Upal Roy

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

Objectives:Long-acting nanoformulated antiretroviral therapy (nanoART) with improved pharmacokinetics, biodistribution and limited systemic toxicities will likely improve drug adherence and access to viral reservoirs. Design:Atazanavir and ritonavir crystalline nanoART were formulated in a poloxamer-188 excipient by high-pressure homogenization. These formulations were evaluated for antiretroviral and neuroprotective activities in humanized NOD/scid-IL-2Rgcnull (NSG) mice. Methods:NanoART-treated NSG mice were evaluated for drug biodistribution, pharmacodynamics and toxicity. CD34+ human hematopoietic stem cells were transplanted at birth in replicate NSG mice. The mice were infected with HIV-1ADA at 5 months of age. Eight weeks later, the infected animals were treated with weekly subcutaneous injections of nanoformulated ATV and RTV. Peripheral viral load, CD4+ T-cell counts and lymphoid and brain histopathology and immunohistochemistry tests were performed. Results:NanoART treatments by once-a-week injections reduced viral loads more than 1000-fold and protected CD4+ T-cell populations. This paralleled high ART levels in liver, spleen and blood that were in or around the human minimal effective dose concentration without notable toxicities. Importantly, examination of infected brain subregions showed that nanoART elicited neuroprotective responses with detectable increases in microtubule-associated protein-2, synaptophysin and neurofilament expression when compared to untreated virus-infected animals. Therapeutic interruptions produced profound viral rebounds. Conclusion:Long-acting nanoART has translational potential with sustained and targeted efficacy and with limited systemic toxicities. Such success in drug delivery and distribution could improve drug adherence and reduce viral resistance in infected people.

MRP (ABCC) transporters-mediated efflux of anti-HIV drugs, saquinavir and zidovudine, from human endothelial cells

The constituents of highly active anti-retroviral therapy (HAART) include HIV-1 protease inhibitors (HPIs) and nucleoside reverse transcriptase inhibitors (NRTIs). Endothelial cell (EC) barriers, especially the blood-brain-barrier (BBB) suppresses the entry of HAART drugs to subendothelial HIV-1 reservoirs. The ATP binding cassette (ABC) transporter family members, multidrug resistant-1 (MDR-1) and multidrug resistance-associated proteins (MRPs) can efflux both HPIs and NRTIs from intracellular compartments. Using brain derived ECs from non-human sources, previous studies suggested a dominant role for MDR-1 in HAART efflux from the BBB. However, due to species variations in ABC-transporter expression, drug-efflux functions using human brain ECs need to be investigated. Furthermore, roles of ABC-transporters in drug-efflux from systemic EC barriers need to be studied. We monitored the expression of ABC-transporters in primary human ECs obtained from brain (HBMVECs), aorta (HAECs), pulmonary-artery (HPAECs), dermal-microvessel (HDMVECs) and umbilical vein (HUVECs). Gene expression for MDR-1 and MRPs (MRP-1 to MRP-5) were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Drug efflux functions were determined by calcein retention assays. Intracellular accumulation of both 3H-saqui-navir (an HPI) and 3H-zidovudine (an NRTI) were also monitored in HAECs and HBMVECs. Both assays were carried out in presence of verapamil (20–60 μM) or MK-571 (12.5–50 μM) inhibitors of MDR-1 and MRPs, respectively in presence of verapamil or MK-571. The HBMVECs expressed higher levels of MRPs than MDR-1 and only MK-571 significantly (P < 0.01) suppressed calcein efflux from these cells. However, both HAECs and HPAECs showed MDR-1 and MRP expression and calcein efflux was inhibited by both verapamil and MK-571. Both inhibitors suppressed 3H-saqubinavir efflux from HAECs, but only MK-571 suppressed saquinavir efflux from HBMVECs. In both ECs, 3H-zidovudine efflux was only suppressed by MK-571. Thus, primary human ECs, especially brain derived ECs, predominantly express MRPs and their specific inhibition may enhance HAART efficacy in subendothelial HIV-1 reservoirs.

Pharmacodynamic and Antiretroviral Activities of Combination Nanoformulated Antiretrovirals in HIV-1–Infected Human Peripheral Blood Lymphocyte–Reconstituted Mice

Lack of adherence, inaccessibility to viral reservoirs, long-term drug toxicities, and treatment failures are limitations of current antiretroviral therapy (ART). These limitations lead to increased viral loads, medicine resistance, immunocompromise, and comorbid conditions. To this end, we developed long-acting nanoformulated ART (nanoART) through modifications of existing atazanavir, ritonavir, and efavirenz suspensions in order to establish cell and tissue drug depots to achieve sustained antiretroviral responses. NanoARTs abilities to affect immune and antiviral responses, before or following human immunodeficiency virus type 1 infection were tested in nonobese severe combined immune-deficient mice reconstituted with human peripheral blood lymphocytes. Weekly subcutaneous injections of drug nanoformulations at doses from 80 mg/kg to 250 mg/kg, 1 day before and/or 1 and 7 days after viral exposure, elicited drug levels that paralleled the human median effective concentration, and with limited toxicities. NanoART treatment attenuated viral replication and preserved CD4(+) Tcell numbers beyond that seen with orally administered native drugs. These investigations bring us one step closer toward using long-acting antiretrovirals in humans.

Preclinical Pharmacokinetics and Tissue Distribution of Long-Acting Nanoformulated Antiretroviral Therapy

ABSTRACT Long-acting injectable nanoformulated antiretroviral therapy (nanoART) was developed with the explicit goal of improving medicine compliance and for drug targeting of viral tissue reservoirs. Prior nanoART studies completed in humanized virus-infected mice demonstrated sustained antiretroviral responses. However, the pharmacokinetics (PK) and tissue distribution of nanoART were not characterized. To this end, the PK and tissue distribution of nanoformulated atazanavir (ATV) and ritonavir (RTV) injected subcutaneously or intramuscularly in mice and monkeys were evaluated. Fourteen days after injection, ATV and RTV levels were up to 13-, 41-, and 4,500-fold higher than those resulting from native-drug administration in plasma, tissues, and at the site of injection, respectively. At nanoART doses of 10, 50, 100, and 250 mg/kg of body weight, relationships of more- and less-than-proportional increases in plasma and tissue levels with dose increases were demonstrated with ATV and RTV. Multiple-dose regimens showed serum and tissue concentrations up to 270-fold higher than native-drug concentrations throughout 8 weeks of study. Importantly, nanoART was localized in nonlysosomal compartments in tissue macrophages, creating intracellular depot sites. Reflective data were obtained in representative rhesus macaque studies. We conclude that nanoART demonstrates blood and tissue antiretroviral drug levels that are enhanced compared to those of native drugs. The sustained and enhanced PK profile of nanoART is, at least in part, the result of the sustained release of ATV and RTV from tissue macrophases and at the site of injection.

Mononuclear phagocyte intercellular crosstalk facilitates transmission of cell-targeted nanoformulated antiretroviral drugs to human brain endothelial cells.

Despite the successes of antiretroviral therapy (ART), HIV-associated neurocognitive disorders remain prevalent in infected people. This is due, in part, to incomplete ART penetration across the blood–brain barrier (BBB) and lymph nodes and to the establishment of viral sanctuaries within the central nervous system. In efforts to improve ART delivery, our laboratories developed a macrophage-carriage system for nanoformulated crystalline ART (nanoART) (atazanavir, ritonavir, indinavir, and efavirenz). We demonstrate that nanoART transfer from mononuclear phagocytes (MP) to human brain microvascular endothelial cells (HBMEC) can be realized through cell-to-cell contacts, which can facilitate drug passage across the BBB. Coculturing of donor MP containing nanoART with recipient HBMEC facilitates intercellular particle transfer. NanoART uptake was observed in up to 52% of HBMEC with limited cytotoxicity. Folate coating of nanoART increased MP to HBMEC particle transfer by up to 77%. To translate the cell assays into relevant animal models of disease, ritonavir and atazanavir nanoformulations were injected into HIV-1-infected NOD/scid-γcnull mice reconstituted with human peripheral blood lymphocytes. Atazanavir and ritonavir levels in brains of mice treated with folate-coated nanoART were three- to four-fold higher than in mice treated with noncoated particles. This was associated with decreased viral load in the spleen and brain, and diminished brain CD11b-associated glial activation. We postulate that monocyte-macrophage transfer of nanoART to brain endothelial cells could facilitate drug entry into the brain.

Comparative manufacture and cell-based delivery of antiretroviral nanoformulations.

Nanoformulations of crystalline indinavir, ritonavir, atazanavir, and efavirenz were manufactured by wet milling, homogenization or sonication with a variety of excipients. The chemical, biological, immune, virological, and toxicological properties of these formulations were compared using an established monocyte-derived macrophage scoring indicator system. Measurements of drug uptake, retention, release, and antiretroviral activity demonstrated differences amongst preparation methods. Interestingly, for drug cell targeting and antiretroviral responses the most significant difference among the particles was the drug itself. We posit that the choice of drug and formulation composition may ultimately affect clinical utility.

Magnetically guided central nervous system delivery and toxicity evaluation of magneto-electric nanocarriers

Least component-based delivery of drug-tagged-nanocarriers across blood-brain-barriers (BBB) will allow site-specific and on-demand release of therapeutics to prevent CNS diseases. We developed a non-invasive magnetically guided delivery of magneto-electric nanocarriers (MENCs), ~20 nm, 10 mg/kg, across BBB in C57Bl/J mice. Delivered MENCs were uniformly distributed inside the brain, and were non-toxic to brain and other major organs, such as kidney, lung, liver, and spleen, and did not affect hepatic, kidney and neurobehavioral functioning.

Microglia-derived HIV Nef+ exosome impairment of the blood–brain barrier is treatable by nanomedicine-based delivery of Nef peptides

The negative factor (Nef) of human immunodeficiency virus (HIV) is an accessory protein that is thought to be integral to HIV-associated immune- and neuroimmune pathogenesis. Here, we show that nef-transfected microglia-released Nef+ exosome (exNef) disrupts the apical blood–brain barrier (BBB) and that only nef-transfected microglia release Nef in exosomes. nef–gfp-transduced neurons and astrocytes release exosomes but did not release exNef in the extracellular space. Apical administration of exNef derived from nef-transfected 293T cells reduced transendothelial electrical resistance (TEER) and increased permeability of the BBB. Microglia-derived exNef applied to either the apical/basal BBB significantly reduced expression of the tight junction protein, ZO-1, suggesting a mechanism of exNef-mediated neuropathogenesis. Microglia exposed to exNef release elevated levels of Toll-like receptor-induced cytokines and chemokines IL-12, IL-8, IL-6, RANTES, and IL-17A. Magnetic nanoparticle delivery of Nef peptides containing the Nef myrisolation site across an in vitro BBB ultimately reduced nef-transfected microglia release of Nef exosomes and prevented the loss of BBB integrity and permeability as measured by TEER and dextran-FITC transport studies, respectively. Overall, we show that exNef is released from nef–gfp-transfected microglia; exNef disrupts integrity and permeability, and tight junctions of the BBB, and induces microglial cytokine/chemokine secretion. These exNef-mediated effects were significantly restricted by Nef peptides. Taken together, this study provides preliminary evidence of the role of exNef in HIV neuroimmune pathogenesis and the feasibility of a nanomedicine-based therapeutics targeting exNef to treat HIV-associated neuropathogenesis.

UPLC-MS/MS quantification of nanoformulated ritonavir, indinavir, atazanavir, and efavirenz in mouse serum and tissues.

Animal pharmacokinetic and tissue distribution assays of antiretroviral therapeutic drugs require accurate drug quantification in biological fluids and tissues. Here we report a simple, rapid, and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of commonly used antiretroviral drugs ritonavir (RTV), indinavir (IDV), atazanavir (ATV), and efavirenz (EFV) in mouse serum and tissues (liver, kidney, lung, and spleen). These antiretroviral drugs are currently the cornerstones of common therapeutic regimens for human immunodeficiency virus (HIV) infection. Chromatographic separation was achieved using a gradient mobile phase (5% acetonitrile in methanol and 7.5mM ammonium acetate (pH 4.0)) on an ACQUITY UPLC(®)BEH Shield RP 18 column. All compounds eluted within a 7 min run time. Lopinavir was used as an internal standard. Detection was achieved by dual positive and negative ionization modes on a quadrupole linear ion trap hybrid mass spectrometer with an electrospray ionization (ESI) source. The dynamic range was 0.2-1000 ng/mL for RTV, IDV, and ATV, and 0.5-1000 for EFV. The method was validated and showed high and consistent intra-day and inter-day accuracy and precision for all analytes. This method is used to support the preclinical development studies of targeted- and sustained-release combination ART (nanoART). The current data demonstrate a 1.5-4 fold increase in serum and tissue AUC of nanoformulated ATV, RTV, and EFV administered to mice when compared to native drug. In addition, the tested formulation enhanced exposure of the same anti-HIV drugs in mouse tissues.

The potential of HIV-1 nanotherapeutics: from in vitro studies to clinical trials

Since its discovery almost three decades ago, HIV-1 has grown into the most aggressive pandemic of modern time. Following the implementation of combination antiretroviral therapy, the pathological outcome of HIV infection has substantially improved. However, combination antiretroviral therapy is limited by several factors including, long-term toxicity, serious side effects and complex dosing regimens, and so on. In this regard, researchers have directed their attention toward enhancing current treatment strategies and/or developing alternative HIV-1 therapeutics. In recent years, this attention has fixated on nanomedicine-based anti-HIV therapeutics (HIV-1 nanotherapeutics). In the present study, we have reviewed several HIV-1 nanotherapeutics that have shown success at the preclinical level and/or Phase I/II clinical trials. We also discuss the possible benefits of these nanomedicine-based approaches and their future outlook.