European Journal of Pharmaceutics and Biopharmaceutics (v.59, #3)

APV diary (S1-S2).

This paper reviews the major factors that are closely involved in peptide and protein degradation during the preparation of biodegradable nano- and microparticles. The various means usually employed for overcoming these obstacles are described, in order to bring to the fore the strategies for protein stabilization. Both processing and formulation parameters can be modified and are distinctly considered from a strategic point of view. We describe how partial or full protein stability retention within the carriers and during drug release might be achieved by individual or combined optimized strategies. Additionally, problems commonly encountered during protein quantification, stability determination and release are briefly reviewed. Artefacts that might occur during sampling and analytical procedures and which might hinder critical interpretation of results are discussed.
Keywords: Biodegradable polymers; Nanoparticles; Microparticles; Peptides; PLGA; PLA; Proteins; Stabilization;

The therapeutic antibodies market to 2008 by Alex K. Pavlou; Mark J. Belsey (389-396).
The therapeutic biologics market is currently dominated by recombinant protein products. However, many of these products are mature, and growth of the biologics market will increasingly rely on the expansion of the therapeutic monoclonal antibody sector. Successive technology waves have driven the growth of the monoclonal antibody sector, which is currently dominated by chimeric antibodies. Chimeric products, led by Remicade and Rituxan, will continue to drive market share through to 2008. However, over the forecast period, humanized and fully human monoclonal antibodies, together with technologies such as Fabs and conjugated antibodies, will play an increasingly important role, driving monoclonal antibody market growth at a forecast compound annual growth rate of 20.9%, to reach $16.7 billion by 2008. In terms of therapeutic focus, the monoclonal antibody market is heavily focused on oncology and arthritis, immune and inflammatory disorders, and products within these therapeutic areas are set to continue to be the key growth drivers over the forecast period. Underlying the growth of the market is the evolution of the monoclonal antibody company business model, set to transition towards the highly successful innovator model.
Keywords: Monoclonal antibody; Chimeric; Murine; Humanized; Fabs; Fully human; Market analysis;

Biopharmaceuticals product development is a broad and multidisciplinary field. Science and technology are combined with new manufacturing, regulatory and commercial challenges. However, although there is ample literature on the molecular biology and biochemistry of products, the implementation of processes from test tube to commercial scale has not received similar attention. Consequently, the present study aims to highlight, from practical point of view, some of the key issues involved with manufacturing technologies of biopharmaceuticals at a commercial scale. Regulatory requirements and investments are also addressed based on the practical experiences of start-up and small companies. Finland is used as a case-example of such companies as this is a EU-member state with strong technological growth and rapidly increasing number of biotech companies.
Keywords: Biopharmaceuticals; Biotech engineering; Manufacturing; FDA/EMEA regulations; Scale-up; GMP;

Induction and analysis of aggregates in a liquid IgG1-antibody formulation by Hanns-Christian Mahler; Robert Müller; Wolfgang Frieβ; Aurelie Delille; Susanne Matheus (407-417).
The objective of this study was to compare different agitation stress methods (stirring in Reacti VialsTM versus horizontal shaking) in their effect on protein destabilization, to assess several analytical techniques (light obscuration, turbidimetric and light scattering analysis) for detection of aggregates of various sizes and to evaluate the protecting effect of polysorbate 80 on protein aggregation. A monoclonal IgG1 antibody was used as model protein.Both mechanical stress methods can provoke aggregate formation. The method of stirring induces particles in the range of 10–25 μm comparable to shaking stress. However, stirred samples show a much higher absorbance and reveal a second particle species in DLS analysis, suggesting that stirring stress induces a higher amount of smaller protein aggregates. Addition of polysorbate 80 protects the antibody against aggregation. Only in stirred samples a slight increase in sub-visible particles and turbidity was noted. However, a greater extent of aggregation products was detected by DLS as compared to surfactant-free formulations. Thus, polysorbate 80 appears to stabilise small aggregates and prevents further proceeding of the aggregation process. The induction of aggregates by stirring stress in Reacti VialsTM analysed by absorbance measurement seems to be a good combination for high-throughput formulation studies.
Keywords: Protein stability; Mechanical stress; Aggregation; Association; Light obscuration; Turbidity; Dynamic light scattering; Antibody;

Albumin–protamine–oligonucleotide nanoparticles as a new antisense delivery system. Part 1: Physicochemical characterization by Dirk Lochmann; Jörg Weyermann; Christiane Georgens; Ruth Prassl; Andreas Zimmer (419-429).
In this paper, a ternary system of albumin–protamine–oligonucleotide nanoparticles (AlPrO-NP) recently developed by Vogel et al. [V. Vogel, D. Lochmann, J. Weyermann, G. Mayer, C. Tziatzios, J.A. van den Broek, W. Haase, D. Wouters, U.S. Schubert, J. Kreuter, A. Zimmer, D. Schubert, Oliganucleotide–protamin–albumin nanoparticles: preparation, physical properties and intracellular processing, J. Controlled Rel. (in press)] which could serve as a potential drug delivery system for antisense oligonucleotides. Former studies of binary protamine-oligonucleotide nanoparticles showed two main disadvantages: (i) aggregation of the particles within a few minutes in the presence of salt; (ii) low intracellular dissociation between protamine and oligonucleotide, especially phosphorothioates. To overcome these problems, human serum albumin (HSA) as a non-toxic, biodegradable macromolecule was introduced as protective colloid. The assembly process of AlPrO-NP was investigated by small angle X-ray scattering (SAXS), fluorescence correlation spectroscopy (FCS), photon correlation spectroscopy (PCS) measurements and scanning electron microscopy (SEM). ‘Initial complexes’ of HSA and protamine sulphate with a mean hydrodynamic diameter (d h) of about 10–14 nm were found. After adding oligonucleotides (unmodified, phosphorothioate DNA and small interfering RNA), nanoparticles (NPs) were assembled in water and in isotonic media with a d h in a range of 230–320 nm for most preparations. The chemical composition of the particles was investigated by high performance liquid chromatography and fluorescence spectrometry. The whole amount of oligonucleotides (30 μg) was entrapped into the particles at a 1:2 mass ratio (oligonucleotide/protamine). Approximately 7–10% (w/w) of the HSA was bound to the particles. The surface charge of the particles ranged from about +12 to −60 mV depending on the protamine concentration and the ionic conditions. The size and the molecular weight of the components, initial complexes and two model NP preparations were calculated from FCS data. These data verified the PCS, SEM and SAXS measurements.
Keywords: Nanoparticles; Albumin; Protamine; Oligonucleotides; Small interfering RNA; Antisense;

Albumin–protamine–oligonucleotide-nanoparticles as a new antisense delivery system. Part 2: cellular uptake and effect by Jörg Weyermann; Dirk Lochmann; Christiane Georgens; Andreas Zimmer (431-438).
Antisense oligonucleotides have been used as a specific tool to inhibit the expression of disease associated genes for many years. Unfortunately, oligonucleotides are polyanionic macromolecules which have a weak permeability through biological membranes and are rapidly degraded by nucleases. The purpose of this work is to characterise a new drug delivery system developed by [V. Vogel, D.Lochmann, J. Weyermann, G. Mayer, C. Tziatios, J.A. van der Brock, W. Haase, D. Wouters, U.S. Schubert, J. Kreuter, A. Zimmer, D. Schubert, Oligonucleotide–protamine–albumin nanoparticles preparation, physical properties and intracellular processing, J. Controlled Rel. (in press)] which allows an increased cellular uptake and an intracellular dissociation of the oligonucleotides. The new system based on nanoparticles (NPs) consists of human serum albumin, protamine sulphate and antisense-oligonucleotides (AlPrO). We tested these new nanoparticles on mouse fibroblasts which were stably transfected with a N-methyl-d-aspartate (NMDA) receptor (NR). This cell line enabled us to perform in vitro studies of cellular uptake, intracellular dissociation and effect of the antisense-oligonucleotide in a simple excitotoxicity model. We compared our findings with free oligonucleotides and a commercial available liposomal preparation (DOTAP). We found a 12-fold increased cellular uptake of oligonucleotides in comparison to free oligonucleotides while 100% of the cells were transfected. The AlPrO-NPs showed very low cytotoxic side effects during a 24 h application. We saw an antisense effect of about 35% in a functional assay as well as on the protein level (western blot). The results of the cell penetration and the antisense assay demonstrated that AlPrO nanoparticles are promising carriers for oligonucleotide administration.
Keywords: Nanoparticle; Antisense; Oligonucleotide; Liposomes; DOTAP; Albumin; Protamine;

Semisolid phospholipid dispersions of vesicular morphology, so-called vesicular phospholipid gels (VPGs), were prepared by high-pressure homogenisation and tested in vitro for their suitability as implantable sustained release system for the decapeptide cetrorelix, a potent LH-RH antagonist. The VPGs contained 300–500 mg/g egg phosphatidylcholine (E80) and 0.5–10 mg/g cetrorelix acetate (CXA). The in vitro release experiments showed a wide variability of the system in release, ranging from complete release within less than 24 h (0.5 mg/g CXA; 400 mg/g E80) to a predicted 80% sustained release over 3 months (8.6 mg/g CXA; 280 mg/g E80). Erosion of the phospholipid matrix, i.e. release of phospholipid vesicles was found to be the main release mechanism, following zero order or first order kinetics depending on the composition of the VPG. CXA-concentration dependent drug–drug or drug–lipid interactions are assumed to be responsible for the change in release kinetics and the decrease of CXA release at high concentrations of the peptide. Multivariate analysis revealed that both lipid concentration and peptide concentration and also the interactions between the two factors are significant factors for the release rate of the peptide. In summary: based on the presented in vitro release data sustained release of therapeutically relevant CXA doses over up to 6 weeks appears feasible. VPGs are thus considered as a promising new approach for the sustained release of peptide hormones.
Keywords: Cetrorelix; Peptide; Vesicular phospholipid gel; Liposome; Depot system; Sustained release; In vitro release;

Baclofen-loaded microspheres: preparation and efficacy testing in a new rabbit model by Frederic Lagarce; Pascal Renaud; Nathalie Faisant; Guillaume Nicolas; Annie Cailleux; Joel Richard; Philippe Menei; Jean-Pierre Benoit (449-459).
Intrathecal baclofen is the reference treatment for severe spasticity. This drug has to be injected chronically in the intrathecal space by implanted pumps which are very expensive, uncomfortable and sometimes lead to side effects. Previous work has been performed by our group to assess the feasibility of encapsulating baclofen into poly(lactide-co-glycolide) (PLGA) microspheres and injecting these preparations in the intrathecal space of rabbits. The aims of the present study were to improve the encapsulation process for industrial application (scale-up), and to set up an animal model to assess the duration of effect of the new formulations. Modifications included the replacement of methylene chloride by a less toxic solvent, ethyl acetate, and the use of high molecular weight polymers to extend the release rate of the drug. The temperature and organic solvent extraction rate were fully controlled during the whole manufacturing process. All these modifications resulted in high quality microsphere batches with a CV inferior to 5% for encapsulation efficiency and drug loading. Encapsulation efficiency and release patterns were dependent on the drug payload and the polymer used. A formulation displaying a sustained release of baclofen over 174 days and a moderate burst effect of 16% in the first day in vitro was evaluated in a new reliable model of baclofen activity based on electrophysiological measurement of H-reflex in the rabbit. The activity of a very low dose of baclofen microspheres in vivo was sustained over 35 days. Furthermore, the preparation was well tolerated. These newly developed preparations are a very promising approach for enhancing the efficacy and comfort of patients undergoing spasticity treatment.
Keywords: Spasticity; Intrathecal baclofen; Rabbit; H-reflex; Electromyography; Microsphere; Ethyl acetate; Sustained release; Poly(lactide co-glycolide);

Stability of different formulations and ion pairs of hypericin by Ann Huygens; Appolinary R. Kamuhabwa; Peter A.M. de Witte (461-468).
Hypericin, solubilized in an instillation fluid consisting of an aqueous buffer supplemented with 1% plasma proteins, is currently used as a clinical diagnostic tool for the detection of superficial TCC (transitional cell carcinoma) tumors. However, the development of a sterile and stable hypericin stock formulation, excluding the presence of plasma constituents, would be an important factor in a more general clinical application of the method. Therefore, we investigated the stability of several heat sterilized hypericin formulations and ion pairs. Besides sodium hypericinate (in distilled water, in phosphate buffer, in polyethyleneglycol (PEG) 400), several other hypericinate salts (potassium, lysine, TRIS or hexylamine) were investigated. As to that, the physical appearance of different hypericin concentrates stored at 4 and 37 °C was investigated. Besides, after dilution into cell culture medium, the ability of hypericin remaining to accumulate in tumor cells and demonstrating photocytotoxic effects upon light irradiation was assessed. These findings suggest that PEG 400 is an excellent hypericin formulation, since it maintained the stability of the compound for at least 120 d when stored at either 4 or 37 °C. PEG 400 therefore is a suitable vehicle for the storage of hypericin prior to preparation of the bladder instillation solution.
Keywords: Bladder cancer; Formulations; Hypericin; Ion pairs; Stability;

Determination of hypericin in human plasma by high-performance liquid chromatography after intravesical administration in patients with transitional cell carcinoma of the bladder by Appolinary A.R. Kamuhabwa; José Diana Di Mavungu; Luc Baert; Marie-Ange D'Hallewin; Jos Hoogmartens; Peter A.M. de Witte (469-474).
In the present study, the systemic absorption of hypericin was investigated after intravesical instillation of the compound in nine patients with superficial transitional cell carcinoma (TCC) bladder tumors. Hypericin (8 μM) was instilled in the bladder for 2–3 h before photodynamic diagnosis of bladder tumors. Blood was then collected from a peripheral vein 1 h after termination of the instillation. Solid phase extraction with ammonium acetate buffer and methanol was used to extract hypericin from the plasma. A reversed-phase high performance liquid chromatographic method with fluorescence detection was used to identify and quantify hypericin in the extracts from plasma samples. Analysis of standard plasma samples, which were spiked with known amounts of hypericin, indicated that the pH of the buffer was a determining factor in the extraction yield. The results obtained using ammonium buffer (pH 3.5) and methanol showed the mean extraction recovery of hypericin to be 64% (RSD=12%, n=6). The limits of detection and quantification were 6 and 20 nM, respectively. Extraction and analysis of the plasma of patients after intravesical administration showed hypericin concentrations below the detection limit (<6 nM). In addition, photodynamic treatment of in vitro cultured HeLa cells incubated with 1–100 nM hypericin concentrations showed that lower concentrations (1–20 nM) of hypericin do not induce significant photocytotoxic effects. Taken together, these results imply that photosensitization or other systemic side effects in patients are not to be expected after photodynamic diagnosis of TCC bladder tumors with hypericin.
Keywords: Hypericin; Bladder carcinoma; Plasma; Photodynamic therapy; HPLC;

A comparison between povidone-ethylcellulose and povidone-eudragit transdermal dexamethasone matrix patches based on in vitro skin permeation by Biswajit Mukherjee; Sushmita Mahapatra; Ritu Gupta; Balaram Patra; Amit Tiwari; Priyanka Arora (475-483).
The present study was designed to develop a suitable matrix type transdermal drug delivery system (TDDS) of dexamethasone using blends of two different polymeric combinations, povidone (PVP) and ethylcellulose (EC) and Eudragit with PVP. Physical studies including moisture content, moisture uptake, flatness to study the stability of the formulations and in vitro dissolution of the experimental formulations were performed to determine the amount of dexamethasone present in the patches were performed and scanning electron microscopy (SEM) photographs of the prepared TDDS were taken to see the drug distribution pattern. Drug–excipient interaction studies were carried out using Fourier transform infrared (FTIR) spectroscopic technique. In vitro skin permeation study was conducted in a modified Franz's diffusion cell. All the formulations were found to be suitable for formulating in terms of physicochemical characteristics and there was no significant interaction noticed between the drug and polymers used. In vitro dissolution studies showed that the drug distribution in the matrix was homogeneous and the SEM photographs further demonstrated this. The formulations of PVP:EC provided slower and more sustained release of drug than the PVP:Eudragit formulations during skin permeation studies and the formulation PVP:EC (1:5) was found to provide the slowest release of drug. Based on the above observations, it can be reasonably concluded that PVP–EC polymers are better suited than PVP–Eudragit polymers for the development of TDDS of dexamethasone.
Keywords: Dexamethasone; Transdermal patches; Eudragit; PVP; Ethylcellulose; In vitro skin permeation studies;

Proniosomes as a drug carrier for transdermal delivery of ketorolac by Ibrahim A. Alsarra; A.A. Bosela; S.M. Ahmed; G.M. Mahrous (485-490).
Niosomes are nonionic surfactant vesicles that have potential applications in the delivery of hydrophobic and hydrophilic drugs. Permeation of a potent nonsteroidal anti-inflammatory, ketorolac, across excised rabbit skin from various proniosome gel formulations was investigated using Franz diffusion cells. Each of the prepared proniosomes significantly improved drug permeation and reduced the lag time (P<0.05). Proniosomes prepared with Span 60 provided a higher ketorolac flux across the skin than did those prepared with Tween 20 (7- and 4-fold the control, respectively). A change in the cholesterol content did not affect the efficiency of the proniosomes, and the reduction in the lecithin content did not significantly decrease the flux (P>0.05). The encapsulation efficiency and size of niosomal vesicles formed by proniosome hydration were also characterized by specific high performance liquid chromatography method and scanning electron microscopy. Each of the prepared niosomes achieved about 99% drug encapsulation. Vesicle size was markedly dependent on the composition of the proniosomal formulations. Proniosomes may be a promising carrier for ketorolac and other drugs, especially due to their simple production and facile up.
Keywords: Niosomes; Proniosomes; Ketorolac; Transdermal delivery; Permeation;

Development and characterization of PLGA nanospheres and nanocapsules containing xanthone and 3-methoxyxanthone by Maribel Teixeira; Maria J. Alonso; Madalena M.M. Pinto; Carlos M. Barbosa (491-500).
The aim of the present work was to develop and characterize two different nanosystems, nanospheres and nanocapsules, containing either xanthone (XAN) or 3-methoxyxanthone (3-MeOXAN), with the final goal of improving the delivery of these poorly water-soluble compounds. The xanthones-loaded nanospheres (nanomatrix systems) and nanocapsules (nanoreservoir systems), made of poly(dl-lactide-co-glycolide) (PLGA), were prepared by the solvent displacement technique. The following characteristics of nanoparticle formulations were determined: particle size and morphology, zeta potential, incorporation efficiency, thermal behaviour, in vitro release profiles and physical stability at 4 °C. The nanospheres had a mean diameter <170 nm, a narrow size distribution (polydispersity index <0.1), and a negative surface charge (zeta potential <−36 mV). Their incorporation efficiencies were 33% for XAN and 42% for 3-MeOXAN. The presence of the xanthones did not affect the nanospheres size and zeta potential. DSC studies indicated that XAN and 3-MeOXAN were dispersed at a molecular level within the polymeric nanomatrix. Nanocapsules were also nanometric (mean size <300 nm) and exhibited a negative charge (zeta potential <−36 mV). Their incorporation efficiency values (>77%) were higher than those corresponding to nanospheres for both xanthones. The release of 3-MeOXAN from nanocapsules was similar to that observed for the correspondent nanoemulsion, indicating that drug release is mainly governed by its partition between the oil core and the external aqueous medium. In contrast, the release of XAN from nanocapsules was significantly slower than from the nanoemulsion, a behaviour that suggests an interaction of the drug with the polymer. Nanocapsule formulations exhibited good physical stability at 4 °C during a 4-month period for XAN and during a 3-month period for 3-MeOXAN.
Keywords: Xanthone; 3-Methoxyxanthone; Nanoparticles; Nanospheres; Nanocapsules; PLGA;

Riboflavin (RF) in combination with light, in the wavelength range of 310–800 nm, is used to induce degradation of alginic acid gels. Light irradiation of alginate solutions in the presence of RF under aerobic conditions causes scission of the polymer chains. In the development process of a new drug delivery system, RF photosensitized degradation of alginic acid gels is studied by monitoring changes in the turbidity and rheological parameters of alginate/glucono-δ-lactone (GDL) systems with different concentrations of GDL. Addition of GDL induces gel formation of the samples by gradually lowering the pH-value of the system. The turbidity is measured and the cloud point determined. The turbidity starts to increase after shorter times with enhanced concentration of GDL. Enhanced viscoelasticity is detected with increasing GDL concentration in the post-gel regime, but small differences are detected at the gel point. The incipient gel is ‘soft’ and has an open structure independent on the GDL concentration. In the post-gel regime solid-like behavior is observed, this is more distinct for the systems with high GDL concentrations. The effect of photosensitized RF on alginate/GDL systems decreases with increasing amount of GDL in the system. The same trend is detected whether the systems are irradiated in the pre-gel or in the post-gel regime.
Keywords: Alginate; Photosensitation; Degradation; Drug delivery; Hydrogel; Viscoelasticity;

Starch–dextrin mixtures as base excipients for extrusion–spheronization pellets by S. Almeida Prieto; J. Blanco Méndez; F.J. Otero Espinar (511-521).
Extrusion–spheronization pellets are generally produced with microcrystalline cellulose (MCC) as the principal excipient, giving rise to particles of very high quality. A number of alternative excipients have been proposed and evaluated, mostly other cellulose derivatives (e.g. different grades of Avicel), or mixtures of MCCs and other excipients. In the present study, we evaluated the possible use of starch+agglutinant mixtures as principal excipients for extrusion–spheronization pellets, with the aim of producing pellets with more suitable properties for certain types of release. We first characterized the different excipients in terms of morphometry and basic physical properties. Subsequently, torque-rheometry was used to characterize the rheology of wetted masses of the different excipients and excipient mixtures, with the aim of determining optimal amount of wetting agent (water). We also evaluated the water absorption and water retention capacities of each excipient. In view of the results obtained, we produced pellets with the different starch+agglutinant mixtures (but without drug), and used image analysis to characterize pellet morphology. Our results show that some of the mixtures—notably starch (corn starch or wheat starch)+20% white dextrin—gave high-quality pellets with good size and shape distributions. In addition, the properties of the different materials tested suggest that it may be possible to obtain pellets with very different properties.
Keywords: Pelletization; Extrusion–spheronization; Torque-rheometry; Starchs; Dextins; Image analysis;

Spectrofluorimetric study of eflucimibe-γ-cyclodextrin inclusion complex by Nathalie Mesplet; Philippe Morin; Jean-Paul Ribet (523-526).
Eflucimibe, a novel and highly potent acyl-coenzyme A cholesterol O-acyl-transferase (ACAT) inhibitor, is sparingly soluble in aqueous media and exhibits a very weak natural fluorescence. However, when increasing concentrations of γ-cyclodextrin (γ-CD) are added, an increase in the fluorescence signal is observed, attesting the formation of a non-covalent inclusion complex between eflucimibe and the γ-CD. In this work, the stoichiometry of the complex and the corresponding association constant have been determined from fluorescence data by Benesi–Hildebrand's method (double reciprocal plots). As a result, a 1:1 stoichiometric ratio and a 20 M−1 formation constant were obtained. This apparent formation constant was determined in water containing 10% methanol, which was needed to improve ‘aqueous’ solubility of the drug in a CD-free medium. Owing to the extreme hydrophobicity of eflucimibe, these results provide valuable information for pharmaceutical formulation studies.
Keywords: Eflucimibe; Cyclodextrin; Spectrofluorimetry; Inclusion complex; Stoichiometry; Association constant;

Effects of roller compaction settings on the preparation of bioadhesive granules and ocular minitablets by Wim Weyenberg; An Vermeire; Jo Vandervoort; Jean Paul Remon; Annick Ludwig (527-536).
An experimental factorial design was employed to evaluate bioadhesive granules and bioerodible ocular minitablets (6 mg and Ø 2 mm). The purpose of this study was to compare minitablets prepared using roller compacted granules with an optimised minitablet formulation, manufactured on laboratory scale by direct compression. The formulation consisted of drum dried waxy maize starch, Carbopol® 974P, and ciprofloxacin in a ratio of 90.5/5/3 (w/w/w). Three roller compactor parameters were varied, i.e. the roller speed, the horizontal screw speed and the compaction force, while the vertical screw speed was kept constant. Afterwards, the ribbons were milled to obtain granules suitable for compression. The friability, the flow properties, the bulk material characteristics (apparent and tap density and porosity) and the particle size distributions of two granule sieve fractions (90–125 and 125–355 μm) were investigated. The roller speed and the compaction force have the largest influence on the granule characteristics, followed by the horizontal screw speed. The physical properties of non- and gamma-irradiated minitablets were determined. From the tablet strength, friability and dissolution results, a low compaction force and a high roller speed were shown to be preferable to prepare granules which can be further tabletted into adequate ocular minitablets.
Keywords: Ocular minitablet; Roller compaction/dry granulation; Bioadhesive formulation; Experimental design; Ciprofloxacin;

A new family of graft copolymers combining hydrophilic and hydrophobic components have recently been proposed as direct compression excipients. Copolymers were synthetised by free radical copolymerisation of starch derivatives with methyl methacrylate (MMA) and were alternatively dried by oven or freeze-drying techniques. The aim of this study was to investigate the water vapour sorption–desorption behaviour of these copolymers, focusing on the influence of variables such as the hydrophobic component and the drying process.Moisture sorption and desorption isotherms were measured at 25 °C and analysed according to GAB and Young–Nelson equations, which distinguish between different physical forms of moisture distribution. The Young–Nelson model gave the best fit to the experimental data. The results obtained showed that the presence of the acrylic component modified not only the total hygroscopicity of the copolymers, as compared with the original starch derivatives, but also the water distribution in the solid, which might have an important role in the effect of moisture content on copolymer characteristics. The main water-sorption mechanism seemed to be absorption into the copolymer particles structure, in agreement with their starching nature. In terms of water sorption–desorption characteristics, no marked differences were found between the two drying methods used.
Keywords: Water vapour sorption–desorption isotherm; Methyl methacrylate; Starch derivatives; Copolymers; Drying method; Water–solid interaction;

The present study was to investigate oral absorption of the two similar flavonoid glycosides, isoquercitrin (IQ, quercetin-3-O-glucoside) and hyperoside (HP, quercetin-3-O-galactoside) in rats. Two groups of male SD rats received an oral dose of either IQ (4.5 mg/kg) or HP (6.0 mg/kg). Blood samples were collected via jugular vein at time intervals after drug administration and the plasma concentrations of the studied compounds were analyzed by HPLC. The stability of IQ and HP in the GI tract was also measured by incubation with various GI contents from rats. The results showed that unchanged IQ was barely detectable whereas the glucuronidated quercetin (the aglycone of IQ) was found to be the major form in plasma after oral administration of IQ. In contrast, HP could not be detected in plasma neither as unchanged form nor its aglycone or conjugated aglycone form. Additional in vitro stability studies demonstrated that HP is more stable than IQ in the GI tract. This suggests that IQ could be hydrolyzed easier than HP to its aglycone in GI tract before being absorbed. In conclusion, IQ, as a flavonoid glucoside, could be rapidly absorbed and transformed into glucuronidated quercetin and such absorption might be related to the hydrolysis of the type of sugar moieties attached to its aglycone molecule.
Keywords: Isoquercitrin; Hyperoside; Flavonoids; Absorption;

The influence of gellan gum on the transfer of fluorescein dextran across rat nasal epithelium in vivo by Björn Jansson; Helene Hägerström; Nelly Fransén; Katarina Edsman; Erik Björk (557-564).
The nasal uptake of a 3000 Da fluorescein dextran (FD3) was investigated in rats, using fluorescence microscopy. The uptake from a formulation containing deacetylated gellan gum, an in situ gelling agent, was compared to that from a mannitol solution. Additionally, the rheological behavior of the gellan gum in water and saline was studied. It was shown that the gellan gum solution was easily administered owing to its low viscosity, and upon contact with the mucosa, a gel was formed. The epithelial uptake and transfer of FD3 appeared to be increased and prolonged using the gellan gum formulation. This increase was not accompanied by qualitative changes of the epithelial FD3 distribution or any visible harmful effects.
Keywords: Intranasal administration; In situ gel; Fluorescein dextran; Deacetylated gellan gum; Nasal mucosa; Olfactory mucosa; Fluorescence microscopy;

Effects of process variables on the powder yield of spray-dried trehalose on a laboratory spray-dryer by Michael Maury; Keith Murphy; Sandeep Kumar; Lei Shi; Geoffrey Lee (565-573).
A systematic examination is presented of the effects of process variables on the powder yield of amorphous trehalose obtained from the Büchi Model 191 laboratory-scale mini spray dryer. By using a specially made, narrow cyclone the powder yield could be greatly improved at all process temperatures examined. Calculations of the separation efficiencies of the improved cyclone and the manufacturer's standard cyclone are given, which show that the former's higher tangential particle velocity at the radius of the exit duct is responsible for the improved performance. The powder yield increases with higher process temperatures, owing to improved droplet drying and reduced droplet/particle deposition on the walls of the drying chamber. A maximum in the powder yield is reached, however, after which it decreases sharply. This is caused by heating of the cyclone wall to >10 °C above the so-called ‘sticky point’ of the trehalose, causing increased particle deposits on the walls of the tower and cyclone. Increasing liquid feed flow rate or decreasing atomizing air flow rate too extensively were both detrimental to powder yield. The drying air flow rate should be as high as possible to ensure sufficient enthalpy throughput to dry the trehalose adequately to give a high powder yield. The enthalpy balance calculation for drying trehalose with the new cyclone was used successfully to interpret the results obtained. Some recommendations for optimizing powder yield of an amorphous material are given.
Keywords: Spray-drying; Protein; Trehalose; Yield; Cyclone; Enthalpy balance;

by Geoffrey Lee (575).