Showing papers in "Molecular Pharmaceutics in 2008"

Factors Affecting the Clearance and Biodistribution of Polymeric Nanoparticles

Abstract: Nanoparticle (NP) drug delivery systems (5−250 nm) have the potential to improve current disease therapies because of their ability to overcome multiple biological barriers and releasing a therapeutic load in the optimal dosage range. Rapid clearance of circulating nanoparticles during systemic delivery is a critical issue for these systems and has made it necessary to understand the factors affecting particle biodistribution and blood circulation half-life. In this review, we discuss the factors which can influence nanoparticle blood residence time and organ specific accumulation. These factors include interactions with biological barriers and tunable nanoparticle parameters, such as composition, size, core properties, surface modifications (pegylation and surface charge), and finally, targeting ligand functionalization. All these factors have been shown to substantially affect the biodistribution and blood circulation half-life of circulating nanoparticles by reducing the level of nonspecific uptake, de...

Pharmacokinetics and biodistribution of nanoparticles.

Abstract: Nanoparticles show their promise for improving the efficacy of drugs with a narrow therapeutic window or low bioavailability, such as anticancer drugs and nucleic acid-based drugs. The pharmacokinetics (PK) and tissue distribution of the nanoparticles largely define their therapeutic effect and toxicity. Chemical and physical properties of the nanoparticles, including size, surface charge, and surface chemistry, are important factors that determine their PK and biodistribution. The intracellular fate of the nanoparticles after cellular internalization that affects the drug bioavailability is also discussed. Strategies for overcoming barriers for intracellular delivery and drug release are presented. Finally, future directions for improving the PK of nanoparticles and perspectives in the field are discussed.

Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects on nanoparticle biodistribution.

Abstract: Nanoparticles have unique physicochemical properties which make them promising platforms for drug delivery. However, immune cells in the bloodstream (such as monocytes, platelets, leukocytes, and dendritic cells) and in tissues (such as resident phagocytes) have a propensity to engulf and eliminate certain nanoparticles. A nanoparticle’s interaction with plasma proteins (opsonins) and blood components (via hemolysis, thrombogenicity and complement activation) may influence uptake and clearance and hence potentially affect distribution and delivery to the intended target sites. Nanoparticle uptake by the immune cells is influenced by many factors. Different nanoparticles have been shown to act on different pathways, while various characteristics/properties also affect which pathway is employed for particle internalization. Nanoparticle protein binding occurs almost instantaneously once the particle enters biological medium, and the physical properties of such a particle−protein complex are often different ...

Biodistribution, clearance, and biocompatibility of iron oxide magnetic nanoparticles in rats.

Abstract: It is essential to determine the biodistribution, clearance, and biocompatibility of magnetic nanoparticles (MNPs) for in vivo biomedical applications to ensure their safe clinical use. We have studied these aspects with our novel iron oxide MNP formulation, which can be used as a magnetic resonance imaging (MRI) agent and a drug carrier system. Changes in serum and tissue iron levels were analyzed over 3 weeks after intravenous administration of MNPs to rats. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP) levels, and total iron-binding capacity (TIBC) were also measured with time to assess the effect of MNPs on liver function. Selected tissues were also analyzed for oxidative stress and studied histologically to determine biocompatibility of MNPs. Serum iron levels gradually increased for up to 1 week but levels slowly declined thereafter. Biodistribution of iron in various body tissues changed with time but greater fraction of the injected iron localized in the liver and spleen than in the brain, heart, kidney, and lung. Magnetization measurements of the liver and spleen samples showed a steady decrease over 3 weeks, suggesting particle degradation. Serum showed a transient increase in ALT, AST, AKP levels, and TIBC over a period of 6-24 h following MNP injection. The increase in oxidative stress was tissue dependent, reaching a peak at approximately 3 days and then slowly declining thereafter. Histological analyses of liver, spleen, and kidney samples collected at 1 and 7 days showed no apparent abnormal changes. In conclusion, our MNPs did not cause long-term changes in the liver enzyme levels or induce oxidative stress and thus can be safely used for drug delivery and imaging applications.

Hydroxypropyl methylcellulose acetate succinate-based spray-dried dispersions: an overview.

Abstract: Spray-dried dispersions (SDDs) of low-solubility drugs have been prepared using the polymer hydroxypropyl methylcellulose acetate succinate (HPMCAS). For a variety of drug structures, these SDDs provide supersaturation in in vitro dissolution determinations and large bioavailability increases in vivo. In bile-salt/lecithin in vitro solutions, these SDDs provide amorphous drug/polymer colloids and an increased concentration of free drug and drug in micelles relative to crystalline or amorphous drug. As dry powders, the SDDs are a single amorphous phase in which the drug remains amorphous and dispersed and does not crystallize over storage times relevant for practical drug products. A melting temperature (Tm)/glass-transition temperature (Tg) (K/K) versus log P map for 139 compounds formulated as SDDs provides a perspective on an appropriate formulation strategy for low-solubility drugs with various physical properties.

Anticancer therapeutics: targeting macromolecules and nanocarriers to hyaluronan or CD44, a hyaluronan receptor.

Abstract: The complex system involved in the synthesis, degradation and binding of the high molecular weight glycosaminoglycan hyaluronic acid (hyaluronan or HA) provides a variety of structures that can be exploited for targeted cancer therapy. In many cancers of epithelial origin there is an upregulation of CD44, a receptor that binds HA. In other cancers, HA in the tumor matrix is overexpressed. Both CD44 on cancer cells and HA in the matrix have been targets for anticancer therapy. Even though CD44 is expressed in normal epithelial cells and HA is part of the matrix of normal tissues, selective targeting to cancer is possible. This is because macromolecular carriers predominantly extravasate into the tumor and not normal tissue; thus CD44-HA targeted carriers administered intravenously localize preferentially into tumors. Anti-CD44 antibodies have been used in patients to deliver radioisotopes or mertansine for treatment of CD44 expressing tumors. In early phase clinical trials, patients with breast or head and neck tumors treated with anti-CD44 conjugates experienced stabilized disease. A dose-limiting toxicity was associated with distribution of the antibody-drug conjugate to the skin, a site in the body with a high level of CD44. HA has been used as a drug carrier and a ligand on liposomes or nanoparticles to target drugs to CD44 overexpressing cells. Drugs can be attached to HA via the carboxylate on the glucuronic acid residue, the hydroxyl on the N-acetylglucosamine or the reducing end which are located on a repeating disaccharide. Drugs delivered in HA-modified liposomes exhibited excellent antitumor activity both in vitro and in murine tumor models. The HA matrix is also a potential target for anticancer therapies. By manipulating the interaction of HA with cell surface receptors, either by degrading it with hyaluronidase or by interfering with CD44-HA interactions using soluble CD44 proteins, tumor progression was blocked. Finally, cytotoxic drugs or prodrug converting enzymes can be attached to the HA matrix to generate a cytotoxic fence around the tumor. This review describes how the complex interplay among cancer biology, the CD44-HA interaction, drug carriers and drug targeting has been used to improve anticancer therapies. As these approaches evolve, they hold forth the prospect of significantly improved targeted anticancer treatments.

Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms.

Abstract: Terpenoids represent a diverse class of molecules that provide a wealth of opportunities to address many human health and societal issues. The expansive array of structures and functionalities that have been evolved in nature provide an excellent pool of molecules for use in human therapeutics. While this class of molecules has members with therapeutic properties including anticancer, antiparasitic, antimicrobial, antiallergenic, antispasmodic, antihyperglycemic, anti-inflammatory, and immunomodulatory properties, supply limitations prevent the large scale use of some molecules. Many of these molecules are only found in ppm levels in nature thus requiring massive harvesting to obtain sufficient amounts of the drug. Synthetic biology and metabolic engineering provide innovative approaches to increase the production of the desired molecule in the native organism, and most importantly, transfer the biosynthetic pathways to other hosts. Microbial systems are well studied, and genetic manipulations allow the optimization of microbial metabolisms for the production of common terpenoid precursors. Using a host of tools, unprecedented advancements in the large scale production of terpenoids have been achieved in recent years. Identification of limiting steps and pathway regulation, coupled with design strategies to minimize terpenoid byproducts wih a high flux to the desired biosynthetic pathways, have yielded greater than 100-fold improvements in the production of a range of terpenoids. This review focuses on the biodiversity of terpenoids, the biosynthetic pathways involved, and engineering efforts to maximize the production through these pathways.

3-D tumor model for in vitro evaluation of anticancer drugs

Abstract: The efficacy of potential anticancer drugs during preclinical development is generally tested in vitro using cancer cells grown in monolayer; however, a significant discrepancy in their efficacy is observed when these drugs are evaluated in vivo. This discrepancy, in part, could be due to the three-dimensional (3-D) nature of tumors as compared to the two-dimensional (2-D) nature of monolayer cultures. Therefore, there is a need for an in vitro model that would mimic the 3-D nature of tumors. With this objective, we have developed surface-engineered, large and porous biodegradable polymeric microparticles as a scaffold for 3-D growth of cancer cells. Using the MCF-7 cell line as model breast cancer cells, we evaluated the antiproliferative effect of three anticancer drugs: doxorubicin, paclitaxel and tamoxifen in 3-D model vs in 2-D monolayer. With optimized composition of microparticles and cell culture conditions, a density of 4.5 × 106 MCF-7 cells/mg of microparticles, which is an 18-fold increase from...

Solid state amorphization of pharmaceuticals.

Abstract: Amorphous solids are conventionally formed by supercooling liquids or by concentrating noncrystallizing solutes (spray-drying and freeze-drying). However, a lot of pharmaceutical processes may also directly convert compounds from crystal to noncrystal which may have desired or undesired consequences for their stability. The purpose of this short review paper is (i) to illustrate the possibility to amorphize one compound by several different routes (supercooling, dehydration of hydrate, milling, annealing of metastable crystalline forms), (ii) to examine factors that favor crystal to glass rather than crystal to crystal transformations, (iii) to discuss the role of possible amorphous intermediates in solid−solid conversions induced by milling, (iv) to address the issue of chemical stability in the course of solid state amorphization, (v) to discuss the nature of the amorphous state obtained by the nonconventional routes, (vi) to show the effect of milling conditions on glasses properties, and (vii) to atte...

Nonribosomal peptide synthetases involved in the production of medically relevant natural products.

Abstract: Natural products biosynthesized wholly or in part by nonribosomal peptide synthetases (NRPSs) are some of the most important drugs currently used clinically for the treatment of a variety of diseases Since the initial research into NRPSs in the early 1960s, we have gained considerable insights into the mechanism by which these enzymes assemble these natural products This review will present a brief history of how the basic mechanistic steps of NRPSs were initially deciphered and how this information has led us to understand how nature modified these systems to generate the enormous structural diversity seen in nonribosomal peptides This review will also briefly discuss how drug development and discovery are being influenced by what we have learned from nature about nonribosomal peptide biosynthesis

Strain improvement of recombinant Escherichia coli for efficient production of plant flavonoids.

Abstract: Plant flavonoid polyphenols continue to find increasing pharmaceutical and nutraceutical applications; however their isolation, especially of pure compounds, from plant material remains an underlying challenge. In the past Escherichia coli, one of the most well-characterized microorganisms, has been utilized as a recombinant host for protein expression and heterologous biosynthesis of small molecules. However, in many cases the expressed protein activities and biosynthetic efficiency are greatly limited by the host cellular properties, such as precursor and cofactor availability and protein or product tolerance. In the present work, we developed E. coli strains capable of high-level flavonoid synthesis through traditional metabolic engineering techniques. In addition to grafting the plant biosynthetic pathways, the methods included engineering of an alternative carbon assimilation pathway and the inhibition of competitive reaction pathways in order to increase intracellular flavonoid backbone precursors a...

Pharmaceutically Active Natural Product Synthesis and Supply via Plant Cell Culture Technology

Abstract: The chemical diversity of plant-derived natural products allows them to function in a multitude of ways including flavor enhancers, agricultural chemicals, and importantly, human medicinals Supply of pharmaceutically active natural products is often a challenge due to the slow growing nature of some species, low yields found in nature, and unpredictable variability in accumulation Several production options are available including natural harvestation, total chemical synthesis, semisynthesis from isolated precursors, and expression of plant pathways in microbial systems However, for some medicinal natural products, such as the anticancer agent paclitaxel, where low yields in nature, chemical complexity and lack of knowledge of the complete biosynthetic pathway, preclude many of these options, plant cell culture technology is an attractive alternative for supply Plant cell suspension cultures are amenable to scale-up, environmental optimization, and metabolic engineering This review focuses on some of the key challenges in utilizing and commercializing plant cell culture suspension technology, with a focus on pharmaceutically active natural products Recent research has been directed toward application of traditional strategies such as reactor design, cell immobilization, and enzyme elicitation as well as emerging strategies such as characterizing cellular heterogeneity and variability through flow cytometric techniques, metabolic engineering, and system-wide analysis

Application of melt extrusion in the development of a physically and chemically stable high-energy amorphous solid dispersion of a poorly water-soluble drug.

Abstract: Formulation of active pharmaceutical ingredients (API) in high-energy amorphous forms is a common strategy to enhance solubility, dissolution rate and, consequently, oral bioavailability of poorly water-soluble drugs. Amorphous APIs are, however, susceptible to recrystallization and, therefore, there is a need to physically stabilize them as solid dispersions in polymeric carriers. Hot melt extrusion has in recent years gained wide acceptance as a method of choice for the preparation of solid dispersions. There is a potential that the API, the polymer or both may degrade if excessively high temperature is needed in the melt extrusion process, especially when the melting point of the API is high. This report details a novel method where the API was first converted to an amorphous form by solvent evaporation and then melt-extruded with a suitable polymer at a drug load of at least 20% w/w. By this means, melt extrusion could be performed much below the melting temperature of the drug substance. Since the gl...

Methotrexate Loaded Polyether-Copolyester Dendrimers for the Treatment of Gliomas: Enhanced Efficacy and Intratumoral Transport Capability

Abstract: Therapeutic benefit in glial tumors is often limited due to low permeability of delivery systems across the blood−brain barrier (BBB), drug resistance, and poor penetration into the tumor tissue. In an attempt to overcome these hurdles, polyether-copolyester (PEPE) dendrimers were evaluated as drug carriers for the treatment of gliomas. Dendrimers were conjugated to d-glucosamine as the ligand for enhancing BBB permeability and tumor targeting. The efficacy of methotrexate (MTX)-loaded dendrimers was established against U87 MG and U 343 MGa cells. Permeability of rhodamine-labeled dendrimers and MTX-loaded dendrimers across the in vitro BBB model and their distribution into avascular human glioma tumor spheroids was also studied. Glucosylated dendrimers were found to be endocytosed in significantly higher amounts than nonglucosylated dendrimers by both the cell lines. IC50 of MTX after loading in dendrimers was lower than that of the free MTX, suggesting that loading MTX in PEPE dendrimers increased its p...

Xenoreceptors CAR and PXR Activation and Consequences on Lipid Metabolism, Glucose Homeostasis, and Inflammatory Response

Abstract: Xenobiotic and drug metabolism and transport are managed by a large number of genes coordinately regulated by at least three nuclear receptors or xenosensors: aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR, NR1I3), and pregnane X receptor (PXR, NR1I2). Initially characterized as xenosensors, it is now evident that CAR and PXR also trigger pleiotropic effects on liver function. Recent studies have shown the existence of crosstalk between xenosensors and other nuclear receptors or transcription factors controlling endogenous signaling pathways which regulate physiological functions. This review is focused on recent observations showing that activation of CAR and PXR alters lipid metabolism, glucose homeostasis, and inflammation by interfering with HNF4alpha, FoxO1, FoxA2, PGC1alpha, or NFkB p65. Such crosstalks explain clinical observations and provide molecular mechanisms allowing understanding how xenobiotics and drugs may affect physiological functions and provoke endocrine disruptions.

Amorphous Compositions Using Concentration Enhancing Polymers for Improved Bioavailability of Itraconazole

Abstract: Amorphous engineered particle compositions of itraconazole (ITZ) and potential concentration enhancing polymers, cellulose acetate phthalate (CAP) and polyvinyl acetate phthalate (PVAP), were produced by ultra-rapid freezing to investigate the effect of these polymers on the bioavailability of ITZ solid dispersions. X-ray diffraction analyses of engineered particle compositions were shown to be amorphous. Modulated differential scanning calorimetry demonstrated that ITZ:CAP engineered particle compositions exhibited a strong correlation with the Gordon-Taylor relationship while ITZ:PVAP formulations exhibited positive deviations from predicted values attributed to hydrogen bonding interactions between the drug and polymer. Energy dispersive spectroscopy mapping demonstrated that the drug was homogenously distributed within all compositions, supporting the miscibility of the drug with the polymers. Scanning electron microscopy imaging of the particles demonstrated that the material existed in two general forms, discrete particles of approximately 5 microm and larger aggregates in excess of 30 microm, with engineered particle compositions having approximately 15 times higher measured specific surfaces areas compared to micronized ITZ. In vitro supersaturated dissolution results showed that all compositions provided significantly lower levels of supersaturation in acidic media and greater extents of supersaturation in neutral media compared to Sporanox pellets. ITZ: CAP formulations provided the greatest degree and extent of supersaturation in neutral media. Dissolution data were fitted to an exponential relationship based on a simplified model of particle growth, allowing for the determination of drug half-life in solution for evaluation of stabilization behavior. 1:2 ITZ:CAP showed superior in vitro performance compared to all other engineered particle compositions and was selected for in vivo testing. Although not fully elucidated, data indicated that the stabilization mechanism was due to interactions between the drug and polymer, primarily attributed to steric hindrance resulting from the molecular weight of the polymer chain and chemical composition of the polymer backbone relative to position of hydrogen bonding sites. In vivo testing conducted in Sprague-Dawley rats (n = 6) demonstrated a significant improvement in oral bioavailability from the 1:2 ITZ:CAP (AUC = 4,516 +/- 1,949 ng x h/mL) compared to the Sporanox pellets (AUC = 2,132 +/- 1,273 ng x h/mL) (p < or = 0.05). Additionally, the more rapid onset of action indicated superior targeting of the upper small intestines, and the prolonged half-life suggested the utility of CAP to maintain supersaturated concentrations, in vivo. These results demonstrated that amorphous compositions of ITZ and enteric concentration enhancing polymers provided improved bioavailability due to enhanced intestinal targeting and increased durations of supersaturation.

The impact of molecular weight and PEG chain length on the systemic pharmacokinetics of PEGylated poly l-lysine dendrimers.

Abstract: The impact of PEGylation on the pharmacokinetics and biodistribution of (3)H-labeled poly l-lysine dendrimers has been investigated after intravenous administration to rats. The volumes of distribution, clearance and consequently the plasma half-lives of the PEGylated dendrimers were markedly dependent on the total molecular weight of the PEGylated dendrimer, but were not specifically affected by the PEG chain length alone. In general, the larger dendrimer constructs (i.e. >30 kDa) had reduced volumes of distribution, were poorly renally cleared and exhibited extended elimination half-lives ( t 1/2 1-3 days) when compared to the smaller dendrimers (i.e. <20 kDa) which were rapidly cleared from the plasma principally into the urine ( t 1/2 1-10 h). At later time points the larger dendrimers concentrated in the organs of the reticuloendothelial system (liver and spleen); however, the absolute extent of accumulation was low. Size exclusion chromatography of plasma and urine samples revealed that the PEGylated dendrimers were considerably more resistant to biodegradation in vivo than the underivatized poly l-lysine dendrimer cores. The results suggest that the size of PEGylated poly l-lysine dendrimer complexes can be manipulated to optimally dictate their pharmacokinetics, biodegradation and bioresorption behavior.

Cationic solid lipid nanoparticles reconstituted from low density lipoprotein components for delivery of siRNA

Abstract: Cationic solid lipid nanoparticles (SLN), reconstituted from natural components of protein-free low-density lipoprotein, were used to deliver small interfering RNA (siRNA). The cationic SLN was prepared using a modified solvent-emulsification method. The composition was 45% (w/w) cholesteryl ester, 3% (w/w) triglyceride, 10% (w/w) cholesterol, 14% (w/w) dioleoylphosphatidylethanolamine (DOPE), and 28% (w/w) 3beta-[ N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-chol). The SLN had a mean diameter of 117+/-12 nm and a surface zeta potential value of +41.76+/-2.63 mV. A reducible conjugate of siRNA and polyethylene glycol (PEG) (siRNA-PEG) was anchored onto the surface of SLN via electrostatic interactions, resulting in stable complexes in buffer solution and in even 10% serum. Under an optimal weight ratio of DC-chol of SLN and siRNA-PEG conjugate, the complexes exhibited higher gene silencing efficiency of GFP and VEGF than that of polyethylenimine (PEI) 25K with showing much reduced cell cytotoxicity. Flow cytometry results also showed that siRNA-PEG/SLN complexes were efficiently taken up by cells. Surface-modified and reconstituted protein-free LDL mimicking SLN could be utilized as noncytotoxic, serum-stable, and highly effective carriers for delivery of siRNA.

Endocytosis inhibitors prevent poly(amidoamine) dendrimer internalization and permeability across Caco-2 cells.

Abstract: Previous studies from our group demonstrated visual evidence that endocytosis mechanism(s) contribute to the internalization and intracellular trafficking of cationic and anionic poly(amidoamine) (PAMAM) dendrimers across Caco-2 cells These dendrimers colocalized with established endocytosis markers, which suggested PAMAM dendrimers may be internalized by a clathrin-dependent endocytosis mechanism and are rapidly trafficked to endosomal and lysosomal compartments In the present study, generation 4 PAMAM-NH2 (G4NH2) dendrimer was labeled with tritium to measure the rate of uptake and permeability in Caco-2 cells The effect of endocytosis inhibitors brefeldin A, colchicine, filipin, and sucrose on G4NH2 absorption and transport was examined to give further insight into the endocytosis mechanisms that transport PAMAM dendrimers across Caco-2 cell monolayers G4NH2 showed linear uptake at lower concentrations, and rapidly increased as a function of concentration The rate of G4NH2 uptake significantly decl

Enhanced bioavailability of a poorly soluble VR1 antagonist using an amorphous solid dispersion approach: a case study.

Abstract: Amorphous solid dispersions (ASD) of a poorly soluble water-soluble VR1 antagonist (AMG 517) were explored for improving physical stability and in vivo exposure. AMG 517 was incorporated at 15 or 50 wt % into polymeric microparticles of hydroxypropyl methylcellulose acetate succinate (HPMCAS) and hydroxypropyl methylcellulose (HPMC) by spray-drying. Solid particles having a collapsed, corrugated structure were observed by SEM. Median particle size ranged from 29 to 40 μm by laser light scattering, and residual solvent levels were below 2% by thermal gravimetric analysis. ASD powders exhibited single glass transition temperatures (Tg) in the range of 98−117 °C by modulated DSC and were amorphous by XRPD. Amorphous stability, characterized at 40 °C/75% RH (open dish) by XRPD, was at least six months for ASD formulations. Drug dissolution and supersaturation testing in a USP-2 apparatus indicated superior performance of ASD formulations over micronized AMG 517. PK of an ASD formulation in capsule (15 wt % AM...

Surface-enhanced crystallization of amorphous nifedipine.

Abstract: Amorphous solids are generally more soluble and faster dissolving than their crystalline counterparts, a property useful for delivering poorly soluble drugs. Amorphous drugs must be stable against crystallization, for crystallization negates their advantages. Recent studies found that crystal growth in amorphous indomethacin is orders of magnitude faster at the free surface than through the bulk and this surface-enhanced crystallization can be inhibited by an ultrathin coating. Herein, we report a second system that exhibits the same phenomena. Crystal growth at the free surface of amorphous nifedipine (NIF) was at least 1 order of magnitude faster than that through the bulk below the glass transition temperature Tg (42 °C). A thin coating of gold (10 nm) reduced the surface crystal growth rate to the bulk crystal growth rate. Surface-enhanced crystal growth was more pronounced near and below Tg than substantially above Tg, which suggests that this growth mode is more important for the glassy state. Our r...

Polyplex micelles with cyclic RGD peptide ligands and disulfide cross-links directing to the enhanced transfection via controlled intracellular trafficking.

Abstract: Thiolated c(RGDfK)-poly(ethylene glycol)-block-poly(lysine) (PEG-PLys), a novel block polymer that has a cyclic RGD peptide in the PEG terminus and thiol groups in the PLys side chain, was prepared and applied to the preparation of targetable disulfide cross-linked polyplex micelles through ion complexation with plasmid DNA (pDNA). The obtained polyplex micelles achieved remarkably enhanced transfection efficiency against cultured HeLa cells possessing αvβ3 integrin receptors, which are selectively recognized by cyclic RGD peptides, demonstrating the synergistic effect of cyclic RGD peptide ligands on the micelle surface and disulfide cross-links in the core to exert the smooth release of pDNA in the intracellular environment via reductive cleavage. This enhancement was not due to an increase in the uptake amount of polyplex micelles but to a change in their intracellular trafficking route. Detailed confocal laser scanning microscopic observation revealed that polyplex micelles with cyclic RGD peptide lig...

Biodistribution and Pharmacokinetic Analysis of Paclitaxel and Ceramide Administered in Multifunctional Polymer-Blend Nanoparticles in Drug Resistant Breast Cancer Model

Abstract: In this study, we have investigated the biodistribution and pharmacokinetic analysis of paclitaxel (PTX) and the apoptotic signaling molecule, C6-ceramide (CER), when administered in a multifunctional polymer-blend nanoparticle formulation to female nude mice bearing an orthotopic drug sensitive MCF7 and multidrug resistant MCF7TR (MDR-1 positive) human breast adenocarcinoma. A polymer-blend nanoparticle system was engineered to incorporate temporally controlled sequential release of the combination drug payload. Hereby, PTX was encapsulated in the pH-responsive rapid releasing polymer, poly(beta-amino ester) (PbAE), while CER was present in the slow releasing polymer, poly(d,l-lactide-co-glycolide) (PLGA) within these blend nanoparticles. When particle formulations were administered intravenously to MCF7 and MCF7TR tumor bearing mice, higher concentrations of PTX were found in the blood due to longer retention time and an enhanced tumor accumulation relative to administration of free drug. In addition, t...

Synthesis, Characterization, and Biological Evaluation of Integrin αvβ3-Targeted PAMAM Dendrimers

Abstract: Ligand size and valency strongly influence the receptor uptake and clearance of tumor angiogenesis imaging agents. The structures of successful imaging agents exhibit a high degree of variability, encompassing small monovalent arginine-glycine-aspartic acid (RGD)-containing peptides, multivalent RGD-oligomers, and a monoclonal antibody against integrin alpha-v-beta-3 (alpha-v-beta-3). We have pursued a nanoscale approach to imaging of angiogenesis using rationally designed polyamidoamine (PAMAM) dendrimers covalently adorned with RGD-cyclopeptides. An orthogonal oxime-ligation strategy was applied to chemoselectively effect conjugation of the PAMAM dendrimers with RGD-cyclopeptides for targeting alpha vbeta 3. Fluorescent dyes for optical imaging and chelates for gadolinium-based magnetic resonance (MR) imaging were subsequently appended to create robust multimodal macromolecular imaging agents. Fluorescence microscopy revealed selective binding of the resulting RGD peptide-bearing dendrimer with empty chelates to alpha-v-beta-3-expressing cells, but somewhat reduced selectivity was observed following Gd(III) complexation. The expected incomplete saturation of chelates with Gd(III) ions permitted radiometal complexation, and an in vivo tissue distribution of the resulting agent in M21 melanoma tumor-bearing mice showed mostly renal and reticuloendothelial accumulation, with the tumor:blood ratio peaking (3.30+/-0.03) at 2 h postinjection.

Thermodynamics, molecular mobility and crystallization kinetics of amorphous griseofulvin.

Abstract: Griseofulvin is a small rigid molecule that shows relatively high molecular mobility and small configurational entropy in the amorphous phase and tends to readily crystallize from both rubbery and glassy states. This work examines the crystallization kinetics and mechanism of amorphous griseofulvin and the quantitative correlation between the rate of crystallization and molecular mobility above and below Tg. Amorphous griseofulvin was prepared by rapidly quenching the melt in liquid N2. The thermodynamics and dynamics of amorphous phase were then characterized using a combination of thermal analysis techniques. After characterization of the amorphous phase, crystallization kinetics above Tg were monitored by isothermal differential scanning calorimetry (DSC). Transformation curves for crystallization fit a second-order John−Mehl−Avrami (JMA) model. Crystallization kinetics below Tg were monitored by powder X-ray diffraction and fit to the second-order JMA model. Activation energies for crystallization wer...

Targeted disruption of peptide transporter Pept1 gene in mice significantly reduces dipeptide absorption in intestine.

Abstract: PEPT1 is a high-capacity, low-affinity peptide transporter that mediates the uptake of di- and tripeptides in the intestine and kidney. PEPT1 also has significance in its ability to transport therapeutic agents and because of its potential as a target for anti-inflammatory therapies. To further understand the relevance of specific peptide transporters in intestinal physiology, pharmacology and pathophysiology, we have generated Pept1 null mice by targeted gene disruption. The Pept1 gene was disrupted by insertion of a lacZ reporter gene under the control of the endogenous Pept1 promoter. Phenotypic profiling of wild-type and Pept1 null mice was then performed, along with in vitro intestinal uptake, in situ intestinal perfusion and in vivo pharmacokinetic studies of glycylsarcosine (GlySar). Pept1 null mice lacked expression of PEPT1 protein in the intestine and kidney, tissues in which this peptide transporter is normally expressed. Pept1-deficient mice were found to be viable, fertile, grew to normal size and weight, and were without any obvious abnormalities. Nevertheless, Pept1 deletion dramatically reduced the intestinal uptake and effective permeability of the model dipeptide GlySar (i.e., by at least 80%), and its oral absorption following gastric gavage (i.e., by about 50%). In contrast, the plasma profiles of GlySar were almost superimposable between wild-type and Pept1 null animals after intravenous dosing. These novel findings provide strong evidence that PEPT1 has a major role in the in vivo oral absorption of dipeptides.

New ways of imaging uptake and intracellular fate of liposomal drug carrier systems inside individual cells, based on Raman microscopy.

Abstract: Recent developments, combining Raman spectroscopy with optical microscopy, provide a new noninvasive technique to assess and image cellular processes. Of particular interest are the uptake mechanisms of various cytologically active compounds. In order to distinguish the species of interest from their cellular environment spectroscopically, compounds may be labeled with deuterium. Here, we apply Raman microspectroscopy to follow the uptake of liposomal drug carrier systems that have been introduced to deliver biologically active compounds to their site of action within human breast adenocarcinoma MCF-7 cells. The distribution patterns of liposomes and liposomes surface-modified with a cell-penetrating peptide (TAT-peptide, TATp) have been imaged over time. The spectroscopic information obtained provides a clear evidence for variable rates, as well as different efficiencies of liposome uptake depending on their surface properties. Depending on the experimental setup, the technique may be applied to fixed or...

Applications of Physiologically Based Absorption Models in Drug Discovery and Development

Abstract: This article describes the use of physiologically based models of intestinal drug absorption to guide the research and development of new drugs. Applications range from lead optimization in the drug discovery phase through clinical candidate selection and extrapolation to human to phase 2 formulation development. Early simulations in preclinical species integrate multiple screening data and add value by transforming these individual properties into a prediction of in vivo absorption. Comparison of simulations to plasma levels measured after oral dosing in animals highlights unexpected behavior, and parameter sensitivity analysis can explore the impact of uncertainties in key properties, point toward factors which are limiting absorption and contribute to assessment of compound developability. Physiological models provide reliable prediction of human absorption and with refinement based on phase 1 data are useful guides to further market formulation development. Improvements in the accuracy of simulations ...

PEG-b-PPS diblock copolymer aggregates for hydrophobic drug solubilization and release: cyclosporin A as an example.

Abstract: Micelles formed from amphiphilic block copolymers have been explored in recent years as carriers for hydrophobic drugs. In an aqueous environment, the hydrophobic blocks form the core of the micelle, which can host lipophilic drugs, while the hydrophilic blocks form the corona or outer shell and stabilize the interface between the hydrophobic core and the external medium. In the present work, mesophase behavior and drug encapsulation were explored in the AB block copolymeric amphiphile composed of poly(ethylene glycol) (PEG) as a hydrophile and poly(propylene sulfide) PPS as a hydrophobe, using the immunosuppressive drug cyclosporin A (CsA) as an example of a highly hydrophobic drug. Block copolymers with a degree of polymerization of 44 on the PEG and of 10, 20 and 40 on the PPS respectively (abbreviated as PEG44-b-PPS10, PEG44-b-PPS20, PEG44-b-PPS40) were synthesized and characterized. Drug-loaded polymeric micelles were obtained by the cosolvent displacement method as well as the remarkably simple method of dispersing the warm polymer melt, with drug dissolved therein, in warm water. Effective drug solubility up to 2 mg/mL in aqueous media was facilitated by the PEG- b-PPS micelles, with loading levels up to 19% w/w being achieved. Release was burst-free and sustained over periods of 9-12 days. These micelles demonstrate interesting solubilization characteristics, due to the low glass transition temperature, highly hydrophobic nature, and good solvent properties of the PPS block.

Mini-review: endocrine actions of fibroblast growth factor 19.

Abstract: Fibroblast growth factor (FGF) 19 is an atypical member of the fibroblast growth factor family of signaling molecules. FGF19, FGF21, and FGF23 comprise a phylogenetic subfamily with attributes that distinguish them from typical FGFs. The FGF19 subfamily has reduced heparin binding resulting from a disrupted β-trefoil domain. Reduced heparin binding allows these FGFs to diffuse beyond their site of origin and act as endocrine hormones. This family of FGFs is regulated, at least in part, by nuclear hormone receptors. FGF19 expression is regulated by the farnesoid X receptor, a nuclear hormone receptor that is a key regulator of bile acid biosynthesis and transport. In line with its regulation by a bile acid receptor, FGF19 is involved in the regulation of bile acid biosynthesis and gallbladder filling. FGF19 originates from intestine and signals to liver via the portal circulation with a pronounced diurnal pattern. FGF19 is the only FGF to not have a closely related mouse homologue. The mouse homologue of F...