Pharmaceutical Research (v.28, #7)

Pharmaceutical Equivalence by Design for Generic Drugs: Modified-Release Products by André Sirota Raw; Robert Lionberger; Lawrence X. Yu (1445-1453).
The Office of Generic Drugs has ensured the high quality of generic products based upon two requirements: pharmaceutical equivalence and bioequivalence to the reference listed drug (RLD). This paradigm has been used with success toward ensuring quality generic drug products that provide the same therapeutic benefit as the RLD. Drug products have increased in design complexity; as a result, approaches to ensure therapeutic equivalence must evolve to provide assurance of quality generic drug products. The Food and Drug Administration quality by design initiative (QbD) provides an enhanced evaluation approach by introducing the concept of a quality target product profile (QTPP). The QTPP introduces, within the context of the current regulatory framework, the quality concept of “pharmaceutical equivalence by design.” This article illustrates through several examples how this QbD element in the evaluation of modified-release drug products enhances the current framework to ensure generic drug product equivalence. It achieves this by complementing the traditional paradigm, “equivalence by testing,” where product equivalence is based upon inferences from a limited bioequivalence study, to one that also considers whether the drug product was developed to be an equivalent to the RLD, using appropriate quality surrogates that target “pharmaceutical equivalence by design.”
Keywords: generic drugs; modified release; pharmaceutical equivalence; quality by design (QbD); quality target product profile (QTPP); therapeutic equivalence

The Alabama Drug Discovery Alliance: A Collaborative Partnership to Facilitate Academic Drug Discovery by Maaike Everts; W. Blaine Knight; David R. Harris; John A. Secrist III; Richard J. Whitley (1454-1459).
The Alabama Drug Discovery Alliance is a collaboration between the University of Alabama at Birmingham and Southern Research Institute that aims to support the discovery and development of therapeutic molecules that address an unmet medical need. The alliance builds on the expertise present at both institutions and has the dedicated commitment of their respective technology transfer and intellectual property offices to guide any commercial opportunities that may arise from the supported efforts. Although most projects involve high throughput screening, projects at any stage in the drug discovery and development pathway are eligible for support. Irrespective of the target and stage of any project, well-functioning interdisciplinary teams are crucial to a project’s progress. These teams consist of investigators with a wide variety of expertise from both institutions to contribute to the program’s success.
Keywords: academic drug discovery; Alabama; University of Alabama at Birmingham; high throughput screening; Southern Research Institute

Mechanistic PKPD models are now advocated not only by academic and industrial researchers, but also by regulators. A recent development in this area is based on the growing realisation that innovation could be dramatically catalysed by creating synergy at the interface between Systems Biology and PKPD, two disciplines which until now have largely existed in ‘parallel universes’ with a limited track record of impactful collaboration. This has led to the emergence of systems pharmacology. Broadly speaking, this is the quantitative analysis of the dynamic interactions between drug(s) and a biological system to understand the behaviour of the system as a whole, as opposed to the behaviour of its individual constituents; thus, it has become the interface between PKPD and systems biology. It applies the concepts of Systems Engineering, Systems Biology, and PKPD to the study of complex biological systems through iteration between computational and/or mathematical modelling and experimentation. Application of systems pharmacology can now impact across all stages of drug research and development, ranging from very early discovery programs to large-scale Phase 3/4 patient studies, and has the potential to become an integral component of a new ‘enhanced quantitative drug discovery and development’ (EQD3) R&D paradigm.
Keywords: modelling and simulation; pharmacokinetics/pharmacodynamics; PKPD; systems biology; systems pharmacology

The purpose of this article is to present the evolution of quality principles and how they have been implemented in the pharmaceutical industry. The article discusses the challenges that the FDA PAT Guidance and the ICH Q8, Q9 and Q10 Guidelines present to industry and provides a comprehensive overview of the basic tools that can be used to effectively build quality into products. The principles of the design of experiments, the main tools for statistical process analysis and control, and the requisite culture change necessary to facilitate statistical, knowledge-based management are also addressed.
Keywords: design of experiments; knowledge management; process analytical technologies; process variability; quality; quality by design; statistical process control and analysis; statistical thinking

Protein Microarrays: Novel Developments and Applications by Luis Berrade; Angie E. Garcia; Julio A. Camarero (1480-1499).
Protein microarray technology possesses some of the greatest potential for providing direct information on protein function and potential drug targets. For example, functional protein microarrays are ideal tools suited for the mapping of biological pathways. They can be used to study most major types of interactions and enzymatic activities that take place in biochemical pathways and have been used for the analysis of simultaneous multiple biomolecular interactions involving protein-protein, protein-lipid, protein-DNA and protein-small molecule interactions. Because of this unique ability to analyze many kinds of molecular interactions en masse, the requirement of very small sample amount and the potential to be miniaturized and automated, protein microarrays are extremely well suited for protein profiling, drug discovery, drug target identification and clinical prognosis and diagnosis. The aim of this review is to summarize the most recent developments in the production, applications and analysis of protein microarrays.
Keywords: drug discovery; protein chips; protein immobilization; protein profiling; proteomics

Designing Dendrimers for Drug Delivery and Imaging: Pharmacokinetic Considerations by Wassana Wijagkanalan; Shigeru Kawakami; Mitsuru Hashida (1500-1519).
Dendrimers have well-organized high branches with a layered architecture providing a series of versatile chemical modification for various purposes. Consequently, this dendrimer nanotechnology explores a new promising class of nanoscale carriers for therapeutic drugs and imaging reagents using passive and active targeting approaches. By controlling dendritic structures, the biological fate of dendrimer/dendrimer-based drugs can be significantly altered based on their intrinsic physicochemical properties, including the hydrophilicity of the unit molecules, particle size, surface charge, and modification. Accordingly, pharmacokinetic aspects play an important role in the design and development of dendrimer systems for successful in vivo application and clinical translation. This review focuses on the recent progress regarding dendritic architectures, structure-related toxicity, and critical factors affecting the pharmacokinetics and biodistribution of dendrimer/dendrimer-based drugs. A better understanding of the basic aspects of dendritic systems and their pharmacokinetics will help to develop a rationale for the design of dendrimers for the controlled delivery of drugs and imaging reagents for therapeutic or diagnostic purposes.
Keywords: biodistribution; dendrimer; drug delivery; imaging; pharmacokinetics

Agarose Surface Coating Influences Intracellular Accumulation and Enhances Payload Stability of a Nano-delivery System by Enrica De Rosa; Ciro Chiappini; Dongmei Fan; Xuewu Liu; Mauro Ferrari; Ennio Tasciotti (1520-1530).
Protein therapeutics often require repeated administrations of drug over a long period of time. Protein instability is a major obstacle to the development of systems for their controlled and sustained release. We describe a surface modification of nanoporous silicon particles (NSP) with an agarose hydrogel matrix that enhances their ability to load and release proteins, influencing intracellular delivery and preserving molecular stability.We developed and characterized an agarose surface modification of NSP. Stability of the released protein after enzymatic treatment of loaded particles was evaluated with SDS-page and HPLC analysis. FITC-conjugated BSA was chosen as probe protein and intracellular delivery evaluated by fluorescence microscopy.We showed that agarose coating does not affect NSP protein release rate, while fewer digestion products were found in the released solution after all the enzymatic treatments. Confocal images show that the hydrogel coating improves intracellular delivery, specifically within the nucleus, without affecting the internalization process.This modification of porous silicon adds to its tunability, biocompatibility, and biodegradability the ability to preserve protein integrity during delivery without affecting release rates and internalization dynamics. Moreover, it may allow the silicon particles to function as protein carriers that enable control of cell function.
Keywords: agarose hydrogel; intracellular delivery; nanopores; porous silicon; protein

Effects of Food on a Gastrically Degraded Drug: Azithromycin Fast-Dissolving Gelatin Capsules and HPMC Capsules by William Curatolo; Ping Liu; Barbara A. Johnson; Angela Hausberger; Ernest Quan; Thomas Vendola; Neha Vatsaraj; George Foulds; John Vincent; Richa Chandra (1531-1539).
Commercial azithromycin gelatin capsules (Zithromax®) are known to be bioequivalent to commercial azithromycin tablets (Zithromax®) when dosed in the fasted state. These capsules exhibit a reduced bioavailability when dosed in the fed state, while tablets do not. This gelatin capsule negative food effect was previously proposed to be due to slow and/or delayed capsule disintegration in the fed stomach, resulting in extended exposure of the drug to gastric acid, leading to degradation to des-cladinose-azithromycin (DCA). Azithromycin gelatin capsules were formulated with “superdisintegrants” to provide fast-dissolving capsules, and HPMC capsule shells were substituted for gelatin capsule shells, in an effort to eliminate the food effect.Healthy volunteers were dosed with these dosage forms under fasted and fed conditions; pharmacokinetics were evaluated. DCA pharmacokinetics were also evaluated for the HPMC capsule subjects. In vitro disintegration of azithromycin HPMC capsules in media containing food was evaluated and compared with commercial tablets and commercial gelatin capsules.When the two fast-dissolving capsule formulations were dosed to fed subjects, the azithromycin AUC was 38.9% and 52.1% lower than after fasted-state dosing. When HPMC capsules were dosed to fed subjects, the azithromycin AUC was 65.5% lower than after fasted-state dosing. For HPMC capsules, the absolute fasting-state to fed-state decrease in azithromycin AUC (on a molar basis) was similar to the increase in DCA AUC. In vitro capsule disintegration studies revealed extended disintegration times for commercial azithromycin gelatin capsules and HPMC capsules in media containing the liquid foods milk and Ensure®.Interaction of azithromycin gelatin and HPMC capsules with food results in slowed disintegration in vitro and decreased bioavailability in vivo. Concurrent measurement of serum azithromycin and the acid-degradation product DCA demonstrates that the loss of azithromycin bioavailability in the fed state is largely (and probably entirely) due to gastric degradation to DCA. Capsules can provide a useful and elegant dosage form for almost all drugs, but may result in a negative food effect for drugs as acid-labile as azithromycin.
Keywords: azithromycin; capsules; des-cladinose-azithromycin; food effect; HPMC capsules

The Effect of Composition and Gastric Conditions on the Self-Emulsification Process of Ibuprofen-Loaded Self-Emulsifying Drug Delivery Systems: A Microscopic and Dynamic Gastric Model Study by Annalisa Mercuri; Antonio Passalacqua; Martin S. J. Wickham; Richard M. Faulks; Duncan Q. M. Craig; Susan A. Barker (1540-1551).
To investigate the physical processes involved in the emulsification of self-emulsifying drug delivery systems (SEDDSs) and the use of the Dynamic Gastric Model (DGM) as a characterisation tool.SEDDSs based on soybean oil, Tween 80, Span 80 and ibuprofen were prepared and their equilibrium phase diagrams established. The emulsification behaviour in a range of media was studied using polarised light microscopy and particle sizing. The behaviour of the SEDDSs in the DGM and conventional testing equipment was assessed.A range of liquid crystalline mesophases was observed, enhanced in the presence of the drug. Polarised light microscopy showed different emulsification processes in the presence and absence of the drug, which was also manifest in different droplet sizes. The droplet size distribution varied between the DGM and the USP II dissolution apparatus.The model SEDDS displays complex liquid crystalline behaviour which may be intimately involved in the emulsification process, which in turn may alter particle size on emulsification, although there remains a question as to the in vivo significance of this effect. Furthermore, we demonstrate that the DGM represents a very promising new method of assessing the biological fate of SEDDSs.
Keywords: droplet size; dynamic gastric model; mechanism of self-emulsification; phase diagram; self-emulsifying systems

Effect of Sugar Molecules on the Viscosity of High Concentration Monoclonal Antibody Solutions by Feng He; Christopher E. Woods; Jennifer R. Litowski; Lauren A. Roschen; Himanshu S. Gadgil; Vladimir I. Razinkov; Bruce A. Kerwin (1552-1560).
To assess the effect of sugar molecules on solution viscosity at high protein concentrations.A high throughput dynamic light scattering method was used to measure the viscosity of monoclonal antibody solutions. The effects of protein concentration, type of sugar molecule (trehalose, sucrose, sorbitol, glucose, fructose, xylose and galactose), temperature and ionic strength were evaluated. Differential scanning fluorimetry was used to reveal the effect of the same sugars on protein stability and to provide insight into the mechanism by which sugars increase viscosity.The addition of all seven types of sugar molecules studied result in a significant increase in viscosity of high concentration monoclonal antibody solutions. Similar effects of sugars were observed in the two mAbs examined; viscosity could be reduced by increasing the ionic strength or temperature. The effect by sugars was enhanced at higher protein concentrations.Disaccharides have a greater effect on the solution viscosity at high protein concentrations compared to monosaccharides. The effect may be explained by commonly accepted mechanisms of interactions between sugar and protein molecules in solution.
Keywords: dynamic light scattering; high throughput; monoclonal antibody; preferential exclusion; preferential hydration; sugar molecule; viscosity

Pharmacokinetic-Pharmacodynamic Modeling of the Inhibitory Effects of Naproxen on the Time-Courses of Inflammatory Pain, Fever, and the Ex Vivo Synthesis of TXB2 and PGE2 in Rats by Elke H. J. Krekels; Marie Angesjö; Ingemo Sjögren; Kristina Ängeby Möller; Odd-Geir Berge; Sandra A. G. Visser (1561-1576).
To quantify and compare the time-course and potency of the analgesic and antipyretic effects of naproxen in conjunction with the inhibition of PGE2 and TXB2.Analgesia was investigated in a rat model with carrageenan-induced arthritis using a gait analysis method. Antipyretics were studied in a yeast-induced fever model using telemetrically recorded body temperature. Inhibition of TXB2 and PGE2 synthesis was determined ex vivo. Pharmacokinetic profiles were obtained in satellite animals. Population PKPD modeling was used to analyze the data.The IC50 values (95% CI) of naproxen for analgesia (27 (0–130) μM), antipyretics (40 (30–65) μM) and inhibition of PGE2 (13 (6–45) μM) were in similar range, whereas inhibition of TXB2 (5 (4–8) μM) was observed at lower concentrations. Variability in the behavioral measurement of analgesia was larger than for the other endpoints. The inhibition of fever by naproxen was followed by an increased rebound body temperature.Due to better sensitivity and similar drug-induced inhibition, the biomarker PGE2 and the antipyretic effect would be suitable alternative endpoints to the analgesic effects for characterization and comparisons of potency and time-courses of drug candidates affecting the COX-2 pathway and to support human dose projections.
Keywords: analgesia; fever; naproxen; pharmacokinetic-pharmacodynamic modeling; prostaglandins

Effective Gene Delivery to Mesenchymal Stem Cells Based on the Reverse Transfection and Three-Dimensional Cell Culture System by Cai-Xia He; Ni Li; Yu-Lan Hu; Xiu-Mei Zhu; Hai-Jie Li; Min Han; Pei-Hong Miao; Zhong-Jie Hu; Gang Wang; Wen-Quan Liang; Yasuhiko Tabata; Jian-Qing Gao (1577-1590).
To enhance the level and prolong the duration of gene expression for gene-engineered rat mesenchymal stem cells (MSCs) using non-viral vector.A novel transfection system based on reverse transfection method and three-dimensional (3D) scaffold was developed. The reverse gene transfection system was evaluated for transfection efficiency compared to conventional methods. Collagen sponge and polyethylene terephthalate non-woven fabric were introduced as scaffolds to perform 3D culture with reverse transfection. pDNA coding TGFβ-1 was delivered to MSCs to assess its ability in inducing chondrogenesis with the 3D non-viral reverse transfection system.The reverse transfection method induced higher transgene levels than the conventional transfection in the presence of serum. The electric charge of the anionic gelatin plays an important role in this system by affecting the release pattern of the gene complexes and through the adsorption of serum protein to the substrate. During a long-time in vitro culture, MSCs cultured on 3D scaffolds exhibited a higher transgene expression level and more sustained transgene expression than those cultured and transfected on the two-dimensional substrate.The combination of reverse transfection system with 3D cell culture scaffold benefits the cell proliferation and long-time gene transfection of MSCs.
Keywords: gene transfection; mesenchymal stem cells; non-viral gene vector; reverse transfection; three dimensional

High-Throughput Screening of Excipients Intended to Prevent Antigen Aggregation at Air-Liquid Interface by Sébastien Dasnoy; Nancy Dezutter; Dominique Lemoine; Vivien Le Bras; Véronique Préat (1591-1605).
The aim was to develop a high-throughput screening method compatible with low protein concentrations, as present in vaccines, in order to evaluate the performance of various excipients in preventing the aggregation at air-liquid interface of an experimental recombinant antigen called Antigen 18A.Aggregation of Antigen 18A was triggered by shaking in a half-filled vial or by air bubbling in a microplate. Size-exclusion chromatography, turbidimetry, Nile Red fluorescence spectroscopy, and attenuated total reflection Fourier-transform infrared spectroscopy were used to assess Antigen 18A aggregation. A high-throughput method, based on tryptophan fluorescence spectroscopy, was set up to screen excipients for their capability to prevent Antigen 18A aggregation at air-liquid interface.While a similar aggregation profile was obtained with both stress tests when using size-exclusion chromatography, spectroscopic and turbidimetric methods showed an influence of the stress protocol on the nature of the aggregates. The high-throughput screening revealed that 7 out of 44 excipients significantly prevented Antigen 18A from aggregating. We confirmed the performance of hydroxypropyl-β-cyclodextrin and hydroxypropyl-γ-cyclodextrin, as well as poloxamers 188 and 407, in half-filled shaken vials.A high-throughput screening approach can be followed for evaluating the performance of excipients against aggregation of a protein antigen at air-liquid interface.
Keywords: excipients; fluorescence spectroscopy; high-throughput screening; protein aggregation; vaccines

To develop a population pharmacokinetic (PK) model which allowed the simultaneous modeling of trans-resveratrol and its glucuronide and sulfate conjugates.Male Sprague–Dawley rats were administered i.v. and p.o. with 2, 10 and 20 mg·kg−1 of trans-resveratrol. Blood was collected at different times during 24 h. An integrated PK model was developed using a sequential analysis, with non-linear mixed effect modeling (NONMEM). A prediction-corrected visual predictive check (pcVPC) was used to assess model performance. The model predictive capability was also evaluated with simulations after the i.v. administration of 15 mg·kg−1 that were compared with an external data set.Disposition PK of trans-resveratrol and its metabolites was best described by a three-linked two-compartment model. Clearance of trans-resveratrol by conversion to its conjugates occurred by a first-order process, whereas both metabolites were eliminated by parallel first-order and Michaelis-Menten kinetics. The pcVPC confirmed the model stability and precision. The final model was successfully applied to the external data set showing its robustness.A robust population PK model has been built for trans-resveratrol and its glucuronide and sulfate conjugates that adequately predict plasmatic concentrations.
Keywords: glucuronide and sulfate conjugates; NONMEM; polyphenols; population pharmacokinetics; trans-resveratrol

Pharmacokinetic-Pharmacodynamic Disease Progression Model for Effect of Etanercept in Lewis Rats with Collagen-Induced Arthritis by Hoi-Kei Lon; Dongyang Liu; Qi Zhang; Debra C. DuBois; Richard R. Almon; William J. Jusko (1622-1630).
To develop a pharmacokinetic-pharmacodynamic disease progression (PK/PD/DIS) model to characterize the effect of etanercept in collagen-induced arthritis (CIA) rats on rheumatoid arthritis (RA) progression.The CIA rats received either 5 mg/kg intravenous (IV), 1 mg/kg IV, or 5 mg/kg subcutaneous (SC) etanercept at day 21 post-disease induction. Effect on disease progression was measured by paw swelling. Plasma concentrations of etanercept were assayed by enzyme-linked immunosorbent assay (ELISA). PK profiles were fitted first; parameter estimates were applied to fit paw edema data for PD and DIS-related parameter estimation using ADAPT 5 software.The model contained a two-compartment PK model with Michaelis-Menten elimination. For SC administration, two additional mathematical functions for absorption were added. The disease progression component was an indirect response model with a time-dependent change in paw edema production rate constant (k in ) assumed to be inhibited by etanercept.Etanercept has modest effects on paw swelling in CIA rats. The PK and PD profiles were well described by the developed PK/PD/DIS model, which may be used for other anti-cytokine biologic agents for RA.
Keywords: arthritis; etanercept; model; pharmacodynamics; pharmacokinetics

“Click” Conjugation of Peptide on the Surface of Polymeric Nanoparticles for Targeting Tumor Angiogenesis by Stéphanie Deshayes; Victor Maurizot; Marie-Claude Clochard; Cécile Baudin; Thomas Berthelot; Stéphane Esnouf; Didier Lairez; Michel Moenner; Gérard Déléris (1631-1642).
Angiogenesis plays a critical role in tumor growth. This phenomena is regulated by numerous mediators such as vascular endothelial growth factor (VEGF). CBO-P11, a cyclo-peptide, has proven to specifically bind to receptors of VEGF and may be used as targeting ligand for tumor angiogenesis. We herein report the design of novel nanoparticles conjugated to CBO-P11 in order to specifically target tumor site.The conjugation of CBO-P11 on the surface of poly(vinylidene fluoride) (PVDF) nanoparticles was investigated using the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition known as “click” reaction. CBO-P11 was modified with a near-infrared cyanine dye bearing an alkyne function, allowing both “click” coupling on azido-modified nanoparticles and fluorescence labelling. Each step of this nanodevice construction was judiciously performed in aqueous solution and successfully characterized. The cytotoxicity of nanoparticles was evaluated in human brain endothelial cell line and their affinity for VEGF receptors was determined via fluorescence-based uptake assays on porcine aortic endothelial cell line.Nanoparticles were found to be spherical, dense, monodisperse and stable. No cytotoxicity was observed after four days of incubation demonstrating the biocompatibility of nanoparticles. Fluorescence highlighted the specific interaction of these functionalized nanoparticles for VEGF receptors, suggesting that the targeting peptide bioactivity was retained.These results demonstrate the potential of these functionalized nanoparticles for targeting tumor angiogenesis and their possible use as multifunctional plateform for cancer treament if coupled with therapeutic agents.
Keywords: angiogenesis; “click” chemistry; cyclo-peptide; nanoparticles; PVDF; tumor targeting

To critically evaluate the effect of submicron and micron-sized organic particulates on the ultraviolet (UV) absorption spectra of aqueous systems and assess the applicability of UV/Vis fiber-optic probes for in-situ concentration monitoring in the presence of particles of different sizes.UV absorbance spectra were obtained for aqueous felodipine suspensions containing a range of particle sizes (300 nm–400 μm) and suspension concentrations and for methanolic solutions of different concentrations and amorphous films of different thicknesses. Select suspensions were further characterized using nuclear magnetic resonance (NMR) experiments. Mie theory was used to provide insight into the role of particle size on scattering and absorption of UV radiation.Large increases in absorbance as a function of total suspension concentration were observed for nanosuspensions but not for the other particle sizes evaluated. NMR measurements of solution concentration indicated that the observed increases in UV absorbance values for these systems were not caused by increases in the concentration of dissolved molecules, implying that nanoparticles of felodipine might absorb UV light. Mie theory-based calculations enabled reconstruction of the experimental observations and supported this hypothesis.For solutions containing small (submicron) felodipine particles, UV spectra were influenced by absorption of the particles and contributions from absorption of dissolved molecules and scattering of the particles. Caution should be applied when using in situ UV/VIS-probes to monitor the amount of dissolved material during dissolution, in particular when small particles are present (e.g. dissolution of nanoparticulate formulations) or generated (e.g. precipitation of supersaturated solutions) in the dissolution medium.
Keywords: concentration measurement; fiber-optic probes; Mie theory; nanoparticles

Antitumour Efficacy of Two Paclitaxel Formulations for Hyperthermic Intraperitoneal Chemotherapy (HIPEC) in an In Vivo Rat Model by Wim Bouquet; Steven Deleye; Steven Staelens; Lieselotte De Smet; Nancy Van Damme; Isabelle Debergh; Wim P. Ceelen; Filip De Vos; Jean Paul Remon; Chris Vervaet (1653-1660).
To evaluate the tumour growth delay of a peritoneal carcinomatosis (PC) of colorectal origin after intraperitoneal chemotherapy with paclitaxel/randomly-methylated-β-cyclodextrin (Pac/RAME-β-CD) versus Taxol® at normo- and hyperthermic conditions in rats.Hyperthermic intraperitoneal chemotherapy (HIPEC) was performed 7 days post implantation of the tumour with both formulations at a Pac concentration of 0.24 mg/ml. Tumour evaluation was performed via positron emission tomography (PET) and magnetic resonance imaging (MRI) imaging, measuring tumour activity and tumour volume, respectively. Scans were taken at 2 and 7 days post treatment.PET and MRI data showed a significant reduction in tumour activity and tumour volume for rats treated with Pac/RAME-β-CD (at normo- and hyperthermic conditions), compared to the control group. Treatment with Taxol® did not result in a significant reduction of tumour activity and tumour volume. No significant differences between the normo- and hyperthermic conditions were observed for both formulations, indicating that hyperthermia and paclitaxel were not synergistic despite the direct cytotoxic effect of hyperthermia.Monitoring tumour growth via PET and MRI indicated that Pac/RAME-β-CD inclusion complexes had a significantly higher efficacy compared to Taxol® in a rat model for peritoneal carcinomatosis.
Keywords: hyperthermic intraperitoneal chemotherapy; paclitaxel; tumour growth delay; β-cyclodextrin

O-Linked Glycosylation Leads to Decreased Thermal Stability of Interferon Alpha 2b as Measured by Two Orthogonal Techniques by Michael James Wilson Johnston; Grant Frahm; Xuguang Li; Yves Durocher; Mary Alice Hefford (1661-1667).
Thermal stability is considered an indication of protein fold and conformational stability. We investigate the influence of glycosylation on the thermal stability of interferon alpha 2b (IFN α-2b).Far ultraviolet light circular dichroism spectroscopy (UV CD) and differential scanning calorimetry (DSC) were used to assess the thermal stability of the European Directorate for the Quality of Medicines IFN α-2b reference standards as well as an O-linked glycosylated IFN α-2b produced in human embryonic kidney cells.Assessment of thermal stability of IFN α-2b and glycosylated IFN α-2b by DSC revealed that non-glycosylated interferon (Tm = 65.7 +/− 0.2°C, n = 3) was more thermally stable than the glycosylated variant (Tm = 63.8 C +/− 0.4°C, n = 3). These observations were confirmed with far UV CD (Tm IFN α-2b = 65.3 +/− 0.4°C, Tm glycosylated IFN α-2b = 63.6 +/− 0.2°C, n = 3). Enzymatic deglycosylation of IFN α-2b resulted in improved thermally stability when assessed with far UV CD and DSC.We demonstrate that O-linked glycosylation decreases the thermal stability of IFN α-2b compared to a non-glycosylated variant of the protein.
Keywords: circular dichroism; differential scanning calorimetry; glycosylation; interferon

Surface Characterisation of Bioadhesive PLGA/Chitosan Microparticles Produced by Supercritical Fluid Technology by Luca Casettari; Enzo Castagnino; Snjezana Stolnik; Andrew Lewis; Steven M. Howdle; Lisbeth Illum (1668-1682).
Novel biodegradable and mucoadhesive PLGA/chitosan microparticles with the potential for use as a controlled release gastroretentive system were manufactured using supercritical CO2 (scCO2) by the Particle Gas Saturated System (PGSS) technique (also called CriticalMixTM).Microparticles were produced from PLGA with the addition of mPEG and chitosan in the absence of organic solvents, surfactants and crosslinkers using the PGSS technique. Microparticle formulations were morphologically characterized by scanning electron microscope; particle size distribution was measured using laser diffraction. Microparticle surface was analyzed using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) to evaluate the presence of chitosan on the surface. Mucoadhesiveness of the microparticles was evaluated in vitro using a mucin assay employing two different kinds of mucin (Mucin type III and I-S) with different degrees of sialic acid contents, 0.5–1.5% and 9–17%, respectively.The two analytical surface techniques (XPS and ToF-SIMS) demonstrated the presence of the chitosan on the surface of the particles (<100 μm), dependent on the polymer composition of the microparticles. The interaction between the mucin solutions and the PLGA/chitosan microparticles increased significantly with an increasing concentration of mucin and chitosan.The strong interaction of mucin with the chitosan present on the surface of the particles suggests a potential use of the mucoadhesive carriers for gastroretentive and oral controlled drug release.
Keywords: chitosan; controlled release; mucoadhesive; PLGA; ToF-SIMS; XPS

Microemulsion Microstructure Influences the Skin Delivery of an Hydrophilic Drug by Wafa Naoui; Marie-Alexandrine Bolzinger; Bernard Fenet; Jocelyne Pelletier; Jean-Pierre Valour; Rafik Kalfat; Yves Chevalier (1683-1695).
We aimed to investigate the influence of microemulsion nanoscale organization as either oil-in-water droplets, water-in-oil droplets, or bicontinuous structures on skin delivery of drugs assisted by microemulsions.Three microemulsions of different microstructure, o/w, w/o, and bicontinuous at the skin temperature (32°C), having the same oil and water contents and containing the same ingredients were selected using the Kahlweit fish phase diagrams method. The microemulsions are quaternary mixtures of the Polysorbate 21 (Tween®21) and Sorbitan monolaurate (Span®20) surfactants, isononyl isononanoate oil and water. The microemulsion nanostructure was characterized by electrical conductivity, Pulsed Field Gradient Spin-Echo NMR and Small-Angle Neutron Scattering measurements. The Franz cell method was used to monitor skin absorption of caffeine loaded in microemulsions over 24 h exposure to excised pig skin.Three microemulsions with the three structures were selected, keeping the same composition but the Tween®21/Span®20 ratio. The transdermal flux of caffeine was in the order aqueous solution ≈ w/o < bicontinuous < o/w microemulsion. The o/w microemulsion allows the permeation of 50% of the applied dose within 24 h.The structure of microemulsions is of relevance for skin absorption. The water-continuous structures allow faster transport of hydrophilic drugs.
Keywords: microemulsion; microstructure; percutaneous penetration; phase diagram; skin absorption

Complex Pharmacokinetics of a Humanized Antibody Against Human Amyloid Beta Peptide, Anti-Abeta Ab2, in Nonclinical Species by Yulia Vugmeyster; Pam Szklut; David Wensel; John Ross; Xin Xu; Michel Awwad; Davinder Gill; Lioudmila Tchistiakov; Garvin Warner (1696-1706).
Anti-Aβ Ab2 (Ab2) is a humanized monoclonal antibody against amino acids 3–6 of primate (but not rodent) amyloid β (Aβ) and is being evaluated for the treatment of Alzheimer’s disease (AD). This study was conducted to predict the human pharmacokinetics of Ab2. In vivo PK profile of Ab2 in preclinical species and in vitro mechanistic studies in preclinical and human systems were used for pharmacokinetic predictions.In Tg2576 and PSAPP mice that have ~100-fold higher circulating levels of human Aβ compared to humans, elimination of Ab2 was target-mediated, such that exposure was 5–10 fold lower compared to wild-type rodents or to PDAPP mice that have human Aβ concentrations in plasma similar to humans. In cynomolgus monkeys, the t1/2 of Ab2 was faster (<2.5 days) compared to that of the control antibody (~13 days). The fast elimination of Ab2 in cynomolgus monkeys was linked to off-target binding to cynomolgus monkey fibrinogen that was also causing incomplete recovery of Ab2 in cynomolgus monkey serum in blood partitioning experiments. Ab2 had significantly weaker to undetectable binding to human (and mouse) fibrinogen and had good recovery in human serum in blood partitioning experiments.These data predict that elimination of Ab2 in healthy or AD humans is expected to be slow, with t1/2 similar to that observed for other humanized antibodies.
Keywords: Alzheimer’s disease; anti-Abeta antibody; fibrinogen; human PK prediction; pharmacokinetics

DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery by Ethlinn V. B. van Gaal; Ronald S. Oosting; Roel van Eijk; Marta Bakowska; Dries Feyen; Robbert Jan Kok; Wim E. Hennink; Daan J. A. Crommelin; Enrico Mastrobattista (1707-1722).
To evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA.A set of DTS was identified and cloned into EGFP-reporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry.Neither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNF-α, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.No beneficial effects of DTS on gene expression or nuclear uptake were observed in this study.
Keywords: DNA; DNA nuclear Targeting Sequences; non-viral gene delivery; nuclear import

Polyelectrolyte complex nanoparticles are a promising vehicle for siRNA delivery but suffer from low stability under physiological conditions. An effective stabilization method is essential for the success of polycationic nanoparticle-mediated siRNA delivery. In this study, sodium triphosphate (TPP), an ionic crosslinking agent, is used to stabilize siRNA-containing nanoparticles by co-condensation.siRNA and TPP were co-encapsulated into a block copolymer, poly(ethylene glycol)-b-polyphosphoramidate (PEG-b-PPA), to form ternary nanoparticles. Physicochemical characterization was performed by dynamic light scattering and gel electrophoresis. Gene silencing efficiency in cell lines was assessed by dual luciferase assay system.The PEG-b-PPA/siRNA/TPP ternary nanoparticles exhibited high uniformity with smaller size (80–100 nm) compared with PEG-b-PPA/siRNA nanoparticles and showed increased stability in physiological ionic strength and serum-containing medium, due to the stabilization effect from ionic crosslinks between negatively charged TPP and cationic PPA segment. Transfection and gene silencing efficiency of the TPP-crosslinked nanoparticles were markedly improved over PEG-b-PPA/siRNA complexes in serum-containing medium. No significant difference in cell viability was observed between nanoparticles prepared with and without TPP co-condensation.These results demonstrated the effectiveness of TPP co-condensation in compacting polycation/siRNA nanoparticles, improving nanoparticle stability and enhancing the transfection and knockdown efficiency in serum-containing medium.
Keywords: block copolymer gene carrier; siRNA; sodium triphosphate; stabilization; ternary nanoparticles

To test the efficacy and viability of poly (lactic-co-glycolic acid) (PLGA) microspheres encapsulating an inclusion complex of prostaglandin E1 (PGE1) and 2-hydroxypropyl-β-cyclodextrin (HPβCD) for pulmonary delivery of PGE1 for treatment of pulmonary arterial hypertension (PAH), a disease of pulmonary circulation.PLGA-based microparticulate formulations of PGE1-HPβCD inclusion complex or plain PGE1 were prepared by a double-emulsion solvent evaporation method. HPβCD was used as a complexing agent to increase the aqueous solubility of PGE1, act as a porosigen to produce large porous particles, and promote absorption of PGE1. Particles were characterized for micromeritic properties, in vivo absorption, metabolic degradation, and acute safety.Incorporation of HPβCD in the microparticles resulted in development of large particles with internal pores, which, despite large mean diameters, had aerodynamic diameters in the inhalable range of 1 to 5 μm. HPβCD incorporation also resulted in a significant increase in the amount of drug released in vitro in simulated interstitial lung fluid, showing a desirable burst release profile required for immediate hemodynamic effects. Compared to plain PLGA microparticles, entrapment efficiency was decreased upon complexation with HPβCD. In vivo absorption profile indicated prolonged availability of PGE1 in circulation following pulmonary administration of the optimized microparticulate formulations, with an extended half-life of almost 4 hours. Metabolic degradation and acute toxicity studies suggested that microparticulate formulations were stable under physiological conditions and safe for the lungs and respiratory epithelium.This study demonstrates the feasibility of PGE1-HPβCD complex encapsulated in PLGA microparticles as a potential delivery system for controlled release of inhaled PGE1.
Keywords: microparticles.; poly (lactic-co-glycolic acid); prostaglandin E1 ; pulmonary arterial hypertension; pulmonary delivery; 2-hydroxypropyl-β-cyclodextrin

Establishing a Link Between Amino Acid Sequences and Self-Associating and Viscoelastic Behavior of Two Closely Related Monoclonal Antibodies by Sandeep Yadav; Alavattam Sreedhara; Sonoko Kanai; Jun Liu; Samantha Lien; Henry Lowman; Devendra S. Kalonia; Steven J. Shire (1750-1764).
To investigate the underlying cause for the observed differences in self-associating and viscoelastic behavior between two monoclonal antibodies, MAb1, and MAb2.Several mutants were designed by swapping charged residues in MAb1 with those present in MAb2 at their respective positions and vice versa. Rheological analysis was done at low and high shear rates. Dynamic light scattering quantified intermolecular interactions in dilute solutions; sedimentation equilibrium analysis determined the corrected weight average molecular weight (M wc ) to assess the self-associating behavior in high concentration. The molecular charge was estimated from electrophoretic mobility measurements.Replacing the charged residues in the CDR of MAb1 resulted in a lower M wc and solution viscosity. The corresponding changes in either just the variable light (VL) or variable heavy (VH) chain showed only a partial decrease in viscosity, whereas changes in both VL and VH chains resulted in a dramatic reduction in viscosity. The converse case where the VL and VH chains of MAb2 were made to look like MAb1 did not self-associate or show increased viscosity.Exposed charged residues in the CDR of MAb1 are critical in determining the self-associating and highly viscous behavior observed at high concentrations.
Keywords: high concentration protein solution; intermolecular interactions; protein viscosity/rheology; self-association

Lyophilization of Protein-Loaded Polyelectrolyte Microcapsules by Marie-Luce De Temmerman; Joanna Rejman; Johan Grooten; Thomas De Beer; Chris Vervaet; Jo Demeester; Stefaan C. De Smedt (1765-1773).
To evaluate if lyophilization can be used to obtain a dry formulation of polyelectrolyte microcapsules, which have emerged as a new class of microparticles for the encapsulation and delivery of biomacromolecules.Microcapsules composed of dextran sulfate and poly-L-arginine were obtained by coating CaCO3 microparticles by means of the layer-by-layer technique. Microcapsules were lyophilized using different stabilizers; intactness was checked by CLSM and SEM. Horseradish peroxidase was encapsulated as model enzyme and retained activity after freeze-drying was determined using a fluorescence assay. Ovalbumin was encapsulated as model antigen; immunogenicity after lyophilization was evaluated in vitro by a T-cell proliferation assay and in vivo by measuring the antibody titer in mice.The results clearly demonstrate the necessity of using polyols in the formulation to prevent rupture of the microcapsules and to preserve the activity of encapsulated enzymes. Lyophilized microcapsules appeared as a promising adjuvant for antigen delivery, as both in vitro as in vivo assays showed higher immune activation compared to free antigen.Lyophilization is a promising strategy towards improved stability of protein-loaded microcapsules.
Keywords: drug delivery; encapsulation; layer-by-layer; lyophilization; microcapsules

The BCS, BDDCS, and Regulatory Guidances by Mei-Ling Chen; Gordon L. Amidon; Leslie Z. Benet; Hans Lennernas; Lawrence X. Yu (1774-1778).
Keywords: BCS; BDDCS; biopharmaceutics classification system; biopharmaceutics drug disposition classification system; drug metabolism; drug permeability

The Evolution of the Manufacturing Science and the Pharmaceutical Industry by Stavros N. Politis; Dimitrios M. Rekkas (1779-1781).
Keywords: manufacturing science; pharmaceutical industry; post mass production paradigm

News (1782-1784).