ABSTRACT
Antiretroviral therapy (ART) suppresses human immunodeficiency virus (HIV) infection. Research seeking to transform viral suppression into elimination has generated novel immune, chemical and molecular antiviral agents. However, none, to date, have excised latent integrated proviral DNA or removed infected cells from infected persons. These efforts included, but are not limited to, broadly neutralizing antibodies, "shock" and "kill" latency-reversing agents, innate immune regulators, and sequential long-acting antiretroviral nanoformulated prodrugs and CRISPR-Cas9 gene editing. While, the latter, enabled the complete excision of latent HIV-1 from the host genome success was so far limited. We contend that improvements in antiretroviral delivery, potency, agent specificity, or combinatorial therapies can provide a pathway towards complete HIV elimination.
Subject(s)
Disease Eradication/methods , HIV Infections/prevention & control , AIDS Vaccines/therapeutic use , Anti-HIV Agents/therapeutic use , HIV Infections/drug therapy , HIV Infections/epidemiology , HIV-1/genetics , HIV-1/immunology , HumansABSTRACT
Antiretroviral therapy (ART) has improved the quality of life in patients infected with HIV-1. However, complete viral suppression within anatomical compartments remains unattainable. This is complicated by adverse side effects and poor adherence to lifelong therapy leading to the emergence of viral drug resistance. Thus, there is an immediate need for cellular and tissue-targeted long-acting (LA) ART formulations. Herein, we describe two LA prodrug formulations of darunavir (DRV), a potent antiretroviral protease inhibitor. Two classes of DRV prodrugs, M1DRV and M2DRV, were synthesized as lipophilic and hydrophobic prodrugs and stabilized into aqueous suspensions designated NM1DRV and NM2DRV. The formulations demonstrated enhanced intracellular prodrug levels with sustained drug retention and antiretroviral activities for 15 and 30 days compared to native DRV formulation in human monocyte-derived macrophages. Pharmacokinetics tests of NM1DRV and NM2DRV administered to mice demonstrated sustained drug levels in blood and tissues for 30 days. These data, taken together, support the idea that LA DRV with sustained antiretroviral responses through prodrug nanoformulations is achievable.
Subject(s)
Darunavir/administration & dosage , HIV Protease Inhibitors/administration & dosage , Prodrugs/administration & dosage , Prodrugs/chemical synthesis , Animals , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/virology , Cell Survival/drug effects , Chromatography, Liquid , Darunavir/chemical synthesis , Darunavir/chemistry , Darunavir/pharmacokinetics , Drug Resistance, Viral/drug effects , HIV Protease Inhibitors/pharmacokinetics , HIV-1/drug effects , HIV-1/enzymology , Humans , Macrophages/drug effects , Macrophages/ultrastructure , Macrophages/virology , Male , Mice , Mice, Inbred BALB C , Microscopy, Electron , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Rats , Tandem Mass SpectrometryABSTRACT
Nucleoside reverse transcriptase inhibitors (NRTIs) require intracellular phosphorylation to active triphosphate (TP) nucleotide metabolites before they can inhibit the HIV reverse transcriptase. However, monitoring these pharmacologically active TP metabolites is challenging due to their instability and their low concentrations at the pg/ml levels in blood and tissues. The combination of lamivudine (3TC) and abacavir (ABC) is one of the first lines for HIV therapy. Therefore, a sensitive, selective, accurate, and precise LC-MS/MS method was developed and validated for the simultaneous quantification of 3TC- and ABC-TP metabolites in mouse blood and tissues. Calibration curves were linear over the range of 10-100,000â¯pg/ml for 3TC-TP and 4-40,000â¯pg/ml for carbovir-TP (CBV-TP; phosphorylated metabolite of ABC). This corresponds to 2.1-21,322 fmol/106 cells for 3TC-TP and 0.8-8000â¯fmol/106 cells for CBV-TP. Accuracy and precision were less than 15% for all quality control sample (QCs), and absolute extraction recovery of were >65% for 3TC-TP and >90% for CBV-TP. The method was optimized to ensure stability of TP samples and standards during sample collection, preparation, analysis, and storage conditions. This method has enhanced sensitivity and requires smaller amounts of blood and tissue samples compared to previous LC-MS/MS methods for 3TC- and CBV-TP quantification. The developed method was successfully applied to characterize the pharmacokinetic profile of TP metabolites in mouse peripheral blood mononuclear cells (PBMCs), spleen, lymph nodes, and liver cells. In addition, another direct, simple, and high-throughput method for the quantification of TP standards was developed and used for the analysis of stability samples.
Subject(s)
Dideoxynucleosides/blood , Lamivudine/blood , Polyphosphates/blood , Animals , Anti-HIV Agents/blood , Chromatography, Liquid/methods , Humans , Leukocytes, Mononuclear/metabolism , Male , Mice , Mice, Inbred BALB C , Reverse Transcriptase Inhibitors/blood , Tandem Mass Spectrometry/methodsABSTRACT
Antiretroviral drug (ARV) metabolism is linked largely to hepatic cytochrome P450 activity. One ARV drug class known to be metabolized by intestinal and hepatic CYP3A are the protease inhibitors (PIs). Plasma drug concentrations are boosted by CYP3A inhibitors such as cobisistat and ritonavir (RTV). Studies of such drug-drug interactions are limited since the enzyme pathways are human specific. While immune-deficient mice reconstituted with human cells are an excellent model to study ARVs during human immunodeficiency virus type 1 (HIV-1) infection, they cannot reflect human drug metabolism. Thus, we created a mouse strain with the human pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 genes on a NOD.Cg-Prkdcscid Il2rgtm1Sug /JicTac background (hCYP3A-NOG) and used them to evaluate the impact of human CYP3A metabolism on ARV pharmacokinetics. In proof-of-concept studies we used nanoformulated atazanavir (nanoATV) with or without RTV. NOG and hCYP3A-NOG mice were treated weekly with 50 mg/kg nanoATV alone or boosted with nanoformulated ritonavir (nanoATV/r). Plasma was collected weekly and liver was collected at 28 days post-treatment. Plasma and liver atazanavir (ATV) concentrations in nanoATV/r-treated hCYP3A-NOG mice were 2- to 4-fold higher than in replicate NOG mice. RTV enhanced plasma and liver ATV concentrations 3-fold in hCYP3A-NOG mice and 1.7-fold in NOG mice. The results indicate that human CYP3A-mediated drug metabolism is reduced compared with mouse and that RTV differentially affects human gene activity. These differences can affect responses to PIs in humanized mouse models of HIV-1 infection. Importantly, hCYP3A-NOG mice reconstituted with human immune cells can be used for bench-to-bedside translation.