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GS-9219 is a novel nucleoside analogue that was designed based on a double-prodrug approach applied to limit plasma exposure to the parent compound, 9-(2-phosphonylmethoxyethyl)guanine (PMEG), as a means of reducing toxicity. Studies in cell lines and chronic lymphocytic leukemia (CLL) cells demonstrated that after GS-9219 entered cells, the bisphosphoramidate groups were hydrolyzed, generating the first intermediate metabolite, N6-cyclopropyl-9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (cpr-PMEDAP). The second, and rate-limiting, metabolic step was the deamination of cpr-PMEDAP to PMEG, which was subsequently phosphorylated by intracellular kinases to the long-lived (>12 hr) active metabolite, PMEG diphosphate (PMEGpp). Successful competition with dGTP for incorporation by DNA polymerases resulted in nascent DNA chain termination due to the lack of a 3’-hydroxyl moiety. Recent studies demonstrated that lymphoid tissues and cells in dogs were enriched with PMEGpp following intravenous GS-9219 administration. Although most CLL cells are not active in DNA replication, they maintain an active excision repair capacity. We hypothesized that the incorporation of PMEGpp into DNA during repair re-synthesis would result in DNA chain termination, inhibition of repair processes, and accumulation of DNA strand breaks that would activate signaling pathways to cell death in CLL cells. Pharmacokinetic studies in CLL cells illustrated that the intracellular metabolism of GS-9219 was proportional to the extracellular concentrations from 3 to 30 μM GS-9219 in a time-dependent manner. After 24 hr incubation with 10 μM GS-9219, the median intracellular concentration of PMEGpp was 3.9 μμ (range, 1.1-8.7 μM; n = 9), a concentration which greatly exceeded that generated by PMEG (<0.01 μM), and was similar to dGTP levels in CLL cells (<5 μM). UV irradiation activated the nucleotide excision repair pathway in CLL cells as indicated by an increase in [3H]thymidine incorporation. Pretreatment with GS-9219 reduced the UV-induced thymidine incorporation in CLL cells. The combination of GS-9219 (10 μμ) with UV (2 J/m2) induced more Annexin-V staining than the sum of using single agents alone (n = 8, p < 0.05). GS-9219 inhibited repair re-synthesis stimulated by 4-hydroperoxycyclophosphamide (4-HC) as well, and was also associated with greater than additive apoptosis induction. Comparisons with 10 μM arabinosylguanine (ara-G) demonstrated similar apoptosis induction as GS-9219 combined with either UV or 4-HC. Studies of signaling pathways activated in response to DNA chain termination are ongoing. GS-9219 is an interesting clinical candidate for B-cell malignancies and clinical trials have recently initiated at multiple centers.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA