Pharmaceutical Research (v.31, #2)

FRAP in Pharmaceutical Research: Practical Guidelines and Applications in Drug Delivery by Hendrik Deschout; Koen Raemdonck; Jo Demeester; Stefaan C. De Smedt; Kevin Braeckmans (255-270).
Fluorescence recovery after photobleaching (FRAP) is a fluorescence microscopy technique that has attracted a lot of interest in pharmaceutical research during the last decades. The main purpose of FRAP is to measure diffusion on a micrometer scale in a non-invasive and highly specific way, making it capable of measurements in complicated biomaterials, even in vivo. This has proven to be very useful in the investigation of drug diffusion inside different tissues of the body and in materials for controlled drug delivery. FRAP has even found applications for the improvement of several medical therapies and in the field of diagnostics. In this review, an overview is given of the different applications of FRAP in pharmaceutical research, together with essential guidelines on how to perform and analyse FRAP experiments.
Keywords: diffusion; drug delivery; FRAP; microscopy

Computational Models for Neglected Diseases: Gaps and Opportunities by Elizabeth L. Ponder; Joel S. Freundlich; Malabika Sarker; Sean Ekins (271-277).
Neglected diseases, such as Chagas disease, African sleeping sickness, and intestinal worms, affect millions of the world’s poor. They disproportionately affect marginalized populations, lack effective treatments or vaccines, or existing products are not accessible to the populations affected. Computational approaches have been used across many of these diseases for various aspects of research or development, and yet data produced by computational approaches are not integrated and widely accessible to others. Here, we identify gaps in which computational approaches have been used for some neglected diseases and not others. We also make recommendations for the broad-spectrum integration of these techniques into a neglected disease drug discovery and development workflow.
Keywords: African sleeping sickness; Chagas disease; computational models; leishmaniasis; malaria; schistosomiasis; tuberculosis

Radioiodinated Hypericin: Its Biodistribution, Necrosis Avidity and Therapeutic Efficacy are Influenced by Formulation by Marlein Miranda Cona; Yeranddy Aguiar Alpizar; Junjie Li; Matthias Bauwens; Yuanbo Feng; Ziping Sun; Jian Zhang; Feng Chen; Karel Talavera; Peter de Witte; Alfons Verbruggen; Raymond Oyen; Yicheng Ni (278-290).
To study whether formulation influences biodistribution, necrosis avidity and tumoricidal effects of the radioiodinated hypericin, a necrosis avid agent for a dual-targeting anticancer radiotherapy.Iodine-123- and 131-labeled hypericin (123I-Hyp and 131I-Hyp) were prepared with Iodogen as oxidant, and formulated in dimethyl sulfoxide (DMSO)/PEG400 (polyethylene glycol 400)/water (25/60/15, v/v/v) or DMSO/saline (20:80, v/v). The formulations with excessive Hyp were optically characterized. Biodistribution, necrosis avidity and tumoricidal effects were studied in rats (n = 42) without and with reperfused liver infarction and implanted rhabdomyosarcomas (R1). To induce tumor necrosis, R1-rats were pre-treated with a vascular disrupting agent. Magnetic resonance imaging, tissue-gamma counting, autoradiography and histology were used.The two formulations differed significantly in fluorescence and precipitation. 123I-Hyp/Hyp in DMSO/PEG400/water exhibited high uptake in necrosis but lower concentration in the lung, spleen and liver (p < 0.01). Tumor volumes of 0.9 ± 0.3 cm3 with high radioactivity (3.1 ± 0.3% ID/g) were detected 6 days post-treatment. By contrast, 131I-Hyp/Hypin DMSO/saline showed low uptake in necrosis but high retention in the spleen and liver (p < 0.01). Tumor volumes reached 2.6 ± 0.7 cm3 with low tracer accumulation (0.1 ± 0.04%ID/g).The formulation of radioiodinated hypericin/hypericin appears crucial for its physical property, biodistribution, necrosis avidity and tumoricidal effects.
Keywords: cancer; formulation; OncoCiDia; radioiodinated hypericin; targeted radiotherapy

Block copolymer micelles are extensively used as drug controlled release carriers, showing promising application prospects. The comb or brush copolymers are especially of great interest, whose densely-grafted side chains may be important for tuning the physicochemical properties and conformation in selective solvents, even in vitro drug release. The purpose of this work was to synthesize novel block copolymer combs via atom transfer radical polymerization, to evaluate its physicochemical features in solution, to improve drug release behavior and to enhance the bioavailablity, and to decrease cytotoxicity.The physicochemical properties of the copolymer micelles were examined by modulating the composition and the molecular weights of the building blocks. A dialysis method was used to load hydrophobic camptothecin (CPT), and the CPT release and stability were detected by UV–vis spectroscopy and high-performance liquid chromatography, and the cytotoxicity was evaluated by MTT assays.The copolymers could self-assemble into well-defined spherical core-shell micelle aggregates in aqueous solution, and showed thermo-induced micellization behavior, and the critical micelle concentration was 2.96–27.64 mg L−1. The micelles were narrow-size-distribution, with hydrodynamic diameters about 128–193 nm, depending on the chain length of methoxy polyethylene glycol (mPEG) blocks and poly(N-isopropylacrylamide) (PNIPAM) graft chains or/and compositional ratios of mPEG to PNIPAM. The copolymer micelles could stably and effectively load CPT but avoid toxicity and side-effects, and exhibited thermo-dependent controlled and targeted drug release behavior.The copolymer micelles were safe, stable and effective, and could potentially be employed as CPT controlled release carriers.
Keywords: copolymer micelles; cytotoxicity; drug release; stability; thermosensitivity

Existing PBPK models incorporating intestinal first-pass metabolism account for effect of drug permeability on accessible absorption surface area by use of “effective” permeability, P eff , without adjusting number of enterocytes involved in absorption or proportion of intestinal CYP3A involved in metabolism. The current model expands on existing models by accounting for these factors.The PBPK model was developed using SAAM II. Midazolam clinical data was generated at GlaxoSmithKline.The model simultaneously captures human midazolam blood concentration profile and previously reported intestinal availability, using values for CYP3A CLu int , permeability and accessible surface area comparable to literature data. Simulations show: (1) failure to distinguish absorbing from non-absorbing enterocytes results in overestimation of intestinal metabolism of highly permeable drugs absorbed across the top portion of the villous surface only; (2) first-pass extraction of poorly permeable drugs occurs primarily in enterocytes, drugs with higher permeability are extracted by enterocytes and hepatocytes; (3) CYP3A distribution along crypt-villous axes does not significantly impact intestinal metabolism; (4) differences in permeability of perpetrator and victim drugs results in their spatial separation along the villous axis and intestinal length, diminishing drug-drug interaction magnitude.The model provides a useful tool to interrogate intestinal absorption/metabolism of candidate drugs.
Keywords: drug-drug interaction; enterocyte; first pass extraction; intestinal availability; intestinal villous

To develop, characterize and exploring the sulfasalazine loaded fucoyslated multi walled carbon nanotubes for Kupffer cell targeting for effective management of cytokine-induce liver damage.Sulfasalazine was loaded into the fucosylated MWCNTs after subsequential functionalization (carboxylation, acylation and amidation) using dialysis membrane technique. The in vitro, in vivo studies were performed on macrophages J 774 cell line for Kupffer cells targeting for the treatment of cytokine-induced liver damage.The loading of SSZ into SSZ-FUCO-MWCNTs was 87.77 ± 0.11% (n = 3). Sustained release was obtained from SSZ-FUCO-MWCNTs, with 89.12 ± 0.71% of SSZ released into medium at 48th hr. SSZ-FUCO-MWCNTs showed the 9.01 ± 0.23% hemolysis was drastically reduced from 21.62 ± 0.24% SSZMWCNTs 21.62 ± 0.24%. In SRB assay, SSZ-FUCO-MWCNTs showed more cytotoxicity than raw and SSZ-MWCNTs. In cytokine assay, SSZ- FUCO-MWCNTs exhibited significantly higher inhibition of IL-12 p40 secretion. In Western blot assay, SSZ-FUCO-MWCNTs significantly inhibit NF-κB activation.The results suggested that the SSZ-FUCO-MWCNTs may be useful nano-carriers for targeted delivery to Kupffer cells in the treatment of cytokine-induced liver damage.
Keywords: carbon nanotubes; fucose; Kupffer cells; liver targeting; sulfasalazine

The impact of efflux transporters in intracellular concentrations of a drug can be predicted with modeling techniques. In Part 1, several compartmental models were developed and evaluated. The goal of Part 2 was to apply these models to the characterization and interpretation of saturation kinetic data.The compartmental models from Part 1were used to evaluate a previously published dataset from cell lines expressing varying levels of P-glycoprotein. Kinetic parameters for the transporter were estimated and compared across models.Fits and errors for all compartmental models were identical. All compartmental models predicted more consistent parameters than the Michaelis-Menten model. The 5-compartment model with efflux out of the membrane predicted differential impact of P-gp upon apical versus basolateral drug exposure. Finally, the saturable kinetics of active efflux along with a permeability barrier was modeled to delineate a relationship between intracellular concentration with or without active efflux versus donor concentration. This relationship was not a rectangular hyperbola, but instead was shown to be a quadratic function.One approach to estimate an in vivo transporter effect is to first model an intracellular Km value from in vitro data, and use this value along with the appropriate tissue transporter expression levels and relative surface area to calculate the relevant apparent Km (or Ki) values. Together with the results from Part 1, these studies suggest that compartmental models can provide a path forward to better utilize in vitro transporter data for in vivo predictions such as physiologically based pharmacokinetic modeling.
Keywords: compartmental models; intracellular concentrations; kinetics; P-glycoprotein; transporters

Compartmental Models for Apical Efflux by P-glycoprotein—Part 1: Evaluation of Model Complexity by Swati Nagar; Jalia Tucker; Erica A. Weiskircher; Siddhartha Bhoopathy; Ismael J. Hidalgo; Ken Korzekwa (347-359).
With the goal of quantifying P-gp transport kinetics, Part 1 of these manuscripts evaluates different compartmental models and Part 2 applies these models to kinetic data.Models were developed to simulate the effect of apical efflux transporters on intracellular concentrations of six drugs. The effect of experimental variability on model predictions was evaluated. Several models were evaluated, and characteristics including membrane configuration, lipid content, and apical surface area (asa) were varied.Passive permeabilities from MDCK-MDR1 cells in the presence of cyclosporine gave lower model errors than from MDCK control cells. Consistent with the results in Part 2, model configuration had little impact on calculated model errors. The 5-compartment model was the simplest model that reproduced experimental lag times. Lipid content and asa had minimal effect on model errors, predicted lag times, and intracellular concentrations. Including endogenous basolateral uptake activity can decrease model errors. Models with and without explicit membrane barriers differed markedly in their predicted intracellular concentrations for basolateral drug exposure. Single point data resulted in clearances similar to time course data.Compartmental models are useful to evaluate the impact of efflux transporters on intracellular concentrations. Whereas a 3-compartment model may be sufficient to predict the impact of transporters that efflux drugs from the cell, a 5-compartment model with explicit membranes may be required to predict intracellular concentrations when efflux occurs from the membrane. More complex models including additional compartments may be unnecessary.
Keywords: compartmental models; intracellular concentrations; kinetics; P-glycoprotein; transporters

Development of a High Efficiency Dry Powder Inhaler: Effects of Capsule Chamber Design and Inhaler Surface Modifications by Srinivas R. B. Behara; Dale R. Farkas; Michael Hindle; P. Worth Longest (360-372).
The objective of this study was to explore the performance of a high efficiency dry powder inhaler (DPI) intended for excipient enhanced growth (EEG) aerosol delivery based on changes to the capsule orientation and surface modifications of the capsule and device.DPIs were constructed by combining newly designed capsule chambers (CC) with a previously developed three-dimensional (3D) rod array for particle deagglomeration and a previously optimized EEG formulation. The new CCs oriented the capsule perpendicular to the incoming airflow and were analyzed for different air inlets at a constant pressure drop across the device. Modifications to the inhaler and capsule surfaces included use of metal dispersion rods and surface coatings. Aerosolization performance of the new DPIs was evaluated and compared with commercial devices.The proposed capsule orientation and motion pattern increased capsule vibrational frequency and reduced the aerosol MMAD compared with commercial/modified DPIs. The use of metal rods in the 3D array further improved inhaler performance. Coating the inhaler and capsule with PTFE significantly increased emitted dose (ED) from the optimized DPI.High efficiency performance is achieved for EEG delivery with the optimized DPI device and formulation combination producing an aerosol with MMAD < 1.5 μm, FPF<5μm/ED > 90%, and ED > 80%.
Keywords: 3D rod array; excipient enhanced growth; high efficiency DPI; low surface energy coatings; PTFE coating

To develop and characterize microspheres using poly (lactic-co-glycolic acid) (PLGA) blends (PLGA5050 (25 KD) and PLGA6535 (70 KD)) for dexamethasone delivery to prevent foreign body response to implantable biosensors.A single emulsion based oil/water solvent evaporation/extraction method was used to prepare microspheres.All the microspheres prepared exhibited the typical triphasic release profile, but with different initial burst release, lag phase and zero order release rates. The burst release was reduced when the two PLGA were mixed at a molecular level, whereas increase in burst release was observed when phase separation occurred. Microspheres prepared using PLGA blends had significantly shorter lag phase. The activation energy (Ea) of dexamethasone release from microspheres was similar to the Ea value of PLGA degradation. The release kinetics were significantly enhanced under accelerated conditions (45 and 53°C) without altering the release mechanism of the post-burst phase. A rank order correlation between accelerated and “real-time” release kinetics was observed.Polymer blends of PLGA can produce microspheres with reduced lag time. The accelerated release testing conditions investigated can discriminate the formulations and predict “real-time” release. Such accelerated release testing can be used as a rapid screening method to facilitate formulation development.
Keywords: “real-time” and accelerated in vitro release; microspheres; PLGA blends

Impact of Different Vehicles for Laser-Assisted Drug Permeation via Skin: Full-Surface versus Fractional Ablation by Woan-Ruoh Lee; Shing-Chuan Shen; Ibrahim A. Aljuffali; Yi-Ching Li; Jia-You Fang (382-393).
This study aimed to assess impact of different vehicles for laser-assisted skin drug delivery. We also tried to uncover the mechanisms by which different vehicles affect laser-aided skin permeation.Full-surface ablative (conventional) and fractional lasers were used to irradiate nude mouse skin. Imiquimod and 5-aminolevulinic acid (ALA) were used as lipophilic and hydrophilic permeants. Vehicles employed included water with 40% polyethylene glycol 400 (PEG 400), propylene glycol (PG), and ethanol. Lipid nanoparticles were also utilized as carriers. In vitro permeation profiles showed improvement in imiquimod flux with conventional laser (2.5 J/cm2) producing a 12-, 9-, and 5-fold increase when loading imiquimod in 40% PEG400, PG, and ethanol, respectively, as compared with intact skin. Nanoparticulate delivery by laser produced a 6-fold enhancement in permeation. Fractional laser produced less enhancement of imiquimod delivery than conventional laser. Laser exposure increased follicular imiquimod accumulation from 0.80 to 5.81 μg/cm2. ALA permeation from aqueous buffer, PEG 400, and PG with conventional laser treatment was 641-, 445-, and 104-fold superior to passive control. In vivo skin deposition of topically applied ALA examined by confocal microscopy indicated the same trend as the in vitro experiment, with aqueous buffer showing the greatest proporphyllin IX signaling. Diffusion of cosolvent molecules into ablated skin and drug partitioning from vehicle to skin are two predominant factors controlling laser-assisted delivery. In contrast to conventional laser, lateral drug diffusion was anticipated for fractional laser.Our results suggest that different drug delivery vehicles substantially influence drug penetration enhanced by lasers.
Keywords: 5-aminolevulinic acid; ablative laser; imiquimod; nanoparticle; vehicle

In Vitro–In Vivo Correlation in Skin Permeation by D. Mohammed; P. J. Matts; J. Hadgraft; M. E. Lane (394-400).
In vitro skin permeation studies have been used extensively in the development and optimisation of delivery of actives in vivo. However, there are few reported correlations of such in vitro studies with in vivo data. The aim of this study was to investigate the skin permeation of a model active, niacinamide, both in vitro and in vivo.Conventional diffusion cell studies were conducted in human skin to determine niacinamide permeation from a range of vehicles which included dimethyl isosorbide (DMI), propylene glycol (PG), propylene glycol monolaurate (PGML), N-methyl 2-pyrrolidone (NMP), Miglyol 812N® (MG), and mineral oil (MO). Single, binary or ternary systems were examined. The same vehicles were subsequently examined to investigate niacinamide delivery in vivo. For this proof-of-concept study one donor was used for the in vitro studies and one volunteer for the in vivo investigations to minimise biovariability. Analysis of in vitro samples was conducted using HPLC and in vivo uptake of niacinamide was evaluated using Confocal Raman spectroscopy (CRS).The amount of niacinamide permeated through skin in vitro was linearly proportional to the intensity of the niacinamide signal determined in the stratum corneum in vivo. A good correlation was observed between the signal intensities of selected vehicles and niacinamide signal intensity.The findings provide further support for the use of CRS to monitor drug delivery into and across the skin. In addition, the results highlight the critical role of the vehicle and its disposition in skin for effective dermal delivery.
Keywords: confocal raman spectroscopy; in vitro - in vivo correlation; niacinamide; permeation; skin

Microfluidic Preparation of Liposomes to Determine Particle Size Influence on Cellular Uptake Mechanisms by Abhay U. Andar; Renee R. Hood; Wyatt N. Vreeland; Don L. DeVoe; Peter W. Swaan (401-413).
This study investigates the cellular uptake and trafficking of liposomes in Caco-2 cells, using vesicles with distinct average diameters ranging from 40.6 nm to 276.6 nm. Liposomes were prepared by microfluidic hydrodynamic flow focusing, producing nearly-monodisperse populations and enabling size-dependent uptake to be effectively evaluated.Populations of PEG-conjugated liposomes of various distinct sizes were prepared in a disposable microfluidic device using a simple continuous-flow microfluidic technique. Liposome cellular uptake was investigated using flow cytometry and confocal microscopy.Liposome uptake by Caco-2 cells was observed to be strongly size-dependent for liposomes with mean diameters ranging from 40.6 nm to 276.6 nm. When testing these liposomes against endocytosis inhibitors, cellular uptake of the largest (97.8 nm and 162.1 nm in diameter) liposomes were predominantly subjected to clathrin-dependent uptake mechanisms, the medium-sized (72.3 nm in diameter) liposomes seemed to be influenced by all investigated pathways and the smallest liposomes (40.6 nm in diameter) primarily followed a dynamin-dependent pathway. In addition, the 40.6 nm, 72.3 nm, and 162.1 nm diameter liposomes showed slightly decreased accumulation within endosomes after 1 h compared to liposomes which were 97.8 nm in diameter. Conversely, liposome co-localization with lysosomes was consistent for liposomes ranging from 40.6 nm to 97.8 nm in diameter.The continuous-flow synthesis of nearly-monodisperse populations of liposomes of distinct size via a microfluidic hydrodynamic flow focusing technique enabled unique in vitro studies in which specific effects of particle size on cellular uptake were elucidated. The results of this study highlight the significant influence of liposome size on cellular uptake mechanisms and may be further exploited for increasing specificity, improving efficacy, and reducing toxicity of liposomal drug delivery systems.
Keywords: endocytosis; liposomes; microfluidics; nanoparticles

Combining Computational Methods for Hit to Lead Optimization in Mycobacterium Tuberculosis Drug Discovery by Sean Ekins; Joel S. Freundlich; Judith V. Hobrath; E. Lucile White; Robert C. Reynolds (414-435).
Tuberculosis treatments need to be shorter and overcome drug resistance. Our previous large scale phenotypic high-throughput screening against Mycobacterium tuberculosis (Mtb) has identified 737 active compounds and thousands that are inactive. We have used this data for building computational models as an approach to minimize the number of compounds tested.A cheminformatics clustering approach followed by Bayesian machine learning models (based on publicly available Mtb screening data) was used to illustrate that application of these models for screening set selections can enrich the hit rate.In order to explore chemical diversity around active cluster scaffolds of the dose–response hits obtained from our previous Mtb screens a set of 1924 commercially available molecules have been selected and evaluated for antitubercular activity and cytotoxicity using Vero, THP-1 and HepG2 cell lines with 4.3%, 4.2% and 2.7% hit rates, respectively. We demonstrate that models incorporating antitubercular and cytotoxicity data in Vero cells can significantly enrich the selection of non-toxic actives compared to random selection. Across all cell lines, the Molecular Libraries Small Molecule Repository (MLSMR) and cytotoxicity model identified ~10% of the hits in the top 1% screened (>10 fold enrichment). We also showed that seven out of nine Mtb active compounds from different academic published studies and eight out of eleven Mtb active compounds from a pharmaceutical screen (GSK) would have been identified by these Bayesian models.Combining clustering and Bayesian models represents a useful strategy for compound prioritization and hit-to lead optimization of antitubercular agents.
Keywords: bayesian models; clustering; Collaborative Drug Discovery Tuberculosis database; dual-event models; function class fingerprints; lead optimization; Mycobacterium tuberculosis ; tuberculosis

We evaluated the controlled release of lysozyme from various poly(D,L-lactic-co-glycolic acid) (PLGA) 50/50-polyethylene glycol (PEG) block copolymers relative to PLGA 50/50.Lysozyme was encapsulated in cylindrical implants (0.8 mm diameter) by a solvent extrusion method. Release studies were conducted in phosphate buffered saline +0.02% Tween 80 (PBST) at 37°C. Lysozyme activity was measured by a fluorescence-based assay. Implant erosion was evaluated by kinetics of polymer molecular weight decline, water uptake, and mass loss.Lysozyme release from an AB15 di-block copolymer (15% 5 kDa PEG, PLGA 28 kDa) was very fast, whereas an AB10 di-block copolymer (with 10% 5 kDa PEG, PLGA 45 kDa) and ABA10 tri-block copolymer (with 10% 6 kDa PEG, PLGA 27 kDa) showed release profiles similar to PLGA. We achieved continuous lysozyme release for up to 4 weeks from AB10 and ABA10 by lysozyme co-encapsulation with the pore-forming and acid-neutralizing MgCO3, and from AB15 by co-encapsulation of MgCO3 and blending AB15 with PLGA. Lysozyme activity was mostly recovered during 4 weeks.These block co-polymers may have utility either alone or as PLGA blends for the controlled release of proteins.
Keywords: controlled release; implants; lysozyme; PLGA-PEG block co-polymers

Use of a Fundamental Approach to Spray-Drying Formulation Design to Facilitate the Development of Multi-Component Dry Powder Aerosols for Respiratory Drug Delivery by Susan Hoe; James W. Ivey; Mohammed A. Boraey; Abouzar Shamsaddini-Shahrbabak; Emadeddin Javaheri; Sadaf Matinkhoo; Warren H. Finlay; Reinhard Vehring (449-465).
A fundamental approach incorporating current theoretical models into aerosol formulation design potentially reduces experimental work for complex formulations. A D-amino acid mixture containing D-Leucine (D-Leu), D-Methionine, D-Tryptophan, and D-Tyrosine was selected as a model formulation for this approach.Formulation design targets were set, with the aim of producing a highly dispersible D-amino acid aerosol. Particle formation theory and a spray dryer process model were applied with boundary conditions to the design targets, resulting in a priori predictions of particle morphology and necessary spray dryer process parameters. Two formulations containing 60% w/w trehalose, 30% w/w D-Leu, and 10% w/w remaining D-amino acids were manufactured.The design targets were met. The formulations had rugose and hollow particles, caused by deformation of a crystalline D-Leu shell while trehalose remained amorphous, as predicted by particle formation theory. D-Leu acts as a dispersibility enhancer, ensuring that both formulations: 1) delivered over 40% of the loaded dose into the in vitro lung region, and 2) achieved desired values of lung airway surface liquid concentrations based on lung deposition simulations.Theoretical models were applied to successfully achieve complex formulations with design challenges a priori. No further iterations to the design process were required.
Keywords: antimicrobial; D-amino acids; in silico ; lung; particle formation model; spray drying

Zanamivir Conjugated to Poly-L-Glutamine is Much More Active Against Influenza Viruses in Mice and Ferrets Than the Drug Itself by Alisha K. Weight; Jessica A. Belser; Terrence M. Tumpey; Jianzhu Chen; Alexander M. Klibanov (466-474).
Previously, polymer-attached zanamivir had been found to inhibit influenza A viruses in vitro far better than did small-molecule zanamivir (1) itself. The aim of this study was to identify in vitro—using the plaque reduction assay—a highly potent 1-polymer conjugate, and subsequently test its antiviral efficacy in vivo.By examining the structure-activity relationship of 1-polymer conjugates in the plaque assay, we have determined that the most potent inhibitor against several representative influenza virus strains has a neutral high-molecular-weight backbone and a short alkyl linker. We have examined this optimal polymeric inhibitor for efficacy and immunogenicity in the mouse and ferret models of infection. 1 attached to poly-L-glutamine is an effective therapeutic for established influenza infection in ferrets, reducing viral titers up to 30-fold for 6 days. There is also up to a 190-fold reduction in viral load when the drug is used as a combined prophylactic/therapeutic in mice. Additionally, we see no evidence that the drug conjugate stimulates an immune response in mice upon repeat administration. 1 attached to a neutral high-molecular-weight backbone through a short alkyl linker drastically reduced both in vitro and in vivo titers compared to those observed with 1 itself. Thus, further development of this polymeric zanamivir for the mitigation of influenza infection seems warranted.
Keywords: influenza virus; mouse model of infection; ferret model of infection; polymeric antiviral agents; zanamivir

Tumor-Specific Delivery and Therapy by Double-Targeted DTX-CMCS-PEG-NGR Conjugates by Fengxi Liu; Min Li; Chunxi Liu; Yongjun Liu; Yanchao Liang; Fengshan Wang; Na Zhang (475-488).
To synthesize and evaluate the antitumor efficacy of double-targeted docetaxel (DTX)-carboxymethyl chitosan (CMCS)-PEG-NGR (DTX-CPN) conjugates that could target to CD13 over-expressed tumor neovascular endothelium cells and tumor cells.DTX was conjugated to CMCS via biodegradable linker and cNGR was applied to endow the conjugates with double targeting ability. The physiochemical properties and stability of this DTX-CPN conjugates were characterized. Cellular uptake study was carried out to evaluate the targeting ability of DTX-CPN conjugates. Cytotoxicity and apoptosis analysis were conducted to evaluate in vitro antitumor effects. In vivo antitumor efficacy was investigated in B16 murine melanoma model.DTX-CPN conjugates could self-assemble into nanoparticles in water and were stable in plasma. cNGR modification could promote the cellular uptake of DTX-CPN conjugates in CD13 positive HUVEC and B16 cells, leading to more significant cytotoxicity and apoptosis effect than non-targeted conjugates. DTX-CPN conjugates also exhibited better antitumor effect than non-targeted conjugates and Duopafei® in a B16 murine melanoma model.Double-targeted DTX-CPN conjugates could efficiently target to tumor neovascular cells and tumor cells, and achieve good antitumor effects. DTX-CPN conjugates may be promising candidate for one-double targeting cancer therapy.
Keywords: Carboxymethyl chitosan; cNGR; Docetaxel; Double-targeted; Polymer-drug conjugates

4-[(1S,2S)-2-(4-cyclobutylpiperazine-1-carbonyl)cyclopropyl]benzamide (“AZ1”) is a histamine 3 (H3) autoreceptor in vivo antagonist. Sleep disturbance is a well-known class-effect for H3 antagonists and associated with high H3 receptor occupancy (RO) at night. The objective of the present work was to investigate if it was possible to obtain large diurnal fluctuations in RO for AZ1 and to suggest suitable doses for a Phase IIa study.Four Phase I studies were pooled and used to build a population pharmacokinetic model in NONMEM. Based on simulations of the PK model and the reported Ki-value for H3 RO from a human PET-study, RO vs. time profiles were simulated.The model well described the AZ1 pharmacokinetics. Simulations predicting plasma concentration and RO vs. time profiles for several doses were explored and doses with a wide range of fluctuation in RO over the dosing interval could be identified.By using population modeling and simulations of PK data and the Ki-value from a human PET study, predictions of RO vs. time for unstudied doses of AZ1 was made. Using this methodology it was possible to suggest doses with expected large diurnal fluctuations in RO.
Keywords: histamine; NONMEM; population modeling; receptor occupancy; simulation

To correlate the polymer’s degree of precipitation inhibition of indomethacin in solution to the amorphous stabilization in solid state.Precipitation of indomethacin (IMC) in presence of polymers was continuously monitored by a UV spectrophotometer. Precipitates were characterized by PXRD, IR and SEM. Solid dispersions with different polymer to drug ratios were prepared using solvent evaporation. Crystallization of the solid dispersion was monitored using PXRD. Modulated differential scanning calorimetry (MDSC), IR, Raman and solid state NMR were used to explore the possible interactions between IMC and polymers.PVP K90, HPMC and Eudragit E100 showed precipitation inhibitory effects in solution whereas Eudragit L100, Eudragit S100 and PEG 8000 showed no effect on IMC precipitation. The rank order of precipitation inhibitory effect on IMC was found to be PVP K90 > Eudragit E100 > HPMC. In the solid state, polymers showing precipitation inhibitory effect also exhibited amorphous stabilization of IMC with the same rank order of effectiveness. IR, Raman and solid state NMR studies showed that rank order of crystallization inhibition correlates with strength of molecular interaction between IMC and polymers.Correlation is observed in the polymers ability to inhibit precipitation in solution and amorphous stabilization in the solid state for IMC and can be explained by the strength of drug polymer interactions.
Keywords: molecular interactions; polymers; poorly soluble drugs; precipitation inhibition; solid dispersion

Effects of Antigen-Expressing Immunostimulatory Liposomes on Chemotaxis and Maturation of Dendritic Cells In Vitro and in Human Skin Explants by Anastasia Lanzi; Cynthia M. Fehres; Tanja D. de Gruijl; Yvette van Kooyk; Enrico Mastrobattista (516-526).
Antigen-Expressing Immunostimulatory Liposomes (AnExILs) represent a novel DNA vaccination platform based on the production of protein antigens from DNA templates inside liposomes mediated by an in vitro transcription and translation (IVTT) mix. The aim of this study was to analyze the effects of AnExILs on different dendritic cells (DCs) models and to better understand the role of the different components of this formulation on its adjuvanticity.The effect of β-galactosidase-expressing AnExILs on maturation and particle uptake by murine DC cell line, fresh human monocyte-derived DCs or human dermal DCs in skin explants was investigated and compared to the effects of either plain liposomes or IVTT mix alone.AnExILs induced efficient DC chemotaxis and promoted up-regulation of maturation markers on murine DCs, due to the presence of IVTT in the formulation. Furthermore, the amount of active βGal associated with DCs was higher for AnExILs than for free βGal expressed in IVTT or βGal encapsulated into non-adjuvanted liposomes. Most interestingly, the same trend was observed with human DCs.Both IVTT mix and liposomal vehicles were shown to be key components of the AnExIL formulation responsible for its adjuvanticity. AnExILs combine antigen production, adjuvanticity and delivery in one system, and can efficiently activate both murine and human DCs.
Keywords: cell-free protein synthesis; chemotaxis; DC activation; DNA-vaccine; liposome

AAPS Connection (527-529).