Long-acting nanoformulated antiretroviral therapy elicits potent antiretroviral and neuroprotective responses in HIV-1-infected humanized mice

Dash, Prasanta K.; Gendelman, Howard E.; Roy, Upal; Balkundi, Shantanu; Alnouti, Yazen; Mosley, R. Lee; Gelbard, Harris A.; McMillan, JoEllyn M; Gorantla, Santhi; Poluektova, Larisa Y.

doi:10.1097/qad.0b013e328357f5ad

Cited by 119 publications

(129 citation statements)

References 33 publications

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“…This is seen in nanoART's abilities to maintain consistent plasma and tissue drug levels, as demonstrated in our previous studies (4). Nonetheless, to facilitate clearance of human immunodeficiency virus type 1 (HIV-1), antiretroviral drugs need to be effectively delivered to viral sanctuaries (5).…”

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confidence: 99%

“…For immunofluorescence staining, cells were washed three times with PBS and fixed with 4% paraformaldehyde (PFA) at room temperature for 30 min. The cells were treated with blocking/permeabilizing solution (0.1% Triton, 5% BSA in PBS) and quenched with 50 mM NH 4 Cl for 15 min. The cells were washed once with 0.1% Triton in PBS and sequentially incubated with primary and secondary antibodies at room temperature.…”

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confidence: 99%

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Endosomal Trafficking of Nanoformulated Antiretroviral Therapy Facilitates Drug Particle Carriage and HIV Clearance

Guo

Zhang

Wysocki

et al. 2014

J Virol

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Limitations of antiretroviral therapy (ART) include poor patient adherence, drug toxicities, viral resistance, and failure to penetrate viral reservoirs. Recent developments in nanoformulated ART (nanoART) could overcome such limitations. To this end, we now report a novel effect of nanoART that facilitates drug depots within intracellular compartments at or adjacent to the sites of the viral replication cycle. Poloxamer 407-coated nanocrystals containing the protease inhibitor atazanavir (ATV) were prepared by high-pressure homogenization. These drug particles readily accumulated in human monocyte-derived macrophages (MDM). NanoATV concentrations were ϳ1,000 times higher in cells than those that could be achieved by the native drug. ATV particles in late and recycling endosome compartments were seen following pulldown by immunoaffinity chromatography with Rab-specific antibodies conjugated to magnetic beads. Confocal microscopy provided cross validation by immunofluorescent staining of the compartments. Mathematical modeling validated drug-endosomal interactions. Measures of reverse transcriptase activity and HIV-1 p24 levels in culture media and cells showed that such endosomal drug concentrations enhanced antiviral responses up to 1,000-fold. We conclude that late and recycling endosomes can serve as depots for nanoATV. The colocalization of nano-ATV at endosomal sites of viral assembly and its slow release sped antiretroviral activities. Long-acting nanoART can serve as a drug carrier in both cells and subcellular compartments and, as such, can facilitate viral clearance. IMPORTANCEThe need for long-acting ART is significant and highlighted by limitations in drug access, toxicity, adherence, and reservoir penetrance. We propose that targeting nanoformulated drugs to infected tissues, cells, and subcellular sites of viral replication may improve clinical outcomes. Endosomes are sites for human immunodeficiency virus assembly, and increasing ART concentrations in such sites enhances viral clearance. The current work uncovers a new mechanism by which nanoART can enhance viral clearance over native drug formulations.

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Endosomal Trafficking of Nanoformulated Antiretroviral Therapy Facilitates Drug Particle Carriage and HIV Clearance

Guo

Zhang

Wysocki

et al. 2014

J Virol

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“…We previously developed, characterized, and tested therapeutic responses of long-acting nanoformulated ART (nanoART) (4)(5)(6)(7)(8)(9)(10)(11). Recent successes have come from encasing hydrophobic crystalline antiretroviral drugs (ARVs) into excipients, decorating the formed particles with ligands that target cells and tissue reservoirs or by pharmacologically boosting intracellular depot formations (4,(11)(12)(13).…”

Section: Introductionmentioning

confidence: 99%

Autophagy facilitates macrophage depots of sustained-release nanoformulated antiretroviral drugs

Gnanadhas¹,

Dash²,

Sillman³

et al. 2017

Journal of Clinical Investigation

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Long-acting anti-HIV products can substantively change the standard of care for patients with HIV/AIDS. To this end, hydrophobic antiretroviral drugs (ARVs) were recently developed for parenteral administration at monthly or longer intervals. While shorter-acting hydrophilic drugs can be made into nanocarrier-encased prodrugs, the nanocarrier encasement must be boosted to establish long-acting ARV depots. The mixed-lineage kinase 3 (MLK-3) inhibitor URMC-099 provides this function by affecting autophagy. Here, we have shown that URMC-099 facilitates ARV sequestration and its antiretroviral responses by promoting the nuclear translocation of the transcription factor EB (TFEB). In monocyte-derived macrophages, URMC-099 induction of autophagy led to retention of nanoparticles containing the antiretroviral protease inhibitor atazanavir. These nanoparticles were localized within macrophage autophagosomes, leading to a 4-fold enhancement of mitochondrial and cell vitality. In rodents, URMC-099 activation of autophagy led to 50-fold increases in the plasma drug concentration of the viral integrase inhibitor dolutegravir. These data paralleled URMC-099-mediated induction of autophagy and the previously reported antiretroviral responses in HIV-1-infected humanized mice. We conclude that pharmacologic induction of autophagy provides a means to extend the action of a long-acting, slow, effective release of antiretroviral therapy. Center for Neural Development and Disease, University of Rochester Medical Center (URMC), Rochester, New York, USA. Conflict of interest:H.A. Gelbard and H.E. Gendelman are members of the scientific advisory board for WavoDyne Therapeutics Inc., which is developing URMC-099 as a firstin-class MLK-3 inhibitor for clinical trials. URMC-099 is owned by URMC (patent nos. US 8,846,909 B2;8,877,772;and 9,181,247 and associated international patents). tion of TFEB mRNA by approximately 4-to 8-fold under different treatment conditions ( Figure 2F). With HIV-1 infection, TFEB mRNA levels were reduced by 2-to 3-fold compared with levels in uninfected controls, and URMC-099 could recover the phenotype. Increased TFEB translocation was delayed and did not reach significant levels until day 7 after URMC-099 treatment (Supplemental Figure 2B). These data confirm that URMC-099 regulates TFEB nuclear translocation in an mTORC1-dependent manner. URMC-099 stimulates autophagy in human MDMs. On the basis of the preceding data sets, we theorized that nuclear translocation of TFEB induces autophagy and lysosomal biogenesis. TFEB serves as the master regulator of both autophagy and lysosomal biogenesis (26), and such regulation could have a profound role in nanoART intracellular sequestration in MDMs. Thus, we examined the autophagosome markers microtubule-associated protein 1 light chain 3 β (LC3B, also known as MAP1LC3B) and beclin 1 (BECN1). Using Western blot assays, we found that each marker was significantly upregulated by URMC-099 ( Figure 3, A-C and Supplemental Figure 3A). URMC-099 increased the expression ...

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“…These efforts primarily have focused on nanomilled formulations of the antiretroviral (ARV) drugs, allowing them to be administered parenterally as injections (25,26). The ARV drugs atazanavir and ritonavir, nanoformulated in poloxamer 188 excipient by high-pressure homogenization, were evaluated preclinically as LA-ART candidates in humanized mouse (28) and nonhuman primate models (29). Parenteral administration of the integrase strand-transfer inhibitor GSK1265744 (GSK744) fully protected rhesus macaques against repeated low-dose intrarectal challenges of SHIV162P3 (30).…”

Section: Discussionmentioning

confidence: 99%

Pharmacokinetics of Injectable, Long-Acting Nevirapine for HIV Prophylaxis in Breastfeeding Infants

Cortez

Quintero

Moss

et al. 2015

Antimicrob Agents Chemother

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c Mother-to-child transmission (MTCT) of HIV-1 remains a global health problem. The World Health Organization (WHO) recommendations advise the administration of a once-daily, oral, prophylactic regimen of the nonnucleoside reverse transcriptase inhibitor nevirapine (NVP) from birth until 4 to 6 weeks of age for infants born to HIV-infected mothers in regions without access to safe and nutritionally adequate alternatives to breast milk. A critical factor driving the successful implementation of the WHO guidelines involves sustaining high adherence to the frequent dosing. With these challenges in mind, we have developed the first injectable, sustained-release NVP formulations with the goal of providing, for 6 weeks or longer, preventative plasma drug levels from a single subcutaneous administration at birth. The long-acting NVP consists of large (>50 m), monodisperse NVP particles coated with biocompatible polymers that control the drug release kinetics. Two lead formulations exhibiting burst-free, sustained-release kinetics for up to 75 days in vitro were developed. Subsequent in vivo studies in rats demonstrated no toxicity related to the formulations. Rat plasma NVP concentrations were above the analytical assay's limit of quantification for up to 28 days. Pharmacokinetic analysis of the rat plasma NVP concentration-time data allowed absorption rate constants to be calculated. These data then were used to simulate infant NVP exposure from a single injected dose (<200 mg) of our longacting formulations, demonstrating preliminary feasibility of the technology to maintain safe, preventative NVP plasma levels (0.2 to 3.0 g ml ؊1 ) for 6 weeks or longer.

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Long-acting nanoformulated antiretroviral therapy elicits potent antiretroviral and neuroprotective responses in HIV-1-infected humanized mice

Cited by 119 publications

References 33 publications

Endosomal Trafficking of Nanoformulated Antiretroviral Therapy Facilitates Drug Particle Carriage and HIV Clearance

Endosomal Trafficking of Nanoformulated Antiretroviral Therapy Facilitates Drug Particle Carriage and HIV Clearance

Autophagy facilitates macrophage depots of sustained-release nanoformulated antiretroviral drugs

Pharmacokinetics of Injectable, Long-Acting Nevirapine for HIV Prophylaxis in Breastfeeding Infants

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