Tot Bui

Lipid-drug association enhanced HIV-1 protease inhibitor indinavir localization in lymphoid tissues and viral load reduction: A proof of concept study in HIV-2287-infected macaques

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 (t1/2&bgr; = 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.

In Vitro-to-in Vivo Prediction of P-glycoprotein-Based Drug Interactions at the Human and Rodent Blood-Brain Barrier

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 (EC50) 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 EC50 of CsA (0.6 μM) for P-gp inhibition was virtually the same as our previously determined in vivo unbound EC50 at the rat BBB (0.5 μM). Moreover, at 2.8 μM CsA (total blood concentration), our in vitro data predicted an increase of 129% in [11C]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.

MDR1 G1199A polymorphism alters permeability of HIV protease inhibitors across P-glycoprotein-expressing epithelial cells

Objective:To evaluate the impact of the human multidrug resistance gene (MDR1) G1199A polymorphism (amino acid change Ser400Asn) on P-glycoprotein (P-gp)-dependent transepithelial permeability and uptake kinetics of HIV protease inhibitors (PI), by using recombinant epithelial cells expressing wild-type MDR1 (MDR1wt) or the G1199A variant (MDR11199A). Methods:Using a recombinant expression system developed previously, the transepithelial permeability and uptake kinetic parameters of five PI, amprenavir, indinavir, lopinavir, ritonavir, and saquinavir were estimated across polarized epithelial cells. Results:For all PI, the transepithelial permeability ratio (basolateral-to-apical transport divided by apical-to-basolateral transport) was significantly greater in MDR11199A cells than MDR1wt cells: amprenavir (1.7-fold), indinavir (1.8-fold), lopinavir (1.5-fold), ritonavir (2.8-fold), and saquinavir (2.1-fold). However, the impact of G1199A on P-gp activity appeared to primarily influence drug permeability in the apical-to-basolateral direction. Kinetic analysis of ritonavir and saquinavir uptake by MDR1wt- and MDR11199A-expressing cells showed that Vmax was similar, while uptake Km was significantly higher in cells expressing the G1199A variant suggesting that alterations in P-gp-dependent efflux mediated by G1199A were due to changes in transporter affinity. Conclusions:Alterations in transepithelial permeability of HIV PI due to the G1199A polymorphism may impact oral bioavailability of PI and penetration into cells and tissues of the lymphoid and central nervous systems.

Novel Gd Nanoparticles Enhance Vascular Contrast for High-Resolution Magnetic Resonance Imaging

Background Gadolinium (Gd), with its 7 unpaired electrons in 4f orbitals that provide a very large magnetic moment, is proven to be among the best agents for contrast enhanced MRI. Unfortunately, the most potent MR contrast agent based on Gd requires relatively high doses of Gd. The Gd-chelated to diethylene-triamine-penta-acetic acid (DTPA), or other derivatives (at 0.1 mmole/kg recommended dose), distribute broadly into tissues and clear through the kidney. These contrast agents carry the risk of Nephrogenic Systemic Fibrosis (NSF), particularly in kidney impaired subjects. Thus, Gd contrast agents that produce higher resolution images using a much lower Gd dose could address both imaging sensitivity and Gd safety. Methodology/Principal Findings To determine whether a biocompatible lipid nanoparticle with surface bound Gd can improve MRI contrast sensitivity, we constructed Gd-lipid nanoparticles (Gd-LNP) containing lipid bound DTPA and Gd. The Gd-LNP were intravenously administered to rats and MR images collected. We found that Gd in Gd-LNP produced a greater than 33-fold higher longitudinal (T1) relaxivity, r1, constant than the current FDA approved Gd-chelated contrast agents. Intravenous administration of these Gd-LNP at only 3% of the recommended clinical Gd dose produced MRI signal-to-noise ratios of greater than 300 in all vasculatures. Unlike current Gd contrast agents, these Gd-LNP stably retained Gd in normal vasculature, and are eliminated predominately through the biliary, instead of the renal system. Gd-LNP did not appear to accumulate in the liver or kidney, and was eliminated completely within 24 hrs. Conclusions/Significance The novel Gd-nanoparticles provide high quality contrast enhanced vascular MRI at 97% reduced dose of Gd and do not rely on renal clearance. This new agent is likely to be suitable for patients exhibiting varying degrees of renal impairment. The simple and adaptive nanoparticle design could accommodate ligand or receptor coating for drug delivery optimization and in vivo drug-target definition in system biology profiling, increasing the margin of safety in treatment of cancers and other diseases.

Effects of garlic on cytochromes P450 2C9- and 3A4-mediated drug metabolism in human hepatocytes

Several reports suggest garlic supplements may inhibit the metabolism of cytochrome P450 (CYP) 2C9 and CYP3A4 substrates, such as warfarin and saquinavir. To characterize the effects of garlic extract on CYP2C9 and CYP3A4 enzyme activity immortalized human hepatocytes (Fa2N-4 cells) were exposed to garlic extract (0–200 μg/mL). CYP2C9 and CYP3A4 enzyme activities were evaluated in parallel with enzymatic activities, expression of respective RNA transcripts was also assessed. Exposure to increasing concentrations of garlic extract led to progressive reduction in Fa2N-4 CYP2C9 activity as detected by diclofenac hydroxylation. CYP2C9 mRNA expression also revealed a concentration-dependent reduction. Greater than 90% reduction in CYP2C9 activity was observed following four days of exposure to 50 μg/mL garlic extract. In contrast, exposure to garlic extract had no effect on the CYP3A4 enzymatic activity or RNA transcript concentration in Fa2N-4. Therefore, suppression of CYP2C9 expression and activity is a heretofore unrecognized mechanism by which garlic extract may modulate CYP activity. Exposure of hepatocytes to garlic extract may reduce the expression and activity of CYP2C9 with no detectible effects on CYP3A4.

Optimization of lipid-indinavir complexes for localization in lymphoid tissues of HIV-infected macaques.

Summary: In HIV-infected persons on highly active antiretroviral therapy, residual virus is found in lymphoid tissues. Indinavir concentrations in lymph node mononuclear cells of patients on highly active antiretroviral therapy were approximately 25% to 35% of those in blood mononuclear cells, suggesting that drug insufficiency contributes to residual virus in lymphoid tissues. Therefore, we developed novel lipid-indinavir nanoparticles targeted to lymphoid tissues. Given subcutaneously, these nanoparticles provided indinavir concentrations 250% to 2270% higher than plasma indinavir concentrations in both peripheral and visceral lymph nodes. Improved indinavir delivery was reflected in reduced viral RNA and CD4+ T-cell rebound. This study optimized lipid nanoparticle formulation with respect to indinavir in lymphoid tissues of HIV-infected macaques. Regardless of lipid characteristic tested (charge, fluidity, and steric modification), indinavir binds completely to lipid at pH 7.4 but is reversed at pH 5.5 or lower. Compared with previous formulations, nanoparticles composed of disteroyl phosphatidylcholine and methyl polyethylene glycol-disteroyl phosphatidylethanolamine (DSPC:mPEG-DSPE) provided 6-fold higher indinavir levels in lymph nodes and enhanced drug exposure in blood. Enhanced anti-HIV activity paralleled improved intracellular drug accumulation. Collectively, these data suggest that indinavir nanoparticles composed of DSPC:mPEG-DSPE provided the most effective lymphoid delivery and could maximally suppress the virus in lymphoid tissues.

Differential Disposition of Soluble and Liposome-Formulated Human Recombinant Interleukin-7: Effects on Blood Lymphocyte Population in Guinea Pigs

The effects of liposome formulation on interleukin-7 (IL-T)-dependent lymphopoietic activity was investigated based on the pharmacokinetics and tissue distribution profile of soluble and liposome-formulated recombinant human IL-7. Using 125I-IL-7, we determined the role of liposome formulation on in vivoIL-7 disposition by analyzing injection site, blood, tissue, and urinary kinetics. Following a 30- to 40-µg subcutaneous dose of soluble IL-7, most of the IL-7 was eliminated through urinary excretion within 24 hr. An equivalent subcutaneous dose of liposome-encapsulated IL-7 resulted in a peak level less than one-tenth that seen with soluble drug but produced sustained blood and urinary levels for 5 days. The bioavailability of liposome-encapsulated IL-7 was comparable to that of soluble IL-7, as determined by both blood and urinary data. Kinetic analysis of IL-7 at the subcutaneous injection site indicated that liposome encapsulation significantly reduced the rate of disappearance at the injection site. Studies with a mixture of 40% liposome-encapsulated and 60% soluble IL-7 gave an intermediate response between that of soluble IL-7 and that of liposome-encapsulated IL-7. Characterization of blood cells from IL-7-treated animals indicated that treatment with two weekly doses of mixed IL-7 liposomes (40% liposome encapsulated IL-7) significantly increased the total numbers of lymphocytes by day 14. In contrast, animals treated with soluble IL-7 on an identical dose and schedule did not produce any effect on blood lymphocytes. Collectively, liposome formulation provided a lower, but significantly sustained blood IL-7 level that enhanced IL-7 effects on blood lymphocyte numbers.

A Novel Human Multidrug Resistance Gene MDR1 Variant G571A (G191R) Modulates Cancer Drug Resistance and Efflux Transport

The human multidrug resistance gene MDR1 encodes a membrane-bound transporter P-glycoprotein (Pgp) that confers the drug resistance of cancer cells by mediating an ATP-dependent drug efflux transport. We and others have reported a number of functionally significant MDR1 variants, including G1199A and G1199T, that modulate cancer drug resistance and intracellular levels of antivirals. In this report, we describe a novel G571A variant of MDR1 detected in 6.4% of leukemia patients. Because this nucleotide modification gives rise to an amino acid change from Gly to Arg at the 191 amino acid position of Pgp, we have developed and characterized the functional affect of the G571A variant in stable, recombinant cells. Using six chemotherapeutic drugs, doxorubicin HCl, daunorubicin HCl, vinblastine sulfate, vincristine sulfate, taxanes (paclitaxel), and epipodophyllotoxin (etoposide, VP-16), we found that the MDR1571A variant selectively reduced the degree of Pgp-mediated resistance in drug-dependent manner. Although there was a minimal effect on doxorubicin and daunorubicin, the MDR1-dependent resistance on vinblastine, vincristine, paclitaxel, and etoposide was reduced by approximately 5-fold. The increased drug sensitivity in MDR1571A, compared with MDR1wt, paralleled the intracellular drug levels. These data suggest that individuals with this novel MDR1 variant, the 571A genotype, may be more sensitive to the specific anticancer drugs that are Pgp substrates.

Augmentation of cell-mediated immunotherapy against herpes simplex virus by interleukins: comparison of in vivo effects of IL-2 and IL-7 on adoptively transferred T cells

We investigated the ability of human recombinant interleukin-7 (IL-7) to enhance cytotoxic T lymphocyte (CTL) activity in vivo using mice infected with herpes simplex virus type-1 (HSV-1). IL-7 or interleukin-2 (IL-2) was administered twice daily to immune naive mice subjected to adoptive transfer of immune T cells after infection with HSV-1. The immunotherapeutic effect was measured by detecting the virus recovered from pinna. Administration of HSV-1 immune T cells to naive mice significantly increased their ability to clear the virus. Twice-daily injections of IL-7 at 200 IU provided an additional 20-fold reduction in virus load, compared with T cell therapy alone (P < 0.0005). Combining IL-2 and T cell therapy provided about a sevenfold reduction compared with T cell therapy alone (P < 0.0009). IL-7 also enhanced the antiviral effects of T cell therapy against HSV-1 through the enhancement of CD8+ CTLs, as observed with IL-2. These results indicate that IL-7 may be used adjunct to adoptive T lymphocyte therapy in modulating human viral diseases and cancer through enhanced immune T cell activities.

MDR1 (ABCB1) G1199A (Ser400Asn) polymorphism alters transepithelial permeability and sensitivity to anticancer agents

PurposeP-glycoprotein (P-gp), encoded by MDR1 (or ABCB1), is important in anticancer drug delivery and resistance. We evaluated alterations in P-gp-mediated transport of anticancer agents due to the MDR1 G1199A polymorphism.MethodsUsing stable recombinant epithelial cells expressing wild-type (MDR1wt) or G1199A (MDR11199A), anticancer drug sensitivity and transepithelial permeability were evaluated.ResultsThe recombinant cells MDR1wt and MDR11199A displayed comparable doxorubicin resistance. However, MDR11199A cells displayed greater resistance to vinblastine, vincristine, paclitaxel, and VP-16 (11-, 2.9-, 1.9-, and 2.9-fold, respectively). Alterations in transepithelial permeability paralleled these changes. Efflux of doxorubicin was similar between MDR1wt- and MDR11199A-expressing cells, while P-gp-mediated transport was greater for vinblastine and vincristine in MDR11199A cells (2.9- and 2.0-fold, respectively).ConclusionsThe occurrence and magnitude of the MDR1 G1199A effect is drug specific. Overall, the MDR1 G1199A polymorphism may impact anticancer efficacy through modulation of drug distribution and delivery to target tumor cells.