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Tenofovir alafenamide and elvitegravir loaded nanoparticles for long-acting prevention of HIV-1 vaginal transmission
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Mandal, Subhra; Prathipati, Pavan K.; Kang, Guobin; Zhou, You; Yuan, Zhe; Fan, Wenjin; Li, Qingsheng; Destache, Christopher J.
AIDS: Feb 20 2017
"This preliminary proof-of-concept study demonstrated TAF + EVG fabricated into a nanoformulation using a FDA-approved polymer resulting in detectable drug concentrations in humanized mice for up to 14 days. HIV-1 efficacy was shown over a prolonged period 100% when challenged at 4 days post-nanoparticle injection and 60% when challenged at 14 days post-nanoparticle injection. Further research using other antiretrovirals encapsulated into nanoformulations would be of interest. Also, an interesting future study could be SubQ delivery of the combination antiretroviral nanoformulations and then rectal challenge with HIV-1......The present study demonstrates SubQ administration of TAF + EVG nanoparticles leads to higher accumulation of TFV in humanized mouse vaginal tissues. Therefore, higher and steady antiretroviral levels within vaginal tissue due to nano-encapsulation could potentially show better protection from HIV-1 infection......Next we evaluated the TAF + EVG nanoparticle protection efficacy against HIV-1 challenge. The hu-NSG mouse is a good model to use to determine tissue drug levels that closely resemble the hu-BLT mice. Median TFV and elvitegravir vaginal drug levels at day 4 averaged 577.7 and 83 ng/g, respectively, resulted in 100% protection. This compares with median TFV and EVG vaginal drug levels at day 14 were 34.6 ng/g and less than 10 ng/g, respectively, resulted in 60% efficacy in hu-BLT mice (Figs. 2 and 4). Moreover, the in-vitro 90% inhibition concentration concentrations in TZM-bl cells demonstrated a low concentration (3.6 ng/ml) for the combined TAF + EVG nanoparticle formulation (Fig. 1). The in-vitro 90% inhibition concentration estimation could explain the 60% prevention efficacy on day 14 challenged mice. Certainly, a larger animal model would lend to a more accurate assessment of HIV-1 protection when assessing the efficacy of these combination nanoparticles.
Through this report, we confirm successful encapsulation of two combination antiretrovirals (TAF + EVG) into nanoparticles (TAF + EVG nanoparticles) for PrEP delivery. We pursued combination antiretroviral (cARV) for encapsulation into the nanoparticle because clinical trials have documented a nonsignificant advantage to more than one drug in prevention trials compared with TFV alone [2,3,5,15]. Present results demonstrate SubQ administration of TAF + EVG nanoparticle shows efficacy in the humanized-bone marrow -liver -thymus (hu-BLT) mouse model against HIV-1 vaginal transmission....Currently, for PrEP against HIV-1 infection a long-acting injectable delivery system is the 'on-demand' requirement. Therefore, the goal of present experiments was to determine the sustained-release efficacy of a novel long-acting nanoformulation to prevent vaginal HIV-1 infection in hu-BLT mouse model. In parallel, we also determined tissue pharmacokinetics at the site of infection in hu-CD34-NSG mice. To the best of our knowledge, this is the first report of combining antiretroviral drugs in a single nano-formulation as a potential long-acting PrEP delivery modality to protect from HIV-1 infection. Furthermore, this is the first report of conducting simultaneous tissue pharmacokinetic study of TAF and EVG in the organs of hu-NSG mouse model."
Abstract
Objective: This report presents tenofovir (TFV) alafenamide (TAF) and elvitegravir (EVG) fabricated into nanoparticles for subcutaneous delivery as prevention strategy.
Design: Prospective prevention study in humanized bone marrow-liver-thymus (hu-BLT) mice.
Methods: Using an oil-in-water emulsion solvent evaporation technique, TAF + EVG drugs were entrapped together into nanoparticles containing poly(lactic-co-glycolic acid). In-vitro prophylaxis studies (90% inhibition concentration) compared nanoparticles with drugs in solution. Hu-BLT (n = 5/group) mice were given 200 mg/kg subcutaneous, and vaginally challenged with HIV-1 [5 x 105 tissue culture infectious dose for 50% of cells cultures (TCID50)] 4 and 14 days post-nanoparticle administration (post-nanoparticle injection). Control mice (n = 5) were challenged at 4 days. Weekly plasma viral load was performed using RT-PCR. Hu-BLT mice were sacrificed and lymph nodes were harvested for HIV-1 viral RNA detection by in-situ hybridization. In parallel, CD34+ humanized mice (3/time point) compared TFV and EVG drug levels in vaginal tissues from nanoparticles and solution. TFV and EVG were analyzed from tissue using liquid chromatograph-tandem mass spectrometry (LC-MS/MS).
Results: TAF + EVG nanoparticles were less than 200 nm in size. In-vitro prophylaxis indicates TAF + EVG nanoparticles 90% inhibition concentration was 0.002 μg/ml and TAF + EVG solution was 0.78 μg/ml. TAF + EVG nanoparticles demonstrated detectable drugs for 14 days and 72 h for solution, respectively. All hu-BLT control mice became infected within 14 days after HIV-1 challenge. In contrast, hu-BLT mice that received nanoparticles and challenged at 4 days post-nanoparticle injection, 100% were uninfected, and 60% challenged at 14 days post-nanoparticle injection were uninfected (P = 0.007; Mantel-Cox test). In-situ hybridization confirmed these results.
Conclusion: This proof-of-concept study demonstrated sustained protection for TAF + EVG nanoparticles in a hu-BLT mouse model of HIV vaginal transmission.
Introduction
Currently, approximately 36.9 million people worldwide are living with HIV-1 [1]. Orally administered antiretroviral drugs for preexposure prophylaxis (PrEP) as a preventive strategy have demonstrated efficacy in diverse groups of high-risk individuals [2-6]. Indeed, oral antiretroviral therapy has shown significant protection from HIV-1 among people who are at high risk such as MSM, injection drug users, and serodiscordant couples [7]. However, several clinical trials with oral tenofovir (TFV) and TFV disoproxil fumurate/emtricitabine (TDF/FTC) have been terminated early due to lack of efficacy [8,9] predominantly due to nonadherence issues. Trial participants have needed to ingested TFV or TDF/FTC daily for HIV prevention. The use of 'on demand' TDF/FTC (Truvada) has also demonstrated significant efficacy for MSM [10]. In the iPERGAY study of 'on demand' antiretrovirals, TDF/FTC were taken before and after sexual activity. However, there are significant side effects with daily Truvada that can negatively impact adherence. An international survey among high-risk individuals has documented positive responses for a long-acting preventive option [11]. Thus, a long-acting injectable delivery system could be the 'on demand' requirement for PrEP against HIV-1 infection. This would promote preventation and provide significant adherence.
Strong evidence has been accumulating that there is a correlation between the antiretroviral tissue concentration and prevention efficacy. In the CAPRISA 004 trial of 1% TFV vaginal gel, efficacy was correlated with cervico-vaginal fluid levels more than 1000 ng/ml [12]. However, there are significant differences in nucleoside reverse transcriptase inhibitor (NRTI) drug accumulation pattern in different organs. Thompson et al. reported that FTC concentration is high in vaginal tissue, whereas rectal tissue shows TFV dominance after oral administration of Truvada [13]. Microbicide Trials Network (MTN-001) clinical trial compared vaginal tissue TFV levels after oral and vaginal preparations in HIV-1 negative women. Also drug administration route has shown differences in antiretroviral accumulation. The results showed TFV gel achieved 130-times higher levels of TFV active metabolite (TFV di-phosphate) in vaginal tissue compared with oral TFV therapy [14]. Yet, preventive results with 1% TFV gel showed only 39% protection. Moreover, the Partners-in-PrEP clinical trial randomized oral TDF or TDF/FTC drugs among HIV-1 uninfected members of serodiscordant couples showed that plasma TFV levels at least 40 ng/ml were associated with an estimated 88% protective efficacy when TFV alone was ingested and 91% protection when consumed TDF/FTC combination [15]. TFV plasma threshold in this clinical trial was similar to the plasma level when patients take the drug daily without any missed doses. Therefore, a major factor is the route of administration and antiretroviral tissue accumulation. Thus our hypothesis is subcutaneous (SubQ) dosing indicates promise over other factors responsible for the less than optimal results. Therefore, current study was designed to investigate if tissue drug levels are among the critical factors that play a role in HIV-1 protection using a long-acting nanomedicines combining TFV alafenamide (TAF) and elvitegravir (EVG).
Our laboratory has been developing nanotechnology-based long-acting delivery systems for HIV treatment and prevention [16-21]. The nano-formulation involved use of a Food and Drug Administration (FDA)-approved polymer to encapsulate antiretrovirals into nanoparticles to develop a novel sustained drug-delivery module for HIV-1 prevention. Through this report, we confirm successful encapsulation of two combination antiretrovirals (TAF + EVG) into nanoparticles (TAF + EVG nanoparticles) for PrEP delivery. We pursued combination antiretroviral (cARV) for encapsulation into the nanoparticle because clinical trials have documented a nonsignificant advantage to more than one drug in prevention trials compared with TFV alone [2,3,5,15]. Present results demonstrate SubQ administration of TAF + EVG nanoparticle shows efficacy in the humanized-bone marrow -liver -thymus (hu-BLT) mouse model against HIV-1 vaginal transmission.
Discussion
Currently, for PrEP against HIV-1 infection a long-acting injectable delivery system is the 'on-demand' requirement. Therefore, the goal of present experiments was to determine the sustained-release efficacy of a novel long-acting nanoformulation to prevent vaginal HIV-1 infection in hu-BLT mouse model. In parallel, we also determined tissue pharmacokinetics at the site of infection in hu-CD34-NSG mice. To the best of our knowledge, this is the first report of combining antiretroviral drugs in a single nano-formulation as a potential long-acting PrEP delivery modality to protect from HIV-1 infection. Furthermore, this is the first report of conducting simultaneous tissue pharmacokinetic study of TAF and EVG in the organs of hu-NSG mouse model.
Thus far the Partners-in-PrEP clinical trial reported that plasma TFV concentrations more than 40 ng/ml had shown significantly higher protection [15]. This along with other studies have also conferred that people receiving TDF/FTC for prevention have shown higher protection against HIV-1 compared with those receiving TDF alone [2,3,5,22]. However, the above-reported maintenance of plasma antiretroviral drug concentration is only possible when the volunteers remain adhered to daily drug intake regime. Due to nonadherence, several clinical trials have shown lack of efficacy leading to early study termination [8,9].
We chose TAF (an FDA-approved NRTI drug) as ester prodrug of TFV over TDF, as TAF has been reported to be as effective as TDF in HIV-1 suppression and less toxic to kidneys and bone. Systemically TAF penetrates into tissue better than TDF [28]. As another component of antiretroviral combination regimen, we chose EVG, an anti-HIV integrase strand transfer inhibitor [29]. However, not much literature could be found regarding the concentration-response relationship for EVG. Oral administered EVG (150 mg daily) was found to have peak plasma concentrations at 4 h, and the plasma half-life corresponds to 7.6 h. Massud et al. reported in macaques administration of 50 mg/kg EVG orally the plasma drug concentrations were similar to humans [29], and it shows the highest penetration in rectal and vaginal fluids. However, EVG administered orally was detectable in vaginal secretions from macaques for only 24 h. Therefore, keeping this in mind we formulated a potential long-acting combination antiretrovirals (TAF + EVG) nanoparticles for PrEP application. These two drugs in the formulation could show positive suppression of vaginal HIV-1 infectivity.
To date, there is no clear report that specifies the vaginal TFV concentration that is necessary for protection against HIV-1 [30]. So far, the vaginal tissue concentrations after a single oral dose of Truvada showed TDF ester prodrug concentration to be 7 ng/g [31]. Veselinovic et al. determined TFV (prodrug form of TDF) levels at steady-state after oral gavage of TDF (61.5 mg/kg) in humanized and nonhumanized mice [31]. They reported a median peak vaginal TFV level to be 729 ng/g after oral administration in Rag [2] knockout mice. The present study demonstrates SubQ administration of TAF + EVG nanoparticles leads to higher accumulation of TFV in humanized mouse vaginal tissues. Therefore, higher and steady antiretroviral levels within vaginal tissue due to nano-encapsulation could potentially show better protection from HIV-1 infection.
Next we evaluated the TAF + EVG nanoparticle protection efficacy against HIV-1 challenge. The hu-NSG mouse is a good model to use to determine tissue drug levels that closely resemble the hu-BLT mice. Median TFV and elvitegravir vaginal drug levels at day 4 averaged 577.7 and 83 ng/g, respectively, resulted in 100% protection. This compares with median TFV and EVG vaginal drug levels at day 14 were 34.6 ng/g and less than 10 ng/g, respectively, resulted in 60% efficacy in hu-BLT mice (Figs. 2 and 4). Moreover, the in-vitro 90% inhibition concentration concentrations in TZM-bl cells demonstrated a low concentration (3.6 ng/ml) for the combined TAF + EVG nanoparticle formulation (Fig. 1). The in-vitro 90% inhibition concentration estimation could explain the 60% prevention efficacy on day 14 challenged mice. Certainly, a larger animal model would lend to a more accurate assessment of HIV-1 protection when assessing the efficacy of these combination nanoparticles.
This preliminary proof-of-concept study demonstrated TAF + EVG fabricated into a nanoformulation using a FDA-approved polymer resulting in detectable drug concentrations in humanized mice for up to 14 days. HIV-1 efficacy was shown over a prolonged period 100% when challenged at 4 days post-nanoparticle injection and 60% when challenged at 14 days post-nanoparticle injection. Further research using other antiretrovirals encapsulated into nanoformulations would be of interest. Also, an interesting future study could be SubQ delivery of the combination antiretroviral nanoformulations and then rectal challenge with HIV-1.
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