Penetration and release studies of positively and negatively charged nanoemulsions--is there a benefit of the positive charge?

The surface of all tissues, including the stratum corneum, carries a negative charge. Following that fact it is assumed that a positively charged topical formulation could lead to an enhanced penetration because of an increased interaction with the negative charge of the membrane. The intention of this study is to prove an enhanced penetration of a positively charged nanoemulsion compared to a negatively charged nanoemulsion, both containing prednicarbate. The release and penetration of these nanoemulsions, produced with the high pressure homogenization method, were investigated. Regarding these results reveals that the release of the negatively charged formulation is higher compared to the positively charged nanoemulsion, while the penetration of the positively charged nanoemulsion is enhanced compared to the negatively charged formulation. The results of the investigated positively charged nanoemulsion containing prednicarbate show that its topical use could be advantageous for the therapy of atopic dermatitis, especially regarding phytosphingosine, which was responsible for the positive charge.

Development of a positively charged prednicarbate nanoemulsion.

A physically and chemically stable positively charged prednicarbate nanoemulsion was developed as a carrier system for the treatment of atopic dermatitis. Phytosphingosine was used to obtain the positive charge and also because of its supportive properties for the restoration of damaged skin. As production method high pressure homogenization was employed. The optimal concentrations of phytosphingosine, the oil phase, and the emulsifiers were investigated. The production was optimized by investigating the influence of homogenization cycles, homogenization pressure, production temperature and type of homogenizer with respect to particle size, physical stability of the emulsions and chemical stability of prednicarbate. From the results the best formulation and the most appropriate production parameters were identified. In addition it could be shown that during high pressure homogenization the drug is relocated from the inner oil phase of the emulsion towards the stabilizer layer, which could be shown by an increase in chemical stability of prednicarbate. The efficiency of incorporation is influenced by the energy input during homogenization (e.g. number of homogenization cycles) but also by the production temperature. It was found that the nanoemulsions should be produced at elevated temperatures, with low homogenization pressures but higher numbers of homogenization cycles (e.g. 300 bar and 10 cycles). The results prove that the efficiency of high pressure homogenization should not only be judged by investigating the particle size and the physical stability of the emulsions alone, but also by assessing the chemical stability of the incorporated drug.

Improving the ex vivo stability of drug ester compounds in rat and dog serum: inhibition of the specific esterases and implications on their identity.

In drug development, it has been noticed that some drug compounds, especially esters, are unstable in serum samples ex vivo. This can lead to a substantial underestimation of the actual drug concentration. The rat and the dog, representing a rodent and non-rodent species, respectively, are widely used in preclinical studies. We studied the degradation of three structurally different drug esters in rat and dog serum. Moreover, the efficiency of selected enzyme inhibitors to prevent these degradations was investigated. Furthermore, we found indications of the identity of the drug-specific esterases by means of their inhibitor sensitivity as well as by protein purification and identification. The studied drugs were sagopilone, drospirenone, and methylprednisolone aceponate (MPA) all of which are used in (pre-)clinical drug development. The sagopilone-cleaving esterases in rat serum were inhibited by serine hydrolase inhibitors. We partly purified these esterases resulting in an activity yield of 5% and a purification factor of 472. Using matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS), the rat carboxylesterase isoenzyme ES-1 was identified in these fractions, thus pointing to its involvement in sagopilone cleavage. Drospirenone cleavage in rat serum was effected by butyrylcholinesterase (BChE) and paraoxonase 1 (PON1) as we deduced from the high efficacy of certain serine hydrolase and metallohydrolase inhibitors, respectively. Likewise, some inhibition characteristics implied that MPA was cleaved in rat serum by BChE and serine proteases. Partial purification of the MPA-specific esterases resulted in activity yields of 1-2%, exhibiting up to 10,000-fold purification. In dog serum, we found that sagopilone was not degraded which was in contrast to MPA and drospirenone. MPA degradation was mainly prevented by serine hydrolase inhibitors. We used a three-step purification to isolate the esterases cleaving MPA. This procedure resulted in an activity yield of 12% and 645-fold purification. By protein identification using liquid chromatography (LC)-electrospray ionization (ESI)-MS, we identified alpha(2)-macroglobulin (alpha(2)M) in the active fractions. We therefore assumed that serine hydrolases, probably butyrylcholinesterase, known to form esteratically active complexes with alpha(2)M, were responsible for MPA cleavage. In contrast, PON1 was assumed to be involved in drospirenone cleavage due to the high efficiency of metallohydrolase inhibitors. This indication was supported by the presence of PON1 in drospirenone-cleaving fractions as we found by affinity chromatography and Western immunoblotting for isolation and detection of PON1, respectively. The identity of the assumed cleaving enzymes remains, however, to be further studied. The inhibitors we found can serve as a tool for stabilizing drug ester compounds in biological samples ex vivo.

Poly(I:C) coated PLGA microparticles induce dendritic cell maturation.

Microparticles from poly(D,L-lactic-co-glycolic acid) [PLGA] are of steadily rising interest for the delivery of antigens to immune cells and the induction of a long-lasting immune response for vaccination or immunological tumor therapy. However, if the desired vaccine contains only weak antigens and fails to activate the antigen presenting cells (APC), the opposite effect, i.e., the induction of immunotolerance may be observed. Therefore, it was the aim of this study to show the ability of protein loaded PLGA microparticles to additionally carry a specific, surface-coated maturation signal to human dendritic cells (DC), i.e., the most potent APC. Polyinosine-polycytidylic acid [poly(I:C)], a ligand of Toll-like receptor (TLR) 3, was efficiently bound either in a single layer or a multilayer attempt to the surface of diethylaminoethyl dextran modified PLGA microparticles. These particles were effectively phagocytized by DC ex vivo and induced a maturation similar to that achieved with a cytokine cocktail or higher concentrations of soluble poly(I:C). In conclusion, the concept of surface coating of biodegradable microparticles with selected TLR ligands might successfully be used in DC-based cell therapies for cancer or in vaccination trials to induce DC maturation and specifically amplify the immunological response to encapsulated antigens.

Determination of rat serum esterase activities by an HPLC method using S-acetylthiocholine iodide and p-nitrophenyl acetate.

Establishing esterase assays allows the determination and comparison of esteratic activities of tissues of one organism and between organisms. We have developed a high-performance liquid chromatography (HPLC) assay for the determination of S-acetylthiocholine (ATC) and p-nitrophenyl acetate (NPA) hydrolyzing activities of rat serum esterases based on ion pair chromatography with on-line radiochemical and ultraviolet (UV) detection. ATC is a substrate for cholinesterases, whereas NPA is cleaved by a variety of esterases and other proteins (e.g., cholinesterases, paraoxonase, carboxylesterase, albumin). Both substrates were incubated, simultaneously or separately, with rat serum to explore potential interferences between the enzymatic hydrolyses of the compounds. The ratio of the peak area of the (14)C-labeled substrates to the total peak area of the substrates and their corresponding cleavage products was compared with the UV quantitation of ATC and p-nitrophenolate (NP), the cleavage product of NPA, measured at 230 and 350 nm, respectively. The peak identity of ATC and NP was confirmed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The reaction rates of the assays using one substrate or both, as well as using radiochemical or UV detection, were equal. Moreover, the correlation between rat serum volumes and reaction rates was shown for both substrates. In conclusion, one can (i) choose between the two detection methods reliably, (ii) take advantage of monitoring both substrate and product by using radiochemical detection, and (iii) combine both substrates to determine esterase activities in rat serum and probably other biological matrices.

Corneal permeation studies of everolimus microemulsion.

PURPOSE: To prevent corneal-graft rejection, the topical application of an immunosuppressive drug is an alternative to the systemic application of immunosuppressive drugs or corticosteroids, which may have adverse side effects. The aim of this study was to determine the permeation rate of everolimus through freshly isolated pig cornea (ex vivo). METHODS: A fluorescence polarization immunoassay with a commercially available assay system was used to quantify everolimus in the acceptor samples of the permeation tests. RESULTS: Everolimus is a poorly soluble drug and was, therefore, incorporated in an eye-administrable microemulsion. The stability of this microemulsion containing 0.1% (1 mg/mL) of the drug was satisfying over a period of 12 months. A concentration of 8.64 ng/mL was already reached 30 min after administration of the microemulsion to the cornea. CONCLUSIONS: This everolimus-containing microemulsion is a promising ocular formulation for preventing corneal-graft rejection.

Changes of the properties in the upper layers of human skin on treatment with models of different pharmaceutical formulations - an ex vivo ESR imaging study.

The noninvasive method of spectral-spatial electron spin resonance imaging (ESRI) was used to obtain a polarity map of human skin. The spin probes TEMPO, TEMPOL, and CAT-1, which are considered to act as drug representatives, were applied as reporter molecules. The polarity in skin layers was described by means of the changes of the hyperfine splitting constant A(iso), which itself is a reflection of interactions at a molecular level, and the effect of polarity on the spatial distribution of spin probes in skin samples was studied. Analyses of ESR tomograms of two-phase systems finalized in a simplified description for the empiric interpretation of values of the isotropic hyperfine coupling constants A(iso) of spin probes in different layers of human skin. The simplified statement provides values for the probability of interactions of water molecules with the NO group of spin probes. This allows conclusions concerning the state of hydration of the spin probes in different layers of the skin and introduces the spatial polarity function as additional and valuable information for existing skin models.

Formulation of sirolimus eye drops and corneal permeation studies.

The aim of this study was the development of eye drops with 1 mg/mL sirolimus and the evaluation of the drug's ability to permeate the freshly isolated pig cornea. Cyclodextrin solutions, liposomes, hydrotrope mixtures, poloxamer gels, and a microemulsion were tested for their suitability to dissolve the extremely hydrophobic drug sirolimus (solubility in water 2.6 microg/mL). The drug content in the formulations was determined by high-performance liquid chromatography, whereas this method is not sensitive enough for the quantification of therapeutic concentrations (7-12 ng/mL). Thus, the acceptor samples of the permeation tests were examined by microparticle enzyme immunoassay. A microemulsion is a suitable vehicle to prepare eye drops with sufficient sirolimus concentrations of 1 mg/mL in a formulation with acceptable tolerance and satisfactory stability over 12 months. However, the drug cannot permeate the intact cornea. After removal of the corneal epithelium, drug concentrations in the acceptor sample reach the lower limit of therapeutical levels. Conclusively, the present sirolimus eye drops might be promising therapeutic tools for the immunomodulatory treatment of ocular surface disorders, such as keratoconjunctivitis sicca, vernal conjunctivitis, or atopical blepharitis. They are not suitable to achieve therapeutic concentrations in the aqueous humour of patients with intact cornea.

ESR imaging investigations of two-phase systems.

The possibilities of electron spin resonance (ESR) and electron spin resonance imaging (ESRI) for investigating the properties of the spin probes TEMPO and TEMPOL in two-phase systems have been examined in the systems water/n-octanol, Miglyol/Miglyol, and Precirol/Miglyol. Phases and regions of the phase boundary could be mapped successfully by means of the isotropic hyperfine coupling constants, and, moreover, the quantification of rotational and lateral diffusion of the spin probes was possible. For the quantitative treatment of the micropolarity, a simplified empirical model was established on the basis of the Nernst distribution and the experimentally determined isotropic hyperfine coupling constants. The model does not only describe the summarized micropolarities of coexisting phases, but also the region of the phase boundary, where solvent molecules of different polarities and tendencies to form hydrogen bonds compete to interact with the NO group of the spin probe.

Antioxidative treatment inhibits the release of thrombogenic tissue factor from irradiation- and cytokine-induced endothelial cells.

OBJECTIVES: The aim of this study was to investigate the effect of the antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC) on the ionizing radiation (IR)- and tumor necrosis factor-alpha (TNF-alpha) induced tissue factor (TF) expression and its release from human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were irradiated with a single dose of either 5 Gy or 10 Gy and stimulated with TNF-alpha (10 ng/mL) in the presence or absence of PDTC and NAC, respectively. Quantitative real-time PCR, ELISA, and TF activity measurements were performed, including TF activity in the supernatant. Apoptosis was detected by flow cytometric active caspase-3 measurement and formation of reactive oxygen species (ROS) by chemiluminescence. RESULTS: We demonstrated a thus far uninvestigated persistent induction of TF expression in HUVECs after treatment with IR and TNF-alpha. Combined stimulation with IR and TNF-alpha led to an immense shedding of microparticle-associated TF which was positively correlated with apoptosis and ROS formation. Antioxidative pre-treatment reduced not only apoptosis and ROS formation, but also the release of thrombogenic microparticles. CONCLUSIONS: Antioxidative treatment inhibited apoptosis and shedding of microparticles, thereby reducing thrombogenicity. Thus, antioxidants may help to prevent late thrombosis after antiproliferative treatment when used in combination with anticoagulants.

Increased sensitivity of chitosan determination by a dye binding method.

Chitosan is a topic of current research in pharmaceutics, medicine, biotechnology, and beyond. This note describes an improved quantification of chitosan using the dye Cibacron Brilliant Red 3B-A. The method is sensitive and of a good reproducibility and linearity in the range of 10-80 microg/mL.

Stable cationic microparticles for enhanced model antigen delivery to dendritic cells.

The objectives of this work were (i) to prepare physically stable cationic microparticles and (ii) to study the impact of the surface properties on microparticle phagocytosis and the phenotype of dendritic cells (DC). Protein loaded biodegradable microparticles from poly(lactic-co-glycolic acid) [PLGA] were produced in a micromixer-based w/o/w solvent evaporation procedure. Anionic particles were obtained by using polyvinyl alcohol (PVA) as stabilizing agent; for cationic surfaces cetyltrimethylammonium bromide (CTAB) and chitosan/PVA or DEAE-dextran/PVA blends were evaluated. In phagocytosis studies human monocytes and monocyte-derived DC were incubated with microparticles and analysed by flow cytometry. While CTAB modified microparticles lost their positive charge and aggregated due to CTAB desorption from the particle surface, the modification with chitosan and DEAE-dextran resulted in stable microparticles without cell toxicity. Due to a very low endotoxin content, phagocytosis of anionic and cationic microparticles did not induce an upregulation of maturation-associated surface markers on DC. DEAE-dextran modified microparticles showed an enhanced model protein delivery into phagocytic cells. Overall, PLGA microparticles are suitable vehicles for protein delivery to DC, which might be used for DC-based cell therapies.

A novel agglutination test for antigen-specific detection of platelet antibodies.

A simple and rapid antigen-specific assay for the identification antibodies to platelets is lacking, yet. Red-dyed polystyrene microbeads were coated with monoclonal antibodies to various platelet glycoprotein complexes, and used for the detection of platelet autoantibodies and alloantibodies. The results were largely identical with those obtained by monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA). The new test is reliable yet less complex and time-consuming than the currently available assays, and it can be implemented in any routine laboratory.

A simple and practical assay for the antigen-specific detection of platelet antibodies.

BACKGROUND: The antigen-specific assays currently used for characterization of platelet (PLT)-reactive auto- and alloantibodies are too technically complex and impracticable for most routine laboratories. Here, a novel antigen-specific particle assay (ASPA) for PLTs similar to that of red blood cells is described. STUDY DESIGN AND METHODS: PLTs were solubilized and then incubated with red-dyed polystyrene particles coated with monoclonal antibodies (MoAbs) to various PLT glycoprotein complexes. These particles were directly tested for coating with autoantibodies (n = 8) or indirectly tested for serum autoantibodies (n = 33) or alloantibodies against HPA-1a (n = 4) or HPA-5b (n = 5). Serum samples from healthy blood donors (n = 100) served as negative controls. RESULTS: Negative reactions were clearly distinguishable from positive reactions, and the results of the particle assay were in concordance with those obtained by the standard MoAb-specific immobilization of PLT antigen assay (MAIPA) in all cases with alloanti-bodies. In three patients, only the ASPA was able to detect autoantibodies that were completely undetectable by the MAIPA. In contrast, in only one patient, the MAIPA detected autoantibodies that the ASPA failed to detect. CONCLUSION: In our opinion, the new ASPA is reliable, yet less complex and time-consuming than the currently available assays, and it can be implemented in any routine laboratory.

Microenvironmental pH and microviscosity inside pH-controlled matrix tablets: an EPR imaging study.

Incorporation of pH modifiers is a commonly used strategy to enhance the dissolution rate of weakly basic drugs from sustained release solid dosage forms. Electron paramagnetic resonance imaging (EPRI) was applied to spatially monitor pH(M) and the rotational correlation time (tau(R)), a parameter which is closely related to the surrounding microviscosity inside HPMC (hydroxypropylmethylcellulose) matrix tablets. Fumaric, citric, and succinic acid were employed as pH modifiers. 4-(methylamino)-2-ethyl-5,5-dimethyl-4-pyridine-2-yl-2,5-dihydro-1H-imidazole-1-oxyl (MEP) was used as spin label. Fumaric and citric acid reduced the pH(M) to equal extents in the initial phase. With the progress of hydration, the more soluble citric acid diffused out from the tablet resulting in an increase in pH(M), originating at the outer layers. In contrast, fumaric acid maintained a constantly reduced pH(M) inside the entire tablet. Due to its lower acidic strength, succinic acid did not reduce the pH(M) as effectively as the other pH modifiers used. The more water-soluble acids stimulated the water penetration into the matrix system, thereby rapidly decreasing tau(R). Once the matrix tablets were hydrated, the included pH modifiers influenced tau(R) insignificantly. EPRI, a novel approach for monitoring pH(M) and tau(R) non-invasively and spatially resolved, was used successfully for the optimization of an pH-controlled formulation.

Effect of lipid-containing, positively charged nanoemulsions on skin hydration, elasticity and erythema--an in vivo study.

Dry skin and other skin disorders such as atopic dermatitis are characterized by impaired stratum corneum (SC) barrier function and by an increase in transepidermal water loss (TEWL) leading to a decrease in skin hydration. The possibility that dermatological and cosmetic products containing SC lipids could play a part in the restoration of disturbed skin barrier function is of great interest in the field of dermatology and cosmetics. The aim of the present study was to evaluate the effect of positively charged oil/water nanoemulsions (PN) containing ceramide 3B and naturally found SC lipids (PNSC) such as ceramide 3, cholesterol, and palmitic acid on skin hydration, elasticity, and erythema. Creams of PNSC were compared to PN creams, to creams with negatively charged o/w nanoemulsion and SC lipids (NNSC) and to Physiogel cream, a SC lipid containing formulation, which is already on the market. The formulations (PN, PNSC, and NNSC) were prepared by high-pressure homogenization. After adding Carbopol 940 as thickener, particle size and stability of the creams were not significantly changed compared to the nanoemulsions. The studies were carried out on three groups, each with 14 healthy female test subjects between 25 and 50 years of age, using Corneometer 825, Cutometer SEM 575 and Mexameter 18 for measurements of skin hydration, elasticity, and erythema of the skin, respectively. The creams were applied regularly and well tolerated throughout the study. All formulations increased skin hydration and elasticity. There was no significant difference between PNSC and Physiogel. However, PNSC was significantly more effective in increasing skin hydration and elasticity than PN and NNSC indicating that phytosphingosine inducing the positive charge, SC lipids and ceramide 3B are crucial for the enhanced effect on skin hydration and viscoelasticity.

Preparation of protein loaded poly(D,L-lactide-co-glycolide) microparticles for the antigen delivery to dendritic cells using a static micromixer.

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 microm 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).

Design of a phytosphingosine-containing, positively-charged nanoemulsion as a colloidal carrier system for dermal application of ceramides.

Positively charged oil/water (o/w) nanoemulsions (PN) are effective vehicles to change the permeability of the skin. This study focused on the preparation and characterisation of phytosphingosine (PS) containing PN (PPN) which serve as colloidal carriers for the dermal application of ceramide IIIB (CIIIB) and the stratum corneum (SC) lipids (PPNSC) such as ceramide III (CIII), cholesterol, and palmitic acid. The investigations were conducted using appropriate emulsification and homogenisation processing conditions to optimise PPNSC with regard to droplet size, physical stability, and solubility of PS, CIII and CIIIB. A decrease in droplet size was observed through eight homogenisation cycles at a pressure of 500 bar and a temperature of 50 degrees C. Above these optimal values, an increase in droplet size was observed. PS and ceramides have low solubilities in oil and water. When Lipoid E-80 (LE80) was added to the oil phase, the solubility of PS and ceramides increased, indicating some interactions shown by DSC measurements. SC lipids and CIIIB could be successfully incorporated in PPN without producing any physical instability. The high stability of PPNSC is probably due to the presence of a hydrophilic (Tween 80) and a lipophilic surfactant (LE80), supported by the lipophilic cosurfactant PS, at the o/w interface. It was shown that PS was responsible for the positive charge and thus supported the high physical stability of PPNSC. This optimised emulsion was selected for further skin absorption evaluation.

In vivo ESR studies on subcutaneously injected multilamellar liposomes in living mice.

PURPOSE: An innovative, noninvasive, low-frequency electron spin resonance (ESR) spectroscopy method was applied and adapted to investigate the integrity of multilamellar liposomes from hydrogenated phospholipids after subcutaneous injection in living mice. Moreover, the fate of the injected liposomal preparations was examined, as well as the possibility to achieve a depot effect. METHODS: Highly concentrated solutions of the spin probe 2,2,6,6-tetramethyl-4-trimethylammoniumpiperidine-1-oxyl-iodide (CAT-1; 138 mM) were encapsulated in liposomes. They were characterized by laser diffraction, and the liberation of spin probe was investigated by ESR spectroscopy. RESULTS: Line shape changes allowed the differentiation between encapsulated and released CAT-1 after subcutaneous injection of liposomes. Multilamellar liposomes form a local depot at the site of injection. A sustained release of the spin probe from the depot was monitored by means of ESR. Whereas 40% of the spin probe was released within the first 96 h after administration, 60% remained in intact liposomes under the skin. No depot formation could be observed after injection of CAT-1 solutions, but a fast signal decrease due to systemic distribution and bioreduction of the nitroxide spin probe. CONCLUSIONS: Noninvasive analysis of liposomal integrity in living animals was successfully accomplished using a new L-Band ESR spectroscopy method. The liberation of CAT-1 from liposomes in vitro and in vivo was monitored by changes in the lineshape of ESR spectra and Heisenberg spin exchange. The significance of liposomal integrity for the formation of a localized drug depot effect was proved.