European Journal of Pharmaceutics and Biopharmaceutics (v.62, #3)

APV Diary (S1-S2).

Purpose: To develop lipid semisolid formulations of EMD 50733, a poorly soluble, neutral drug candidate and to develop an in vitro–in vivo correlation for these formulations using the dog as the in vivo model. Methods: The model drug, EMD 50733, (with BCS Class II properties) was dissolved in molten lipid/surfactant mixtures and the melt was filled into hard capsules and allowed to re-solidify at room temperature. The dissolution profiles in bio-relevant dissolution media and the bioavailability in dogs were measured and compared to that of a standard formulation consisting of a lactose/drug mixture. Results: The best results with respect to dissolution, stability upon storage and bioavailability were obtained with a formulation that contained a commercially available lipid mixture (Gélucire 44/14) and a solubilizing agent (2-vinylpyrrolidone). With this formulation it was possible to dissolve a typical drug dose in a fill volume suitable for a #0 capsule. Additionally, surface tension measurements showed that the formulation formed micelles during dissolution in aqueous media: the molecular dispersion of the drug in this self-micelle forming system is postulated to protect the drug from precipitation in vivo as well as in vitro. For other formulations tested, neither the in vitro nor the in vivo performance indicated sufficient drug solubilizing properties. Conclusion: To achieve adequate and reliable dissolution of poorly soluble drugs in vivo, lipid excipients should not only have appropriate solubilizing properties for the drug in the formulation, but should also assist in maintaining drug in solution during release in the GI tract.
Keywords: Poorly soluble drugs; Gélucire 44/14; Vitamin E TPGS; Lipid excipients; BCS class II; Dissolution; Bioavailability; In vitro–in vivo correlations;

Poorly soluble photodynamic therapy (PDT) agent, meso-tetratphenylporphine (TPP), was effectively solubilized using non-targeted and tumor-targeted polymeric micelles prepared of polyethylene glycol/phosphatidyl ethanolamine conjugate (PEG-PE). Encapsulation of TPP into PEG-PE-based micelles and immunomicelles (bearing an anti-cancer monoclonal 2C5 antibody) resulted in significantly improved anticancer effects of the drug at PDT conditions against murine (LLC, B16) and human (MCF-7, BT20) cancer cells in vitro. For this purpose, the cells were incubated for 6 or 18 h with the TPP or TPP-loaded PEG-PE micelles/immunomicelles and then light-irradiated for 30 min. The phototoxic effect depended on the TPP concentration and specific targeting by immunomicelles. An increased level of apoptosis was shown in the PDT-treated cultures. The attachment of the anti-cancer 2C5 antibodies to TPP-loaded micelles provided the maximum level of cell killing at a given time. The results of this study showed that TPP-containing PEG-PE micelles may represent a useful formulation of the photosensitizer for practical PDT.
Keywords: Photodynamic therapy; meso-Tetraphenylporphine; Micelles; Cancer cells; Cytotoxicity; Apoptosis;

Influence of cyclodextrins and chitosan, separately or in combination, on glyburide solubility and permeability by N. Zerrouk; G. Corti; S. Ancillotti; F. Maestrelli; M. Cirri; P. Mura (241-246).
The effect of chitosan and of different concentrations of β- or hydroxypropyl-β-cyclodextrins, separately or in various (w/w) combinations, on the dissolution characteristics of glyburide (an oral hypoglycemic agent subject to incomplete and variable bioavailability) and on its permeability through Caco-2 cells has been investigated. Cyclodextrins (and particularly the hydroxypropyl-derivative, in virtue of its higher water solubility) were clearly more effective than chitosan in enhancing the drug dissolution properties: the aqueous glyburide solubility was improved 40-fold in the presence of 25 mM hydroxypropyl-β-cyclodextrin, 25-fold in the presence of 13 mM β-cyclodextrin (saturation solubility) and only 3-fold in the presence of chitosan at its saturation concentration (0.5% w/v). When chitosan and cyclodextrin were simultaneously present, a strong reduction of the cyclodextrin solubilizing efficiency towards the drug was observed, and it was attributed to a possible competition effect of polymer and glyburide for the interaction with the macrocycle. By contrast, permeation studies revealed that chitosan was more powerful than cyclodextrins in enhancing the glyburide permeability through Caco-2 cells. This was probably in virtue of the polymer's favourable effect on the tight junctions opening, as demonstrated by the significant decrease in the transepithelial electrical resistance recorded in its presence. Moreover, interestingly, when using the carriers together, conversely from solubility studies, a significant (P<0.05) synergistic effect in enhancing glyburide apparent permeability was revealed in permeation experiments.
Keywords: Glyburide; Solubility; Permeability; Caco-2 cells; Cyclodextrins; Chitosan;

The cellular immune response against tumors, viruses, or intracellular bacteria requires adequate antigen delivery to professional phagocytes, their processing and the presentation of antigenic peptides to T-cells. Biodegradable microparticles to enhance antigen phagocytosis and the response of cytotoxic lymphocytes have been proposed. The aim of the present study was to formulate poly(lactide-co-glycolide) (PLGA) microparticles using a w/o/w solvent evaporation procedure in order to obtain suitable vehicles for vaccination. Bovine serum albumin bearing fluorescein isothiocyanate (FITC–BSA) was used as a model antigen. For microparticle preparation a static micromixer was employed. Microparticles of 2–3 μm can be produced with good reproducibility by applying high flow rates at the micromixer. Microparticles with a smooth surface and only one pore were observed using scanning electron microscopy (SEM). Confocal laser scanning microscopy (CLSM) allowed localisation of the FITC–BSA near the surface of the microparticle. Microencapsulation of FITC–BSA did not altered the polymer characteristics, as determined by measuring the glass transition temperature. Additionally we could determine residual methylene chloride, employed as solvent in microparticle preparation, to be less than 1/1000 of the USP and Ph. Eur. limit. The microparticles described herein were able to deliver the model antigen to human dendritic cells (DC).
Keywords: Static micromixer; Microparticles; PLGA; Residual solvent; Dendritic cells; Vaccination;

Haemolytic activity of maltopyranoside surfactants by Erik Söderlind; Lina Karlsson (254-259).
The haemolytic activity of a number of maltopyranoside surfactants was studied. The study included octyl, nonyl, decyl, dodecyl, tetradecyl, cyclohexyl-propyl, cyclohexyl-hexyl, and dimethyl-heptyl maltopyranoside. The in vitro haemolytic activity was determined by employing a static method in which surfactants are added to an erythrocyte suspension and the released haemoglobin is determined. It was found that alkyl maltopyranosides become more haemolytic the longer the alkyl chain. Branching or presence of cyclic groups clearly decreases haemolytic activity, but it also increases the critical micelle concentration. As a result, the cyclic or branched surfactants do not become better solubilizing excipients than the straight-chain surfactants. The most useful surfactant for pharmaceutical applications appears to be tetradecyl maltopyranoside, which is the least haemolytic surfactant relative to its critical micelle concentration.
Keywords: Haemolysis; Sugar surfactant; Maltopyranoside surfactant; Solubilizing agent;

Evaluation of the potential of air jet milling of solid protein-poly(acrylate) complexes for microparticle preparation by Wolfgang Schlocker; Siegfried Gschließer; Andreas Bernkop-Schnürch (260-266).
It was the aim of this study to evaluate the potential of air jet milling for the preparation of protein-loaded microparticles in industrial quantities. The model protein horseradish peroxidase was incorporated via co-precipitation in carbomer (NaC934P) (1:100) and a poly(methacrylate) (Eudragit®L100-55) (1:100) used as carrier matrix. Co-precipitation of the model protein and each polymer in aqueous solution was achieved either by a pH-shift or by the addition of various non-solvents. Dried protein/polymer complexes (desiccator under vacuumization at 4 °C with silica blue gel) were ground with an air jet mill and resulting microparticles were investigated regarding protein load, remaining protein activity, size distribution and shape. Results of this study showed that the polymer used and the method of co-precipitation has a great impact on protein load. Using carbomer a maximum protein load of 60±1% was achieved, whereas in case of Eudragit® L100-55 the maximum was 78±5% (means±SD; n=3–4). Using petroleum ether, isopropanol or tetrahydrofurane as non-solvents led to significantly higher protein loads than a pH-shift from 7 to 5, 4 and 3.5, respectively. Determination of the remaining protein activity after milling showed, that the grinding air pressure (GAP) has a major impact on protein stability. In case of Eudragit®L100-55 at a GAP of 4.5 bar peroxidase activity was almost completely lost, whereas 42±1% loss in activity was determined at a GAP of 2.5 bar. The mean particle size of protein/carbomer and protein/poly(methacrylate) particles was determined to be 3.6–5.2 and 4.5–8.7 μm at a GAP of 2.5 bar and 2.7–3.1 and 2.4–3.1 μm at a GAP of 4.5 bar, respectively. Generally, 90% of all particles were in the range of 3–16 μm. All particles were of spherical shape exhibiting a non-porous surface.According to these results, air jet milling seems to represent a novel method for the large-scale production of protein drug loaded microparticles.
Keywords: Air jet mill; Carbomer; Microparticles; Eudragit®; Protein complexation; Micronization;

Chitosan drug binding by ionic interaction by Yaowalak Boonsongrit; Ampol Mitrevej; Bernd W. Mueller (267-274).
Three model drugs (insulin, diclofenac sodium, and salicylic acid) with different pI or pKa were used to prepare drug-chitosan micro/nanoparticles by ionic interaction. Physicochemical properties and entrapment efficiencies were determined. The amount of drug entrapped in the formulation influences zeta potential and surface charge of the micro/nanoparticles. A high entrapment efficiency of the micro/nanoparticles could be obtained by careful control of formulation pH. The maximum entrapment efficiency did not occur in the highest ionization range of the model drugs. The high burst release of drugs from chitosan micro/nanoparticles was observed regardless of the pH of dissolution media. It can be concluded that the ionic interaction between drug and chitosan is low and too weak to control the drug release.
Keywords: Chitosan; Ionic interaction; Microparticles; Nanoparticles; Insulin; Diclofenac sodium; Salicylic acid;

Gadolinium (Gd) neutron capture therapy (NCT) is currently under development as a potential approach for tumor therapy. Nanoparticles have been suggested as a potential delivery system to carry or target Gd to tumors for thermal or epithermal neutron irradiation. The reconstituted chylomicron emulsion is an artificial chylomicron remnant prepared using commercially available natural and biocompatible lipids. We proposed to use this nanometer-scale emulsion to deliver Gd to solid tumors by modifying the surface of the emulsion. A lipophilic Gd compound, gadolinium acetylacetonate (GdAcAc), was incorporated into the emulsion, resulting in a final pure Gd concentration of more than 1 mg/mL. The apparent solubility of GdAcAc was enhanced by about 6000-fold by this incorporation. The emulsion particles were shown to be stable in a two-week short-term stability study when stored at 4 °C. In addition, no extensive particle aggregation was observed when the emulsion particles were incubated in simulated biological media such as serum. Also, GdAcAc does not significantly ‘leak’ out from the emulsion particles. Only ∼5% was released in 20 h in a SDS (0.5% w/v) in phosphate buffered saline (pH 7.4, 10 mM) medium. Finally, the emulsion particles were coated with polyethylene glycol (PEG), and injected into Balb/C mice via the tail vein. A significant proportion (71.6±18.4%) of the PEG-coated, GdAcAc-incorporated emulsion remained circulating in the blood 5 h after the injection, while the PEG-free emulsion was mainly accumulated inside the liver. This chylomicron emulsion may be used to deliver Gd into solid tumors for NCT.
Keywords: Nanoparticles; tumor targeting; PEG; biodistribution; lipoprotein;

Spray coated pellets as carrier system for mucoadhesive drug nanocrystals by Jan Möschwitzer; Rainer H. Müller (282-287).
High pressure homogenization can be employed to produce drug nanocrystals with a number of advantages, like improved solubility behaviors, better drug targeting or even increased mucoadhesiveness. To obtain a controlled drug delivery system it is necessary to transform the resulting nanosuspension into a solid dosage form. The present study shows the feasibility to use a mucoadhesive nanosuspension of poorly soluble hydrocortisone acetate produced by high pressure homogenization as layering dispersion in a fluidized bed process, followed by the application of an enteric coating to achieve a controlled drug release. To point out the advantages of drug nanocrystals the new fomulation was compared with a formulation containing micronized drug. Both formulations were characterized with regard to their particle size and crystallinity by using laser diffractometry, photon correlation spectroscopy and X-ray diffraction. The pellet morphology was characterized by using the environmental scanning electron microscopy (ESEM). In the in vitro dissolution tests an accelerated dissolution velocity and an increased drug release could be shown for the pellets containing drug nanocrystals.
Keywords: High pressure homogenization; Poorly soluble drugs; Controlled drug delivery; Drug nanocrystals; Pellets;

Studies on the stability of the chloramphenicol in the microemulsion free of alcohols by Feng-Feng Lv; Na Li; Li-Qiang Zheng; Chen-Ho Tung (288-294).
Microemulsion composed of Span20+Tween20 isopropyl myristate (IPM)+H2O were investigated as potential drug delivery systems for eye drops. The system is important in that all its components are food grade so that the microemulsion is almost free of toxicity and irritation. The phase transition was investigated using the electrical conductivity measurements. The chloramphenicol is used to treat the eye diseases such as trachoma and keratitis. However, this drug in the common eye drops hydrolyzes easily. The main product of the hydrolysis is glycol. Here, the chloramphenicol was trapped into the oil-in-water (o/w) microemulsions free of alcohols. Its stability was investigated by the high performance liquid chromatography (HPLC) assays in the accelerated experiments of 3 months. The location of the chloramphenicol molecules in the microemulsion formulations was determined by means of dynamic light scattering (DLS) and 1H NMR spectroscopy. The results of HPLC revealed that the content of the glycols in the microemulsion formulation was much lower than that in the commercial eye drops at the end of the accelerated experiments. It implied that the stability of the chloramphenicol in the microemulsion formulations was increased remarkably. The results of DLS and NMR confirmed that the chloramphenicol molecules should be trapped into the hydrophilic shells of the microemulsion drops, which were composed of many oxyethylene groups. The benzene rings of the chloramphenicol molecules were near the group of α2-CH2 and the oxyethylene groups of the surfactant molecules. It was this reason that enabled the chloramphenicol molecules in the microemulsions to be screened from the bulk water and its stability to be increased remarkably.
Keywords: Non-ionic surfactants; Microemulsion; Phase behavior; Chloramphenicol eye drops; HPLC; DLS; 1H NMR; Stability;

Chitosan microparticulates were prepared by spray drying from aqueous media containing hydrochloric acid or acetic acid. The medium affected the morphology and degree of acetylation of chitosan, the presence of acetic acid resulting in increased acetylation of the polymer during processing. Co-spray drying salbutamol sulphate/chitosan systems with the crosslinking agent formaldehyde had no detectable effect on particle morphology. However, with increasing salbutamol loading particles became less spherical, taking on a collapsed appearance. Spray dried chitosan–salbutamol sulphate microparticulates were X-ray amorphous. Chitosan–salbutamol sulphate composites were compressed into discs to quantify drug release and showed delayed release of salbutamol sulphate. The general power law equation fitted the data better than the t 0.5, mono- or bi-exponential models and gave n indices greater than 0.5, i.e. in the range 0.53–0.71. Crosslinking did not dramatically alter the drug release behaviour. Both crosslinked and non-crosslinked composites swelled during release, the former to the greater extent. The release data for crosslinked composites gave slightly higher n values than the corresponding non-crosslinked composites, consistent with the increased swelling of these systems. Release studies were also conducted on the microparticulates. Because of the small particle size and large surface area present, the release of the highly soluble drug salt was extremely rapid (>90% release in 5 min). Twin impinger analysis indicated good in vitro deposition of the microparticulates and potential for pulmonary delivery.
Keywords: Chitosan; Salbutamol; Spray drying; Drug release;

The long term goal of this study is to develop an efficient nanoscopic vaccine delivery system, based on the biodegradable and natural polymer gelatin, to deliver therapeutic protein antigens along with adjuvants into dendritic cells (DCs). In this study, gelatin nanoparticles were tested for qualitative and quantitative uptake in murine DCs in vitro. A second aim of this study was to prove that the carrier system is able to deliver tetramethylrhodamine conjugated dextran (TMR–dextran), as a model drug into the DCs. The TMR–dextran was incorporated during the preparation of the gelatin nanoparticles. DCs were generated from murine bone marrow cells by an established ex vivo technique. Flow cytometry showed that 88% of the cells positive for the specific murine DC marker CD11c took up TMR–dextran loaded gelatin nanoparticles, whereas only 4% of the soluble form of TMR–dextran was taken up. Double color confocal laser scanning microscopy (CLSM) showed that gelatin nanoparticles were phagocytosed by DCs and the triple color CLSM showed that the TMR–dextran was localized mainly in lysosomes as expected, but partly also outside the lysosomes, presumably in the cytoplasm. An in vitro release study of TMR–dextran from gelatin nanoparticles demonstrated that there was hardly any release in phosphate buffered saline (PBS), but by trypsin-assisted degradation of gelatin nanoparticles resulted in the release of about 80% of the TMR–dextran from the particles. These results suggest that gelatin nanoparticles hold promise as a new biocompatible tool for vaccine delivery to DCs, with applications in cancer immunotherapy.
Keywords: Dendritic cells; Gelatin; Nanoparticles; Tetramethylrhodamine dextran; Phagocytosis; Intracellular localisation; Cancer vaccination;

A HPLC method with photodiode array detection (PDA) was developed for the determination and a pharmacokinetic study of eleutheroside E (ELU E) and eleutheroside B (ELU B) in rat plasma and tissue following an eleutherococcus injection. The analysis was performed on a Kromasil C18 column, using water-acetonitrile as the gradient mobile phase and 0.8 mL/min flow rate. Detection wavelengths of ELU E and ELU B were 220 and 206 nm, respectively. Protein from the biological sample was deposited using acetonitrile. ELU E and ELU B were extracted from the biological samples using acetonitrile, separated by solid-phase extraction, and eluted from the cartridge using 60% methanol. The extraction recovery of ELU E and ELU B was 91.2 and 88.8%, respectively. The limit of detection was 37.6 ng/mL for ELU E and 37.0 ng/mL for ELU B (S/N=3) in plasma. Blood drug level-time cuvers of ELU E and ELU B in Wister rats following administration of an eleutherococcus injection into femoral vein were shown to fit a three-compartment model. The half-life (t 1/2) was 4.662 h for ELU E and 2.494 h for ELU B. Following administration of a single eleutherococcus injection, the concentration of ELU E and ELU B in the tissue was Cliver>Ckidney>Cspleen>Cheart and Ckidney>Cliver>Cheart. We believe the method described in the present paper is accurate and reliable and can be used for pharmacokinetic studies of ELU E and ELU B in rats. In addition, the method for sample preparation, using solid phase extraction, is precise, simple and rapid.
Keywords: Eleutherococcus injection; Eleutheroside E; Eleutheroside B; Pharmacokinetics; Tissue distribution; High-performance liquid chromatography;

In vitro toxicity to breast cancer cells of microsphere-delivered mitomycin C and its combination with doxorubicin by Richard Y. Cheung; Andrew M. Rauth; Patrick T. Ronaldson; Reina Bendayan; Xiao Yu Wu (321-331).
To better understand and design microsphere systems for the locoregional delivery of anticancer drug combinations to solid tumors, (1) the cytotoxicity of microsphere-delivered mitomycin C (MMC) was evaluated and (2) various schedules of MMC and doxorubicin (Dox) were tested for their toxicity in vitro towards a murine breast cancer cell-line, EMT6. To accomplish the former MMC was loaded onto oxidized sulfopropyl dextran microspheres, released in a pH 7.4 buffer solution and tested for its potency against EMT6 cells versus a standard MMC solution. For the latter EMT6 cells were exposed to MMC or Dox as single agents or together using various drug concentrations and schedules. The efficacy of the treatments was measured using a clonogenic assay. MMC released from the microspheres showed similar activity against EMT6 cells to freshly prepared MMC solutions. Greater-than-additive toxicity was observed when MMC was given either simultaneously or after Dox exposure. In contrast, administration of MMC before Dox exposure resulted in toxicity that ranged from additive to sub-additive; this reduced toxicity was mainly due to increasing cell density arising from the design of the assay. These results help explain our previous in vivo investigations using microsphere-delivered combinations of the same agents in EMT6 solid tumors.
Keywords: Mitomycin C; Doxorubicin; Microspheres; Breast cancer cells; Anticancer drug combinations;