2013

Grants awarded in 2013

A total of SEK 760 000 was awarded in grants from the Swedish Fund for Research without Animal Experiments in 2013. It was divided between 11 projects, five of which were new. Recipients and project titles below. More information about the projects will be added shortly.

New projects

Jenny Almkvist, GU Holding, University of Gothenburg, “InnoVitro-development of an in vitro test for screening substances for contact sensibilization
SEK 50 000

Roland Grafström, IMM, Karolinska Institutet
Definition of the human toxicogenomics space for chemicals toxicity prediction”  SEK 80 000

Maria Karlgren, Uppsala University
Development of a humanized BBB model
SEK 80 000.

Pär Matsson, Uppsala University
Models for predicting intracellular drug exposure
SEK 80 000.

“For a pharmaceutical drug to give the desired effect it must reach the organ and the cells that need treatment. In most cases, the pharmacological effect results from that the drug molecule binds to a specific protein inside the target cell. To predict if a new drug will give the desired effect it is thus necessary to determine the amount of drug that is distributed into the different cells of the body.

Currently, there are no good methods for this, and, typically, drug concentrations in the blood are used as a surrogate for the relevant concentrations inside the target cells. This can lead to over- or underpredictions of up to hundred-fold compared to the true drug levels, and a drug with good results in a test tube or a preclinical animal model can completely lack effect, or result in serious side-effects when given to human patients. We aim to develop a novel experimental method for measuring intracellular drug exposure, which will be the first to provide intracellular unbound drug concentrations – i.e., the concentration that can interact with intracellular targets and metabolizing enzymes.

The method will be used to establish a unique database of high quality intracellular drug accumulation data. Building on the experimental data, computational structure-activity models will be developed that will allow prediction of intracellular concentrations directly from a drug’s chemical structure. Drug-like molecules that do not reach their targets in sufficient amounts, or which result in dangerously high concentrations, can thereby be filtered out in the early stages of the drug development process, avoiding unnecessary experiments in laboratory animals and patients.

The developed models can be used to prioritize candidate drugs for further development, ultimately leading to significantly fewer animal experiments in the pharmacological and toxicological evaluation of new drugs.”

Stina Oredsson, Lund University
Neurotoxicity, Genotoxicity and Metabolism of Salinomycin Analogs
with Cancer Stem Cell Inhibiting Properties

SEK 50 000

“The presence of cells with self-renewal ability and the capacity to give rise to the heterogeneous lineages of cancer cells that comprise a tumour has been demonstrated in a variety of human malignant tumours including breast cancer. These cells that seemingly give rise to a hierarchically organised tumour have been called cancer stem cells (CSC).

The CSCs only comprise a small part of the tumour but unfortunately they seem to harbour mechanisms yielding increased survival to various insults. A major problem is that these properties allow CSCs to survive many of the current therapies and thus seem to be the major cause for tumour recurrence and metastasis. Therefore, the identification and development of small molecules that effectively eradicate CSCs has the potential of being transformative to improve clinical outcome in cancer. We are working together with chemists that have synthesized new small molecules and we have found that these molecules inhibit CSCs.

The specific purpose of this project is to elucidate neurotoxicity, genotoxicity and metabolism of the compounds we are working with. The aim is to get an understanding of structure activity relationships of the compounds in relation to general toxicity not related to CSCs and to use the data to select a few compounds for further work towards clinical use.

Since the ultimate goal is to find a compound that can be used in cancer treatment, the selected compound(s) must unequivocally pass animal testing as decided by regulatory toxicity. By performing extended tests on general toxicity in cell lines we will identify compounds with potential severe off-target effects.

The use of animal-derived products will also be reduced by total exclusion of fetal calf serum (FCS) in all cell culturing including routine culturing. To investigate neurotoxicity we will use the human SH-SY5Y neuroblastoma cell line. Genotoxicity will be investigated using a genotox screen set up by Anthem Biosciences Pvt. Ltd. The human HepaRG cell line will be used to investigate metabolism.

This research will limit the abuse of animals in two ways. Firstly, none of the cell lines will be cultured in medium containing fetal calf serum, not even in routine culturing. The very use of fetal calf serum actually defeats the purpose of using cell culturing as replacement for animals in research. Secondly, we do have compounds that are very efficient in inhibiting CSCs. Instead of testing all compounds (around 20) for CSC activity in animal tumour models, which is an accepted research strategy, we want to apply a number of general toxicity tests on cell lines before choosing compounds for test in animal tumour models. By this action we spare a large number of animals from unnecessary exposure to compounds that have potential toxic off-target effects.”

Projects receiving continued funding

Anna Herland, Karolinska Institutet
Stem cell derived cholinergic neurons as in vitro models to reduce animal experiments within neurodegeneration and analgesia
SEK 80 000.

“The aim of our research is to create better model systems of human neuronal tissue. Such models are essential to increase knowledge in basic scientific questions as well as to facilitate pharmaceutical development.

We are focused on research within analgesia, drugs that relieve pain, and neurodegeneration. These disease conditions are prevalent, it is estimated that one in five Europeans suffer from chronic pain and that about 5.4 million Americans are diagnosed with Alzheimer’s disease.

The available models systems for neuronal tissue used today are animals, especially rodents, and human, cancer derived, cell lines. Both have strikingly low predictivity both of effects and side effects.

The guiding hypothesis of this project is that cholinergic neural differentiation of stem cells in combination with three-dimensional cell culture systems can provide highly relevant models of human basal forebrain. We are using human induced pluripotent cells originating from human skin biopsies.

These cells can be differentiated to virtually any specialized cell in the human body; here we have developed a method to make them in to a neuronal subtype, cholinergic neurons. Cholinergic neurons have strong association with the mentioned pathologies. By culturing the differentiated cholinergic neurons in three-dimensional gel like systems we increase the resemblance with the tissue. We have optimized a gel system that supports the neural growth very well. Moreover we have observed that when we culture neurons in the gel system but not in conventional cell culture we can resemble features of Alzheimer’s disease.

Our next step is evaluate neurons that we have derived from skin biopsies from Alzheimer’s patients. Ultimately such a system could decrease the demand to use animals or animal derived cells both in academic and industrial settings.”

Erik Tesselaar, Linköping University
Development of a microdialysis based microdosing technique for a faster and safer drug development“.
SEK 100 000.

Anna Forsby , Stockholm University
Neuronal stem cells as a substitute to primary, embryonic brain cell cultures for the estimation of neuronal mediated acute systemic toxicity.” SEK 80 000.

Malin Lindstedt, Lund University
A novel testing strategy for assessing sensitizing capacity of chemicals and proteins “. SEK 80 000.

“The objective of this project is to develop a human cell-based assay to test the propensity of new chemicals and proteins, used by e.g. the cosmetic, chemical and pharmaceutical industries, to cause sensitization. The test systems will be based on a myeloid cell line which resembles the orchestrators of the immune response; the dendritic cells (DC). We will utilize the cellular and molecular mechanisms involved in allergen recognition.

Using array technologies, we will identify biomarkers that can be used to distinguish between sensitizers and non-sensitizers, in order to use these as readout for in vitro sensitization testing. We will focus on i) validation of our recently developed genomic test using a blinded panel of skin-sensitizing chemicals and controls, ii) identification of biomarkers involved in occupational respiratory sensitization to chemicals or protein allergen components, iii) describe the basal molecular mechanism and pathways involved in sensitization and iv) transfer assay format from complete genome analysis to focused multiplex RNA assays for routine analysis.

The project is expected to generate a state-of-the-art test strategy for in vitro sensitization. The allergen detection test has the potential to reduce animal experimentation for hazard evaluation, currently performed with the Local Lymph Node Assay (LLNA) in mice, and should exhibit higher prediction accuracy and correlation with human experience than LLNA.”

Lena Palmberg, Karolinska Institutet
A three-dimensional model based on human cells from the airways to replace animals for controlled nanoparticles aerosol exposures “. SEK 80 000.

“Good reliable human airway wall models will most likely replace animal models in the study of nanoparticle exposures, a research field under intense growth. Our model is unique in that it combines the use of human primary respiratory cells with quality exposures to a variety of different aerosols of toxicological and pharmacological significance. The combination mimics realistic exposures of the human airway wall and give good opportunities to study the cell-to-cell and cell-to-matrix interaction in a manner that will likely reduce the need for animal models.

Our primary goal is to develop a 3D-model with primary human cells and culture them under air-liquid interface (ALI). Primary respiratory epithelial cells will be placed on top of a collagen layer including fibroblasts. After initial submerged culturing the model will be airlifted and additives stimulate the epithelial cell layer to differentiate out to ciliated and mucus-producing cells. Secondly, this model will be combined with a newly developed dustgun technique to expose with controlled amounts of palladium nanoparticles, originating from cars. Thirdly, the model will be modified to develop goblet cell hyperplasia; characteristic features in chronic bronchitis.

This model will give us important information regarding the interaction between glucocorticosteroids and innate immune responses, which may have implications of the beneficial effects of steroid treatment of exacerbation in chronic lung disorders.”

 

Michael Stigson, BMC, Uppsala University
Mechanism-based developmental toxicity testing in vitro “. SEK 80 000.

“The aim of this project is to develop completely animal-free high-throughput screening systems that can be used to predict developmental toxicity of new chemical entities during the early phases of preclinical drug development. Such in vitro assays could guide early decision-making and eliminate potentially problematic compounds from further studies so that fewer candidate drugs will later show up with teratogenic effects in the systematic full-scale regulatory tests in mammals that are generally performed after a large number of laboratory animals have already been used to evaluate therapeutic effects and general toxicity.

In vitro methods using mouse embryonic stem cells are currently available but these methods are not readily automated and miniaturized into a system for rapid and cost-effective high throughput screening of large numbers of chemical compounds. Moreover, the currently available in vitro tests do not take into account the underlying mechanisms or pathways of toxicity but rely on the evaluation of late endpoints of manifest toxicity. Our strategy is to study early toxicogenomic responses to known teratogenic compounds in embryo-derived pluripotent stem cells in order to identify mechanistically relevant molecular targets and early response markers of teratogen exposure that can be evaluated as predictive endpoints in screening tests.

Since mouse embryonic stem cells conventionally are cultured on a feeder layer of primary cells (mouse embryonic fibroblasts) and in the presence of fetal bovine serum, we have adapted an established embryonic stem cell line (R1) to serum- and feeder-free culture conditions. Our preliminary studies indicate that the alternatively cultured R1 cells appear to have properties comparable to conventionally cultured R1 cells. These cells are being further evaluated for use in screening systems in an undifferentiated state or under conditions in which cells differentiate along more or less defined lineages.”

Senast uppdaterad: 7 mars 2016