Comparison of the mechanism of cytotoxicity of 2-chloro-9-(2-deoxy-2- fluoro-beta-D-arabinofuranosyl)adenine, 2-chloro-9-(2-deoxy-2-fluoro- beta-D-ribofuranosyl)adenine, and 2-chloro-9-(2-deoxy-2,2-difluoro- beta-D-ribofuranosyl)adenine in CEM cells.

In an effort to understand biochemical features that are important to the selective antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine [Cl-F( upward arrow)-dAdo], we evaluated the biochemical pharmacology of three structurally similar compounds that have quite different antitumor activities. Cl-F( upward arrow)-dAdo was 50-fold more potent as an inhibitor of CEM cell growth than were either 2-chloro-9-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)adenine [Cl-F( downward arrow)-dAdo] or 2-chloro-9-(2-deoxy-2, 2-difluoro-beta-D-ribofuranosyl)adenine [Cl-diF( upward arrow downward arrow)-dAdo]. The compounds were similar as substrates of deoxycytidine kinase. Similar amounts of their respective triphosphates accumulated in CEM cells, and the rate of disappearance of these metabolites was also similar. Cl-F( upward arrow)-dAdo was 10- to 30-fold more potent in its ability to inhibit the incorporation of cytidine into deoxycytidine nucleotides than either Cl-F( downward arrow)-dAdo or Cl-diF( upward arrow downward arrow)-dAdo, respectively, which indicated that ribonucleotide reductase was differentially inhibited by these three compounds. Thus, the differences in the cytotoxicity of these agents toward CEM cells were not related to quantitative differences in the phosphorylation of these agents to active forms but can mostly be accounted for by differences in the inhibition of ribonucleotide reductase activity. Furthermore, the inhibition of RNA and protein synthesis by Cl-F( downward arrow)-dAdo and Cl-diF( upward arrow downward arrow)-dAdo at concentrations similar to those required for the inhibition of DNA synthesis can help explain the poor antitumor selectivity of these two agents because all cells require RNA and protein synthesis.

Excision of 2',2'-difluorodeoxycytidine (gemcitabine) monophosphate residues from DNA.

The activity of gemcitabine (dFdC), an effective agent against solid tumors, depends on the incorporation of its triphosphate into DNA. In vitro investigations demonstrated that, depending on the sequence of template DNA, polymerases may pause after incorporation of gemcitabine nucleotide at either the 3'-terminal or 3'-penultimate position. Proofreading enzymes such as 3'-->5' exonucleases, which are associated with DNA polymerases, can excise mismatched deoxynucleotides from DNA. To model this reaction, we evaluated excision of the gemcitabine nucleotide from oligodeoxynucleotide (19-mer) containing 3'-penultimate dFdC monophosphate (dFdCMP) or dCMP by the 3'-->5' exonuclease of the Klenow fragment. The rate of excision of the 3'-terminal deoxynucleotide was similar, with both primers resulting in formation of primers with terminal dCMP or dFdCMP. The primer containing dCMP was further excised, and by 40 min, more than 75% of total radioactivity was in excision products smaller than 18-mer. In contrast, most of the primers (90%) with terminal dFdCMP were unexcised. When primers terminated with either dFdCMP or dCMP were used as substrates, normal primer was hydrolyzed almost completely by 20 min; however, only 40% of primers containing dFdCMP had excision of dFdCMP molecule. Kinetic studies demonstrated that the enzyme had similar affinity for primers containing penultimate or terminal dFdCMP, but the apparent Vmax for excision was 4-5-fold greater for removal of a 3'-terminal deoxynucleotide than for cleavage of a dFdCMP molecule. Reaction conditions that permitted polymerization of one deoxynucleotide to primers containing either 3'-penultimate dCMP or dFdCMP were used to evaluate excision during DNA synthesis. The excised primers could not be extended because the reaction lacked the requisite deoxynucleotide triphosphate. After 5 min, more than one-half of the dCMP primers were extended, whereas only 15% had been excised. In comparison, 30% of the analogue-containing primers lost the terminal deoxynucleotide, with a proportional lower incidence of extension (30%). Lesser excision of dFdCMP-containing substrate was observed in reactions containing deoxynucleotide triphosphates required to make full-length products. Consistent with this result, in the absence of 3'-->5' exonuclease activity, both primers were extended similarly by the polymerization unit of the Klenow fragment. Taken together, these data demonstrate that dFdCMP residues are difficult to excise from DNA, and DNA polymerase can extend primers with 3'-dFdCMP. This results in the internal incorporation of dFdCMP into DNA, as observed in whole cells.

Ab initio quantum mechanical and X-ray crystallographic studies of gemcitabine and 2'-deoxy cytosine.

Gemcitabine 2',2'-difluoro 2'-deoxy cytosine (GEM) is a novel nucleoside which has demonstrated broad preclinical anti-cancer activity and appears promising in early stage human clinical trials. One purpose of this study was to characterize the energetically favored conformational modes of GEM by means of ab initio quantum mechanical studies with comparison to a novel X-ray crystallographic structure, and to determine the performance of ab initio quantum mechanical theory by comparison with X-ray structural data for GEM and 2'-deoxy cytosine (CYT). Another objective of this study was to attempt to determine key structural and electronic atomic interactions relating to the 2',2'-difluoro substitution in GEM by the application of ab initio quantum mechanical methods. To our knowledge, these are the first reported ab initio quantum mechanical geometry optimizations of nucleosides using large (e.g. 6-31G*) slit valence function basis sets. The development of accurate physicochemical models on a small scale enables us to extend our studies of GEM to more complex studies including DNA incorporation, deamination, ribonucleotide reductase inhibition, and triphosphorylation.

Radiosensitization of pancreatic cancer cells by 2',2'-difluoro-2'-deoxycytidine.

PURPOSE: We have reported that the deoxycytidine analog 2',2'difluoro-2'-deoxycytidine (dFdCyd) is a potent radiosensitizer of HT29 human colon cancer cells probably through its effects on intracellular deoxyribonucleotide (dNTP) pools. Because dFdCyd has activity against pancreatic cancer in clinical trials, we wished to determine if dFdCyd would radiosensitize human pancreatic cancer cells. METHODS AND MATERIALS: We assessed the effect of dFdCyd on radiation sensitivity of two human pancreatic cancer cell lines, Panc-1 and BxPC-3. To begin to investigate the mechanism of sensitization, we determined the effect of dFdCyd on dNTP pools and cell cycle distribution. RESULTS: We found that dFdCyd produced radiation enhancement ratios of 1.7-1.8 under noncytotoxic conditions in both cell lines. Sensitization was not associated with intracellular levels of 2',2'-difluoro-2'-deoxycytidine triphosphate, the cytotoxic metabolite of dFdCyd, but occurred when dATP pools were depleted below the level of approximately 1 micromolar. Although both cell lines showed substantial cell cycle redistribution after drug treatment, the flow cytogram of the BxPC-3 cells would not, by itself, be anticipated to result in increased radiation sensitivity. CONCLUSIONS: These findings demonstrate that dFdCyd is a potent radiation sensitizer of human pancreatic cancer cells and support the development of a clinical protocol using combined dFdCyd and radiation therapy in the treatment of pancreatic cancer.

Comparison of the antitumor activity of gemcitabine and ara-C in a panel of human breast, colon, lung and pancreatic xenograft models.

Gemcitabine is a new deoxycytidine analog that exhibits significant cytotoxicity against a variety of cultured murine and human tumor cells. The cytotoxic action of gemcitabine appears to be due to the inhibition of DNA synthesis by inhibition of ribonucleotide reductase and by competition with dCTP for incorporation into DNA. We have previously shown that gemcitabine, but not cytosine arabinoside (ara-C), has a broad spectrum of antitumor activity against 7 different types of murine solid tumors. The activity of gemcitabine was schedule dependent. To further characterize its activity, gemcitabine was tested against 12 human carcinoma xenografts. When given on an every 3 day x 4 schedule, the following percent inhibitions (at maximally tolerated doses [MTD]; MTD/2) in tumor growth were seen: MX-1 mammary (93%; 80%), CX-1 colon (92%; 82%), HC-1 colon (96%; 92%), GC3 colon (98%; 94%), VRC5 colon (99%; 100%), LX-1 lung (76%; 61%), CALU-6 lung (75%; 38%), NCI-H460 lung (45%; 46%), HS766T pancreatic (73%; not tested), PaCa-2 pancreatic (69%; 40%), PANC-1 pancreatic (70%; 60%), and BxPC-3 pancreatic (9%; 19%). In contrast, only the LX-1 lung carcinoma xenograft was responsive to ara-C treatment, which inhibited tumor growth by a marginal 62 percent. Thus, like its activity against murine solid tumors, gemcitabine has excellent antitumor activity against a broad spectrum of human solid tumors.

Medicinal chemistry of difluoropurines.

A series of over 70 difluoropurine analogs was synthesized by varying the C-2, 6 and 8 substituents about the purine ring system. After initial in vitro and in vivo screening, testing concentrated on the 2,6-diaminopurine analog (dFdAP) and the guanosine analog (dFdG). dFDAP appears to be a prodrug for dFdG. Both compounds significantly inhibited mammary tumor growth in mice, caused a moderate inhibition in ovarian and lymphosarcoma models, and demonstrated no activity in lung and melanoma models. This is a narrower spectrum of activity than that of gemcitabine (dFdC). The antitumor activity of dFdAP in human xenografts that are refractory to standard clinical agents was comparable or superior to that of gemcitabine. However, during the preliminary toxicology testing, dFdG was associated with several deaths caused by cardiac toxicity. Therefore, although dFdG is a potentially useful oncolytic, further investigation is required.

Difluorodeoxyguanosine: cytotoxicity, metabolism, and actions on DNA synthesis in human leukemia cells.

The success of gemcitabine (2',2'-difluorodeoxycytidine; dFdC) resulted in new interest in its purine congeners. Based on the structure-activity relationship studies of catabolism and anabolism, 2',2'-difluorodeoxyguanosine (dFdG) emerged as a lead candidate among the difluoropurine analogs. The cytotoxicity, metabolism, and actions of dFdG on DNA synthesis were studied in the human leukemia lymphoblastoid line CCRF-CEM. The IC50 values of dFdG after a 72-hour continuous incubation were 0.01, 0.03, and 0.28 mumol/L for CCRF-CEM, K562, and HL-60 cells, respectively. A cell line deficient in dCyd kinase was equally sensitive to dFdG, suggesting that, in contrast to dFdC, dFdG may be activated by other deoxynucleoside kinase(s). Consistent with these data, coincubation with dGuo spared the dFdG-mediated toxicity; however, up to 500 mumol/L dCyd failed to reverse the toxicity of dFdG. These observations indicated that dGuo kinase, which phosphorylates arabinosylguanine, also appears to play a major role in activating dFdG. CCRF-CEM cells incubated with varying concentrations of [3H]dFdG accumulated dFdGTP in a dose-dependent manner; a 3-hour incubation with 1 mmol/L dFdG resulted in more than 600 mumol/L intracellular dFdGTP. This is in contrast to the gemcitabine triphosphate accumulation, which is saturated at 10 to 20 mumol/L of exogenous dFdC. dFdG metabolites affected ribonucleotide reductase, resulting in a lowering of the dCTP pool; this is in agreement with the effect of dFdC on dNTP pools in leukemia cell lines. The major effect of dFdG on macromolecular synthesis was inhibition of DNA synthesis. DNA primer extension over a defined template revealed that dFdGTP was a good substrate for DNA polymerase alpha and incorporated opposite C sites of the template. Unlike arabinosyl analogs, but similar to gemcitabine triphosphate, dFdGTP incorporation caused DNA polymerase to pause after one normal deoxynucleotide was incorporated beyond the analog. The unique activation requirements of dFdG, its novel mode of inhibition of DNA synthesis, and its potent toxicity to human leukemia cells make it a promising new antimetabolite.

Cytotoxicity, metabolism, and mechanisms of action of 2',2'-difluorodeoxyguanosine in Chinese hamster ovary cells.

The emerging clinical success of gemcitabine (2',2'-difluorodeoxycytidine) stimulated interest in the synthesis and evaluation of purine congeners. The cytotoxicity, metabolism, and mechanisms of action of the lead candidate, 2',2'-difluorodeoxyguanosine (dFdGuo), were studied in Chinese hamster ovary cells. Unlike the natural nucleoside deoxyguanosine (dGuo), dFdGuo was not a substrate for purine nucleoside phosphorylase. Wild-type Chinese hamster ovary cells and a mutant line deficient in deoxycytidine (dCyd) kinase were similarly affected by dFdGuo (50% inhibitory concentration, 7.5 and 6.5 microM, respectively), suggesting that unlike gemcitabine, dCyd kinase was not responsible for activation of dFdGuo. This was further confirmed by separation of nucleoside kinases (adenosine kinase, dGuo kinase, and dCyd kinase) of Chinese hamster ovary cells on DEAE-cellulose column chromatography. The kinase activity that phosphorylated dGuo also converted dFdGuo to its monophosphate, suggesting that dGuo kinase activated dFdGuo. Consistent with this result, coincubation with dGuo spared the dFdGuo-mediated toxicity; however, addition of up to 10 mM dCyd did not reverse the toxicity of dFdGuo. Intracellularly, dFdGuo was phosphorylated to its mono-, di-, and triphosphates; dFdGuo triphosphate (dFdGTP) was the major metabolite and accumulated to 45 microM after a 6-h incubation with 30 microM dFdGuo. The elimination of dFdGTP was monophasic with a t1/2 of about 6 h. Deoxynucleotides were decreased in cells incubated with dFdGuo, suggesting that ribonucleotide reductase was inhibited. dATP, which decreased 78% after a 4-h incubation with 30 microM dFdGuo, was most affected. dFdGuo was a potent inhibitor of DNA synthesis. Extension of a DNA primer over a defined template in the presence of dFdGTP revealed that dFdGTP was a good substrate for incorporation opposite C sites of the template by DNA polymerase alpha. dFdGTP incorporation caused DNA polymerase alpha to pause after the polymerization of one additional deoxynucleotide. This pattern of inhibition, which is shared by gemcitabine, distinguishes 2',2'-difluoronucleosides from arabinosylnucleosides which halt primer extension at the incorporation site. dGTP competed effectively with dFdGTP for incorporation by DNA polymerase alpha. The unique activation requirements and patterns of inhibition of DNA synthesis distinguish this promising new antimetabolite from other nucleoside analogues.

Comparison of the inhibition of type A and type B S-adenosylhomocysteine hydrolase: effects of cofactor content on inhibition behavior and nucleoside binding.

The enzyme S-adenosylhomocysteine hydrolase (E.C.3.3.1.1) occurs in two forms in bovine liver: Type A, which carries four moles of NAD+ per mole of enzyme tetramer, and Type B, which carries two moles of NAD+ per mole of tetramer. The inhibition of these two forms of the enzyme with 2',2'-difluoro-2'-deoxyadenosine has been investigated. The studies examined the binding stoichiometry and stability of the enzyme-inhibitor complexes formed from each type of the enzyme, the degree of NAD+ reduction and NAD+ release, and the possibility of covalent bond formation between the enzyme and the inhibitor. Significant differences in the behavior of the two forms of the enzyme were encountered which may have important implications for the design of S-adenosylhomocysteine hydrolase inhibitors as therapeutic agents.

Metabolism of 2',2'-difluoro-2'-deoxycytidine and radiation sensitization of human colon carcinoma cells.

Difluorodeoxycytidine (dFdCyd) is a new antimetabolite with clinical activity in patients with solid tumors but not leukemias. We have studied the metabolism, cytotoxicity, and radiosensitizing properties of dFdCyd in HT-29 human colon carcinoma cells. The results demonstrated that dFdCyd rapidly accumulated as the 5'-triphosphate dFdCTP in HT-29 cells, which was eliminated slowly in the absence of dFdCyd with a half-life of > 12 h. Accumulation of dFdCTP was associated with rapid depletion of cellular dATP pools. Exposure to the concentration that reduces cell survival by 50% of 30 nM dFdCyd decreased dATP levels by > 80% within 4 h. dGTP pools were depleted at higher concentrations of dFdCyd, whereas smaller decreases were effected in dTTP and dCTP pools. These results contrast with previous reports in leukemic cells which demonstrated that dFdCyd exposure depleted the endogenous dCTP pool to a greater extent than the dTTP, dATP, or dGTP pools. Based on these data, we suggest that the profound depletion by dFdCyd of dATP and dGTP pools in HT-29 compared to leukemic cells accounts for the superiority of this agent in solid tumors versus leukemias. Additional studies demonstrated that dFdCyd was a potent radiosensitizer in HT-29 cells. Maximal radiosensitization was observed when cells were irradiated immediately following dFdCyd exposure instead of prior to or in the middle of drug treatment. Radiation sensitization was dose and time dependent, with a noncytotoxic exposure to 10 nM dFdCyd for 24 h or 30 nM dFdCyd for 16 h producing a radiation enhancement ratio of approximately 2. Under these conditions, only the cellular dATP pool was depleted. When cells were exposed to higher concentrations of dFdCyd for 4 h, equivalent radiosensitization with a radiation enhancement ratio of 1.4 was obtained using 0.1, 1.0, or 10 microM dFdCyd. Despite a 15-fold increase in dFdCTP and depletion of dGTP and dCTP pools to < 25% of the control value with 10 microM compared to 0.1 microM dFdCyd, no increase in radiosensitization was observed. These results suggest that dATP depletion is an important factor in the radiosensitizing property of this promising new antitumor compound.

Effect of a template-located 2',2'-difluorodeoxycytidine on the kinetics and fidelity of base insertion by Klenow (3'-->5'exonuclease-) fragment.

Gemcitabine [2',2'-difluorodeoxycytidine (dFdCyd)], a potent antitumor agent, inhibits DNA synthesis and is incorporated internally into DNA. The effect of a template-incorporated dFdCyd molecule (dFdCyd-) on DNA polymerase function was examined. Two 25-base deoxyoligonucleotides were synthesized with either a single dFdCyd- or template-incorporated deoxycytidine molecule (dCyd-) at the same position. Each was annealed separately to an identical complementary 5'-32P-labeled primer and extended by the Klenow fragment (3'-->5' exo-) of DNA polymerase I. "Correct" insertion of dGMP was 80-fold less efficient opposite dFdCyd- than dCyd-. A comparison of misinsertion efficiencies opposite template dFdCyd gave values of 2.7 x 10(-2) for dAMP insertion, 1.1 x 10(-3) for dTMP insertion, and 5.9 x 10(-4) for dCMP insertion. A similar measurement opposite template dC gave values of 1.8 x 10(-4), 1.7 x 10(-4), and 2.9 x 10(-6) for dAMP, dTMP, and dCMP insertion, respectively. Thus, the presence of dFdCyd on the template strand inhibited "normal" DNA synthesis and increased deoxyribonucleotide misinsertion frequencies. Pausing during DNA synthesis occurred directly opposite template dFdCyd suggesting that dFdC.dG base pairs might be less stable than normal dC.dG pairs, resulting in a decreased rate of primer extension beyond this site. Consistent with kinetic data, thermal denaturation measurements using comparable surrounding sequences showed that dFdC.dG "correct" pairs were less stable than dC.dG base pairs. Measurements on base mispairs showed that dFdC.dC was more stable than dC.dC, while no measurable Tm differences were found between polymers containing dFdC.dA and dC.dA or dFdC.dT, and dC.dT.

Effects of 2',2'-difluorodeoxycytidine (Gemcitabine) on wild type and variant mouse leukemia L1210 cells.

2',2'-Difluorodeoxycytidine (Gemcitabine, dFdCyd) is a cytotoxic agent which is active toward a variety of tumor cells. It has been shown that there are multiple intracellular sites of action which include ribonucleotide reductase and DNA polymerase. In these studies, the effects of dFdCyd on wild-type mouse leukemia L1210 cells and variant L1210 cell lines which had alterations at the ribonucleotide reductase site or at the deoxyribonucleoside kinase site were studied. For cell growth, the IC50 value for dFdCyd in wild-type L1210 cells was 3.1 nM. In the variant cell lines, the IC50 values were: hydroxyurea-resistant (HU), 3.3 nM; deoxyadenosine-resistant (Y8), 1.8 nM; pyrazoloimidazole/deoxyadenosine-resistant (ED2), 1.9 nM; and deoxyguanosine-resistant (dGuo-R), 44.7 nM. The dGuo-R cell line had a relatively specific loss of the deoxyribonucleoside kinase responsible for phosphorylating deoxyguanosine and cytosine arabinoside with little loss of the deoxycytidine kinase activity. DFdCyd had no effect on the total uptake of [14C]cytidine into the cells or incorporation into RNA. DFdCyd inhibited the conversion of [14C]cytidine to deoxycytidine nucleotides and incorporation into DNA. However, the incorporation of cytidine into DNA was inhibited to a greater extent than was the inhibition of in situ ribonucleotide reductase activity. Ribonucleotide reductase activity in cell-free extracts prepared from L1210 cells treated with dFdCyd (20 nM) overnight was reduced by 50%. These results show that cell lines which have increased levels of ribonucleotide reductase activity (HU and ED2) or loss of feedback inhibition by dATP (ED2 and Y8) are still sensitive to dFdCyd. The findings indicate that ribonucleotide reductase is not the primary site of inhibition by dFdCyd.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of new anticancer agents against the MIA PaCa-2 and PANC-1 human pancreatic carcinoma xenografts.

Human pancreatic carcinoma xenograft models were developed from established MIA PaCa-2 and PANC-1 cell lines (ATCC, Rockville, MD). Tumors were maintained by serial trocar implantation in CD1 nu/nu mice, and attempts were made to test all drugs under optimal schedules at maximum tolerated doses. In both models, adriamycin, cisplatin, and 5-fluorouracil were inactive (< 60% inhibition of tumor weight), whereas gemcitabine (LY188011] produced modest activity (69% inhibition in MIA PaCa-2 and 76% inhibition in PANC-1. Major differences in tumor sensitivity were noted with diarylsulfonylureas (DSU) and taxol. The DSU (Sulofenur [LY186641] and LY295501) produced complete inhibition in the MIA PaCa-2 xenograft, but were inactive in PANC-1. Conversely, taxol produced 80% inhibition of PANC-1 tumor growth, but was inactive against MIA PaCa-2. In general, in vivo antitumor activity roughly correlated with in vitro tumor cytotoxicity with the exception of DSU. We have previously shown that DSU are extensively bound to albumin and that in vitro cytotoxic activity in serum-containing medium is not predictive of in vivo antitumor activity. The MIA PaCa-2 and PANC-1 xenograft models may be useful for selecting potential candidates for therapy of human pancreatic cancer.

Synthesis and restriction enzyme analysis of oligodeoxyribonucleotides containing the anti-cancer drug 2',2'-difluoro-2'-deoxycytidine.

The anti-cancer drug 2',2'-difluoro-2'-deoxycytidine (dFdC) is internally incorporated into DNA in vitro. To determine the effects of this incorporation on DNA structure and function, the beta-cyanoethyl phosphoramidite of dFdC was synthesized and oligodeoxyribonucleotides containing dFdC were made using automated solid phase DNA synthesis techniques. Extension of the coupling time was required to achieve high coupling efficiency, suggesting a significant reduction in the rate of phosphotriester formation. Insertion of dFdC 5' into the recognition sequence of restriction enzymes HpaII and KpnI reduced the rate of cutting by 4% and 14% over 60 minutes. This reduction is similar to the effects seen with arabinofuranosylcytidine (ara-C) but small compared to the reductions caused by base analogues and phosphothioates. Insertion of dFdC into the BamHI recognition sequence, but not 5' to the cut site, did not alter the rate of cutting/recognition. The presence of a single dFdC reduced the Tm's of oligomers by 2-4 degrees C, depending on sequence and location. These results demonstrate that, once incorporated into DNA, dFdC does not greatly alter recognition between DNA and restriction enzymes; however, it does significantly alter duplex stability.

Cellular elimination of 2',2'-difluorodeoxycytidine 5'-triphosphate: a mechanism of self-potentiation.

2',2'-Difluorodeoxycytidine (dFdC, Gemcitabine) is a deoxycytidine analogue which, after phosphorylation to the 5'-di- and 5'-triphosphate (dFdCTP), induces inhibition of DNA synthesis and cell death. We examined the values for elimination kinetics of cellular dFdCTP and found they were dependent on cellular concentration after incubation of CCRF-CEM cells with dFdC and washing into drug-free medium. When the drug was washed out at low cellular dFdCTP levels (less than 50 microM), dFdCTP elimination was linear (t1/2 = 3.3 h), but it became biphasic at intracellular dFdCTP levels greater than 100 microM. Although the initial elimination rate was similar at all concentrations, at higher concentrations the terminal elimination rate increased with increasing cellular dFdCTP concentration, with a nearly complete inhibition of dFdCTP elimination at 300 microM. The deamination product 2',2'-difluorodeoxyuridine was the predominant extracellular catabolite at low cellular dFdCTP concentrations, whereas at high dFdCTP concentrations dFdC was the major excretion product. The dCMP deaminase inhibitor 3,4,5,6-tetrahydrodeoxyuridine transformed the monophasic dFdCTP degradation seen at low dFdCTP levels into a biphasic process, whereas the deoxycytidine deaminase inhibitor 3,4,5,6-tetrahydrouridine had no effect on dFdCTP elimination. An in situ assay indicated that dCMP deaminase activity was inhibited in whole cells, an action that was associated with a decreased dCTP:dTTP value. In addition, dFdCTP inhibited partially purified dCMP deaminase with a 50% inhibitory concentration of 0.46 mM. We conclude that dFdC-induced inhibition of dCMP deaminase resulted in a decrease of dFdCTP catabolism, contributing to the concentration-dependent elimination kinetics. This action constitutes a self-potentiation of dFdC activity.

Action of 2',2'-difluorodeoxycytidine on DNA synthesis.

The action of the new deoxycytidine analogue 2',2'-difluorodeoxycytidine (dFdC) on DNA synthesis was investigated in whole cells and in vitro assay systems with purified DNA polymerases. DNA synthesis in human lymphoblastoid CEM cells was inhibited by dFdC in a concentration-dependent manner that could not be reversed by exogenous deoxynucleosides. The analogue was incorporated into cellular DNA; most of the incorporated dFdC 5'-monophosphate (dFdCMP) residues were in internucleotide linkage. In vitro DNA primer extension assays demonstrated that dFdC 5'-triphosphate (dFdCTP) competed with deoxycytidine triphosphate for incorporation into the C sites of the growing DNA strand. The ratios of the apparent Km values for the incorporation of dFdCTP and dCTP into a C site of M13mp19 DNA were 21.8 and 22.9 for DNA polymerases alpha and epsilon, respectively. The apparent Ki values of dFdCTP were 11.2 microM for DNA polymerase alpha and 14.4 microM for polymerase epsilon. After dFdCMP incorporation, the primer was extended by one deoxynucleotide before a major pause in the polymerization process was observed. This was in contrast to the action of arabinosylcytosine 5'-triphosphate, which caused both DNA polymerases alpha and epsilon to pause at the site of incorporation. The 3'----5' exonuclease activity of DNA polymerase epsilon was essentially unable to excise nucleotides from DNA containing dFdCMP at either the 3'-end or at an internal position, whereas arabinosylcytosine monophosphate was removed from the 3'-terminus at 37% the rate for deoxynucleotides. The cytotoxic activity of dFdC was strongly correlated with the amount of dFdCMP incorporated into cellular DNA. Our results demonstrate qualitative and quantitative differences in the molecular actions of dFdC and arabinosylcytosine on DNA metabolism, but are consistent with an important role for such incorporation in the toxicity of dFdC.

Inhibition of ribonucleotide reduction in CCRF-CEM cells by 2',2'-difluorodeoxycytidine.

The new deoxycytidine analogue 2',2'-difluorodeoxycytidine (dFdC) is a specific inhibitor of DNA synthesis that has marked cytotoxicity and therapeutic activity. A 2-hr incubation with 0.1-10 microM dFdC decreased cellular viability 78-97%. This treatment reduced deoxynucleoside triphosphate pools, similar to the action of the ribonucleotide reductase inhibitor hydroxyurea. The most pronounced decrease occurred in the dCTP pool, quantitatively followed by the decrease of dATP, dGTP, and dTTP. In contrast, inhibition of DNA synthesis by arabinosylcytosine did not affect the dCTP level, whereas dATP, dGTP, and dTTP pools increased, but less than 2-fold. The incorporation of [5-3H]cytidine into the dCTP pool, a measure of ribonucleotide reductase activity in whole cells, was reduced to 3% of controls by 0.1 microM dFdC, but to only 40% by 0.1 microM ara-C. Each drug decreased incorporation of [5-3H]cytidine into DNA to a similar extent (greater than 94%), suggesting limitation by a reaction proximal to this step. The cellular concentration of dFdC 5'-diphosphate was 0.3 microM at 50% inhibition of the in situ activity of ribonucleotide reductase. Direct assays of partially purified ribonucleoside diphosphate reductase (EC 1.17.4.1) demonstrated 50% inhibition by 4 microM dFdC 5'-diphosphate; dFdC 5'-triphosphate was much less inhibitory. We conclude that dFdC 5'-diphosphate acts as an inhibitor of ribonucleoside diphosphate reductase.

Evaluation of the antitumor activity of gemcitabine (2',2'-difluoro-2'-deoxycytidine).

A new pyrimidine antimetabolite, 2',2'-difluorodeoxycytidine, Gemcitabine (LY188011, dFdCyd) has been synthesized and evaluated in experimental tumor models. dFdCyd is a very potent and specific deoxycytidine analogue. The concentration required for 50% inhibition of growth is 1 ng/ml in the CCRF-CEM human leukemia cell culture assay. Concurrent addition of deoxycytidine to the cell culture system provides about a 1000-fold decrease in biological activity. The inhibition of growth of human leukemia cells in culture led to the in vivo evaluation of this compound as a potential oncolytic agent. Maximal activity in vivo was seen with dFdCyd when administered on an every third day schedule. 1-beta-D-Arabinofuranosylcytosine, administered on a daily for 10-day schedule, was directly compared to dFdCyd in this evaluation. dFdCyd demonstrated good to excellent antitumor activity in eight of the eight murine tumor models evaluated. 1-beta-D-Arabinofuranosylcytosine was substantially less active or had no activity in these same tumor models. This in vivo activity against murine solid tumors supports the conclusion that dFdCyd is an excellent candidate for clinical trials in the treatment of cancer.

Comparison of the cellular pharmacokinetics and toxicity of 2',2'-difluorodeoxycytidine and 1-beta-D-arabinofuranosylcytosine.

2',2'-Difluorodeoxycytidine (dFdC) is a new deoxycytidine analogue with good activity against human leukemic cell lines and murine solid tumors, while the activity of 1-beta-D-arabinofuranosylcytosine (ara-C) is established in experimental systems and for the treatment of human adult leukemia. This study compared the cellular metabolism and cytotoxic properties of dFdC and ara-C in Chinese hamster ovary cells. In wild-type cells, dFdC was significantly more cytotoxic than ara-C after both 4- and 18-h incubations. The 5'-triphosphate of dFdC (dFdCTP) was the major cellular metabolite (85-90%), reaching cellular concentrations up to 20-fold greater than those observed for ara-C 5'-triphosphate at equimolar concentrations of the parent drug. A deoxycytidine kinase-deficient mutant neither accumulated dFdCTP nor showed any cytotoxic response up to drug concentrations of 100 microM. The cytotoxicity of dFdC could be competitively reversed by deoxycytidine further suggesting that dFdC, like ara-C, required phosphorylation by deoxycytidine kinase for biological activity. Several explanations for the different cellular accumulation of the drug triphosphates were established: (a) nucleoside transport studies demonstrated that the membrane permeation of dFdC was 65% more rapid than that of ara-C; (b) deoxycytidine kinase had a higher affinity for dFdC (Km = 3.6 microM) than for ara-C (Km = 8.8 microM), while the Km for deoxycytidine was 1.4 microM; (c) the elimination of intracellular dFdCTP was biphasic with t1/2 alpha = 3.9 and t1/2 beta greater than 16 h while the degradation of ara-CTP was monophasic and significantly faster (t1/2 = 0.7 h). The comparatively long half-life of dFdCTP was related to the prolonged inhibition of DNA synthesis after removal of exogenous nucleoside. Together these factors contribute to the more potent cytotoxicity of dFdC compared with ara-C.