Metabolism, mechanism of action and sensitivity profile of fluorocyclopentenylcytosine (RX-3117; TV-1360)

Abstract

A novel cytidine analog fluorocyclopentenylcytosine (RX-3117; TV-1360) was characterized for its cytotoxicity in a 59-cell line panel and further characterized for cytotoxicity, metabolism and mechanism of action in 15 additional cancer cell lines, including gemcitabine-resistant variants. In both panels sensitivity varied 75-fold (IC50: 0.4- > 30 μM RX-3117). RX-3117 showed a different sensitivity profile compared to cyclopentenyl-cytosine (CPEC) and azacytidine, substrates for uridine-cytidine-kinase (UCK). Dipyridamole, an inhibitor of the equilibrative-nucleoside-transporter protected against RX-3117. Uridine and cytidine protected against RX-3117, but deoxycytidine (substrate for deoxycytidine-kinase [dCK]) not, although it protected against gemcitabine, demonstrating that RX-3117 is a substrate for UCK and not for dCK. UCK activity was abundant in all cell lines, including the gemcitabine-resistant variants. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). RX-3117 was rapidly metabolised to its nucleotides predominantly the triphosphate, which was highest in the most sensitive cells (U937, A2780) and lowest in the least sensitive (CCRF-CEM). RX-3117 did not significantly affect cytidine and uridine nucleotide pools. Incorporation of RX-3117 into RNA and DNA was higher in sensitive A2780 and low in insensitive SW1573 cells. In sensitive U937 cells 1 μM RX-3117 resulted in 90% inhibition of RNA synthesis but 100 μM RX-3117 was required in A2780 and CCRF-CEM cells. RX-3117 at IC50 values did not affect the integrity of RNA. DNA synthesis was completely inhibited in sensitive U937 cells at 1 μM, but in other cells even higher concentrations only resulted in a partial inhibition. At IC50 values RX-3117 downregulated the expression of DNA methyltransferase. In conclusion, RX-3117 showed a completely different sensitivity profile compared to gemcitabine and CPEC, its uptake is transporter dependent and is activated by UCK. RX-3117 is incorporated into RNA and DNA, did not affect RNA integrity, depleted DNA methyltransferase and inhibited RNA and DNA synthesis. Nucleotide formation is related with sensitivity.

The Cytidine Analog Fluorocyclopentenylcytosine (RX-3117) Is Activated by Uridine-Cytidine Kinase 2

“…Non-solid tumor cell lines CCRF-CEM (Lymphoblastic leukemia) and U937 (Leukemic monocyte lymphoma) were cultured in RPMI1640 medium (Lonza) supplemented with 10% fetal bovine serum and 20 mM HEPES. The sources of the cell lines have been described earlier [3]. Cells were maintained in an experimental growth phase for all experiments and were tested negative for mycoplasma.…”

“…Cytidine analogs, a subclass of nucleoside analogs that are inserted into RNA and DNA replacing cytidine, are used to treat a wide variety of cancer types. Examples of successful cytidine analogs in anti-cancer applications are cytarabine and gemcitabine [2, 3], the latter drug is predominantly used for treatment of patients with non-small cell lung cancer (NSCLC) [4]. Nevertheless, the inter- and intra-tumor heterogeneity can imply for resistance to drugs in patients.…”

“…A previous study provided preliminary information on its mechanism of action and metabolism [3]. Uptake of RX-3117 was shown to be mediated by human equilibrative nucleoside transporter 1 (hENT1) and its cytotoxic activity was exerted via its phosphorylated metabolites.…”

“…Furthermore, this study showed that RX-3117, contrary to a drug such as gemcitabine, is not deaminated by cytidine deaminase (CDA) and that RX-3117 causes both inhibition of DNA and RNA synthesis, although the inhibition of the former is more pronounced. RX-3117 also targets DNA methyltransferase (DNMT) [3, 5] of which there are multiple variants [7]. DNMT3a and DNMT3b establish de novo DNA methylation patterns in DNA, which is important during embryogenesis [7], while DNMT1 differs in that its role is to maintain the established DNA methylation pattern through cell division and thus DNA replication [8].…”

Fluorinated Nucleosides as an Important Class of Anticancer and Antiviral Agents

“…Moreover, RX-3117 can act as an effective demethylating agent by inhibiting DNA methyltransferase (DNMT). Interestingly, unlike gemcitabine, RX-3117 is a poor substrate for cytidine deaminase (CDA) as shown by Peters et al in their in vitro studies in the U397 cell line simultaneously treated with RX-3117 and tetrahydrouridine (THU), an inhibitor of CDA enzyme [69]. A first-in-human trial of RX-3117 in patients with solid tumours has been completed and demonstrated encouraging evidence of the single agent safety and tolerability [71].…”