Regina Scherließ

The MTT assay as tool to evaluate and compare excipient toxicity in vitro on respiratory epithelial cells

There are not many excipients already approved in drug products for the use in the respiratory tract. In this study, a rapid in vitro screening procedure to assess and compare acute toxicity of soluble excipient substances on respiratory epithelial cells utilising the Calu-3 cell line is presented. The test substances are either dissolved in HBSS+HEPES buffer or are directly applied to the cellular surface. After 4h incubation, the substances are removed and the cell viability is assessed using an MTT assay. The tested excipients include polysorbate 20 and 80, lactose and povidone 30 as well as glycerol and propylene glycol as examples of excipients already being used in formulations for application in the respiratory tract. These substances are sorted according to their toxic effect and new excipients not yet used in the respiratory tract like HPMC can be classified in this scheme. With this, besides information from systemic toxicity tests, a first valuation of the acute toxic effect of the substance on respiratory epithelial cells is gained. This can aid in the choice of new excipients being necessary for modern respiratory formulations comprising new active compounds as biomolecules or new delivery strategies such as sustained or prolonged delivery.

In vivo evaluation of chitosan as an adjuvant in subcutaneous vaccine formulations.

Vaccines utilising pure antigens instead of whole pathogens and alternative administration routes require the use of potent adjuvants and effective antigen delivery systems. Chitosan has been reported to act as both an adjuvant as well as a matrix for delivery systems. Chitosan is a natural product produced predominantly from crab shell and commercially available preparations vary in molecular weight, degree of deacetylation and purity. In this study, the impact of chitosan characteristics (molecular weight, degree of deacetylation, particle size, viscosity and impurities) on adjuvant activity were examined. It could be shown that the degree of immune response differed if different chitosan qualities were used and this could be attributed to different characteristics of the chitosan qualities: the immunoadjuvant effect of chitosan probably is a result of an interplay between chemical properties such as molecular weight and degree of deacetylation and physical properties such as particle size and preparation technique, which impacts characteristics such as solubility and viscosity. Hence, the chitosan quality to be used as adjuvant in vaccine preparations needs to be selected carefully.

Stabilisation of proteins via mixtures of amino acids during spray drying.

Biologicals are often formulated as solids in an effort to preserve stability which generally requires stabilising excipients for proper drying. The purpose of this study was to screen amino acids and their combinations for their stabilising effect on proteins during spray drying. Catalase, as model protein, was spray dried in 1+1 or 1+2 ratios with amino acids. Some amino acids namely arginine, glycine and histidine showed good retention of catalase functionality after spray drying and subsequent storage stress. A 1+1 combination of arginine and glycine in a 1+2 ratio with catalase resulted in a tremendously good stabilising effect. Storage at high temperature/humidity also showed beneficial effects of this combination. To evaluate whether this was a general principle, these findings were transferred to an antigenic protein of comparable size and supramolecular structure (haemagglutinin) as well as to a smaller enzyme (lysozyme). Upon spray drying with the combination of amino acids it could be shown that both proteins remain more stable especially after storage compared to the unprotected protein. The combination of arginine and glycine is tailored to the needs of protein stabilisation during spray drying and may hence be utilised in dry powder formulation of biomolecules with superior stability characteristics.

Induction of protective immunity against H1N1 influenza A(H1N1)pdm09 with spray-dried and electron-beam sterilised vaccines in non-human primates

Currently, the need for cooled storage and the impossibility of terminal sterilisation are major drawbacks in vaccine manufacturing and distribution. To overcome current restrictions a preclinical safety and efficacy study was conducted to evaluate new influenza A vaccine formulations regarding thermal resistance, resistance against irradiation-mediated damage and storage stability. We evaluated the efficacy of novel antigen stabilizing and protecting solutions (SPS) to protect influenza A(H1N1)pdm09 split virus antigen under experimental conditions in vitro and in vivo. Original or SPS re-buffered vaccine (Pandemrix) was spray-dried and terminally sterilised by irradiation with 25 kGy (e-beam). Antigen integrity was monitored by SDS-PAGE, dynamic light scattering, size exclusion chromatography and functional haemagglutination assays. In vitro screening experiments revealed a number of highly stable compositions containing glycyrrhizinic acid (GA) and/or chitosan. The most stable composition was selected for storage tests and in vivo assessment of seroconversion in non-human primates (Macaca fascicularis) using a prime-boost strategy. Redispersed formulations with original adjuvant were administered intramuscularly. Storage data revealed high stability of protected vaccines at 4°C and 25°C, 60% relative humidity, for at least three months. Animals receiving original Pandemrix exhibited expected levels of seroconversion after 21 days (prime) and 48 days (boost) as assessed by haemagglutination inhibition and microneutralisation assays. Animals vaccinated with spray-dried and irradiated Pandemrix failed to exhibit seroconversion after 21 days whereas spray-dried and irradiated, SPS-protected vaccines elicited similar seroconversion levels to those vaccinated with original Pandemrix. Boost immunisation with SPS-protected vaccine resulted in a strong increase in seroconversion but had only minor effects in animals treated with non SPS-protected vaccine. In conclusion, utilising the SPS formulation technology, spray-drying and terminal sterilisation of influenza A(H1N1)pdm09 split virus vaccine is feasible. Findings indicate the potential utility of such formulated vaccines e.g. for needle-free vaccination routes and delivery to countries with uncertain cold chain facilities.

First in vivo evaluation of particulate nasal dry powder vaccine formulations containing ovalbumin in mice.

In this study three different dry powder vaccine formulations containing the model antigen ovalbumin were evaluated for their immune effects after nasal administration to C57Bl/6 mice in an adoptive cell transfer model. The formulations were chitosan nanoparticles in a mannitol matrix, chitosan microparticles and agarose nanoparticles in a mannitol matrix. Dry powder administration to mice was well tolerated and did not result in any adverse reactions. No translocation of the dry powder formulations to the lung could be detected. The local cellular immune response in the cervical lymph nodes was modest and only for the chitosan microparticles and the agarose nanoparticles was there a significant difference compared to s.c. injection of ovalbumin in alum. No humoral response could be measured after nasal administration. The results provide some evidence that nasal administration of dry powder formulations can stimulate an immune response, but the response was modest. This is probably due to a low antigen dose and low immunogenicity of the formulations. Further studies will aim at enhancing the antigen load and improving adjuvant activity.

Solvent Free Fabrication of Micro and Nanostructured Drug Coatings by Thermal Evaporation for Controlled Release and Increased Effects

Nanostructuring of drug delivery systems offers many promising applications like precise control of dissolution and release kinetics, enhanced activities, flexibility in terms of surface coatings, integration into implants, designing the appropriate scaffolds or even integrating into microelectronic chips etc. for different desired applications. In general such kind of structuring is difficult due to unintentional mixing of chemical solvents used during drug formulations. We demonstrate here the successful solvent-free fabrication of micro-nanostructured pharmaceutical molecules by simple thermal evaporation (TE). The evaporation of drug molecules and their emission to a specific surface under vacuum led to controlled assembling of the molecules from vapour phase to solid phase. The most important aspects of thermal evaporation technique are: solvent-free, precise control of size, possibility of fabricating multilayer/hybrid, and free choice of substrates. This could be shown for twenty eight pharmaceutical substances of different chemical structures which were evaporated on surfaces of titanium and glass discs. Structural investigations of different TE fabricated drugs were performed by atomic force microscopy, scanning electron microscopy and Raman spectroscopy which revealed that these drug substances preserve their structurality after evaporation. Titanium discs coated with antimicrobial substances by thermal evaporation were subjected to tests for antibacterial or antifungal activities, respectively. A significant increase in their antimicrobial activity was observed in zones of inhibition tests compared to controls of the diluted substances on the discs made of paper for filtration. With thermal evaporation, we have successfully synthesized solvent-free nanostructured drug delivery systems in form of multilayer structures and in hybrid drug complexes respectively. Analyses of these substances consolidated that thermal evaporation opens up the possibility to convert dissoluble drug substances into the active forms by their transfer onto a specific surface without the need of their prior dissolution.

Spatial separation of the processing and MHC class I loading compartments for cross-presentation of the tumor-associated antigen HER2/neu by human dendritic cells

Cross-presentation is the process by which professional antigen presenting cells (APCs) (B cells, dendritic cells (DCs) and macrophages) present endocytosed antigens (Ags) via MHC-I to CD8+ T cells. This process is crucial for induction of adaptive immune responses against tumors and infected cells. The pathways and cellular compartments involved in cross-presentation are unresolved and controversial. Among the cells with cross-presenting capacity, DCs are the most efficient, which was proposed to depend on prevention of endosomal acidification to block degradation of the epitopes. Contrary to this view, we show in this report that some cargoes induce strong endosomal acidification following uptake by human DCs, while others not. Moreover, processing of the tumor-associated antigen HER2/neu delivered in nanoparticles (NP) for cross-presentation of the epitope HER2/neu369–377 on HLA-A2 depended on endosomal acidification and cathepsin activity as well as proteasomes, and newly synthesized HLA class I. However, the HLA-A*0201/HER2/neu369–377 complexes were not found in the endoplasmic reticulum (ER) nor in endolysosomes but in hitherto not described vesicles. The data thus indicate spatial separation of antigen processing and loading of MHC-I for cross-presentation: antigen processing occurs in the uptake compartment and the cytosol whereas MHC-I loading with peptide takes place in a distinct subcellular compartment. The findings further elucidate the cellular pathways involved in the cross-presentation of a full-length, clinically relevant tumor-associated antigen by human DCs, and the impact of the vaccine formulation on antigen processing and CD8+ T cell induction.

Powder flow analysis: A simple method to indicate the ideal amount of lactose fines in dry powder inhaler formulations

Many efforts have been made in the past to understand the function of lactose fines which are given as a ternary component to carrier-based dry powder inhaler formulations. It is undisputed that fines can significantly improve the performance of such formulations, but choosing the right amount of fines is a crucial point, because too high concentrations can have negative effects on the dispersion performance. The aim of this study was to indicate the optimal concentration of fines with a simple test method. For this purpose, mixtures with salbutamol sulfate and two different lactose carriers were prepared with a high shear mixer, measured with a FT4 powder rheometer and tested for fine particle delivery with two different inhaler devices. A correlation between the fluidization energy, measured with the aeration test set up, and the fine particle fractions (FPF) could be proven. This also applied for the aeration ratio, as well as the permeability of the powder samples. In addition, drug-free mixtures hardly differed in their rheological properties from mixtures containing the active pharmaceutical ingredient (API), which indicates that the method could be suitable for cost-saving screening trials. Furthermore, important aspects that explain the function of fines, such as the saturation of active sites, the formation of agglomerates and an increase in fluidization energy, could be shown in this study.

Antigen delivery via hydrophilic PEG-b-PAGE-b-PLGA nanoparticles boosts vaccination induced T cell immunity.

Here, we evaluate the use of hydrophilic PEG-b-PAGE-b-PLGA (PPP) for the preparation of antigen loaded nanoparticles (NPs) as a platform for prophylactic vaccination. To investigate the suitability of PPP-NPs for antigen delivery, we used the double emulsion evaporation technique to prepare NPs of different sizes, antigen-loading efficiencies and -release kinetics for the model antigen Ovalbumin (OVA). Prior to applying the PPP-NPs in biological in vitro or in vivo models, all materials were tested for absence of cytotoxicity and endotoxins. While the uptake of NPs in antigen presenting cells was size but not polymer dependent, the efficiency of cross presentation of NP-associated antigen on MHC I molecules for CD8 T cell activation depended on the polymer type. T cell activation by antigen-presenting cells was significantly increased in vitro if antigen was delivered via PPP NPs compared to PLGA NPs or soluble OVA, although antigen content was the same in all tested formulations. Subcutaneous application of PPP-OVA-NPs even without adjuvants led to generation of potent CD8 T cell-mediated OVA-specific cytotoxicity in vivo that was more pronounced than after application of OVA alone or PLGA-OVA-NPs. Our data suggest that PPP-NPs can serve as platform for antigen-delivery in future vaccination formulations. Although PPP-NPs already bear intrinsic adjuvant-function, the complementation with TLR ligands loaded inside NPs may further strengthen the immune response to a point, where it might be possible to use it as a therapeutic vaccine to break immune tolerance in chronic disease states.

Evaluation and prediction of powder flowability in pharmaceutical tableting

Abstract The focus of this study is to establish a characterization method determining the powder flowability in context of tableting. At first, flowability of different materials is measured using the ring shear tester, and its prediction from particle size is established. Next, the model die-filling system is presented which is a modified version of previous studies. Using this system, flowability of different materials is measured at varying die speeds. A new curve fit to assess die fill ratio vs die speed is suggested improving predictability, and a novel flowability metric, “Die Fill Index” (DFI), is derived. The DFI is appropriate to describe flowability for most of the tested materials, and sensitivity of a material with respect to tableting speed. A correlation is generated predicting DFI from particle size. Additionally, it is shown that model die filling is the preferable method to assess flowability for tableting compared to ring shear tester.