In the absence of a cure or vaccine for HIV-1 infection, the identification of novel drug targets and the development of new drugs is the best approach to address the emergence of resistance as well as the complications associated with current therapies. Drugs that target cellular proteins are less likely to be susceptible to the emergence of drug resistance compared to the current anti-HIV therapies which target viral proteins. However, drugs that target cellular proteins are likely to be associated with an increase in toxicity which may limit their clinical use. For example, hydroxyurea has been used alone and in combination with nucleoside reverse transcriptase inhibitors (NRTIs) to decrease viral loads in HIV-1 infected individuals. The side effects associated with hydroxyurea has significantly curtailed its clinical use. Less toxic alternatives to hydroxyurea would offer important alternatives for the treatment of HIV-1 as well as other retroviral infections.
A recent study demonstrated that two clinically approved drugs, decitabine and gemcitabine have potent anti-HIV activity in cell culture when used alone or in combination 
. Importantly, based on gemcitabine's potency and lack of toxicity, the data suggest that gemcitabine may be a clinically relevant alternative to hydroxyurea for the treatment of retroviral infections.
In this study, we examined the antiretroviral activity of gemcitabine in vivo and ex vivo using the LP-BM5 MuLV model (a murine AIDS model). Murine AIDS is caused by a combination of three murine leukemia viruses including BM5-eco, BM5def, and mink cell focus inducing virus (MCF) 
. BM5def is a retrovirus that is unable to replicate due to deletion of most of the pol gene. However, its intact gag gene is thought to be responsible for MAIDS pathogenesis. Although LP-BM5 is not a perfect model for AIDS pathogenesis, it is well characterized, safe, inexpensive, and has been validated with a number of clinically approved anti-HIV-1 drugs and has been useful for identifying drugs with broad antiretroviral activity (for review see 
Our studies found that gemcitabine decreased replication of MuLV in cell culture as well as in vivo using the MAIDS model. In fact, all measures of MAIDS-associated pathogenesis including splenomegaly, histopathology of spleen and lymph nodes and levels of IgM and provirus indicated reduced viral replication. The reduction of provirus by gemcitabine further supported the gemcitabine-mediated reduction in viral replication. There are at least two mechanisms that could account for the decrease in disease progression. First, gemcitabine could decrease dNTP levels enough to decrease proliferation of infected cells. Second, gemcitabine could alter dNTP levels such that viral replication is inhibited without inhibition of cellular proliferation. These mechanisms are likely to be dose dependent with low gemcitabine doses preferentially inhibiting viral replication and higher doses inhibiting both viral replication and cell proliferation. In support of this, 2 mg/kg/day of gemcitabine caused a significant reduction in IgM levels compared to the uninfected animals. This is likely due to gemcitabine-mediated decrease in B cell proliferation. In contrast, IgM levels from mice treated with 1 mg/kg/day had levels comparable to the uninfected animals which suggest there was not a significant reduction in B cell numbers compared to the uninfected mice. The cell culture data also supported this assertion as the decrease in replication was not associated with a significant loss of cell numbers (). These data indicate that gemcitabine's antiviral activity is distinct from its activity used to treat human cancers. Furthermore, doses of gemcitabine used clinically in cancer treatment are significantly higher than those used in our study. For example, gemcitabine's anti-cancer activity is achieved with a dosing regimen that includes 1000 mg/m2
given once every week for seven weeks, followed by one week without drug and additional rounds of treatment as needed 
. In our study, an antiviral effect was seen in animals at doses as low as 1 mg/kg which correlates to 3 mg/m2
when using the BSA method to convert the mouse dose to the human equivalent dose (mouse dose multiplied by mouse km of 3) 
. Although the metabolism of gemcitabine in mice is likely different compared to humans, the significant difference in dosing further supports that the antiviral activity seen here is not due to inhibition of cell proliferation.
Toxicity of gemcitabine was significant at higher doses. In fact, all mice treated with 3 and 4 mg/kg/day lost 15% or greater of their body weight during the study and as a result were prematurely euthanized. However, the 1 mg/kg/day dose of gemcitabine decreased disease pathology with no detectable toxicity. Specifically, all of these animals gained body weight similar to the uninfected animals. Additionally, these animals did not show any signs of hepatotoxicity as detected by histopathological analysis (data not shown). The 2 mg/kg/day dose was also effective at decreasing disease progression. However, some of the mice in this treatment group showed signs of toxicity in the loss of body weight while others in this group appeared to tolerate gemcitabine well. Like the animals treated with 1 mg/kg/day gemcitabine, there was no hepatotoxicity observed in animals treated with 2 mg/kg/day gemcitabine. The extent to which gemcitabine effected the animals in the 2 mg/kg group was not limited to toxicity as the efficacy of gemcitabine's antiviral activity also varied (see ). Although it is not clear why the mice could have such different responses to the drug, the affect of this variation could be minimized by increasing the number of animals in each treatment group.
Although gemcitabine has been used as cancer chemotherapy for many years, it has likely been neglected as an antiviral due to its poor oral bioavailability, which would necessitate drug delivery by injection. However, a prodrug for gemcitabine has been described recently, making gemcitabine a more attractive candidate for use in treatment of retroviral infections, including highly drug-resistant HIV-1 
. If its bioavailability is improved, gemcitabine could serve as a novel anti-HIV drug for those resistant to the current therapies.
In summary, the findings of our study indicate that gemcitabine has potent antiretroviral activity in vivo and ex vivo using the LP-BM5 MuLV model. These findings, along with previous ex vivo HIV-1 studies with gemicitabine an decitabine, suggest that gemcitabine has broad antiretroviral activity and could be particularly useful in vivo when used in combination drug therapy.