European Journal of Pharmaceutics and Biopharmaceutics (v.59, #1)

APV Diary (S3-S4).

Editorial by Sven Stegemann (v).

In vitro adsorption of plasma proteins onto the surface (charges) modified-submicron emulsions for intravenous administration by Shunmugaperumal Tamilvanan; Sven Schmidt; Rainer H. Müller; Simon Benita (1-7).
Surface (charge) modified submicron emulsions (cationic and anionic) were prepared following the well established combined emulsification techniques and characterized for their droplet size distribution and surface charge. The effect of these emulsions on in vitro adsorption of plasma proteins was investigated by means of two dimensional polyacrylamide gel electrophoresis (2D PAGE). The presence of poloxamer 188 in tested emulsions effectively eliminated the adsorption of the larger proteins like immunoglobulins, fibrinogen, etc. However, depending on the type of surface charges, the smaller proteins such as apolipoproteins and albumin were almost completely adsorbed onto the submicron emulsions. Indeed, when compared to marketed lipofundin MCT 10%—and deoxycholic acid—based anionic emulsions, the adsorption of apolipoprotein, especially apoA-1, was approximately three times more on stearylamine—and oleylamine—based cationic emulsions and oleic acid-based anionic emulsions. In addition, the ratio between the apoA-1 and apoA-IV was found to be 1 for lipofundin MCT 10% whereas it was about 0.26 for deoxycholic acid-based anionic emulsion and above 5 for oleic acid-based anionic emulsions and cationic emulsions. This indicates that emulsions having similar surface/interfacial charge imparted by different anion-forming stabilizers (oleic or deoxycholic acids) exhibited markedly different protein adsorption patterns.
Keywords: Cationic; Anionic; Emulsion; In vitro; Plasma protein; Adsorption;

Three formulation techniques were compared in order to develop a multi-particulate formulation of viable, interleukin-10 producing Lactococcus lactis Thy12. First, freeze-dried L. lactis was compacted into mini-tablets. Next, liquid L. lactis culture was used as the granulation fluid for the production of pellets by extrusion/spheronisation. Finally, liquid L. lactis culture was layered on inert pellets as an alternative technique for the production of pellets. L. lactis viability and interleukin-10 production was evaluated. Viability dropped to 15.7% after compaction of freeze-dried L. lactis and to 1.0% after pelletisation of liquid L. lactis by extrusion/spheronisation. The viability in the mini-tablets and pellets, stored for 1 week at RT and 10% RH was reduced to 23 and 0.5% of initial viability, respectively. Storage for 1 week at RT and 60% RH resulted in complete loss of viability. Layering of L. lactis on inert pellets resulted in low viability (4.86%), but 1 week after storage at RT and 10% RH, 68% of initial viability was maintained. Increasing product temperature and cell density of L. lactis in the layering suspension did not significantly change viability after layering and storage. Interleukin-10 production capacity of L. lactis Thy12 was maintained after layering.
Keywords: Extrusion/spheronisation; Layering; Compaction; Viability; L. lactis; Recombinant;

The activity of the renal peptide transporters PEPT2 and PEPT1 determines—among other factors such as metabolic stability in liver and plasma—the circulatory half-life of penicillins and cephalosporins during therapy. This study was initiated to examine systematically the interaction of β-lactam antibiotics with PEPT2. Interaction of 31 cephalosporins and penicillins with the carrier protein was characterized by measuring their ability to inhibit the uptake of [14C]Gly-Sar into renal SKPT cells. Cefadroxil, cefaclor, cyclacillin, cephradine, cephalexin and moxalactam were recognized by PEPT2 with very high affinity comparable to that of natural dipeptides (K i=3–100 μM). Ceftibuten, dicloxacillin, amoxicillin, metampicillin, cloxacillin, ampicillin, cefixime, cefamandole, oxacillin and cefmetazole interacted with PEPT2 with medium affinity (K i=0.1–5 mM). For the other β-lactam antibiotics studied interaction was very low or not measurable (K i>5 mM). The affinity constants of β-lactam antibiotics at rPEPT2 and hPEPT1 are significantly correlated, but the rank orders are not identical. Decisive differences between PEPT1 and PEPT2 recognition of the N-terminal part of the compounds became evident. Moreover, this large data set of affinity constants of β-lactam antibiotics will be useful for structure–transport (binding) analyses of PEPT2.
Keywords: Peptide transport; β-lactam antibiotics; SKPT cells; Caco-2 cells; PEPT1 and PEPT2;

FK888 is a candidate selective NK1 receptor antagonist, and it exhibits poor absorption from the gastrointestinal tract in healthy volunteers. In a previous study, the optimized dry powder inhaler (DPI) formulation with carrier lactose using the Spinhaler® was developed, although the maximum dose per capsule was only 5 mg because the fine particle fraction (FPF) was reduced at doses over 5 mg. The objective of this study was to develop an optimized DPI formulation for higher doses, such as 40 mg, with proportional systemic absorption. The Spinhaler® and E-haler® were used as the inhalation devices, and the in vitro deposition was evaluated using a multistage cascade impactor at different flow rates (28.3 and 60 l/min). When hydroxypropyl methylcellulose (HPMC) capsules were used as the container, and spherical soft agglomerates of fine FK888 particles (soft pellets) and the E-haler® were used, the fraction of particles emitted from the inhalation system (Em) was significantly improved, to over 80% of the nominal dose, and no significant difference was found between the airflow rates (84.3±2.3% for 28.3 l/min, 88.1±3.6% for 60 l/min). It was also found that the E-haler® was an extremely suitable device for obtaining the higher respirable particle percentage of emitted particles (RP) in the 40 mg formulation with the soft pellets contained in HPMC capsules (35.0±1.8% for 28.3 l/min and 42.5±3.5% for 60 l/min), compared with the Spinhaler® (13.8±3.0% for 28.3 l/min and 28.9±1.0% for 60 l/min). Using the formulations with the E-haler®, proportional systemic absorption was achieved up to 40 mg FK888 in healthy volunteers (62.91±27.58, 103.70±40.19 and 254.79±85.01 ng h/ml as AUCs for 10, 20 and 40 mg FK888, respectively; R 2=0.9641). It is also expected that the E-haler® will act as an efficient device when a higher dose, such as 40 mg, is required in clinical situations.
Keywords: Dry powder inhaler; E-haler; Multistage cascade impactor; Pulmonary absorption; Soft pellets, HPMC capsule;

Large-scale production of lipoplexes with long shelf-life by Jule Clement; Karin Kiefer; Andrea Kimpfler; Patrick Garidel; Regine Peschka-Süss (35-43).
The instability of lipoplex formulations is a major obstacle to overcome before their commercial application in gene therapy. In this study, a continuous mixing technique for the large-scale preparation of lipoplexes followed by lyophilisation for increased stability and shelf-life has been developed. Lipoplexes were analysed for transfection efficiency and cytotoxicity in human aorta smooth muscle cells (HASMC) and a rat smooth muscle cell line (A-10 SMC). Homogeneity of lipid/DNA-products was investigated by photon correlation spectroscopy (PCS) and cryotransmission electron microscopy (cryo-TEM). Studies have been undertaken with DAC-30®, a composition of 3β-[N-(N,N′-dimethylaminoethane)-carbamoyl]-cholesterol (DAC-Chol) and dioleylphosphatidylethanolamine (DOPE) and a green fluorescent protein (GFP) expressing marker plasmid. A continuous mixing technique was compared to the small-scale preparation of lipoplexes by pipetting. Individual steps of the continuous mixing process were evaluated in order to optimise the manufacturing technique: lipid/plasmid ratio, composition of transfection medium, pre-treatment of the lipid, size of the mixing device, mixing procedure and the influence of the lyophilisation process.It could be shown that the method developed for production of lipoplexes on a large scale under sterile conditions led to lipoplexes with good transfection efficiencies combined with low cytotoxicity, improved characteristics and long shelf-life.
Keywords: Scale-up; Lipoplex; Non-viral gene transfer; Cytotoxicity; DAC-30®;

Permeability modulation of human intestinal Caco-2 cell monolayers by interferons by Hiroko Kawaguchi; Yukiko Akazawa; Yoshihiko Watanabe; Yoshinobu Takakura (45-50).
We investigated the effects of interferon-β (IFN-β) and IFN-γ on the drug efflux activity of the human intestinal Caco-2 cell line, expressing the P-glycoprotein (P-gp) on the apical membrane. The cells grown on Transwell plates were pretreated with 1000 U/ml IFN-β, IFN-γ or a combination of both for 3 days, and then the transepithelial electrical resistance (TEER) and the vectorial transport of rhodamine-123 (Rho-123) across the cell monolayers were evaluated. Exposure to IFN-γ reduced substantially the TEER, but the effect of IFN-β was minimal? The apparent permeability of Rho-123 in both the basolateral-to-apical and apical-to-basolateral directions was significantly increased by IFN-γ but scarcely by IFN-β. The combination of IFN-γ and IFN-β showed similar effects to IFN-γ alone. Meanwhile, the cellular uptake of Rho-123 from the apical side was not affected by any IFN treatment. The uptake level was increased approximately three times in the presence of verapamil, a P-gp inhibitor, and the increased level was not affected by any IFN treatment, indicating that the efflux activity mediated by P-gp in the monolayers is not altered by these cytokines. Taken together, these results suggest that IFNs modulate the permeability of Caco-2 monolayer through effect on paracellular transport rather than effect on P-gp activity.
Keywords: P-glycoprotein; Transepithelial electrical resistance; Interferon-β; Interferon-γ; Rhodamine-123; Caco-2 cell;

Conversion of cyclosporine A prodrugs in human tears vs rabbits tears by F. Lallemand; O. Felt-Baeyens; S. Rudaz; A.R. Hamel; F. Hubler; R. Wenger; M. Mutter; K. Besseghir; R. Gurny (51-56).
The aim of this study was to evaluate the rate and mechanism of conversion of two water-soluble prodrugs of cyclosporine A (CsA) intended for topical delivery to the eye. The new molecules were designed according to the double prodrug concept: a solubilizing moiety was grafted onto CsA via an ester function, which could be hydrolysed via a two-step process (enzymatic and chemical). Prodrug solutions were prepared extemporaneously in an isotonic and neutral aqueous medium compatible with ophthalmic use. The rates of conversion into the parent molecule were determined by incubating the prodrugs in fresh rabbit or human tears or in a phosphate buffer solution (PBS) at pH 7.4. Both prodrugs were converted into CsA within the first minute in the presence of rabbit tears with rate constants of k=5.9×10−3  min−1 and k=3.8×10−3  min−1, respectively, for UNIL088 and UNIL089, whereas chemical conversion in PBS was negligible (k=0.5×10−3  min−1 for both molecules). Incubation of UNIL088 in human tears showed a significantly high conversion rate. It is concluded that the developed double prodrugs underwent a bioconversion in physiological media and thus represent promising candidates for topical delivery of CsA to the eye.
Keywords: Cyclosporine A; Prodrug; Ex vivo model; Ocular drug delivery; Conversion; Rabbit tears; Human tears;

Non-phospholipid vesicles for pulmonary glucocorticoid delivery by Claudio Terzano; Luigi Allegra; Franco Alhaique; Carlotta Marianecci; Maria Carafa (57-62).
In the formulation of inhaled drugs for the treatment of asthma and chronic obstructive pulmonary disease (COPD), considerable attention has been devoted to new aerosol morphologies which can either enhance the local effect and/or increase the penetration through the mucus, secreted in bronchial inflammatory diseases. In diseases characterized by bronchial hypersecretion, lipophilic substances, such as corticosteroids, can be remarkably impeded in reaching their receptors, which are localized within the cytoplasm of bronchial epithelial cells. Vesicles consisting of one or more surfactant bilayers enclosing aqueous spaces, are of particular interest because they offer several advantages with regard to chemical stability, lower cost and availability of materials compared to conventional liposomes. With the purpose of carrying out research leading to an innovative formulation for lung delivery capable of permeating the mucous layer, beclomethasone dipropionate, clinically used for the treatment of asthma and COPD, was entrapped in non-phospholipid vesicles. The composition providing the highest entrapment efficiency was chosen. The vesicles obtained after jet nebulization were characterized by means of freeze-fracture microscopy and dynamic light scattering. The efficiency of this new drug delivery system was evaluated in vitro with simulated mucus by means of diffusion experiments (three compartment cell apparatus), using 0.1% mucin gel-like dispersion as a barrier to drug permeation.
Keywords: Non-phospholipid vesicles; Lung delivery; Beclomethasone dipropionate; Asthma; Chronic obstructive pulmonary disease; Mucus permeation;

Multivariate methods are utilized to compare nicotinic acid and dihydropyridine as a drug carrier. Nicotinic acid and dihydropyridine form ester groups on 10 β-lactam antibiotics with an oxymethyl group forming a linkage between the antibiotic and the drug carrier (nicotinic acid or dihydropyridine). Calculated molecular properties are analyzed by self-organizing tree algorithm (SOTA), bivariate regression, cluster analysis, factor analysis, discriminant analysis, hierarchical classification, and principal coordinates analysis. Ten important pharmacological properties for each of the nicotinic acid and dihydropyridine antibiotic derivatives are numerically similar and highly correlated. Calculated molecular properties include molar refractivity, molar volume, parachor, index of refraction, partition coefficient (log  P), polarizability, and polar surface area. Dermal permeability coefficients (K p) for nicotinic acid derivatives are similar to values for dihydropyridine derivatives. Dermal permeabliity coefficients analyzed by hierarchical classification and SOTA analysis were shown to be closely interrelated and highly correlated. Ten properties of the nicotinic acid and dihydropyridine were compared by Passing–Bablok regression analysis and shown to be highly correlated (r=0.9879). Box plot analysis of 10 properties, inclusive of both groups of derivatives, showed narrow ranges in values. Cluster analysis of derivative properties showed the nicotinic acid derivatives to be highly similar to the dihydropyridine derivatives of the same antibiotics. Cluster analysis was performed by single linkage, complete linkage, and centroid linkage. Factor analysis showed the nicotinic acid derivatives to be interrelated and similar to the dihydropyridine derivatives. Discriminant analysis performed on all derivatives formed a single highly cohesive and non-differentiated cluster, demonstrating strong similarity among nicotinic acid and dihydropyridine derivatives. Principal coordinates analysis (determines similarity) of K p values showed high similarity between the nicotinic acid and dihydropyridine antibiotic derivatives.
Keywords: Nicotinic acid; Dihydropyridine; Pattern recognition; Antibiotics; Multivariate methods;

Drug release from tableted wet granulations comprising cellulosic (HPMC or HPC) and hydrophobic component by S. Kiortsis; K. Kachrimanis; Th. Broussali; S. Malamataris (73-83).
The effects of component nature, proportion and processing on the release rate and mechanism were investigated for tablets comprising drug, cellulosic polymer and hydrophobic components. Four drugs differing in solubility (diclofenac sodium, ibuprofen, naproxen and indomethacin), two cellulosic polymers (HPC and HPMC) and hydrophobic Emvelop® were used in two levels of mass fraction and weight ratio of drug:carrier and of cellulosic–hydrophobic component. Compression was applied after granulation or physical mixing. Drug release was evaluated in pH 6.5 phosphate buffer BP and elucidation of the release mechanism was attempted by fitting kinetic models. Statistical significance of the effects of formulation variables on the release rate and mechanism expressed by the coefficient, k, and exponent, n, of the power law kinetic model, respectively, was evaluated by ANOVA. It was found that for the release mechanism most significant is the effect of drug solubility followed by cellulosic polymer type, mixing procedure and drug mass fraction. Significant interaction between drug solubility and type of cellulosic polymer indicated that alteration in the swelling of HPMC and HPC is caused by the drug solubility. Weight ratio of cellulosic–hydrophobic component does not affect the release mechanism, but only the release rate. Similarly, for the release rate most significant was found the effect of drug solubility, followed by cellulosic polymer type, weight ratio of cellulosic–hydrophobic component, mixing method and drug mass fraction. Also significant were the interactions of drug solubility with the type and proportion of the cellulosic polymer and the processing applied. Depending on the drug solubility and type of polymer present, wet granulation can increase or decrease the release rate.
Keywords: Controlled release; Cellulosic–hydrophobic (gel forming) matrix; Wet granulation; Solubility; Diffusion–erosion kinetics;

Properties of hot-melt extruded tablet formulations for the colonic delivery of 5-aminosalicylic acid by L. Diane Bruce; Navnit H. Shah; A. Waseem Malick; Martin H. Infeld; James W. McGinity (85-97).
Hot-melt extruded tablets were prepared using Eudragit® S 100 as the polymeric carrier to target delivery of 5-aminosalicylic acid (5-ASA) to the colon. Scanning electron microscopy, modulated differential scanning calorimetry and X-ray diffraction analysis of the hot-melt tablet extrudates demonstrated that 5-ASA remained crystalline and was homogeneously dispersed throughout the polymer matrix. A pre-plasticization step was necessary when incorporating triethyl citrate (TEC) into the formulation in order to achieve uniform mixing of the polymer and plasticizer, effectively reduce the polymer glass transition temperature (T g), and to lower the processing temperatures. The concentration of TEC in the extrudates not only influenced the processing temperature, but also influenced the drug release rates from the extruded tablets due to leaching of the TEC during dissolution testing. Citric acid monohydrate was found to plasticize Eudragit® S 100, and when combined with TEC in the powder blend, the temperatures required for processing were reduced. Tablets containing citric acid released drug at a slower rate as a result of the suppression of polymer ionization due to a decrease in the micro-environmental pH of the tablet. The drug release profiles of the extruded tablets were found to fit both diffusion and surface erosion models.
Keywords: Hot-melt extrusion; Colonic drug delivery; 5-ASA; Mesalamine; Thermal processing; Eudragit® S 100; Micro-environmental pH;

Solid-state characterization and dissolution properties of Naproxen–Arginine–Hydroxypropyl-β-cyclodextrin ternary system by Paola Mura; Gian Piero Bettinetti; Marzia Cirri; Francesca Maestrelli; Milena Sorrenti; Laura Catenacci (99-106).
The effect of ternary complexation of naproxen, a poorly water soluble anti-inflammatory drug, with hydroxypropyl-β-cyclodextrin and the basic aminoacid L-arginine on the drug dissolution properties has been investigated. Equimolar binary (drug–cyclodextrin or drug–arginine) and ternary (drug–cyclodextrin–arginine) systems were prepared by blending, cogrinding, coevaporation, and characterized by differential scanning calorimetry, thermogravimetric analysis, FT-IR spectroscopy, X-ray diffractometry. The dissolution behavior of naproxen from the different products was evaluated by means of a continuous flow through method. The results of solid state studies indicated the presence of strong interactions between the components in ternary coevaporated and coground systems, which were both of totally amorphous nature. In contrast, the presence of either free drug or free arginine was detected when the third component (cyclodextrin or aminoacid) was physically mixed, respectively, to the drug–arginine binary system (as physical mixture, coevaporate, or coground product) or to the drug–cyclodextrin binary system (as physical mixture, coevaporate, or coground product). All ternary combinations were significantly (P<0.001) more effective than the corresponding binary drug-cyclodextrin and drug–arginine systems in improving the naproxen dissolution rate. The best performance in this respect was given by the ternary coevaporate, with about 15 times increase in terms of both drug relative dissolution rate and dissolution efficiency. The synergistic effect of the simultaneous use of arginine and cyclodextrin on the dissolution rate of naproxen was attributed to the combined effects of inclusion in cyclodextrin and salt formation, as well as to a specific role played by arginine in this interaction.
Keywords: Hydroxypropyl-β-cyclodextrin; Naproxen; Arginine; Ternary system; Dissolution rate;

The incorporation of a drug in a carrier by melt embedding may either result in a solid solution or in a solid suspension of the active ingredient within the carrier material. As the dispersivity of the drug is of outstanding importance for its dissolution characteristics, parameters which are supposed to influence crystallinity and dispersivity, e.g. cooling rate during preparation and storage conditions like temperature and relative humidity are investigated. It is found that the absence of crystalline drug material in solid dispersions containing nimodipine and polyethylene glycol 2000 is the prerequisite for a high dissolution rate and a remarkable supersaturation in the dissolution medium. Shock freezing during the preparation process, low storage temperatures and low relative humidities are identified to prevent recrystallisation. Furthermore, emphasis is put on the physico-chemical characterisation of solid dispersions. It is shown that the determination of crystallinity and dispersivity of the drug in solid dispersions can only be successful by combining different investigation methods like differential scanning calorimetry, hot stage microscopy, X-ray diffraction as well as macroscopic observation.
Keywords: Dissolution; Nimodipine; Polyethylene glycol; Recrystallisation; Relative humidity; Solid dispersions; Supersaturation; Stability; Storage; Temperature;

Study of the physicochemical properties and stability of solid dispersions of loperamide and PEG6000 prepared by spray drying by Ilse Weuts; Dieter Kempen; Geert Verreck; Annelies Decorte; Koen Heymans; Jef Peeters; Marcus Brewster; Guy Van den Mooter (119-126).
Solid dispersions of PEG6000 and loperamide—a poorly water-soluble agent—were prepared by spray drying. Their physicochemical properties were evaluated immediately after preparation. The dissolution was higher than that of pure crystalline loperamide. DSC- and XRD-measurements revealed that in the dispersions, loperamide is partially present in the crystalline state. A eutectic state diagram was obtained. The samples containing 20% loperamide were stored under different conditions (40 °C and 0% RH, 25 °C and 52% RH, 4 °C and 0% RH) to investigate their stability as a function of time. The dissolution properties deteriorate upon storage at high temperature (40 °C and 0% RH) and in conditions of higher relative humidity (25 °C and 52% RH). The DSC-curves clearly indicate an increase in the amount of crystalline compound under these conditions. From these observations it could be concluded that loperamide, which is partially crystalline and partially amorphous in the freshly prepared samples, continues to crystallize under these conditions, resulting in progressively poorer dissolution properties.
Keywords: Solid dispersions; Dissolution properties; Differential scanning calorimetry; Stability; Physicochemical properties; PEG6000; Loperamide;

Preliminary assessment of carrageenan as excipient for extrusion/spheronisation by Martin Bornhöft; Markus Thommes; Peter Kleinebudde (127-131).
The current study pursues the suitability of different types of carrageenan as a novel extrusion aid. The aim was to find out a suitable substitution to the commonly used microcrystalline cellulose (MCC). The types of κ-carrageenan were found to be the most appropriate material and the required fraction to produce acceptable pellets in the formulation was determined. The investigation showed that 5% of κ-carageenan was necessary to produce pellets without MCC. Similar formulations produced with MCC or κ-carrageenan were compared with respect to size and shape of the pellets. κ-Carrageenan required higher water content for the formation of pellets, but the formulation was more robust as the optimal range of water content was much broader. Hence, κ-Carrageenan seems to be a suitable and promising extrusion aid. The study showed that the substitution of MCC by κ-carrageenan in formulations is possible and the produced pellets were of high quality.
Keywords: Carrageenan; Microcrystalline cellulose; Extrusion/spheronisation; Pellet; Image analysis;

Compaction mechanism and tablet strength of unlubricated and lubricated (silicified) microcrystalline cellulose by B. van Veen; G.K. Bolhuis; Y.S. Wu; K. Zuurman; H.W. Frijlink (133-138).
This paper describes the differences in compaction properties between microcrystalline cellulose (MCC) and microcrystalline cellulose co-processed with colloidal silicon dioxide (SMCC). The different compaction parameters are not only compared for the pure materials, but also for the lubricated powders with magnesium stearate. Neither magnesium stearate, nor colloidal silicon dioxide, facilitates extensively the densification of (silicified) microcrystalline cellulose during compaction. The difference in tablet relaxation of MCC and SMCC indicates a small negative effect of colloidal silicon dioxide on the interparticle bonding strength of unlubricated MCC. However, for lubricated MCC a larger increase in tablet relaxation at a high compression speed was found than for lubricated SMCC tablets. Accordingly, the decrease in tablet strength was larger for the MCC tablets than for the SMCC tablets when lubrication was applied. The examination of the tablet strengths of tablets compressed from physical mixtures of MCC with increasing concentrations of colloidal silicon dioxide proved the slightly negative influence of silicon dioxide on the tablet strength of unlubricated MCC tablets and the positive effect of colloidal silicon dioxide addition on the tensile strength of lubricated MCC tablets. Co-processing of MCC with colloidal silicon dioxide showed no extra contribution on the tablet strength of lubricated tablets above the physical mixtures. The interactions between the different materials were further supported by the interaction parameters based on partial solubility parameters.
Keywords: Microcrystalline cellulose; Silicified microcrystalline cellulose; Colloidal silicon dioxide; Direct compression; Lubricant;

Effects of gamma-irradiation on trehalose–hydroxyethylcellulose microspheres loaded with vancomycin by A. Bartolotta; M.C. D'Oca; M. Campisi; V. De Caro; G. Giandalia; L.I. Giannola; M. Brai; E. Calderaro (139-146).
Ionizing radiation can be used as a drug sterilization technique, provided that the drug itself is not modified and that no toxic products are produced; moreover, if the irradiated product is a drug delivery system, the drug release characteristics must not be significantly altered by radiation. The aim of this work was to study the effects of sterilization by ionizing radiation on hydroxyethylcellulose/trehalose spherical micromatrices, containing the antibiotic vancomycin. Our experimental results showed that gamma-rays did not alter the chromophore groups of vancomycin (UV measurements), and did not modify the kinetic behavior of drug release from microspheres. Moreover, no significant changes in the shape and in the size distribution of microspheres were found after irradiation. The electron spin resonance (ESR) spectroscopy was proven to be a valid identification method of the executed radiation treatment, even after 5 years. The experimental results showed that the therapeutic application of the pharmacological system investigated was not compromised by irradiation, and that ESR spectroscopy can be used to distinguish irradiated from non-irradiated products.
Keywords: Drug sterilization; ESR; Quality control; Ionizing radiation; Drug delivery system; Microspheres; Gamma-irradiation;

The objective of this study was to investigate the influence of a hydrophilic polymer, hydroxyethylcellulose (HEC), on the release properties of theophylline from pellets coated with Eudragit® RS 30 D, and the physicochemical properties of Eudragit® RS 30 D cast films. The release rate of theophylline from Eudragit® RS 30 D coated pellets decreased during storage at 25 °C/60% RH due to the further coalescence of colloidal acrylic particles. In addition, water-vapor permeability and tensile strength of Eudragit® RS 30 D cast film decreased after 1-month storage at 25 °C/60% RH. The presence of 10% hydroxyethylcellulose in the coating formulation was shown to stabilize the drug release rate from coated pellets, the water-vapor permeability and the tensile strength of free films. Atomic force microscopy and scanning electronic microscopy were used to demonstrate that the HEC was immiscible with Eudragit® RS 30 D in the cast films. The stabilization effect of HEC was investigated and determined to be due to the formation of an incompatible phase between the latex particles which impaired further coalescence of the colloidal acrylic particles.
Keywords: Eudragit® RS 30 D; Hydroxyethylcellulose; Stabilization; Coalescence; Drug release rate;

Synthesis and properties of polysaccharide prodrugs of 5-aminosalicylic acid as potential colon-specific delivery systems by Meijuan Zou; Hirokazu Okamoto; Gang Cheng; Xiuhua Hao; Jin Sun; Fude Cui; Kazumi Danjo (155-160).
The drug release of the polymer prodrugs of 5-aminosalicylic acid (5-ASA) was not only dependent on the property of the polymers but also dependent on the solubility of the prodrugs. We prepared several polysaccharide prodrugs of 5-ASA to examine the effect of solubility of prodrugs on the release characteristics of 5-ASA in the gastrointestinal contents of rats. The amide prodrug, chitosan-5-ASA (ChT-5-ASA), did not release the 5-ASA in the cecal and colonic contents. The ester prodrugs, hydroxypropyl cellulose-5-ASA (HPC-5-ASA), being poor solubility in 0.05 mol/l acetic acid solution also did not release the 5-ASA in any of gastrointestinal contents of rats. Whereas the 5-ASA release from cyclodextrins-5-ASA (CyDs-5-ASA) in cecal and colonic contents was significantly higher than that in stomach and small intestine contents. And furthermore, with the decrease in the degree of substitution, the solubility of CyD-5-ASA increased, and the release of 5-ASA in the gastrointestinal contents was also higher at the same time interval of incubation. When the ratio of cyclodextrin (CyD) and 5-formylaminosalicylic acid (5-fASA), a precursor of 5-ASA prodrugs, was 1:10, CyD-5-ASA was very slightly soluble, and no release of 5-ASA was observed within 48 h in gastrointestinal contents. The present results suggested that the ester prodrugs of 5-ASA with certain solubility could release 5-ASA in the cecal and colonic contents of rat.
Keywords: Polysaccharide prodrug; Cyclodextrin; Hydroxypropyl cellulose; Chitosan; 5-Aminosalicylic acid; Incubation; Colon-specific;

Liposomal incorporation of Artemisia arborescens L. essential oil and in vitro antiviral activity by Chiara Sinico; Alessandro De Logu; Francesco Lai; Donatella Valenti; Maria Manconi; Giuseppe Loy; Leonardo Bonsignore; Anna Maria Fadda (161-168).
The effect of liposomal inclusion on the in vitro antiherpetic activity of Artemisia arborescens L. essential oil was investigated. In order to study the influence of vesicle structure and composition on the antiviral activity of the vesicle-incorporated oil, multilamellar (MLV) and unilamellar (SUV) positively charged liposomes were prepared by the film method and sonication. Liposomes were obtained from hydrogenated (P90H) and non-hydrogenated (P90) soy phosphatidylcholine. Formulations were examined for their stability for over one year, monitoring the oil leakage from vesicles and the average size distribution. The antiviral activity was studied against Herpes simplex virus type 1 (HSV-1) by a quantitative tetrazolium-based colorimetric method. Results showed that Artemisia essential oil can be incorporated in good amounts in the prepared vesicular dispersions. Stability studies pointed out that vesicle dispersions were very stable for at least six months and neither oil leakage nor vesicle size alteration occurred during this period. After one year of storage oil retention was still good, but vesicle fusion was present. Antiviral assays demonstrated that the liposomal incorporation of A. arborescens essential oil enhanced its in vitro antiherpetic activity especially when vesicles were made with P90H. On the contrary, no significant difference in antiviral activity was observed between the free and SUV-incorporated oil.
Keywords: Artemisia arborescens L.; Essential oil; Liposomes; Vesicles; Antiviral agents; HSV-1 virus;

Evaluation of in vitro and in vivo antitumor activity of BCNU-loaded PLGA wafer against 9L gliosarcoma by Jin Soo Lee; Tae Kun An; Gang Soo Chae; Je Kyo Jeong; Sun Hang Cho; Hai Bang Lee; Gilson Khang (169-175).
The purpose of the present study was to develop implantable BCNU-loaded poly(d,l-lactide-co-glycolide) (PLGA) wafer for the controlled release of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and to evaluate its in vitro and in vivo antitumor activity. The release rate of BCNU from PLGA wafer increased with the increase of BCNU amount loaded and the release was continued until 7 days. In vitro and in vivo antitumor activity of BCNU-loaded PLGA wafer was investigated using in vitro cytotoxicity against 9L gliosarcoma cells and a subcutaneous (s.c.) solid tumor model of 9L gliosarcoma, respectively. The wafers containing BCNU showed more effective cytotoxicity than BCNU powder due to its short half-life and inhibited the proliferation of 9L gliosarcoma cells. BCNU-loaded PLGA wafer delayed the growth of the tumors significantly and increasing the dose of BCNU in the wafer resulted in a substantial regression of the tumor. These results of antitumor activity of BCNU-loaded PLGA wafer demonstrate the feasibility of the wafers for clinical application.
Keywords: Malignant gliomas; BCNU; PLGA wafer; 9L gliosarcoma; Antitumor activity;

Development and characterization of a novel Cremophor® EL free liposome-based paclitaxel (LEP-ETU) formulation by J. Allen Zhang; Gopal Anyarambhatla; Lan Ma; Sydney Ugwu; Tong Xuan; Tommaso Sardone; Imran Ahmad (177-187).
Taxol® is a marketed product for the treatment of ovarian, breast, non-small cell lung cancer and AIDS-related Kaposi's Sarcoma. It is thus far one of the most effective anticancer drugs available on the market. However, paclitaxel is only sparingly soluble in water and therefore, intravenous administration depends on the use of the non-ionic surfactant Cremophor® EL (polyethoxylated castor oil) to achieve a clinically relevant concentrated solution. Unfortunately, Cremophor® EL increases toxicity and leads to hypersensitivity reactions in certain individuals. We have developed a well characterized novel lyophilized liposome-based paclitaxel (LEP-ETU) formulation that is sterile, stable and easy-to-use. The mean particle size of the liposomes is about 150 nm before and after lyophilization, and the drug entrapment efficiency is greater than 90%. Stability data indicated that the lyophilized LEP-ETU was physically and chemically stable for at least 12 months at 2–8 and 25 °C. Moreover, the formulation can be diluted to about 0.25 mg/ml without drug precipitation or change in particle size. In vitro drug release study in phosphate-buffered saline (PBS, pH 7.4) showed that less than 6% of the entrapped paclitaxel was released after 120 h, indicating that the drug is highly stable in an entrapped form at physiologic temperature.
Keywords: Liposome-based formulation; Paclitaxel; Liposomes; Stability; Lyophilization; Taxol®; Entrapment;

Characterization of cellulosic hot-melt extruded films containing lidocaine by Michael A. Repka; Kavitha Gutta; Suneela Prodduturi; Manish Munjal; Steven P. Stodghill (189-196).
Hot-melt extrusion technology was used to produce thin films containing a model drug, lidocaine, and the cellulosic polymers hydroxypropyl cellulose (HPC) and hydroxypropyl methyl cellulose (HPMC). Two film formulations were extruded and compared, one containing only HPC and the other containing HPC:HPMC (80:20). Thermal analysis of the films using differential scanning calorimetry (DSC) suggested that the drug existed in the amorphous condition, which was confirmed by wide angle X-ray diffractometry. Sustained release of the drug was observed from both of the polymer matrices. Dissolution profiles suggested that HPMC retarded the drug release from HPC:HPMC (80:20) films. However, the mechanism of drug release from both of the films was predominantly diffusion of the drug through the polymer matrices. Incorporation of HPMC also increased both adhesive strength and work of adhesion as compared to the HPC-only films.
Keywords: Hot-melt extrusion; Lidocaine; Texture analyzer; Muco-adhesive films; Hydroxypropyl cellulose; Hydroxypropyl methyl cellulose;

The effectiveness of press-on forces (defined as the adhesive forces between drug and carrier particles) in relation to carrier payload as the result of collisions between carrier particles during the mixing process of an adhesive mixture, has been investigated. Three different carriers of the same size fraction (250–355 μm), but with completely different surface rugosity were studied. It could be shown that this effectiveness depends on the carrier rugosity. The fraction of drug detached from the carrier particles during inhalation appeared to decrease faster with increasing carrier payload for crystalline carriers than for granular carriers. Apparently, increasing the volume of the carrier surface cavities increases the drug mass that can find shelter from the press-on forces during mixing. By measuring the size distribution in the aerosol, it could also be shown that the press-on forces may increase the size of the particles that are detached. This seems to be the result of drug particle re-agglomeration on the carrier surface during mixing. On the other hand, when press-on forces are highly ineffective, an increase in the size of detached particles may also be the result of incomplete break-up of natural drug agglomerates. Finally, it could be shown that when the press-on forces are highly effective, the effect of mixing time is small.
Keywords: Adhesive mixtures; Carrier rugosity; Budesonide; Mixing time; Lactose; Carrier payload;

The aim of this study was to develop mucoadhesive microspheres that can be utilised for the controlled release of triclosan in oral-care formulations, specifically dental pastes. Using a double-emulsion solvent evaporation technique, triclosan was incorporated into microspheres that were prepared from Gantrez™ MS-955, Carbopol™ 974P, polycarbophil or chitosan and the profiles for its release were established under simulated ‘in use’ conditions. Triclosan was rapidly released into a sodium lauryl sulphate containing buffer from all but the chitosan microspheres. The release of triclosan from microspheres suspended in a non-aqueous paste, was found to be sustained over considerable time-periods, which were influenced strongly by the nature of the polymeric carrier. For microspheres that were fabricated from Gantrez, Carbopol or polycarbophil, the release appeared to obey zero-order kinetics whereas in the case of chitosan-derived vehicles, the release profile fitted the Baker and Lonsdale model. The work has demonstrated that these polymeric microspheres, particularly those of chitosan, are promising candidates for the sustained release of triclosan in the oral cavity.
Keywords: Polymer microspheres; Triclosan; Double emulsion; Bioadhesion; Oral cavity; Release kinetics;

Based on the free-volume theory of diffusion in rubbers it is shown that the diffusion coefficient D of a permeating drug can be written as the product of two probabilities Dw 1 w 2. The polymer chains in a rubber are subject to thermal vibrations. Hence collisions between vibrating polymer segments and permeating drug molecules can occur. w 1 describes the probability that by such a collision a drug molecule experiences an energy exchange which is large enough to overcome its interactions with its neighborhood. w 2 describes the probability that a permeating drug molecule will find in its immediate neighborhood a free volume being equal or larger than its own volume. Diffusion takes place if the drug molecule experiences a sufficiently large exchange of momentum and finds at the same time a sufficiently large free volume. As both events are independent their overall probability is given by the product of the two probabilities. For a given rubber and a given species of drug molecules in a first approximation w 1 can be considered as constant. This means the diffusion coefficient is mainly determined by w 2. This probability, however, is strongly determined by the microstructure of the rubber. In this study a procedure is developed allowing for a straightforward synthesis of silicone rubbers with defined network density.
Keywords: Diffusion; Polymers; Therapeutic systems; Silicon rubbers; Controlled release; Response surfaces;

A linear system comprising n compartments is completely defined by the rate constants between any of the compartments and the initial condition in which compartment(s) the drug is present at the beginning. The generalized solution is the time profiles of drug amount in each compartment, described by polyexponential equations. Based on standard matrix operations, an Excel worksheet computes the rate constants and the coefficients, finally the full time profiles for a specified range of time values.
Keywords: IVIVC; Excel; Systems analysis; Pharmacokinetics; Compartmental models;

Reverse iontophoresis of lithium: electrode formulation using a thermoreversible polymer by Valentine Wascotte; Benoît Leboulanger; Richard H. Guy; M. Begoña Delgado-Charro (237-240).
This work investigated the use of a thermoreversible gel as a collector vehicle in reverse iontophoresis applications. A 20% (w/w) aqueous gel of Pluronic F127 was a suitable receptor medium to be used at the cathodal chamber. In vitro iontophoresis experiments investigated the simultaneous extraction of lithium (analyte of interest) and sodium (used as an internal standard) into either a control buffer or a gelled receptor. The gelification process at room temperature provided a suitable consistency and contact with the skin surface during the iontophoresis experiments. Subsequent cooling of the gelled solution to 4 °C allows an easy recovery of lithium and sodium for later quantification. Both the lithium extraction fluxes and the lithium to sodium ratio of extraction fluxes were linearly related to the subdermal lithium concentration. On the whole, the results show that thermoreversible polymer solutions offer a simple and convenient way to handle samples in reverse iontophoresis studies.
Keywords: Pluronic F-127; Thermoreversible polymer; Reverse iontophoresis; Lithium; Non-invasive monitoring;

by Bernd W. Müller (241-242).

by Richard Süverkrüp (241).

by J. Kreuter* (242).

by Peter Gmeiner (243).

Doctoral theses (245-246).