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Introduction: RX-3117 is an oral, small molecule cytidine analog anticancer agent with an improved pharmacological profile relative to gemcitabine and other nucleoside analogs. The agent has excellent activity against various cancer cell lines and xenografts including gemcitabine-resistant variants and it has excellent oral bioavailability; it is not a substrate for the degradation enzyme cytidine deaminase. RX-3117 is being evaluated at a daily oral schedule of 700 mg (5 days/week for 3 weeks) which results in plasma levels in the micromolar range that have been shown to be cytotoxic to cancer cells. It has shown clinical activity in refractory bladder cancer and pancreatic cancer. Areas covered: The review provides an overview of the relevant market and describes the mechanism of action, main pharmacokinetic/pharmacodynamic features and clinical development of this investigational small molecule. Expert opinion: RX-3117 is selectively activated by uridine-cytidine kinase 2 (UCK2), which is expressed only in tumors and has a dual mechanism of action: DNA damage and inhibition of DNA methyltransferase 1 (DNMT1). Because of its tumor selective activation, novel mechanism of action, excellent oral bioavailability and candidate biomarkers for patient selection, RX-3117 has the potential to replace gemcitabine in the treatment of a spectrum of cancer types.

Fluorocyclopentenylcytosine (RX-3117) is an orally available cytidine analog, currently in Phase I clinical trial. RX-3117 has promising antitumor activity in various human tumor xenografts including gemcitabine resistant tumors. RX-3117 is activated by uridine-cytidine kinase (UCK). Since UCK exists in two forms, UCK1 and UCK2, we investigated which form is responsible for RX-3117 phosphorylation. For that purpose we transfected A549 and SW1573 cell lines with UCK-siRNAs. Transfection of UCK1-siRNA efficiently downregulated UCK1-mRNA, but not UCK2-mRNA expression, and did not affect sensitivity to RX-3117. However, transfection of UCK2-siRNA completely downregulated UCK2-mRNA and protein and protected both A549 and SW1573 against RX-3117. UCK enzyme activity in two panels of tumor cell lines and xenograft cells correlated only with UCK2-mRNA expression (r = 0.803 and 0.915, respectively), but not with UCK1-mRNA. Moreover, accumulation of RX-3117 nucleotides correlated with UCK2 expression. In conclusion, RX-3117 is activated by UCK2 which may be used to select patients potentially sensitive to RX-3117.

Variant factor X in an individual with a mild bleeding tendency was suspected based on deficient procoagulant activity (10-20% of normal) and antigen (30-35% of normal) levels of plasma factor X. Heteroduplex analysis of factor X gene exons indicated heterozygosity for mutations in both exons 6 and 4, confirmed by direct sequencing of the amplified exons. Substitution of C by T at nucleotide position 13,984 (Arg-139 to Cys) was found in the factor X gene exon 6 of the propositus. This mutation creates a BsmI site and the patient tested heterozygous for the BsmI cleavage involved, as did one of his two daughters. In addition, exon 4 was found to have the normal A and a novel C (Asn-57 to Thr) at nucleotide position 9338. The exon 4 mutation creates a BsaJI site, detectable after amplification mismatch to remove an existing BsaJI site. Both the patient and the second of his two daughters were heterozygous for this cleavage. The two variant proteins are called factors XWenatchee I (Arg-139 to Cys) and II (Asn-57 to Thr). A mixed variant isolate derived from the plasma of the propositus exhibited heavy/light chains of normal size, as well as an apparent single-chain molecule not dissociable by reducing agent. A single-chain molecule would be predicted for form I, if the mutation blocks processing cleavages that normally remove a tripeptide interposed between the heavy and light chains. A Western blot of partially purified factor X from the daughter who inherited the form I defect revealed a component migrating the same as the putative single-chain species. Based upon the factor X activity vs. antigen ratios for the propositus and both daughters, both forms I and II are probably dysfunctional molecules.

RX-3117 (fluorocyclopentenyl-cytosine) is a novel cytidine analog currently being evaluated in a Phase Ib clinical trial in cancer patients with solid tumors. The radiosensitizing effect of RX-3117 was studied in A2780 ovarian cancer cells and non-small cell lung cancer cell lines and related to cell survival and the effect on cell cycle and cell cycle proteins. RX-3117 has a schedule-dependent radiosensitizing effect, but only at pre-incubation (dose modifying factors: 1.4-1.8), observed at pulse and fractionated irradiation. Radiosensitizion was also seen in a three-dimensional spheroid model. At the low radiosensitizing concentration, RX-3117 in combination with radiation led to an accumulation of cells in S-phase, which was accompanied with an increase of cell cycle proteins such as p-Chk2 and p-cdc25C. In addition, RX-3117 caused DNA damage and increased apoptosis. In conclusion, our in vitro experiments showed a radiosensitizing effect of RX-3117.