Pharmacodynamics of long-acting folic acid-receptor targeted ritonavir-boosted atazanavir nanoformulations

Abstract

Long-acting nanoformulated antiretroviral therapy (nanoART) that targets monocyte-macrophages could improve the drug's half-life and protein-binding capacities while facilitating cell and tissue depots. To this end, ART nanoparticles that target the folic acid (FA) receptor and permit cell-based drug depots were examined using pharmacokinetic and pharmacodynamic (PD) tests. FA receptor-targeted poloxamer 407 nanocrystals, containing ritonavir-boosted atazanavir (ATV/r), significantly increased drug bioavailability and PD by five and 100 times, respectively. Drug particles administered to human peripheral blood lymphocyte reconstituted NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice and infected with HIV-1ADA led to ATV/r drug concentrations that paralleled FA receptor beta staining in both the macrophage-rich parafollicular areas of spleen and lymph nodes. Drug levels were higher in these tissues than what could be achieved by either native drug or untargeted nanoART particles. The data also mirrored potent reductions in viral loads, tissue viral RNA and numbers of HIV-1p24+ cells in infected and treated animals. We conclude that FA-P407 coating of ART nanoparticles readily facilitates drug carriage and antiretroviral responses.

Keywords: Folic acid receptor; Human immunodeficiency virus type one; Long-acting nanoformulated antiretroviral therapy; Non-obese diabetic severe combined immunodeficient mice; Pharmacodynamics; Pharmacokinetics.

Figure 1

(A) Treatment paradigm to determine pharmacokinetics, biodistribution, efficacy and toxicology profiles of nanoformulated antiretrovirals in human-peripheral blood lymphocyte reconstituted non-obese diabetic, severe combined immunodeficient mice (Hu-PBL-NSG mice); Cartoon schematic showing the advantage of targeting folate receptor-β on macrophage. A representative photomicrograph shows the expression of mouse folate receptor β (mFRβ) in spleen 36 hrs after treatment with (B) PBS and (C) nanoART. Balb/cJ mice were injected intramuscularly with PBS or nanoATV/r (100 mg/kg). Mice were sacrificed 36 hrs after treatment, and spleen sections were probed with a mFRβ-specific antibody using the Vertex M.O.M kit. Nuclei were stained with hematoxylin. The cartoon represents expression of FRβ on macrophages in spleen following (D) PBS and (E) nanoART treatment.

Figure 2

Pharmacokinetics of FA-P407- and P407- ATV/r in Balb/cJ mice. FA-P407-ATV/r and P407-ATV/r were administered at a dose of 50 mg/kg or 100 mg/kg by intramuscular injection on day 0, and mice were sacrificed on day 14. (A) Plasma was collected at indicated time points and ATV and RTV levels were quantified by UPLC-MS/MS. Data are expressed as mean ± SEM. * Statistically different from P407-ATV/r at P < 0.05 by 2-way ANOVA and Bonferroni’s posthoc test. (B) Comparison of tissue distribution of ATV and RTV after FA-P407-ATV/r and P407-ATV/r treatment in Balb/cJ mice. The indicated tissues were collected on day 14 after sacrifice, and ATV and RTV levels were quantified by UPLC-MS/MS. Data are expressed as mean ± SEM and *, **statistically different from P407-ATV/r at P < 0.05 and P < 0.01 by student’s unpaired t-test respectively.

Figure 3

Flow cytometric evaluation of FA-P407-ATV/r and P407-ATV/r in hu-PBL reconstituted NSG mice infected with HIV-1_ADA. On day 0, PBS, Free drug or nanoATV/r (a combination of ATV and rRTV) was delivered to PBL–reconstituted NSG mice at doses (A) 100 mg/kg, (B) 50 mg/kg or (C) 10 mg/kg. Mice were then infected with HIV-1_ADA on day 1 and sacrificed on day 14. Fluorescence-activated cell sorting analyses show the ratio of human CD4+ to CD8+ T cells among total CD3+ T cells for peripheral blood and spleen from individual mice. Data are means ± SEM and *, **statistically different at P < 0.05 and P < 0.01 by Mann-Whitney U-test.

Figure 4

Levels of HIV-1p24 antigen in liver and spleen of HIV-1 infected huPBL-reconstituted NSG mice treated with FA-P407-ATV/r and P407-ATV/r. FA-P407-ATV/r and P407-ATV/r (100 mg/kg each drug) were administered intramuscularly on day 0 to huPBL- NSG mice. Mice were infected with HIV-1_ADA on day 1 and sacrificed on day 14. Livers and spleens were collected on day 14 after drug injection, then sectioned and immunostained with antibodies specific for HLA-DR or HIV-1p24, and visualized by DAB.

Figure 5

Comparison of antiretroviral activities of FA-P407-ATV/r and P407-ATV/r in hu-PBL reconstituted NSG mice infected with HIV-1_ADA. Mice were treated on day 0 with PBS, free drug ATV/r, P407-ATV/r or FA-P407-ATV/r at doses of (A) 100 mg/kg or (B) 50 mg/kg. Mice were then infected with HIV-1_ADA on 24 hours later and sacrificed on day 14. Paraffin embedded sections of liver, spleen and lung were probed for HIV-1p24 and HLA-DR. Antiretroviral efficacy was determined by counting the number of HIV-1p24+ cells per section and expressed as percent of HLA-DR+ cells. Data are expressed as mean ± SEM and *, **statistical differences were determined at P < 0.05 and P < 0.01 by the Mann-Whitney U-test.

Figure 6

Comparison of antiretroviral activities of FA-P407-ATV/r and P407-ATV/r in hu-PBL reconstituted NSG mice infected with HIV-1_ADA. Mice were treated on day 0 with PBS (untreated), free drug ATV/r, P407-ATV/r or FA-P407-ATV/r at doses of (A) 100 mg/kg or (B) 50 mg/kg. Mice were then infected with HIV-1_ADA 24 hours later and sacrificed on day 14. HIV-1gag RNA expression in spleen was determined and normalized to human CD45 expression as a marker for human cell reconstitution. Data are expressed as mean ± SEM and considered *, ** statistically different, at P < 0.05 and P < 0.01, by the Mann-Whitney U-test.