2',2'-Difluorodeoxycytidine (gemcitabine; dFdC) is a nucleoside analog with promising antitumor activity. To be active it must be phosphorylated by deoxycytidine kinase (dCK). We induced resistance to gemcitabine in the human ovarian carcinoma cell line A2780 by exposure to increasing concentrations of gemcitabine. At 72 hours' exposure the IC50, defined as the concentration of gemcitabine causing 50% growth inhibition, increased from 0.6 nmol/L gemcitabine in A2780 to 92 mumol/L in the resistant variant, AG6000. AG6000 is cross-resistant to other drugs that require activation by dCK, such as I-beta-D-arabinofuranosylcytosine, 5-aza-2'-deoxycytidine, and 2-chlorodeoxyadenosine. AG6000 was also cross-resistant to 2',2'-difluorodeoxyuridine (dFdU), the deamination product of gemcitabine. In addition, cross-resistance to the multidrug-resistance drugs doxorubicin and vincristine was observed. This was not associated with induction of P-glycoprotein. No accumulation of gemcitabine triphosphate could be detected in AG6000 cells, in contrast to the parental A2780 cells. There was no specific dCK activity in extracts from AG6000 cells. Western blot analysis using a polyclonal anti-dCK antibody did not reveal any dCK protein in AG6000 cell extracts. Reverse transcribed and polymerase chain reaction amplified mRNA, using specific dCK primers, demonstrated that AG6000 expressed a normal length amplicon of 701 base pairs, besides an aberrant amplicon of 500 base pairs. Although the resistant cell line is routinely cultured in 6 mumol/L gemcitabine, the resistant phenotype can be maintained for at least 10 passages without gemcitabine. These results indicate that the gemcitabine resistance phenotype is stable and mainly due to dCK deficiency.

Schedule-dependent antitumor effect of gemcitabine in in vivo model system.

Gemcitabine (2',2'-difluorodeoxycytidine, dFdC, LY188011) is a new deoxycytidine analog with preclinical antitumor activity in solid tumors from murine and human origin. Of particular importance is the fact that the therapeutic effects of gemcitabine at the maximum tolerated dose level are dependent on the administration schedule. This paper describes the sensitivity pattern of gemcitabine in human head and neck squamous cell carcinoma, ovarian carcinoma, and soft tissue sarcoma, all growing as xenografts in athymic nude mice. The drug was injected intraperitoneally in various schedules at equitoxic, maximum tolerated dose levels, resulting in a reversible weight loss that varied between 5% and 15%. Generally, it was found that treatment with 120 mg/kg gemcitabine, injected four times at 3-day intervals, was more effective than the schedules of daily (five times 2.5 to 3.5 mg/kg) or weekly (two times 240 mg/kg) injections. Other workers have shown that this 3-day interval schedule also was active in human pancreas and lung carcinoma xenografts. Additional experiments were performed on normal mice bearing the colon 26-10 murine colon carcinoma. The effect of a continuous intravenous infusion system was investigated by giving two injections of 15 mg/kg gemcitabine for 24 hours at a 7-day interval. Interestingly, the efficacy of treatment increased dramatically with this infusion schedule, producing complete remissions in most tumors. In conclusion, our data on the effect of gemcitabine in animal tumor models indicate that (1) the time interval between push injections is important when intermittent schedules are used and (2) continuous infusions over a 24-hour period can be very effective in in vivo models.

Interaction between cisplatin and gemcitabine in vitro and in vivo.

Both gemcitabine (2',2'-difluorodeoxycytidine; dFdC) and cisplatin (cis-diammine dichloroplatinum; CDDP) are active against several solid malignancies, including ovarian cancer and head and neck squamous cell carcinoma. Because of differences in mechanisms of action and toxicity profiles, combination of the two drugs has enormous clinical potential. We studied possible synergism between the drugs: in vitro using three variants of the human ovarian cancer cell line A2780, and in vivo using gemcitabine- and cisplatin-sensitive and -resistant tumors, the head and neck cancer xenografts HNX-22B and HNX-14C and the murine syngeneic colon 26-10 tumor. In vitro, cells were cultured for 72 hours and exposed to the drugs for 1 to 72 hours; synergy was evaluated by multiple drug-effect analysis. In wild-type A2780 and cisplatin-resistant ADDP cells, simultaneous exposure for 24 and 72 hours was synergistic, as well as preincubation with cisplatin for 4 hours followed by gemcitabine. Preincubation with gemcitabine for 4 hours followed by gemcitabine and cisplatin was synergistic in ADDP and A2780 cells. Cisplatin did not enhance the accumulation of gemcitabine triphosphate in A2780 and ADDP cells. Cisplatin caused a marginal decrease of the number of double strand breaks in the DNA caused by gemcitabine. In vivo, gemcitabine at the maximum tolerated dose of 100 or 120 mg/kg could be combined with cisplatin at 4 mg/kg. When injected simultaneously this resulted in at least additive anti-tumor activity in HNX-22B, but not in HNX-14C and colon 26-10 tumors. Cisplatin, injected 4 hours before or after gemcitabine, was equally active as the simultaneous schedule in HNX-22B tumors, but more toxic. In conclusion, the combination of gemcitabine and cisplatin can be synergistic in vitro and at least additive in vivo; this synergism is schedule dependent. The mechanism cannot be explained by gemcitabine triphosphate accumulation or DNA damage studies.

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.

2',2'-Difluoro-deoxycytidine (gemcitabine) incorporation into RNA and DNA of tumour cell lines.

Gemcitabine (dFdC) is a new cytidine analogue which is active mainly by the incorporation of its triphosphate (dFdCTP) into DNA, leading to cell death. We determined incorporation of dFdC into nucleic acids of two solid tumour cell lines: the murine colon carcinoma cell line Colon 26-10, the human ovarian carcinoma cell line A2780, and the human leukemic cell line CCRF-CEM. dFdC was not only incorporated into DNA, but also into RNA. The extent of incorporation into DNA was highest in A2780 cells and lowest in CCRF-CEM cells (2-4-fold difference). The same pattern was observed for incorporation into RNA, but with a 10-20-fold difference. In A2780, incorporation into DNA was about twice that of the incorporation into RNA, in CEM cells 10-20-fold that of RNA. Incorporation into RNA was verified using two methods for separation of RNA and DNA, acid precipitation and CsCl-gradient centrifugation. Incorporation into DNA was time and concentration dependent, but incorporation into RNA seemed to be only concentration dependent. We also determined the effect of dFdC on DNA and RNA synthesis by measurement of thymidine and uridine incorporation, respectively, using similar conditions as for the incorporation studies. In all three cell lines DNA synthesis was inhibited almost completely, even at 0.1 microM dFdC and at 4-hr exposure. RNA synthesis inhibition did not exceed 50% in both solid tumour cell lines, even at 1 microM dFdC exposure for 24 hr. A clear concentration effect was only observed in the CCRF-CEM cell line and only after 24 hr exposure. At a 1 microM dFdC exposure for 24 hr, RNA synthesis was completely inhibited in these cells. Incorporation of dFdC into RNA and inhibition of RNA synthesis represent an unrecognized but possibly important mechanism of action of this drug.

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.

Decreased resistance to gemcitabine (2',2'-difluorodeoxycitidine) of cytosine arabinoside-resistant myeloblastic murine and rat leukemia cell lines: role of altered activity and substrate specificity of deoxycytidine kinase.

We determined the potential activity of 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) in 1-beta-D-arabinofuranosylcytidine (ara-C)-sensitive and-resistant leukemia cell lines. Both drugs are phosphorylated by deoxycytidine kinase (dCK); the triphosphates, dFdCTP and ara-CTP, respectively, are incorporated into DNA. In the murine leukemia cell line L1210, induction of resistance to ara-C resulted in the 2200-fold resistant subline L4A6. The Brown Norway rat myelocytic leukemia ara-C-sensitive cell line (BCLO) was >300-fold more sensitive to ara-C than its variant Bara-C. In L1210 cells, gemcitabine was 8-fold more active than ara-C; in L4A6, BCLO, and Bara-C cells, gemcitabine was 16-, 28-, and more than 3-fold more active than ara-C, respectively. A partial explanation for these differences may be the higher dCK activity in the parental cell lines L1210 and BCLO with gemcitabine compared to ara-C as a substrate. DCK activity was not or hardly detectable in the resistant L4A6 and Bara-C cell. In the rat leukemia cell lines, deoxycytidine (dCyd) phosphorylation activity showed an aberrant pattern, since the activity with dCyd was 1.5-fold higher in the Bara-C cell line compared with BCLO, possibly due to thymidine kinase 2. The wild-type L1210 cells accumulated at least 3-fold more ara-CTP and dFdCTP than the rat leukemia cell line BCLO. The ara-C-resistant variants L4A6 and Bara-C did not accumulate dFdCTP or ara-CTP. In conclusion, gemcitabine was more active than ara-C in all leukemia cell lines tested. The sensitivity of the wild-type cell lines correlates with the accumulation of dFdCTP and ara-CTP, but is independent of dCK. However, both resistant variants had decreased dCK activities, but were relatively more sensitive to dFdC than to ara-C.

Antitumor activity of prolonged as compared with bolus administration of 2',2'-difluorodeoxycytidine in vivo against murine colon tumors.

2',2'-Difluorodeoxycytidine (gemcitabine) is a cytidine analogue with established antitumor activity against several experimental tumor types and against human ovarian and non-small-cell lung cancer. Both preclinical studies and most clinical trials involving patients with solid tumors have focused on short-term administration schedules; however, mechanistic studies indicate that a continuous-infusion schedule may be more effective. We determined the maximal tolerated dose (MTD) of gemcitabine in mice using various schedules. At these MTDs we observed considerably better antitumor activity of gemcitabine in two of three murine colon carcinoma lines using a prolonged administration as compared with a standard bolus protocol (i.p. 120 mg/kg q3d x 4). On the latter schedule, Colon 26-10 grown in BALB/c mice was the most sensitive tumor line, showing a growth-delay factor (GDF, number of doubling times gained by the treatment) of 6.7, whereas Colon 38 (grown in C57/B16 mice) was the least sensitive tumor, displaying a GDF of 0.9. Prolonged treatment (q3d x 6) of Colon 26-10 at a lower dose (100 mg/kg) enhanced the antitumor activity (GDF 9.6) while producing similar toxicity. A similar weight loss was found following the continuous infusion (c.i.) of gemcitabine using Alzet osmotic pumps s.c. for 3 or 7 days (2 mg/kg), but the GDF increased to 2.4 in Colon 38 (C57/B16) as compared with that provided by the bolus injections. Continuous infusion of gemcitabine at 15 mg/kg per 24 h q7d x 2 i.v. via the tail vein was more effective than bolus injection against Colon 26-10, with the GDF being > 17.7 and 73% of the tumors regressing completely. However, against Colon 38 tumors this schedule was not effective (GDF 0.4), even with a 25% higher dose. The plasma pharmacokinetics of gemcitabine was determined after one bolus dose (120 mg/kg). The peak concentration of gemcitabine was 225 microM and that of the deaminated catabolite 2',2'-difluorodeoxyuridine (dFdU) was 79 microM. The elimination of gemcitabine was much faster than that of dFdU, with the t1/2 beta values being 15 min and 8 h, respectively. For the c.i. schedules, plasma concentrations were below the detection limit of the assay (< 0.5 microM). Our results suggest that prolonged infusion of gemcitabine can give a better antitumor activity than bolus injections and shows promise of being active in clinical trials.

New targets for pyrimidine antimetabolites for the treatment of solid tumours. 2: Deoxycytidine kinase.

Deoxycytidine kinase is an enzyme required for the activation of, for example, cytarabine, the most widely used agent for the chemotherapy of haematological malignancies. However, deoxycytidine kinase also plays an important role in the activation of several new agents used in the treatment of leukaemia, such as cladribine. Recently, a new cytidine analogue, gemcitabine, has shown impressive activity as a single agent against several solid malignancies (ovarian cancer, non-small cell lung cancer), demonstrating that in solid tumours deoxycytidine kinase can be an important target for the activation of antimetabolites. Studies on the regulation of deoxycytidine kinase have shown that the enzyme has a complicated regulation (feedback inhibition by the product and regulation by ribonucleotides). Modulation of deoxycytidine kinase activity has already been shown to be an effective way to improve the effect of cytarabine and will probably be a target for new therapies.