Pharmaceutical Research (v.32, #9)

Obituary: Paul M. Bummer (1955 – 2015) by Peter W. Swaan (2813-2813).

A New Vision for the Eye: Unmet Ocular Drug Delivery Needs by Hongwen M. Rivers; Siladitya Ray Chaudhuri; Jaymin C. Shah; Sachin Mittal (2814-2823).
Age related macular degenerationATP-binding cassette transporter, sub-family G (WHITE), member 2Chemistry, manufacturing and controlsCytomegalovirusChoroidal neovascularizationDiabetic macular edemaDiabetic retinopathyGanciclovirIntraocular pressureIntravitrealMicro-electro-mechanical systemsMultidrug resistance-associated protein transportersOrganic anion transportersOrganic cation transportersOptical coherence tomographyPlatelet-derived growth factorp-glycoproteinPoly-lactic-co-glycolic acidRetinal pigment epitheliumTriamcinolone acetonide suspensionVascular endothelial growth factor

Advancements in Tumor Targeting Strategies for Boron Neutron Capture Therapy by Micah John Luderer; Pilar de la Puente; Abdel Kareem Azab (2824-2836).
Boron neutron capture therapy (BNCT) is a promising cancer therapy modality that utilizes the nuclear capture reaction of epithermal neutrons by boron-10 resulting in a localized nuclear fission reaction and subsequent cell death. Since cellular destruction is limited to approximately the diameter of a single cell, primarily only cells in the neutron field with significant boron accumulation will be damaged. However, the emergence of BNCT as a prominent therapy has in large part been hindered by a paucity of tumor selective boron containing agents. While L-boronophenylalanine and sodium borocaptate are the most commonly investigated clinical agents, new agents are desperately needed due to their suboptimal tumor selectivity. This review will highlight the various strategies to improve tumor boron delivery including: nucleoside and carbohydrate analogs, unnatural amino acids, porphyrins, antibody-dendrimer conjugates, cationic polymers, cell-membrane penetrating peptides, liposomes and nanoparticles.
Keywords: Boron neutron capture therapy; BNCT; Cancer; Neutron radiation; Targeted delivery

Immunopotentiator-Loaded Polymeric Microparticles as Robust Adjuvant to Improve Vaccine Efficacy by Weifeng Zhang; Lianyan Wang; Tingyuan Yang; Yuan Liu; Xiaoming Chen; Qi Liu; Jilei Jia; Guanghui Ma (2837-2850).
Adjuvants are required to ensure the efficacy of subunit vaccines. Incorporating molecular immunopotentiators within particles could overcome drawbacks of molecular adjuvants (such as solubility and toxicity), and improve adjuvanticity of particles, achieving stronger adjuvant activity. Aim of this study is to evaluate the adjuvanticity of immunopotentiator-loaded polymeric particles for subunit vaccine.PLGA microparticles (PMPs) and imiquimod (TLR-7 ligand)-loaded PLGA microparticles (IPMPs) were prepared by SPG premix membrane emulsification. In vitro and in vivo studies were performed to their adjuvant activity, using ovalbumin and H5N1 influenza split vaccine as antigens.Incorporating imiquimod into microparticles significantly improved the efficacy of PLGA microparticles in activating BMDCs and pMΦs, and antigen uptake by pMΦs was also promoted. IPMPs showed stronger adjuvanticity to augment OVA-specific immune responses than PMPs. IgG subclass profiles and cytokine secretion levels by splenocytes indicated that IPMPs elicited more Th1-polarized immune response, compared to PMPs. In vivo study using H5N1 influenza split vaccine as antigen also confirmed the effects of IPMPs on antigen-specific cellular immunity.Considering adjuvanticity and safety profiles (PLGA and IMQ, both approved by FDA), we conclude that IMQ-loaded PLGA microparticles are promising robust adjuvant for subunit vaccines.
Keywords: adjuvant; imiquimod; immune response; microparticles; subunit vaccine

Local Sustained Delivery of 25-Hydroxyvitamin D3 for Production of Antimicrobial Peptides by Jiang Jiang; Guojun Chen; Franklin D. Shuler; Chi-Hwa Wang; Jingwei Xie (2851-2862).
This study seeks to develop fiber membranes for local sustained delivery of 25-hydroxyvitamin D3 to induce the expression and secretion of LL-37 at or near the surgical site, which provides a novel therapeutic approach to minimize the risk of infections.25-hydroxyvitamin D3 loaded poly(L-lactide) (PLA) and poly(ε-caprolactone) (PCL) fibers were produced by electrospinning. The morphology of obtained fibers was characterized using atomic force microscope (AFM) and scanning electron microscope (SEM). 25-hydroxyvitamin D3 releasing kinetics were quantified by enzyme-linked immunosorbent assay (ELISA) kit. The expression of cathelicidin (hCAP 18) and LL-37 was analyzed by immunofluorescence staining and ELISA kit. The antibacterial activity test was conducted by incubating pseudomonas aeruginosa in a monocytes’ lysis solution.AFM images suggest that the surface of PCL fibers is smooth, however, the surface of PLA fibers is relatively rough, in particular, after encapsulation of 25-hydroxyvitamin D3. The duration of 25-hydroxyvitamin D3 release can last more than 4 weeks for all the tested samples. Plasma treatment can promote the release rate of 25-hydroxyvitamin D3. Human keratinocytes and monocytes express significantly higher levels of hCAP18/LL-37 after incubation with plasma treated and 25-hydroxyvitamin D3 loaded PCL fibers than the cells incubated with around ten times amount of free drug. After incubation with this fiber formulation for 5 days LL-37 in the lysis solutions of U937 cells can effectively kill the bacteria.Plasma treated and 25-hydroxyvitamin D3 loaded PCL fibers induce significantly higher levels of antimicrobial peptide production in human keratinocytes and monocytes without producing cytotoxicity.
Keywords: 25-hydroxyvitamin D3 ; electrospinning; fibers; local sustained delivery; surgical site infection

A Flow-Cytometry-Based Approach to Facilitate Quantification, Size Estimation and Characterization of Sub-visible Particles in Protein Solutions by Christian Lubich; Mantas Malisauskas; Thomas Prenninger; Thomas Wurz; Peter Matthiessen; Peter L. Turecek; Friedrich Scheiflinger; Birgit M. Reipert (2863-2876).
Sub-visible particles were shown to facilitate unwanted immunogenicity of protein therapeutics. To understand the root cause of this phenomenon, a comprehensive analysis of these particles is required. We aimed at establishing a flow-cytometry-based technology to analyze the amount, size distribution and nature of sub-visible particles in protein solutions.We adjusted the settings of a BD FACS Canto II by tuning the forward scatter and the side scatter detectors and by using size calibration beads to facilitate the analysis of particles with sizes below 1 μM. We applied a combination of Bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt) and DCVJ (9-(2,2-dicyanovinyl)julolidine) to identify specific characteristics of sub-visible particles.The FACS technology allows the analysis of particles between 0.75 and 10 μm in size, requiring relatively small sample volumes. Protein containing particles can be distinguished from non-protein particles and cross-β-sheet structures contained in protein particles can be identified.The FACS technology provides robust and reproducible results with respect to number, size distribution and specific characteristics of sub-visible particles between 0.75 and 10 μm in size. Our data for number and size distribution of particles is in good agreement with results obtained with the state-of-the-art technology micro-flow imaging.
Keywords: cross-β-sheet structures; flow cytometry; protein aggregates; protein therapeutics; sub-visible particles

To study composition and heterogeneity of insoluble subvisible particles in Mab formulations resulting from degradation of polysorbate 20 and to develop a better understanding of the mechanisms of polysorbate degradation leading to particle formation.In this study, we exploit the potential of Raman microscopy for chemical identification of particles in monoclonal antibody formulations. Through a combination of experiments and density functional theory (DFT) calculations, we identified unique spectral marker bands for insoluble degradation products of polysorbate 20. We first applied our methodology to identify particles in model systems containing complex mixtures of fatty acids and then to subvisible particles in antibody formulations stored at 5°C for several years.Most of the subvisible particles identified were comprised of mixtures of fatty acids with no observable signal from fatty acid esters consistent with hydrolysis being the predominant degradation mechanism leading to particulate formation under these storage conditions.Our methodology is generally applicable for identification of particles in antibody formulations and, in particular, has the potential to give detailed information about particle heterogeneity and insight into mechanistic aspects of particle formation.
Keywords: degradation; fatty acid; particle identification; polysorbate 20; Raman spectroscopy

Combined Microdialysis-Tumor Homogenate Method for the Study of the Steady State Compartmental Distribution of a Hydrophobic Anticancer Drug in Patient-Derived Xenografts by Carles Monterrubio; Sonia Paco; Monica Vila-Ubach; Eva Rodríguez; Romina Glisoni; Cinzia Lavarino; Paula Schaiquevich; Alejandro Sosnik; Jaume Mora; Angel M. Carcaboso (2889-2900).
To develop a reproducible microdialysis-tumor homogenate method for the study of the intratumor distribution of a highly hydrophobic anticancer drug (SN-38; 7-ethyl-10-hydroxycamptothecin) in neuroblastoma patient-derived xenografts.We studied the nonspecific binding of SN-38 to the microdialysis tubing in the presence of 2-hydroxypropyl-beta-cyclodextrin (HPBCD) in the perfusate. We calibrated the microdialysis probes by the zero flow rate (ZFR) method and calculated the enhancement factor (f = extrapolated SN-38 concentration at the ZFR / SN-38 concentration in the dialysed solution) of HPBCD. We characterized the extravasation of HPBCD to tumors engrafted in mice. In vivo microdialysis and terminal homogenate data at the steady state (subcutaneous pump infusions) were used to calculate the volume of distribution of unbound SN-38 (Vu,tumor) in neuroblastoma.HPBCD (10% w/v) in the perfusate prevented the nonspecific binding of SN-38 to the microdialysis probe and enhanced SN-38 recovery (f = 1.86). The extravasation of HPBCD in the tumor during microdialysis was lower than 1%. Vu,tumor values were above 3 mL/g tumor for both neuroblastoma models and suggested efficient cellular penetration of SN-38.The method contributes to overcome the limitations of the microdialysis technique in hydrophobic drugs and provides a powerful tool to characterize compartmental anticancer drug distribution in xenografts.
Keywords: drug distribution; drug penetration; hydrophobic anticancer drugs; microdialysis; neuroblastoma; patient-derived xenograft (PDX); SN-38; solid tumor; steady state; tumor homogenate; tumor microenvironment

Novel Self-assembled, Gel-core Hyaluosomes for Non-invasive Management of Osteoarthritis: In-vitro Optimization, Ex-vivo and In-vivo Permeation by Wessam M. El-Refaie; Yosra S. R. Elnaggar; Magda A. El-Massik; Ossama Y. Abdallah (2901-2911).
Hyaluronic acid (HA) is an imperative biomaterial with desirable rheological properties to alleviate symptoms of osteoarthritis. Nevertheless, scantly percutaeous permeation of this macromolecule handicaps its effective use for orthopedics and triggers intra-articular injection as the only surrogate. This study presents novel self-assembeld HA-based gel core elastic nanovesicles, (hyaluosomes; GC-HS), for non-invasive transdermal delivery of HA.GC-HS were prepared with 1% HA using simple film hydration technique. Their size, zeta potential, percentage entrapment efficiency (% EE), elasticity, and ex-vivo transdermal permeation were evaluated compared to conventional liposomes CL. Structure elucidation of the formed novel system was performed using light, polarizing and transmission electron microscopy. In-vivo permeation of GC-HS through knee joints of female Sprague Dawley rats was compared to CL and HA alone.GC-HS showed nanosize (232.8 ± 7.2), high negative zeta potential (−45.1 ± 8.3) and higher elasticity (size alteration 5.43%) compared to CL. This novel system has self-penetration enhancing properties compared to CL and plain gel. GC-HS showed self-assembled properties and high physically stable for at least 6 months at 4°C. Ex-vivo permeation of HS was significantly higher than CL and plain HA gel alone. In-vivo study exhibited significant six folds increase in transdermal permeation of HA to knee joints from GC-HS compared to plain HA gel.Novel GC-HS are promising nanogels for topical management of osteoarthritis surrogating the need for intra-articular injection.
Keywords: elastic nanovesicles; gel-core; hyaluronic acid; osteoarthritis; transdermal

Modulation of Pharmacokinetic and Cytotoxicity Profile of Imatinib Base by Employing Optimized Nanostructured Lipid Carriers by Biki Gupta; Bijay Kumar Poudel; Tuan Hiep Tran; Roshan Pradhan; Hyuk-Jun Cho; Jee-Heon Jeong; Beom Soo Shin; Han-Gon Choi; Chul Soon Yong; Jong Oh Kim (2912-2927).
To prepare, optimize and characterize imatinib-loaded nanostructured lipid carriers (IMT-NLC), and evaluate their pharmacokinetic and cytotoxicity characteristics.IMT-NLC was prepared by hot homogenization method, and optimized by an approach involving Plackett-Burman design (PBD) and central composite design (CCD). An in vivo pharmacokinetic study was conducted in rats after both oral and intravenous administration. The in vitro cytotoxicity was evaluated by MTT assay on NCI-H727 cell-lines.PBD screening, followed by optimization by CCD and desirability function, yielded an optimized condition of 0.054, 6% w/w, 2.5% w/w and 1.25% w/v for organic-to-aqueous phase ratio (O/A), drug-to-lipid ratio (D/L), amount of lecithin (Lec) and amount of Tween® 20 (Tw20) respectively. The optimized IMT-NLC exhibited a particle size (Sz) of 148.80 ± 1.37 nm, polydispersity index (PDI) 0.191 ± 0.017 of and ζ-potential of −23.0 ± 1.5 mV, with a drug loading (DL) of 5.48 ± 0.01% and encapsulation efficiency (EE) of 97.93 ± 0.03%. IMT-NLC displayed sustained IMT release in vitro, significantly enhanced in vivo bioavailability of IMT after intravenous and oral administration, and greater in vitro cytotoxicity on NCI-H727 cells, compared with free IMT.A combined DoE approach enabled accurate optimization and successful preparation of IMT-NLC with enhanced in vivo pharmacokinetic and in vitro cytotoxicity characteristics.
Keywords: nanostructured lipid carrier; imatinib base; Plackett-Burman design; central composite design; bioavailability

The Effect of Active Pharmaceutical Ingredients on Aerosol Electrostatic Charges from Pressurized Metered Dose Inhalers by Yang Chen; Paul M. Young; David F. Fletcher; Hak Kim Chan; Edward Long; David Lewis; Tanya Church; Daniela Traini (2928-2936).
This study investigated the effect of different active pharmaceutical ingredients (API) on aerosol electrostatic charges and aerosol performances for pressurized metered dose inhalers (pMDIs), using both insulating and conducting actuators.Five solution-based pMDIs containing different API ingredients including: beclomethasone dipropionate (BDP), budesonide (BUD), flunisolide (FS), salbutamol base (SB) and ipratropium bromide (IPBr) were prepared using pressure filling technique. Actuator blocks made from nylon, polytetrafluoroethylene (PTFE) and aluminium were manufactured with 0.3 mm nominal orifice diameter and cone nozzle shape. Aerosol electrostatics for each pMDI formulation and actuator were evaluated using the electrical low-pressure impactor (ELPI) and drug depositions were analysed using high performance liquid chromatography (HPLC).All three actuator materials showed the same net charge trend across the five active drug ingredients, with BDP, BUD and FS showing positive net charges for both nylon and PTFE actuators, respectively. While SB and IPBr had significantly negative net charges across the three different actuators, which correlates to the ionic functional groups present on the drug molecule structures.The API present in a pMDI has a dominant effect on the electrostatic properties of the formulation, overcoming the charge effect arising from the actuator materials. Results have shown that the electrostatic charges for a solution-based pMDI could be related to the interactions of the chemical ingredients and change in the work function for the overall formulation.
Keywords: aerosols; APIs; electrostatics charges; metered dose inhalers

Unstirred Water Layers and the Kinetics of Organic Cation Transport by Takahiro Shibayama; Mark Morales; Xiaohong Zhang; Lucy J. Martínez-Guerrero; Alfred Berteloot; Timothy W. Secomb; Stephen H. Wright (2937-2949).
Unstirred water layers (UWLs) present an unavoidable complication to the measurement of transport kinetics in cultured cells, and the high rates of transport achieved by overexpressing heterologous transporters exacerbate the UWL effect. This study examined the correlation between measured Jmax and Kt values and the effect of manipulating UWL thickness or transport Jmax on the accuracy of experimentally determined kinetics of the multidrug transporters, OCT2 and MATE1.Transport of TEA and MPP was measured in CHO cells that stably expressed human OCT2 or MATE1. UWL thickness was manipulated by vigorous reciprocal shaking. Several methods were used to manipulate maximal transport rates.Vigorous stirring stimulated uptake of OCT2-mediated transport by decreasing apparent Kt (Ktapp) values. Systematic reduction in transport rates was correlated with reduction in Ktapp values. The slope of these relationships indicated a 1500 μm UWL in multiwell plates. Reducing the influence of UWLs (by decreasing either their thickness or the Jmax of substrate transport) reduced Ktapp by 2-fold to >10-fold.Failure to take into account the presence of UWLs in experiments using cultured cells to measure transport kinetics can result in significant underestimates of the apparent affinity of multidrug transporters for substrates.
Keywords: kinetics; organic cation; transport; unstirred water layers

Particle Image Velocimetry Evaluation of Fluid Flow Profiles in USP 4 Flow-Through Dissolution Cells by Hiroyuki Yoshida; Akemi Kuwana; Hiroko Shibata; Ken-ichi Izutsu; Yukihiro Goda (2950-2959).
To evaluate fluid flow profiles in the flow-through cell (FTC, USP apparatus 4) system with pulsatile and non-pulsatile pumps.Instantaneous velocity vectors in the dissolution cells were obtained from images sequentially captured by a particle image velocimetry (PIV) system. The data were sorted to follow the pump pulse cycle.The analysis showed changes in the flow profiles during a pump pulse (0.5 s) at a 0.025-s interval in two sizes of cells installed in the FTC system. Supplying a slow flow from the pulsatile pump induced instantaneous downward (inner layer) and upward (outer layer) flow in the larger cell during the suction phase. Analysis at varied medium and cell temperatures strongly suggested a contribution of natural convection to the complex flow caused by relatively high cell temperature. Uniform upward flow was observed in other cells and flow rate conditions. The time-averaged vertical velocities in the cells were similar in the pulsatile and non-pulsatile pump systems.The PIV analysis provides information on how flow rate and pump pulse affect fluid flow profiles at multiple points in flow-through dissolution cells. An appropriate temperature control should reduce the complex flow of the medium in the FTC system.
Keywords: dissolution testing; flow-through cell system; hydrodynamics; particle image velocimetry; pulsatile pump

Vaginal Microbicide Film Combinations of Two Reverse Transcriptase Inhibitors, EFdA and CSIC, for the Prevention of HIV-1 Sexual Transmission by Wei Zhang; Minlu Hu; Yuan Shi; Tiantian Gong; Charlene S. Dezzutti; Bernard Moncla; Stefan G. Sarafianos; Michael A. Parniak; Lisa C. Rohan (2960-2972).
EFdA is a potent nucleoside reverse transcriptase inhibitor (NRTI) with activity against a wide spectrum of wild-type and drug resistant HIV-1 variants. CSIC is a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) with demonstrated anti-HIV properties important for use in topical prevention of HIV transmission. The objective of this study was to develop and characterize film-formulated EFdA and CSIC for use as a female-controlled vaginal microbicide to prevent sexual transmission of HIV.Assessments of EFdA- and CSIC-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity studies, compatibility with the normal vaginal Lactobacillus flora and anti-HIV bioactivity evaluations.No significant change in physicochemical properties or biological activity of the combination films were noted during 3 months storage. In vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) of either EFdA or CSIC was several orders of magnitude higher than the 50% effective concentration (EC50) values. Film-formulated EFdA and CSIC combination showed additive inhibitory activity against wild type and drug-resistant variants of HIV. Epithelial integrity studies demonstrated that the combination vaginal film had a much lower toxicity to HEC-1A monolayers compared to that of VCF®, a commercial vaginal film product containing nonoxynol-9. Polarized ectocervical explants showed films with drug alone or in combination were effective at preventing HIV infection.Our data suggest that vaginal microbicide films containing a combination of the NRTI EFdA and the NNRTI CSIC have potential to prevent HIV-1 sexual transmission.
Keywords: CSIC; EFdA; HIV prevention; microbicide; vaginal film

Characterization of efflux transport proteins of the human choroid plexus papilloma cell line HIBCPP, a functional in vitro model of the blood-cerebrospinal fluid barrier by Alexandra Bernd; Melanie Ott; Hiroshi Ishikawa; Horst Schroten; Christian Schwerk; Gert Fricker (2973-2982).
To characterize the human choroid plexus (CP) papilloma cell line HIBCPP with respect to ABC export protein expression and function in order to evaluate its use as an in vitro model to study carrier-mediated transport processes at the CP.Expression profiles of ABC transporters were studied by quantitative real-time PCR and Western Blot analysis. Functionality of transporters was investigated by means of uptake experiments and permeation studies carried out on permeable filter systems. In addition, immunohistochemistry served to study localization of ABCC1 and ABCC4.Both qPCR and Western Blot revealed that ABC transporters known to be expressed in CP are also expressed in HIBCPP cells. Immunohistochemistry confirmed basolateral expression of ABCC1. Functionality of ABCC1, ABCC4, ABCB1 and ABCG2 could be shown in uptake assays.Altogether, the HIBCPP cells promise to be a functional and relevant in vitro tool to investigate transport processes at the blood-cerebrospinal fluid barrier.
Keywords: ABC transporter; barrier function; blood-cerebrospinal fluid barrier; HIBCPP; human cell line

Preparation and Evaluation of Multiple Nanoemulsions Containing Gadolinium (III) Chelate as a Potential Magnetic Resonance Imaging (MRI) Contrast Agent by Estelle Sigward; Yohann Corvis; Bich-Thuy Doan; Kadri Kindsiko; Johanne Seguin; Daniel Scherman; Denis Brossard; Nathalie Mignet; Philippe Espeau; Sylvie Crauste-Manciet (2983-2994).
The objective was to develop, characterize and assess the potentiality of W1/O/W2 self-emulsifying multiple nanoemulsions to enhance signal/noise ratio for Magnetic Resonance Imaging (MRI).For this purpose, a new formulation, was designed for encapsulation efficiency and stability. Various methods were used to characterize encapsulation efficiency ,in particular calorimetric methods (Differential Scanning Calorimetry (DSC), thermogravimetry analysis) and ultrafiltration. MRI in vitro relaxivities were assessed on loaded DTPA-Gd multiple nanoemulsions.Characterization of the formulation, in particular of encapsulation efficiency was a challenge due to interactions found with ultrafiltration method. Thanks to the specifically developed DSC protocol, we were able to confirm the formation of multiple nanoemulsions, differentiate loaded from unloaded nanoemulsions and measure the encapsulation efficiency which was found to be quite high with a 68% of drug loaded. Relaxivity studies showed that the self-emulsifying W/O/W nanoemulsions were positive contrast agents, exhibiting higher relaxivities than those of the DTPA-Gd solution taken as a reference.New self-emulsifying multiple nanoemulsions that were able to load satisfactory amounts of contrasting agent were successfully developed as potential MRI contrasting agents. A specific DSC protocol was needed to be developed to characterize these complex systems as it would be useful to develop these self-formation formulations.
Keywords: differential scanning calorimetry; magnetic resonance imaging; multiple nanoemulsions; self-emulsifying; ultrafiltration

Focused Ultrasound as a Scalable and Contact-Free Method to Manufacture Protein-Loaded PLGA Nanoparticles by Stefan Schiller; Andrea Hanefeld; Marc Schneider; Claus-Michael Lehr (2995-3006).
Although nanomaterials are under investigation for a very broad range of medical applications, only a small fraction of these are already commercialized or in clinical development. A major challenge for the translation of nanomedicines into the clinic is the missing scalability of the available lab scale preparation methods and, ultimately, non-identical samples during early and late research.Protein-loaded PLGA nanoparticles using focused ultrasound in an emulsion solvent diffusion method were prepared in different batch sizes to evaluate achievable mean size, protein loading, and yield.Using the same equipment, nanoparticles could be prepared in batch sizes from 1 mg to 2.5 g. Size and yield were directly controllable by the amount of incident energy with good reproducibility. The nanoparticles displayed similar mean size, protein loading, and nanoparticle yield in batch sizes over three orders of magnitude. A scalable purification method based on diafiltration was established.The proposed method enables for feasibility studies during early research using just a small amount of polymer and protein, while at the same time it allows for larger scale production at later stages. As the proposed method further relies on contact-free energy transmission, it is especially suited for the preparation of clinical research samples.
Keywords: drug delivery; focused ultrasound; nanoparticles; nanotechnology; protein

Production of Highly Charged Pharmaceutical Aerosols Using a New Aerosol Induction Charger by Laleh Golshahi; P. Worth Longest; Landon Holbrook; Jessica Snead; Michael Hindle (3007-3017).
Properly charged particles can be used for effective lung targeting of pharmaceutical aerosols. The objective of this study was to characterize the performance of a new induction charger that operates with a mesh nebulizer for the production of highly charged submicrometer aerosols to bypass the mouth-throat and deliver clinically relevant doses of medications to the lungs.Variables of interest included combinations of model drug (albuterol sulfate) and charging excipient (NaCl) as well as strength of the charging field (1–5 kV/cm). Aerosol charge and size were measured using a modified electrical low pressure impactor system combined with high performance liquid chromatography.At the approximate mass median aerodynamic diameter (MMAD) of the aerosol (~0.4 μm), the induction charge on the particles was an order of magnitude above the field and diffusion charge limit. The nebulization rate was 439.3 ± 42.9 μl/min, which with a 0.1% w/v solution delivered 419.5 ± 34.2 μg of medication per minute. A new correlation was developed to predict particle charge produced by the induction charger.The combination of the aerosol induction charger and predictive correlations will allow for the practical generation and control of charged submicrometer aerosols for targeting deposition within the lungs.
Keywords: charged particle generation; charged submicrometer aerosols; induction charging; pharmaceutical aerosols; respiratory drug delivery

Reducing the Visibility of the Vector/DNA Nanocomplexes to the Immune System by Elastin-Like Peptides by Faranak S. Nouri; Xing Wang; Xuguang Chen; Arash Hatefi (3018-3028).
One of the major hurdles facing nanomedicines is the antibody production against nanoparticles that subsequently results in their opsonization and clearance by macrophages. The objective of this research was to examine and identify the sequence of a low-immunogenic peptide based on recombinant elastin-like polypeptides (ELPs) that does not evoke IgG response and can potentially be used for masking the surfaces of the nanoparticles.Biopolymers composed of a DNA condensing domain in fusion with anionic, neutral and cationic elastin-like peptides were genetically engineered. The biopolymers were used to complex with plasmid DNA and form ELP-coated nanoparticles. Then, the potential immunogenicity of nanoparticles in terms of IgM/IgG response after repeated injections was evaluated in Balb/c immunocompetent mice.The results revealed the sequence of a non-immunogenic ELP construct that in comparison to control group did not elicit any significant IgG response, whereas the vector/DNA complexes that were coated with polyethylene glycol (PEG) did elicit significant IgG response under the same conditions.The identification of the sequence of an ELP-based peptide that does not induce IgG response opens the door to more focused in-depth immunotoxicological studies which could ultimately lead to the production of safer and more effective drug/gene delivery systems such as liposomes, micelles, polymeric nanoparticles, viruses and antibodies.
Keywords: elastin; elastin-like polypeptides; ELP; PEG immunogenicity; recombinant polymer

To assess the antiangiogenic effect of bumetanide with dynamic contrast enhanced (DCE)-MRI and a biodegradable macromolecular MRI contrast agent.A new polydisulfide containing macrocyclic gadolinium (Gd(III)) chelates, poly([(Gd-DOTA)-DETA]-co-DTBP) (GODP), was synthesized as a safe biodegradable macromolecular MRI contrast agent for DCE-MRI. Nude mice bearing flank HT29 colon cancer xenografts were then treated daily with either bumetanide or saline for a total of 3 weeks. DCE-MRI was performed before and after the treatment weekly. The DCE-MRI data were analyzed using the adiabiatic approximation to the tissue homogeneity (AATH) model to assess the change of tumor vascularity in response to the treatment. Immunohistochemistry (IHC) and western blot were performed to study tumor angiogenic biomarkers and hypoxia.DCE-MRI with GODP revealed that bumetanide reduced vascular permeability and plasma volume fraction by a significantly greater extent than the saline control therapy after 3 weeks of therapy. These changes were verified by the significant decline of CD31 and VEGF expression in the bumetanide treatment group. Despite a significant regression in vascularity, the tumors remained highly proliferative. Overexpression of the transcription factor HIF-1α in response to elevated hypoxia is thought to be the driving force behind the uninterrupted tumor expansion.This study demonstrated the effectiveness of DCE-MRI with GODP in detecting vascular changes following the administration of bumetanide. Bumetanide has the potential to curtail growth of the tumor vasculature and can be employed in future therapeutic strategies.
Keywords: anti-angiogenesis; bumetanide; cancer therapy; DCE-MRI; polydisulfide

Identification and Assessment of Octreotide Acylation in Polyester Microspheres by LC–MS/MS by Mehrnoosh Shirangi; Wim E. Hennink; Govert W. Somsen; Cornelus F. van Nostrum (3044-3054).
Polyesters with hydrophilic domains, i.e., poly(d,l-lactic-co-glycolic-co-hydroxymethyl glycolic acid) (PLGHMGA) and a multiblock copolymer of poly(ε-caprolactone)-PEG-poly(ε-caprolactone) and poly(l-lactide) ((PC-PEG-PC)-(PL)) are expected to cause less acylation of encapsulated peptides than fully hydrophobic matrices. Our purpose is to assess the extent and sites of acylation of octreotide loaded in microspheres using tandem mass spectrometry analysis.Octreotide loaded microspheres were prepared by a double emulsion solvent evaporation technique. Release profiles of octreotide from hydrophilic microspheres were compared with that of PLGA microspheres. To scrutinize the structural information and localize the actual modification site(s) of octreotide, liquid chromatography ion-trap mass spectrometry (LC-ITMS) was performed on the acylated adducts.Hydrophilic microspheres showed less acylated adducts in comparison with PLGA microspheres. LC-MS/MS showed that besides the N-terminus and primary amine of lysine, the primary hydroxyl of the end group of octreotide was also subjected to acylation. Nucleophilic attack of the peptide can also occur to the carbamate bond presented in (PC-PEG-PC)-(PL) since 1,4-butanediisocyanate was used as the chain extender.Hydrophilic polyesters are promising systems for controlled release of peptide because substantially less acylation occurs in microspheres based on these polymers. LC-ITMS provided detailed structural information of octreotide modifications via mass analysis of ion fragments.
Keywords: acylation; aliphatic polyester; LC–MS/MS; octreotide

Experimental Blood–Brain Barrier (BBB) permeability models for drug molecules are expensive and time-consuming. As alternative methods, several traditional Quantitative Structure-Activity Relationship (QSAR) models have been developed previously. In this study, we aimed to improve the predictivity of traditional QSAR BBB permeability models by employing relevant public bio-assay data in the modeling process.We compiled a BBB permeability database consisting of 439 unique compounds from various resources. The database was split into a modeling set of 341 compounds and a validation set of 98 compounds. Consensus QSAR modeling workflow was employed on the modeling set to develop various QSAR models. A five-fold cross-validation approach was used to validate the developed models, and the resulting models were used to predict the external validation set compounds. Furthermore, we used previously published membrane transporter models to generate relevant transporter profiles for target compounds. The transporter profiles were used as additional biological descriptors to develop hybrid QSAR BBB models.The consensus QSAR models have R2 = 0.638 for five-fold cross-validation and R2 = 0.504 for external validation. The consensus model developed by pooling chemical and transporter descriptors showed better predictivity (R2 = 0.646 for five-fold cross-validation and R2 = 0.526 for external validation). Moreover, several external bio-assays that correlate with BBB permeability were identified using our automatic profiling tool.The BBB permeability models developed in this study can be useful for early evaluation of new compounds (e.g., new drug candidates). The combination of chemical and biological descriptors shows a promising direction to improve the current traditional QSAR models.
Keywords: biological descriptors; blood–brain barrier; hybrid model; permeability

Multilayered Thin Films from Boronic Acid-Functional Poly(amido amine)s by Sry D. Hujaya; Johan F. J. Engbersen; Jos M. J. Paulusse (3066-3086).
To investigate the properties of phenylboronic acid-functional poly(amido amine) polymers (BA-PAA) in forming multilayered thin films with poly(vinyl alcohol) (PVA) and chondroitin sulfate (ChS), and to evaluate their compatibility with COS-7 cells.Copolymers of phenylboronic acid-functional poly(amido amine)s, differing in the content of primary amine (DAB-BA-PAA) or alcohol (ABOL-BA-PAA) side groups, were synthesized and applied in the formation of multilayers with PVA and ChS. Biocompatibility of the resulting films was evaluated through cell culture experiments with COS-7 cells grown on the films.PVA-based multilayers were thin, reaching ~100 nm at 10 bilayers, whereas ChS-based multilayers were thick, reaching ~600 nm at the same number of bilayers. All of the multilayers are stable under physiological conditions in vitro and are responsive to reducing agents, owing to the presence of disulfide bonds in the polymers. PVA-based films were demonstrated to be responsive to glucose at physiological pH at the investigated glucose concentrations (10–100 mM). The multilayered films displayed biocompatibility in cell culture experiments, promoting attachment and proliferation of COS-7 cells.Responsive thin films based on boronic acid functional poly(amido amine)s are promising biocompatible materials for biomedical applications, such as drug releasing surfaces on stents or implants. Graphical Abstract Layer-by-Layer Assembly
Keywords: biodegradable polymers; chondroitin sulfate; layer-by-layer assembly; multilayered thin films; poly(vinyl alcohol)

In Vitro and In Vivo Efficacy of Self-Assembling RGD Peptide Amphiphiles for Targeted Delivery of Paclitaxel by Poonam Saraf; Xiaoling Li; Lisa Wrischnik; Bhaskara Jasti (3087-3101).
The objective of this work was to compare the efficacy of self-assembling cyclic and linear RGD peptide amphiphiles as carriers for delivering paclitaxel to αvβ3 integrin overexpressing tumors.Linear (C18-ADA5-RGD) and cyclic (C18-ADA5-cRGDfK) peptide amphiphiles were synthesized and characterized for CMC, aggregation number and micelle stability using fluorescence spectroscopy methods. Size and morphology of micelles was studied using TEM. Fluorescence polarization and confocal microscopy assays were established to compare binding and internalization of micelles. The targeting efficacy was studied in A2058 cells using cytotoxicity assay as well as in vivo in melanoma xenograft mouse model.The linear and cyclic RGD amphiphiles exhibited CMC of 25 and 8 μM, respectively, formed nano-sized spherical micelles and showed competitive binding to αvβ3 integrin protein. FITC-loaded RGD micelles rapidly internalized into A2058 melanoma cells. Paclitaxel-loaded RGD micelles exhibited higher cytotoxicity compared with free drug in A2058 cells in vitro as well as in vivo.Cyclic RGD micelles exhibited better targeting efficacy but were less effective compared to linear RGD micelles as drug delivery vehicle due to lower drug solubilization capacity and lesser kinetic stability. Results from the study proved the effectiveness of self-assembling low molecular weight RGD amphiphiles as carriers for targeted delivery of paclitaxel.
Keywords: micelles; paclitaxel; RGD; self-assembly; targeting

The large increase in viscosity of highly concentrated monoclonal antibody solutions can be challenging for downstream processing, drug formulation, and delivery steps. The objective of this work was to examine the viscosity of highly concentrated solutions of a high purity IgG1 monoclonal antibody over a wide range of protein concentrations, solution pH, ionic strength, and in the presence / absence of different excipients.Experiments were performed with an IgG1 monoclonal antibody provided by Amgen. The steady-state viscosity was evaluated using a Rheometrics strain-controlled rotational rheometer with a concentric cylinder geometry.The viscosity data were well-described by the Mooney equation. The data were analyzed in terms of the antibody virial coefficients obtained from osmotic pressure data evaluated under the same conditions. The viscosity coefficient in the absence of excipients was well correlated with the third osmotic virial coefficient, which has a negative value (corresponding to short range attractive interactions) at the pH and ionic strength examined in this work.These results provide important insights into the effects of intermolecular protein-protein interactions on the behavior of highly concentrated antibody solutions.
Keywords: antibody; protein interactions; rheology; virial coefficients; viscosity

Salt Stability – The Effect of pHmax on Salt to Free Base Conversion by Yi-Ling Hsieh; Jeremy M. Merritt; Weili Yu; Lynne S. Taylor (3110-3118).
The aim of this study was to investigate how the disproportionation process can be impacted by the properties of the salt, specifically pHmax.Five miconazole salts and four sertraline salts were selected for this study. The extent of conversion was quantified using Raman spectroscopy. A mathematical model was utilized to estimate the theoretical amount of conversion.A trend was observed that for a given series of salts of a particular basic compound (both sertraline and miconazole are bases), the extent of disproportionation increases as pHmax decreases. Miconazole phosphate monohydrate and sertraline mesylate, although exhibiting significantly different pHmax values (more than 2 units apart), underwent a similar extent of disproportionation, which may be attributed to the lower buffering capacity of sertraline salts.This work shows that the disproportionation tendency can be influenced by pHmax and buffering capacity and thus highlights the importance of selecting the appropriate salt form during the screening process in order to avoid salt-to-free form conversion.
Keywords: buffering capacity; mathematical modelling; pHmax ; salt disproportionation

Erratum to: Curdlan-conjugated PLGA Nanoparticles possess macrophage stimulant activity and drug delivery capabilities by Matshawandile Tukulula; Rose Hayeshi; Pascaline Fonteh; Debra Meyer; Abongile Ndamase; Michael T. Madziva; Vincent Khumalo; Philip Labuschagne; Brendon Naicker; Hulda Swai; Admire Dube (3119-3119).

AAPSConnection (3120-3122).