Nucleoside analogues are antimetabolites that can be used to treat AML. These compounds mimic the effects of physiological nucleotides in terms of uptake and metabolism and become incorporated into nascent DNA resulting in synthesis inhibition and chain termination. Nucleoside drugs often suffer from poor metabolism to the active triphosphate forms, deactivation and clinical toxicities. Phosphate prodrugs (protides) have modified monophosphate groups on the drug which potentially enables them to enter cells and subsequently become metabolised into the monophosphate form. Production of the monophosphate form of the drug is the rate limiting step in the production of the active triphosphate form.
Cladribine (2-chlorodeoxyadenosine) has been used as a parent drug to produce a series of protides with altered monophosphate groups. The parent drug and the protides were used at concentrations between 10μM to 0.002μM on a panel of six myeloid cell lines (HL-60, NB4, NB4R2, U937, KG1 and K562) for 72 hours. The effects of the protides were measured by proliferative assays (MTS) and IC50 values calculated. Two protides, CC122 and CC124, were assessed in combination with cytarabine (Ara-C) at ratios of 1:1 and 1:10 in the same manner as previously described.
Fourteen protides of cladribine were analysed in addition to cladribine itself and Ara-C. The average IC50 value for each protide, across all the cell lines, tested were reduced by 2 to 11 fold compared to cladribine and Ara-C. One protide (CPF193) had no effect on any of the cell lines, whilst CPF194 was found only to be active in the KG1 cell line with a 2.7 fold reduction in the concentration required kill 50% of the cells treated compared to cladribine. In general, K562 cells did not respond to treatment with Ara-C, cladribine or 6 of the protides although they did respond to the remaining protides with IC50 values slightly higher than the other cell lines.
The IC50 values for each protide in each cell line individually was also calculated and some cell lines, particularly NB4, showed dramatic reductions in IC50. In particular protide CC124 showed over 40-fold reduction from 9.4μM for cladribine to 0.23μM for CC124. This was also lower than the IC50 for Ara-C in this cell line (0.53μM).
Ara-C and cladribine combinations, at a ratio of 1:1, gave an average combination index (CI) at the IC50 of 0.9857 across all the cell lines, excluding K562, indicating an additive effect only. However, protide CC124 and Ara-C in combination, at the same ratio, produced an average CI of 0.6334 indicating synergistic activity.
This study has shown that altered monophosphate groups on the nucleoside inhibitor cladribine reduces the IC50, particularly when used in combination with Ara-C. The reduced doses would be beneficial in a clinical environment.