Pharmacodynamic and antiretroviral activities of combination nanoformulated antiretrovirals in HIV-1-infected human peripheral blood lymphocyte-reconstituted mice

Abstract

Lack of adherence, inaccessibility to viral reservoirs, long-term drug toxicities, and treatment failures are limitations of current antiretroviral therapy (ART). These limitations lead to increased viral loads, medicine resistance, immunocompromise, and comorbid conditions. To this end, we developed long-acting nanoformulated ART (nanoART) through modifications of existing atazanavir, ritonavir, and efavirenz suspensions in order to establish cell and tissue drug depots to achieve sustained antiretroviral responses. NanoART's abilities to affect immune and antiviral responses, before or following human immunodeficiency virus type 1 infection were tested in nonobese severe combined immune-deficient mice reconstituted with human peripheral blood lymphocytes. Weekly subcutaneous injections of drug nanoformulations at doses from 80 mg/kg to 250 mg/kg, 1 day before and/or 1 and 7 days after viral exposure, elicited drug levels that paralleled the human median effective concentration, and with limited toxicities. NanoART treatment attenuated viral replication and preserved CD4(+) Tcell numbers beyond that seen with orally administered native drugs. These investigations bring us one step closer toward using long-acting antiretrovirals in humans.

Figure 1.

Experimental protocols used in studies of nanoformulated antiretroviral therapy (nanoART) efficacy and toxicology profiles. A, Treatment paradigm to determine pharmacokinetics and tissue distribution of nanoART (M2001 [atazanavir, ATV] + M3001 [ritonavir, RTV]; 80, 150, or 250 mg/kg each drug) by subcutaneous administration to NSG mice on days 0 and 7. B, Treatment paradigm to determine preinfection antiviral activity of nanoART (H2001 [ATV] + H3001 [RTV]; 250 mg/kg each drug) by subcutaneous administration on day 0 to human peripheral blood lymphocyte (huPBL)–reconstituted NSG mice 24 h prior to human immunodeficiency virus type 1 (HIV-1) infection. C, Treatment paradigm to determine antiviral activity of nanoART (H2001 + H3001, 250 mg/kg each drug or H2001 + H3001 + H4001 [efavirenz, EFV], 100 mg/kg each drug) by subcutaneous administration on days 0 and 7 to PBL-reconstituted, HIV-1–infected NSG mice.

Figure 2.

Morphology, in vitro activity, and tissue biodistribution of nanoformulated antiretroviral therapy (nanoART). Serum drug and tissue levels in mice treated with 2 weekly injections of different doses of nanoART (atazanavir [ATV] and ritonavir [RTV]). A, Scanning electron micrographs (×15 000) of nanoformulations of ATV formulated by wet-milling (M3001) or homogenization (H3001), RTV formulated by wet-milling (M2001) or homogenization (H2001), and homogenized efavirenz (EFV) (H4001) on 0.2-µm polycarbonate filter membranes. Bar = 1 μm. B, Selection criteria for formulations used. P188, poloxamer 188; mPEG₂₀₀₀-DSPE, 1,2-distearoyl-phosphatidylethanolamine-methyl-polyethyleneglycol conjugate-2000; PDI, polydispersity index; PK, pharmacokinetics. ^aFormulations were produced by wet-milling (M) or high-pressure homogenization (H). ^bScoring of PDI, in vitro activity (MDM nanoART uptake, 15-day drug retention, drug release, and antiviral activities) and cytotoxicity (alamarBlue reduction and tumor necrosis factor α MDM production were determined as a decade weighted ratio (see Methods). Scoring of PK was determined as a function of the highest serum drug levels 7 days after nanoART administration (250 mg/kg subcutaneously) to BALB/cJ mice. Drug levels of ATV and RTV were determined from serum on days 1, 6, and 14 (C) and tissues on day 14 (D) after 2 weekly subcutaneous injections of combined nanoART (ATV/RTV; M2001 + M3001; 80, 150, or 250 mg/kg each) given to nonreconstituted, noninfected NSG mice on days 0 and 7. Data are expressed as median ± 25th (serum) and 75th (serum and tissues) percentiles for 5 mice per group. Differences were determined by analysis of variance of rank-transformed values and Tukey post hoc analysis. ^†Significantly different from values for other doses at that day or tissue at P ≤ .05.

Figure 3.

Tissue histology following nanoformulated antiretroviral therapy (nanoART) injections in mice. A, Hematoxylin and eosin staining of sections from liver, spleen, lung, and kidney in nonreconstituted, noninfected NSG mice treated with 2 weekly subcutaneous injections of 250 mg/kg each atazanavir (ATV)/ritonavir (RTV) (M3001 + M2001). B, Tissue histology of peripheral blood lymphocyte (PBL)–reconstituted, human immunodeficiency virus type 1 (HIV-1)–infected NSG mice treated with 2 weekly subcutaneous injections of either ATV/RTV (H3001 and H2001, 250 mg/kg each) or ATV/RTV/efavirenz (EFV) (H3001, H2001, and H4001, 100 mg/kg each) or untreated (PBS). All images at ×200 magnification. Bar = 100 μm.

Figure 4.

Serum and tissue drug levels in human immunodeficiency virus type 1 (HIV-1)–infected peripheral blood lymphocyte (PBL)–NSG mice treated with 2 weekly injections of nanoformulated antiretroviral therapy (nanoART) combinations (atazanavir [ATV] and ritonavir [RTV] or ATV, RTV, and efavirenz [EFV]). Following subcutaneous injections of either ATV and RTV (H3001 and H2001; 250 mg/kg each) or ATV, RTV, and EFV (H3001, H2001, and H4001; 100 mg/kg each) given on days 0 and 7 to PBL-reconstituted, HIV-1–infected mice, drug levels of ATV, RTV, and EFV were determined in serum on days 1, 6 and 14 (A), and in liver, spleen, kidney, lung, and brain on day 14 (B). Data are expressed as median ± 25th (serum) and 75th (serum and tissues) percentiles for 8 mice per group.

Figure 5.

Antiretroviral activities of nanoformulated antiretroviral therapy (nanoART) administered prior to human immunodeficiency virus type 1 (HIV-1) exposure. NanoART was delivered on day 0 as a combination of atazanavir (ATV) and ritonavir (RTV) (250 mg/kg each H3001 and H2001, subcutaneously) to peripheral blood lymphocyte (PBL)–reconstituted NSG mice. Native drugs (250 mg/kg each ATV and RTV in PBS) were delivered by oral gavage on day 0. Mice were infected with HIV-1_ADA on day 1. Spleens were harvested on day 9 after drug injection. A, Fluorescence-activated cell sorting analysis shows the percentage of human CD4⁺ among total CD3⁺ T cells (left panel) and CD4⁺/CD8⁺ T-cell ratios (right panel) for spleens from individual mice. B, Percentages of HIV-1p24–expressing cells among human HLA-DR⁺ cells were determined from cell counts of immunohistochemistry from splenic tissues (left panel). HIV-1gag RNA expression normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA expression in spleen (right panel). A and B, Data are shown as individual data points and median ± 25th and 75th percentiles (bar ± whisker) for 5–8 mice per group, analysis of variance of rank-transformed values, and significant differences determined by Tukey post hoc analyses, whereby P < .01 compared to native drug–treated, PBL-reconstituted, HIV-1–infected mice. C, Tissue and serum drug levels of ATV (left panel) and RTV (right panel) on day 9 following subcutaneous injection of nanoART or native drugs to mice. Data are expressed as median ± 25th (serum) and 75th (serum and tissue) percentiles for 8 mice per group, analyzed by Mann-Whitney test and significant differences determined by Tukey post hoc analyses. *Significantly different from nanoART-treated, PBL-reconstituted, HIV-1–infected NSG mice at P ≤ .01.

Figure 6.

Antiretroviral activities of nanoformulated antiretroviral therapy (nanoART). NanoART was delivered on days 0 and 7 as a combination of atazanavir (ATV) and ritonavir (RTV) (250 mg/kg each H3001 and H2001, subcutaneously) or a combination of ATV, RTV, and efavirenz (EFV) (100 mg/kg each H3001, H2001, and H4001, subcutaneously) to peripheral blood lymphocyte (PBL)–NSG mice infected intraperitoneally with human immunodeficiency virus type 1 (HIV-1)_ADA 12 h before initial nanoART dose. Peripheral blood and spleens were collected on day 14 after initial nanoART injection. Fluorescence-activated cell sorting analyses show the percentage of human CD4⁺ among total CD3⁺ T cells (left panels) and CD4⁺/CD8⁺ T-cell ratios (right panels) for peripheral blood (A) and spleen (B) from individual mice. C, Percentages of HIV-1p24–expressing cells among human HLA-DR⁺ cells were determined from cell counts of immunostained serial sections through spleens of PBL-reconstituted, HIV-1_ADA–infected PBL-NSG mice. D, HIV-1gag RNA expression in spleen normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA expression. E, Expression of human HLA-DR and HIV-1p24 in alternate serial sections of splenic tissues from HIV-1–infected NSG mice treated with PBS vehicle (top panels), ATV and RTV (middle panels), or ATV, RTV, and EFV (bottom panels). Spleens were collected on day 14 after initial nanoART injection, sectioned, immunostained with antibodies specific for HLA-DR or HIV-1p24, followed by secondary antibodies conjugated with horseradish peroxidase, and were visualized by 3,3-diaminobenzidine . A–D, Data are shown as individual data points and median ± 25th and 75th percentiles (bar ± whisker) for 3–8 mice per group, analyzed by analysis of variance of rank-transformed values and significant differences determined by Tukey post hoc analysis, whereby P ≤ .05 compared to (*) nontreated, nonreconstituted, noninfected NSG mice and (^#) nontreated, HIV-1–infected PBL-NSG mice.