Rodney J. Y. Ho

Emerging Research and Clinical Development Trends of Liposome and Lipid Nanoparticle Drug Delivery Systems

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.

(Section A: Molecular, Structural, and Cellular Biology of Drug Transporters) The Role of MDR1 Genetic Polymorphisms in Interindividual Variability in P-glycoprotein Expression and Function

The human multidrug resistance gene (MDR1), spanning greater than 200 kb, encodes for the ATP-dependent membrane efflux transporter, P-glycoprotein (Pgp). Significant progress has been made in the discovery of MDR1 polymorphisms and the assessment of allelic frequencies. The search for key genetic determinants that predispose individuals to drugs that are substrates or inhibitors of Pgp has just begun. Reports in the literature, particularly focusing on the C3435T polymorphism, have provided discordant results with respect to functional modification in vitro, and Pgp expression and disposition of probe drugs in vivo. Due to the large size of the MDR1 gene, genotyping based on individual single nucleotide polymorphism (SNPs) analysis is not sufficient to predict functional consequences. Strong linkage disequilibrium has been detected between several MDR1 polymorphisms, and discrepancies in the literature may be due to the focus on the influence of single nucleotide variations instead of on linked nucleotide variations. Multiple SNPs found on the same chromosome are assigned to a specific haplotype, and some attempts have been made to determine the role of MDR1 haplotypes in Pgp variability. Most of the data for MDR1 haplotype have been predicted based on computational or mathematical models. However, molecular haplotyping techniques, analysis of linkages on the same chromosome directly by biophysical and biochemical means, may be needed to characterize haplotypes in individuals with a highly polymorphic and large gene like MDR1. Haplotype identification may prove to be vital in identifying the functional significance of MDR1 polymorphisms on disease susceptibility and drug disposition.

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.

Characterization of the humanMDR1 gene

P-glycoprotein (Pgp), an ATP-dependent efflux transporter that protects the body from environmental toxins and xenobiotics, is encoded by the humanMDR1 gene. HumanMDR1 is located on chromosomal region 7q21. Although several different single nucleotide polymorphisms were shown to influence Pgp expression and activity, the reported length of theMDR1 gene in Genbank and other databases continues to evolve and varies between 6.3 kilobases (kb) and 210 kb. With DNA derived from human cell lines and tissues, we have characterized theMDR1 genomic sequence to be 209 kb.

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.

Inhibition of P-glycoprotein activity at the primate blood-brain barrier increases the distribution of nelfinavir into the brain but not into the cerebrospinal fluid.

P-glycoprotein (P-gp) expression at the rodent blood-brain barrier (BBB) limits the central nervous system (CNS) distribution of anti-human immunodeficiency virus (HIV) protease inhibitors (PIs). However, it is not clear whether P-gp activity at the human BBB is as effective as that in rodents in preventing the distribution of PIs into the CNS. If it is, inhibition of P-gp at the human BBB could increase the distribution of the PIs into the CNS and, therefore, their efficacy against HIV-associated dementia. Because the distribution of the PIs into the human brain cannot be directly measured, we conducted studies in a more representative animal, the nonhuman primate. Specifically we investigated the distribution of nelfinavir (a PI and a P-gp substrate; 6 mg/kg i.v.) into the brain and cerebrospinal fluid (CSF) of nonhuman primates (cynomolgus monkeys, Macaca fascicularis) in the presence and absence of the potent and selective P-gp inhibitor, zosuquidar, and whether changes in brain nelfinavir concentration, after inhibition of P-gp, paralleled those in the CSF. Our data indicate that nelfinavir has poor penetration into the macaques brain and CSF, and P-gp inhibition at the BBB by zosuquidar enhanced the distribution of nelfinavir into the brain by 146-fold. However, the concentration of nelfinavir in the CSF was unaffected by coadministration of zosuquidar (p > 0.05). In conclusion, P-gp inhibition at the nonhuman primate BBB significantly enhanced the distribution of nelfinavir into the brain, and this effect was not observed in the CSF. Therefore, as is common in human studies investigating P-gp inhibition at the BBB, CSF concentration of a drug should not be used as a surrogate marker for brain drug concentration.

Interactions of indocyanine green and lipid in enhancing near-infrared fluorescence properties: the basis for near-infrared imaging in vivo.

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.

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.