Finding ways to effectively control HIV-1 infection remains a singular goal for ART. While current antiretroviral drugs reduce morbidity and mortality associated with viral infection, there are limitations that prevent ART from eradicating the virus in infected people. Indeed, a combination of drug-related biochemical, physiological, as well as human factors, continue to lead to treatment failure in a proportion of patients (Fellay et al., 2001
; Hawkins, 2006
). Notable is the formation of viral sanctuaries, such as the CNS, where ART compounds are unable to access and virus is allowed to replicate albeit at limited levels (Best et al., 2009
; Varatharajan and Thomas, 2009
NanoART provides an important means to overcome this and other limitations for CNS drug delivery. In this report, we show that nanoART can be manufactured and optimized in laboratory models of HIV infection. Second
, combinations of nanoART can improve antiretroviral efficacy without cytotoxicity. Viral breakthrough was seen 20 days after monotherapy for each drug while combination therapy still provided total viral inhibition at 20 days. The improvement in antiretroviral activities with combination therapy could potentially be due to the presence of more drugs and to having drugs with different mechanisms of action. Third
, the particles were located within the same sub-cellular compartments. Interestingly, there was no reduction in the absolute amount of each drug taken up when cells were treated with combo nanoART compared to when each was used alone. Fourth
, this work serves to bridge animal studies being developed to assess safety and efficacy with the final goal being human clinical trials. The fact that MDM are able to take up all three nanoART formulations simultaneously provides an important step in this direction. Also, that multiple nanoART can be loaded simultaneously and that uptake is not competitive among formulations supports its utility in an infected human host.
The cell-based delivery system developed in the current report is based on monocyte-macrophage function and viral dissemination in an infected human host. One focus of such infection is the brain. Indeed, HIV infection of the brain can result in both functional and cognitive deficits, a condition known as HIV-1 associated neurocognitive disorder (HAND)(Gray et al., 2001
). Some form of neurological dysfunction is displayed in nearly half of people infected with HIV; this makes HAND a not infrequent cause of dementia worldwide (Valcour and Paul, 2006
; Ellis et al., 2007
; Varatharajan and Thomas, 2009
). This is primarily due to the inability of ART medications to effectively cross the blood-brain barrier (BBB) or to persist within the parenchyma upon entering the CNS (reviewed by (Nowacek et al., 2009b
)). Successful delivery of antiretroviral drugs to the brain and purging of this viral sanctuary could effectively eliminate this form of dementia.
Our laboratory has focused its research efforts on developing long-acting parenteral drug formulations that can be maintained inside cells for extended periods and travel specifically to viral sanctuaries where ART compounds can be released. In these past studies (Dou et al., 2006
; Dou et al., 2007
; Dou et al., 2009
; Nowacek et al., 2009b
), we demonstrated that antiretroviral compounds could be manufactured into stable formulations of nanometer sized drug crystals coated in surfactant polymers, that the physical properties of these particles could be modified, and that these changes had an affect upon cellular handling. Most importantly, we demonstrated that MDM pretreated with nanoART could release drug and inhibit HIV-1 replication for up to 15 days after treatment. However, in all prior studies the effect of only one formulation was explored at a time. In the clinic, ART involves the use of multiple drugs of different classes simultaneously. We hypothesized that we could further extend the time of drug release and improve viral inhibition over our previous findings by exposing MDM to a “nanoART cocktail”. The fact that multiple formulations showed substantial improvements over a single nanoART preparation was an unexpected finding, as we believed that each would affect uptake of the others. This was not the case.
In the clinic, ART therapy commonly consists of a drug “cocktail”; this improves pharmacokinetics (PK), more effectively reduces viral loads, and decreases the risk of viral resistance (Zolopa, 2009
). For example, RTV (a protease inhibitor, PI) can be used to “boost” serum levels of other PIs, such as ATV (Horberg et al., 2008
). In fact, ATV is commonly prescribed in conjunction with a low dose of RTV in order to increase its serum levels (Johnson et al., 2005
; Gisslen et al., 2006
). A possible addition to ATV-RTV therapy is EFV, a nonnucleoside reverse transcriptase inhibitor. In 2008, the FDA recommended a combination of ATZ with RTV and EFV once daily as a possible treatment in ART-naive patients(Horberg et al., 2008
In addition, increasing the overall drugs taken up by MDM also extended the time of drug release. A potential explanation for this finding involves intracellular drug metabolism. RTV is a very potent inhibitor of cytochrome P450 enzymes, such as CYP2B6, CYP2D6, CYP3A4/5, and others (Kumar et al., 1999
; Hesse et al., 2001
; Zeldin and Petruschke, 2004
; Xu and Desai, 2009
). In the clinic RTV is used in ART drug combinations to both increase bioavailability and reduce hepatic clearance of other antiretroviral compounds (Zeldin and Petruschke, 2004
; Feldt et al., 2005
; Gianotti et al., 2007
; Xu and Desai, 2009
). This allows for improved dosing schedules and better viral inhibition (Zeldin and Petruschke, 2004
; Xu and Desai, 2009
). The expression of CYP2B6, CYP2D6, and CYP3A4/5 mRNAs have been identified in alveolar macrophages suggesting that macrophages are capable of metabolizing antiretroviral medications(Anttila et al., 1997
; Hukkanen et al., 1997
; Raunio et al., 1999
; Piipari et al., 2000
; Hukkanen et al., 2002
). Therefore, after nanoART are taken up by MDM, a portion of the drugs may be metabolized by the cells. By including RTV in the nanoART combination, we may be inhibiting nanoART metabolism and improving intracellular stability, thereby extending the time of drug release.
A major challenge for medicine is to improve clinical outcomes for CNS disorders related to aging, infection and degeneration. These conditions include, but are not limited to: stroke, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, and HAND. In all cases disease progression could be slowed, halted or potentially reversed with targeted drug delivery. Potentially useful medications exist for these diseases; unfortunately, like ART compounds, many are unable to effectively cross the BBB, enter the CNS, and potentiate a positive change(Kabanov and Gendelman, 2007
; Nowacek et al., 2009a
). Many neurologic diseases have an inflammatory component (Perry et al., 1995
), thereby allowing immunocytes, such as macrophages, to migrate into sites of inflammation via diapedesis and chemotaxis (Kumagai et al., 1987
). Therefore, we anticipate that our model of cell-mediated drug delivery of crystalline NP may be applicable to other major CNS disorders besides HAND.
This work brings us yet one step closer to realizing the use of nanoART in humans, but much work still needs to be done. In the future, we plan to elucidate mechanisms of nanoART uptake, study NP trafficking, and identify sub-cellular compartments in which nanoART are stored within MDM. In addition, we plan to investigate in vivo PK, biodistribution, and efficacy upon a single parenteral administration.