International Journal of Pharmaceutics (v.431, #1-2)

Immunogenicity of protein aggregates—Concerns and realities by Wei Wang; Satish K. Singh; Ning Li; Maria R. Toler; Kevin R. King; Sandeep Nema (1-11).
Protein aggregation is one of the key challenges in the development of protein biotherapeutics. It is a critical product quality issue as well as a potential safety concern due to the increased immunogenicity potential of these aggregates. The overwhelming safety concern has led to an increased development effort and regulatory scrutiny in recent years. The main purposes of this review are to examine the literature data on the relationship between protein aggregates and immunogenicity, to highlight the linkage and existing inconsistencies/uncertainties, and to propose directions for future investigations/development.
Keywords: Aggregation; Particulates; Subvisible; Vaccine; Adjuvant;

Effect of moving solid–liquid interface on dissolution kinetics: dissolution phenomena governed by diffusion in liquid phase as explored by Josef Stefan.Display Omitted► An extended original solution of the Stefan dissolution-diffusion problem is presented. ► The dissolution-diffusion boundary conditions at moving interface are stated. ► The continuity requirements for interfacial fluxes are illustrated. ► The Stefan solution, not known from literature, is made available.In a series of papers published from 1871 to 1889, Jožef (Josef) Stefan dealt with several diffusion processes, including also multicomponent systems. In his last paper on diffusion, which appeared in 1889, he studied the dissolution-diffusion process with a moving interface, and gave an analytical solution to this problem. However, Stefan's dissolution-diffusion analysis is not mentioned in literature, and its existence seems to be unknown in scientific community. The present paper summarizes the main Stefan ideas on dissolution of solids governed by diffusion of solute in the adjacent solvent phase thus making his results accessible to wider scientific circles.
Keywords: Stefan problem; Dissolution; Diffusion; Moving interface; Interface conditions; Mass transfer;

Improving the stability of the EC1 domain of E-cadherin by thiol alkylation of the cysteine residue by Maulik Trivedi; Jennifer S. Laurence; Todd D. Williams; C. Russell Middaugh; Teruna J. Siahaan (16-25).
The objective of this work was to improve chemical and physical stability of the EC1 protein derived from the extracellular domain of E-cadherin. In solution, the EC1 protein has been shown to form a covalent dimer via a disulfide bond formation followed by physical aggregation and precipitation. To improve solution stability of the EC1 protein, the thiol group of the Cys13 residue in EC1 was alkylated with iodoacetate, iodoacetamide, and maleimide-PEG-5000 to produce thioether derivatives called EC1-IA, EC1-IN, and EC1-PEG. The physical and chemical stabilities of the EC1 derivatives and the parent EC1 were evaluated at various pHs (3.0, 7.0, and 9.0) and temperatures (0, 3, 70 °C). The structural characteristics of each molecule were analyzed by circular dichroism (CD) and fluorescence spectroscopy and the derivatives have similar secondary structure as the parent EC1 protein at pH 7.0. Both EC1-IN and EC1-PEG derivatives showed better chemical and physical stability profiles than did the parent EC1 at pH 7.0. EC1-PEG had the best stability profile compared to EC1-IN and EC1 in solution under various conditions.
Keywords: E-cadherin; EC1 domain; Pegylation; Thiol acetylation; Physical stability; Chemical stability;

Prediction of drug-packaging interactions via molecular dynamics (MD) simulations by Peter Feenstra; Michael Brunsteiner; Johannes Khinast (26-32).
The interaction between packaging materials and drug products is an important issue for the pharmaceutical industry, since during manufacturing, processing and storage a drug product is continuously exposed to various packaging materials. The experimental investigation of a great variety of different packaging material–drug product combinations in terms of efficacy and safety can be a costly and time-consuming task. In our work we used molecular dynamics (MD) simulations in order to evaluate the applicability of such methods to pre-screening of the packaging material-solute compatibility. The solvation free energy and the free energy of adsorption of diverse solute/solvent/solid systems were estimated. The results of our simulations agree with experimental values previously published in the literature, which indicates that the methods in question can be used to semi-quantitatively reproduce the solid-liquid interactions of the investigated systems.
Keywords: Leaching; Sorption; Adsorption; Molecular dynamics; Polymers;

Evaluation of subcutaneous forms in the improvement of pharmacokinetic profile of warfarin by J. Scala-Bertola; L. Javot; J.A. Camargo; F. Bonneaux; T. Lecompte; P. Maincent; A. Sapin (33-38).
We attempted to prepare a subcutaneous pharmaceutical form of warfarin based on a suspension or poly(ɛ-caprolactone) microparticles to improve patient adherence. The warfarin suspension had a mean particle size of 20.0 μm and in vitro release close to 100% in 72 h. Microparticle size and encapsulation efficiencies ranged from 54.0 to 80.0 μm and 37.0 to 47.0%, respectively. After 72 h, warfarin microparticles exhibited in vitro drug release ranging from 62.0 to 80.0%. Warfarin subcutaneous dosage forms were administered to rabbits. Plasma concentration of warfarin was determined and biological activity was measured by prothrombin time monitoring. The observed relative bioavailabilities calculated from plasma concentrations and prothrombin times were 54.2 and 92.1%, and 61.8 and 61.4% for suspension and microparticles, respectively.
Keywords: Warfarin delivery; Microparticles; Polycaprolactone; Drug release; Prothrombin time monitoring; Bioavailability;

Pulmonary and nasal deposition of ketorolac tromethamine solution (SPRIX) following intranasal administration by Rachel Bacon; Steve Newman; Lisa Rankin; Gary Pitcairn; Roger Whiting (39-44).
Visual spread pattern of 99mTc-diethylenetriaminepenta acetic acid (DTPA) radiolabelled ketorolac tromethamine formulation (31.5 mg) following nasal administration in healthy subjects. The majority of the radiolabelled intranasal dose was deposited in the nasal cavity. The fraction of the dose recorded from the lung regions averaged <0.5%, and was considered to represent scattered radiation rather than true pulmonary deposition. The visual spread patterns within the nasal cavity were most uniform following administration in the upright position regardless of inhalation manoeuvre.Ketorolac tromethamine is a racemic, non-steroidal, anti-inflammatory drug (NSAID). An intra-nasal (IN) formulation, SPRIX®, is approved for the treatment of short term (up to 5 days) acute moderate to moderately severe pain. The primary objective of this study was to determine whether 99mTc-diethylenetriaminepenta acetic acid (DTPA) radiolabelled ketorolac tromethamine formulation (31.5 mg) was deposited in the lungs of healthy subjects (4 men and 9 women) following nasal inhalation of different intensities (gentle or vigorous sniff) and under different postural conditions (upright or semi-supine). The secondary objectives were to determine the deposition pattern of radiolabelled ketorolac solution in the nasal cavity and the clearance of the radiolabel over a 6 h period post-administration. The nasal spray pump delivery device used showed a droplet size distribution with a volume mean diameter (VMD) of 50 μm and approximately 85% of the aerosol mass contained in droplets >10 μm diameter. The fraction of the dose recorded from the lung regions averaged <0.5%, and was considered to represent scattered radiation rather than true pulmonary deposition. This fraction was not affected by posture or by inhalation manoeuvre. The majority of the radiolabelled intranasal dose was deposited in the nasal cavity. The visual spread patterns within the nasal cavity were most uniform following administration in the upright position regardless of inhalation manoeuvre. Clearance from the nasal cavity was initially very rapid, with only 16–30% of the dose remaining after 10 min and 6–14% after 6 h. Retention was greatest following gentle inhalation.
Keywords: Intranasal ketorolac; Nasal deposition; Clearance;

The development of a novel dry powder inhaler by Xi Zhang; Yingliang Ma; Liqiang Zhang; Jingxu Zhu; Fang Jin (45-52).
A novel active and multi-dose dry powder inhaler (DPI) was developed and evaluated to deliver a small quantity (100–500 μg) of pure drug without any excipient. This dry powder inhaler utilized two compressed air flows to dispense and deliver drug powder: the primary flow aerosolizes the drug powder from its pocket and the secondary flow further disperses the aerosol.In vitro tests by Anderson Cascade Impactor (ACI) indicated that the fine particle fraction (FPF) (<4.7 μm) of drug delivery could reach over a range of 50–70% (w/w). Emitted dose tests showed that delivery efficiency was above 85% and its relative standard deviation (RSD) was under 10%. Confocal microscopy was used to confirm the deposition of fluorescently labeled spray-dried powder in rabbit lungs. Also, a chromatographic method was used to quantify drug deposition. The results of animal tests showed that 57% of aerosol deposited in the rabbit lung and 24% deposited in its trachea. All the results implied that this novel active dry powder inhaler could efficiently deliver a small quantity of fine drug particles into the lung with quite high fine particle fraction.
Keywords: Dry powder inhaler; Active inhaler; In vitro evaluation; Animal test; Fluorescent image;

Diffusion and binding of 5-fluorouracil in non-ionic hydrogels with interpolymer complexation by Wenbin Zhou; Ping Lu; Li Sun; Changzhu Ji; Jian Dong (53-60).
Hydrogen-bonded interpolymer complexes can be used for development of novel dosage forms. In this study, two types of crosslinked hydrogels, copolymer networks of N-vinyl pyrrolidone and acrylamide (PVP-co-PAM) and interpenetrating polymer networks (IPN) composed of crosslinked PVP-co-PAM and poly(vinyl alcohol) (PVA), were synthesized at three different degrees of crosslinking. The side chain groups in such polymers can form non-ionic complexes through H-bonding, resulting in additional “crosslinks” in the hydrogels. Both kinds of hydrogels have significantly larger swelling sensitivities than the networks formed with ionizable side chains. In the IPNs, introduction of the PVA chains into the PVP-co-PAM networks raises the permeability, indicating more open pores. The permeability decreases with the increasing degree of crosslinking of the copolymer. For probing the drug binding in the hydrogels, Fourier transform infrared spectra (FTIR) difference spectroscopy indicated the presence of significant H-bonding interactions between 5-fluorouracil (5-FU) and the side chains of the polymers. Such interactions are larger in the PVP-co-PAM copolymers than in the IPN hydrogels, thereby causing an additional source of the slower release kinetics in the copolymer hydrogels as revealed by the Peppas model, albeit both types of the networks followed a non-Fickian transport mechanism.
Keywords: Hydrogel; Interpenetrating polymer network; Controlled release; Permeation; 5-Fluorouracil;

Development of drug delivery systems for the dermal application of therapeutic DNAzymes by Thomas Schmidts; Kay Marquardt; Peggy Schlupp; Dorota Dobler; Florian Heinz; Ulf Mäder; Holger Garn; Harald Renz; Jana Zeitvogel; Thomas Werfel; Frank Runkel (61-69).
DNAzymes are potent novel drugs for the treatment of inflammatory diseases such as atopic dermatitis. DNAzymes represent a novel class of pharmaceuticals that fulfil a causal therapy by interruption of the inflammation cascade at its origin. There are two challenges regarding the dermal application of DNAzymes: the large molecular weight and the sensitivity to DNases as part of the natural skin flora. To overcome these limitations suitable carrier systems have to be considered. Nano-sized drug carrier systems (submicron emulsions, microemulsions) are known to improve the skin uptake of drugs due to their ability to interact with the skin's lipids. To protect the drug against degradation, the hydrophilic drug may be incorporated into the inner aqueous phase of carrier systems, such as water-in-oil-in-water multiple emulsions. In the present study various emulsions of pharmaceutical grade were produced. Their physicochemical properties were determined and the influence of preservation systems on stability was tested. Drug release and skin uptake studies using various skin conditions and experimental set-ups were conducted. Furthermore, cellular uptake was determined by flow cytometric analysis. The investigations revealed that the developed multiple emulsion is a suitable and promising drug carrier system for the topical application of DNAzyme.
Keywords: DNAzyme; Oligonucleotides; W/O/W multiple emulsion; Submicron emulsion; Skin penetration;

Preparation and evaluation of granules with pH-dependent release by melt granulation by Kai Shiino; Yasunori Iwao; Yukari Fujinami; Shigeru Itai (70-77).
A combination of melt granulation and AMCE polymer for preparing granules with pH-dependent release.This study had two objectives: (1) to prepare, by melt granulation in a high-shear mixer, granules containing acetaminophen (APAP) as a model drug and aminoalkyl methacrylate copolymer E (AMCE) as a pH-sensitive polymer that readily dissolves at pH values lower than 5, and (2) to investigate the effects of AMCE loading (5–15%) on granule properties and the in vitro release profile of drug from the granules. Compared with polymer-free granules, the granules containing 5% and 10% AMCE were found to have higher median diameters and wider particle size distributions. For the formulation containing 15% AMCE, on the other hand, the diameters and distribution were similar to those for polymer-free granules. From compression testing, load–displacement curves revealed that AMCE enhanced particle strength at ambient temperature and induced plastic strain, while suppressing fragmentation of the granules. In addition, from dissolution testing using media with pH 4.0 and pH 6.5, granules containing AMCE, except 15% AMCE loading, exhibited drug release with significant pH dependence. When the pH 4.0 and pH 6.5 dissolution profiles were further compared by calculating the difference factor (f 1), the 5% AMCE granules showed the strongest pH dependence of drug release among all formulations in this study. Large cracks and breakage were observed on the surface of 10% AMCE granules after they were used in dissolution testing. The obtained results are attributed to the plastic strain properties of AMCE above its glass transition temperature, and to the irregular distribution of AMCE within granules. Hence, this study has demonstrated for the first time that the combination of melt granulation and AMCE incorporation enables the formulation of novel functional granules that exhibit pH-dependent release of the active ingredient.
Keywords: Melt granulation; Aminoalkyl methacrylate copolymer E; pH-dependent release behavior; Granules properties;

Radioiodinated dechloro-4-iodofenofibrate: A hydrophobic model drug for molecular imaging studies by Sandra Breyer; Angelika Semmler; Tobias Miller; Alexandra Hill; Simon Geissler; Uwe Haberkorn; Walter Mier (78-83).
Radiolabeling is a valuable option for tracking drug molecules in biodistribution experiments. In the development of innovative drug delivery systems the influence of the pharmaceutical formulation on the drugs’ pharmacokinetics has to be investigated. The hypolipidemic agent fenofibrate is an ideal model drug for testing the performance of drug delivery systems designed for poorly soluble compounds. Herein, we report a de novo synthesis of a fenofibrate derivative, dechloro-4-iodofenofibrate, as well as its conversion into its radioiodinated derivatives containing 125I or 131I. The enzymatic stability of the radiolabeled compounds synthesized was determined in vitro. A scintigraphic imaging study supplemented by biodistribution experiments and analysis of excreted metabolites revealed the stability required for in vivo applications and its similarity to fenofibrate. Therefore a convenient method is presented to synthesize radioiodinated derivatives of fenofibrate. These tracers show excellent in vitro and in vivo properties to study the behavior of lipophilic drugs.
Keywords: Drug delivery; Radiolabeling; Fenofibrate; Hydrophobic drugs; Iodination;

Systematic evaluations regarding interparticular mass transfer in spheronization by Martin Koester; Emilie Willemsen; Cornelia Krueger; Markus Thommes (84-89).
Pellets are frequently used in pharmaceutical applications. The extrusion-spheronization process is a well-established technique used to produce pellets of a spherical shape and narrow size distribution. In this process, cylindrical extrudates are transformed into spherical pellets by spheronization. Most established mechanisms consider only breakage and deformation to explain pellet formation. An interaction between the rounding extrudates via adhesion of fine particles was not considered for many years.This study deals with the evolution of pellet properties over time during the spheronization process in order to quantify the influence of pellet interactions on their properties. Therefore the most important pelletization aids (MCCI, MCCII and κ-carrageenan) were investigated using acetaminophen as a model drug and lactose as a filler. In the first seconds of the spheronization process, a high fine fraction was seen which decreased during the process. Simultaneously, the material transferred between the pellets increased. However the fine fraction is not high enough to explain the mass transfer; therefore a direct transfer between the pellets was assumed. The pelletization aid has a huge influence on the amount of mass transferred. Whereas κ-carrageenan leads to a quite low mass transfer of 15%, MCCI and MCCII show higher values up to 25%.
Keywords: Microcrystalline cellulose; κ-Carrageenan; Wet extrusion; Spheronization; Mechanism; Mass transfer;

Decoupling the role of image size and calorie intake on gastric retention of swelling-based gastric retentive formulations: Pre-screening in the dog model by Anita K. Lalloo; Emma L. McConnell; Lan Jin; Richard Elkes; Christian Seiler; Yunhui Wu (90-100).
Gastric retention is postulated as an approach to improve bioavailability of compounds with narrow absorption windows. To elucidate the role of image size on gastric retention and pharmacokinetics, formulations with different image sizes and swelling kinetics but similar dissolution rates were designed and imaged in dogs. Diet had a clear effect, with increasing calorific intake prolonging retention in the dog model. In contrast to clinical observations, no obvious effect of image size on gastric retention was observed in the dog, with the larger gastric retentive (GR) and smaller controlled release (CR) formulations both demonstrating similar gastric emptying. Comparable pharmacokinetic profiles were observed for the two formulations, corroborating the imaging data and providing evidence of similar in vivo dissolution rates and dosage form integrity in the dog. Food, specifically meal composition, resulted in comparable enhancements in exposure in the dog and clinic due to prolonged gastric retention. However, differentiating retention based on image size in the dog was not feasible due to the smaller pyloric aperture compared to humans. This work illustrates that the dog is capable of determining the pharmacokinetic advantage of gastric retention relative to immediate release (IR) or CR formulations, however, has limited value in differentiating between CR and GR formulations.
Keywords: Gastric retention; Image size; Swelling mechanism; Imaging; Calorie content; Pharmacokinetics;

Enhanced stability of horseradish peroxidase encapsulated in acetalated dextran microparticles stored outside cold chain conditions by Naveen Kanthamneni; Sadhana Sharma; Samantha A. Meenach; Beau Billet; Ji-Cheng Zhao; Eric M. Bachelder; Kristy M. Ainslie (101-110).
Micro- and nanoparticles have been shown to improve the efficacy of safer protein-based (subunit) vaccines. Here, we evaluate a method of improving the vaccine stability outside cold chain conditions by encapsulation of a model enzyme, horseradish peroxidase (HRP), in an acid-sensitive, tunable biodegradable polymer, acetalated dextran (Ac-DEX). Vaccines that are stable outside the cold chain would be desirable for use in developing nations. Ac-DEX particles encapsulating HRP were prepared using two different methods, probe sonication and homogenization. These particles were stored under different storage conditions (−20 °C, 4 °C, 25 °C or 45 °C) for a period of 3 months. On different days, the particles were characterized for various physical and chemical measurements. At all conditions, Ac-DEX particles remained spherical in nature, as compared to PLGA particles that fused together starting at day 3 at 45 °C. Furthermore, our results indicated that encapsulation of HRP in Ac-DEX reduces its storage temperature dependence and enhances its stability outside cold chain conditions. Homogenized particles performed better than probe sonicated particles and retained 70% of the enzyme's initial activity as compared to free HRP that retained only 40% of the initial activity after 3 months of storage at 25 °C or 45 °C. Additionally, HRP activity was more stable when encapsulated in Ac-DEX, and the variance in enzyme activity between the different storage temperatures was not observed for either particle preparation. This suggests that storage at a constant temperature is not required with vaccines encapsulated in Ac-DEX particles. Overall, our results suggest that an Ac-DEX based micro-/nanoparticles system has wide applications as vaccines and drug delivery carriers, including those in developing nations.
Keywords: Acid sensitive polymer; Horseradish peroxidase; Microparticle; Storage; Vaccines;

Insight into the solubility and dissolution behavior of piroxicam anhydrate and monohydrate forms by Urve Paaver; Andres Lust; Sabiruddin Mirza; Jukka Rantanen; Peep Veski; Jyrki Heinämäki; Karin Kogermann (111-119).
Hydrate formation during solubility testing was quantitatively monitored with Raman spectroscopy together with partial least squares (PLS) regression. This method helped to explain the solubility-time profiles of anhydrous piroxicam (PRX) in different environments and in the presence of sodium lauryl sulphate during testing.The aim of the present study was two-fold: (1) to investigate the effect of pH and presence of surfactant sodium lauryl sulphate (SLS) on the solubility and dissolution rate of two solid-state forms of piroxicam (PRX), anhydrate (PRXAH) and monohydrate (PRXMH), and (2) to quantitatively assess the solid-phase transformation of PRXAH to PRXMH in slurry with a special interest to the impact on the solubility and dissolution behavior of the drug. X-ray powder diffractometry (XRPD), Raman spectroscopy and scanning electron microscopy (SEM) were used for characterization of the solid-state forms. Phase transformation was monitored in slurry by means of in-line Raman spectroscopy, and the partial least squares (PLS) regression model was used for predicting the amount of PRXMH. The results showed that the solubility and dissolution rate of PRXAH were higher compared to PRXMH at different pHs. The pH and presence of SLS together affected the solubility and dissolution rate of different PRX forms. The lowest solubility values and dissolution rates for PRX forms were observed in distilled water (pH 5.6) at 37 °C. The changes in the dissolution rate could be explained by the hydrate formation during solubility testing. The rate of hydrate formation was also dependent on the pH of the dissolution medium.
Keywords: Piroxicam; Dissolution; Hydrate formation; Solubility; Supersaturation; Nucleation;

Investigation on the stability of saquinavir loaded liposomes: Implication on stealth, release characteristics and cytotoxicity by Lakshmi Narashimhan Ramana; Shilpee Sharma; Swaminathan Sethuraman; Udaykumar Ranga; Uma Maheswari Krishnan (120-129).
Although anti-retroviral therapy is the most efficient disease management strategy for HIV-AIDS, its applications are limited by several factors including the low bioavailability and first pass metabolism of the drugs. Nanocarriers such as liposomes have been developed to circumvent some of these problems. We report here preparation of novel liposome formulations for efficient delivery of anti-retroviral drugs to mammalian cells in culture. The liposomes were prepared and surface was modified using poly (ethylene glycol). Encapsulation efficiency of the anti-retroviral drug saquinavir was found to be approximately 33% and also exhibited sustained release of the drug. Although PEGylated liposomes were more stable in protein-supplemented media, had better colloidal stability and exhibited lesser sonochemical stability due to lower cavitation threshold. The cell viability studies using Jurkat T-cells revealed that the PEGylated liposomes loaded with saquinavir were less cytotoxic as compared to the non-PEGylated liposomes or free drug confirming the potential of the liposomes as a sustained drug-release system. The drug delivery potential of the liposomes loaded with Alexa flour 647 was evaluated using Jurkat T-cells and flow cytometry showing uptake upto 74%. Collectively, our data demonstrate efficient targeting of mammalian cells using novel liposome formulations with insignificant levels of cytotoxicity.
Keywords: PEGylated liposomes; Non-PEGylated liposomes; Saquinavir;

In-situ phase transition from microemulsion to liquid crystal with the potential of prolonged parenteral drug delivery by Xiazhong Ren; Darren Svirskis; Raid G. Alany; Sara Zargar-Shoshtari; Zimei Wu (130-137).
This study is the first to investigate and demonstrate the potential of microemulsions (MEs) for sustained release parenteral drug delivery, due to phase transition behavior in aqueous environments. Phase diagrams were constructed with Miglyol 812N oil and a blend of (co)surfactants Solutol HS 15 and Span 80 with ethanol. Liquid crystal (LC) and coarse emulsion (CE) regions were found adjacent to the ME region in the water-rich corner of the phase diagram. Two formulations were selected, a LC-forming ME and a CE-forming ME and each were investigated with respect to their rheology, particle size, drug release profiles and particularly, the phase transition behavior. The spreadability in an aqueous environment was determined and release profiles from MEs were generated with gamma-scintigraphy. The CE-forming ME dispersed readily in an aqueous environment, whereas the LC-forming ME remained in a contracted region possibly due to the transition of ME to LC at the water/ME interface. Gamma-scintigraphy showed that the LC-forming ME had minimal spreadability and a slow release of 99mTc in the first-order manner, suggesting phase conversion at the interface. In conclusion, owing to the potential of phase transition, LC-forming MEs could be used as extravascular injectable drug delivery vehicles for prolonged drug release.
Keywords: Microemulsion; Phase transition; Parenteral drug delivery; Liquid crystal; Prolonged drug release; Release kinetics;

Clotrimazole was formulated in nanoemulsion based system with the aim of improving its solubility and dissolution, which can further used for its preclinical evaluation. Clotrimazole nanoemulsion was prepared using spontaneous nanoemulsification method. Preformulation studies were preformed to evaluate drug-excipient compatibility, solution state pH stability and pH solubility profile. Solubility of clotrimazole in oils, surfactants and cosurfactants was determined to identify nanoemulsion components. Surfactants and cosurfactants were screened for their ability to emulsify selected oily phases. Phase diagrams were constructed to identify area of nanoemulsification. Influence of clotrimazole and pH of dilution medium on phase behavior were assessed. Drug-excipient chemical compatibility study facilitated to anticipate acid catalyzed degradation of clotrimazole. The pH of nanoemulsion was adjusted to 7.5, which could stabilize clotrimazole. Nanoemulsion composed of Capryol 90, Solutol HS 15 and Gelucire 44/14 enhanced solubility of clotrimazole up to 25 mg/ml. The optimized clotrimazole nanoemulsion could withstand the extensive dilution and did not show any phase separation or drug precipitation. The nanoemulsion exhibited mean globule size <25 nm, which was not affected by pH of dilution medium. Dissolution profile of clotrimazole nanoemulsion in various media showed 100% drug release within 15 min irrespective of pH of medium.
Keywords: Clotrimazole; Preformulation; Solubility; Phase behaviors; Nanoemulsion; Stability;

The aim of present investigation was to evaluate the potential of clotrimazole as antimalarial drug. Due to poor aqueous solubility and high lipophilicity, it was previously formulated in a nanoemulsion based system. The intrinsic effects of nanoemulsion on improvement of antimalarial activity of clotrimazole were assessed in mice infected with Plasmodium berghei and compared to its suspension formulation. In four-day suppressive test, mice treated with 10 mg/kg clotrimazole nanoemulsion showed the highest suppression of parasitemia and; parasitemia was significantly lower than that of 10 mg/kg clotrimazole suspension. In onset of activity and recrudescence test, percent reduction of parasitemia was significantly higher in 10 and 15 mg/kg clotrimazole nanoemulsion groups compared to 15 mg/kg suspension group. In both murine models, survival of mice treated with nanoemulsion was significantly prolonged compared to suspension at equivalent doses. The inhibition of parasite growth by clotrimazole in the nanoemulsion was dose dependent as determined by test for linear trend. In repeated dose oral toxicity, levels of serum liver enzymes and biomarkers of hepatotoxicity did not vary significantly from control. Six-month stability testing of the clotrimazole nanoemulsion exhibited no changes in various physiochemical attributes of drug product compared to initial analysis.
Keywords: Clotrimazole; Malaria; Nanoemulsion; Plasmodium berghei; Repeated dose toxicity; Stability;

Design and optimization of self-nanoemulsifying drug delivery systems (SNEDDS) for enhanced dissolution of gemfibrozil by Ana Maria Sierra Villar; Beatriz Clares Naveros; Ana Cristina Calpena Campmany; Monserrat Aróztegui Trenchs; Coloma Barbé Rocabert; Lyda Halbaut Bellowa (161-175).
Self-nanoemulsifying drug delivery systems of gemfibrozil were developed under Quality by Design approach for improvement of dissolution and oral absorption. Preliminary screening was performed to select proper components combination. Box–Behnken experimental design was employed as statistical tool to optimize the formulation variables, X 1 (Cremophor® EL), X 2 (Capmul® MCM-C8), and X 3 (lemon essential oil). Systems were assessed for visual characteristics (emulsification efficacy), turbidity, droplet size, polydispersity index and drug release. Different pH media were also assayed for optimization. Following optimization, the values of formulation components (X 1, X 2, and X 3) were 32.43%, 29.73% and 21.62%, respectively (16.22% of gemfibrozil). Transmission electron microscopy demonstrated spherical droplet morphology. SNEEDS release study was compared to commercial tablets. Optimized SNEDDS formulation of gemfibrozil showed a significant increase in dissolution rate compared to conventional tablets. Both formulations followed Weibull mathematical model release with a significant difference in t d parameter in favor of the SNEDDS. Equally amodelistic parameters were calculated being the dissolution efficiency significantly higher for SNEDDS, confirming that the developed SNEDDS formulation was superior to commercial formulation with respect to in vitro dissolution profile. This paper provides an overview of the SNEDDS of the gemfibrozil as a promising alternative to improve oral absorption.
Keywords: Gemfibrozil; Self-nanoemulsifying drug delivery systems; SNEDDS; Pseudoternary phase diagrams; Box–Behnken design;

Long-chain cationic derivatives of PTA (1,3,5-triaza-7-phosphaadamantane) as new components of potential non-viral vectors by Rita Cortesi; Paola Bergamini; Laura Ravani; Markus Drechsler; Andrea Costenaro; Mirko Pinotti; Matteo Campioni; Lorenza Marvelli; Elisabetta Esposito (176-182).
The purpose of this study was to investigate the potential of new positively charged solid lipid nanoparticles (SLN) to convey nucleic acids. The cationic character of SLN was obtained by adding as cationic molecules two different long-chain cationic phosphines (CP), namely hexadecyl-PTA iodide (CP16) and octadecyl-PTA iodide (CP18). The obtained CP-SLN are characterized by a positive charge on the surface and reproducible dimensions around 220 nm. These nanosystems are able to efficiently bind nucleic acid molecules and to protect DNA from the activity of serum nucleases up to 120 min. Lastly, in vitro experiments demonstrated that CP-SLN exhibit a quite pronounced antiproliferative effect on cultured human K562 erythroleukemic cells and a limited effect as transfecting adjuvant.These data, and particularly the ability of CP-SLN to protect DNA from degradation, encourages further studies aimed at proposing these nanosystems as a potential approach to deliver nucleic acid to cells in living organisms.
Keywords: PTA; Non-viral gene delivery systems; Cationic solid lipid nanoparticles (cationic SLN); Transfection;

The in vivo antitumor activity of LHRH targeted methotrexate–human serum albumin nanoparticles in 4T1 tumor-bearing Balb/c mice by Azade Taheri; Rassoul Dinarvand; Fatemeh Ahadi; Mohammad Reza Khorramizadeh; Fatemeh Atyabi (183-189).
The use of targeted drug delivery systems is a growing trend in cancer treatment to decrease the adverse effect of anti-cancer drugs. In this study, we sought to conjugate methotrexate–human serum albumin nanoparticles (MTX–HSA NPs) with luteinizing-hormone releasing hormone (LHRH). The LHRH was intended to target LHRH receptors overexpressed on the several types of tumors. The expression of LHRH receptors on the 4T1 breast cancer cells was confirmed by FITC conjugated LHRH receptor antibody using fluorescence microscopy. Female Balb/c mice bearing 4T1 breast cancer tumor were treated with a single i.v. injection of free MTX, non-targeted MTX–HSA NPs and LHRH targeted MTX–HSA NPs. LHRH targeted MTX–HSA nanoparticles showed stronger anti-tumor activity in vivo. By 7 days after treatment, average tumor volume in the LHRH targeted MTX–HSA NPs treated group decreased to 8.67% of the initial tumor volume when the number of attached LHRH molecules on MTX–HSA NPs was the highest, while the average tumor volume in non-targeted MTX–HSA NPs treated mice grew rapidly and reached 250.7% of the initial tumor volume 7 days after the treatment. LHRH targeted MTX–HSA NPs could significantly extend the survival time of tumor bearing mice compared with the non-targeted MTX–HSA NPs and free MTX formulations.
Keywords: Nanoparticles; Methotrexate; Human serum albumin; LHRH; In vivo tumor targeting;

Enhanced antitumor efficacy of cisplatin by tirapazamine–transferrin conjugate by Lin Wu; Jinhui Wu; Yuanyuan Zhou; Xiaolei Tang; Yanan Du; Yiqiao Hu (190-196).
Combination of tirapazamine (TPZ) with cisplatin has been studied extensively in clinical trial for tumor therapy. However, in phase III clinical trial, the combination therapy did not show overall survival improvement in patients. To decrease the side effects and increase the efficacy of the combination therapy, TPZ was conjugated with transferrin (Tf-G-TPZ) for targeted delivery and co-administered with cisplatin. In vitro toxicity study showed that the combination of Tf-G-TPZ with cisplatin induced substantially higher cytotoxicity of tumor cells than the combination of TPZ and cisplatin. After Tf-G-TPZ was intravenously injected into tumor-bearing mice, its total accumulation in tumor was 2.3 fold higher than that of the unmodified TPZ, suggesting transferrin-mediated target delivery of TPZ into the tumor tissue. With the increased accumulation of Tf-G-TPZ in tumor, the synergistic anti-tumor effects of Tf-G-TPZ and cisplatin were also enhanced as showed by the 53% tumor inhibition rate. Meanwhile, the side effects such as body weight lost were not significantly increased. Therefore, Tf-G-TPZ holds great promise to a better substitute for TPZ in the combination therapy with cisplatin.
Keywords: Transferrin; Tirapazamine; Cisplatin; Targeting anti-tumor effect; Tissue distribution;

Suppression of tumor growth in xenograft model mice by small interfering RNA targeting osteopontin delivery using biocompatible poly(amino ester) by Arash Minai-Tehrani; Hu-Lin Jiang; You-Kyoung Kim; Youn-Sun Chung; Kyeong-Nam Yu; Ji-Eun Kim; Ji-Young Shin; Seong-Ho Hong; Jae-Ho Lee; Hye-Joon Kim; Seung-Hee Chang; Sungjin Park; Bit Na Kang; Chong-Su Cho; Myung-Haing Cho (197-203).
Gene therapy using small interfering RNA (siRNA) is a novel strategy for effective anticancer therapies. However, low gene transfection efficiency and technical difficulties linked to siRNA delivery limit their practical application for gene delivery. Therefore, development of effective siRNA carriers is required. Overexpression of osteopontin (OPN) and its association with tumorigenesis has been reported in a majority of breast cancers. In this study, we used siRNA against OPN (siOPN) and investigated the possible OPN-dependent signaling pathway and the potential use of poly(amino ester) (PAE) based on glycerol propoxylate triacrylate (GPT) and spermine (SPE) for siRNA delivery. The GPT–SPE could effectively condense siRNA and protect the siRNA from RNaseA enzyme degradation. GPT–SPE/siRNA complexes showed good intracellular uptake and high gene silencing efficiency in vitro. Furthermore, in the breast cancer xenograft model, intratumoral injection of GPT–SPE/siOPN significantly inhibited tumor growth. These results demonstrated that silencing of OPN effectively suppressed the growth of breast cancer cells and further suggested that delivery of siRNA using GPT–SPE may act as an effective gene carrier for cancer therapy.
Keywords: Cancer gene therapy; Osteopontin; siRNA; Poly(amino ester);

Photodynamic therapy using glycol chitosan grafted fullerenes by Dong Sup Kwag; Nam Muk Oh; Young Taik Oh; Kyung Taek Oh; Yu Seok Youn; Eun Seong Lee (204-209).
Glycol chitosan (GC)-grafted fullerene (GC-g-C60) conjugates were developed for use in photodynamic therapy of tumor cells. GC-g-C60 was synthesized in anhydrous benzene/dimethylsulfoxide (DMSO) co-solvent via the chemical conjugation of free amine groups of GC to C=C double bonds of C60. The GC-g-C60 with 5 × 10−4 C60 molecules per one repeating unit of GC was soluble in water. As C60 molecules conjugated to GC increased to 0.16 molecules per one repeating unit of GC, GC-g-C60 started to form supramolecular assemblies (∼30 nm) stabilized in phosphate buffer saline (PBS, 150 mM, pH 7.4). Upon 670 nm light illumination, photo-responsive properties of GC-g-C60 allowed tremendous singlet oxygen generation in tumor cells for super phototoxicity. GC-g-C60 also showed highly increased tumor accumulation ability for in vivo tumor of KB tumor-bearing nude mice. It is expected that our GC-g-C60 conjugate may be a good candidate for in vivo photodynamic therapy in various malignant tumor cells.
Keywords: Fullerene conjugates; Glycol chitosan; Photodynamic therapy;

Calcium phosphate embedded PLGA nanoparticles: A promising gene delivery vector with high gene loading and transfection efficiency by Jie Tang; Jin-Ying Chen; Jing Liu; Min Luo; Ying-Jing Wang; Xia-wei Wei; Xiang Gao; Bi-lan Wang; Yi-Bo Liu; Tao Yi; Ai-Ping Tong; Xiang-Rong Song; Yong-Mei Xie; Yinglan Zhao; Mingli Xiang; Yuan Huang; Yu Zheng (210-221).
In the purpose of increasing incorporation efficiency and improving the release kinetics of plasmid DNA (pDNA) from poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles, a facile method for the fabrication of calcium phosphate (CaPi) embedded PLGA nanoparticles (CaPi-pDNA-PLGA-NPs) was developed. The effect of several preparation factors on the particle size, incorporation efficiency, pDNA release and transfection efficiency in vitro was studied by Single Factor Screening Method. These preparation factors included the molecular weight (MW), hydrolysis degree (HD) of polyvinyl alcohol (PVA), sonication power and time, composition of organic phase, initial concentration of calcium phosphate and calcium (Ca) to phosphate ion (P) ratio (Ca/P ratio), etc. The CaPi-pDNA-PLGA-NPs made according to the optimal formulation were spherical in shape observed by transmission electron microscopy (TEM) with a mean particle size of 207 ± 5 nm and an entrapment efficiency of 95.7 ± 0.8%. Differential scanning calorimetry (DSC) suggested that there existed interaction between the DNA-calcium-phosphate (CaPi-pDNA) complexes and the polymeric matrices of PLGA. X-ray diffractometry (XRD) further proved the conclusion and indicated that the CaPi-pDNA was in weak crystallization form inside the nanoparticles. The Brunauer–Emmett–Teller (BET) surface area measurement demonstrated that the CaPi-pDNA-PLGA-NPs are mesoporous with specific surface area of 57.5 m2/g and an average pore size of 96.5 Å. The transfection efficiency of the CaPi-pDNA-PLGA-NPs on human embryonic kidney 293 (HEK 293) cells in vitro was 22.4 ± 1.2%, which was much higher than those of both the pDNA loaded PLGA nanoparticles (pDNA-PLGA-NPs) and the CaPi-pDNA embedded PLGA microparticles (CaPi-pDNA-PLGA-MPs). The CaPi-pDNA-PLGA-NPs are promising vectors for gene delivery.
Keywords: CaPi-pDNA-PLGA-NPs; PLGA nanoparticle; Calcium phosphate; Plasmid DNA; HEK293;

The objective of this study was to increase the potency of doxorubicin against adriamycin-resistant NCI/ADR-RES cells by concurrent treatment with doxorubicin and MBO-asGCS loaded solid-lipid nanoparticles (SLN). Loading doxorubicin as ion-pair complex with deoxytaurocholate into cationic and neutral SLN was investigated. Fast release and poor entrapment were observed in cationic nanoparticles, which were corrected by entrapping the complex in neutral polyoxyethylene (20) stearyl ether (Brij® 78)/VitE-TPGS nanoparticles. Slow doxorubicin release confirmed the influence of charge and electrolytes on the dissociation of ion-pair complexes. To evaluate antitumor activity, NCI/ADR-RES cells were treated with separate SLN: one loaded with doxorubicin and another carrying MBO-asGCS oligonucleotide. The viability of cells treated with 5 μM doxorubicin was reduced to 17.2% whereas viability was reduced to 2.5% for cells treated with both 5 μM doxorubicin SLN and 100 nM MBO-asGCS SLN. This suggested enhanced apoptosis due to sensitization and effective intracellular delivery of MBO-asGCS and doxorubicin by SLN.
Keywords: Oligonucleotide; Solid lipid nanoparticles; Drug delivery; Gene delivery; Multidrug resistance;

Effect of isotonic solutions and peptide adsorption on zeta potential of porous silicon nanoparticle drug delivery formulations by Martti Kaasalainen; Ermei Mäkilä; Joakim Riikonen; Miia Kovalainen; Kristiina Järvinen; Karl-Heinz Herzig; Vesa-Pekka Lehto; Jarno Salonen (230-236).
Zeta potential of porous silicon nanoparticles is highly dependent on the nature of the loaded peptide and the isotonic medium.Recently, highly promising results considering the use of porous silicon (PSi) nanoparticles as a controlled and targeted drug delivery system have been published. Drugs are typically loaded into PSi nanoparticles by electrostatic interactions, and the drug-loaded nanoparticles are then administered parenterally in isotonic solutions. Zeta potential has an important role in drug adsorption and overall physical stability of nanosuspensions. In the present study, we used zeta potential measurements to study the impact of the formulation components to the nanosuspension stability. The impact of medium was studied by measuring isoelectric points (IEP) and zeta potentials in isotonic media. The role of drug adsorption was demonstrated with gastrointestinal peptides GLP-1(7-37) and PYY (3-36) and the selection of isotonic additive was demonstrated with peptide-loaded PSi nanoparticles. The results show the notable effect of isotonic solutions and peptide adsorption on zeta potential of PSi nanosuspensions. As a rule of thumb, the sugars (sucrose, dextrose and mannitol) seem to be good media for negatively charged peptide-loaded particles and weak acids (citric- and lactic acid) for positively charged particles. Nevertheless, perhaps the most important rule can be given for isotonic salt solutions which all are very poor media when the stability of nanosuspension is considered.
Keywords: Porous silicon; Nanoparticles; Gastrointestinal peptide; Parenteral peptide delivery; Isotonic formulation; Zeta potential;

Formation mechanism of a new carbamazepine/malonic acid cocrystal polymorph by Waree Limwikrant; Aiko Nagai; Yumi Hagiwara; Kenjirou Higashi; Keiji Yamamoto; Kunikazu Moribe (237-240).
A new 2/1 carbamazepine (CBZ)/malonic acid (MA) cocrystal polymorph form C was formed using a vibrational rod mill, whereas the known cocrystal polymorph form A was prepared using a ball mill. IR measurements showed that the interaction between CBZ and MA in cocrystal form C was formed by amide–carboxylic acid heterosynthons, similar to that in cocrystal form A. However, NMR results showed that the molecular states of CBZ at the dibenzazepine ring appeared to be different, which could be due to variation in either the conjugation of the aromatic rings or the π–π interaction of CBZ. Factors affecting the formation of cocrystal polymorphs, such as heat and force, were investigated to clarify the formation mechanism.
Keywords: Cocrystal; Polymorphism; Grinding; Carbamazepine; Transition;

Corrosion inhibitors from expired drugs by Nicolae Vaszilcsin; Valentin Ordodi; Alexandra Borza (241-244).
This paper presents a method of expired or unused drugs valorization as corrosion inhibitors for metals in various media. Cyclic voltammograms were drawn on platinum in order to assess the stability of pharmaceutically active substances from drugs at the metal–corrosive environment interface. Tafel slope method was used to determine corrosion rates of steel in the absence and presence of inhibitors. Expired Carbamazepine and Paracetamol tablets were used to obtain corrosion inhibitors. For the former, the corrosion inhibition of carbon steel in 0.1 mol L−1 sulfuric acid solution was about 90%, whereas for the latter, the corrosion inhibition efficiency of the same material in the 0.25 mol L−1 acetic acid–0.25 mol L−1 sodium acetate buffer solution was about 85%.
Keywords: Expired drugs; Corrosion inhibitors; Inhibition efficiency; Carbamazepine; N-Acetyl-4-aminofenol;