Pharmacodynamics of folic acid receptor targeted antiretroviral nanotherapy in HIV-1-infected humanized mice

doi:10.1016/j.antiviral.2015.05.009

Antiviral Research

Volume 120, August 2015, Pages 85-88

https://doi.org/10.1016/j.antiviral.2015.05.009 Get rights and content

Highlights

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Improved pharmacodynamics of FA-nanoATV/r were shown during chronic HIV-1 infection of CD34+ HSC-NSG mice.
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In mice treated every other week with 100 mg/kg FA-nanoATV/r showed viral RNA copies/ml below limit of detection.
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FA-nanoATV/r enabled dosing reductions of 2.5 times over nondecorated nanoATV/r to achieve viral suppression.

Abstract

Long-acting nanoformulated antiretroviral therapy (nanoART) can sustain plasma drug levels and improve its biodistribution. Cell targeted-nanoART can achieve this and bring drug efficiently to viral reservoirs. However, whether such improvements affect antiretroviral responses remains unknown. To these ends, we tested folic acid (FA)-linked poloxamer407-coated ritonavir-boosted atazanavir (FA-nanoATV/r) nanoparticles for their ability to affect chronic HIV-1 infection in humanized mice. Following three, 100 mg/kg FA-nanoATV/r intramuscular injections administered every other week to infected animals, viral RNA was at or below the detection limit, cell-associated HIV-1p24 reduced and CD4+ T cell counts protected. The dosing regimen improved treatment outcomes more than two fold from untargeted nanoATV/r. We posit that these nanoformulations have potential for translation to human use.

Section snippets

Physicochemical characterization

FA-nanoATV/r nanoformulations (FA-P407-ATV/r) were prepared by high-pressure homogenization (Puligujja et al., 2013). Physicochemical characteristics including particle size, charge, polydispersity (PDI) and shape were determined. Particle size, polydispersity and zeta potential ranged from 257 to 433 nm, 0.17 to 0.33 and −8.9 to −12.1 mV, respectively for FA-nanoATV and FA-nanoRTV.

Infection and nanoART treatments

The University of Nebraska Medical Center Institutional Review Board approved human fetal tissue usage. CD34+ hematopoietic stem cells (HSC) were isolated from human fetal liver by immune selection (Miltenyl Biotec Inc., Auburn, CA) then transplanted into NSG mice at birth (Gorantla et al., 2007). At 22 weeks of age mice were infected with a 10⁴ tissue culture infective dose 50 (TCID₅₀)/mouse of HIV-1_ADA by intraperitoneal injection. Ten weeks later mice were administered 100 mg/kg FA-nanoATV/r

Plasma and tissue drug distribution

Plasma samples were collected at weeks 2, 4 and 6 after drug administration. Mouse tissues were collected after sacrifice. Drug concentrations were determined by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) (Huang et al., 2011). Following treatment with FA-nanoATV/r, plasma ATV concentrations were maintained above the human minimum effective concentration (MEC) of 150 ng/ml (la Porte et al., 2006) throughout the study. At 2, 4 and 6 weeks ATV levels were 285 ± 84,

Viral load determinations

To determine antiviral efficacy of FA-nanoATV/r, pre- and post-treatment viral loads were determined in blood from HIV-1 infected-CD34+ HSC-NSG mice. Viral load (RNA copies/ml) was determined in plasma using COBAS Amplicor System v1.5 kit (Roche Molecular Diagnostics, Switzerland) (Dash et al., 2012). Individual mouse viral loads before treatment with FA-nanoATV/r were 0.7 × 10⁵, 5.0 × 10⁵, 2.0 × 10⁵, 3.2 × 10⁵, and 0.7 × 10⁵ viral RNA copies/ml. Treatment with FA-P407-ATV/r produced up to 3 log decrease

Flow cytometric analyses

To assess whether FA-nanoATV/r treatment provided immune protection, we determined the percent of human CD45, CD3, CD4 and CD8 positive cells in blood, spleen and bone marrow. The number of human CD45+, CD3+, CD4+ and CD8+ cells (BD Pharmingen, San Diego, CA) were determined by fluorescence-activated cell sorting (FACS) using a BD FACSDiva® (BD Immunocytometry Systems, Mountain View, CA) system. CD4+ lymphocyte percentages were determined from the CD3+ gated cells. After sacrifice, CD4+ and

Immunohistochemistry

Spleens collected at sacrifice were fixed with 10% neutral buffered formalin (Fisher Scientific, Kalamazoo, MI) and paraffin embedded. Five μm thick serial sections were collected and immunostained (Fig. 2C) with human leukocyte antigen; HLA-DR (clone CR3/43; 1:100) and mouse monoclonal antibody against HIV-1p24 (clone Kal-1; 1:10) (Puligujja et al., 2015). Antiretroviral activity was determined by dividing the total HIV-1p24+ cells by total HLA-DR+ cells in five random microscopic

Acknowledgments

This work was supported by the University of Nebraska Foundation which includes individual donations from Carol Swarts and Frances and Louie Blumkin, the Vice Chancellor’s office of the University of Nebraska Medical Center, ViiV Healthcare and National Institutes of Health grants P01 DA028555, R01 NS36126, P01 NS31492, 2R01 NS034239, P01 MH64570, P01 NS43985, P30 MH062261 and R01 AG043540. The authors thank Jaclyn Knibbe, Selena Dickinson and Edward Makarov for expert technical assistance.

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Short CommunicationPharmacodynamics of folic acid receptor targeted antiretroviral nanotherapy in HIV-1-infected humanized mice

Highlights

Abstract

Section snippets

Physicochemical characterization

Infection and nanoART treatments

Plasma and tissue drug distribution

Viral load determinations

Flow cytometric analyses

Immunohistochemistry

Acknowledgments

J. Chromatogr. B, Anal. Technol. Biomed. Life Sci.

Nanomed. Nanotechnol. Biol. Med.

Biomaterials

Comparative manufacture and cell-based delivery of antiretroviral nanoformulations

Int. J. Nanomed.

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

Aids

Long-acting HIV drugs advanced to overcome adherence challenge

Nat. Med.

Short Communication
Pharmacodynamics of folic acid receptor targeted antiretroviral nanotherapy in HIV-1-infected humanized mice