The novel anti-cancer fluoropyrimidine NUC-3373 is a potent inhibitor of thymidylate synthase and an effective DNA-damaging agent.

INTRODUCTION: Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU. METHODS: Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC-MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage. RESULTS: We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU. CONCLUSION: Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.

Thymineless Death by the Fluoropyrimidine Polymer F10 Involves Replication Fork Collapse and Is Enhanced by Chk1 Inhibition.

We are developing the fluoropyrimidine polymer F10 to overcome limitations of 5-fluorouracil (5-FU) that result from inefficient metabolism to 5-fluoro-2'-deoxyuridine-5'-mono- and tri-phosphate, the deoxyribonucleotide metabolites that are responsible for 5-FU's anticancer activity. F10 is much more cytotoxic than 5-FU to colorectal cancer (CRC) cells; however, the mechanism of enhanced F10 cytotoxicity remains incompletely characterized. Using DNA fiber analysis, we establish that F10 decreases replication fork velocity and causes replication fork collapse, while 1000-fold excess of 5-FU is required to achieve similar endpoints. Treatment of HCT-116 cells with F10 results in Chk1 phosphorylation and activation of intra-S-phase checkpoint. Combining F10 with pharmacological inhibition of Chk1 with either PF-477736 or prexasertib in CRC cells enhanced DNA damage relative to single-agent treatment as assessed by γH2AX intensity and COMET assay. PF-477736 or prexasertib co-treatment also inhibited upregulation of Rad51 levels in response to F10, resulting in reduced homologous repair. siRNA knockdown of Chk1 also increased F10-induced DNA damage assessed and sensitized CRC cells to F10. However, Chk1 knockdown did not inhibit Rad51 upregulation by F10, indicating that the scaffolding activity of Chk1 imparts activity in DNA repair distinct from Chk1 enzymatic activity. Our results indicate that F10 is cytotoxic to CRC cells in part through DNA damage subsequent to replication fork collapse. F10 is ~1000-fold more potent than 5-FU at inducing replication-mediated DNA damage which correlates with the increased overall potency of F10 relative to 5-FU. F10 efficacy can be enhanced by pharmacological inhibition of Chk1.

The applications of the novel polymeric fluoropyrimidine F10 in cancer treatment: current evidence.

F10 is a novel polymeric fluoropyrimidine drug candidate with strong anticancer activity in multiple preclinical models. F10 has strong potential for impacting cancer treatment because it displays high cytotoxicity toward proliferating malignant cells with minimal systemic toxicities thus providing an improved therapeutic window relative to traditional fluoropyrimidine drugs, such as 5-fluorouracil. F10 has a unique mechanism that involves dual targeting of thymidylate synthase and Top1. In this review, the authors provide an overview of the studies that revealed the novel aspects of F10's cytotoxic mechanism and summarize results obtained in preclinical models of acute myeloid leukemia, acute lymphocytic leukemia, glioblastoma and prostate cancer that demonstrate the strong potential of F10 to improve treatment outcomes.

The cytotoxic and pro-apoptotic activities of the novel fluoropyrimidine F10 towards prostate cancer cells are enhanced by Zn(2+) -chelation and inhibiting the serine protease Omi/HtrA2.

BACKGROUND: Intracellular Zn(2+) levels decrease during prostate cancer progression and agents that modulate intracellular Zn(2+) are cytotoxic to prostate cancer cells by an incompletely described mechanism. F10 is a new polymeric fluoropyrimidine drug-candidate that displays strong activity with minimal systemic toxicity in pre-clinical models of prostate cancer and other malignancies. The effects of exogenous Zn(2+) or Zn(2+) chelation for enhancing F10 cytotoxicity are investigated as is the role of Omi/HtrA2, a serine protease that promotes apoptosis in response to cellular stress. METHODS: To test the hypothesis that the pro-apoptotic effects of F10 could be enhanced by modulating intracellular Zn(2+) we investigated cell-permeable and cell-impermeable Zn(2+) chelators and exogenous Zn(2+) and evaluated cell viability and apoptosis in cellular models of castration-resistant prostate cancer (CRPC; PC3, C4-2). The role of Omi/HtrA2 for modulating apoptosis was evaluated by pharmacological inhibition and Western blotting. RESULTS: Exogenous Zn(2+) initially reduced prostate cancer cell viability but these effects were transitory and were ineffective at enhancing F10 cytotoxicity. The cell-permeable Zn(2+) -chelator tetrakis-(2-pyridylmethl) ethylenediamine (TPEN) induced apoptosis in prostate cancer cells and enhanced the pro-apoptotic effects of F10. The pro-apoptotic effects of Zn(2+) -chelation in combination with F10 treatment were enhanced by inhibiting Omi/HtrA2 implicating this serine protease as a novel target for prostate cancer treatment. CONCLUSIONS: Zn(2+) -chelation enhances the pro-apoptotic effects of F10 and may be useful for enhancing the effectiveness of F10 for treatment of advanced prostate cancer. The serine protease Omi/HtrA2 modulates Zn(2+) -dependent apoptosis in prostate cancer cells and represents a new target for treatment of CRPC. Prostate 75:360-369, 2015. © 2014 Wiley Periodicals, Inc.

Replication-dependent irreversible topoisomerase 1 poisoning is responsible for FdUMP[10] anti-leukemic activity.

Previous studies have indicated that 5-Fluoro-2'-deoxyuridine-5'-O-monophosphate 10mer (FdUMP[10]) displays strong antileukemic activity through the dual targeting of thymidylate synthase (TS) and DNA topoisomerase 1 (Top1). The present studies were undertaken to clarify the relationship between the induction of a thymineless state and the formation of Top1 cleavage complexes (Top1CC) for inducing cell death and to clarify the role of DNA replication for induction of lethal DNA double-strand breaks (DSBs) in FdUMP[10]-treated acute myeloid leukemia (AML) cells. Human promyelocytic (HL60) and AML (KG1a, Molm13, THP-1) cells were synchronized by serum starvation and treated with FdUMP[10] with thymidine (Thy) rescue. Cells were assayed for TS inhibition, DNA DSBs, Top1CC, and apoptosis to clarify the interrelationship of TS inhibition and Top1CC for cell death. FdUMP[10] induced a thymineless state in AML cells and exogenous Thy administered within the first 18 hours of treatment rescued FdUMP[10]-induced Top1CC formation, γH2AX phosphorylation, and apoptosis induction. Exogenous Thy was not effective after cells had committed to mitosis and undergone cell division in the presence of FdUMP[10]. FdUMP[10] treatment resulted in Chk1 activation, and Chk1 inhibition enhanced FdUMP[10]-induced DNA damage and apoptosis. Jnk-signaling was required for FdUMP[10]-induced apoptosis in promyelocytic HL60 cells and in THP1 cells, but was antiapoptotic in Molm13 and to a lesser extent KG1a AML cells. The results are consistent with FdUMP[10] inducing a thymineless state, leading to misincorporation of FdU into genomic DNA of proliferating cells. Top1CC form in cells upon re-entry into S-phase, resulting in DNA double-strand breaks, and initiating apoptotic signaling that can be either muted or enhanced by Jnk-signaling depending on cell type.

Cytotoxicity of new duplex drugs linking 3'-C-ethynylcytidine and 5-fluor-2'-deoxyuridine against human melanoma cells.

Melanoma is an increasingly common and potentially fatal malignancy of the skin and some mucous membranes. As no cure exists for metastatic disease, there is an urgent need for novel drugs. 2'-Deoxy-5-fluorouridylyl-(3'-5')-3'-C-ethynylcytidine [5-FdU(3'-5')ECyd] and 3'-C-ethynylcytidinylyl-(5' → 1-O)-2-O-octadecyl-sn-glycerylyl-(3-O → 5')-2'-deoxy-5-fluorouridine [ECyd-lipid-5-FdU] represent cytostatic active duplex drugs, which can be metabolized into various active antimetabolites. We evaluated the cytotoxicity of these heterodinucleoside phosphate analogs, their corresponding monomers ECyd and 5-FdU and combinations thereof on six metastatic melanoma cell lines and six ex vivo patient-derived melanoma cells in comparison to current standard cytostatic agents and the BRAF V600E inhibitor Vemurafenib. In vitro (real-time)-proliferation assays demonstrated that 5-FdU(3'-5')ECyd and ECyd-lipid-5-FdU had a high cytotoxic efficacy causing 75% melanoma cell death at concentrations in the nanomolar and micromolar range. Cytotoxicity was conducted by induction of DNA cleavage indicating apoptotic cells. Chicken embryotoxicity demonstrated that the duplex drugs were less toxic than 5-FdU at 0.01 µM. In vivo the duplex drug 5-FdU(3'-5')ECyd was efficacious in the murine LOX IMVI melanoma xenograph model on administration of 11.2 mg/kg/injection every fourth day. Both duplex drugs are promising novel cytostatic agents for the treatment of malignant melanoma meriting clinical evaluation.

Genome-wide mRNA and microRNA profiling of the NCI 60 cell-line screen and comparison of FdUMP[10] with fluorouracil, floxuridine, and topoisomerase 1 poisons.

A profile of microRNA (miRNA) and mRNA expression patterns across the NCI-60 cell-line screen was analyzed to identify expression signatures that correlate with sensitivity to FdUMP[10], fluorouracil (5FU), floxuridine (FdU), topotecan, and irinotecan. Genome-wide profile analyses revealed FdUMP[10] resembles FdU most closely and shows dissimilarities with 5FU. FdUMP[10] had the largest dynamic range of any of these drugs across the NCI-60 indicative of cancer cell-specific activity. Genes involved in endocytosis, such as clathrin (CLTC), SNF8, annexin A6 (ANXA6), and amyloid protein-binding 2 (APPBP2) uniquely correlated with sensitivity to FdUMP[10], consistent with a protein-mediated cellular uptake of FdUMP[10]. Genes involved in nucleotide metabolism were enriched for the three fluoropyrimidine drugs, with the expression profile for 5FU correlated to an RNA-mediated cytotoxic mechanism, whereas expression of glycosyltransferases (XYLT2) that use UDP sugars as substrates and the nucleoside diphosphatase and metastasis suppressor NM23 (NME1) were associated with FdUMP[10] sensitivity. Topotecan and irinotecan had significant negative correlations with miR-24, a miRNA with a high aggregate P(CT) score for topoisomerase 1 (Top1). Our results reveal significant new correlations between FdUMP[10] and Top1 poisons, as well as new information on the unique cytotoxic mechanism and genomic signature of FdUMP[10].

The role of thymidine phosphorylase in the induction of early growth response protein-1 and thrombospondin-1 by 5-fluorouracil in human cancer carcinoma cells.

Thymidine phosphorylase (TP) is an enzyme involved in reversible conversion of thymidine to thymine. TP is identical to an angiogenic factor, pletelet-derived endothelial cell growth factor (PD-ECGF) and the expression levels of TP in a variety of malignant tumors were higher than the adjacent non-neoplastic tissues. To investigate the molecular basis for the effect of TP on the metabolic process and the anticancer effect of 5-fluorouracil (5-FU), human gastric carcinoma AZ521 cells and epidermoid carcinoma KB cells were transfected with TP cDNA, and AZ521/TP and KB/TP were cloned. AZ521/TP and KB/TP cells overexpressed TP and were more sensitive to 5-FU than the counterpart parental cells. TPI, a newly synthesized inhibitor for TP (Ki=2.36 x 10(-9) M), decreased the sensitivity to 5-FU of the TP expressing cells but not of the parental cells. 5-Formyl-tetrahydrofolate (leucovorin; LV) stabilized the complex of thymidylate synthase (TS) and 5-fluoro-deoxyuridine-monophosphate (FdUMP), increased the sensitivity to 5-FU of TP expressing AZ521 cells, but not of the parental cells. The levels of FdUMP in TP expressing cells were significantly higher than in parental cells and TPI considerably decreased FdUMP to the level comparable to that in the parental cells. 5-FU increased the expression of early growth response protein-1 (Egr-1) and an angiogenesis inhibitor, thrombospondin-1 (TSP-1), in KB/TP cells but only slightly in KB/CV cells, if any. TPI attenuated the induction of Egr-1 and TSP-1 mRNA by 5-FU, while LV increased the expression of Egr-1 and TSP-1 mRNA in KB/TP cells. These findings demonstrate that the TP has a principal role in the production of FdUMP and the enhanced responses to 5-FU by leucovorin in TP-overexpressing KB and AZ521 cells, and FdUMP but not FUTP is implicated in the induction of Egr-1 and TSP-1 in KB cells.

Differential expression of a novel gene BRE (TNFRSF1A modulator/BRCC45) in response to stress and biological signals.

Stress-responsive genes play critical roles in many biological functions that includes apoptosis, survival, differentiation and regeneration. We have identified a novel stress-responsive gene called BRE which interacts with TNF-receptor-1 and blocks the apoptotic effect of TNF-alpha. BRE enhances tumor growth in vivo and is up-regulated in hepatocellular and esophageal carcinomas. BRE also regulates the ubiquitination of the DNA repair complex BRCC, and the synthesis of steroid hormones. Here, we examined BRE-mRNA in cells after treatments with UV and ionizing radiation (IR). UV and IR treatment alone suppressed BRE-mRNA levels by more than 90% at 24 h, while hydroxyurea, fluorodeoxyuridine, aphidicolin, known inhibitors of S-phase DNA synthesis, had no significant effect. BRE protein expression was unaltered in cells treated with TNF-alpha, Interleukin-1 and Dexamethasone, while a threefold increase was observed following chorionic gonadotropin exposure. Although BRE plays a regulatory role in many different pathways, yet its expression is apparently under very stringent control.

Design and characterization of a mutation outside the active site of human thymidylate synthase that affects ligand binding.

Owing to its central role in DNA synthesis, human thymidylate synthase (hTS) is a well-established target for chemotherapeutic agents, such as fluoropyrimidines. The use of hTS inhibitors in cancer therapy is limited by their toxicity and the development of cellular drug resistance. Here, with the aim of shedding light on the structural role of the A-helix in fluoropyrimidine resistance, we have created a fluoropyrimidine-resistant mutant by making a single point mutation, Glu30Trp. We postulated that residue 30, which is located in the A-helix, close to but outside the enzyme active site, could have a long-range effect on inhibitor binding. The mutant shows 100 times lower specific activity with respect to the wild-type hTS and is resistant to the classical inhibitor, FdUMP, as shown by a 6-fold higher inhibition constant. Circular dichroism experiments show that the mutant is folded. The results of molecular modeling and simulation suggest that the Glu30Trp mutation gives rise to resistance by altering the hydrogen-bond network between residue 30 and the active site.

Mechanisms of action of FdUMP[10]: metabolite activation and thymidylate synthase inhibition.

FdUMP[10] is a multimer of FdUMP, a suicide inhibitor of thymidylate synthase (TS), and was designed to bypass resistance to 5-fluorouracil (5FU). The aim of the study was to compare the effect of FdUMP[10] with 5FU and 5-fluoro-2-deoxyuridine (FUdR) in their efficacy to inhibit their target TS in resistant cells. Therefore cell lines FM3A/0, FM3A/TK- (deficient in thymidine kinase) and FM3A/TS- (deficient in thymidylate synthase) were used to determine TK dependency and specificity for TS inhibition. FdUMP[10] inhibited cell growth with greater potency than 5FU and FdUMP. Direct folate-based inhibitors Raltitrexed, GW1843U89 and Pemetrexed were also evaluated using these cell lines. In TK-deficient cells these folate-based inhibitors had greater potency than the fluoropyrimidines (FPs). Surprisingly, Pemetrexed even inhibited cell growth in TS-deficient cells. Incubation with nucleotidase and phosphatase inhibitors resulted in a reduction of cytotoxicity of FdUMP[10], indicating that the drug can be degraded outside the cells. In the TS in situ inhibition assay (TSIA) 24 h exposure of FM3A cells to 0.5 microM FdUMP and 0.05 microM FdUMP[10] decreased TSIA to 7 and 1% of control. Inhibition of nucleotidase and phosphatase activities reduced the effect of FdUMP[10], while the inhibitory effect was lower in cells lacking TK. FdUMP[10] can enter the cells intact, but also to some extent after dephosphorylation. In conclusion, FdUMP[10] can bypass resistance to FUdR by direct inhibition of TS.

Sulfonyl-containing nucleoside phosphotriesters and phosphoramidates as novel anticancer prodrugs of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP).

A series of sulfonyl-containing 5-fluoro-2'-deoxyuridine (FdU) phosphotriester and phosphoramidate analogues were designed and synthesized as anticancer prodrugs of FdUMP. Stability studies have demonstrated that these compounds underwent pH dependent beta-elimination to liberate the corresponding nucleotide species with half-lives in the range of 0.33-12.23 h under model physiological conditions in 0.1 M phosphate buffer at pH 7.4 and 37 degrees C. Acceleration of the elimination was observed in the presence of human plasma. Compounds with an FdUMP moiety (4-9) were considerably more potent than those without (1-3) as well as 5-fluorouracil (5-FU) against Chinese hamster lung fibroblasts (V-79 cells) in vitro. Addition of thymidine (10 microM) reversed the growth inhibition activities of only 5-FU and the compounds with an FdUMP moiety, but had no effect on those without. These results are consistent with thymidylate synthase as the target of the prodrugs.

A novel polypyrimidine antitumor agent FdUMP[10] induces thymineless death with topoisomerase I-DNA complexes.

FdUMP[10], a 10mer of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), the thymidylate synthase inhibitory metabolite of 5-fluorouracil (FU), is most closely correlated with the DNA topoisomerase I (Top1) inhibitor camptothecin in the National Cancer Institute COMPARE analysis, but not with FU. FdUMP[10] exhibits more potent antiproliferative activity than FdUMP or 5-fluoro-2'-deoxyuridine (FdU) and is markedly more active than FU. Camptothecin-resistant P388/CPT45 cells lacking Top1 are cross-resistant to FdUMP[10] as well as to FdUMP, FdU, and the thymidylate synthase inhibitor raltitrexed (Tomudex). FdUMP[10] induces DNA single-strand breaks and cellular Top1-DNA complexes. Such complexes are also observed in response to FdUMP, FdU, raltitrexed, and FU. The FdUMP[10]-induced Top1-DNA complexes are not inhibited by the caspase inhibitor z-VAD-fmk and form independently of apoptotic DNA fragmentation, indicating that they do not correspond to apoptotic Top1-DNA complexes. In biochemical assay, Top1 is directly trapped at uracil and FdU misincorporation sites. We propose that FdUMP[10] damages DNA by trapping Top1 at uracil and FdU misincorporation sites resulting from thymidylate synthase inhibition and thymine depletion.

Synthesis and anticancer activities of amphiphilic 5-fluoro-2'-deoxyuridylic acid prodrugs.

Amphiphilic anticancer prodrugs of 5'-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP) were synthesized according to the hydrogen phosphonate method by coupling lipophilic cytosine derivatives or a phospholipid with 5-fluoro-2'-deoxyuridine (5-FdU). Studies within the in vitro Anticancer Screen Program of the National Cancer Institute have demonstrated high anticancer activities of the heterodinucleoside phosphates: N4-palmitoyl-2'-deoxycytidylyl-(3' --> 5')-3'-O-acetyl-5-fluoro-2'-deoxyuridine (dC(pam)-5-FdU(Ac), N4-palmitoyl-2',3'-dideoxycytidylyl-(5' --> 5')-3'-O-acetyl-5-fluoro-2'-deoxyuridine (ddC(pam)-(5' --> 5')-5-FdU(Ac), 5-fluoro-2'-deoxyuridylyl-(3' --> 5')-5-fluoro-N4-hexadecyl-2'-deoxycytidine (5-FdU-5-FdC(hex)), and of the new liponucleotide 1-O-octadecyl-rac-glycerylyl-(3 --> 5')-5-fluoro-2'-deoxyuridine (Oct1Gro-(3 --> 5')-5-FdU). The anticancer activities of these prodrugs are comparable to those of 5-FdU and the tumor specificities are modulated by their structures. The highest cytotoxic activity being even superior to 5-FdU was expressed by the dimer 5-FdU-5-FdC(hex).

A trojan horse approach for silencing thymidylate synthase.

In this paper we present a new and possibly more effective way of inhibiting thymidylate synthase (TS) in cells than through the use of substrate analogue inhibitors. An inactive double mutant of TS (DM), Arg(126)Glu/Cys(146)Trp, is shown to progressively impair the reactivation of native Escherichia coli TS when the two are denatured together in vitro. The individual single mutant proteins Arg(126)Glu and Cys(146)Trp showed little or no inhibition. When the DM is introduced into E. coli and induced from an expression plasmid, the mutant subunits act as a decoy in deceiving newly formed native TS subunits to fold with them to yield inactive heterodimers. As a consequence of the depletion of TS, the cells die a "thymineless" death when grown in medium devoid of thymine. Addition of thymine to the medium enables the cells to grow normally, although only very low levels of TS activity could be detected in those cells containing induced DM. The individual single-site mutations of the DM, Arg(126)Glu and Cys(146)Trp, did not inhibit growth, as might be expected from the in vitro studies. However, when a nontoxic level of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) is added to growing DM-transformed cells, the combination is lethal to the cells. These experiments suggest that a similar dominant-negative response to the DM of TS could be affected in tumor cells, for which preliminary evidence is presented. This technique, either alone or combined with other modalities, suggest a new approach to targeting cells for chemotherapy.

Designing a pronucleotide stratagem: lessons from amino acid phosphoramidates of anticancer and antiviral pyrimidines.

Phosphoramidate pronucleotides have proven to be an effective strategy for the intracellular delivery of nucleoside 5'-monophosphates. This review will summarize our efforts to understand the in vitro and in vivo behavior of phosphoramidate monoesters of 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-3'-deoxythymidine (FLT) and 5-fluoro-2'-deoxyuridine (FUdR). Insights drawn from these studies have proved valuable for the future design of phosphoramidate-based pronucleotides.

Enhanced DNA-directed effects of FdUMP[10] compared to 5FU.

FdUMP[N] molecules and conjugates are much more effective at inhibiting the proliferation of human tumor cells than is the widely used anticancer drug 5-fluorouracil (5FU). We have evaluated the inhibition of thymidylate synthase (TS), the extent of DNA damage, cell cycle arrest, and the induction of apoptosis by FdUMP[10] and 5FU in the human colorectal cancer cell line HT29. The magnitude and duration of TS inhibition following exposure of HT29 cells to FdUMP[10] at 1 x 10(-8) M was greater than that which occurred following exposure of these cells to 5FU at 1 x 10(-6) M. FdUMP[10] exposure also resulted in much more extensive DNA damage to HT29 cells than occurred following exposure to 100-fold higher concentrations of 5FU. Although exposure of HT29 cells to both drugs resulted in S-phase arrest, more complete accumulation of cells in S-phase was achieved following FdUMP[10] exposure at much lower drug concentrations. FdUMP[10] was also much more effective at inducing apoptosis in HT29 cells than was 5FU. The results are consistent with FdUMP[10] being much more efficient that 5FU at inducing DNA damage that results in apoptotic cell death in colon cancer cells.

Human herpesvirus 8 gene encodes a functional thymidylate synthase.

We demonstrate that human herpesvirus 8, obtained from the lymphoma cell line BC-3 as well as from Kaposi's sarcoma lesions, carries a gene that encodes a functional thymidylate synthase (TS). The particular characteristics of this enzyme are studied and compared to the characteristics of TSs encoded by other organisms.

Cytotoxic targeting of F9 teratocarcinoma tumours with anti-ED-B fibronectin scFv antibody modified liposomes.

We prepared small unilamellar liposomes derivatised with single chain antibody fragments specific for the ED-B domain of B-fibronectin. This extracellular matrix associated protein is expressed around newly forming blood vessels in the vicinity of many types of tumours. The single chain antibody fragments were functionalised by introduction of C-terminal cysteines and linked to liposomes via maleimide groups located at the terminal ends of poly(ethylene glycol) modified phospholipids. The properties of these anti-ED-B single chain antibody fragments-liposomes were analysed in vitro on ED-B fibronectin expressing Caco-2 cells and in vivo by studying their biodistribution and their therapeutic potential in mice bearing subcutanous F9 teratocarcinoma tumours. Radioactively labelled ((114m)Indium) single chain antibody fragments-liposomes accumulated in the tumours at 2-3-fold higher concentrations during the first 2 h after i.v. injection compared to unmodified liposomes. After 6-24 h both liposome types were found in similar amounts (8-10% injected dose g(-1)) in the tumours. Animals treated i.v. with single chain antibody fragments-liposomes containing the new cytotoxic agent 2'-deoxy-5-fluorouridylyl-N(4)-octadecyl-1-beta-D-arabinofuranosylcytosine (30 mg kg(-1) per dose, five times every 24 h) showed a reduction of tumour growth by 62-90% determined on days 5 and 8, respectively, compared to animals receiving control liposomes. Histological analysis revealed a marked reduction of F9 tumour cells and excessive deposition of fibronectin in the extracellular matrix after treatment with single chain antibody fragments-2-dioxy-5-fluorouridylyl-N(4)-octadecyl-1-beta-D-arabinofuranosylcytosine-liposomes. Single chain antibody fragments-liposomes targeted to ED-B fibronectin positive tumours therefore represent a promising and versatile novel drug delivery system for the treatment of tumours.

Synthesis and biological studies of novel nucleoside phosphoramidate prodrugs.

A novel approach to the intracellular delivery of nucleotides using phosphoramidate-based prodrugs is described. Specifically, we have developed phosphoramidate prodrugs of the anticancer nucleotide 5-fluoro-2'-deoxyuridine-5'monophosphate (FdUMP). These phosphoramidate prodrugs contain an ester group that undergoes intracellular activation liberating phosphoramidate anion, which undergoes spontaneous cyclization and P-N bond cleavage to yield the nucleoside monophosphate quantitatively. In vitro evaluation of 5-fluoro-2'-deoxyuridine phosphoramidate prodrugs 2a and 3b against L1210 mouse leukemia cells show potent inhibition of cell growth (IC(50) 0.5-3 nM). Cell-based thymidylate synthase inhibition studies show that, in contrast to FUdR, the nitrofuran compound 2a is of comparable potency in wild type vs thymidine kinase deficient LM cells. This result indicates that the activation of this novel prodrug occurs via the proposed mechanism of intracellular delivery. However, naphthoquinone 3b has an IC(50) value for thymidylate synthase inhibition that is comparable to FUdR in thymidine kinase deficient cells. Further studies revealed that 3b rapidly decomposes to the nucleotide in cell culture medium, suggesting that the naphthoquinone analogue is not sufficiently stable to function as a nucleotide prodrug.