European Journal of Pharmaceutics and Biopharmaceutics (v.69, #3)

APV Diary (S1-S2).

Stabilization of IgG1 in spray-dried powders for inhalation by S. Schüle; T. Schulz-Fademrecht; P. Garidel; K. Bechtold-Peters; W. Frieß (793-807).
The protein stabilizing capabilities of spray-dried IgG1/mannitol formulations were evaluated. The storage stability was tested at different residual moisture levels prepared by vacuum-drying or equilibration prior to storage. Vacuum-drying at 32 °C/0.1 mbar for 24 h reduced the moisture level below 1%, constituting an optimal basis for improved storage stability. The crystalline IgG1/mannitol powders with a weight ratio of 20/80 up to 40/60 failed to prevent the antibody aggregation as assessed by size exclusion chromatography during storage. Ratios of 60/40 up to 80/20 IgG1/mannitol provided superior stability of the antibody and the powders could be produced with high yields. The lower the residual moisture, the better was the stabilizing capability. An amount of 20% mannitol provided the best stabilization. Storage stability of 60/40, 70/30, and 80/20 IgG1/mannitol formulations over one year was adequate at 2–8 °C and 25 °C. Closed storage (sealed in vials) at 40 °C/75% RH and open storage at 25 °C/60% RH revealed that the stability still required optimization. The lower the protein content, the better was the powder flowability. The aerodynamic properties of powders spray-dried with 10% solids content were inadequate, as the particle size ranged between 5.1 and 7.2 μm and the fine particle fraction accounted for only 4–11%. Reduction of the solids content to 2.5% did improve the aerodynamic properties as the mass mean aerodynamic diameter was reduced to 3.6 μm and the fine particle fraction was increased to about 14%. The reduction of the solids content did not influence the storage stability significantly. Also spray-drying at higher temperatures had no significant impact on the storage stability, despite a higher tendency to form amorphous systems. In order to improve the storage stability and to maintain the good flowability of 70/30 IgG1/mannitol powder or to keep the storage stability but to improve the flowability of the 80/20 IgG1/mannitol powder, mannitol was partially substituted by a second excipient such as trehalose, sucrose, glycine, lactose, lactosucrose, or dextran 1. Differences in the stabilizing capability were noticeable upon closed storage at 40 °C/75% RH and open powder storage. Protein stabilization was improved by the addition of glycine but trehalose and sucrose were most effective in preventing aggregation, which can be primarily attributed to the water replacement properties of the sugars. The addition of another excipient, isoleucine had positive effects on both flowability and protein stability.
Keywords: Spray-drying; Antibody; Powder inhalation; Protein stability;

The aim of this study was to characterize factor(s) in rat bronchoalveolar lavage fluid (BALF) that enhance(s) insulin transport across primary rat alveolar epithelial cell monolayers (RAECM) in primary culture. BALF was concentrated 7.5-fold using the Centricon device and the retentate was used to characterize the factor(s) involved in enhancing apical-to-basolateral transport of intact 125I-insulin across various epithelial cell monolayers. These factor(s) enhanced transport of intact insulin across type II cell-like RAECM (3-fold increase) and type I cell-like RAECM (2-fold increase), but not across Caco-2 or MDCK cell monolayers. The insulin transport-enhancing factor(s) were temperature- and trypsin-sensitive. The mechanism of enhancement did not seem to involve paracellular transport or fluid-phase endocytosis, since fluxes of sodium fluorescein and FITC-dextran (70 kDa) were not affected by the factor(s) in the apical bathing fluid. BALF enhancement of intact 125I-insulin transport was abolished at 4 °C and in the presence of monensin, suggesting involvement of transcellular pathways. Sephacryl S-200 purification of BALF retentate, followed by LC-MS/MS, indicated that the high molecular weight (>100 kDa) fractions (which show some homology to alpha-1-inhibitor III, murinoglobulin gamma 2, and pregnancy-zone protein) appear to facilitate transcellular transport of insulin across RAECM.
Keywords: Cell culture; Peptide delivery; Pulmonary; Insulin; Transcytosis;

Cationic lipid/DNA complexes induce TNF-α secretion in splenic macrophages by Caroline Lonez; Michel Vandenbranden; Abdelatif Elouahabi; Jean-Marie Ruysschaert (817-823).
Cationic lipids are widely used as vectors to deliver DNA into mammalian cells in vitro and in vivo. However, cationic lipid/DNA lipoplexes induce an inflammatory response, characterized by pro-inflammatory cytokine secretion, which severely limits their use. The main goal of this work is to identify the organs and the cell type involved in TNF-α secretion after lipoplex injection. We determined the kinetics of distribution of the cationic lipid/DNA complex in blood, lung, liver and spleen and quantified the TNF-α amount in organ homogenates and in the serum at different points of times. Increase in TNF-α production was only observed in the spleen and no significant increase of TNF-α production could be observed in the other organs. Fractionation of spleen cells revealed that macrophages were mainly responsible for TNF-α secretion. This observation was verified in vivo by using macrophage-removing agents. In conclusion, we show here that the TNF-α secreted in the serum after intravenous injection of lipoplexes comes mainly from the splenic macrophages.
Keywords: Cationic liposomes; Lipoplexes; Macrophage; DiC14-amidine; Spleen; Transfection;

Moisture-induced aggregation has been identified as a key problem in the long term storage stability of therapeutic proteins. In the present work, we have investigated the impact of the disaccharide trehalose on the aggregation behavior of a model protein, bovine serum albumin (BSA) under moist conditions. About 50% aggregation of BSA was observed at a moisture level of 8 μl/10 mg protein. Including trehalose in the protein sample caused a significant reduction in aggregation. We address the probable mechanisms for the protective effect of trehalose by considering the various hypotheses that have been proposed in the literature. The techniques that have been used include denaturing and non-denaturing gel electrophoresis and tryptophan intrinsic fluorescence. The nature of the aggregates was studied by carrying out electrophoresis of the aggregated protein in the presence of reducing and chaotropic reagents. The interaction studies of aggregated BSA with Thioflavin T and CongoRed indicate the possibility of amyloid type of character in the former. These studies may explain the protective role of trehalose under conditions where the storage stability of therapeutic proteins is compromised.
Keywords: Aggregation mechanism; Biopharmaceuticals; Moisture-induced aggregation; Protein stability; Trehalose;

From adsorption kinetics and interfacial rheological studies performed by using a pendant-drop method, i.e. in conditions close to those of the primary emulsion of the water-in-oil-in-water emulsion-encapsulation technique, it was shown that adsorption of the hen egg-white lysozyme (HEWL) at the water/dichloromethane (DCM) interface can be efficiently slowed down by modulating some parameters. It was shown that a decrease of the ionic strength of the aqueous phase, and the optimization of the density of the poly(ethylene glycol) (PEG) adsorbed film by increasing the PEG concentration or by modulating the polymer chain length, can significantly decrease the rate of adsorption of HEWL at the water/DCM interface. Moreover, it was shown that the choice of the dissolution phase of PEG (DCM or water) clearly influences the results.
Keywords: Poly(ethylene glycol); Hen egg-white lysozyme; Emulsion; Pendant-drop tensiometer; Adsorption;

Sustained release of bioactive glycosylated glial cell-line derived neurotrophic factor from biodegradable polymeric microspheres by E. Garbayo; E. Ansorena; J.L. Lanciego; M.S. Aymerich; M.J. Blanco-Prieto (844-851).
Glial cell-line derived neurotrophic factor (GDNF), a potent neurotrophic factor for dopaminergic neurons, appeared as a promising candidate for treating Parkinson’s disease. GDNF microencapsulation could ensure protection against degradation due to the fragile nature of the protein. Poly(lactide-co-glycolide) (PLGA) microparticles loaded with recombinant glycosylated GDNF obtained in a mammalian cell line were prepared by TROMS, a semi-industrial technique capable of encapsulating fragile molecules maintaining their native properties. The effects of several parameters as PLGA copolymer type, PEG 400 quantity co-encapsulated with GDNF or drug loading, on the properties of the particles were investigated. Microparticles showed a mean diameter between 8 and 30 μm, compatible with their stereotaxic implantation. The drug entrapment efficiency ranged from 50.6% to 100% depending on the microsphere composition. GDNF was better encapsulated using hydrophilic polymers with high molecular weight such as RG 503H. In vitro drug release was influenced by the polymer type as well as by the amount of PEG 400 co-encapsulated with GDNF. Microparticles prepared using PLGA RG 503H released 67% of the total protein content within 40 days. Moreover, very low concentrations of poly(vinyl alcohol) were detected after microparticles washing and freeze-drying. Finally, a PC-12 bioassay demonstrated that the in vitro GDNF released was bioactive.
Keywords: Rat recombinant GDNF; Biodegradable microparticles; PLGA; TROMS; PEG 400;

Transport and structural analysis of molecular imprinted hydrogels for controlled drug delivery by Siddarth Venkatesh; Jishnu Saha; Shondra Pass; Mark E. Byrne (852-860).
Molecular imprinting provides a rational design strategy for the development of controlled release drug delivery systems. We demonstrate that imprinting a hydrogel network results in macromolecular memory for the template molecule, indicated by the two or more times greater partitioning into these networks as compared to non-imprinted networks. Partitioning of drug into networks synthesized from multiple functional monomers was 8 times greater than networks synthesized from single monomers. One-dimensional permeation studies showed that the gel with maximum incorporated chemical functionality had the lowest diffusion coefficient, which was one to two orders of magnitude lower than all other gels studied. All imprinted networks had significantly lower diffusion coefficients than non-imprinted networks, in spite of comparable mesh sizes and equilibrium polymer volume fractions in the swollen state, which to our knowledge, is the first time that such a study has been conducted in the literature. We propose the “tumbling hypothesis”, wherein a molecule tumbling through an imprinted network with multiple, organized functionalities and an appropriate mesh size, experiences heightened interactions with memory sites and shows delayed transport kinetics. Thus, the structural plasticity of polymer chains, i.e. the organization of functional groups into memory sites, may be responsible for enhanced loading and extended release.
Keywords: Drug delivery; Controlled release; Sustained release; Molecular imprinting; Structural plasticity; Macromolecular memory;

The determination of stratum corneum thickness An alternative approach by Lisa M. Russell; Sandra Wiedersberg; M. Begoña Delgado-Charro (861-870).
The individual thickness of the stratum corneum is required to normalise drug permeation profiles in dermato-pharmacokinetic studies. The thickness is often estimated using tape-stripping combined with transepidermal water loss measurements. A linear transformation of Fick’s first law is used to relate the progressively thinner barrier with the corresponding increase in transepidermal water loss and to estimate the thickness by linear regression. However, the data from an important subset of subjects are poorly fitted to this linear model. This is typically due to the removal of loose outer layers of stratum corneum, which do not contribute significantly to barrier function. This work proposes two alternative non-linear models. All three models were used to fit data from 31 in vivo tape-stripping experiments and their outcomes and goodness-of-fit compared. The results suggest that the linear model may overestimate the stratum corneum thickness and is open to subjectivity regarding the selection of data points to be fitted. The non-linear models satisfactorily fitted all the data, including all data points. No significant differences were found between the thicknesses derived from the two non-linear models. However, the analysis of the goodness-of-fit of the models to the data suggests a preference for a baseline-corrected approach.
Keywords: Tape-stripping; Transepidermal water loss (TEWL); Stratum corneum; Thickness; Dermato-pharmacokinetics;

Dissolution rate enhancement of the novel antitumoral β-lapachone by solvent change precipitation of microparticles by Marcílio S.S. Cunha-Filho; Ramón Martínez-Pacheco; Mariana Landín (871-877).
β-Lapachone [βLAP] is a novel antitumor drug, which was recently on clinical trials with promising preliminary results. Problems derived from its low water solubility, its instability in solution and its high therapeutic dose constitute some challenges for pharmaceutical researchers. The purpose of the present work is to enhance the limited dissolution rate of βLAP by the design of particles using a solvent change precipitation process. The procedure induces the spontaneous crystalline growth of the βLAP in the presence of a stabilizing polymer (Hydroxypropylmethylcellulose) that limits the size of the particles generated. Physicochemical characterization of microparticles and the βLAP dissolution rate was carried out. The utility of the βLAP microcrystals in the development of tablets with adequate dissolution properties was also stated. The procedure was optimized in order to obtain stable and homogeneous particles with a small mean particle size (∼3 μm) and a narrow particle size distribution. There were no differences between the drying methods evaluated (in an oven and freeze-drying) with regard to particle morphology or dissolution behaviour, which is almost instantaneous. Tablets having suitable mechanical properties were produced by dry granulation prior to compression. The compression process did not compromise βLAP dissolution characteristics.
Keywords: β-Lapachone; Solvent change precipitation; Stabilizing agent; Dissolution rate; Tablets;

Bioprocess of uniform-sized crosslinked chitosan microspheres in rats following oral administration by Wei Wei; Lian-Yan Wang; Lan Yuan; Xiao-Da Yang; Zhi-Guo Su; Guang-Hui Ma (878-886).
Chitosan microspheres have a great potential in pharmaceutical application. In this study, uniform-sized chitosan microspheres crosslinked with glutaraldehyde (CG microspheres) were prepared by Shirasu Porous Glass (SPG) membrane emulsification technique. Based on the characterizations of uniform size and autofluorescence, it was possible to develop a new detecting system for observing and quantifying the CG microspheres in rats with three different diameters (2.1, 7.2 and 12.5 μm) synchronously after oral administration. This system was a combination of scanning electron microscopy (SEM), laser scanning confocal microscope (LSCM) and flow cytometer technique, which showed the advantages of being simple, intuitionistic, repeatable and sensitive. After oral administration of three kinds of particles with different diameters, bioadhesion in gastrointestinal tract, absorption in gastrointestinal tract, distribution in systemic tissues, and biodegradation in reticuloendothelial system (RES) were studied firstly in detail. The CG microspheres showed different fates in bioadhesion, absorption and distribution according to their diameters, while the biodegradation also varied due to the different locations in RES. These original results would indicate a better way for the CG microspheres in the clinical application.
Keywords: Chitosan; Microsphere; Bioadhesion; Absorption; Distribution; Biodegradation;

In vitro–in vivo correlations of self-emulsifying drug delivery systems combining the dynamic lipolysis model and neuro-fuzzy networks by Dimitrios G. Fatouros; Flemming Seier Nielsen; Dionysios Douroumis; Leontios J. Hadjileontiadis; Anette Mullertz (887-898).
The aim of the current study was to evaluate the potential of the dynamic lipolysis model to simulate the absorption of a poorly soluble model drug compound, probucol, from three lipid-based formulations and to predict the in vitro–in vivo correlation (IVIVC) using neuro-fuzzy networks. An oil solution and two self-micro and nano-emulsifying drug delivery systems were tested in the lipolysis model. The release of probucol to the aqueous (micellar) phase was monitored during the progress of lipolysis. These release profiles compared with plasma profiles obtained in a previous bioavailability study conducted in mini-pigs at the same conditions. The release rate and extent of release from the oil formulation were found to be significantly lower than from SMEDDS and SNEDDS. The rank order of probucol released (SMEDDS ∼ SNEDDS > oil formulation) was similar to the rank order of bioavailability from the in vivo study. The employed neuro-fuzzy model (AFM-IVIVC) achieved significantly high prediction ability for different data formations (correlation greater than 0.91 and prediction error close to zero), without employing complex configurations. These preliminary results suggest that the dynamic lipolysis model combined with the AFM-IVIVC can be a useful tool in the prediction of the in vivo behavior of lipid-based formulations.
Keywords: In vitro–in vivo correlations (IVIVC); In vitro dynamic lipolysis model; Self-emulsifying drug delivery systems; Mathematical modelling; Neuron-fuzzy networks;

Self-microemulsifying drug delivery system (SMEDDS) improves anticancer effect of oral 9-nitrocamptothecin on human cancer xenografts in nude mice by Juan-Li Lu; Jian-Cheng Wang; Shu-Xin Zhao; Xiao-Yan Liu; Hui Zhao; Xuan Zhang; Shu-Feng Zhou; Qiang Zhang (899-907).
9-Nitrocamptothecin (9-NC) is an orally administered topoisomerase-I inhibitor for the treatment of pancreatic carcinoma, but its oral absorption and bioavailability are poor. The main objective of this study was to develop optimal 9-nitrocamptothecin (9-NC) microemulsion prepared by self-microemulsifying drug delivery system (SMEDDS). Two SMEDDS formulations of 9-NC prepared from a mixture of ethyl oleate, Tween-80 (T-form) or Cremophor EL (C-form), and PEG-400/ethanol were formed as microemulsions under dilution with aqueous phase. The resulting microemulsions were evaluated in vitro and in vivo, including the kinetics and antitumor effects in SKOV-3 human ovarian cancer xenograft in nude mice. Following 1:10 aqueous dilution of optimal 9-NC SMEDDS, the droplet sizes of resulting microemulsions were (30.8 ± 4.6) nm and (39.8 ± 8.2) nm for SMEDDS T-form and C-form, respectively, and the zeta potential values were −(4.3 ± 0.5) mV and −(5.7 ± 0.5) mV, respectively. In SKOV-3 cells, the growth inhibition (IC50) of various 9-NC formulations was greatest with SMEDDS T-form (3.5 ± 0.7 nM) followed by SMEDDS C-form (4.6 ± 0.4 nM), 9-NC solution (6.6 ± 1.4 nM) and 9-NC suspension (26.0 ± 2.9 nM) (P  < 0.01). It was indicated that the area under the plasma concentration–time curve (AUC0→8 h) values of various formulations of 9-NC after oral administration ranked as the following sequence: SMEDDS T-form (360.12 ± 19.44 ng h/ml) ≈ SMEDDS C-form (351.71 ± 33.66 ng h/ml) > 9-NC solution (241.21 ± 24.67 ng h/ml) > 9-NC suspension (161.24 ± 24.31 ng h/ml). The 9-NC SMEDDS formulations also produced significantly more tumor shrinkage (P  < 0.01) when compared to 9-NC suspension in nude mice bearing human ovarian cancer xenografts. The results suggest that SMEDDS is a promising drug delivery system to increase the oral bioavailability and antitumor effects of 9-NC and may be applied to other lipophilic drugs. 9-NC SMEDDS represents a novel 9-NC therapy for cancer patients.
Keywords: 9-Nitrocamptothecin; Self-microemulsifying drug delivery system (SMEDDS); Pharmacokinetic; Antitumor activity;

Pre-formulation of liposomes against Helicobacter pylori: Characterization and interaction with the bacteria by Pierre-Louis Bardonnet; Vincent Faivre; Paul Boullanger; Jean-Claude Piffaretti; Françoise Falson (908-922).
This paper deals with the formulation of targeted liposome against Helicobacter pylori. We describe the characterization of liposomes loaded with antimicrobial agents (ampicillin and metronidazole) and the quantification of the interactions between such formulations and bacteria. If the encapsulation rate of ampicillin seems not strongly affected by the change of phospholipidic composition, the encapsulation of metronidazole drastically decreased in epikuron 170 liposomes compared to DPPC ones. Furthermore, as observed with X-ray diffraction measurements, the presence of metronidazole results in the disorganisation of the phospholipid bilayers. Concerning the liposome–bacteria interactions, it has been observed that the incorporation of fucosyled glycolipids in the vesicle membrane leads to liposomes that are able to interact with the bacteria either in their spiral or in their coccoid forms. Since coccoid forms are occasionally found in vivo, their recognition by the liposomes we have formulated seems promising in the fight against Helicobacter pylori.
Keywords: Liposome; Helicobacter pylori; Glycolipid; Targeting; BabA2;

Novel micelle-forming poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) block copolymers bearing doxorubicin (DOX) side groups (PEO-b-P(CL-DOX)) on the PCL block were synthesized. Prepared block copolymers were characterized, assembled to polymeric micellar drug conjugates and assessed for the level of DOX release at pH 7.4 and pH 5.0 using a dialysis membrane to separate released and conjugated drug. The possibility for the degradation of PCL backbone for PEO-b-P(CL-DOX) micelles was investigated using gel permeation chromatography. Micelle-forming DOX conjugate did not show any signs of DOX release at 37 °C within 72 h of incubation at both pHs, but revealed signs of poly(ester) core degradation at pH 5.0. In further studies, PEO-b-PCL micelles bearing benzyl, carboxyl or DOX groups in the core were also used as micellar nano-containers for the physical encapsulation of DOX, where maximum level of drug-loading and control over the rate of DOX release was achieved by polymeric micelles containing benzyl groups in their core, i.e., PEO-b-poly(α-benzylcarboxylate-ε-caprolactone) (PEO-b-PBCL) micelles. The in vitro cytotoxicity of chemically conjugated DOX as part of PEO-b-P(CL-DOX) and physically encapsulated DOX in PEO-b-PBCL against B16F10 murine melanoma cells was assessed and compared to that of free DOX. Consistent with the results of in vitro release study, cytotoxicity of micellar PEO-b-P(CL-DOX) conjugate (IC50 of 3.65 μg/mL) was lower than that of free and physically encapsulated DOX in PEO-b-PBCL (IC50 of 0.09 and 3.07 μg/mL, respectively) after 24 h of incubation. After 48 h of incubation, the cytotoxicity of conjugated DOX (IC50 of 0.50 μg/mL) was still lower than the cytotoxicity of free DOX (IC50 of 0.03 μg/mL), but surpassed that of physically encapsulated DOX in PEO-b-PBCL (IC50 of 1.54 μg/mL). The results point to a potential for PEO-b-P(CL-DOX) and PEO-b-PBCL as novel polymeric micellar drug conjugates and nano-containers bearing hydrolyzable cores for DOX delivery.
Keywords: Doxorubicin; Cytotoxicity; Polymeric micelles; Polymer–drug conjugate; Poly(ethylene oxide)-block-poly(ε-caprolactone);

A special class of porous silica materials, silica aerogels, was recently shown to be a potential candidate for oral drug delivery systems. It was demonstrated, that stability of drugs and their dissolution rate can essentially be improved through the adsorption on to these materials. In this work, drug loaded silica aerogels are firstly applied as dermal drug delivery systems. Dithranol is used as a representative drug since there is a need to enhance its dermal availability. The unstable and nearly water-insoluble drug exhibits a poor penetration. Release of dithranol from aerogels into various semi-solid formulations and its dissolution as well as the release and penetration into artificial membranes were investigated by Fourier-transform infrared attenuated total reflection (FTIR-ATR) spectroscopy. Two model membranes (one hydrophilic and one lipophilic) were applied. Several formulations were tested and the most promising one was used in order to study the penetration of dithranol into human stratum corneum (SC). Dithranol adsorbed on hydrophilic silica aerogels exhibited superior penetration behaviour compared to that of the standard ointment (dithranol in white soft paraffin).
Keywords: Hydrophilic silica aerogels; Dithranol; FTIR-ATR spectroscopy; Human stratum corneum; Model membranes; Release; Penetration;

Cutaneous concentration of lycopene correlates significantly with the roughness of the skin by Maxim Darvin; Alexa Patzelt; Saskia Gehse; Sabine Schanzer; Christian Benderoth; Wolfram Sterry; Juergen Lademann (943-947).
Antioxidant substances in the skin are expected to slow down photo ageing. We therefore developed the hypothesis that high levels of antioxidant substances may be correlated to lower levels of skin roughness.By utilizing modern optical non-invasive in vivo methods, the structures of the furrows and wrinkles as well as the concentration of lycopene were analyzed quantitatively on the forehead skin of 20 volunteers aged between 40 and 50 years.In a first step, the age of the volunteers was correlated to their skin roughness. Here, no significant correlation was found. In a second step, a significant correlation was obtained between the skin roughness and the lycopene concentration (R  = 0.843).These findings indicate that higher levels of antioxidants in the skin effectively lead to lower levels of skin roughness, and therefore support our hypothesis.
Keywords: Roughness; Furrows; Wrinkles; Lycopene; Free radicals; Skin ageing;

Quercetin in w/o microemulsion: In vitro and in vivo skin penetration and efficacy against UVB-induced skin damages evaluated in vivo by Fabiana T.M.C. Vicentini; Thaís R.M. Simi; José O. Del Ciampo; Nilce O. Wolga; Dimitrius L. Pitol; Mamie M. Iyomasa; M. Vitória L.B. Bentley; Maria J.V. Fonseca (948-957).
The present study evaluated the potential of a w/o microemulsion as a topical carrier system for delivery of the antioxidant quercetin. Topical and transdermal delivery of quercetin were evaluated in vitro using porcine ear skin mounted on a Franz diffusion cell and in vivo on hairless-skin mice. Skin irritation by topical application of the microemulsion containing quercetin, and the protective effect of the formulation on UVB-induced decrease of endogenous reduced glutathione levels and increase of cutaneous proteinase secretion/activity were also investigated. The w/o microemulsion increased the penetration of quercetin into the stratum corneum and epidermis plus dermis at 3, 6, 9 and 12 h post-application in vitro and in vivo at 6 h post-application. No transdermal delivery of quercetin occurred. By evaluating established endpoints of skin irritation (erythema formation, epidermis thickening and infiltration of inflammatory cells), the study demonstrated that the daily application of the w/o microemulsion for up to 2 days did not cause skin irritation. W/o microemulsion containing quercetin significantly prevented the UVB irradiation-induced GSH depletion and secretion/activity of metalloproteinases.
Keywords: Microemulsion; Quercetin; Skin penetration; Skin irritation; UVB radiation;

Passive and iontophoretic permeation of glipizide by Ashish Jain; Bijaya Ghosh; Naresh Rajgor; B.G. Desai (958-963).
In vitro iontophoretic delivery of glipizide across the pigskin was investigated. The experiment was carried out at three different donor drug concentrations using cathodal iontophoresis (current density 0.5 mA cm−2) with corresponding passive controls. At all concentration levels, iontophoresis showed enhanced permeation rate compared to passive controls (P  < 0.01). For passive permeation, the steady-state flux significantly increased with the increase in donor drug concentration (P  < 0.01). Passive process followed zero-order profile while the profile was nonlinear in iontophoresis. Competition by chloride ions released in the cathode compartment could be the reason. Flux enhancement was highest at the lowest drug load and lowest at the highest drug load. The target flux of glipizide was calculated to be 0. 4147 μmol h−1. As the highest flux obtained was 0.2727 μmol cm−2  h−1, it can be said that glipizide is a promising candidate for iontophoretic delivery.
Keywords: Glipizide; Iontophoresis; Pigskin; Current density; In vitro;

Direct compression properties of chitin and chitosan by Viviana García Mir; Jyrki Heinämäki; Osmo Antikainen; Ofelia Bilbao Revoredo; Antonio Iraizoz Colarte; Olga Maria Nieto; Jouko Yliruusi (964-968).
Deformation and compaction properties of native amino poly-saccharides chitin and chitosan were studied and compared with those obtained with established pharmaceutical direct compression excipients. An instrumented single-punch tablet machine was used for tablet compaction. The following compression parameters were evaluated: a ratio of crushing strength and compression pressure, plasticity and elasticity factor (PF and EF), tensile strength and R-value. Chitin and chitosan were found to have a marked tendency to plastic deformation, and both showed a good compression behaviour compared with the other direct compression excipients including microcrystalline cellulose. It is concluded that chitin and chitosan are potential co-excipients for direct compression applications.
Keywords: Chitin; Chitosan; Direct compression; Plastic deformation; Elasticity; Tablets;

Layering of pellets with recombinant Lactococcus lactis Thy 12 was optimised for the production of a dosage form with a high load of viable recombinant L. lactis. Shear stress induced during the atomisation and the type of carrier used for the layering process did not influence the viability. A 5% lactose matrix resulted in the highest viability of L. lactis (8.9 ± 1.7%) which could be maintained for at least 12 months at −20 °C. A higher bacterial cell load on the pellets was obtained using a longer process time, but the addition of 10% skim milk was essential to maintain the stabilising capacity of the matrix. Increasing the load of viable L. lactis was also possible using a higher bacterial cell concentration of the layering suspension and increasing the amount of stabilising matrix to 10% lactose/20% skim milk, yielding a formulation with 1.7 × 109  cfu/100 mg pellets. To protect the bacteria during gastric passage and to obtain ileum targeting, the formulation was enteric coated with 5% Eudragit® FS30D, but after coating and gastric residence for 2 h HCl about 1% of the bacteria remained viable. Application of a subcoating, previous to enteric coating, did not result in a higher viability.
Keywords: Layering; Viability; Recombinant Lactococcus lactis; Ileum targeting; Pellets; Eudragit; Lactose; Skim milk;

The present research is based on the hypothesis that leaky enteric-coated pellets formulations are able to provide sustained input for drugs that have an absorption window, such as ranitidine hydrochloride, without jeopardizing their bioavailability. Leaky enteric-coated pellets formulations are defined as enteric-coated pellets that allow some of the drug to be released from the formulation in gastric fluid. Different approaches to making leaky enteric-coated pellets were investigated using extrusion–spheronization followed by spray coating. Leaky enteric coats were formulated using a commonly used enteric polymer, Eudragit® L 30 D-55, combined with soluble compounds including lactose, PEG 8000 and surfactants (Span 60 (hydrophobic) or Tween 80 (hydrophilic)). The rate of drug release from the formulations in simulated gastric fluid can be tailored by varying the additive’s amount or type. All leaky enteric-coated formulations studied completely released the drugs within 30 min after changing dissolution medium to phosphate buffer, pH 6. Predictions of plasma concentration–time profiles of the model drug ranitidine hydrochloride from leaky enteric-coated pellets in fasted conditions and from immediate-release formulations were performed using computer simulations. Simulation results are consistent with a hypothesis that leaky enteric-coated pellets formulations provide sustained input for drugs shown to have an absorption window without decreasing bioavailability. The sustained input results from the combined effects of the formulation and GI transit effects on pellets.The present research demonstrates a new application of knowledge about gastrointestinal transit effects on drug formulations. It also shows that enteric-coating polymers have new applications in areas other than the usual enteric-coated formulations. The hypothesis that a leaky enteric-coated pellets formulation may maintain or increase the bioavailability of drugs that have a window of absorption is still to be confirmed by further in vivo studies.
Keywords: Leaky enteric-coating; Pellets; Ranitidine hydrochloride; Extrusion–spheronization; Computer simulations; Absorption window; GI transit;

Effect of the type of lubricant on the characteristics of orally disintegrating tablets manufactured using the phase transition of sugar alcohol by Yoshio Kuno; Masazumi Kojima; Hiroaki Nakagami; Etsuo Yonemochi; Katsuhide Terada (986-992).
The aim of this study was to evaluate the effect of lubricants on the characteristics of orally disintegrating (OD) tablets manufactured using the phase transition of sugar alcohol. OD tablets were produced by directly compressing a mixture containing lactose–xylitol granules, disintegrant, glidant and lubricant, and subsequent heating. The effect of the type of lubricant on the tablet characteristics was evaluated using magnesium stearate (Mg-St), sodium stearyl fumarate (SSF), and talc as lubricants. The hardness of the tablets increased to ca. 6 kp as a result of heating, regardless of the kind of lubricant. The oral disintegration time of the tablets containing Mg-St or SSF increased with an increase in the hardness. In contrast, the oral disintegration time of the tablets containing talc was not changed despite of an increase in hardness. The water absorption rate of the tablets containing talc was much faster than that of the tablets containing other lubricants. The surface free energy measurement showed that the polarity of the tablet components containing talc was remarkably increased by heating. The water absorption rate of the tablets containing talc was also increased by heating. These results indicate that a more hydrophilic surface might be attained by heating the talc. Consequently, talc was demonstrated to be the most desirable lubricant for the preparation of OD tablets based on the principle of the phase transition of sugar alcohol.
Keywords: Orally disintegrating tablets; Phase transition; Sugar alcohol; Saccharide; Lubricant; Talc;

Although famotidine was reported to be 7.5 and 20 times more potent than ranitidine and cimetidine, respectively, its oral bioavailability is low and variable; due mainly to its poor aqueous solubility. The purpose of this study was to improve famotidine dissolution through its formulation into liquisolid systems and then to investigate the in vitro and in vivo performance of the prepared liquisolid tablets. The new mathematical model was utilized to formulate various liquisolid powder systems. Both DSC and XRD suggested loss of famotidine crystallinity upon liquisolid formulation which was further confirmed by SEM indicating that even though the drug existed in a solid dosage form, it is held within the powder substrate in a solubilized, almost molecularly dispersed state, which contributed to the enhanced drug dissolution properties. All the tested liquisolid tablet formulations showed higher drug dissolution rates (DR) than the conventional, directly compressed tables. In addition, the selected optimal formula released 78.36% of its content during the first 10 min which is 39% higher than that of the directly compressed tablets. Further, the bioavailability study indicated that the prepared optimal liquisolid formula did not differ significantly from the marketed famotidine tablets concerning C max, t max, and AUC (0–8) at P  < 0.05.
Keywords: Famotidine; Liquisolid system; New formulation mathematical model; In vitro dissolution; Bioavailability study;

Modulation of drug release kinetics of shellac-based matrix tablets by in-situ polymerization through annealing process by Sontaya Limmatvapirat; Chutima Limmatvapirat; Satit Puttipipatkhachorn; Jurairat Nunthanid; Manee Luangtana-anan; Pornsak Sriamornsak (1004-1013).
A new oral-controlled release matrix tablet based on shellac polymer was designed and developed, using metronidazole (MZ) as a model drug. The shellac-based matrix tablets were prepared by wet granulation using different amounts of shellac and lactose. The effect of annealing temperature and pH of medium on drug release from matrix tablets was investigated. The increased amount of shellac and increased annealing temperature significantly affected the physical properties (i.e., tablet hardness and tablet disintegration) and MZ release from the matrix tablets. The in-situ polymerization played a major role on the changes in shellac properties during annealing process. Though the shellac did not dissolve in acid medium, the MZ release in 0.1 N HCl was faster than in pH 7.3 buffer, resulting from a higher solubility of MZ in acid medium. The modulation of MZ release kinetics from shellac-based matrix tablets could be accomplished by varying the amount of shellac or annealing temperature. The release kinetics was shifted from relaxation-controlled release to diffusion-controlled release when the amount of shellac or the annealing temperature was increased.
Keywords: Shellac; Matrix tablets; Release kinetics; Annealing; In-situ polymerization;

Increasing sodium pantoprazole photostability by microencapsulation: Effect of the polymer and the preparation technique by R.P. Raffin; L.M. Colomé; E.E.S. Schapoval; A.R. Pohlmann; S.S. Guterres (1014-1018).
Pantoprazole sodium is a proton pump inhibitor, used in acid-related disorders, like peptic ulcers and gastroesophageal reflux. This drug is unstable in acid solution and in the presence of salts. The aim of this work was to study the photostability under UVC radiation of pantoprazole and to determine its kinetics. A methanol solution and the solid pantoprazole were evaluated by HPLC within 120 min and 10 days, respectively. The work was also dedicated to evaluate and compare the ability of microencapsulation in stabilizing pantoprazole after UVC radiation. Pantoprazole-loaded microparticles prepared by emulsification/solvent evaporation or spray drying were compared. Pantoprazole was encapsulated using Eudragit S100® or its blend with poly(ε-caprolactone) or HPMC. In methanol solution, pantoprazole was completely degraded after 120 min and presented zero-order kinetics with t 1/2 of 6.48 min. In the solid form, after 10 days, pantoprazole concentration was reduced to 27% following zero-order kinetic. The microparticles prepared only with Eudragit S100® demonstrated an increase of the drug photostability. After 10 days of irradiation, 56 and 44% of the drug was stable when encapsulated by emulsification/solvent evaporation and spray drying, respectively. The use of polymer blends did not improve the pantoprazole photostability.
Keywords: Pantoprazole; Photostability; HPLC; Microencapsulation; Spray drying; Solvent evaporation;

The purpose of this work was to develop an extended release matrix tablet of nicorandil; a freely water soluble drug used in cardiovascular diseases. Chitosan (CH)/hyaluronate sodium (HA), pectin (PE) or alginate sodium (AL) interpolymer complexes (IPCs) were prepared. The optimum IPCs (CH:HA, 40:60), (CH:PE, 30:70) and (CH:AL, 20:80) were characterized by Fourier transform infrared spectroscopy. The IPCs were based on electrostatic interactions between protonated amine groups of CH and carboxylate groups of HA, PE or AL. Nicorandil matrix tablets were prepared using the optimum IPCs, alone or in combination with Imwitor® 900 K. Evaluations such as weight variation, thickness, content uniformity, friability, disintegration and in vitro release studies were performed. The tablets showed acceptable pharmacotechnical properties and complied with compendial requirements. Results of the dissolution studies revealed that formula F11 (CH:AL, 20:80) IPC:Imwitor® 900 K, 3:1) could extend drug release >8 h. Most formulae exhibited non-Fickian diffusion drug release profiles. When compared to the immediate release Ikorel® tablet, the duration of effective nicorandil therapeutic concentration from formula F11, in healthy human volunteers, was significantly (P  < 0.05) extended from 4 to 8 h with expected lowering in side effects potential.
Keywords: Nicorandil; Chitosan; Hyaluronic acid sodium salt; Pectin; Alginate; Interpolymer complex; Imwitor® 900 K; Matrix tablet;

Application of the SeDeM Diagram and a new mathematical equation in the design of direct compression tablet formulation by Josep M. Suñé-Negre; Pilar Pérez-Lozano; Montserrat Miñarro; Manel Roig; Roser Fuster; Carmen Hernández; Ramon Ruhí; Encarna García-Montoya; Josep R. Ticó (1029-1039).
Application of the new SeDeM Method is proposed for the study of the galenic properties of excipients in terms of the applicability of direct-compression technology. Through experimental studies of the parameters of the SeDeM Method and their subsequent mathematical treatment and graphical expression (SeDeM Diagram), six different DC diluents were analysed to determine whether they were suitable for direct compression (DC). Based on the properties of these diluents, a mathematical equation was established to identify the best DC diluent and the optimum amount to be used when defining a suitable formula for direct compression, depending on the SeDeM properties of the active pharmaceutical ingredient (API) to be used. The results obtained confirm that the SeDeM Method is an appropriate system, effective tool for determining a viable formulation for tablets prepared by direct compression, and can thus be used as the basis for the relevant pharmaceutical development.
Keywords: Expert system; SeDeM; Formulation; Bulk Density (Da); Tapped Density (Dc); Inter-particle Porosity (Ie); Angle of Repose (α); Flowability (t′′); Particle Size (%Pf); Direct compression (DC);

Influence of the delivery systems using a microneedle array on the permeation of a hydrophilic molecule, calcein by Jae-Ho Oh; Hyoun-Hyang Park; Ki-Young Do; Manhee Han; Dong-Hun Hyun; Chang-Gyu Kim; Chang-Hyeon Kim; Seung S. Lee; Sung-Joo Hwang; Sang-Chul Shin; Cheong-Weon Cho (1040-1045).
Despite the advantages of drug delivery through the skin, such as easy accessibility, convenience, prolonged therapy, avoidance of the liver first-pass metabolism and a large surface area, transdermal drug delivery is only used with a small subset of drugs because most compounds cannot cross the skin at therapeutically useful rates. Recently, a new concept was introduced known as microneedles and these could be pierced to effectively deliver drugs using micron-sized needles in a minimally invasive and painless manner. In this study, biocompatible polycarbonate (PC) microneedle arrays with various depths (200 and 500 μm) and densities (45, 99 and 154 ea/cm2) were fabricated using a micro-mechanical process. The skin permeability of a hydrophilic molecule, calcein (622.5D), was examined according to the delivery systems of microneedle, drug loading, depth of the PC microneedle, and density of the PC microneedle. The skin permeability of calcein was the highest when the calcein gel was applied to the skin with the 500 μm-depth PC microneedle, simultaneously. In addition, the skin permeability of calcein was the highest when 0.1 g of calcein gel was coupled to the 500 μm-depth PC microneedle (154 ea/cm2) as well as longer microneedles and larger density of microneedles. Taken together, this study suggests that a biocompatible PC microneedle might be a suitable tool for transdermal drug delivery system of hydrophilic molecules with the possible applications to macromolecules such as proteins and peptides.
Keywords: Microneedle; Calcein; Permeation; Gel; Delivery systems;

Physicochemical characterization of five glyburide powders: A BCS based approach to predict oral absorption by Hai Wei; Chad Dalton; Marie Di Maso; Isadore Kanfer; Raimar Löbenberg (1046-1056).
The purpose of this study was to investigate the suitability of physicochemical parameters of Active Pharmaceutical Ingredients (APIs) as input functions for the Advanced Compartmental Absorption and Transit Model (ACAT) to predict the oral absorption of drug products. Five different glyburide APIs were characterized using X-ray powder diffraction (XRPD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), Raman spectroscopy, particle size and particle size distribution, specific surface area and true density measurements, as well as dissociation constant (pK a), partition coefficient (log  P) and distribution coefficient (log  D). The computer simulations were performed using GastroPlus™. The results of XPRD, DSC and Raman spectroscopy indicated that no significant differences in crystal form were present in the five APIs. However, significant differences in particle size and particle size distribution were observed. A basic in vitro/in vivo relationship between the APIs’ particle size and clinically observed plasma time profiles was established. The study demonstrates that in silico methods can assist the formulation scientist to set meaningful API specifications. Computer simulations could shorten the drug development process since appropriate bioawaivers, based on data from simulation studies, may be justified.
Keywords: Glyburide/glibenclamide; Oral absorption; Material characterization; Particle size; Advanced Compartmental Absorption and Transit model;

Nebulization of nanoparticulate amorphous or crystalline tacrolimus – Single-dose pharmacokinetics study in mice by Prapasri Sinswat; Kirk A. Overhoff; Jason T. McConville; Keith P. Johnston; Robert O. Williams (1057-1066).
Developing a pulmonary composition of tacrolimus (TAC) provides direct access to the graft in lung transplant offering the possibility of high drug levels. The objective of this study was to investigate the physicochemical and pharmacokinetic characteristics of the nanostructured aggregates containing amorphous or crystalline nanoparticles of TAC produced by ultra-rapid freezing (URF). TAC and lactose (1:1 ratio; URF-TAC:LAC) and TAC alone (URF-TAC) were investigated for pulmonary delivery and compared to unprocessed TAC. X-ray diffraction (XRD) results indicated that URF-TAC was crystalline, whereas URF-TAC:LAC was amorphous. In vitro results revealed the superior physiochemical characteristics of both URF formulations compared to unprocessed TAC. The surface area of URF processed TAC was higher (25–29 m2/g) than that of the unprocessed TAC (0.53 m2/g) and subsequently enhanced dissolution rates. In addition, URF-TAC:LAC displayed the ability to supersaturate in the dissolution media to about 11 times the crystalline equilibrium solubility. Similar aerodynamic particle sizes of 2–3 μm, and fine particle fraction between 70% and 75% were found in both formulations. The local and systemic pharmacokinetic studies in mice showed similar AUC(0–24), higher C max, and lower T max for the URF-TAC:LAC compared to the URF-TAC. Nanostructured aggregates containing amorphous or crystalline nanoparticles of TAC were demonstrated to be effectively delivered via nebulization, with similar in vitro and in vivo performances.
Keywords: Tacrolimus (TAC); Ultra-rapid freezing (URF); Nanoparticles; Amorphous; Crystalline; Pulmonary delivery;

Thermodynamic behavior of glassy state of structurally related compounds by Aditya Mohan Kaushal; Arvind Kumar Bansal (1067-1076).
Thermodynamic properties of amorphous pharmaceutical forms are responsible for enhanced solubility as well as poor physical stability. The present study was designed to investigate the differences in thermodynamic parameters arising out of disparate molecular structures and associations for four structurally related pharmaceutical compounds – celecoxib, valdecoxib, rofecoxib, and etoricoxib. Conventional and modulated temperature differential scanning calorimetry were employed to study glass forming ability and thermodynamic behavior of the glassy state of model compounds. Glass transition temperature of four glassy compounds was in a close range of 327.6–331.8 K, however, other thermodynamic parameters varied considerably. Kauzmann temperature, strength parameter and fragility parameter showed rofecoxib glass to be most fragile of the four compounds. Glass forming ability of the compounds fared similar in the critical cooling rate experiments, suggesting that different factors were determining the glass forming ability and subsequent behavior of the compounds in glassy state. A comprehensive understanding of such thermodynamic facets of amorphous form would help in rationalizing the approaches towards development of stable glassy pharmaceuticals.
Keywords: Differential scanning calorimetry; Amorphous; Glass transition temperature; Fragility; Heat capacity;

Non-invasive diagnosis and monitoring of chronic kidney disease by reverse iontophoresis of urea in vivo by Valentine Wascotte; Eric Rozet; Ana Salvaterra; Philippe Hubert; Michel Jadoul; Richard H. Guy; Véronique Préat (1077-1082).
Reverse iontophoresis uses a small current to extract molecules and ions through the skin. The aim of the study was to determine whether reverse iontophoresis of urea can be used (i) to diagnose and monitor non-invasively chronic kidney disease (CKD), and (ii) to track urea levels closely during a hemodialysis session.A current of 0.8 mA was applied for 2 h in 10 healthy volunteers, in 9 patients with CKD, and in 10 patients undergoing hemodialysis. Urea fluxes extracted by reverse iontophoresis and urea concentrations in the blood were measured.Extracted urea fluxes discriminated healthy volunteers from patients with CKD within 90 min. A non-invasive measure of blood urea concentrations can be achieved after 120 min. A urea reservoir in the skin interferes with the extraction and a pre-hemodialysis “depletion” period is required. Mild and transient sensation and erythema induced by iontophoresis were significantly lower in the CKD group. Gelling the formulation of the iontophoresis reservoir gave similar results to those obtained when using a simple aqueous solution.Reverse iontophoresis can be used to non-invasively diagnose individuals with CKD and to monitor urea concentrations in blood.
Keywords: Reverse iontophoresis; Urea; Non-invasive monitoring; Diagnosis; Chronic kidney disease;

Improved formulation of photosensitizer chlorin e6 polyvinylpyrrolidone for fluorescence diagnostic imaging and photodynamic therapy of human cancer by William Wei Lim Chin; Paul Wan Sia Heng; Patricia Soo Ping Thong; Ramaswamy Bhuvaneswari; Werner Hirt; Sebastian Kuenzel; Khee Chee Soo; Malini Olivo (1083-1093).
An improved formulation of the photosensitizer chlorin e6 (Ce6) in combination with the hydrophilic polymer polyvinylpyrrolidone (PVP) was investigated for its potential clinical applications in fluorescence diagnosis and photodynamic therapy (PDT) of cancer. This study reports the comparative preclinical biodistribution and efficacy of Ce6 delivered with or without PVP versus dimethyl sulfoxide (DMSO). The safety and fluorescence pharmacokinetics of Ce6–PVP in humans was also accessed. Biodistribution results showed that Ce6–PVP had higher tumor to normal tissue ratio compared to the other formulations. The sensitivity and specificity derived from the area under the receiver operating characteristics curves showed that the formulations were able to discriminate tumor from peritumoral muscle in the following order: Ce6–PVP > Ce6 > Ce6–DMSO. In vitro PDT results showed that Ce6–PVP was found to induce selective phototoxicity in leukemic cells compared to peripheral mononuclear blood cells. In addition, in vivo light irradiation at 1 h after Ce6–PVP was found to induce greater tumor necrosis without causing animal toxicity. In patients, preferential accumulation of Ce6–PVP was observed in angiosarcoma lesions compared to normal skin following intravenous administration. In conclusion, PVP significantly enhanced the Ce6 concentration in tumors compared with Ce6 alone and increased the therapeutic index of PDT without any side effects in animal model. No serious adverse events were observed in human as well.
Keywords: Chlorin e6; Polyvinylpyrrolidone; Photosensitizer; Photodynamic therapy; Fluorescence diagnosis; Biodistribution; Imaging; Formulation;

Knowledge about the microstructure and the release rate of hydrophilic macromolecules is required for a rational development of comfortable and safe contact lenses. Semi-interpenetrating networks of poly(hydroxyethyl methacrylate) (pHEMA) with poly(vinyl pyrrolidone) (PVP) were prepared by free radical polymerization of HEMA in the presence of PVP K30 or K90F, under anhydrous conditions or after addition of water, and evaluated in terms of swelling, porosity, PVP release rate, air–water surface tension, and friction coefficient. The greater water content was during polymerization, the higher was the swelling degree and porosity. Microphase separation above a certain volume of water resulted in hydrogels with bumpy surface and interconnected pores. All hydrogels showed a high optical clarity and slowly released PVP (20% after 9 days). In general, the greater the content of PVP or the higher its molecular weight was, the lower the friction coefficients were. In the case of hydrogels prepared with water, the friction was influenced by the balance between the ability to hold water in the network (which contributes to the sliding and PVP release) and the deleterious effect of an irregular surface. Controlled delivery of PVP revealed as a critical factor for improving the frictional behavior of pHEMA contact lenses.
Keywords: Semi-IPNs; Sustained release of poly(vinyl pyrrolidone); Comfortable lenses; Rheological evaluation of surface friction; Porosity;

Physicochemical stability of phospholipid-dispersed suspensions of crystalline itraconazole by Jane Werling; Sabine Graham; Heather Owen; Lakshmy Nair; Thomas Gonyon; Phillip W. Carter (1104-1113).
The physicochemical stability of an aqueous, phospholipid-based dispersion of itraconazole microcrystals was studied as a model water-insoluble drug suspension. The particle size, phospholipid concentrations, free fatty acid (FFA) content, pH, and zeta potential of two test suspensions were followed over 63 days at 5 and 40 °C storage conditions. Hydrolysis of a control suspension containing Lipoid E80 led to rapid FFA formation, pH drop, and subsequent particle aggregation. In the second suspension, sodium oleate used in conjunction with Lipoid E80 significantly enhanced the suspension physicochemical stability. Oleate anions effectively (1) increased the anionic charge of the phospholipid surface layer, (2) buffered the suspension near pH 7, and (3) reduced the specific production of oleic acid as a phosphatidylcholine (PC) degradant. The observed hydrolysis rate constants k obs  ∼ 2 × 10−7 (Lipoid only) and k obs  ∼ 5 × 10−8 (Lipoid and oleate) were consistent with the pH dependent behavior reported for saturated soybean PC solutions. Mechanistically, FFA formed initially in the control suspension partitioned to the aqueous phase with limited influence on the phospholipid microenvironment at the itraconazole particle surface. Phospholipid stabilization of water-insoluble drugs was demonstrated with clear benefits from fatty acid anions as co-additives to influence the surface microenvironment, reduce hydrolysis kinetics, and enhance suspension physicochemical stability.
Keywords: Phospholipid; Hydrolysis; Physicochemical stability; Oleate; Crystalline suspensions; Itraconazole;

Characterization of ternary solid dispersions of Itraconazole in polyethylene glycol 6000/polyvidone-vinylacetate 64 blends by Sandrien Janssens; Hector Novoa de Armas; Ward D’Autry; Ann Van Schepdael; Guy Van den Mooter (1114-1120).
The good compatibility between Itraconazole and polyvidone-vinylacetate 64 (PVPVA 64) was pointed out previously. However, the dissolution properties of these systems left room for improvement. Therefore polyethylene glycol 6000 (PEG 6000), known for its solubilizing and wetting properties, was added to the PVPVA 64 matrix. Physicochemical analysis showed that up to 10% of PEG 6000 could be mixed with PVPVA 64. Addition of 10%, 20% or 40% of Itraconazole rendered amorphous solid dispersions consisting of a ternary mixed phase and a PVPVA 64 rich amorphous phase. If the PEG 6000 fraction was elevated up to 25% of the carrier, the PEG 6000 crystallinity degree was around 73 ± 0.6%. Up to 20% of Itraconazole could be molecularly dispersed in the 25/75 w/w polymer blend. An Itraconazole melting peak could be detected for the sample containing 40% of drug. Dissolution experiments showed that no benefit was obtained by adding PEG 6000 to the PVPVA 64 matrix for samples containing up to 20% of Itraconazole. The dissolution of the ternary dispersions with 40% of Itraconazole on the other hand showed improvement compared to binary Itraconazole/PVPVA 64 dispersions.
Keywords: Ternary solid dispersion; MDSC; Powder X-ray diffraction; Dissolution; GC–MS; Itraconazole; PEG 6000; PVPVA 64;

Two sets of copolymers comprising of styrene and either methyl or ethyl methacrylate as comonomer were conveniently synthesized by microemulsion copolymerization. The purified materials were characterized by GPC-MALLS and were shown to form artificial nanolatexes in THF. ATR-FTIR analysis revealed differences in copolymer composition and based on the copolymer properties, a selection of copolymers was chosen to cast drug-loaded, microporous films that exhibit microencapsulation of drug agglomerates. The contact angles of the copolymers suggested potential applications in medical devices to prevent the formation of bacterial biofilms that commonly result in infections. Additionally, the different copolymeric films showed two phases of drug release characterized by a rapid initial drug release followed by a zero-order phase. Depending on the application, one could select the copolymer films that best suited the application i.e. for short-term drug release applications such as urinary catheters or long-term applications such as artificial implants.
Keywords: Methacrylate copolymer; Microencapsulation; Film; Rifampin; Controlled release;

Modulation of gel formation and drug-release characteristics of lidocaine-loaded poly(vinyl alcohol)-tetraborate hydrogel systems using scavenger polyol sugars by Ryan G. Loughlin; Michael M. Tunney; Ryan F. Donnelly; Diarmaid J. Murphy; Mark Jenkins; Paul A. McCarron (1135-1146).
Polyol sugars, displaying a plurality of hydroxyl groups, were shown to modulate tetrahydroxyborate (borate) cross-linking in lidocaine hydrochloride containing poly(vinyl alcohol) semi-solid hydrogels. Without polyol, demixing of borate cross-linked PVA hydrogels into two distinct phases was noticeable upon lidocaine hydrochloride addition, preventing further use as a topical system. d-Mannitol incorporation was found to be particularly suitable in circumventing network constriction induced by ionic and pH effects upon adding the hydrochloride salt of lidocaine. A test formulation (4% w/v lidocaine HCl, 2% w/v d-mannitol, 10% w/v PVA and 2.5% w/v THB) was shown to constitute an effective delivery system, which was characterised by an initial burst release and a drug release mechanism dependent on temperature, changing from a diffusion-controlled system to one with the properties of a reservoir system. The novel flow properties and innocuous adhesion of PVA–tetrahydroxyborate hydrogels support their application for drug delivery to exposed epithelial surfaces, such as lacerated wounds. Furthermore, addition of a polyol, such as d-mannitol, allows incorporation of soluble salt forms of active therapeutic agents by modulation of cross-linking density.
Keywords: Poly(vinyl alcohol); Borate; Hydrogel; Lidocaine hydrochloride; Mannitol; Polyol; Topical delivery;

Characterisation and controlled drug release from novel drug-loaded hydrogels by Luke M. Geever; Ciaran C. Cooney; John G. Lyons; James E. Kennedy; Michael J.D. Nugent; Sinead Devery; Clement L. Higginbotham (1147-1159).
Hydrogel based devices belong to the group of swelling controlled drug delivery systems. Temperature responsive poly(N-isopropylacrylamide)–poly(vinylpyrrolidinone) random copolymers were produced by free radical polymerisation, using 1-hydroxycyclohexylphenyketone as an ultraviolet-light sensitive initiator, and poly(ethylene glycol) dimethacrylate as the crosslinking agent (where appropriate). The hydrogels were synthesised to have lower critical solution temperatures (LCST) near body temperature, which is favourable particularly for ‘smart’ drug delivery applications. Two model drugs (diclofenac sodium and procaine HCl) were entrapped within these xerogels, by incorporating the active agents prior to photopolymerisation. The properties of the placebo samples were contrasted with the drug-loaded copolymers at low levels of drug integration. Modulated differential scanning calorimetry (MDSC), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and atomic force microscopy (AFM) were used to investigate the influence of the drugs incorporated on the solid-state properties of the xerogels. MDSC and swelling studies were carried out to ascertain their effects on the LCST and swelling behaviour of the hydrated samples. In all cases, drug dissolution analysis showed that the active agent was released at a slower rate at temperatures above the phase transition temperature. Finally, preliminary in vitro cytotoxicity evaluations were performed to establish the toxicological pattern of the gels.
Keywords: Hydrogels; Photopolymerisation; Lower critical solution temperature; Temperature sensitive; Drug delivery;

Application of PGSTE-NMR technique to characterize the porous structure of pharmaceutical tablets by Virginie Busignies; Patrice Porion; Bernard Leclerc; Pierre Evesque; Pierre Tchoreloff (1160-1170).
Direct compaction of pharmaceutical tablets is a complex process that results in a heterogeneous density distribution inside the compact. In the present study, we have used a non-invasive and non-destructive technique: the pulsed-gradient stimulated-echo (PGSTE) NMR method to access to topological information (connectivity, tortuosity) about the porous structure of the tablets obtained with three different pharmaceutical excipients: the microcrystalline cellulose, the lactose and the anhydrous calcium phosphate. These materials were chosen since their mechanical properties under pressure are highly differentiated. To probe the pore space with the PGSTE-NMR technique, the tablets were initially impregnated with silicone oil that is NMR sensitive (1H NMR). The time-dependent apparent self-diffusion coefficient was measured over a suitable range of diffusion time in the directions perpendicular and parallel to the compression axis, from which the tortuosity factor and the anisotropy of the porous structure can be studied. These results show that the porous structure varies with pressure and depends on the excipient behaviour under pressure. Then, this work demonstrates that PGSTE-NMR could be an alternative and a very interesting technique to obtain useful information on the structural properties of such compacted materials.
Keywords: Compaction; Tablets; Porosity; Compressibility; Pulsed-gradient stimulated-echo NMR; Diffusion; Self-diffusion coefficient; Tortuosity; Anisotropy;

In vivo infrared spectroscopy studies of alkanol effects on human skin by M. Dias; A. Naik; R.H. Guy; J. Hadgraft; M.E. Lane (1171-1175).
Many studies investigate the permeation of actives through the skin and ignore the role of excipients. The solvents used in formulations will undoubtedly penetrate the skin where they can have a number of effects. They can extract skin lipids, they can alter the fluidity of the lipids and they can alter the polarity of the skin. The degree to which they do this and the depth into the skin where this occurs will depend on the uptake kinetics. The problem is to distinguish the different effects. Using ATR-FTIR and deuterated materials this can be achieved in vivo. The aim of the present study was to study the higher alkanols (hexanol, octanol, decanol) in vivo using a combination of ATR-FTIR spectroscopy and tape stripping. Studies conducted in vivo using deuterated vehicles confirmed the lipid extraction effects of d-hexanol and d-octanol, whereas d-decanol did not change skin lipid content. The uptake of d-decanol was higher than for the other vehicles consistent with previous observations on mouse skin for alkanols of increasing chain length. In general, solvent uptake was proportional to the induced shift in the C–H stretching frequency. Lipid disorder was induced by all vehicles studied in vivo and was proportional to the amount of vehicle present in the skin.
Keywords: Infrared spectroscopy; Skin; Alkanols; Lipid extraction; Lipid disorder; Penetration enhancement;

Disodium norcantharidate (DSNC) loaded poly(ε-caprolactone) (PCL) microspheres were prepared by s/o/w solvent evaporation technique, and the formation mechanism and release behavior of the microspheres were investigated. The particle formation of the microspheres was influenced by the osmotic effect of DSNC. During the microsphere preparation, water diffused into the emulsion droplets and dissolved the particles of DSNC. Thereafter, DSNC generated osmotic effect and drove the water to flow in the emulsion droplets more quickly, thus forming an inner water phase. As the water influx proceeded, the state of the emulsion was transferred from s/o/w to w/o/w, thus resulting in the porosity of the microspheres. The release tests were carried out in the release media of different osmotic pressures achieved by adding different amounts of dextrose. The results indicated that the initial release of DSNC from the microspheres was controlled by a combination of osmotic effect and diffusion, but the release after the initial was mainly controlled by diffusion. This study demonstrated that the osmotic effect of DSNC not only was responsible for the particle formation but also contributed to the release from the microspheres.
Keywords: Particle formation; Porosity; Microspheres; Release behavior; Disodium norcantharidate;

Characterisation of excipient-free nanoporous microparticles (NPMPs) of bendroflumethiazide by A.M. Healy; B.F. McDonald; L. Tajber; O.I. Corrigan (1182-1186).
The purpose of this study was to prepare excipient-free porous microparticles of bendroflumethiazide by spray drying and to characterise the physicochemical properties of the particles produced. Solutions of bendroflumethiazide in ethanol/water, ethanol/water/ammonium carbonate or methanol/water/ammonium carbonate were spray dried using a laboratory spray dryer. Spray dried products were characterised by scanning electron microscopy, X-ray powder diffraction, differential scanning calorimetry, FTIR, laser diffraction particle sizing and density measurement. Nanoporous microparticles (NPMPs) were prepared from the alcoholic solutions containing ammonium carbonate. NPMPs were amorphous in nature, had median particles sizes less than 3 μm and densities that were significantly reduced compared to non-porous spray dried bendroflumethiazide powder. The novel process may be used to produce excipient-free amorphous microparticles with desirable physical properties such as amorphous solid state, porosity and low bulk density. This new engineering technology has applications in the design of other therapeutic agents such as those used in pulmonary delivery.
Keywords: Spray drying; Porous microparticles; Amorphous; Excipient-free; Nanopores;

Validation of reference genes for qPCR studies on Caco-2 cell differentiation by Claudia Piana; Michael Wirth; Stefan Gerbes; Helmut Viernstein; Franz Gabor; Stefan Toegel (1187-1192).
Validation of reference gene expression stabilities is a prerequisite for reliable normalization of qPCR data. The present study assessed the variation of six reference genes (ACTB, GAPDH, B2M, HPRT1, SDHA, YWHAZ) in Caco-2 cells under the influence of different growth supports and cultivation periods. Genes were ranked according to their stability using the geNorm software. To verify the influence of reference gene selection, ALPI gene expression during differentiation was quantified using the most or the least stable reference gene for normalization. Experimental conditions significantly affected the expression levels of reference genes. Whereas GAPDH and ACTB were revealed as most stable genes, SDHA was the least stable one. The extent of ALPI gene expression was significantly changed by the selection of the reference gene. This study provides a basis for qPCR studies related to both the differentiation process of Caco-2 cells and the elucidation of cell behaviour influenced by surface modifications.
Keywords: Caco-2; Differentiation; Cell-surface interaction; qPCR; Normalization; Reference genes; geNorm;

by Ulrich F. Schaefer (1193).

by Wolfgang Kreis (1193-1194).

by Marc Schneider (1194-1195).