The high mutation rate combined with the rapid replication of HIV-1 drives the emergence of drug resistance under suboptimal treatment conditions. Resistance to one drug often confers drug resistance to other drugs in the same drug class, emphasizing the need for new drugs that exploit novel drug targets. Our previous studies demonstrated the anti-HIV-1 activity of two FDA-approved drugs, decitabine and gemcitabine (8
), that appear to inhibit HIV-1 replication through lethal mutagenesis, a process where the mutation rate prevents the virus from replicating with enough fidelity to remain viable. In this study, we examined the antiretroviral activity of decitabine and gemcitabine in vivo
using the LP-BM5 MuLV model (a murine AIDS model). This is an efficient model with which to examine the in vivo
efficacy and toxicity of potential anti-HIV-1 compounds and has been validated with approved anti-HIV-1 drugs (10
). The disease induced by LP-BM5, the infectious agent of MAIDS, has striking similarities to that induced by HIV-1, including (i) a dependence on CD4+
T cells for disease initiation, (ii) early onset of hypergammaglobulinemia, (iii) loss of B and T cell responses with disease progression, (iv) splenomegaly, and (v) increased susceptibility to opportunistic infections with disease progression. However, there are notable differences in the diseases induced by MuLV and HIV-1, which emphasizes the need to confirm the anti-MuLV activity in cell culture before moving into the animal model.
The results presented here show that decitabine and gemcitabine inhibited MuLV in cell culture at nanomolar concentrations ( and ), indicating that the LP-BM5/MAIDS model would be a suitable model for examining the efficacies and toxicities of these drugs in vivo
. While the potency of the individual drugs was similar when comparing MuLV and HIV-1, the combination of decitabine and gemcitabine did not synergistically inhibit MuLV replication, as determined by the FIC method of evaluating synergy (11
). This is in contrast to HIV-1, where the combination of decitabine and gemcitabine synergistically inhibits HIV-1 replication (8
). This difference may indicate that one or both drugs use a different mechanism of action to inhibit MuLV compared to the mechanism used to inhibit HIV-1. Similar to the cell culture data, the antiviral activities of decitabine and gemcitabine were potentiated in vivo
according to certain indices of disease progression, including the histopathology of spleen and lymph nodes. While the average spleen weight () was reduced in animals treated with the combination of decitabine and gemcitabine, the provirus levels were not statistically significantly different from those in animals treated with gemcitabine alone. Similarly, the provirus levels were not statistically significantly different when comparing the animals treated with gemcitabine alone to the animals treated with both decitabine and gemcitabine. This lack of potentiation in vivo
may be due to the variation within each group as well as the small number of animals per treatment group, or it may indicate a different mechanism of action when comparing the mechanism for these drugs in MuLV infection to those in HIV infection.
Although MuLV and HIV-1 have similar life cycles and mechanisms of replication, they also demonstrate some notable differences that may explain why the combination of decitabine and gemcitabine does not act synergistically to inhibit MuLV replication in vivo. Since each drug exhibited similar (decitabine) or lower (gemcitabine) potencies when comparing activities against MuLV and HIV-1 (), the difference in the ability of these drugs to work in combination suggests a difference in their antiviral mechanism of action when comparing the mechanism against HIV-1 to that against MuLV.
As previously described (8
), decitabine and gemcitabine are proposed to inhibit HIV-1 replication by lethal mutagenesis, a process where the mutation rate is increased to a level that prevents viral replication with enough fidelity for the virus to remain viable. In the proposed model, decitabine is incorporated into HIV-1 DNA during reverse transcription, where it forms noncanonical base pairs, thereby increasing the mutation frequency, which can be detected experimentally as an increase in G-to-C mutations in the provirus. Gemcitabine's anti-HIV-1 activity is attributed to its inhibition of ribonucleotide reductase, which alters deoxynucleoside triphosphate (dNTP) pools, where it potentiates decitabine in one of two ways: (i) gemcitabine reduces dCTP levels, thereby increasing incorporation of decitabine, a cytidine analog, or (ii) gemcitabine increases mutation frequency simply by altering dNTP pools, as would be suggested by previous studies that showed that alterations in dNTP pools alone increased HIV-1 mutation frequency (2
While gemcitabine's reduction or alteration of dNTP pools is likely to increase the HIV-1 mutation frequency and/or increase incorporation of decitabine, gemcitabine may have a different effect on MuLV replication. For example, the Km of MuLV reverse transcriptase (RT) for dNTPs is higher than that for HIV RT, indicating that MuLV is less efficient at reverse transcribing viral DNA under conditions of low dNTP levels. Thus, gemcitabine's reduction in dNTP pools may inhibit MuLV reverse transcription since MuLV is unable to efficiently reverse transcribe under lower dNTP concentrations. In contrast, HIV-1 is able to efficiently reverse transcribe viral DNA in the presence of low dNTP levels, supporting the suggestion that gemcitabine's anti-HIV activity is likely due to an increase in mutation frequency either through alterations in dNTP pools or by increases in the incorporation of decitabine. Further biochemical studies will be performed to determine if gemcitabine's anti-MuLV activity is due to inhibition of the enzymatic activity of reverse transcriptase.
While the antiviral mechanism of gemcitabine is likely to differ when comparing MuLV to HIV-1, we do not believe that the antiviral activity of decitabine would differ between the two viruses. Decitabine is a cytidine analog that is incorporated into viral DNA during reverse transcription. Since the EC50
s of decitabine for MuLV and HIV-1 are similar () and decitabine does not have an altered sugar moiety (a common feature of nucleoside RT inhibitors that are less potent in MuLV) (3
), there is no reason to believe that decitabine would have a different affinity for MuLV RT than HIV-1 RT.
Although both decitabine and gemcitabine are used as anticancer drugs, the concentrations needed to inhibit viral replication are significantly lower (100 to 1,000 times lower) than the concentrations needed to inhibit cell growth in vitro
. Our results show that the doses needed to inhibit viral replication in vivo
were well tolerated, with no signs of toxicity. The dose of gemcitabine used in cancer chemotherapy in humans is 1,000 mg/m2
once per week for 7 weeks with a 1-week break (26
). In contrast, the dose of gemcitabine used in this study, 1 mg/kg, is equivalent to 3 mg/m2
in humans when using the body surface area method to convert mouse dosing to human dosing (23
). The standard decitabine dosing is 15 mg/m2
every 8 h for 3 days, with this cycle repeated every 6 weeks for a minimum of 4 cycles. In contrast, the dose of decitabine used in this study, 0.1 mg/kg, is equivalent to 0.3 mg/m2
, which, like the dose of gemcitabine, is well below the therapeutic dose used to inhibit cell growth in humans.
One limitation for the use of decitabine and gemcitabine as antiretrovirals is that both are currently administered intravenously. However, Eli Lilly has developed a gemcitabine prodrug that is currently in clinical trials. Although no decitabine prodrugs are currently in clinical trials, it has been demonstrated that with appropriate oral dosing, it is possible to achieve pharmacologically relevant plasma concentrations for cancer treatment (17
). Since the dose of decitabine needed to inhibit HIV replication is expected to be lower, it is possible that a prodrug would not be needed, although without a prodrug, it is likely that decitabine would have to be taken multiple times per day, which is not reasonable for anti-HIV drugs.
In summary, the findings presented here indicate that the combination of decitabine and gemcitabine has potent antiretroviral activity in vivo and ex vivo using the LP-BM5/MAIDS model. These findings, along with previous HIV-1 studies in cell culture with gemcitabine and decitabine, suggest that this drug combination has broad antiretroviral activity that is well tolerated at doses that exhibit antiviral activity.