European Journal of Pharmaceutics and Biopharmaceutics (v.59, #2)

APV Diary (S1-S2).

Spray-drying of proteins: effects of sorbitol and trehalose on aggregation and FT-IR amide I spectrum of an immunoglobulin G by Michael Maury; Keith Murphy; Sandeep Kumar; Alexander Mauerer; Geoffrey Lee (251-261).
An immunoglobulin G (IgG) was spray-dried on a Büchi 190 laboratory spray-dryer at inlet and outlet air temperatures of 130 and 190 °C, respectively. The IgG solution contains initially 115 mg/ml IgG plus 50 mg/ml sorbitol. After dialysis, at least 80% of low molecular weight component was removed. After spray-drying the dialyzed IgG and immediate redissolution of the powder, an increase in aggregates from 1 to 17% occurred. A major shift towards increase β-sheet structure was detected in the spray-dried solid, which, however, reverted to native structure on redissolution of the powder. A correlation between aggregation determined by size exclusion chromatography and alterations in secondary structure determined by Fourier transformation infra-red spectroscopy could not therefore be established. On spray-drying a non-dialyzed, sorbitol-containing IgG only some 0.7% aggregates were formed. The sorbitol is therefore evidently able to stabilize partially the IgG during the process of spray-drying. Addition of trehalose to the liquid feed produced quantitatively the same stabilizing action on the IgG during spray-drying as did the sorbitol. This finding again points towards a water replacement stabilization mechanism. The IgG spray-dried powder prepared from the dialyzed liquid feed showed continued substantial aggregation on dry storage at 25 °C. This was substantially less in the non-dialyzed, sorbitol-containing spray-dried powder. Addition of trehalose to both dialyzed and non-dialyzed system produced substantial improvement in storage stability and reduction in aggregate formation in storage. The quantitative stabilizing effect of the trehalose was only slightly higher than that of the sorbitol. Taken together, these results indicate that both the sorbitol and trehalose stabilize the IgG primarily by a water replacement mechanism rather than by glassy immobilization. The relevance of this work is its questioning of the importance of the usually considered dominance of glassy stabilization of protein in dried systems of high glass transition temperature, such as trehalose. The low glass transition temperature sorbitol produces almost equal process and storage stability in this case.
Keywords: Immunoglobulin; Spray-drying; Stabilization; Sorbitol; Trehalose; Water replacement;

Novel d-penicillamine carrying nanoparticles for metal chelation therapy in Alzheimer's and other CNS diseases by Zhengrong Cui; Paul R. Lockman; Craig S. Atwood; Cheng-Hsuan Hsu; Anshul Gupte; David D. Allen; Russell J. Mumper (263-272).
Metal ions accumulate in the brain with aging and in several neurodegenerative diseases. Aside from the copper storage disease, Wilson's disease, recent attention has focused on the accumulation of zinc, copper and iron in the Alzheimer's disease (AD) brain and the accumulation of iron in Parkinson's disease. In particular, the parenchymal deposition of β-amyloid (Aβ) and its interaction with metal ions has been postulated to play a role in the progression of AD. Thus, the strategy of lowering brain metal ions and targeting the interaction of Aβ peptide and metal ions through the administration of chelators has merit. Our recent finding that nanoparticle delivery systems can cross the blood–brain barrier has led us to investigate whether chelators delivered conjugated to nanoparticles could act to reverse metal ion induced protein precipitation. In the present studies, the Cu (I) chelator d-penicillamine was covalently conjugated to nanoparticles via a disulfide bond or a thioether bond. Nanoparticle-chelator conjugates were stable between pH 6–8 in aqueous suspension if stored at 4 °C, and did not aggregate when challenged with salts and serum. Release of d-penicillamine from the nanoparicles was achieved using reducing agents such as dithiothreitol (as a model for glutathione). Nanoparticles treated only under reducing conditions that released the conjugated d-penicillamine were able to effectively resolubilize copper–Aβ (1–42) aggregates. These results indicate that nanoparticles have potential to deliver d-penicillamine to the brain for the prevention of Aβ (1–42) accumulation, as well as to reduce metal ion accumulation in other CNS diseases.
Keywords: Wilson's disease; Alzheimer's disease; Metal chelation; β-Amyloid; Copper; Aggregates; Resolubilization;

A multi-particulate formulation of F4 fimbriae was developed for oral vaccination of suckling piglets against enterotoxigenic Escherichia coli infections. A feasibility test showed that incorporation of F4 fimbriae in a disintegrating pellet formulation consisting of 87.5% Pharmatose® 200 M, 2.5% Avicel® CL 611 and 10% Explotab® by extrusion/spheronisation and subsequent fluid bed drying resulted in the maintenance of 69±12% of the biological activity. But subsequent coating resulted in pellets with poor enteric properties, although good in vivo immunising results were obtained after administration to piglets. From the economical point of view, a pellet formulation was optimised to decrease vaccine dose and dosing frequency. After disintegration testing, pellets consisting of lactose (α-lactose monohydrate 90 mesh/β-lactose 75/25 (w/w)) and microcrystalline cellulose in a ratio of 80/20 (w/w) showed a sponge-like structure from which F4 fimbriae could be released. Coating of these pellets resulted in good enteric properties. To improve disintegrating properties of the pellets, the lactose concentration was increased or sodium carboxymethyl starch was added. But this resulted in poor enteric properties after coating. Dissolution test showed that F4 fimbriae were released from the optimised enteric-coated pellets but interaction between F4 fimbriae and the coating polymer was seen. This incompatibility leads to unpredictable in vitro quantification of F4 biological activity.
Keywords: Pellets; Enteric coating; Oral vaccination; F4 fimbriae; Piglet;

Studies on a new device for drug delivery to the eye by Rachel T. Pijls; Trudi Sonderkamp; Gert W. Daube; Ralph Krebber; Hans H.L. Hanssen; Rudy M.M.A. Nuijts; Leo H. Koole (283-288).
Delivery of drugs to the anterior side of the eye is routinely done with eye drops, but this method results in low bioavailability and low patient compliance. Herein, we describe a new device for the delivery of drugs to the eye. The device, called the OphthaCoil, consists of a drug-loaded adherent hydrogel coating on a thin metallic wire, which is coiled. The drug release rates of the dye fluorescein and the antibiotic chloramphenicol have already been evaluated in vitro. In this report the drug release rate of the anti-infective pradofloxacin was evaluated in vitro and in vivo. The data show that the OphthaCoil is capable of sustained drug delivery to the tear film in dogs. Drug levels in the tear fluid of the dogs were well above the MIC-values of relevant bacteria after 16 h, but it should be noted that pradofloxacin has an exceptionally high antimicrobial activity. The study indicates that the OphthaCoil holds promise as a platform for sustained release of drugs to the eye. The device was well tolerated, but the devices were lost when left overnight. Most probably, this is due to the third eyelid pushing the device out of the conjunctival sac during sleep. It should be noted that this complication has no immediate implication for extended wear of the OphthaCoil in humans, as humans do not have third eyelids.
Keywords: Drug delivery; Hydrogel; Pradofloxacin; In vivo; Ophthalmology;

Application of an electronic nose system for evaluation of unpleasant odor in coated tablets by Shinji Ohmori; Yasuo Ohno; Tadashi Makino; Toshio Kashihara (289-297).
The purpose of this study was to apply an electronic nose system for evaluation of unpleasant odor in tablets containing l-cysteine, an unpleasant odor drug, and demonstrate the odor masking ability of thin-layer sugarless coated tablets, which we have newly developed, by both electronic nose system and sensory evaluations. We demonstrated the qualitative evaluation of the unpleasant odor using air as a reference indicator and the quantitative evaluation of the unpleasant odor using the distances between air and samples in the electronic nose system evaluation. The electronic nose system evaluation was positively and well-correlated with the sensory evaluation by volunteers. We suggest that the electronic nose system evaluation is appropriate as an alternative or a support method for sensory evaluation by volunteers. As the results of both electronic nose system and sensory evaluations, we demonstrated that the thin-layer sugarless coated tablets have excellent masking ability of the unpleasant odor, equivalent to that of sugar-coated tablets due to the dense coating layers.
Keywords: Electronic nose system; Odor; Sensory evaluation; l-cysteine; Air; Distance; Thin-layer sugarless coated tablets;

Many pharmaceutical tablets are based on hydrophilic polymers, which, after exposure to water, form a gel layer around the tablet that limits the dissolution and diffusion of the drug and provides a mechanism for controlled drug release. Our aim was to determine the thickness of the swollen gel layer of matrix tablets and to develop a method for calculating the polymer concentration profile across the gel layer. MR imaging has been used to investigate the in situ swelling behaviour of cellulose ether matrix tablets and NMR spectroscopy experiments were performed on homogeneous hydrogels with known polymer concentration. The MRI results show that the thickest gel layer was observed for hydroxyethylcellulose tablets, followed by definitely thinner but almost equal gel layer for hydroxypropylcellulose and hydroxypropylmethylcellulose of both molecular weights. The water proton NMR relaxation parameters were combined with the MRI data to obtain a quantitative description of the swelling process on the basis of the concentrations and mobilities of water and polymer as functions of time and distance. The different concentration profiles observed after the same swelling time are the consequence of the different polymer characteristics. The procedure developed here could be used as a general method for calculating polymer concentration profiles on other similar polymeric systems.
Keywords: Matrix tablet; Swelling; Gel layer; Polymer concentration profile; MRI; NMR; Cellulose ether;

pH- and temperature-sensitive release behaviors from polyelectrolyte complex films composed of chitosan and PAOMA copolymer by Toru Yoshizawa; Yoshitsune Shin-ya; Kyung-Jin Hong; Toshio Kajiuchi (307-313).
This paper describes the pH and temperature effects on drug release from polyelectrolyte complex (PEC) films composed of a cationic polymer, chitosan, and an anionic polymer, polyalkyleneoxide–maleic acid copolymer (PAOMA). In this study, we prepared and investigated PEC films in terms of the drug release properties as pH- and temperature-sensitive drug carriers. Drug release rates were tested at pH 3.8 and 7.2, and at 25 and 50 °C. Salicylic acid and phenol were selected as model drugs. An increase in pH from 3.8 to 7.2 resulted in an increase in the rate of drug release because of the repulsive forces between carboxyl groups in PAOMA and anionic groups in model drugs. When the hydrophobic PAOMA was used as a polyanion, the drug release rate increased at 50 °C. This is attributed to the increase of release area due to the phase transition of PAOMA and the increase of repulsive forces between carboxyl groups in PAOMA and anionic groups in model drugs.
Keywords: Chitosan; Drug release; Polyalkyleneoxide–maleic acid copolymer; pH- and temperature-sensitivities; Polyelectrolyte complex film;

The purpose of this research was to correlate the state of agglomeration determined by the modeling of dissolution and de-agglomeration profiles, using mixtures of micronised indomethacin designed to have different dissolution rates and extents of particle agglomeration in dissolution media. Dissolution profiles were determined using the USP paddle method. De-agglomeration profiles were obtained from laser diffraction particle sizing of mixtures of indomethacin in dissolution media under non-sink conditions. Data were modeled and key parameters estimated using a non-linear least squares estimation algorithm. The key parameters of initial apparent volume concentrations as dispersed and agglomerated particles, and dissolution rate constants (for dissolution modeling), and the apparent volume concentrations of dispersible and non-dispersible agglomerates and the de-agglomeration rate constant (for de-agglomeration modeling) were related to indomethacin and sodium lauryl sulphate concentrations in the lactose–povidone mixtures. Micronised sodium lauryl sulphate added to the mixture was more effective in de-agglomeration than equivalent concentrations in the dissolution media. An excellent correlation existed between the total initial apparent volume concentration of agglomerates determined by dissolution and de-agglomeration (P=0.98). The use of key parameters estimated from the modeling of dissolution and de-agglomeration profiles provides a useful tool in dosage form development of formulations of poorly water soluble drugs.
Keywords: Dissolution modeling; De-agglomeration; Non-linear least squares parameter estimation; Laser diffraction particle sizing; Poorly water soluble drugs; Indomethacin;

A novel isothermal titration calorimetry method was used to determine the complexation thermodynamics for hydroxypropyl-β-cyclodextrin with artemisinin and naproxen at varying temperature and pH. The new method is very useful for studying complexation reactions between cyclodextrin and drugs with poor solubility and all the thermodynamic parameters of the cyclodextrin complexation were determined. The analysis of the thermodynamic data reveals involvement of hydrophobic bonding in the cyclodextrin complexes studied. The data also reveals the presence of enthalpy–entropy compensation in the system and provide information as to the orientation of the drug molecule inside the cyclodextrin cavity. From the thermodynamic parameters for dissociation of HPBCD complexes of artemisinin and naproxen at pH 2 it is concluded that the complexation is primarily driven by enthalpy with entropic assistance at all temperatures studied. From the dissociation studies of HPBCD complexes of naproxen at pH 10 it is concluded that the complexation is predominantly driven by entropy and moderately by enthalpy at lower temperatures and by enthalpy with entropic assistance at higher temperatures.
Keywords: Isothermal titration calorimetry; Complexation thermodynamics; Enthalpy–entropy compensation; Hydroxypropyl-β-cyclodextrin; Artemisinin; Naproxen;

Viscoelastic and structural properties of pharmaceutical hydrogels containing monocaprin by Thórunn Ó. Thorgeirsdóttir; Anna-Lena Kjøniksen; Kenneth D. Knudsen; Thórdis Kristmundsdóttir; Bo Nyström (333-342).
The purpose of this study was to characterize the rheological and structural properties of a pharmaceutical multicomponent hydrogel formulation. This formulation consists of a hydrogel-forming poly(acrylic acid) polymer (Carbopol® 974P), microbicide (monocaprin), non-ionic surfactant (Tween® 20 or Tween® 40), and preservatives. The effects of surfactant addition, monocaprin concentration, and pH on the formulation are investigated with the aid of various rheological methods and small angle neutron scattering (SANS). A change of pH from 4 (reduced electrostatic interactions) to higher pH values (prominent electrostatic forces) resulted in a marked impact on both the structural and rheological characteristics, with higher values of the dynamic moduli. At pH 4, the rheological features were strongly influenced by the addition of surfactant and monocaprin concentration, whereas at higher values of pH the effect of additives was modest. The picture that emerges from SANS and rheology is that enhanced association structures evolve at pH 4, while at higher pH the tendency to form associations is inhibited. At all the conditions, the rheological results suggest a viscoelastic solid behavior, which is typical for many gels.
Keywords: Monocaprin; Hydrogel; Rheology; SANS; Pharmaceutical formulation; Drug;

Physical solid-state properties and dissolution of sustained-release matrices of polyvinylacetate by Gelsys Ananay Gonzalez Novoa; Jyrki Heinämäki; Sabir Mirza; Osmo Antikainen; Antonio Iraizoz Colarte; Alberto Suzarte Paz; Jouko Yliruusi (343-350).
Solid-state compatibility and in vitro dissolution of direct-compressed sustained-release matrices of polyvinylacetate (PVAc) and polyvinylpyrrolidone (PVP) containing ibuprofen as a model drug were studied. Polyvinylalcohol (PVA) was used as an alternative water-soluble polymer to PVP. Differential scanning calorimetry (DSC) and powder X-ray diffractometry (PXRD) were used for characterizing solid-state polymer–polymer and drug–polymer interactions. The mechanical treatment for preparing physical mixtures of polyvinyl polymers and the drug (i.e. simple blending or stressed cogrinding) was shown not to affect the physical state of the drug and the polymers. With the drug–polymer mixtures the endothermic effect due to drug melting was always evident, but a considerable modification of the melting point of the drug in physical binary mixtures (drug:PVP) was observed, suggesting some interaction between the two. On the other hand, the lack of a significant shift of the melting endothermic peak of the drug in physical tertiary drug–polymer mixtures revealed no evidence of solid-state interaction between the drug and the present polymers. Sustained-release dissolution profiles were achieved from the direct-compressed matrices made from powder mixtures of the drug and PVAc combined with PVP, and the proportion of PVAc in the mixture clearly altered the drug release profiles in vitro. The drug release from the present matrix systems is controlled by both diffusion of the drug through the hydrate matrix and the erosion of the matrix itself.
Keywords: Sustained-release matrix; Polyvinylacetate; Polyvinylpyrrolidone; Ibuprofen; Solid-state compatibility; Dissolution;

Use of roughness maps in visualisation of surfaces by Paulus Seitavuopio; Jukka Rantanen; Jouko Yliruusi (351-358).
In this study we will present a new method to describe surface roughness. This method builds a roughness map of the studied area. The roughness map can give information of localised roughness. The test surfaces used in the evaluation of the method were tablets, which were made of lactose monohydrate, theophylline anhydrate, sodium chloride and potassium chloride. The roughness determinations were made by a laser profilometer. The new matrix method gives detailed roughness maps, which are able to show local variations in surface roughness values and provide an illustrative picture of the heterogeneity of surface roughness of various materials.
Keywords: Laser profilometer; Scanning electron microscopy (SEM); Tablet; Surface; Roughness; Roughness map;

Targeting of sterically stabilised pH-sensitive liposomes to human T-leukaemia cells by Cristina Fonseca; João N. Moreira; Carlos J. Ciudad; Maria C. Pedroso de Lima; Sérgio Simões (359-366).
The main aim of this work was to develop novel targeted sterically stabilised pH-sensitive liposomes tailored to promote efficient intracellular delivery of therapeutic molecules into human T-leukaemia cells. Our results indicate that the targeting moiety (thiolated transferrin) was successfully coupled to the distal reactive maleimide terminus of poly(ethylene glycol)–phospholipid conjugates incorporated in the liposomal bilayer. Results from atomic force microscopy studies, performed to characterise vesicle surface topology, indicated that, to a certain extent, thiolated transferrin has the ability to associate in a non-specific manner with the lipid membrane of pegylated liposomes. This is an issue not commonly reported in the literature but which is crucial to demonstrate the targeting proof of principle. Nevertheless, fluorimetric studies together with confocal microscopy clearly demonstrate that liposomes bearing covalently coupled transferrin associate more extensively to human T-leukaemia cells in vitro than non-targeted liposomes. Cell mechanistic studies indicate that targeted liposomes bind specifically to transferrin receptors and are internalised via receptor-dependent endocytotic pathway. In addition, the biophysical features exhibited by the developed liposomes, namely their ability to promote pH-triggered cytoplasmic delivery of loaded material, make them promising delivery systems for in vivo targeting of therapeutic molecules to tumours.
Keywords: Nanotechnology; pH-sensitive liposomes; Targeting; Transferrin; Human T-leukaemia cells; Cancer therapy;

Observations in simultaneous microencapsulation of 5-fluorouracil and leucovorin for combined pH-dependent release by Alf Lamprecht; Hiromitsu Yamamoto; Hirofumi Takeuchi; Yoshiaki Kawashima (367-371).
5-Fluorouracil (5-FU) in combination with leucovorin (LV) is nowadays the standard treatment in colon cancer and would be a candidate to be delivered orally to the colon. Eudragit P-4135F or Eudragit RS100 were used separately to prepare microspheres by an oil/oil emulsification process trapping 5-FU and LV simultaneously. Scanning electron microscopy permitted a structural analysis, process parameters were analyzed and drug loading and release profiles were recorded. Particle size varied between 123 (RS100) and 146 μm (P-4135F). Generally, higher encapsulation rates were found with RS100 (5-FU, 60.3±9.7%; LV, 81.4±8.6%) compared to P-4135F (5-FU, 48.3±2.0%; LV, 55.4±2.7%). Microparticles made from Eudragit RS100 released the incorporated drug combination within 8 h not exhibiting general differences between the kinetics of both drugs. P-4135F was found to maintain the undesired 5-FU release at pH 6.8 lower than 25% within 4 h while at pH 7.4, a nearly immediate release (within 15 min) was observed. Although the release was similar at pH 7.4, at pH 6.8 LV showed a distinct initial drug loss of about 60% and a complete release within 2 h. SEM analyses revealed a substantial presence of LV crystals on the particle surface provoking a distinct burst effect of LV. These observations were concluded to be related to the high lipophilicity of P-4135F provoking a separation between P-4135F and LV during the preparation process.
Keywords: Colon delivery; Colorectal cancer; Microspheres; 5-Fluorouracil; Leucovorin;