No abstract

Liposomes are spherical-enclosed membrane vesicles mainly constructed with lipids. Lipid nanoparticles are loaded with therapeutics and may not contain an enclosed bilayer. The majority of those clinically approved have diameters of 50–300 nm. The growing interest in nanomedicine has fueled lipid–drug and lipid–protein studies, which provide a foundation for developing lipid particles that improve drug potency and reduce off-target effects. Integrating advances in lipid membrane research has enabled therapeutic development. At present, about 600 clinical trials involve lipid particle drug delivery systems. Greater understanding of pharmacokinetics, biodistribution, and disposition of lipid–drug particles facilitated particle surface hydration technology (with polyethylene glycol) to reduce rapid clearance and provide sufficient blood circulation time for drug to reach target tissues and cells. Surface hydration enabled the liposome-encapsulated cancer drug doxorubicin (Doxil) to gain clinical approval in 1995. Fifteen lipidic therapeutics are now clinically approved. Although much research involves attaching lipid particles to ligands selective for occult cells and tissues, preparation procedures are often complex and pose scale-up challenges. With emerging knowledge in drug target and lipid–drug distribution in the body, a systems approach that integrates knowledge to design and scale lipid–drug particles may further advance translation of these systems to improve therapeutic safety and efficacy.

Indocyanine green (ICG) is a near-infrared (NIR) contrast agent commonly used for in vivo cardiovascular and eye imaging. For medical diagnosis, ICG is limited by its aqueous instability, concentration-dependent aggregation, and rapid degradation. To overcome these limitations, scientists have formulated ICG in various liposomes, which are spherical lipid membrane vesicles with an aqueous core. Some encapsulate ICG, while others mix it with liposomes. There is no clear understanding of lipid–ICG interactions. Therefore, we investigated lipid–ICG interactions by fluorescence and photon correlation spectroscopy. These data were used to design stable and maximally fluorescent liposomal ICG nanoparticles for NIR optical imaging of the lymphatic system. We found that ICG binds to and is incorporated completely and stably into the lipid membrane. At a lipid:ICG molar ratio of 250:1, the maximal fluorescence intensity was detected. ICG incorporated into liposomes enhanced the fluorescence intensity that could be detected across 1.5 cm of muscle tissue, while free ICG only allowed 0.5 cm detection. When administered subcutaneously in mice, lipid-bound ICG in liposomes exhibited a higher intensity, NIR image resolution, and enhanced lymph node and lymphatic vessel visualization. It also reduced the level of fluorescence quenching due to light exposure and degradation in storage. Lipid-bound ICG could provide additional medical diagnostic value with NIR optical imaging for early intervention in cases of lymphatic abnormalities.

Analysis of indinavir levels in HIV-positive patients indicated that drug concentrations in lymph node mononuclear cells (LNMCs) were about 25-35% of mononuclear cells in blood. To enhance lymphatic delivery of anti-HIV drugs, a novel drug delivery strategy was designed consisting of lipid-associated indinavir (50-80 nm in diameter) complexes in suspension for subcutaneous (SC) injection. Due to the pH-dependent lipophilicity of indinavir, practically all the drug molecules are incorporated into lipid phase when formulated at pH 7.4 and 5:1 lipid-to-drug (m/m) ratio. At pH 5.5, about 20% of drugs were found in lipid-drug complexes. Effects of lipid association on the time course of plasma indinavir concentrations were determined in macaques (Macaca nemestrina) administered with either soluble or lipid-associated formulation of indinavir (10 mg/kg, SC). Results yielded about a 10-fold reduction in peak plasma concentration and a 6-fold enhancement in terminal half-life (t1/2beta = 12 vs. 2 hours). In addition, indinavir concentrations in both peripheral and visceral lymph nodes were 250-2270% higher than plasma (compared with <35% with soluble lipid-free drug administration in humans). Administration of lipid-associated indinavir (20 mg/kg daily) to HIV-2287-infected macaques (at 30-33 weeks after infection) resulted in significantly reduced viral RNA load and increased CD4 T cell number concentrations. Collectively, these data indicate that lipid association greatly enhances delivery of the anti-HIV drug indinavir to lymph nodes at levels that cannot be achieved with soluble drug, provides significant virus load reduction, and could potentially reverse CD4 T cell depletion due to HIV infection.

No abstract

Objective:The aim of the present study was to determine whether a combination of anti-HIV drugs – tenofovir (TFV), lopinavir (LPV) and ritonavir (RTV) – in a lipid-stabilized nanosuspension (called TLC-ART101) could enhance and sustain intracellular drug levels and exposures in lymph node and blood cells above those in plasma.Design:Four macaques were given a single dose of TLC-ART101 subcutaneously. Drug concentrations in plasma and mononuclear cells of the blood (PBMCs) and lymph nodes (LNMCs) were analysed using a validated combination LC-MS/MS assay.Results:For the two active drugs (TFV, LPV), plasma and PBMC intracellular drug levels persisted for over 2 weeks; PBMC drug exposures were three- to four-fold higher than those in plasma. Apparent terminal half-lives (t1/2) of TFV and LPV were 65.3 and 476.9 h in plasma, and 169.1 and 151.2 h in PBMCs. At 24 and 192 h, TFV and LPV drug levels in LNMCs were up to 79-fold higher than those in PBMCs. Analysis of PBMC intracellular TFV and its active metabolite TFV-diphosphate (TFV-DP) indicated that intracellular exposures of total TFV and TFV-DP were markedly higher and persisted longer than in humans and macaques dosed with oral TFV prodrugs, tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF).Conclusions:A simple, scalable three-drug combination, lipid-stabilized nanosuspension exhibited persistent drug levels in cells of lymph nodes and the blood (HIV host cells) and in plasma. With appropriate dose adjustment, TLC-ART101 may be a useful HIV treatment with a potential to impact residual virus in lymph nodes.

ABSTRACT:In vitro inhibition of P-glycoprotein (P-gp) expressed in cells is routinely used to predict the potential of in vivo P-gp drug interactions at the human blood-brain barrier (BBB). The accuracy of such predictions has not been confirmed because methods to quantify in vivo P-gp drug interactions at the human BBB have not been available. With the development of a noninvasive positron emission topography (PET) imaging method by our laboratory to determine P-gp-based drug interactions at the human BBB, an in vitro-in vivo comparison is now possible. Therefore, we developed a high throughput cell-based assay to determine the potential of putative P-gp inhibitors [including cyclosporine A (CsA)] to inhibit (EC 50 ) the efflux of verapamil-bodipy, a model P-gp substrate. LLCPK1-MDR1 cells, expressing recombinant human P-gp, or control cells lacking P-gp (LLCPK1) were used in our assay. Using this assay, quinine, quinidine, CsA, and amprenavir were predicted to be the most potent P-gp inhibitors in vivo at their respective therapeutic maximal unbound plasma concentrations. The in vitro EC 50 of CsA (0.6 M) for P-gp inhibition was virtually the same as our previously determined in vivo unbound EC 50 at the rat BBB (0.5 M). Moreover, at 2.8 M CsA (total blood concentration), our in vitro data predicted an increase of 129% in [11 C]verapamil distribution into the human brain, a value similar to that observed by us (79%) using PET. These data suggest that our high throughput cell assay has the potential to accurately predict P-gp drug interactions at the human BBB.The in vivo importance of P-glycoprotein (P-gp) at the BBB has been well demonstrated by studies in mdr1a/b (Ϫ/Ϫ) mice. For example, compared with the wild-type mouse, in the mdr1a/b (Ϫ/Ϫ) mice, the brain/plasma concentration ratio (or the brain uptake) of the anti-human immunodeficiency virus protease inhibitors is increased 7-to 36-fold and anti-cancer taxanes, paclitaxel, or docetaxel are increased 6-to 28-fold, whereas that of verapamil is increased 8.5-fold (Endres et al., 2006). Similar data have been obtained in mice and rats where P-gp has been chemically ablated with selective inhibitors of P-gp such as PSC833, GF120918, and LY335979 (Lin and Yamazaki, 2003;Endres et al., 2006). For example, the brain/plasma ratio of verapamil is increased 24.1-fold when the rat is pretreated with cyclosporine A (CsA) (Hendrikse and Vaalburg, 2002). Based on these data and others, it has been widely postulated that P-gp plays a vital role in limiting drug distribution at the human BBB and that P-gp-based drug interactions will result in a profound increase in brain concentrations of the affected drugs and, therefore, their CNS efficacy or toxicity.Although rodent studies make a compelling case for the importance of P-gp at the BBB in the CNS distribution of drugs, their ability to predict the magnitude of P-gp-based drug interactions at the human BBB has not been investigated. Due to safety and ethical reasons, it has not been possible to measure in vivo human BB...

The significance of the human multidrug resistance gene (MDR1) G1199A polymorphism, resulting in a Ser400Asn modification in P-glycoprotein (P-gp), remains unclear. We have developed stable recombinant LLC-PK1 epithelial cells expressing either MDR1 wt or MDR1 1199 to evaluate functional consequences of G1199A [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide]. P-gp activity observed in MDR1 wt and MDR1 1199 cells was completely inhibited in the presence of the specific P-gp inhibitor GF120918. Comparable expression of mRNA and protein in the MDR1-expressed cells and correct localization of P-gp in the apical membrane of recombinant cells was verified. . Therefore, the G1199A polymorphism alters the efflux and transepithelial permeability of a fluorescent substrate and sensitivity to select cytotoxic agents, which may influence drug disposition and therapeutic efficacy of some P-gp substrates.The human multidrug resistance gene (MDR1) encodes a 170-kDa integral membrane protein that mediates ATP-dependent substrate efflux. The protein product, P-glycoprotein (P-gp), a member of the ATP-binding cassette superfamily of transporters, resides in the plasma membrane and functions as an efflux transporter of a variety of natural compounds and lipophilic xenobiotics (for review, see Lin, 2003). Although the contribution of P-gp in multidrug resistance for cancer chemotherapy is well documented, the role of P-gp in drug disposition is not fully understood and has continued to generate significant debate. P-gp mediates the energy-dependent efflux of a broad range of xenobiotics in epithelial tissues throughout the human body, including the intestinal mucosa, liver canalicular membrane, kidney proximal tubules, blood-brain barrier, and placenta (Schinkel, 1997).P-gp efflux may, therefore, act to decrease intestinal absorption, enhance biliary excretion and renal tubular secretion, and limit drug distribution to the fetus and brain. Because P-gp is found in tissues important in drug disposition, variation in expression and function of P-gp due to genetic polymorphisms of MDR1 may influence pharmacokinetics and, in turn, pharmacodynamics.Recent progress has been made in identifying genetic polymorphisms in the MDR1 gene in normal human tissues. The first major screen of the MDR1 gene in 188 healthy Caucasian subjects, identified 15 single nucleotide polymorphisms; however, only one, a C 3 T transition at nucleotide position 3435 (C3435T), was shown to correlate with decreased intestinal P-gp expression and digoxin exposure in vivo (Hoffmeyer et al., 2000). Because the C3435T polymorphism in exon 26 is a synonymous polymorphism that does not modify the amino acid sequence of P-gp, several investigators have searched for clues to the significance of C3435T. Another study reported that C3435T is linked to a nonsynonymous G2677T polymorphism, resulting in an alanine-to-serine transition at amino acid 893, and another synonymous SNP, C1236T (Kim ...