International Journal of Pharmaceutics (v.307, #1)

In vivo study of liposomes as drug carriers to oral mucosa using EPR oximetry by V. Erjavec; Z. Pavlica; M. Sentjurc; M. Petelin (1-8).
The purpose of this study was to select the best types of liposomes for use as drug carriers for topical treatment of oral mucosal lesions. Electron paramagnetic resonance (EPR) oximetry, using the paramagnetic probe lithium phthalocyanine, was used in vivo to measure the effects of a hyperemic drug, benzyl nicotinate (BN) which was incorporated into liposomes of varying size and composition. The liposomes were made from either hydrogenated or non-hydrogenated soy lecithin and mixed with polymethyl methacrylate ointment for application. EPR oximetry was used to measure the partial pressure of oxygen (pO2) in the oral mucosa before and after application of liposomes. It was found that the most pronounced changes of pO2 in oral mucosa and also the longest action of the drug occurred after the topical application of BN in multi-lamellar liposomes made from hydrogenated soy lecithin (p  < 0.0001). When these liposomes were applied to oral mucosa over 3 successive days it was found that pO2 increased the most on the first day, the effect gradually decreased following application on the second and third days. The duration of the resulting hyperemia was the longest on the second day (p  < 0.01). Among the examined carriers, multi-lamellar liposomes made from hydrogenated soy lecithin appear to be the most appropriate for local drug delivery to oral mucosa.
Keywords: Drug delivery; Liposomes; In vivo EPR oximetry; Oral mucosa; Rat;

Nasal administration of Carbamazepine using chitosan microspheres: In vitro/in vivo studies by E. Gavini; A.B. Hegge; G. Rassu; V. Sanna; C. Testa; G. Pirisino; J. Karlsen; P. Giunchedi (9-15).
The nasal route is used both for local therapies and, more recently, for the systemic administration of drugs, as well as for the delivery of peptides and vaccines. In this study the nasal administration of Carbamazepine (CBZ) has been studied using microspheres constituted by chitosan hydrochloride (CH) or chitosan glutamate (CG). Blank microspheres were also prepared as a comparison. The microspheres were produced using a spray-drying technique and characterized in terms of morphology (scanning electron microscopy, SEM), drug content, particle size (laser diffraction method) and thermal behaviour (differential scanning calorimetry, DSC). In vitro drug release studies were performed in phosphate buffer (pH 7.0). In vivo tests were carried out in sheep using the microparticles containing chitosan glutamate, chosen on the basis of the results of in vitro studies. The results were compared to those obtained after the nasal administration of CBZ (raw material) alone. For the evaluation of in vivo data statistical analysis was carried out using the unpaired t-test.Spray-drying was a good technique of preparation of CBZ-loaded microspheres. The loading of the drug into the polymeric network always led to an increase in the dissolution rate compared to CBZ raw material. The microspheres obtained using chitosan glutamate had the best behaviour both in vitro and in vivo. They increased the drug concentration in the serum when compared to the nasal administration of the pure drug (C max 800 and 25 ng/ml for microspheres and pure drug, respectively). The results obtained indicate that the loading of CBZ in chitosan glutamate microspheres increases the amount of the drug absorbed through the nose.
Keywords: Carbamazepine; Chitosan salts; Spray-dried microspheres; Nasal route; In vivo study; Sheep;

Influence of post-emulsification drying processes on the microencapsulation of Human Serum Albumin by Majella E. Lane; Fiona S. Brennan; Owen I. Corrigan (16-22).
In the present work, methods used to microencapsulate Human Serum Albumin (HSA) in a biodegradable polymer were compared for their effects on the physicochemical characteristics of HSA-loaded microparticles and on the release and integrity of encapsulated HSA. The polymer used was poly(d,l-lactide-co-glycolide) (75:25) (PLGA) (Boehringer Ingelheim, Resomer RG 752, MW 20,900). Microparticles were formulated by (i) w/o/w emulsification and freeze-drying (EFD) or (ii) w/o/w emulsification and spray-drying (ESD). Particle morphology and size were evaluated by scanning electron microscopy and by laser diffraction analysis. Loading, encapsulation efficiency and protein release were determined using a commercial protein assay kit. Protein integrity was evaluated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. Particles produced by emulsification/spray-drying exhibited greater diversity in shape than those produced by emulsification/freeze-drying. Additionally, protein loading values were significantly higher for particles produced by emulsification/spray-drying rather than particles produced by emulsification/freeze-drying. The structural integrity of encapsulated protein was confirmed for particles produced by both processes. The fraction of HSA released was similar for both formulations. The emulsification/spray-drying technique described appears to be a rapid and efficient method for the preparation of PLGA microparticles loaded with a model protein.
Keywords: PLGA; Human Serum Albumin; Water-in-oil-in-water emulsion; Freeze-drying; Spray-drying;

Release of bioactive BMP from dextran-derived microspheres: A novel delivery concept by Fa-ming Chen; Zhi-fen Wu; Hai-hua Sun; Hong Wu; Su-ning Xin; Qin-tao Wang; Guang-ying Dong; Zhi-wei Ma; Sha Huang; Yong-jie Zhang; Yan Jin (23-32).
Recent developments of biotechnology have produced a great variety of protein and bioactive drugs. For these drugs to be used therapeutically, suitable drug delivery systems have become increasingly essential. Dextran-derived biomaterials have been considered to be compatible matrices for protein and bioactive drugs because of their hydrophilic properties and ability to control drug dissolution and permeability. A novel class of dextran–glycidylmethacrylate (Dex–GMA)/poly(ethylene glycol) (PEG) microspheres were designed and synthesized by polymerization of Dex–GMA emulsified in an aqueous PEG solution. Dex–GMA was prepared by substituting the hydroxyl groups in Dex by GMA. The drug loading and in vitro drug release was evaluated by routine procedure and the biological activity of BMP-loaded microspheres was studied by experimental cytology methods. Recombinant human bone morphogenetic protein-2 (rhBMP-2) were entrapped in dextran-derived microspheres quantitatively and with full preservation of their biological activity. In vitro release kinetics indicated that dextran-derived microspheres could retain rhBMP-2 in a variable manner depending on the preparation and degradation of the microspheres. The release profiles of rhBMP-2 from microspheres as a function of time showed that rhBMP-2 releasing kinetics in vitro fitted to first-order and Higuchi equations. The release profile in vitro was in accord with two phases kinetics law and more than 60% drug were released during 20 days. Cytology studies showed rhBMP-2 microspheres have good biological effects on cultured periodontal ligament cells, and could achieve a longer action time than concentration of rhBMP-2 solution. These properties make those microspheres interesting osteo-conductive BMP carriers, allowing to decrease the amount of implanted factor required for tissue regeneration.
Keywords: Bone morphogenetic proteins; Drug delivery system; Microspheres; In vitro release; Functionalized dextran; Poly(ethylene glycol); Periodontal ligament cells;

Mathematical modelling of in situ and in vitro efflux of ciprofloxacin and grepafloxacin by M. Rodríguez-Ibáñez; G. Sánchez-Castaño; M. Montalar-Montero; T.M. Garrigues; M. Bermejo; V. Merino (33-41).
The efflux process due to p-glycoprotein-like mechanisms of ciprofloxacin (CIP) and grepafloxacin (GRX) has been studied “in situ” in rats and “in vitro” in Caco-2 cells. The results were modelled by a curve fitting procedure which allowed the characterization of the passive (P d) and carrier mediated parameters (V m and K m) from the raw data without initial velocities estimation. CIP absorption in rat was characterized as a passive diffusion at the assayed concentrations. Although the involvement of an efflux transporter cannot be ruled out, its relevance in the transport of the fluoroquinolone is negligible. In GRX absorption, an efflux process is implicated and it is detected in both absorption models. GRX permeability depends on the intestinal segment, reflecting the previously reported different expression level of the efflux transporters along the gut in rat. A first attempt to correlate the “in vitro” and the “in situ” data has been done. The mathematical model has been constructed using very simplistic assumptions and it will require further refinement but, nevertheless, the results are promising and demonstrate that a good modelling approach helps to identify the system critical parameters and how the system behaviour change when the parameters are modified as it happens when we move from the “in vitro” to the “in situ” level. Predicted versus experimental permeability values show a good correlation, demonstrating that the relevance of the secretion process “in situ” in rat can be predicted from the “in vitro” cell results.
Keywords: Kinetic modelling; Fluoroquinolones; Intestinal permeability; Caco-2 model; Efflux; Absorption;

The aim of this study was to correlate the taxonomy of grapefruit, pummelo, orange, lime and lemon with fruit juice-mediated cytotoxicity, modulation of epithelial permeability and P-glycoprotein (P-gp)-mediated efflux using 0–50% juice concentrations. Lime and lemon juices at 30% enhanced the absorption of [14C]-mannitol across Caco-2 cell monolayers by six- and eight-fold, respectively, but grapefruit and pummelo juices did not modulate the paracellular [14C]-mannitol transport even at 50%. Orange juice at 30% increased mannitol absorption to a comparable level as lime juice, but had minimal effects on TEER. All five juices did not modulate the passive diffusional pathway as exemplified by their negligible effects on [3H]-propranolol absorption. Grapefruit, pummelo and orange juices showed P-gp inhibitory activity by reducing rhodamine-123 (R-123) efflux and elevating R-123 cellular accumulation, but lime and lemon juices did not. Lime and lemon juices at ≥30% were cytotoxic towards Caco-2 cells. Grapefruit and pummelo juices at 10% did not affect Caco-2 cell viability, but they enhanced cell growth at concentrations of ≥30%. Orange juice increased cell viability only at lower concentrations. On the basis of these data, lime and lemon juices could be regarded as a group distinct from grapefruit and pummelo juices, while orange juice appeared to belong to a bridging group. This grouping was consistent with the categorization of the citrus fruits according to their dominant flavonoid pattern and taxonomy.
Keywords: Citrus fruit juices; Taxonomy; Mannitol; Propranolol; Rhodamine-123; Caco-2;

Previous studies have confirmed that the phase transition of vesicular gels of hydrogenated phospholipids to the less ordered fluid vesicular state was induced by the increase of the β-sitosterol ratio in the whole gel system and consequently in the lipid bilayer. The purpose of the present study was to evaluate the influence of the β-sitosterol portion in the lipid bilayer and the effect of high pressure homogenization on the structural characteristics of the prepared gel systems. In addition the influence of β-sitosterol on the consequent chlorhexidine release from the obtained vesicles and liposomes was also examined. Lipid mixtures were prepared from different molar ratios of lecithin:sterol components (90:10–65:35 mol%). The obtained mixtures were hydrated with the aqueous solution of chlorhexidine digluconate in order to achieve a 30% (w/w) final concentration of the lipid mixtures and a 4% (w/w) concentration of the drug. One portion of the resultant multilamellar vesicles was homogenized by using high pressure. To characterize the homogenized and non-homogenized systems, transmission electron microscopy of the freeze-fractured samples and differential scanning calorimetry (DSC) were carried out. A vertical type diffusion cell was applied to determine the amount of released chlorhexidine digluconate. Along with the increase in β-sitosterol concentration, the fluidity of the membrane as well as its permeability also increased. The increased permeability—caused by the higher β-sitosterol concentration—and the high pressure homogenization, which increased the dispersity and therefore the surface area, enabled a higher amount of chlorhexidine to be released. The increase of drug release was more pronounced in the case of samples prepared with high pressure homogenization.
Keywords: Multilamellar vesicles (MLV); Vesicular phospholipid gel (VPG); High pressure homogenization; β-sitosterol; Chlorhexidine digluconate release; Transmission electron microscopy; Differential scanning calorimetry;

Conformational study of ketoprofen by combined DFT calculations and Raman spectroscopy by M.L. Vueba; M.E. Pina; F. Veiga; J.J. Sousa; L.A.E. Batista de Carvalho (56-65).
A conformational study of ketoprofen was carried out by both density functional theory (DFT) calculations and Raman spectroscopy. Nine different geometries were found to correspond to energy minimum conformations but only one of them was experimentally detected in the condensed phase spectra.Those rotations which interconvert the five most stable conformers were studied and the intramolecular interactions governing the corresponding conformational preferences were assessed.A thorough vibrational analysis was performed, leading to the assignment of both the solid and liquid spectra. Evidence for formation of intermolecular hydrogen bonds between carboxylic groups of adjacent ketoprofen molecules, leading to dimeric entities, was obtained.
Keywords: Ketoprofen; DFT calculations; Raman spectroscopy; FTIR spectroscopy; Conformational analysis; Rotational isomerism;

PEO and MPEG in high drug load extruded and spheronized beads that are devoid of MCC by Matthew A. Howard; Steven H. Neau; Marvin J. Sack (66-76).
A means to produce extruded–spheronized beads, devoid of microcrystalline cellulose (MCC) and with a high drug load (greater than 80%, w/w), is presented. Immediate release bead product with a high yield (greater than 60% of 1 mm diameter beads) and low friability (mass loss less than 4.0%) that were spherical to the naked eye (roundness score less than 1.20) were obtained. The formulation consists only of water-soluble components, taking advantage of the properties of soluble polyethylene oxide (PEO) and methoxypolyethylene glycol (MPEG). This approach incorporates minimal processing aids, with wetted PEO providing the apparent plasticity and cohesiveness, and MPEG550 providing the apparent self-lubricating characteristics necessary for successful extrusion and subsequent spheronization into beads. The success of this approach has important implications in cases where high drug load beads are desired, but where MCC cannot be used due to chemical incompatibility or where complete release cannot be achieved with MCC-containing beads.
Keywords: Extrusion–spheronization; Pellets; Polyethylene oxide; Polyoxyethylene; Statistical design;

The preparation of silybin–phospholipid complex and the study on its pharmacokinetics in rats by Xiao Yanyu; Song Yunmei; Chen Zhipeng; Ping Qineng (77-82).
The aim of the present study was to find a way of prepare silybin–phospholipid complex to make oral bioavailability of silybin increase and to study its physicochemical properties and to compare the pharmacokinetic characteristics and bioavailability after oral administration of silybin–phospholipid complex and silybin-N-methylglucamine in rats. Using ethanol as a reaction medium, silybin and phospholipids were resolved into the medium, after the organic solvent was removed under vacuum condition, silybin–phospholipid complex was formed. The new complex's physicochemical properties including scanning electron microscopy (SEM), transmission electron microscopy (TEM), differential scanning calorimetry (DSC), solubility, dissolution, etc., were tested. The concentrations of silybin after oral administration of silybin–phospholipid complex and silybin-N-methylglucamine at different time in rats were determined by RP-HPLC. The pharmacokinetic parameters were computed by software program 3p97. Our data showed that silybin and phospholipids in the silybin–phospholipid complex were combined by non-covalent-bond, not forming a new compound and the solubility of silybin–phospholipid complex in water and in n-octanol was effectively enhanced. We found that mean plasma concentration–time curve of silybin after oral administration of silybin–phospholipid complex and silybin-N-methylglucamine in rats was both in accordance with open single-compartment model with first-order absorption. Pharmacokinetic parameters of silybin in rats were T max 10 and 5 min; C max 126.72 and 104.29 ng ml−1; AUC0–∞ 1020.33 and 235.81 ng ml−1  h, respectively. The bioavailability of silybin in rats was increased remarkably after oral administration of silybin–phospholipid complex comparing to silybin-N-methylglucamine. This was mainly due to an impressive improvement of the lipophilic property of silybin–phospholipid complex and improvement of the biological effect of silybin.
Keywords: Silybin–phospholipid complex; Silybin-N-methylglucamine; Physicochemical properties; Oral bioavailability; Pharmacokinetics; Silybin;

Biodegradable microparticulate system of captopril by P.M. Dandagi; V.S. Mastiholimath; M.B. Patil; M.K. Gupta (83-88).
Albumin microparticles have found many applications in diagnosis and treatment in recent years and more than 100 diagnostic agents and drugs have been incorporated into albumin microparticles. In the present study, bovine serum albumin (BSA) based microparticles bearing captopril were prepared by an emulsification–heat stabilization technique. Four batches of microparticles with varying ratio of drug and polymer were prepared. The prepared microparticles were studied for drug loading, particle size distribution, in vitro release characteristics, in vivo tissue distribution study and stability studies. The microparticles had mean diameter between 2 and 11 μm of which more than 70% were below 5 μm and incorporation efficiency of 41–63% was obtained. In vitro release profile for formulations containing captopril-loaded albumin microparticles with heat stabilizing technique shows slow controlled release up to 24 h. The in vivo result of drug-loaded microparticles showed preferential drug targeting to liver followed by lungs, kidneys and spleen. Stability studies showed that maximum drug content and closest in vitro release to initial data were found in the formulation stored at 4 °C. In the present study, captopril-loaded BSA microparticles were prepared and targeted to various organs to a satisfactory level and were found to be stable at 4 °C.
Keywords: Microparticles; Captopril; Bovine serum albumin; Captopril-loaded microparticles; Emulsification–heat stabilization technique;

Lung surfactant as a drug delivery system by C. Vermehren; S. Frokjaer; T. Aurstad; J. Hansen (89-92).
Lung surfactant is a complex mixture of mainly phospholipids and proteins. The composition leads to a unique spreading effect of the surfactant as well as spontaneous vesicle formation, which may be favourable characteristics of a drug delivery system for pulmonary delivery.The aim of study was to investigate the potential use of the surfactant extract, HL10 (LeoPharma, DK) as a drug delivery system. Studies involved incorporation of hydrophilic- and amphipathic model drugs (sucrose and acylated peptides) into HL10 and elucidation of the influence of surfactant proteins on the HL10 behaviour.Results showed that HL10 vesicles did not retain sucrose indicating formation of leaky vesicles. Studying the influence of surfactant proteins on release from DPPC-liposomes showed tendencies toward a protein-induced release. Hence, the surfactant proteins may influence the membrane lipid packing and characteristics resulting in leakiness of the membranes. Incorporation of acylated peptides into HL10 depended on the chain length rendering a successful incorporation of the peptide acylated with C14-acyl chains.This study suggests that HL10 may be a promising drug delivery system for the pulmonary delivery of amphipathic drug substances, e.g. therapeutically active acylated peptides (e.g. acylated insulin).
Keywords: Drug delivery; Lung surfactant; HL10;

The process of opsonization is one of the most important biological barriers to controlled drug delivery. Injectable polymeric nanoparticle carriers have the ability to revolutionize disease treatment via spatially and temporally controlled drug delivery. However, opsonin proteins present in the blood serum quickly bind to conventional non-stealth nanoparticles, allowing macrophages of the mononuclear phagocytic system (MPS) to easily recognize and remove these drug delivery devices before they can perform their designed therapeutic function. To address these limitations, several methods have been developed to mask or camouflage nanoparticles from the MPS. Of these methods, the most preferred is the adsorption or grafting of poly(ethylene glycol) (PEG) to the surface of nanoparticles. Addition of PEG and PEG-containing copolymers to the surface of nanoparticles results in an increase in the blood circulation half-life of the particles by several orders of magnitude. This method creates a hydrophilic protective layer around the nanoparticles that is able to repel the absorption of opsonin proteins via steric repulsion forces, thereby blocking and delaying the first step in the opsonization process.
Keywords: Opsonization; Poloxamer; Poloxamine; Poly(ethylene glycol); PEGylation; Stealth nanoparticles;

Nanoparticle formulation may affect the stabilization of an antiischemic prodrug by E. Leo; C. Contado; F. Bortolotti; B. Pavan; A. Scatturin; G. Tosi; S. Manfredini; A. Angusti; A. Dalpiaz (103-113).
The prodrug 5′-octanoyl-CPA (Oct-CPA) of the antiischemic N 6-cyclopentyladenosine (CPA) has been encapsulated by nanoprecipitation in poly(lactic acid) nanoparticles, which have been recovered by gel-filtration, ultra-centrifugation or dialysis. We have analysed how different surfactants and purification methods can influence the nanoparticle characteristics. The particle sizes have been obtained by scanning electron microscope, whereas a SdFFF system was employed to detect their distributions. The Oct-CPA release from nanoparticles and stabilities in human blood of free and encapsulated prodrug have been analysed by HPLC techniques. The effects of nanoparticles on CPA interaction toward adenosine A1 receptor (its action site) have been analysed using radiolabelled drugs. The smallest nanoparticles and the best degree of homogeneity have been obtained using sodium cholate; the best recovery has been achieved by dialysis, whereas gel-filtration and ultra-centrifugation have induced the greatest removal of surfactants. The release of Oct-CPA was better controlled from the nanoparticles obtained using Pluronic F68 and purified by gel-filtration or ultra-centrifugation. Similarly, these nanoparticles better increased the stability of the prodrug in human blood. In particular, the nanoparticles purified by ultra-centrifugation induced a strong stability to a fraction of the encapsulated Oct-CPA. Any interference by unloaded nanoparticles has been registered for CPA-adenosine A1 receptor interaction.
Keywords: Controlled release; N 6-cyclopentyladenosine; Nanoparticles; Prodrug; SdFFF; Stability;

Noticeboard (114-115).

Guide for Authors (116-121).