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

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.

Macrophage folate receptor-targeted antiretroviral therapy facilitates drug entry, retention, antiretroviral activities and biodistribution for reduction of human immunodeficiency virus infections.

Macrophages serve as vehicles for the carriage and delivery of polymer-coated nanoformulated antiretroviral therapy (nanoART). Although superior to native drug, high drug concentrations are required for viral inhibition. Herein, folate-modified ritonavir-boosted atazanavir (ATV/r)-encased polymers facilitated macrophage receptor targeting for optimizing drug dosing. Folate coating of nanoART ATV/r significantly enhanced cell uptake, retention and antiretroviral activities without altering cell viability. Enhanced retentions of folate-coated nanoART within recycling endosomes provided a stable subcellular drug depot. Importantly, up to a five-fold enhanced plasma and tissue drug levels followed folate-coated formulation injection in mice. Folate polymer encased ATV/r improves nanoART pharmacokinetics bringing the technology one step closer to human use. FROM THE CLINICAL EDITOR: This team of authors describes a novel method for macrophage folate receptor-targeted antiretroviral therapy. Atazanvir entry, retention, and antiretroviral activities were superior using the presented method, and so was its biodistribution, enabling a more efficient way to address human immunodeficiency virus infections, with a hoped for clinical application in the near future.

Preclinical pharmacokinetics and tissue distribution of long-acting nanoformulated antiretroviral therapy.

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.

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-2Rgc (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.

Pharmacotoxicology of monocyte-macrophage nanoformulated antiretroviral drug uptake and carriage.

Limitations inherent to antiretroviral therapy (ART) in its pharmacokinetic properties remain despite over 15 years of broad use. Our laboratory has pioneered a means to improve ART delivery through monocyte-macrophage carriage of nanoformulated drug-encapsulated particles (nanoART). To this end, our prior works sought to optimize nanoART size, charge, and physical properties for cell uptake and antiretroviral activities. To test the functional consequences of indinavir, ritonavir, and efavirenz formulations we investigated relationships between human monocyte and macrophage cytotoxicities and nanoART dose, size, surfactant, and preparation. Wet-milled particles were more cytotoxic to monocytes-macrophages than those prepared by homogenization; with concurrent induction of tumor necrosis factor-alpha. Interestingly, pure suspensions of indinavir and ritonavir at 0.5 mM, and efavirenz at 0.1 mM and 0.5 mM also proved cytotoxic. Individual surfactants and formulated fluconazole neither affected cell function or viability. Although nanoART did not alter brain tight junction proteins ZO-2 and occludin, 0. 5mM ritonavir formulations did alter brain transendothelial electric resistance. These results underscore the potential importance of evaluating the physicochemical and functional properties of nanoART before human evaluations.

Analyses of nanoformulated antiretroviral drug charge, size, shape and content for uptake, drug release and antiviral activities in human monocyte-derived macrophages.

Long-term antiretroviral therapy (ART) for human immunodeficiency virus type one (HIV-1) infection shows limitations in pharmacokinetics and biodistribution while inducing metabolic and cytotoxic aberrations. In turn, ART commonly requires complex dosing schedules and leads to the emergence of viral resistance and treatment failures. We posit that the development of nanoformulated ART could preclude such limitations and affect improved clinical outcomes. To this end, we wet-milled 20 nanoparticle formulations of crystalline indinavir, ritonavir, atazanavir, and efavirenz, collectively referred to as "nanoART," then assessed their performance using a range of physicochemical and biological tests. These tests were based on cell-nanoparticle interactions using monocyte-derived macrophages and their abilities to uptake and release nanoformulated drugs and affect viral replication. We demonstrate that physical characteristics such as particle size, surfactant coating, surface charge, and most importantly shape are predictors of cell uptake and antiretroviral efficacy. These studies bring this line of research a step closer to developing nanoART that can be used in the clinic to affect the course of HIV-1 infection.

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.

Methods development for blood borne macrophage carriage of nanoformulated antiretroviral drugs.

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To this end, our laboratory has applied the principles of nanomedicine to simplify ART regimens and as such reduce toxicities while improving compliance and drug pharmacokinetics. Simple and reliable methods for manufacturing nanoformulated ART (nanoART) are shown. Particles of pure drug are encapsulated by a thin layer of surfactant lipid coating and produced by fractionating larger drug crystals into smaller ones by either wet milling or high-pressure homogenization. In an alternative method free drug is suspended in a droplet of a polymer. Herein, drug is dissolved within a polymer then agitated by ultrasonication until individual nanosized droplets are formed. Dynamic light scattering and microscopic examination characterize the physical properties of the particles (particle size, charge and shape). Their biologic properties (cell uptake and retention, cytotoxicity and antiretroviral efficacy) are determined with human monocyte-derived macrophages (MDM). MDM are derived from human peripheral blood monocytes isolated from leukopacks using centrifugal elutriation for purification. Such blood-borne macrophages may be used as cellular transporters for nanoART distribution to human immunodeficiency virus (HIV) infected organs. We posit that the repackaging of clinically available antiretroviral medications into nanoparticles for HIV-1 treatments may improve compliance and positively affect disease outcomes.

Registered bioimaging of nanomaterials for diagnostic and therapeutic monitoring.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Layer-by-Layer-Coated Gelatin Nanoparticles as a Vehicle for Delivery of Natural Polyphenols.

Natural polyphenols with previously demonstrated anticancer potential, epigallocatechin gallate (EGCG), tannic acid, curcumin, and theaflavin, were encased into gelatin-based 200 nm nanoparticles consisting of a soft gel-like interior with or without a surrounding LbL shell of polyelectrolytes (polystyrene sulfonate/polyallylamine hydrochloride, polyglutamic acid/poly-l-lysine, dextran sulfate/protamine sulfate, carboxymethyl cellulose/gelatin, type A) assembled using the layer-by-layer technique. The characteristics of polyphenol loading and factors affecting their release from the nanocapsules were investigated. Nanoparticle-encapsulated EGCG retained its biological activity and blocked hepatocyte growth factor (HGF)-induced intracellular signaling in the breast cancer cell line MBA-MD-231 as potently as free EGCG.

(-)-Epigallocatechin gallate/gelatin layer-by-layer assembled films and microcapsules.

A new type of protein/polyphenol microcapsules on the basis of naturally occurring polyphenol (-)-epigallocatechin gallate (EGCG) and gelatin, type A, was obtained using the layer-by-layer (LbL) assembly method. The microcapsules show a more pronounced dependence of permeability on molecular weight of permeating substances than commonly used polyallylamine/polystyrene sulfonate capsules. The regularities of EGCG adsorption in alternation with type A and B gelatins have been investigated using quartz crystal microbalance and electrophoretic mobility measurements on microparticles and found to be dependent on gelatin properties. EGCG in the LbL assemblies retains its antioxidant activity. The kinetics of the reaction of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) cation-radicals with films consisting of 1-10 gelatin/EGCG bilayers is affected by film structure. The EGCG content in the protein/polyphenol film material is as high as 30% w/w. Encapsulation of EGCG via its alternated adsorption with gelatins can be a perspective way to new formulations containing the polyphenol for drug delivery applications.

Polypeptide multilayer films: role of molecular structure and charge.

The role of molecular structure, charge, and hydrophobicity in polyelectrolyte layer-by-layer assembly (LbL) of thin films has been studied using the model polypeptides poly-L-glutamatic acid (PLGA) and poly-L-lysine (PLL), quartz crystal microbalance (QCM), and circular dichroism spectroscopy (CD). The adsorption behavior of PLGA and PLL has been compared with the structure of these molecules in aqueous solution under the same conditions. The data show that the deposition of polypeptide per adsorption step scales with average secondary structure content, whether alpha helix or beta sheet. This is contrary to the expectation based on the view that hydrogen bonds are crucial to polypeptide film assembly, because secondary structure formation in a polypeptide reduces its intermolecular hydrogen-bonding potential. The data also show that polypeptide adsorption scales with ionic strength and chain length. Taken together, the results increase knowledge of polypeptide-based LbL thin film fabrication and will help to provide a firmer foundation for the use of natural or designed polypeptides in LbL.