European Journal of Pharmaceutics and Biopharmaceutics (v.66, #3)

APV Diary (S1-S2).

The low solubility in biological fluids displayed by about 50% of the drugs still remains the main limitation in oral, parenteral, and transdermal administration. Among the existing strategies to overcome these drawbacks, inclusion of hydrophobic drugs into polymeric micelles is one of the most attractive alternatives. Amphiphilic poly(ethylene oxide)–poly(propylene oxide) block copolymers are thermoresponsive materials that display unique aggregation properties in aqueous medium. Due to their ability to form stable micellar systems in water, these materials are broadly studied as hydrosolubilizers for poorly water-soluble drugs. The present review provides a concise description of the most important applications of PEO–PPO-based copolymers in the Pharmaceutical Technology field as means for attaining improved solubility, stability, release, and bioavailability of drugs.
Keywords: Poly(ethylene oxide)–poly(propylene oxide) block copolymers; Poloxamer; Poloxamine; Drug solubilization; Drug stability; Bioavailability; Drug delivery;

Chitosan microspheres enhance the immunogenicity of an Ag85B-based fusion protein containing multiple T-cell epitopes of Mycobacterium tuberculosis by Bing dong Zhu; Ya qing Qie; Jiu ling Wang; Ying Zhang; Qing zhong Wang; Ying Xu; Hong hai Wang (318-326).
To develop novel delivery system for tuberculosis (TB) subunit vaccine, biodegradable chitosan microspheres were prepared and used to deliver a fusion protein, Ag85B–MPT64190–198–Mtb8.4 (AMM for short), made from three Mycobacterium tuberculosis genes. AMM-loaded microspheres were first characterized for their morphology, size, zeta potential, loading efficiency, and in vitro release of AMM. C57BL/6 mice were immunized at weeks 1, 3 and 5 subcutaneously with AMM formulated in chitosan microspheres, in incomplete Freund’s adjuvant (IFA), or in phosphate-buffered saline (PBS), respectively. Three weeks after the last immunization, humoral and cell-mediated immune responses were examined. It was shown that the microspheres bound AMM quite efficiently (loading efficiency: >99%). AMM-loaded chitosan microspheres were observed as aggregated shapes with the average particle size of 5.78 ± 0.65 μm and zeta potential of 32.77 ± 1.51 mV. In vitro release studies revealed that only small amount of antigen was released in 16 days. Following subcutaneous administration, splenocytes immunized with AMM in chitosan microspheres produced higher levels of IFN-γ compared to administration of AMM in PBS upon stimulation with Ag85B and synthetic peptide MPT64190–198. The levels of Ag85B-specific IgG (H+L), IgG1 and IgG2a in sera of mice immunized with AMM in chitosan microspheres were also higher than those with AMM in PBS. These results indicate that chitosan microspheres when used as a carrier for fusion protein AMM could elicit strong humoral and cell-mediated immune responses.
Keywords: Mycobacterium tuberculosis; Subunit vaccine; Fusion protein; Chitosan microspheres; Adjuvant;

An efficient vector for gene delivery: α,β-poly (3-dimethylaminopropyl-d,l-aspartamide) by Weihua Xue; Huajia Diao; Xi Chen; Chunming Wang; Jiangning Chen; Junfeng Zhang (327-333).
PSI, as the potential peptide-like intermediate, is subject to simple chemical modification in order to obtain good non-viral carriers for gene delivery. This paper describes the facile synthesis and preliminary evaluation of α,β-poly (3-dimethylaminopropyl-d,l-aspartamide) (PDAI) as a vector. Reaction of PSI with 3-dimethylamino-1-propylamine afforded PDAI in N,N-dimethylformamide (DMF) solution. Such biophysical properties of PDAI/DNA complexes as the particle size and the zeta potential were determined by dynamic light scattering assay. The complexes prepared at weight ratios ranging from 2 to 3 have an average size of around 200 nm and a zeta potential of around 10.0 mV. Gel electrophoresis assays confirmed that PDAI could compact DNA to form the complexes and protect DNA from enzymatic degradation by DNase I at the weight ratio above 2.0. Furthermore, PDAI was found to transfect HepG2 cells at a much higher efficiency than commercially available polyethylenimine (PEI) (W w  = 75,000 Da). MTT cytotoxicity assay demonstrated that PDAI also showed much less toxicity than did PEI, suggesting that PDAI is a new class of transfection reagent to be used as a safe vector.
Keywords: PDAI; PDAI/DNA complex; Gene delivery; Transfection efficiency;

In the present study the release mechanism of the sparingly water-soluble drug felodipine (FELO) from particulate solid dispersions in PVP or PEG was investigated. FT-IR data indicated that a N–H⋯O hydrogen bond is formed between FELO and polymers. The drug–polymer interaction was theoretically studied with the density functional theory with the B3LYP exchange correlation function. The interaction energies have been estimated at −31.8 kJ/mol for PVP and −18.8 kJ/mol for PEG. Also, detailed vibrational analysis of the complexes showed that the red shift of the N–H bond stretching in FELO molecule due to H-bonding was higher in the FELO–PVP complex than in the FELO–PEG complex. Both the experimental and theoretical data indicated that a stronger interaction of FELO with PVP than with PEG was developed. The interactions of FELO with the polymer appeared to control the physical state (amorphous or crystalline) and the particle size of FELO in the solid dispersions. In the FELO/PVP dispersions, the drug is found as amorphous nanoparticles whereas in FELO/PEG dispersions the drug is dispersed as crystalline microparticles. The size of drug particles in the dispersion was also influenced by drug proportion, with an increase in drug content of the dispersion resulting in increased drug particle size. The particle size of drug, the proportion of drug in the dispersion and the properties of the polymer (molecular weight) appeared to determine the mechanism of drug release from the solid dispersions, which was drug diffusion (through the polymer layer)-controlled at low drug contents and drug dissolution-controlled at high drug contents. In situ DLS measurements indicate that the large initial particles of FELO/PVP and FELO/PEG solid dispersions with low drug content (10–20 wt%) are very rapidly decreased to smaller particles (including nanoparticles) during dissolution, leading to the observed impressive enhancement of FELO release rate from these dispersions.
Keywords: Felodipine; PVP; PEG; Solid dispersion; Sparingly soluble drugs; Dissolution enhancement;

Enhanced in vitro permeation of furosemide loaded into thermally carbonized mesoporous silicon (TCPSi) microparticles by Ann Marie Kaukonen; Leena Laitinen; Jarno Salonen; Jaani Tuura; Teemu Heikkilä; Tarja Limnell; Jouni Hirvonen; Vesa-Pekka Lehto (348-356).
The combined release and permeation behavior of furosemide loaded into thermally carbonized mesoporous silicon (TCPSi) microparticles was studied in order to evaluate the potential of TCPSi-loading to improve permeation of furosemide, a BCS class IV compound. Permeation was studied across Caco-2 monolayers at pH 5.5, 6.8 and 7.4 from drug solutions and TCPSi particles. TCPSi-loaded furosemide (39% w/w) exhibited improved dissolution from the microparticles with greatly diminished pH dependence. At pH 5.5, where furosemide solubility restricted the amount that could be dissolved in the control solution to less than 30% of the dose contained in the TCPSi particles, the flux of TCPSi-loaded furosemide across Caco-2 monolayers was over fivefold compared to pre-dissolved furosemide. The improved permeation could be confirmed also from dose-corrected (% dose-permeated) results. At pH 6.8 and pH 7.4, where corresponding doses could be used in control solutions, more than fourfold permeability values were obtained with TCPSi-loaded furosemide. Effects on transepithelial electrical resistance (TEER) and mannitol permeability were monitored and suggest that monolayer integrity was not compromised by the drug-loaded TCPSi microparticles. The improved permeation observed from furosemide-loaded TCPSi particles suggests that the high local concentrations provided by the enhanced dissolution properties of TCPSi-loaded furosemide could prove beneficial for absorption.
Keywords: Porous silicon; Microparticles; Dissolution; Permeation; Thermally carbonized silicon; Non-erosive drug matrix; Poorly soluble compounds;

Loading and delivery of sertraline using inorganic micro and mesoporous materials by Carla D. Nunes; Pedro D. Vaz; Ana C. Fernandes; Paula Ferreira; Carlos C. Romão; Maria José Calhorda (357-365).
Sertraline hydrochloride (designated as sertraline from now on) is an antidepressive drug with unpleasant effects in the gastric tract. Therefore, improved means of delivery allowing for a more controlled and efficient release were looked for. Two different porous materials, montmorillonite-K10 and MCM-41, were chosen as hosts. The drug was intercalated in the interlayer spacing of the clay by cation exchange and was loaded inside the MCM-41 channels by pore volume impregnation means. Spectroscopic evidence (UV/vis, FTIR, powder X-ray diffraction (XRD) and 13C CP/MAS and 29Si CP/MAS and MAS solid-state NMR), as well as elemental analysis, complemented by DFT calculations, demonstrated the presence of sertraline in the composite materials. The release processes were monitored under in vitro conditions using a simulated body fluid. The release profile from the clay is fast, indicating that a concentration peak is reached in a short period of time, while the release profile from MCM is slower but lasts longer. These differences are discussed on the basis of different therapeutic indications for both materials.
Keywords: Microporous materials; Mesoporous materials; Sertraline; Drug delivery systems; Controlled release;

Preliminary studies of the physical stability of a glucagon-like peptide-1 derivate in the presence of metal ions by Stina Christensen; Eva Horn Moeller; Claude Bonde; Anne-Mette Lilleoere (366-371).
The physical stability and the secondary structure of a glucagon-like peptide-1 derivative were investigated in the presence of the metal ions Al3+, Zn2+, Mg2+, and K+, known as possible leachables from container-closure systems. Metal ions were investigated in concentrations of 0–50 ppm. Test solutions of the peptide were exposed to elevated temperature (25 °C) and rotation (37 °C) for up to 4 weeks. The samples were examined by nephelometry, thioflavine T fluorescence, and Fourier-transform infrared spectroscopy. Readily prepared test solutions were examined by tryptophan fluorescence. The stability profiles were unchanged after addition of Mg2+ and K+ in 0–50 ppm concentrations. However, a concentration-dependent increase in thioflavine intensities was observed after addition of Al3+ and Zn2+. The destabilising effect of Al3+ and Zn2+ was furthermore confirmed by FTIR as the secondary structure of the peptide changed from predominantly α-helix to a higher β-sheet content. Additionally Al3+ changed the secondary structure of the peptide using Trp fluorescence.
Keywords: GLP-1 derivate; Metal ions; Physical stability; Tryptophan fluorescence; Thioflavine T test; Fourier-transform infrared spectroscopy;

Monolayers of porcine alveolar epithelial cells in primary culture as an in vitro model for drug absorption studies by Anne Steimer; Helmut Franke; Eleonore Haltner-Ukomado; Michael Laue; Carsten Ehrhardt; Claus-Michael Lehr (372-382).
Filter-grown monolayers of porcine alveolar epithelial cells (pAEpC) in primary culture have been characterized as an in vitro model for pulmonary absorption screening of xenobiotics, including substrates of efflux systems.Experimental conditions and a protocol for transport experiments were optimized using transepithelial electrical resistances (TEER) and permeability of marker compounds as acceptance criteria. Since new drugs often feature poor water solubility, monolayer integrity in the presence of a solubilizer (dimethyl sulfoxide) was tested. Transport studies were carried out with budesonide and triamcinolone acetonide, i.e., two drugs commonly administered to the lungs. Furthermore, expression of P-glycoprotein (P-gp) was assessed by immunofluorescence microscopy and transport studies employing the substrates rhodamine 123 and digoxin.Hydrocortisone-supplemented (0.5 μg/ml) small airway basal medium as transport buffer and a maximal solubilizer concentration of 1.5% dimethyl sulfoxide were found to provide suitable conditions for drug transport studies across pAEpC, as reflected, e.g., by a minimum TEER of 600 Ω cm2. Permeation of marker compounds was reproducible throughout several cell preparations and proved the model successful in distinguishing between low- and high-permeable drugs. P-gp expression was confirmed by immunocytochemistry, even though transport studies revealed no polarity in transepithelial marker transport.In conclusion, our results demonstrate that filter-grown monolayers of pAEpC can be used to study drug transport across alveolar epithelial barrier and thus, may represent a suitable in vitro model for pulmonary drug absorption and delivery.
Keywords: Primary cell culture; P-glycoprotein; Porcine lung; Transepithelial permeability; Pulmonary drug absorption;

Injectable in situ forming drug delivery system for cancer chemotherapy using a novel tissue adhesive: Characterization and in vitro evaluation by Sachiro Kakinoki; Tetsushi Taguchi; Hirofumi Saito; Junzo Tanaka; Tetsuya Tateishi (383-390).
Injectable polymers that are biocompatible and biodegradable are important biomaterials for drug delivery system (DDS) and tissue engineering. We have already developed novel tissue adhesives consisting of biomacromolecules and organic acid derivatives with active ester groups. The resulting tissue adhesive forms in situ as a gel and has high bonding strength for living tissue as well as it has good biocompatibility and biodegradability. Here, we report on the physicochemical properties and in vitro evaluation of this novel tissue adhesive consisting of human serum albumin (HSA) and tartaric acid derivative (TAD) containing doxorubicin hydrochloride (DOX). The results of the measurement of physicochemical characteristics indicate that the gelation time and gel strength of HSA–TAD gels can be controlled according to the material composition. The bonding strength of HSA–TAD adhesives was found to be sufficient to adhere at focus and to correspond with the cross-linking density of HSA–TAD gels. Furthermore, the release of DOX from HSA–TAD gels was sustained for approximately 100 h in an in vitro evaluation. The novel tissue adhesive, therefore, is expected to be applicable for use as an injectable in situ forming DDS.
Keywords: Tissue adhesive; Hydrogel; Drug delivery; Human serum albumin; Tartaric acid; Injectable; In situ forming; N-Hydroxysuccinimide; Doxorubicin;

Paclitaxel/β-cyclodextrin complexes for hyperthermic peritoneal perfusion – Formulation and stability by Wim Bouquet; Wim Ceelen; Bernd Fritzinger; Piet Pattyn; Marc Peeters; Jean Paul Remon; Chris Vervaet (391-397).
Due to its low aqueous solubility paclitaxel is currently formulated in a Cremophor EL®/ethanol mixture. However, the vehicle of this formulation causes several side-effects. Our objective was to formulate a tensioactive-free and solvent-free paclitaxel solution, which can be used for a hyperthermic intraperitoneal chemoperfusion procedure (HIPEC). The potential of chemically modified β-cyclodextrins to form complexes with paclitaxel was investigated as a means to increase the aqueous solubility of paclitaxel. Methylated β-CDs (randomly methylated and 2,6-dimethylated) showed the best ability to solubilise paclitaxel compared to sulfobutyl-ether- and hydroxypropyl-β-CD. The minimal ratio of paclitaxel versus randomly methylated-β-cyclodextrin (RAME-β-CD) yielding 100% inclusion efficiency was 1/20 (mol/mol). Paclitaxel/RAME-β-CD inclusion complexes prepared via freeze drying were stable for at least 6 months when stored at 4 °C. A 5 mg/ml paclitaxel solution was formulated using paclitaxel/RAME-β-CD-complexes. Upon dilution of these solutions, no precipitation was seen. After 24 h storage at room temperature or 2 h at HIPEC conditions (41.5 °C) the 1/40 (mol/mol) ratio showed the highest stability at paclitaxel concentrations of 0.1 and 0.5 mg/ml. When hydroxypropyl methylcellulose (HPMC) was added to the reconstitution medium, the stability significantly increased, offering the opportunity to reduce the amount of RAME-β-CDs in the formulation.
Keywords: Paclitaxel; β-Cyclodextrins; Hyperthermic intraperitoneal chemoperfusion (HIPEC); HPMC;

Chitosan/starch fibers and their properties for drug controlled release by Qun Wang; Na Zhang; Xianwen Hu; Jianhong Yang; Yumin Du (398-404).
Fibers of chitosan and starch, with salicylic acid (SA) as model drug incorporated in different concentrations, were obtained by spinning their solution through a viscose-type spinneret into a coagulating bath containing aqueous tripolyphosphate (TPP) and ethanol. Chemical, morphological and mechanical properties characterization was carried out, as well as the studies of the factors that influence the drug releasing from chitosan/starch fibers. These factors included the component ratio of chitosan and starch, the loaded amount of SA, the pH and the ionic strength of the release solution and others. The diameter of the fibers is around 15 ± 3 μm. The best values of the tensile strength at 12.21 cN/tex and breaking elongation at 25.13% of blend fibers were obtained when the starch content was 30 wt%; the water-retention value (WRV) of blend fibers increased as the composition of starch was raised. The results of controlled release tests showed that the amount of SA released increased with an increase in the proportion of starch present in the fiber. Moreover, the release rate of drug decreased as the amount of drug loaded in the fiber increased, but the cumulative release amount is increasing. The chitosan/starch fibers were also sensitive to pH and ionic strength. The release rate was being accelerated by a lower pH and a higher ionic strength, respectively. All the results indicated that the chitosan/starch fiber was potentially useful in drug delivery systems.
Keywords: Chitosan; Starch; Salicylic acid; Blend fibers; Drug delivery systems;

Thiolated polymers: Evaluation of the influence of the amount of covalently attached l-cysteine to poly(acrylic acid) by Thomas F. Palmberger; Karin Albrecht; Brigitta Loretz; Andreas Bernkop-Schnürch (405-412).
It was the aim of this study to investigate the influence of the amount of thiol groups being covalently attached to poly(acrylic acid) 450 kDa on its properties. Five different PAA450l-cysteine conjugates (PAA450–Cys) were synthesized bearing 53.0 (PAA I), 113.4 (PAA II), 288.8 (PAA III), 549.1 (PAA IV) and 767.0 (PAA V) μmol immobilized thiol groups per gram polymer. Mucoadhesion studies utilizing the rotating cylinder method, tensile studies and disintegration studies were performed. Self-crosslinking properties were measured by the increase in viscosity. Permeation studies were performed on rat small intestine and Caco-2 monolayers using sodium fluorescein as model drug. Following residence times on the rotating cylinder could be identified: PAA I 3.1; PAA II 5.2; PAA III 22.0; PAA IV 33.8; PAA V 53.7; control 1.3 [h]. The disintegration time of all PAA450–Cys tablets was strongly dependent on the degree of thiolation of the polymer. Self-crosslinking studies showed that the different PAA450–Cys conjugates (3% m/v) in phosphate buffer, pH 6.8, formed intramolecular disulfide bonds. In case of Caco-2 monolayer transport studies following P app-values could be identified: PAA I 9.8; PAA II 10.1; PAA III 11.1; PAA IV 8.9; PAA V 8.2; control 6.4 [P app  × 10−6, cm s−1]. Mucoadhesive and self-crosslinking properties are strongly dependent on the degree of thiolation of the polymer and with respect to transport studies, an optimum amount of covalently attached l-cysteine could be identified.
Keywords: Poly(acrylic acid); Thiomer; Mucoadhesion; Disintegration; Self-crosslinking; Permeation enhancement;

Preparation and characterization of simvastatin/hydroxypropyl-β-cyclodextrin inclusion complex using supercritical antisolvent (SAS) process by Seoung Wook Jun; Min-Soo Kim; Jeong-Soo Kim; Hee Jun Park; Sibeum Lee; Jong-Soo Woo; Sung-Joo Hwang (413-421).
In the present study, the practically insoluble drug, simvastatin (SV), and its inclusion complex with hydroxypropyl β-cyclodextrin (HP-β-CD) prepared using supercritical antisolvent (SAS) process were investigated to improve the aqueous solubility and the dissolution rate of drug, thus enhancing its bioavailability. Inclusion complexation in aqueous solution and solid state was evaluated by the phase solubility diagram, differential scanning calorimetry (DSC), powder X-ray diffractometry (PXRD), Fourier-transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). The phase solubility diagram with HP-β-CD was classified as AL-type at all temperatures investigated, indicating the formation of 1:1 stoichiometric inclusion complex. The apparent complexation constants (K 1:1) calculated from phase solubility diagram were 774, 846 and 924 M−1 at 25, 37 and 45 ± 0.5 °C, respectively. No endothermic and characteristic diffraction peaks corresponding to SV was observed for the inclusion complex in DSC and PXRD. FT-IR study demonstrated the presence of intermolecular hydrogen bonds between SV and HP-β-CD in inclusion complex, resulting in the formation of amorphous form. Aqueous solubility and dissolution studies indicated that the dissolution rates were remarkably increased in inclusion complex, compared with the physical mixture and drug alone. Moreover, SV/HP-β-CD inclusion complex performed better than SV in reducing total cholesterol and triglyceride levels. This could be primarily attributed to the improved solubility and dissolution associated with inclusion complex between drug and HP-β-CD. In conclusion, SAS process could be a useful method for the preparation of the inclusion complex of drug with HP-β-CD and its solubility, dissolution rate and hypolipidemic activity were significantly increased by complexation between SV and HP-β-CD.
Keywords: Simvastatin; Hydroxypropyl-β-cyclodextrin; Inclusion complex; Supercritical antisolvent (SAS) process;

Single-layer transdermal film containing lidocaine: Modulation of drug release by Cristina Padula; Sara Nicoli; Paolo Colombo; Patrizia Santi (422-428).
We have recently described an innovative drug delivery system, a water-based and vapor permeable film intended for dermal and/or transdermal delivery. The aim of this work was to modulate the delivery of the model drug lidocaine hydrochloride from the transdermal film across rabbit ear skin. The effect of drug loading, of film-forming polymer type and content, of adhesive and plasticizer on lidocaine transport across the skin was evaluated. Additional objective was to evaluate the effect of occlusion on the kinetics of lidocaine transport, by applying an occlusive backing on the surface of the transdermal film. From the data obtained it can be concluded that the transdermal film acts as a matrix controlling drug delivery. The film-forming polymer molecular weight had a negligible effect on drug penetration, while its content was more effective. The choice of the adhesive seems to be the most important variable governing drug transport. In particular, the presence of lauric acid combined with a basic drug, such as lidocaine, can produce a relevant improvement in permeation, because of the formation of an ion pair. Concerning the kinetics, drug depletion is responsible for the declining permeation rates observed in the late times of permeation.
Keywords: Transdermal; Patch; Permeation; Rabbit ear skin; Lidocaine;

Poly(2-hydroxy-3-phenoxypropylacrylate, 4-hydroxybutyl acrylate, dibutyl maleate) membrane was synthesized by UV curing method in our laboratory for the first time. When above-mentioned monomers were in the weight ratio of 4:4:2, the membrane not only had perfect permeation property but also had excellent plasticity, so the membrane made from monomers in the ratio of 4:4:2 was chosen as an optimized membrane. The optimized membrane provided perfect linear permeation properties in clonidine transdermal drug delivery system. The permeation rate decreased in proportion to the thickness of membrane. When the concentrations of clonidine were in the range of 0.5–7.0 mg/ml, the permeation rate was proportional to the square root of clonidine concentrations. The optimized membrane was characterized by FTIR, DSC and SEM.
Keywords: Transdermal drug delivery systems; UV curing; Clonidine; Controlled release membranes; Polyacrylates;

Swelling, erosion and release behavior of alginate-based matrix tablets by Pornsak Sriamornsak; Nartaya Thirawong; Kingkarn Korkerd (435-450).
Hydrophilic matrix tablets based on the alginate system have been used in relation to their possible function in modified drug delivery formulations using metronidazole as a model drug. The matrix tablets were prepared by direct compression using different grades of alginate. The effect of some factors (i.e. particle size of drug, additive used, and pH of medium) on drug release from alginate-based matrix tablets was also investigated. Swelling, erosion, and in vitro release studies of the matrix tablets were carried out in 0.1 N HCl or phosphate buffer (pH 6.8). The alginate-based matrix tablets swelled or eroded while in contact with the aqueous medium and formed a continuous gel layer or underwent combination of swelling and erosion. The swelling action of alginate matrices is controlled by the rate of its hydration in the medium. Different grades of alginate insignificantly influenced the matrix swelling in acidic medium but significantly influenced in neutral medium. The presence of ammonium or calcium salts induced tablet disintegration in acidic medium. However, incorporation of calcium acetate and sodium bicarbonate can alter the tablet swelling in acidic medium. Release studies showed that all investigated factors influence the drug release. The extent of matrix swelling, erosion, and diffusion of drug determined the kinetics as well as mechanism of drug release from alginate-based matrix tablets. Most of the release data in acidic medium showed a good fit into Korsmeyer–Peppas equation but fitted well with zero-order release model, in neutral medium.
Keywords: Alginate; Swelling; Hydration; Erosion; Drug release; Matrix tablet;

A topical formulation was added with different concentrations of superoxide dismutase (SOD) alone or in association with α-tocopherol (α-TOC). The physical stability was evaluated by rheological behavior of formulations stored at 4 °C, 30 °C/60% RH and 40 °C/70% RH for 6 months. SOD alone and formulations containing SOD 0.2%, 0.4% or 0.6% or SOD and α-TOC were stored in the same conditions and the enzymatic activity was evaluated by the superoxide anion scavenging using chemiluminescence measurement. In vitro release study was carried out using modified Franz diffusion cell and SOD formulations photoprotection against skin erythema was observed for 72 h. SOD and α-TOC formulation proved to be instable, since the interaction between the antioxidants led to both physical and enzymatic activity instability. SOD formulations showed to be physically stable and maintained the enzymatic activity for 6 months when stored at 4 and 30 °C/60% RH. Despite the fact of low SOD release from the formulation, it was effective in inhibiting the UVB-induced skin erythema for 48 h after a single application. Topical administration of antioxidants provides an efficient way to enrich the endogenous cutaneous protection system, and SOD formulations could be used for improving photoprotection of skin.
Keywords: Superoxide dismutase; Antioxidant; Stability; Formulation; Chemiluminescence; Release; Erythema;

The purpose of this study was to develop a taste-masked quinine sulphate dosage form as a flexible pediatric formulation tool. Pellets were produced as they offer more flexibility to body weight dose adaptation and therefore represent an alternative to tablet breaking in pediatrics. Quinine sulphate pellets were produced via extrusion-spheronisation. Next pellets were coated using Eudragit® E PO to obtain a taste-masked formulation. Using 15% dibutyl sebacate (based on polymer weight) as a plasticizer in the formulation caused rapid pellet agglomeration during storage at 40 °C and 75% relative humidity. Using stearic acid (15% based on polymer weight) as plasticizer yielded pellets which were less sensitive to sticking. Quinine sulphate release in water within the first 5 min of dissolution testing: 9.2%, 5.9% and 2.1% of the drug dose was released from pellets coated with 10%, 20% and 30% (w/w) Eudragit® E PO, respectively. These observations correlated well with the bitterness score of the formulations determined via the Astree electronic tongue and its Bitterness Prediction Module, showing that 20% (w/w) Eudragit® E PO was required to obtain a homogeneous film and to delay quinine sulphate release sufficiently to mask the bitterness after drug administration. In acid medium immediate quinine sulphate release was obtained.
Keywords: Pediatric formulation; Quinine sulphate; Pellets; Extrusion-spheronisation; Electronic tongue; Taste-masking; Eudragit® E PO;

Influence of sample characteristics on quantification of carbamazepine hydrate formation by X-ray powder diffraction and Raman spectroscopy by F. Tian; F. Zhang; N. Sandler; K.C. Gordon; C.M. McGoverin; C.J. Strachan; D.J. Saville; T. Rades (466-474).
This study aimed to assess the suitability of two widely utilized solid state characterization techniques namely powder X-ray diffraction (XRPD) and Raman spectroscopy, in polymorph detection and quantification for carbamazepine anhydrate and dihydrate mixtures. The influences of particle size, particle morphology, mixing, and in particular, surface bias on quantitation were investigated. Binary mixtures of carbamazepine anhydrate (form III) and dihydrate were prepared and analyzed using both XRPD and Raman spectroscopy in combination with partial least squares analysis. It was found that in principle both XRPD and Raman spectroscopy could be used to build calibration models for quantitative analysis, and a satisfactory correlation between the two techniques could be achieved. However, Raman spectroscopy appeared to be a more reliable quantification method because problems such as different particle size, morphology, and special distribution of the two solid state forms of the drug seemed to have no significant influence on Raman scattering in this study. The robust nature of Raman analysis greatly facilitates the whole quantification process from the preparation of calibration models to the quantification of in situ CBZ–DH conversion.
Keywords: Raman spectroscopy; X-ray powder diffraction; Carbamazepine; Carbamazepine dihydrate; Quantification;

Liquid chromatography (LC) is considered to be the gold standard in pharmaceutical analysis. Today, there is a need for fast and ultra-fast methods with good efficiency and resolution for achieving separations in few minutes or even seconds. The present work describes a simple methodology for performing a successful method transfer from conventional LC to fast and ultra-fast LC.In order to carry out fast separations, short columns (20–50 mm) packed with small particles (3.5 and 1.7 μm) were used and their chromatographic performance was compared to that of a conventional column (150 mm, 5 μm). For that purpose, an optimized LC system was employed to limit extra-column volumes which can have a dramatic impact on efficiency and resolution.This paper reports the fundamental equations used for transferring an isocratic chromatographic separation performed with a given column geometry and chemistry to a smaller column packed with similar or identical stationary phase, without influence on chromatographic performance. For this purpose, the flow rate and the injected volume need to be adapted.The effect of column length and particle size reduction on chromatographic resolution and analysis time was described for an isocratic separation. Using the method transfer equations, it is possible to predict the new conditions to be used, for fast and ultra-fast separations. In this work, ultra-fast separations were achieved thanks to a new generation of instrumentation (ultra performance liquid chromatography, UPLC) which uses simultaneously short column packed with sub-2 μm particles and ultra-high pressure (up to 1000 bar). This work demonstrates an analysis time reduction up to a factor 12, compared to a conventional LC separation, without affecting the quality of separation. Therefore, the complete resolution of a pharmaceutical formulation was achieved in only a few seconds.
Keywords: Fast liquid chromatography; Isocratic transfer; Short columns; UPLC; Sub-2 μm particles;

Solid lipid microparticles containing the sunscreen agent, octyl-dimethylaminobenzoate: Effect of the vehicle by Rosanna Tursilli; Géraldine Piel; Luc Delattre; Santo Scalia (483-487).
Solid lipid microparticles (SLMs) loaded with the sunscreen agent, octyl-dimethylaminobenzoate (ODAB), were prepared in order to achieve enhanced sunscreen photostability. The microparticles were produced by the melt dispersion technique using glyceryl behenate as lipidic material and poloxamer 188 as the emulsifier. The obtained SLMs showed proper features in terms of morphology, size distribution (1.67–15.81 μm) and ODAB loading (16.15 ± 0.11%, w/w). The sunscreen release from the SLMs was slower than its dissolution rate and the photodecomposition of ODAB was markedly decreased (>51.3%) by encapsulation into the lipid microparticles. The efficacy of the SLM carrier system was also evaluated after their introduction in model topical formulations (i.e., hydrogel and oil-in-water emulsion). Further in vitro release measurements, performed using Franz diffusion cells with polycarbonate membranes, indicated that the retention capacity of the microparticles was lost after their incorporation into the emulsion, whereas it was retained in the hydrogel. Moreover, the SLMs achieved a reduction of the sunscreen photodegradation in the hydrogel vehicle (the ODAB loss decreased from 87.4% to 59.1%), whereas no significant photoprotective effect was observed in the emulsion. Therefore, the efficacy of the ODAB-loaded SLMs was markedly affected by the vehicle.
Keywords: Solid lipid microparticles; Sunscreen agents; Octyl-dimethylaminobenzoate; Release; Photostability; Topical formulations;

Comparative study of doxorubicin-loaded poly(lactide-co-glycolide) nanoparticles prepared by single and double emulsion methods by F. Tewes; E. Munnier; B. Antoon; L. Ngaboni Okassa; S. Cohen-Jonathan; H. Marchais; L. Douziech-Eyrolles; M. Soucé; P. Dubois; I. Chourpa (488-492).
This study describes how the control of doxorubicin (DOX) polarity allows to encapsulate it inside poly(lactide-co-glycolide) (PLGA) nanoparticles formulated either by a single oil-in-water (O/W) or a double water-in-oil-in-water (W/O/W) emulsification method (SE and DE, respectively). DOX is commercially available as a water soluble hydrochloride salt, which is useful for DE. The main difficulty related to DE approach is that the low affinity of hydrophilic drugs to the polymer limits entrapment efficiency. Compared to DE method, SE protocol is easier and should provide an additional gain in entrapment efficiency. To be encapsulated by SE technique, DOX should be used in a more lipophilic molecular form. We evaluated the lipophilicity of DOX in terms of apparent partition coefficient (P) and modulated it by adjusting the pH of the aqueous phase. The highest P values were obtained at pH ranging from 8.6 to 9, i. e. between two DOX pK a values (8.2 and 9.6). The conditions favorable for the drug lipophilicity were then used to formulate DOX-loaded PLGA nanoparticles by SE method. DOX encapsulation efficiency as well as release profiles were evaluated for these nanoparticles and compared to those with nanoparticles formulated by DE. Our results indicate that the encapsulation of DOX in nanoparticles formulated by SE provides an increased drug entrapment efficiency and decreases the burst effect.
Keywords: Doxorubicin; Partition coefficient; PLGA nanoparticles; Single emulsion; Double emulsion; Release kinetics;

by Wolfgang Friess (493).