That inhibition of recombin ation is important for the observed sensitization is also suggested by the TK10 cells which were sensitive to gem citabine alone, and were not further sensitized by MK 8776. This cell line has been reported TNF-�� inhibitor to have a defect in recombination which would explain this observation. The requirement for only a brief incubation with MK 8776 to enhance cytotoxicity is an important observation given that, in clinical trials, the plasma concentration of MK 8776 was shown to exceed 1 umol L for only about 6 h. MK 8776 dissociates rapidly from Chk1 when the drug is removed, so it is unlikely that Chk1 will remain inhibited significantly beyond 6 h. We extended these experiments to more closely reflect the clinical situation by incubating cells briefly with gemcitabine, and then permitting the cells to recover.
Because ribonucleotide reductase remains inhibited for a long time, it took several days for the cells to recover. the rate of recovery depended on the concentration of gemcitabine. Cells in G1 also progressed into S phase during this time, so the number of cells potentially sus ceptible to Chk1 inhibition continued to increase. Hence there are two reasons why delayed addition of MK 8776 can enhance sensitivity to gemcitabine first, there is an increased number of cells arrested in S phase, and sec ond, the arrested cells have been given adequate time to become Chk1 dependent. The current experiments indicated that addition of MK 8776 at 18 h provided the greatest decrease in IC50 for gemcitabine in four cell lines.
However, these experiments only reflect growth inhibition, and the S phase arrest at these low concentrations was very tran sient. Higher concentrations of gemcitabine induce a longer arrest with more cells accumulating in S phase. Consequently, it is possible that later addition of MK 8776 may have improved cell killing as the cells newly arrested in S phase at 18 h may not yet have become Chk1 dependent. To more directly assess the relevance of these in vitro observations, we assessed the S G2 phase arrest that oc curred in two different tumor models in vivo. This was quantified as the ratio of geminin positive to Ki67 positive cells. Eighteen hours after administration of 150 mg kg gemcitabine, there was a marked increase in geminin positive cells suggesting that up to 83 95% of the Ki67 positive cells were in S or G2 phase.
By 42 h, this percentage had partially reverted to the starting value reflecting recovery of the cells. This dose of gemci tabine is considered equivalent to a dose of 450 mg m2 in patients, which is about half the standard dose admin istered. Entinostat We are currently performing a clinical trial to assess the S G2 phase arrest that occurs in patients receiving gemcitabine as a guide for subse quent administration of a Chk1 inhibitor.