International Journal of Pharmaceutics (v.311, #1-2)

Alginate microspheres prepared by internal gelation: Development and effect on insulin stability by Catarina M. Silva; António J. Ribeiro; Isabel Vitória Figueiredo; Alexandra Rocha Gonçalves; Francisco Veiga (1-10).
Recombinant human insulin was encapsulated within alginate microspheres by the emulsification/internal gelation technique with the objective of preserving protein stability during encapsulation procedure. The influence of process and formulation parameters was evaluated on the morphology and encapsulation efficiency of insulin. The in vitro release of insulin from microspheres was studied under simulated gastrointestinal conditions and the in vivo activity of protein after processing was assessed by subcutaneous administration of extracted insulin from microspheres to streptozotocin-induced diabetic rats. Microspheres mean diameter, ranging from 21 to 287 μm, decreased with the internal phase ratio, emulsifier concentration, mixer rotational speed and increased with alginate concentration. Insulin encapsulation efficiency, near 75%, was not affected by emulsifier concentration, mixer rotational speed and zinc/insulin hexamer molar ratio but decreased either by increasing internal phase ratio and calcium/alginate mass ratio or by decreasing acid/calcium molar ratio and alginate concentration. A high insulin release, above 75%, was obtained at pH 1.2 and under simulated intestinal pH a complete dissolution of microspheres occurred. Extracted insulin from microspheres decreased hyperglycemia of diabetic rats proving to be bioactive and showing that encapsulation in alginate microspheres using the emulsification/internal gelation is an appropriate method for protein encapsulation.
Keywords: Alginate; Bioactivity; Insulin; Internal gelation; Microspheres;

Niosomes as carriers for tretinoin by Maria Manconi; Chiara Sinico; Donatella Valenti; Francesco Lai; Anna M. Fadda (11-19).
The influence of drug thermodynamic activity and niosome composition, size, lamellarity and charge on the (trans)dermal delivery of tretinoin (TRA) was studied. For this purpose, tretinoin was incorporated at saturated and unsaturated concentrations in both multilamellar (MLV) and unilamellar (UV) vesicular formulations using two different commercial mixtures of alkyl polyglucosides: octyl-decyl polyglucoside and decyl polyglucoside. Positively and negatively charged vesicular formulations were prepared using either stearylamine or dicetylphosphate as a charge inducer. Niosomes made with polyoxyethylene (4) lauryl ether and liposomes made with soy phosphatidylcholine were also prepared and studied. Vesicular formulations were characterised by transmission electron microscopy and optical and light polarized microscopy for vesicle formation and morphology, and by dynamic laser light scattering for size distribution. The effect of the vesicular incorporation of tretinoin on its (trans)dermal delivery through the newborn pig skin was also investigated in vitro using Franz cells, in comparison with a commercial formulation of the drug (RetinA®). The amount of tretinoin delivered through and accumulated in the several skin layers was detected by HPLC. Overall, obtained results showed that tretinoin cutaneous delivery is strongly affected by vesicle composition and thermodynamic activity of the drug. In particular, small, negatively charged niosomal formulations, which are saturated with tretinoin, have shown to give higher cutaneous drug retention than both liposomes and commercial formulation. Moreover, interactions between skin and vesicles seem to depend on physico-chemical properties of the main component of the vesicular bilayer.
Keywords: Niosomes; Non-ionic surfactants; Alkylpolyglucosides; Tretinoin; In vitro skin permeation; In vitro skin delivery;

Esophageal transit of the weekly film-coated risedronate (Actonel®) placebo tablet in subjects with Kyphosis by Alan C. Perkins; Malcolm Frier; P. Elaine Blackshaw; Robin C. Spiller; K. Julia Fairbairn; Richard J. Dansereau; Thomas Kinghorn; Pat San; David Hosking (20-25).
Risedronate sodium is a pyridinyl bisphosphonate of proven effectiveness for the treatment and prevention of osteoporosis and Paget's disease of the bone. The aim of this study was to compare the esophageal transit and gastric emptying of the placebo film-coated risedronate tablet when taken with 50 or 120 mL of water in subjects with Kyphosis. A total of 23 patients with radiologically documented osteoporosis participated in a single-center, open-label, crossover gamma scintigraphy study. The mean esophageal transit times were 15.6 s (50 mL) and 12.0 s (120 mL) and the mean gastric emptying half-times were 20.5 min (50 mL) and 14.3 min (120 mL). There was no relationship between the degree of Kyphosis measured from lateral standing radiographs and the esophageal transit time. This study demonstrated that even when taken with a minimal volume of water the esophageal transit and gastric emptying of the film-coated 35 mg weekly risedronate placebo tablet was similar in kyphotic subjects to previously obtained results from healthy control subjects.
Keywords: Risedronate; Esophageal transit; Scintigraphy; Kyphosis;

The compression and compaction properties of plasticised high molecular weight USP2208 HPMC were investigated with the aim of improving tablet formation in HPMC matrices. Experiments were conducted on binary polymer–plasticiser mixtures containing 17 wt.% plasticiser, and on a model hydrophilic matrix formulation. A selection of common plasticisers, propylene glycol (PG) glycerol (GLY), dibutyl sebacate (DBS) and triacetin (TRI), were chosen to provide a range of plasticisation efficiencies. T g values of binary mixtures determined by Dynamic Mechanical Thermal Analysis (DMTA) were in rank order PG > GLY > DBS > TRI > unplasticised HPMC. Mean yield pressure, strain rate sensitivity (SRS) and plastic compaction energy were measured during the compression process, and matrix properties were monitored by tensile strength and axial expansion post-compression. Compression of HPMC:PG binary mixtures resulted in a marked reduction in mean yield pressure and a significant increase in SRS, suggesting a classical plasticisation of HPMC analogous to that produced by water. The effect of PG was also reflected in matrix properties. At compression pressures below 70 MPa, compacts had greater tensile strength than those from native polymer, and over the range 35 and 70 MPa, lower plastic compaction values showed that less energy was required to produce the compacts. Axial expansion was also reduced. Above 70 MPa tensile strength was limited to 3 MPa. These results suggest a useful improvement of HPMC compaction and matrix properties by PG plasticisation, with lowering of T g resulting in improved deformation and internal bonding. These effects were also detectable in the model formulation containing a minimal polymer content for an HPMC matrix. Other plasticisers were largely ineffective, matrix strength was poor and axial expansion high. The hydrophobic plasticisers (DBS, TRI) reduced yield pressure substantially, but were poor plasticisers and showed compaction mechanisms that could be attributed to phase separation. The effect of different plasticisers suggests that the deformation characteristics of this HPMC in the solid state is dominated by hydroxyl mediated bonding, rather than by hydrophobic interactions between methoxyl-rich regions.
Keywords: Hydroxypropylmethylcellulose; HPMC; Plasticiser; Matrix; Compaction; Mechanical properties;

Elimination of metformin–croscarmellose sodium interaction by competition by W.X. Huang; M. Desai; Q. Tang; R. Yang; R.V. Vivilecchia; Y. Joshi (33-39).
During analytical method development and validation, a strong charge interaction between metformin and croscarmellose sodium was observed when the aqueous solution containing metformin was spiked with croscarmellose sodium. The charge interaction resulted in the retention of metformin in croscarmellose sodium and caused a serious drug recovery problem. The percent recovery of metformin in the solution was much lower than its theoretical values, especially in the low metformin concentration range. To overcome the metformin–croscarmellose interaction, arginine was selected as a competitor for the binding sites on croscarmellose sodium. Because of the competition and stronger interaction between arginine and croscarmellose sodium than metformin and croscarmellose sodium, a complete recovery of metformin in presence of arginine in both low and high concentration ranges was achieved. The effect of arginine on the recovery of metformin and the competition mechanism are discussed in this paper.
Keywords: Metformin; Croscarmellose sodium; Arginine; Drug–excipient interaction; Competitor–excipient interaction;

The objective of this study was to develop a poly(d,l-lactic-co-glycolic acid) (PLGA)-based coating system for producing biologically-inspired delivery profiles. Protein-loaded microspheres were made from PLGA (50:50) terminated with carboxylic acid groups (PLGA-2A) blended either with more hydrophobic PLGA (50:50) having lauryl ester endcaps (PLGA-LE) or with the more hydrophilic Pluronic F-127 (PF-127). Dense coatings were formed by pressure-sintering the microspheres. Altering hydrophobicity changed the water concentration within coatings, and consequently the time to onset of polymer degradation and protein release was modulated. After blending up to 8% Pluronic, degradation by-products began accumulating immediately upon incubation in saline, whereas, degradation was delayed for up to 14 days with blending of up to 30% PLGA-LE. Primary protein release peaks from one-layer coatings could be created from 7 to 20 days using 8% PF-127 or 30% PLGA-LE blends, respectively. Multilayered coatings of different blends generated several release peaks, with their temporal occurrence remaining approximately the same when layers of other hydrophobicity were added above or below. To allow design of coatings for future use, results were used to construct a model based on Fourier analysis. This polymer blend system and model can be used to mimic temporally varying profiles of protein expression.
Keywords: Polymer blending; PLGA; Protein release; Multilayered; Biodegradable;

Preparation of ethyl cellulose/methyl cellulose blends by supercritical antisolvent precipitation by Ana Rita C. Duarte; M.D. Gordillo; M. Margarida Cardoso; Ana Luísa Simplício; Catarina M.M. Duarte (50-54).
The supercritical antisolvent (SAS) technique was used to prepare ethyl cellulose/methyl cellulose blends, two biocompatible polymers commonly used as drug carriers in controlled delivery systems. Ethyl cellulose is widely used as a drug carrier. The drug release of the delivery devices can be controlled to some extent by addition of a water-soluble or water swellable polymer, such as methyl cellulose. This leads to the solubility enhancement of poorly water-soluble molecules. SAS experiments were carried out at different operational conditions and microspheres with mean diameters ranging from 5 to 30 μm were obtained. The effect of CO2 and liquid flow, temperature and pressure on particle size and particle size distribution was evaluated. The microspheres were precipitated from a mixture of dichloromethane (DCM) and dimethylsulfoxide (DMSO) (4:1 ratio). The best process conditions for this mixture were according to our study 40 °C and 80 bar.
Keywords: Biocompatible polymers; Micronization; Supercritical fluid antisolvent (SAS); Release systems;

The objective of this study was to evaluate the transdermal delivery potential of diclofenac-containing microemulsion system in vivo and in vitro. It was found that the transdermal administration of the microemulsion to rats resulted in 8-fold higher drug plasma levels than those obtained after application of Voltaren Emulgel. After s.c. administration (3.5 mg/kg), the plasma levels of diclofenac reached a peak of 0.94 μg/ml at t  = 1 h and decreased rapidly to 0.19 μg/ml at t  = 6 h, while transdermal administration of the drug in microemulsion maintained constant levels of 0.7–0.9 μg/ml for at least 8 h. The transdermal fluxes of diclofenac were measured in vitro using skin excised from different animal species. In three rodent species, penetration fluxes of 53.35 ± 8.19 (furry mouse), 31.70 ± 3.83 (hairless mouse), 31.66 ± 4.45 (rat), and 22.89 ± 6.23 μg/cm2/h (hairless guinea pig) were obtained following the application of the microemulsion. These fluxes were significantly higher than those obtained by application of the drug in aqueous solution. In contrast to these results, a ‘flip-flop’ phenomenon was observed when frozen porcine skin (but not fresh skin) was significantly more permeable to diclofenac-in-water than to the drug-in-microemulsion. In fact, the drug penetration from the microemulsion was not affected by the skin storage conditions, but it was increased when an aqueous solution was applied. However, this unusual phenomenon observed in non-freshly used porcine skin places a question mark on its relevancy for in vitro penetration studies involving aqueous vehicle systems.
Keywords: Microemulsion; Transdermal drug delivery; Percutaneous penetration; Porcine skin model; Diclofenac;

The pharmacokinetic parameters, i.e. peak concentration (c max); peak time (t max); area under the curve (AUC); elimination rate constant (k); absorption rate constant (k a); Drug clearance (clt), and the volume of distribution ( v d ) of sodium salicylate administered in fractionated coconut oil (FCO) have been compared with that from an aqueous and glycerin vehicles using a three-way crossover study in 12 rabbits. The results of the study show that all of the pharmacokinetic parameters tested differ significantly when administered in oily rather than aqueous or glycerin vehicles. No statistically significant difference was found between any of the above mentioned parameters when comparison was made between aqueous and glycerin formulations. The results indicate that sodium salicylate is absorbed at a lower rate but to a greater extent from oily formulation. Possible reasons for these differences are discussed and is suggested, therefore, that the oily formulation might be used as a sustained release preparation.
Keywords: Gastric emptying rate (GER); Fractionated coconut oil (FCO); Drug clearance (clt); Volume of distribution ( v d ) ; Elimination rate constant (k); Absorption rate constant (k a); Area under the curve (AUC);

Production of GMP-grade radioactive holmium loaded poly(l-lactic acid) microspheres for clinical application by S.W. Zielhuis; J.F.W. Nijsen; R. de Roos; G.C. Krijger; P.P. van Rijk; W.E. Hennink; A.D. van het Schip (69-74).
Radioactive holmium-166 loaded poly(l-lactic acid) microspheres are promising systems for the treatment of liver malignancies. The microspheres are loaded with holmium acetylacetonate (HoAcAc) and prepared by a solvent evaporation method. After preparation, the microspheres (Ho-PLLA-MS) are activated by neutron irradiation in a nuclear reactor. In this paper, the aspects of the production of a (relatively) large-scale GMP batch (4 g, suitable for treatment of 5–10 patients) of Ho-PLLA-MS are described.The critical steps of the Ho-PLLA-MS production process (sieving procedure, temperature control during evaporation and raw materials) were considered and the pharmaceutical quality of the microspheres was evaluated.The pharmaceutical characteristics (residual solvents, possible bacterial contaminations and endotoxins) of the produced Ho-PLLA-MS batches were in compliance with the requirements of the European Pharmacopoeia. Moreover, neutron irradiated Ho-PLLA-MS retained their morphological integrity and the holmium remained stably associated with the microspheres; it was observed that after 270 h (10 times the half-life of Ho-166) only 0.3 ± 0.1% of the loading was released from the microspheres in an aqueous solution.In conclusion, Ho-PLLA-MS which are produced as described in this paper, can be clinically applied, with respect to their pharmaceutical quality.
Keywords: Holmium; Microspheres; PLLA; GMP; CFU; Endotoxins; Stability;

Study of the critical points of HPMC hydrophilic matrices for controlled drug delivery by Antonia Miranda; Mónica Millán; Isidoro Caraballo (75-81).
The knowledge of the percolation thresholds of a system results in a clear improvement of the design of controlled release dosage forms such as inert matrices. Despite hydrophilic matrices are one of the most used controlled delivery systems in the world, but actuality, the mechanisms of drug release from these systems continue to be a matter of debate nowadays. The objective of the present paper is to apply the percolation theory to study the release and hydration rate of hydrophilic matrices. Matrix tablets have been prepared using KCl as a drug model and HPMC K4M as matrix-forming material, employing five different excipient/drug particle size ratios (ranging from 0.42 to 2.33). The formulations studied containing a drug loading in the range of 20–90% (w/w). Dissolution studies were carried out using the paddle method and the water uptake measurements were performed using a modified Enslin apparatus. In order to estimate the percolation threshold, the behaviour of the kinetic parameters with respect to the volumetric fraction of each component at time zero, was studied. The percolation theory has been applied for the first time to the study of matrix type controlled delivery systems. The application of this theory allowed to explain changes in the release and hydration kinetics of these matrices. The critical points observed in dissolution and water uptake studies can be attributed to the excipient percolation threshold, being this threshold one of the main factors governing the gel layer formation and consequently, the drug release control from hydrophilic matrices.
Keywords: Gel layer; HPMC; Swelling; Percolation theory; Percolation threshold;

Short (14–20-mer range) synthetic oligodeoxynucleotides (ODNs) allow specific modulation of cellular gene expression at various stages, thus providing a versatile tool for fundamental studies and a rational approach to anticancer chemotherapy. However, several problems, such as metabolic stability, efficient cell internalization of ODNs and their efficient entrapment into liposomes continue to markedly limit this approach. To improve the target specificity and biological activity of ODN, three different length of poly(l-lysine) (PLL) were conjugated to ODN and these conjugates were encapsulated in N-stearyllactobionamide (N-SLBA)-modified liposomes, N-SLBA is a ligand for the asialoglycoprotein receptor. Then, we investigated their effects on cell cycle and survivin protein levels of HepG2 cells. The results showed that the encapsulation efficiency was improved because the polycationic charges of PLL neutralized the polyanionic charges of ODN. Among them, PLL (M W 2000 and 10,000)-conjugated ODN encapsulated in N-SLBA liposomes induced apoptosis of HepG2 cells and highly inhibited survivin gene expression.
Keywords: ODN; PLL; N-SLBA liposomes; Survivin protein expression;

Batch grinding kinetics of Ethenzamide particles by fluidized-bed jet-milling by Tadashi Fukunaka; Boris Golman; Kunio Shinohara (89-96).
Ethenzamide solids as a representative active pharmaceutical ingredient (API) were batch-ground by means of a fluidized-bed jet-mill which is a relatively new equipment and promising for production in the pharmaceutical field. Thus, the characteristic grinding mechanism was investigated. As a result, the variation of the residual ratio with grinding time after milling was expressed simply by a mathematical model using only the first Kapur function, and it was consistent with experimental data satisfactorily. As the shape of the function was much different from that of inorganic compound and peculiar to API, a cubic function with respect to particle diameter was defined newly and well fitted to the experimental data. The function was also found to be affected by the operating parameters as the grinding gas pressure, the charge weight of raw material and the linear velocity at the grinding nozzle. According to the assessments of the breakage and the selection functions derived from the first Kapur function, it was found that the grinding mechanism of Ethenzamide particles was related with particle attrition mainly.
Keywords: Fluidized-bed jet-mill; Kinetics; Batch grinding; Mathematical model; Ethenzamide;

PLGA microcapsules with novel dimpled surfaces for pulmonary delivery of DNA by Farahidah Mohamed; Christopher F. van der Walle (97-107).
We describe the fabrication of DNA-loaded poly(lactic-co-glycolic acid) (PLGA) microcapsules with novel surface morphologies that will be of use in pulmonary delivery. Our approach was to examine surface morphology and DNA encapsulation efficiency as a function of primary emulsion stability; using two surfactant series based on hydrophile–lipophile balance and hydrophobe molecular weight. Hydrophilic non-ionic surfactants yielded the most stable water-in-dichloromethane emulsions (HLB values >8). These surfactants normally favor convex (o/w) interfacial curvatures and therefore this atypical behavior suggested a relatively high surfactant solvation in the dichloromethane ‘oil’ phase. This was consistent with the large fall in the glass transition temperature for microspheres prepared with Tween 20, which therefore efficiently penetrated the PLGA matrix and acted as a plasiticizer. Blends of Pluronic triblock copolymers performed poorly as water-in-dichloromethane emulsifiers, and were therefore used to generate hollow microspheres (‘microcapsules’) with low densities (0.24 g/cm3). Although the Pluronic-stabilized emulsions resulted in lower DNA loading (15–28%), microspheres (∼8 μm) with novel dimpled surfaces were fabricated. The depth and definition of the dimples was greatest for triblock copolymers with high MW hydrophobe blocks. By cascade impaction, the geometric mean weight diameter of the microcapsules was 3.43 μm, suggesting that they will be of interest as biodegradable pulmonary delivery vehicles.
Keywords: PLGA microcapsules; Non-ionic surfactants; Triblock copolymers; DNA encapsulation; Pulmonary delivery vehicles;

The extent of the protein binding of a drug is an important pharmacokinetic parameter. In this study, the determination of the protein binding of the antibiotics gatifloxacin, moxifloxacin, linezolid and telithromycin to bovine serum albumin (BSA) and human serum albumin (HSA) was performed by means of an automated continuous ultrafiltration method. The continuous ultrafiltration has several advantages compared to classical methods like equilibrium dialysis or discontinuous ultrafiltration. The method is appropriate for a fast determination of the extent of the protein binding of a drug. In one single experiment the calculation of the protein binding over a wide range of different drug/protein ratios is possible. Comparing the results obtained with the continuous ultrafiltration with binding data reported in literature shows good correlation and proves the reliability of the method.
Keywords: Protein binding; Automated continuous ultrafiltration; Antibiotics; HSA; BSA;

Antimycotic influence of β-cyclodextrin complexes—In vitro measurements using laser nephelometry in microtiter plates by Moustafa M.G. Fouda; Dierk Knittel; Uta-Christina Hipler; Peter Elsner; Eckhard Schollmeyer (113-121).
To determine the in vitro susceptibility of fungal organisms to β-cyclodextrin (CD) complexes with the antifungal agents econazole-nitrate (EC) and ciclopirox-olamine (CI), a fast, rapid and simple method using laser nephelometry in 96-microtiter plate is used. The antimycotic influence of the complexes against Candida albicans DSM 11225 and Candida krusei ATCC 6258 species was determined using this method. A rapid inhibition and even killing of both fungi was observed only above certain concentrations of complex ranged between 12.5 and 100 μg/ml for β-CD–econazole complex (CD–EC), while for the complex with ciclopirox-olamine (CD–CI) the range was between 150 and 400 μg/ml. The stability constants of the CD complexes with the two antimycotic derivatives are given. In addition, the nephelometric method allows the determination of solubilities of active agents. Thus, the improvement of solubility of both antimycotic agents in PBS buffer solution was observed by complexation with CD.
Keywords: β-Cyclodextrin; Econazole; Ciclopirox; Antimycotic; Laser nephelometry;

A depot drug delivery system, fibrin encapsulated liposome-in-chitosan matrix (FLCM), has been developed to deliver a water-soluble drug which is configured by a porous chitosan matrix containing a bovine fibrin network encapsulated different surface properties of liposomes. Quinacrine (QR), a water-soluble, low-molecular weight fluorescent marker, is used as a model drug to evaluate the delivery characteristics of the system.The SEM photographs show that the fibrin network adheres to the surfaces and pores of the chitosan matrix of a FLCM system. The QR release periods of the FLCM are sustained for about four times longer than those of QR encapsulated into the liposomes. However, the QR release periods and profiles of the FLCM are influenced by the surface properties of liposomes. The release of QR from FLCM is sustained for 9 days for neutral liposomes and only 5 days for PEG modified liposomes (PEG-liposome). After crosslinking the fibrin network of the FLCM with 0.5% of glutaldehyde, the release of QR is further sustained for 17 days with good linear profiles (e.g., 13 days) and with 50% of reduced burst release compared with those of without crosslinking, indicating that the stability of the fibrin network plays an important role on QR release of the system. More interestingly, the release periods and profiles of QR of the FLCM system are highly similar to those of Tirofiban, low-molecular weight of a water-soluble clinical cardiovascular drug, although the study has been done by human platelet poor plasma instead of bovine fibrinogen as a source of fibrin network. It suggests that the QR is a suitable model for investigating the drug delivery behaviors for water-soluble, low-molecular weight drugs of the FLCM. In conclusion, with QR as a model drug, FLCM with crosslinked fibrin network can effectively sustain the release of QR for 17 days but the release profiles are influenced by the surface properties of encapsulated liposomes. This study suggests that FLCM may have the potential as a depot drug delivery system for water-soluble drugs.
Keywords: Depot drug delivery system; Liposomes; Fibrin; Chitosan; Crosslinked;

Release behaviour and biocompatibility of drug-loaded pH sensitive particles by Oya Sipahigil; Ayla Gürsoy; Fulya Çakalağaoğlu; İmer Okar (130-138).
The purpose of this work was to investigate the physical properties of drug-loaded poly(methacrylic acid-g-ethylene glycol) {P(MAA-g-EG)} particles, their biocompatibility with the gastrointestinal tract of rats and also the effects of these particles on the tight junctions of the rat intestinal epithelium. Model drugs such as diltiazem HCl, diclofenac Na, ciprofloxacin HCl and isoniazid were used in this study. P(MAA-g-EG) particles were prepared by free radical solution polymerization of methacrylic acid (MAA) and poly(ethylene glycol) (PEG). The loading efficiency of the model drugs in the particles and in vitro release profiles were investigated in pH 7.4 phosphate buffer and in gradually pH changing buffers (pH 1.2, 5.8, 6.8 and 7.4). The stability of free particles and drug-loaded particles was established by Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC). In conclusion, P(MAA-g-EG) particles controlled the release rate of small molecular weight model drugs according to the pH of the medium. Stability of those particles loaded with drugs did not change in accelerated stability conditions. Histopathological results indicated that loading drugs to the particles prevented cell and tissue damage after 20 h. Free particles showed no change of tight junctions after 2 and 10 h. The results of TEM showed that increasing the amount of P(MAA-g-EG) particles from 100 to 385 mg clearly opened the tight junction, but with serious epithelial cell disruption.
Keywords: P(MAA-g-EG) particles; Histopathology; Tight junction; Diltiazem HCl; Diclofenac Na; Ciprofloxacin HCl; Isoniazid;

This is the second in a series of papers that report on the release and transport of a range of anionic drugs (diclofenac, salicylic acid) from liquid crystalline gels and ultimately on their use in transdermal delivery. The previous paper [Fitzpatrick, D., Corish, J., 2005. Release characteristics of anionic drug compounds from liquid crystalline gels for transdermal delivery. Part I. Passive release across non-rate limiting membranes. Int. J. Pharm. 301, 226–236] investigated passive release profiles across a non-rate-limiting membrane: here we report on the search for a suitable model enhancer (benzyl dimethyldodecyl ammonium bromide) for the transdermal delivery of anionic compounds. The results presented reveal a significant role for ion pairing and for buffering, analogous to those found in the investigations of cationic species (salbutamol) by Nolan, L.M.A., Corish, J., Corrigan, O.I., Fitzpatrick, D., 2003. Iontophoretic and chemical enhancement of drug delivery. Part I. Across artificial membranes. Int. J. Pharm. 12, 41–55. The method of vehicle preparation is also investigated. It is shown that ion pairing of the drug with the enhancer decreases the amount of drug available for transport from the liquid crystalline gels into aqueous receptor media. This decrease is directly related to the ratio of the concentration of drug to that of the enhancer. Buffering the vehicle inhibits the ion-pair formation to some extent. Vehicle preparation was also found to influence the degree of ion-pair association. The inclusion of a similarly charged enhancer (oleic acid) to the drug was found not to impede the diffusion of the drug from the gels.
Keywords: Diclofenac; Liquid crystalline gel; Transdermal; Buffering; Vehicle preparation; Enhancer;

In this investigation a novel oral pulsatile drug delivery system based on a core-in-cup dry coated tablet, where the core tablet surrounded on the bottom and circumference wall with inactive material, is proposed. The system consists of three different parts, a core tablet, containing the active ingredient, an impermeable outer shell and a top cover layer-barrier of a soluble polymer. The core contained either diclofenac sodium or ketoprofen as model drugs. The impermeable coating cup consisted of cellulose acetate propionate and the top cover layer of hydrophilic swellable materials, such as polyethylene oxide, sodium alginate or sodium carboxymethyl cellulose. The effect of the core, the polymer characteristics and quantity at the top cover layer, on the lag time and drug release was investigated. The results show that the system release of the drug after a certain lag time generally due to the erosion of the top cover layer. The quantity of the material, its characteristics (viscosity, swelling, gel layer thickness) and the drug solubility was found to modify lag time and drug release. The lag time increased when the quantity of top layer increased, whereas drug release decreased. The use of sodium carboxymethyl cellulose resulted in the greatest swelling, gel thickness and lag time, but the lowest drug release from the system. Polyethylene oxide showed an intermediate behaviour while, the sodium alginate exhibited the smallest swelling, gel thickness and the shortest lag time, but the fastest release. These findings suggest that drug delivery can be controlled by manipulation of these formulations.
Keywords: Pulsatile release tablets; Lag time; Diclofenac sodium; Ketoprofen; Swellable polymers; Erosion;

Limonene GP1/PG organogel as a vehicle in transdermal delivery of haloperidol by Perry Fung Chye Lim; Xiang Yang Liu; Lifeng Kang; Paul Chi Lui Ho; Yew Weng Chan; Sui Yung Chan (157-164).
Penetration enhancers are a classical means for improving transdermal drug delivery (TDD). Enhancers permeate into the skin and reversibly decrease the barrier resistance. Basically, our aim is to formulate a transdermal gel containing an appropriate enhancer for a controlled drug release. Terpenes, namely limonene, linalool and cineole, in propylene glycol (PG) were first investigated in vitro for their capacity to enhance the percutaneous release of an anti-psychotic drug, haloperidol (HP). Relative to oxygenated linalool and cineole, hydrocarbon limonene was more effective as a skin enhancer; it increased human skin permeability and decreased lag time. Limonene was thus incorporated in an organogel comprised of gelator GP1 and PG. This skin-friendly gel in a transdermal patch could act as a long-acting formulation that delivers HP at a sustained percutaneous rate. The microscopic framework of the organogel is a branched network of interlocking fibres. Varying the gelator content modulates the fibre density and gel stiffness, and presents different degrees of resistance to drug diffusion on the vehicle side. Rheological and permeation studies demonstrated that an increase in gelator concentration increased gel moduli and decreased drug flux simultaneously. The rheology of the gel matrix influenced drug release rate in a manner described by several experimentally-derived correlations.
Keywords: Penetration enhancers; Transdermal; Terpenes; Haloperidol; Organogel; Rheology;

Colloidal emulsion aphrons (CEAs) are considered as the micron-sized water-in-oil (W/O) emulsion-cores encapsulated by a “soapy shell” consisting of multi-layer surfactant molecules. In this dispersion, the emulsion-core sizes are mainly in 10–100 μm and that of the inner phase droplets are in 1–5 μm. CEAs not only behave analogously to emulsion liquid membrane (ELM) in extraction with advantages of high concentration ratio, counter-concentration extraction and combination of extraction with backwash together, but also have the large interface areas, easy scatteration and quick extraction which colloidal liquid aphrons (CLAs) possess. CEA extraction overcomes the restriction of partition equilibrium between the water and the oil phase that CLAs have. They have greater extraction capacity than CLAs. In this study, the application of CEAs to drug overdose treatment was studied using salicylic acid as the model drug, paraffin oil as the membrane phase, PEG-30 dipolyhydroxystearate (P135) as the hydrophobic surfactant, nonylphenol ethoxylate-10 (NP10) as the hydrophilic surfactant and NaOH solution as the receptor phase. Also some factors affecting the stability of this dispersion and extraction ratio were investigated. In order to prepare CEAs successfully, the concentrations of NP10 and P135 should be in 1.5–3.0% (w/v) and 0.25–1.0% (w/v), respectively, together with the ratio of the volume of oil phase to the volume of inner aqueous phase of CEAs, R oi  ≥ 1:1. For the extraction of salicylic acid, the pH value of the feed phase was supposed to be lower than 2.0 and the suitable NaOH concentration of the receptor phase was higher than 0.02 mol/L. Under this condition, more than 98.7% of salicylic acid was transported into receptor phase in half a minute.
Keywords: Colloidal emulsion aphron; Detoxification; Drug overdose; Extraction;

Development of liposomal capreomycin sulfate formulations: Effects of formulation variables on peptide encapsulation by Maurizio Ricci; Stefano Giovagnoli; Paolo Blasi; Aurelie Schoubben; Luana Perioli; Carlo Rossi (172-181).
The aim of this work was the investigation of the effects of preparation variables on drug content for the development of capreomycin sulfate (CS) liposomal formulations as potential aerosol antitubercular agents.Dipalmitoylphosphatidylcholine (DPPC), hydrogenated phosphatidylcholine (HPC) and distearoylphosphatidylcholine (DSPC) were used for liposome preparation. A freeze–thawing method was chosen for CS encapsulation. Peptide entrapment, size and morphology were evaluated by UV spectrophotometry, photocorrelation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. A 23 full factorial protocol was designed to evaluate the conditions for CS encapsulation improvement.Peptide content ranged between 1 and 8%. Vesicles showed a narrow size distribution, with average diameters around 1 μm and a good morphology. A mathematical model was generated for each liposomal system and check point analyses revealed good agreement between experimental and predicted values. DPPC liposomes were found to provide the highest CS content.Peptide content was successfully increased by assessing formulation variable effects using a 23 factorial design that proved to be a time saving method helpful in developing new CS liposomal formulations for a possible application in aerosol antitubercular therapies.
Keywords: Capreomycin sulfate; Liposomes; Freeze–thawing; Factorial design;

Effect of vehicles and enhancers on the topical delivery of cyclosporin A by Hongzhuo Liu; Sanming Li; Yongjun Wang; Huimin Yao; Yan Zhang (182-186).
Topical delivery of cyclosporin a (CysA) is of great interest for the treatment of autoimmune skin disorders. The purpose of this study was to investigate the effect of various vehicles and enhancers on the topical delivery across rat skin. The topical (to the skin) delivery of CysA was evaluated in vitro using rat skin mounted in a Franz diffusion cell. CysA was analyzed by UV-HPLC. As vehicles, CysA vehicle containing 40% ethanol showed significantly enhanced deposition of CysA into the stratum corneum (SC) and deeper skin, as compared to other vehicles. The efficiency of the vehicles to improve the topical delivery of CysA was sequenced in the order of: 40% ethanol > ethyl oleate > Transcutol > isopropyl myristate > ethanol > Labrasol > propylene glycol > Lauroglycol FCC. Next, we tested effect of pre-treatment with chemical enhancers on the penetration of CysA. The permeation-enhancer effect of enhancers was in the following order: 10% menthol ≈ 0.05% SLS > 5% Azone > 5% NMP > 5% DEMO. Moreover, chemical enhancers shortened the lag time of the penetration of CysA into deeper skin. The present study suggests that the suspension of 40% ethanol containing 0.5% drug can more effectively enhance the topical delivery of CysA after skin pre-treatment with 10% menthol or 0.05% SLS.
Keywords: Cyclosporin A; Vehicle; Enhancer; Pre-treatment; Topical;

Preparation and improvement of release behavior of chitosan microspheres containing insulin by Lian-Yan Wang; Yong-Hong Gu; Zhi-Guo Su; Guang-Hui Ma (187-195).
Chitosan microsphere has potential applications in orally and other mucosally administration of protein and peptide drug, because it shows excellent mucoadhesive and permeation enhancing effect across the biological surfaces. The control of the size and size distribution of chitosan microsphere is necessary in order to improve its reproducibility, bioavailability and repeatable release behavior. Furthermore, it is a big challenge how to maintain the chemical stability of protein drug and improve its release behavior in the preparation of chitosan microspheres, because conventional crosslinking method by glutaraldehyde cannot be used in encapsulation of protein drug containing amino group. In this study, we established a method to prepare uniform-sized microsphere, and solve above problems by combining a special membrane emulsification technique and a step-wise crosslinking method. The preparative condition was optimized, and the chemical stability of protein, encapsulation efficiency, and release behavior were compared with conventional preparative method of drug-loaded chitosan microspheres. As a result, fairly uniform chitosan microspheres were obtained with a coefficient of variation (C.V.) value less than 11%, and the step-wise crosslinking method developed specially for membrane emulsification method provided the microspheres with higher encapsulation efficiency (80%), higher chemical stability of insulin (>95%), lower burst release and steady release behavior.
Keywords: Chitosan microspheres; Uniform size; Membrane emulsification; Step-wise crosslinking; Insulin;

In vivo human buccal permeability of nicotine by Charlotte L. Adrian; Helle B.D. Olin; Kim Dalhoff; Jette Jacobsen (196-202).
The aim was to examine the in vivo buccal pH-dependent permeability of nicotine in humans and furthermore compare the in vivo permeability of nicotine to previous in vitro permeability data. The buccal permeability of nicotine was examined in a three-way cross-over study in eight healthy non-smokers using a buccal perfusion cell. The disappearance of nicotine from perfusion solutions with pH 6.0, 7.4, and 8.1 was studied for 3 h. The apparent permeability of nicotine (P app) was determined at each pH value. Parotid saliva was collected in an attempt to assess systemic levels of nicotine. The disappearance rate of nicotine increased significantly as the pH increased, which resulted in P app values of 0.57 ± 0.55 × 10−4, 2.10 ± 0.23 × 10−4, and 3.96 ± 0.54 × 10−4  cm s−1 (mean ± S.D.) at pH 6.0, 7.4, and 8.1, respectively. A linear relationship (R 2  = 0.993) was obtained between the P app values and non-ionised nicotine, which indicates that the nicotine transfer occurred by means of passive diffusion. P app values of 0.60 × 10−4 and 6.18 × 10−4  cm s−1 were obtained for the mono-protonated and non-ionised species of nicotine, respectively. The analysis of the parotid saliva samples indicated that these samples might be useful in the assessment of systemic absorption of nicotine. Previous buccal in vitro models underestimated the in vivo human permeability of nicotine. However, the in vitro models were capable of predicting the effect of pH on the nicotine permeability.
Keywords: Human buccal permeability; Perfusion cell; Nicotine; pH; In vivo; Saliva;

Sterility is required as stated by compendial requirements and registration authorities worldwide for an injectable drug carrier system. In this study, injectable nanospheres and nanocapsules prepared from amphiphilic β-cyclodextrin, β-CDC6, were assessed for their in vitro properties such as particle size distribution, zeta potential, nanoparticle yield (%), drug entrapment efficiency and in vitro drug release profiles. Different sterilization techniques such as gamma irradiation and autoclaving were evaluated for their feasibility regarding the maintenance of the above mentioned nanoparticle properties after sterilization. It was found that amount these techniques, sterilization with gamma irradiation seemed to be the most appropriate technique with no effect on particle size, drug loading and drug release properties. Gamma irradiation causes some chemical changes on β-CDC6 observed as changes in zeta potential but this does not lead to any significant changes for nanoparticle properties. Autoclaving caused massive aggregation for the nanoparticles followed by precipitation, which led to the conclusion that excessive heat disrupted nanoparticle integrity. Sterile filtration was not feasible since nanoparticle sizes were larger than the filter pore size and the yield after sterilization was very low. Thus, it can be concluded that blank and drug loaded β-CDC6 nanospheres and nanocapsules are capable of being sterilized by gamma irradiation.
Keywords: Amphiphilic cyclodextrin; Autoclaving; Gamma irradiation; Nanoparticle; Tamoxifen;

Our previous studies demonstrated that cationic nanoparticles composed of well-defined poly(methyl methacrylate) (PMMA) cores surrounded by a hairly poly(ethyleneimine) (PEI) shells have comparative advantages over the PEI system for gene delivery. In this study, we focused on the intracellular uptake and release of PEI–PMMA nanoparticle/pDNA complexes. The behavior of the nanoparticle/pDNA complexes in recipient cells was monitored by using confocal laser scanning microscopy. We found that the nanoparticle/pDNA complexes were internalized very effectively by endocytosis. In the recipient cells the nanoparticles were found localized in the cytoplasm. At the same time, the pDNA carried by the nanoparticles successfully detached from the nanoparticles and localized in the nucleus of the HeLa cells.
Keywords: Cationic PEI–PMMA nanoparticles; Nanoparticle/pDNA complexes; Intracellular behavior; Confocal laser scanning microscopy;

Nanoparticles comprising copolymers of aminoethylcarbamoyl-β-cyclodextrin (AEC-β-CD) and ethylene glycol diglycidyl ether (EGDGE) are prepared by an interfacial polyaddition reaction in a miniemulsion system. Polymers are formed in a W/O emulsion containing 0.25–10.0% (w/w) water and 5.0% (w/w) surfactant (MO-3S, tetraglycerin monoester, HLB 8.8), where simple particles are predominantly obtained when the water content is 1.0% and 5.0%. Notably, nano-size small particles (diameter: 0.3 μm) are formed under the condition of 5.0% water and 5.0% surfactant, which have the highest β-CD contents (75.5 wt.%) and the most positive ζ-potential (53.6 mV). The ζ-potential measurement indicates that the obtained particles have positive charge due to protonation of their amino groups below around pH 10. Actually, uptake of 8-anilino-1-naphthalenesulfonic acid (ANS) bearing negative charge (SO3 ) and moderate hydrophobicity depends on the magnitude of ζ-potential of the particles; viz., the particles with ζ-potential of 53.6 mV show the highest efficiency of uptake. The diameter and the β-CD contents are closely related with the water/surfactant ratio, and the ζ-potentials are dependent on both the diameter and the β-CD contents. Inclusion of ANS into the CD cavity of EGDGE/AEC-β-CD particles can be controlled by electrostatic interaction between ANS (negatively charged) and the particle (positively charged). Namely, synergistic effect of cavity-inclusion and electrostatic interaction can dominate the uptake of guest molecules by the particles.
Keywords: Cyclodextrin; Nanoparticle; Interfacial polyaddition reaction;

A new formulation method was developed for preparing poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles loaded with recombinant human granulocyte colony-stimulating factor (rhG-CSF). Lyophilized rhG-CSF powder and PLGA polymer were directly co-dissolved in a single organic phase, and the resulting solution was dispersed into an aqueous solution. PLGA nanoparticles encapsulating rhG-CSF were produced by a spontaneous emulsion/solvent diffusion method. In this manner, rhG-CSF was molecularly dissolved in the polymer phase. Release profile of rhG-CSF from PLGA nanoparticles was compared with those from two kinds of PLGA microparticles which were separately prepared by either single oil-in-water (O/W) or double water-in-oil-in-water (W/O/W) emulsion technique. The sizes of rhG-CSF loaded nanoparticles, O/W microparticles, and W/O/W microparticles were about 257 nm, 4.7 μm, and 4.3 μm, respectively. For rhG-CSF nanoparticles, about 90% of encapsulated rhG-CSF was released out in a sustained manner from PLGA nanoparticles over a 1 week period, but for rhG-CSF microparticles, only about 20% of rhG-CSF could be released out during the same period. Reversed phase and size exclusion chromatograms revealed that the structural integrity of released rhG-CSF from nanoparticles was nearly intact, compared to that of native rhG-CSF.
Keywords: rhG-CSF; Biodegradable; PLGA; Nanoparticles; Sustained release;

T lymphocytes as potential therapeutic drug carrier for cancer treatment by Ute Steinfeld; Christine Pauli; Nikolas Kaltz; Christian Bergemann; Hyeck-Hee Lee (229-236).
The aim of our research is the application of human immune cells (T lymphocytes) as target directed drug carrier. Thereby, the inclusion of therapeutical nanoparticles into immune cells is a new strategy for a localized chemotherapy. The autonomous targeting of diseased sites makes immune cells to perfectly controlled drug delivery systems.The study's aim was to demonstrate the feasibility to mobilise immune cells as therapeutic drug carrier systems which can be combined with existing immunotherapies.Therefore, Jurkat cells as well as T lymphocytes were used to identify the smoothest procedure for loading nanoparticles into immune cells. Different loading processes, incubation times and nanoparticle concentrations were compared. Nanoparticles coated with cytotoxic antibiotic doxorubicin were used in first release experiments. A time dependent liberation of doxorubicin from carrier cells was discussed as first therapeutic approach.
Keywords: T lymphocytes; Nanoparticles; Doxorubicin;

The choice of a suitable oligosaccharide to prevent aggregation of PEGylated nanoparticles during freeze thawing and freeze drying by W.L.J. Hinrichs; F.A. Manceñido; N.N. Sanders; K. Braeckmans; S.C. De Smedt; J. Demeester; H.W. Frijlink (237-244).
In a previous study we have shown that the oligosaccharide inulin can prevent aggregation of poly(ethylene glycol) (PEG) coated plasmid DNA/cationic liposome complexes (“PEGylated lipoplexes”) during freeze thawing and freeze drying [Hinrichs et al., 2005. J. Control. Release 103, 465]. By contrast, dextran clearly failed as stabilizer. These results were ascribed to the fact that inulin and PEG are compatible while dextran and PEG are not. In this study the stabilizing capacities of inulin and dextran (of various molecular weights) during freeze thawing and freeze drying of four different types of nanoparticles, each type with different amounts of PEG at their surface, were investigated. Freeze drying and freeze thawing of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/dioleoyl-phosphatidyl-ethanolamine (DOPE) liposomes and egg phosphatidyl choline (EPC)/cholesterol (CHOL) liposomes showed that inulins are excellent stabilizers even for highly PEGylated liposomes while (especially higher molecular weight) dextrans dramatically lost their stabilizing capacity when increasing the degree of PEGylation of the liposomes. The same results were obtained for plasmid DNA/DOTAP/DOPE complexes. Finally, both inulin and dextran could prevent full aggregation of plasmid DNA/polyethylenimine (PEI) complexes independent whether PEI was PEGylated or not. It is concluded that inulins are preferred as stabilizers over dextrans for various types of PEGylated nanoparticles due to their compatibility with PEG.
Keywords: Freeze drying; Inulin; Nanoparticles; PEGylation; Particle size;

Noticeboard (245-247).