Development of a sprayable hydrogel formulation for the skin application of therapeutic antibodies.

A formulation of an antibody with antibacterial properties for topical use on Staphylococcal skin infections was developed and characterized. The best formulation was obtained with 1.5% (w/v) sodium carboxymethylcellulose containing 10 mg/ml immunoglobulin. Spraying forces and rheological behavior were measured in order to characterize the hydrogel formulation. The percentage of antibody aggregates in gel as well as the antibody release, folding and target binding properties of the released antibody were analyzed to proof an acceptable shelf life and no significant changes in the activity of the antibody over time. No microbial contamination was observed in the chosen non-airless application container. Functional testing of the topical skin formulation was performed with an ex vivo biopsy culture model of dog skin. Histological analysis indicated efficacy in protection from Staphylococcus mediated skin damage and antibody delivery restricted to the epidermal surface. The results demonstrate that this hydrogel is suitable for cutaneous antibody applications in the medical field.

Structure determination and crystal chemistry of large repeat mixed-layer hexaferrites.

Hexaferrites are an important class of magnetic oxides with applications in data storage and electronics. Their crystal structures are highly modular, consisting of Fe- or Ba-rich close-packed blocks that can be stacked in different sequences to form a multitude of unique structures, producing large anisotropic unit cells with lattice parameters typically >100 Šalong the stacking axis. This has limited atomic-resolution structure solutions to relatively simple examples such as Ba2Zn2Fe12O22, whilst longer stacking sequences have been modelled only in terms of block sequences, with no refinement of individual atomic coordinates or occupancies. This paper describes the growth of a series of complex hexaferrite crystals, their atomic-level structure solution by high-resolution synchrotron X-ray diffraction, electron diffraction and imaging methods, and their physical characterization by magnetometry. The structures include a new hexaferrite stacking sequence, with the longest lattice parameter of any hexaferrite with a fully determined structure.

Preliminary evaluation of cytosine-phosphate-guanine oligodeoxynucleotides bound to gelatine nanoparticles as immunotherapy for canine atopic dermatitis.

Cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN) are a promising new immunotherapeutic treatment option for canine atopic dermatitis (AD). The aim of this uncontrolled pilot study was to evaluate clinical and immunological effects of gelatine nanoparticle (GNP)-bound CpG ODN (CpG GNP) on atopic dogs. Eighteen dogs with AD were treated for 8 weeks (group 1, n=8) or 18 weeks (group 2, n=10). Before inclusion and after 2 weeks, 4 weeks, 6 weeks (group 1+2), 8 weeks, 12 weeks and 16 weeks (group 2) 75 µg CpG ODN/dog (bound to 1.5 mg GNP) were injected subcutaneously. Pruritus was evaluated daily by the owner. Lesions were evaluated and serum concentrations and mRNA expressions of interferon-γ, tumour necrosis factor-α, transforming growth factor-ß, interleukin (IL) 10 and IL-4 (only mRNA expression) were determined at inclusion and after 8 weeks (group 1+2) and 18 weeks (group 2). Lesions and pruritus improved significantly from baseline to week 8. Mean improvements from baseline to week 18 were 23 per cent and 44 per cent for lesions and pruritus, respectively, an improvement of ≥50 per cent was seen in six out of nine and three out of six dogs, respectively. IL-4 mRNA expression decreased significantly. The results of this study show a clinical improvement of canine AD with CpG GNP comparable to allergen immunotherapy. Controlled studies are needed to confirm these findings.

Target interaction profiling of midostaurin and its metabolites in neoplastic mast cells predicts distinct effects on activation and growth.

Proteomic-based drug testing is an emerging approach to establish the clinical value and anti-neoplastic potential of multikinase inhibitors. The multikinase inhibitor midostaurin (PKC412) is a promising new agent used to treat patients with advanced systemic mastocytosis (SM). We examined the target interaction profiles and the mast cell (MC)-targeting effects of two pharmacologically relevant midostaurin metabolites, CGP52421 and CGP62221. All three compounds, midostaurin and the two metabolites, suppressed IgE-dependent histamine secretion in basophils and MC with reasonable IC(50) values. Midostaurin and CGP62221 also produced growth inhibition and dephosphorylation of KIT in the MC leukemia cell line HMC-1.2, whereas the second metabolite, CGP52421, which accumulates in vivo, showed no substantial effects. Chemical proteomic profiling and drug competition experiments revealed that midostaurin interacts with KIT and several additional kinase targets. The key downstream regulator FES was recognized by midostaurin and CGP62221, but not by CGP52421 in MC lysates, whereas the IgE receptor downstream target SYK was recognized by both metabolites. Together, our data show that the clinically relevant midostaurin metabolite CGP52421 inhibits IgE-dependent histamine release, but is a weak inhibitor of MC proliferation, which may have clinical implications and may explain why mediator-related symptoms improve in SM patients even when disease progression occurs.

Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

REASONS FOR PERFORMING STUDY: New therapeutic strategies to modulate immune responses in human and equine allergic airway diseases are under extensive investigation. Stimulation of Treg cells with immune modulating agents is a novel therapeutic option. OBJECTIVES: The aim of this field study was to compare the effects of a nebulised nanoparticulate CpG immunotherapy (CpG-GNP) with and without specific allergens. STUDY DESIGN: Longitudinal clinical study comparing 2 therapeutic options. METHODS: Twenty RAO-affected horses were divided into 2 treatment groups (CpG alone and CpG with allergens). Two specific allergens were selected for each horse according to anamnesis and a functional in vitro test. Treatments were given by nebulisation 7 times and the horses were examined 3 times: baseline (I), after the treatment course (II), and after 6 weeks later (III). Clinical parameters, indirect intrapleural measurement, arterial blood gas, amount of tracheal mucus and neutrophil percentage were evaluated. RESULTS: CpG alone resulted in a significant improvement in clinical parameters and a significant reduction of tracheal mucus after treatment and at 6 weeks post treatment. After CpG plus specific allergens, there was significant improvement of 70% of examined parameters. However, there were no significant differences in the results compared with CpG-GNP treatment alone. CONCLUSIONS: There were no significant differences between treatment groups. CpG-GNP immunotherapy alone produced a potent and persistent effect on allergic and inflammatory parameters and may have potential as for treatment of equine and human allergic inflammatory airway diseases. Ethical animal research: The study was approved by the regional legal agency for animal experiments of the Government of Bavaria, Germany (No. 55.2-1-54-2531-31-10). Owners gave informed consent for their horses' inclusion in the study. Sources of funding: Partly supported by the Deutsche Forschungsgemeinschaft (DFG) (Germany) (GE'2044/4-1). The AeroNeb Go™ vibrating mesh nebuliser (Aerogen, Galway, Ireland) was sponsored by Inspiration Medical (Bochum, Germany). Competing interests: None declared.

A simple methodology for characterization of germanium coaxial detectors by using Monte Carlo simulation and evolutionary algorithms.

The determination in a sample of the activity concentration of a specific radionuclide by gamma spectrometry needs to know the full energy peak efficiency (FEPE) for the energy of interest. The difficulties related to the experimental calibration make it advisable to have alternative methods for FEPE determination, such as the simulation of the transport of photons in the crystal by the Monte Carlo method, which requires an accurate knowledge of the characteristics and geometry of the detector. The characterization process is mainly carried out by Canberra Industries Inc. using proprietary techniques and methodologies developed by that company. It is a costly procedure (due to shipping and to the cost of the process itself) and for some research laboratories an alternative in situ procedure can be very useful. The main goal of this paper is to find an alternative to this costly characterization process, by establishing a method for optimizing the parameters of characterizing the detector, through a computational procedure which could be reproduced at a standard research lab. This method consists in the determination of the detector geometric parameters by using Monte Carlo simulation in parallel with an optimization process, based on evolutionary algorithms, starting from a set of reference FEPEs determined experimentally or computationally. The proposed method has proven to be effective and simple to implement. It provides a set of characterization parameters which it has been successfully validated for different source-detector geometries, and also for a wide range of environmental samples and certified materials.

Quorum-sensing linked RNA interference for dynamic metabolic pathway control in Saccharomyces cerevisiae.

Some of the most productive metabolic engineering strategies involve genetic modifications that cause severe metabolic burden on the host cell. Growth-limiting genetic modifications can be more effective if they are 'switched on' after a population growth phase has been completed. To address this problem we have engineered dynamic regulation using a previously developed synthetic quorum sensing circuit in Saccharomyces cerevisiae. The circuit autonomously triggers gene expression at a high population density, and was linked with an RNA interference module to enable target gene silencing. As a demonstration the circuit was used to control flux through the shikimate pathway for the production of para-hydroxybenzoic acid (PHBA). Dynamic RNA repression allowed gene knock-downs which were identified by elementary flux mode analysis as highly productive but with low biomass formation to be implemented after a population growth phase, resulting in the highest published PHBA titer in yeast (1.1mM).

Nanoparticulate CpG immunotherapy in RAO-affected horses: phase I and IIa study.

BACKGROUND: Recurrent airway obstruction (RAO), an asthma-like disease, is 1 of the most common allergic diseases in horses in the northern hemisphere. Hypersensitivity reactions to environmental antigens cause an allergic inflammatory response in the equine airways. Cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODN) are known to direct the immune system toward a Th1-pathway, and away from the pro-allergic Th2-line (Th2/Th1-shift). Gelatin nanoparticles (GNPs) are biocompatible and biodegradable immunological inert drug delivery systems that protect CpG-ODN against nuclease degeneration. Preliminary studies on the inhalation of GNP-bound CpG-ODN in RAO-affected horses have shown promising results. OBJECTIVES: The aim of this study was to evaluate the clinical and immunological effects of GNP-bound CpG-ODN in a double-blinded, placebo-controlled, prospective, randomized clinical trial and to verify a sustained effect post-treatment. ANIMALS AND METHODS: Twenty-four RAO-affected horses received 1 inhalation every 2 days for 5 consecutive administrations. Horses were examined for clinical, endoscopic, cytological, and blood biochemical variables before the inhalation regimen (I), immediately afterwards (II), and 4 weeks post-treatment (III). RESULTS: At time points I and II, administration of treatment rather than placebo corresponded to a statistically significant decrease in respiratory effort, nasal discharge, tracheal secretion, and viscosity, AaDO2 and neutrophil percentage, and an increase in arterial oxygen pressure. CONCLUSION AND CLINICAL IMPORTANCE: Administration of a GNP-bound CpG-ODN formulation caused a potent and persistent effect on allergic and inflammatory-induced clinical variables in RAO-affected horses. This treatment, therefore, provides an innovative, promising, and well-tolerated strategy beyond conventional symptomatic long-term therapy and could serve as a model for asthma treatment in humans.

A chemical biology approach identifies AMPK as a modulator of melanoma oncogene MITF.

The microphthalmia-associated transcription factor (MITF) is indispensable for the viability of melanocytic cells, is an oncogene in melanoma and has a cell type-specific expression pattern. As the modulation of MITF activity by direct chemical targeting remains a challenge, we assessed a panel of drugs for their ability to downregulate MITF expression or activity by targeting its upstream modulators. We found that the multi-kinase inhibitors midostaurin and sunitinib downregulate MITF protein levels. To identify the target molecules shared by both the drugs in melanocytic cells, a chemical proteomic approach was applied and AMP-activated kinase (AMPK) was identified as the relevant target for the observed phenotype. RNA interference and chemical inhibition of AMPK led to a decrease in MITF protein levels. Reduction of MITF protein levels was the result of proteasomal degradation, which was preceded by enhanced phosphorylation of MITF mediated by ERK. As expected, downregulation of MITF protein levels by AMPK inhibition was associated with decreased viability. Together, these results identify AMPK as an important regulator for the maintenance of MITF protein levels in melanocytic cells.

Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins III: collapse during storage at elevated temperatures.

This study investigates the effect of lyophilizate collapse on the stability of pharmaceutical proteins. Recently, it was shown that collapse during freeze-drying has no major negative impact on protein stability during storage at elevated temperatures when compared to non-collapsed cakes [1,2]. In this part of the study, lyophilizates that collapsed during the freeze-drying process were compared to cakes that were initially non-collapsed but collapsed during subsequent storage under accelerated stress conditions. Collapsed and non-collapsed lyophilizates of identical formulation and comparable residual moisture levels, containing a monoclonal IgG antibody, were stored at 40 °C and 50 °C for up to 3 months. Protein stability was monitored using a comprehensive set of analytical techniques assessing the formation of soluble and insoluble aggregates as well as protein conformation. The properties of the freeze-dried cake, namely the glass transition temperature, excipient crystallinity, sucrose degradation, reconstitution behavior, and the residual moisture content, were analyzed as well. The incorporated protein was significantly better stabilized in cakes that collapsed during the freeze-drying process when compared to lyophilizates that collapsed during subsequent storage. This effect can be related to the onset of crystallization and hydrolysis of the stabilizer and non-enzymatic browning.

Controlled ice nucleation in the field of freeze-drying: fundamentals and technology review.

In the scientific community as well as in commercial freeze-drying, controlled ice nucleation has received a lot of attention because increasing the ice nucleation temperature can significantly reduce primary drying duration. Furthermore, controlled ice nucleation enables to reduce the randomness of the ice nucleation temperature, which can be a serious scale-up issue during process development. In this review, fundamentals of ice nucleation in the field of freeze-drying are presented. Furthermore, the impact of controlled ice nucleation on product qualities is discussed, and methods to achieve controlled ice nucleation are presented.

A novel strategy of mesenchymal stem cells delivery in the uterus of mares with endometrosis.

Mesenchymal stem cells (MSCs), because of their immunomodulation and trophic activities, in addition to their capacity to regenerate damaged tissues, have potential for treatment of many diseases. The success of stem cell therapies depends, in part, on the method of cell delivery, which should provide wide cell distribution and homing in to injured sites. The objective of the present study was to developing a novel strategy for delivery of MSCs into the uterus of mares with endometrosis (degenerative alteration of uterine glands and surrounding stroma). Endometrosis was confirmed in all mares (N = 6) used in this study. To trace multipotent equine adipose tissue-derived MSCs (eAT-MSCs) in endometrial tissue, before transplantation, cells were stained with a fluorescent dye. During a synchronized estrus, the eAT-MSCs (2 × 10(7) diluted in 20 mL of sodium chloride 0.9%) were inoculated into uterus using a simple technique, similar to artificial insemination (AI) in mares. At 7 and 21 days after transplantation, homing of fluorescently labeled eAT-MSCs was observed by confocal microscopy of uterine biopsies collected from the uterine body and in both uterine horns, including glandular and periglandular spaces, in three of four treated mares. Herein, we propose a new method of MSCs delivery in uterus of mares with endometrosis, which was minimally invasive and technically simple.

KRAS mutation analysis on low percentage of colon cancer cells: the importance of quality assurance.

KRAS mutation testing is mandatory for patients with metastatic colorectal cancer who are eligible for treatment with an epidermal growth factor receptor targeting agent, since tumors with a mutation are not sensitive to the drug. Several methods for mutation testing are in use and the need for external quality assurance has been demonstrated. An often little addressed but important issue in external quality assurance schemes is a low percentage of tumor cells in the test samples, where the analytical sensitivity of most tests becomes critical. Using artificial samples based on a mixture of cell lines with known mutation status of the KRAS gene, we assessed the reliability of a series of commonly used methods (Sanger sequencing, high resolution melting, pyrosequencing, and amplification refractory mutation system-polymerase chain reaction) on samples with 0, 2.5, 5, 10, and 15 % mutated cells. Nine laboratories throughout Europe participated and submitted a total of ten data sets. The limit of detection of each method differed, ranging from >15-5 % tumor cells. All methods showed a decreasing correct mutation call rate proportionally with decreasing percentage of tumor cells. Our findings indicate that laboratories and clinicians need to be aware of the decrease in correct mutation call rate proportionally with decreasing percentage of tumor cells and that external quality assurance schemes need to address the issue of low tumor cell percentage in the test samples.

Tratamiento combinado de campo magnético, láser, masaje y ejercicio en la parálisis facial periférica idiopática / Combined treatment of magnetic field, laser, massage and exercise in idiopathic peripheral facial paralysis

Introducción Las diferencias de criterios en cuanto a la utilización de agentes físicos en la parálisis facial periférica ha fomentado la realización de diversas investigaciones que permiten proponer nuevas alternativas de tratamiento, como son el uso de nuevos agentes físicos que han probado su eficacia en otras patologías con similitud de fisiopatogenia. Los tratamientos actuales buscan acelerar la mejoría y aumentar el porcentaje de personas recuperadas. Objetivos Evaluar la efectividad que tendría el tratamiento con campo magnético y láser en pacientes con parálisis facial periférica idiopática con menos de una semana de evolución, desde la instalación de los síntomas, sin tratamiento fisioterapéutico previo. Métodos Se realizó un estudio prospectivo, experimental, aleatorizado y controlado a simple ciego en el Servicio de Terapia Física y Rehabilitación del Instituto de Neurología y Neurocirugía (INN) en La Habana, en un periodo comprendido entre enero de 2009 y enero de 2011.ResultadosLos pacientes fueron evaluados al inicio, al mes y a los 3 meses del tratamiento, después de lo cual se comprobó una recuperación más rápida en el grupo experimental con respecto al grupo control, al mes (p=0,004075) y (Z=2,87232) y a los 3 meses (p=0,007859) y (Z=2,65810).Conclusiones Los pacientes que recibieron la terapia combinada de campo magnético, láser, masaje y ejercicios tuvieron una recuperación más rápida respecto al grupo que solo recibió masaje y ejercicio, lo cual corrobora que la terapia propuesta es efectiva en los pacientes con parálisis facial periférica idiopática(AU)

Introduction The different criteria regarding the use of physical agents in peripheral facial paralysis has fostered the performance of various investigations that make it possible to propose new treatment alternatives, such as the use of new physical agents that have proven effective in other conditions with similarity to pathogenesis. Current treatments are aimed at accelerating improvement and increasing the number of people who recovered. Objectives To evaluate the effectiveness that the low frequency electromagnetic field and low level laser therapy would have in patients with idiopathic peripheral facial paralysis with less than one week's evolution from the initiation of the symptoms, without previous physiotherapy. Methods A prospective, experimental, randomized, controlled single blind study was conducted in the Department of Physiotherapy and Rehabilitation, Institute of Neurology and Neurosurgery (INN) in Havana, in a period between January 2009 and January 2011.ResultsPatients were evaluated at baseline, one month and three months of treatment, after which faster recovery was verified in the experimental group compared to the control group at one month (P=.004075) and (Z=2.87232) and at three months (P=.007859) and (Z=2.65810).Conclusions The patients who received combined therapy, magnetic field, laser, massage and exercise had a faster recovery than the group that received massage and exercise alone. This collaborates that the proposed therapy is effective in patients with idiopathic peripheral facial paralysis (AU)

Optimization of a pharmaceutical freeze-dried product and its process using an experimental design approach and innovative process analyzers.

The aim of the present study was to examine the possibilities/advantages of using recently introduced in-line spectroscopic process analyzers (Raman, NIR and plasma emission spectroscopy), within well-designed experiments, for the optimization of a pharmaceutical formulation and its freeze-drying process. The formulation under investigation was a mannitol (crystalline bulking agent)-sucrose (lyo- and cryoprotector) excipient system. The effects of two formulation variables (mannitol/sucrose ratio and amount of NaCl) and three process variables (freezing rate, annealing temperature and secondary drying temperature) upon several critical process and product responses (onset and duration of ice crystallization, onset and duration of mannitol crystallization, duration of primary drying, residual moisture content and amount of mannitol hemi-hydrate in end product) were examined using a design of experiments (DOE) methodology. A 2-level fractional factorial design (2(5-1)=16 experiments+3 center points=19 experiments) was employed. All experiments were monitored in-line using Raman, NIR and plasma emission spectroscopy, which supply continuous process and product information during freeze-drying. Off-line X-ray powder diffraction analysis and Karl-Fisher titration were performed to determine the morphology and residual moisture content of the end product, respectively. In first instance, the results showed that - besides the previous described findings in De Beer et al., Anal. Chem. 81 (2009) 7639-7649 - Raman and NIR spectroscopy are able to monitor the product behavior throughout the complete annealing step during freeze-drying. The DOE approach allowed predicting the optimum combination of process and formulation parameters leading to the desired responses. Applying a mannitol/sucrose ratio of 4, without adding NaCl and processing the formulation without an annealing step, using a freezing rate of 0.9°C/min and a secondary drying temperature of 40°C resulted in efficient freeze-drying supplying end products with a residual moisture content below 2% and a mannitol hemi-hydrate content below 20%. Finally, using Monte Carlo simulations it became possible to determine how varying the factor settings around their optimum still leads to fulfilled response criteria, herewith having an idea about the probability to exceed the acceptable response limits. This multi-dimensional combination and interaction of input variables (factor ranges) leading to acceptable response criteria with an acceptable probability reflects the process design space.

Transglutaminase: new insights into gelatin nanoparticle cross-linking.

Gelatin nanoparticles (GNPs) have demonstrated to be beneficial as a biodegradable and biocompatible delivery system. So far, nanoparticles prepared by the two-step desolvation technique were subsequently cross-linked by glutaraldehyde to guarantee storage stability. Although in vivo and in vitro toxicological studies have not revealed any glutaraldehyde related undesired effects, an alternative to chemical cross-linking could ease future clinical use in humans. Therefore, the recombinant enzyme microbial transglutaminase was used to examine its cross-linking abilities in nanoparticle production. Various process parameters, such as incubation time, temperature, medium, pH and the particle purification were evaluated regarding their impact on particle size and its distribution. Cross-linking reactions were best at 25°C using an ion-free solvent at a neutral pH and have been terminated after 12 h. Preliminary storage stability testing indicated adequate consistency of particle size and particle distribution making transglutaminase a potential candidate for glutaraldehyde substitution in future GNP production.

Vesicular phospholipid gel-based depot formulations for pharmaceutical proteins: development and in vitro evaluation.

The object of this study was to evaluate the potential of vesicular phospholipid gels (VPGs) as an alternative delivery system for therapeutic proteins. Therefore, the model protein erythropoietin (EPO) was incorporated into various VPG formulations by dual asymmetric centrifugation. In order to characterize and to quantify the incorporated protein, different extraction protocols were investigated. Among several detergents and organic solvents an extraction method applying chloroform was found most suitable. SDS-PAGE analysis of EPO extracted from VPGs revealed excellent protein stability which was maintained in the VPGs' matrix after an incorporation process with dual asymmetric centrifuge. Irrespective of the investigated formulation all VPGs delivered the protein over prolonged periods of time at close to linear kinetics without any burst effect. Both the lipid content and the lipid charge had a strong influence on the release behavior. For instance formulations based on 300 mg/g lipid delivered 83% EPO after 280 h while gels based on 550 mg/g lipid liberated 64% within 400 h. From the parallelism of release and erosion kinetics found for all formulations it was concluded that erosion rather than diffusion is the dominant release controlling mechanism for these macromolecule-loaded VPGs. For the first time the present study presents VPGs as promising alternative depot systems for protein drugs.

Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins I: stability after freeze-drying.

The objective of this work was to investigate the effect of cake collapse during freeze-drying on the stability of protein lyophilizates containing a monoclonal IgG(1)-antibody or a second pharmaceutically relevant protein, referred to as PA01. In addition, L-lactic dehydrogenase was investigated because of its well-documented sensitivity towards freeze-drying stresses. Collapse was induced by two different means. First, by varying the ratio of the crystalline bulking agent mannitol to the amorphous stabilizer sucrose, different extents of collapsed cakes were generated. Second, formulations were freeze-dried using an aggressive collapse-cycle and a conventional freeze-drying protocol and collapsed and noncollapsed cakes of identical formulation were produced. Lyophilizates were analyzed using a comprehensive set of analytical techniques to monitor protein stability in terms of formation of soluble and insoluble aggregates, the biological activity and the conformational stability. The stability of excipients, namely the glass transition temperature, crystallinity, reconstitution behavior, and the residual moisture content was analyzed as well. In addition, the extent of collapse was quantified using the decrease of the specific surface area (SSA). Collapsed cakes had comparable residual moisture levels to noncollapsed lyophilizates. Reconstitution times were not increased. Protein stability was not relevantly different between collapsed and noncollapsed cakes.