Pharmaceutical Research (v.31, #11)

Many drugs and desirable phytochemicals are bitter, and bitter tastes are aversive. Food and pharmaceutical manufacturers share a common need for bitterness-masking strategies that allow them to deliver useful quantities of the active compounds in an acceptable form and in this review we compare and contrast the challenges and approaches by researchers in both fields. We focus on physical approaches, i.e., micro- or nano-structures to bind bitter compounds in the mouth, yet break down to allow release after they are swallowed. In all of these methods, the assumption is the degree of bitterness suppression depends on the concentration of bitterant in the saliva and hence the proportion that is bound. Surprisingly, this hypothesis has only rarely been fully tested using a combination of adequate human sensory trials and measurements of binding. This is especially true in pharmaceutical systems, perhaps due to the greater experimental challenges in sensory analysis of drugs.
Keywords: bitter; emulsion; encapsulation; food; taste

Modulating Protein Release Profiles by Incorporating Hyaluronic Acid into PLGA Microparticles Via a Spray Dryer Equipped with a 3-Fluid Nozzle by Feng Wan; Morten Jonas Maltesen; Sune Klint Andersen; Simon Bjerregaard; Stefania G. Baldursdottir; Camilla Foged; Jukka Rantanen; Mingshi Yang (2940-2951).
The purpose of this study was to modulate the release profiles of the model protein drug from spray dried poly(DL-lactic-co-glycolic acid) (PLGA) microparticles by incorporating hyaluronic acid (HA) in the formulation.Bovine serum albumin (BSA)-loaded PLGA microparticles with or without HA were prepared using a spray dryer equipped with a 3-fluid nozzle. The effects of HA on the surface tension and the rheological behavior of the inner feed solution were investigated. The physicochemical properties of the resulting microparticles were characterized using scanning electron microscopy (SEM), laser diffraction (LD), confocal laser scanning microscopy (CLSM) and X-ray photoelectron spectroscopy (XPS). Circular dischoism (CD) was used to characterize conformational integrity of BSA released from the microparticles.Spherical microparticles with D50 of 5–10 μm were obtained. Addition of HA in inner feed solutions increased the feed viscosity, but with no influence on the surface tension. All inner feed solutions showed non-Newtonian shear thinning behavior and the rheological properties were not time dependent. The CLSM and XPS analyses suggested a core-shell like structure of the microparticles when HA was incorporated. The release profiles of BSA were extended and the initial burst releases were suppressed with an increase in HA in the microparticle formulations. In addition, HA seemed to protect BSA from degradation upon the spray-drying process.The present work demonstrates the potential of HA to modulate protein release profile from PLGA microparticle formulations produced via spray drying using 3-fluid nozzle.
Keywords: controlled release; hyaluronic acid; microencapsulation; PLGA microparticles; protein; spray drying

Cerium Oxide Nanoparticles Inhibit Adipogenesis in Rat Mesenchymal Stem Cells: Potential Therapeutic Implications by Antonella Rocca; Virgilio Mattoli; Barbara Mazzolai; Gianni Ciofani (2952-2962).
Cerium oxide nanoparticles (nanoceria, NC) have extraordinary antioxidant activity that made them suitable as a therapeutic agent for several diseases where reactive oxygen species (ROS) act by impairing the normal redox balance. Among different functions, it has been proven that ROS are cellular messengers involved in the adipogenesis: we thus investigated the implication of NC administration in the potential inhibition of adipogenic differentiation of mesenchymal stem cells (MSCs) used as a model of adipogenesis.We evaluated cytotoxic effects and adipogenic maturation of mesenchymal stem cells following in vitro NC administration, both at gene and at phenotype level.Overall, our results demonstrated that NC efficiently inhibit the maturation of MSCs toward adipocytes owing to their ability to reduce the production of the ROS necessary during adipogenesis.These findings, even if preliminary, represent an important step toward the potential pharmaceutical application of NC in the treatment of obesity.
Keywords: Adipogenesis; Cerium oxide nanoparticles; Mesenchymal stem cells; Reactive oxygen species

A Novel High-Speed Imaging Technique to Predict the Macroscopic Spray Characteristics of Solution Based Pressurised Metered Dose Inhalers by Nicolas A. Buchmann; Daniel J. Duke; Sayed A. Shakiba; Daniel M. Mitchell; Peter J. Stewart; Daniela Traini; Paul M. Young; David A. Lewis; Julio Soria; Damon Honnery (2963-2974).
Non-volatile agents such as glycerol are being introduced into solution-based pMDI formulations in order to control mean precipitant droplet size. To assess their biopharmaceutical efficacy, both microscopic and macroscopic characteristics of the plume must be known, including the effects of external factors such as the flow generated by the patient’s inhalation. We test the hypothesis that the macroscopic properties (e.g. spray geometry) of a pMDI spray can be predicted using a self-similarity model, avoiding the need for repeated testing.Glycerol-containing and glycerol-free pMDI formulations with matched mass median aerodynamic diameters are investigated. High-speed schlieren imaging is used to extract time-resolved velocity, penetration and spreading angle measurements of the pMDI spray plume. The experimental data are used to validate the analytical model.The pMDI spray develops in a manner characteristic of a fully-developed steady turbulent jet, supporting the hypothesis. Equivalent glycerol-containing and non glycerol-containing formulations exhibit similar non-dimensional growth rates and follow a self-similar scaling behaviour over a range of physiologically relevant co-flow rates.Using the proposed model, the mean leading edge penetration, velocity and spreading rate of a pMDI spray may be estimated a priori for any co-flow conditions. The effects of different formulations are captured in two scaling constants. This allows formulators to predict the effects of variation between pMDIs without the need for repeated testing. Ultimately, this approach will allow pharmaceutical scientists to rapidly test a number of variables during pMDI development.
Keywords: co-flow; glycerol; HFA; high-speed schlieren imaging; modelling; pMDI

New strategy to surface functionalization of polymeric nanoparticles: one-pot synthesis of scFv anti-LDL(−)-functionalized nanocapsules by Eduardo A. Bender; Marcela F. Cavalcante; Márcia D. Adorne; Letícia M. Colomé; Sílvia S. Guterres; Dulcinéia S. P. Abdalla; Adriana R. Pohlmann (2975-2987).
In general, the surface functionalization of polymeric nanoparticles is carried out by covalently bounding ligands to the nanoparticle surface. This process can cause a lack or decrease of the ligand specificity to its target receptor, besides the need of purification steps. We proposed a ligand-metal-chitosan-lecithin complex as a new strategy to functionalize the surface of biodegradable nanoparticles.One pot synthesis of scFv anti-LDL(−)-functionalized nanocapsules was carried out by self-assembly and interfacial reactions. Particle sizing techniques, lipid peroxidation and molecular recognition by enzyme linked immuno sorbent assays were carried out.The selected formulation had unimodal size distribution with mean diameter of about 130 nm. The metals in the complex did not enhance the oxidative stress, and the scFv anti-LDL(−)-functionalized nanocapsules recognized LDL(−) and did not react with native LDL indicating the maintenance of the active site of the fragment.The one pot synthesis, using the ligand-metal-chitosan-lecithin complex to functionalize the surface of the biodegradable nanocapsules, maintained the active site of the antibody fragment making the device interesting for applications in nanomedicine.
Keywords: LDL single-chain fragment variable; ligand-metal-chitosan-lecithin; lipid-core nanocapsules; polycaprolactone

Hyaluronan-Based Nanocarriers with CD44-Overexpressed Cancer Cell Targeting by Shuangshuang Song; Huan Qi; Jingwen Xu; Pan Guo; Fen Chen; Fei Li; Xinggang Yang; Naicheng Sheng; Yingliang Wu; Weisan Pan (2988-3005).
The objective of the work was to evaluate the potential of hyaluronan-based nanoparticles as tumor-targeting nano-systems for CD44-overexpressed cancer therapy.The synthesized amphiphilic cholesteryl succinoyl hyaluronan (Chol-Suc-HA) conjugates self-assembled into docetaxel(DTX)-loaded nanoparticles in the aqueous environment. The physiochemical properties of Chol-Suc-HA-DTX NPs were characterized. The in vitro cytotoxicity of Chol-Suc-HA-DTX NPs against MCF-7, 4T1, A549 and L929 cells was evaluated using MTT and LDH assays. Moreover, the cellular uptake mechanism was investigated using the CLSM and flow cytometry. The in vivo animal experiments of Chol-Suc-HA-DTX NPs including pharmacokinetic evaluation, bio-distribution observed by EX vivo NIRF imaging and antitumor efficacy were also carried out in SD rats or 4T1 tumor-bearing BALB/c mice.The self-assembled Chol-Suc-HA-DTX NPs with different degree of substitution (DS) of hydrophobic moiety exhibited high drug loading, uniform particle size distribution and excellent in vitro stability. However, the plasma stability of Chol-Suc-HA-DTX NPs was significantly influenced by the DS of hydrophobic moiety. The higher the DS was, the more stable the NPs were. Cellular uptake demonstrated that Chol-Suc-HA-DTX NPs were internalized into cancer cells via CD44 receptor-mediated endocytosis. Compared with Taxotere®, Chol-Suc-HA-DTX NPs displayed remarkably higher cytotoxicity to CD44-positive cancer cells (MCF-7, 4T1, A549 cells). In vivo animal experiments confirmed that Chol-Suc-HA-DTX NPs with relatively high DS values exhibited prolonged circulation time, excellent tumor-targeting properties and efficient antitumor effects with extremely low systemic toxicity. In addition, blank Chol-Suc-HA NPs also slightly suppressed the tumor growth.Chol-Suc-HA NPs with a suitable DS value portend to be promising drug vehicles for systemic targeting of CD44-overexpressed cancers.
Keywords: CD44-mediated endocytosis; hyaluronan; interference with endogenous HA-CD44 interaction; tumor-targeting

Stabilization of HAC1 Influenza Vaccine by Spray Drying: Formulation Development and Process Scale-Up by Changcheng Zhu; Yoko Shoji; Scott McCray; Michael Burke; Caitlin E. Hartman; Jessica A. Chichester; Jeff Breit; Vidadi Yusibov; Dexiang Chen; Manjari Lal (3006-3018).
Stable vaccines with long shelf lives and reduced dependency on the cold chain are ideal for stockpiling and rapid deployment during public emergencies, including pandemics. Spray drying is a low-cost process that has potential to produce vaccines stable at a wide range of temperatures. Our aim was to develop a stable formulation of a recombinant H1N1 influenza hemagglutinin vaccine candidate and take it to pilot-scale spray-drying production.Eight formulations containing different excipients were produced and assayed for antigen stability, powder characteristics, and immunogenicity after storage at a range of temperatures, resulting in the identification of four promising candidates. A pilot-scale spray-drying process was then developed for further testing of one formulation.The pilot-scale process was used to reproducibly manufacture three batches of the selected formulation with yields >90%. All batches had stable physical properties and in vitro potency for 6 months at temperatures from −20°C to +50°C. Formulations stored for 3 months elicited immunogenic responses in mice equivalent to a frozen lot of bulk vaccine used as a stability control.This study demonstrates the feasibility of stabilizing subunit vaccines using a spray-drying process and the suitability of the process for manufacturing a candidate product.
Keywords: pilot scale-up; spray drying; thermostable; vaccines

We aimed to synthesize novel ceramide-chitosan (CS-CE) conjugate that forms stable polymeric nanoparticle capable of functioning as efficient carriers of hydrophobic drug such as Paclitaxel (PTX) for oral delivery.Chitosan (3–5 kDa) was conjugated with ceramide by using a DSC coupling reagent to improve its hydrophobic drug entrapment capacity. The structure of the conjugate was determined by proton (1H) NMR and FT-IR spectrometry. Size distribution and zeta potential were measured by DLS and PTX content in the cells and plasma was determined by HPLC and LC-MS.Under suitable conditions, the CS-CE self assembled to form colloidally stable nanoparticles with a mean diameter of ~300 nm. Further, PTX was incorporated into the CS-CE nanoparticle with 96.9% loading efficiency and 12.1% loading capacity via an emulsion-solvent evaporation method. The PTX-loaded CS-CE (PTX-CS-CE) showed sustained release of PTX and a comparable cytotoxic efficacy to that of free PTX on B16F10 melanoma and MCF-7 human breast adenocarcinoma cell lines. The empty nanoparticles showed no toxicity, indicating that the copolymer is safe to use in drug delivery. In addition, higher cellular uptake and slightly better pharmacokinetic parameters were obtained for PTX-CS-CE nanoparticle compared to free PTX.The polymeric nanoparticle of CS-CE represents a promising nanocarrier of hydrophobic drug for oral delivery.
Keywords: ceramide; chitosan; paclitaxel; polymeric nanoparticle; self assembling

The Development of Drug-Free Therapy for Prevention of Dental Caries by Fu Chen; Zhenshan Jia; Kelly C. Rice; Richard A. Reinhardt; Kenneth W. Bayles; Dong Wang (3031-3037).
The purpose of this study was to develop a novel, drug-free therapy that can reduce the over-accumulation of cariogenic bacteria on dental surfaces.We designed and synthesized a polyethylene glycol (PEG)-based hydrophilic copolymer functionalized with a pyrophosphate (PPi) tooth-binding anchor using “click” chemistry. The polymer was then evaluated for hydroxyapatite (HA) binding kinetics and capability of reducing bacteria adhesion to artificial tooth surface.The PPi-PEG copolymer can effectively inhibit salivary protein adsorption after rapid binding to an artificial tooth surface. As a result, the in vitro S. mutans adhesion study showed that the PPi-PEG copolymer can inhibit saliva protein-promoted S. mutans adhesion through the creation of a neutral, hydrophilic layer on the artificial tooth surface.The results suggested the potential application of a PPi-PEG copolymer as a drug-free alternative to current antimicrobial therapy for caries prevention.
Keywords: biofilm; caries; click chemistry; hydroxyapatite; tooth-binding

A novel bifunctional liposome with long-circulating and pH-sensitive properties was constructed using poly(2-ethyl-oxazoline)-cholesteryl methyl carbonate (PEtOz-CHMC) in this study.PEtOz-CHMC was synthesized and characterized by TLC, IR and 1H-NMR. The obtained PEtOz lipid was inserted into liposomes by the post-insertion method. Through a series of experiments, such as drug release, tumor cell uptake, cytotoxicity, calcium-induced aggregation, pharmacokinetic experiments, etc., the pH-sensitive and long-circulating properties of PEtOzylated liposomes was identified.PEtOz-CHMC modified liposomes (PEtOz-L) showed increased calcein release at low pH. Flow cytometric analysis results showed that the fusion and cellular uptake of PEtOz-L could be promoted significantly at pH 6.4 compared with those at pH 7.4. Confocal laser scanning microscope observations revealed that PEtOz-L could respond to low endosomal pH and directly released the fluorescent tracer into the cytoplasm. MTT assays in HeLa cells demonstrated that doxorubicin hydrochloride (DOX) loaded PEtOz-L exhibited stronger anti-tumor activity in a medium at pH 6.4 than in a medium pH 7.4. PEtOz-L remained stable when these liposomes were incubated in calcium chloride solution. The cumulative calcein release rate of PEtOz-L was significantly lower than that of CL when the liposomes were dialysed in PBS. The pharmacokinetic experiments of liposomes in rats showed that t 1/2 and AUC of PEtOz-L were 4.13 times and 4.71 times higher than those of CL.PEtOzylated liposomes exhibits excellent long-circulating and pH-sensitive properties. Our results suggest that PEtOz is a promising biomaterial for the modification of liposome in drug delivery.
Keywords: bifunctional; liposomes; long-circulating; pH-sensitive; poly(2-ethyl-oxazoline)

Acid-Responsive Polymeric Nanocarriers for Topical Adapalene Delivery by Chenchen Guo; Rajeshree H. Khengar; Mingjing Sun; Zheng Wang; Aiping Fan; Yanjun Zhao (3051-3059).
The acne skin is characteristic of a relatively lower pH microenvironment compared to the healthy skin. The aim of this work was to utilize such pH discrepancy as a site-specific trigger for on-demand topical adapalene delivery.The anti-acne agent, adapalene, was encapsulated in acid-responsive polymer (Eudragit® EPO) nanocarriers via nanoprecipitation. The nanocarriers were characterized in terms of particle size, surface morphology, drug-carrier interaction, drug release and permeation.Adapalene experienced a rapid release at pH 4.0 in contrast to that at pH 5.0 and 6.0. The permeation study using silicone membrane revealed a significant higher drug flux from the nanocarrier (6.5 ± 0.6 μ−2.h−1) in comparison to that (3.9 ± 0.4 μ−2.h−1) in the control vehicle (Transcutol®). The in vitro pig skin tape stripping study showed that at 24 h post dose-application the nanocarrier delivered the same amount of drug to the stratum corneum as the positive control vehicle did.The acid-responsive nanocarriers hold promise for efficient adapalene delivery and thus improved acne therapy. Figure pH liable nanocarriers enhance the adapalene delivery to acne skin.
Keywords: acid-responsive; acne; adapalene; nanocarrier; topical

Pharmacokinetics and Efficacy of the Spleen Tyrosine Kinase Inhibitor R406 after Ocular Delivery for Retinoblastoma by Eleanor M. Pritchard; Elizabeth Stewart; Fangyi Zhu; Cori Bradley; Lyra Griffiths; Lei Yang; Praveen Kumar Suryadevara; Jiakun Zhang; Burgess B. Freeman III; R. Kiplin Guy; Michael A. Dyer (3060-3072).
Retinoblastoma is a childhood cancer of the retina. Clinical trials have shown that local delivery of broad spectrum chemotherapeutic agents is efficacious. Recent studies characterizing the genomic and epigenomic landscape of retinoblastoma identified spleen tyrosine kinase (SYK) as a promising candidate for targeted therapy. The purpose of this study was to conduct preclinical testing of the SYK antagonist R406 to evaluate it as a candidate for retinoblastoma treatment.The efficacy of the SYK antagonist R406 delivered locally in a human orthotopic xenograft mouse model of retinoblastoma was tested. Intraocular exposure of R406 was determined for various routes and formulations.There was no evidence of efficacy for subconjunctival. R406. Maximal vitreal concentration was 10-fold lower than the minimal concentration required to kill retinoblastoma cells in vitro. Dosage of R406 subconjunctivally from emulsion or suspension formulations, direct intravitreal injection of the soluble prodrug of R406 (R788), and repeated topical administration of R406 all increased vitreal exposure, but failed to reach the exposure required for retinoblastoma cell death in culture.Taken together, these data suggest that R406 is not a viable clinical candidate for the treatment of retinoblastoma. This study highlights the importance of pharmacokinetic testing of molecular targeted retinoblastoma therapeutics.
Keywords: Ocular drug delivery; R406; Retinoblastoma; Spleen tyrosine kinase

Opening Up the Optical Imaging Window Using Nano-Luciferin by Apurva R. Patel; Ed Lim; Kevin P. Francis; Mandip Singh (3073-3084).
The objective of this study was to formulate nanoparticles of D-luciferin (Nano-Luc), DiR (Nano-DiR) and dual functional nanoparticles with DiR and luciferin (Nano-LucDiR) for in-vivo imaging as well as tracking of the nanoparticles in tumors.Nano-Luc and Nano-LucDiR were prepared using different lipids, and subsequently characterized for loading and entrapment efficiency, physical properties, release profile, toxicity and stability. We utilized Response Surface Methodology (RSM) to optimize the nanoparticles using design of experiment (DOE Vr.8.0). Nano-Luc was evaluated against free luciferin to establish its pharmacokinetic parameters in mice. In-vivo imaging of tumors and tracking of nanoparticles was carried out with an IVIS® Spectrum-CT (Caliper) using xenograft, orthotopic and metastatic tumor models in BALB/c nude mice with different cell lines and different routes of nanoparticle administration (subcutaneous, intraperitoneal and intravenous).Particle size of both Nano-Luc and Nano-LucDiR were found to be <200 nm. Nano-Luc formulation showed a slow and controlled release upto 72 h (90%) in vitro. The optimized Nano-Luc had loading efficiency of 5.0 mg/ml with 99% encapsulation efficiency. Nano-Luc and Nano-LucDiR formulations had good shelf stability. Nano-Luc and Nano-LucDiR enhanced plasma half-life of luciferin compared to free luciferin thus providing longer circulation of luciferin in plasma enabling imaging of tumors for more than 24 h. Nano-LucDiR allowed simultaneous bioluminescent and fluorescent imaging to be conducted, with three-dimensional reconstruct of tumors without losing either signal during the acquisition time.Nano-Luc and Nano-LucDiR allowed prolonged reproducible in-vivo imaging of tumors, especially during multimodality 3D imaging.
Keywords: enhanced In-vivo imaging; luciferin; nanoparticles; theranostic; tumor imaging

NanoXCT: A Novel Technique to Probe the Internal Architecture of Pharmaceutical Particles by Jennifer Wong; Dexter D’Sa; Matthew Foley; John Gar Yan Chan; Hak-Kim Chan (3085-3094).
To demonstrate the novel application of nano X-ray computed tomography (NanoXCT) for visualizing and quantifying the internal structures of pharmaceutical particles.An Xradia NanoXCT-100, which produces ultra high-resolution and non-destructive imaging that can be reconstructed in three-dimensions (3D), was used to characterize several pharmaceutical particles. Depending on the particle size of the sample, NanoXCT was operated in Zernike Phase Contrast (ZPC) mode using either: 1) large field of view (LFOV), which has a two-dimensional (2D) spatial resolution of 172 nm; or 2) high resolution (HRES) that has a resolution of 43.7 nm. Various pharmaceutical particles with different physicochemical properties were investigated, including raw (2-hydroxypropyl)-beta-cyclodextrin (HβCD), poly (lactic-co-glycolic) acid (PLGA) microparticles, and spray-dried particles that included smooth and nanomatrix bovine serum albumin (BSA), lipid-based carriers, and mannitol.Both raw HβCD and PLGA microparticles had a network of voids, whereas spray-dried smooth BSA and mannitol generally had a single void. Lipid-based carriers and nanomatrix BSA particles resulted in low quality images due to high noise-to-signal ratio. The quantitative capabilities of NanoXCT were also demonstrated where spray-dried mannitol was found to have an average void volume of 0.117 ± 0.247 μm3 and average void-to-material percentage of 3.5%. The single PLGA particle had values of 1993 μm3 and 59.3%, respectively.This study reports the first series of non-destructive 3D visualizations of inhalable pharmaceutical particles. Overall, NanoXCT presents a powerful tool to dissect and observe the interior of pharmaceutical particles, including those of a respirable size.
Keywords: Inhalation aerosols; NanoCT; Porous particles; Pulmonary drug delivery; X-ray computed tomography

Cysteinyl leukotrienes (CysLTs) propagate inflammatory reactions that result from allergen exposure in asthma. Montelukast, a CysLT type-1 receptor antagonist, disrupts mediator–receptor interactions and minimizes inflammatory response. In this study, we have evaluated anti-asthmatic efficacy of inhalable montelukast-loaded large porous particulate formulations in ovalbumin-induced rat airway inflammation model that mimics asthma.The anti-inflammatory effects of a montelukast-loaded formulation were investigated in rats by measuring the total protein content, levels of injury markers and number of inflammatory cells in the bronchoalveolar lavage fluid (BALF). The histopathological studies assessed the morphological and structural changes that occur in asthmatic lungs. Animals were also challenged with methacholine to examine the airway hyper-reactivity.Compared with healthy animals, asthmatic animals showed a 3.8- and 4.77-fold increase in the protein content and number of inflammatory cells in BALF, respectively. Intratracheal montelukast particles reduced the protein content by 3.3-fold and the number of inflammatory cells by 2.62-fold. Also, montelukast particles reduced the lactate dehydrogenase (LDH) and myeloperoxidase (MPO) levels by a 4.87- and 6.8-fold in BALF, respectively. Montelukast particles reduced the airway wall thickness by 2.5-fold compared with untreated asthmatic lungs. Further, particulate formulation protected the lungs against methacholine-induced bronchial provocation (p < 0.05).Respirable large porous particles containing montelukast alleviated allergen-induced inflammatory response in an animal model and prevented histological changes associated with asthma. Thus montelukast-loaded large porous polylactic acid (PLA) particles could be an aerosolized delivery approach for administration of currently available oral montelukast.
Keywords: anti-inflammatory; asthma; microparticles; montelukast; PLA

Although Cu complexes have been investigated as anticancer agents, there has been no description of Cu itself as a cancer killing agent. A stealth liposomal Cu formulation (LpCu) was studied in vitro and in vivo.LpCu was evaluated in prostate cancer origin PC-3 cells by a metabolic cytotoxicity assay, by monitoring ROS, and by flow cytometry. LpCu efficacy was evaluated in vivo using intratumoral and intravenous injections into mice bearing PC-3 xenograft tumors. Toxicology was assessed by performing hematological and blood biochemistry assays, and tissue histology and Cu distribution was investigated by elemental analysis.LpCu and free Cu salts displayed similar levels of cell metabolic toxicity and ROS. Flow cytometry indicated that the mechanisms of cell death were both apoptosis and necrosis. Animals injected i.t. with 3.5 mg/kg or i.v. with 3.5 and 7.0 mg/kg LpCu exhibited significant tumor growth inhibition. Kidney and eye were the main organs affected by Cu-mediated toxicities, but spleen and liver were the major organs of Cu deposition.LpCu was effective at reducing tumor burden in the xenograft prostate cancer model. There was histological evidence of Cu toxicity in kidneys and eyes of animals treated at the maximum tolerated dose of LpCu 7.0 mg/kg.
Keywords: liposomes; copper; reactive oxygen species; cancer therapy; toxicity

Hyperthermia Sensitization and Proton Beam Triggered Liposomal Drug Release for Targeted Tumor Therapy by R. Fernando; D. Maples; L. K. Senavirathna; Y. Zheng; J. C. Polf; E. R. Benton; K. E. Bartels; D. Piao; A. Ranjan (3120-3126).
The objectives of this study were to: 1) determine if mild hyperthermia (40–42°C) can sensitize tumor cells for more effective proton beam radiotherapy (PBRT); 2) characterize the survival fraction of cells exposed to PBRT; and 3) characterize release of the drug doxorubicin (Dox) from low temperature sensitive liposomes (LTSLs) without exposure to mild hyperthermia in combination with PBRT.Dox was actively loaded in LTSLs. A549 monolayer cells were incubated with 100–200 nM of Dox-LTSL (±mild hyperthermia). Cell irradiation (0–6 Gy) was performed by placing the cell culture plates inside a solid water phantom and using a clinical proton treatment beam with energy of 150 MeV. End points were survival fraction, radiation-mediated Dox release, and reactive oxygen species (ROS) production.Hyperthermia effectively sensitized cells for PBRT and lowered the cell survival fraction (SF) by an average of 9.5%. The combination of 100 nM Dox-LTSL and PBRT (1–6 Gy) achieved additive to synergistic response at various dose combinations. At higher radiation doses (>3 Gy), the SF in the Dox and Dox-LTSL groups was similar (~20%), even in the absence of hyperthermia. In addition, 30% of the Dox was released from LTSLs and a 1.3–1.6 fold increase in ROS level occurred compared to LTSL alone therapy.The combination of LTSLs and PBRT achieves additive to synergistic effect at various dose combinations in vitro. Concurrent PBRT and Dox-LTSL treatment significantly improved the cytotoxic outcomes of the treatment compared to PBRT and Dox chemotherapy without LTSLs. We hypothesize that PBRT may induce drug release from LTSL in the absence of hyperthermia.
Keywords: Hyperthermia sensitization; Nanoparticle; Radiation guided drug delivery

Hemocompatibility Assessment of two siRNA Nanocarrier Formulations by Afrouz Yousefi; Marianne Lauwers; Reka Nemes; Thijs van Holten; Negar Babae; Mark Roest; Gert Storm; Raymond Schiffelers; Enrico Mastrobattista (3127-3135).
Since the discovery of RNAi and its therapeutic potential, carrier systems have been developed to deliver small RNAs (particularly siRNA) for modulation of gene expression at the post-transcriptional level. An important factor determining the fate and usability of these systems in vivo is interaction with blood components, blood cells, and the immune system. In this study, a lipid-based and a polymer-based carrier system containing siRNA have been investigated in vitro in terms of their hemocompatibility.The nanocomplexes studied were Angiplex, a targeted lipid-based system, and pHPMA-MPPM polyplex, a formulation based on a cationic polymer. siVEGFR-2 was encapsulated in both carriers and activation of platelets, coagulation, and complement cascade as well as induction of platelet aggregation were evaluated in vitro.Both systems had been shown before to cause significant silencing in vitro. Our findings indicated that pHPMA-MPPM polyplex triggered high platelet activation and aggregation although it did not stimulate coagulation substantially. Angiplex, on the other hand, provoked insignificant activation and aggregation of platelets and activated coagulation minimally. Complement system activation by Angiplex was in general low but stronger than pHPMA-MPPM polyplex.Taken together, these in vitro assays may help the selection of suitable carriers for systemic delivery of siRNA in early preclinical investigations and reduce the use of laboratory animals significantly.
Keywords: Coagulation; Complement activation; Hemocompatibility; Platelet activation; siRNA delivery system

New Thermoresistant Polymorph from CO2 Recrystallization of Minocycline Hydrochloride by Miguel A. Rodrigues; João M. Tiago; Luis Padrela; Henrique A. Matos; Teresa G. Nunes; Lídia Pinheiro; António J. Almeida; Edmundo Gomes de Azevedo (3136-3149).
To prepare and thoroughly characterize a new polymorph of the broad-spectrum antibiotic minocycline from its hydrochloride dehydrate salts.The new minocycline hydrochloride polymorph was prepared by means of the antisolvent effect caused by carbon dioxide. Minocycline recrystallized as a red crystalline hydrochloride salt, starting from solutions or suspensions containing CO2 and ethanol under defined conditions of temperature, pressure and composition.This novel polymorph (β-minocycline) revealed characteristic PXRD and FTIR patterns and a high melting point (of 247 ºC) compared to the initial minocycline hydrochloride hydrates (α-minocycline). Upon dissolution the new polymorph showed full anti-microbial activity. Solid-state NMR and DSC studies evidenced the higher chemical stability and crystalline homogeneity of β-minocycline compared to the commercial chlorohydrate powders. Molecular structures of both minocyclines present relevant differences as shown by multinuclear solid-state NMR.This work describes a new crystalline structure of minocycline and evidences the ability of ethanol-CO2 system in removing water molecules from the crystalline structure of this API, at modest pressure, temperature and relatively short time (2 h), while controlling the crystal habit. This process has therefore the potential to become a consistent alternative towards the control of the solid form of APIs.
Keywords: Antisolvent effect; Minocycline; Physical stability; Structural characterization; Supercritical CO2

Safe, targeted delivery of therapeutics remains a focus of drug/gene delivery, the aim being to achieve optimal efficacy while minimising off-target delivery. Dendrimers have a vast array of potential applications and have great potential as gene and drug delivery tools. We previously reported the development of peptide dendrimers that effectively complexed DNA and that have distinct advantages over conventional spherical dendrimers. Here, to expand the application of peptide-based low generation dendrimers we tested their capacity to be transformed into linkers for antibody-based targeting of diverse payloads.Peptide-based low-generation asymmetric dendrimers were generated and conjugated to partially-reduced antibodies specific for B cell surface antigens or an irrelevant antigen. Preservation of antigen binding by the antibodies and targeting of the conjugated dendrimers carrying a small molecule (biotin) or plasmid DNA payloads was tested.Peptide-based low generation dendrimers were efficiently and site-specifically conjugated to antibodies with retention of antigen-binding capacity. Altering the branching termini of dendrimers facilitated delivery of diverse payloads in vitro and in vivo.We propose that safe, non-toxic peptide dendrimers, which are readily synthesised and modifiable for a variety of applications, form the basis of a new family of biocompatible “linkers” with substantial potential for targeted delivery applications.
Keywords: B cell; Dendrimer; DNA; Monoclonal antibody; Targeting

Concentration Dependent Viscosity of Monoclonal Antibody Solutions: Explaining Experimental Behavior in Terms of Molecular Properties by Li Li; Sandeep Kumar; Patrick M. Buck; Christopher Burns; Janelle Lavoie; Satish K. Singh; Nicholas W. Warne; Pilarin Nichols; Nicholas Luksha; Davin Boardman (3161-3178).
Early identification of monoclonal antibody candidates whose development, as high concentration (≥100 mg/mL) drug products, could prove challenging, due to high viscosity, can help define strategies for candidate engineering and selection.Concentration dependent viscosities of 11 proprietary mAbs were measured. Sequence and structural features of the variable (Fv) regions were analyzed to understand viscosity behavior of the mAbs. Coarse-grained molecular simulations of two problematic mAbs were compared with that of a well behaved mAb.Net charge, ξ-potential and pI of Fv regions were found to correlate with viscosities of highly concentrated antibody solutions. Negative net charges on the Fv regions of two mAbs with poor viscosity behaviors facilitate attractive self-associations, causing them to diffuse slower than a well-behaved mAb with positive net charge on its Fv region. An empirically derived equation that connects aggregation propensity and pI of the Fv region with high concentration viscosity of the whole mAb was developed.An Fv region-based qualitative screening profile was devised to flag mAb candidates whose development, as high concentration drug products, could prove challenging. This screen can facilitate developability risk assessment and mitigation strategies for antibody based therapeutics via rapid high throughput material-free screening.
Keywords: concentration; electrostatics; formulation; molecular modeling; monoclonal antibodies; subcutaneous delivery; viscosity

Pharmacokinetics and Efficacy of Bioerodible Dexamethasone Implant in Concanavalin A-induced Uveitic Cataract Rabbit Model by Srinivas Rao Chennamaneni; Austin Bohner; Ashlie Bernhisel; Balamurali K. Ambati (3179-3190).
To advance therapy for the treatment of concurrent uveitis and post-cataract surgical inflammation; we evaluated pharmacokinetics and pharmacodynamics of Bioerodible Dexamethasone Implant (BDI) containing 0.3 mg of dexamethasone (DXM) in Concanavalin A (Con A) induced uveitis followed by phacoemulsification in New Zealand White (NZW) rabbits.The BDI was implanted in the inferior fornix of the capsular bag after intravitreal injection of Con A and ensuing phacoemulsification in NZW rabbits; standard-of-care topical 0.1% dexamethasone drops served as control. DXM was quantified by liquid chromatography-tandem mass spectrometry and pharmacokinetics of DXM in disease vs. healthy eyes was compared. All eyes were assessed clinically using slit lamp biomicroscopy and Draize scoring scale. Retinal thickness and histological analyses were performed to evaluate retinal edema, inflammation and implant biocompatibility respectively.In Con A-induced inflammatory uveitic cataract model the BDI controlled anterior and posterior segment inflammation as well as retinal thickening more effectively than topical drops. The exposure (AUC0–t) of DXM with BDI is superior in all ocular tissues, while topical drops did not achieve therapeutic posterior segment levels and did not control inflammation nor prevent retinal edema and architectural disruption.Our results demonstrate the superiority of the BDI in suppressing Con A-induced inflammation and retinal edema in NZW rabbits and highlight the need for sustained bidirectional delivery of potent anti-inflammatory agents for 5 to 6 weeks to optimize clinical outcomes.
Keywords: bioerodible dexamethasone implant; cystoid macular edema; pharmacokinetics; topical drops; uveitis

The phase response of Miconazole-PVP VA64 solid dispersions upon compression was investigated. This would allow understanding the phase behavior of these solid dispersions upon application of a different kind of stress (other than humidity and temperature) and ultimately lead to mechanistic perception of the phase changes taking place.Miconazole and PVP VA64 were chosen as a model drug and polymer, respectively and solid dispersions were prepared by spray drying. Dried solid dispersions were compressed using different compression pressure but constant dwell time. MDSC and XRPD were used to characterize and study the effect of compression on the system.The solid dispersions showed a single Tg till 20% drug loading after which two Tg’s were observed. Application of compression to the phase separated 30 and 40% compositions induced mixing resulting in only a single Tg. This reduction in number of Tg’s upon compression is a result of mixing which can be attributed to polymer flow resulting in reduction of the domain size of different phases in the solid dispersions.Application of compression can influence the phase behavior of Miconazole-PVP VA64 solid dispersions. This observation may have drastic impact on the formulation development approach for solid dispersions to be administered as tablets.
Keywords: amorphous solid dispersions; compression; miconazole; phase separation; polymer flow

Pulsatile delivery of proteins, in which release occurs over a short time after a period of little or no release, is desirable for many applications. This paper investigates the effect of biodegradable polymer shell thickness on pulsatile protein release from biodegradable polymer microcapsules.Using precision particle fabrication (PPF) technology, monodisperse microcapsules were fabricated encapsulating bovine serum albumin (BSA) in a liquid core surrounded by a drug-free poly(lactide-co-glycolide) (PLG) shell of uniform, controlled thickness from 14 to 19 μm.When using high molecular weight PLG (Mw 88 kDa), microparticles exhibited the desired core-shell structure with high BSA loading and encapsulation efficiency (55–65%). These particles exhibited very slow release of BSA for several weeks followed by rapid release of 80–90% of the encapsulated BSA within 7 days. Importantly, with increasing shell thickness the starting time of the pulsatile release could be controlled from 25 to 35 days.Biodegradable polymer microcapsules with precisely controlled shell thickness provide pulsatile release with enhanced control of release profiles.
Keywords: bovine serum albumin; controlled release; monodisperse microcapsules; poly(lactide-co-glycolide); pulsatile release

Dissolution Testing of Powders for Inhalation: Influence of Particle Deposition and Modeling of Dissolution Profiles by Sabine May; Birte Jensen; Claudius Weiler; Markus Wolkenhauer; Marc Schneider; Claus-Michael Lehr (3211-3224).
The aim of this study was to investigate influencing factors on the dissolution test for powders for pulmonary delivery with USP apparatus 2 (paddle apparatus).We investigated the influence of dose collection method, membrane holder type and the presence of surfactants on the dissolution process. Furthermore, we modeled the in vitro dissolution process to identify influencing factors on the dissolution process of inhaled formulations based on the Nernst-Brunner equation.A homogenous distribution of the powder was required to eliminate mass dependent dissolution profiles. This was also found by modeling the dissolution process under ideal conditions. Additionally, it could be shown that influence on the diffusion pathway depends on the solubility of the substance.We demonstrated that the use of 0.02% DPPC in the dissolution media results in the most discriminating and reproducible dissolution profiles.In the model section we demonstrated that the dissolution process depends strongly on saturation solubility and particle size. Under defined assumptions we were able show that the model is predicting the experimental dissolution profiles.
Keywords: aerodynamic diameter (MMAD); Andersen cascade impactor; Nernst Brunner equation; paddle apparatus

AAPS Connection (3225-3227).