2017

13 projects receive grants in 2016

A total of 2 million SEK has been awarded in grants from the Swedish Fund for Research without Animal Experiments in 2017. It was divided between 13 projects. Se recipients and project titles below. More information about the projects will be added shortly.

 

Martin Andersson, SP Technical Research Institute of Sweden
Animal-free method for prediction of eye-irritation 
In an internal project SP / RISE, we have surprisingly found that it seems possible to predict whether a liquid is an Eye-irritant or not via the liquids so-called Hansen parameters. Each liquid has three such parameters describing the solubility properties of the liquid, and therefore each fluid can be assigned a place in ”Hansen-space” where the three parameters are x, y and z coordinates. Also polymers have specific Hansen parameters and quids / polymers that have similar parameters are miscible / dissolve in each other. By viewing the cornea as a polymer, we have been able to develop a first animal free-test method that looks promising – based both on in-silico predictions as well as on a test where a specific polymer thread is dipped into the liquid to be asessed, followed by measurement of the elongation of the thread. In case of a Eye irritant that irritates via impregnation into/swelling of the cornea we have seen thread-elongations of up to 50%.
In this project we will further develop this method so that we can ensure that it works and correlates with the real eye-irritation data and we will also try to develop it further so that it can predict the clouding of the cornea and even eye damage caused by acidic / basic or oxidizing / reducing substances

Kunal Bhattacharya, IMM, Karolinska institute: Development of a lung-on-chip model for testing of nanotoxicity
Engineered nanomaterials (ENMs) are being produced for an increasing number of smart technology applications. Therefore, it is important to perform safety assessments of these ENMs to mitigate possible human exposures and health-risks. Due to their small size the ENMs have larger and more reactive surface as compared to their bulk counterparts, and may have yet to be discovered mechanisms of inducing toxicity in humans. In 2013, the Swedish Government issued a report on the safe handling of ENMs and recommended to set aside funding for research and development of testing and risk assessment methodology. Currently, simple 2-D in vitro cell culture models dominate the field of nanotoxicology research, which do not mimic the complex 3-D multicellular microenvironment of tissues and organs. Mechanistic studies performed in small animals such as rats and mice also do not represent a real-life human exposure due to phenotypic, genotypic and physiological differences. Finally, the local microbiota cooperates with the host environment and this is missed in conventional in vitro 2D cell cultures. Therefore, in the proposed research, we aim to develop a microfluidics-based complex tissue system representing human lungs (lung-on-chip devices) using human cell lines grown in serum-free conditions. These models will be used for testing the toxicity of silver and silica nanoparticles. Results generated using the lung-on-chip model will be used to identify the mechanistic toxicity of these ENMs and will be correlated to previously published research work using 2D in vitro models, as well as published in vivo results. The study outcome will provide evidence regarding the usefulness of the lung-on-chip model as a replacement for the existing 2D in vitro and in vivo study models used in the field of nanotoxicology and help in enhancing knowledge on the toxicity of ENMs on human health and development of chronic/acute diseases.

Gunnar Cedersund, Linköping university: Knowledge-driven drug development without animal experiments
In the last few years, mathematical modelling has displayed an unprecedented impact on drug development. For instance, recent specific approvals, and even more recent general guidelines, imply that computer simulations now can be used for regulatory approvals. Since model simulations are cheaper and faster, such approvals typically end the use of test animals for that application. We have modified such an approved type 1 diabetes
model to also describe type 2 diabetes, a much more widespread and rapidly growing disease. Our award-winning model has been developed through numerous experimental/modelling iterations, and is already at use in several major pharmaceutical companies. However, this usage is still mostly done in traditional drug development pipelines, involving a series of cell and animal systems, which often display highly different outcomes. To allow us to move to a radically new, knowledge-driven, more efficient, and increasingly human-centered pipeline, I will use uniquely informative data to do the four most important additions still needed in the model: i) storage and release of fatty acids, ii) specific drug-targets in heart and adipose tissue, iii) multi-level and long-term translations, iv) mapping from other animal-free systems. For these groundbreaking developments, the Swedish Fund for Research without Animal Experiments awarded me their newest award – ”Nytänkaren” – and I am suggested for the board of the new government-initiated National Institute on 3R.

Pernilla Eliasson, Linköping university: How do tendons heal? – and what happens when they don´t heal?
The ethology behind tendon pathology is multifactorial, however different drugs might influence this. Tendon injuries are common and require a long rehabilitation time. Mechanical loading improves healing, but it is unclear how. I have introduced a model for 3D cell culture studies of human primary tendon fibroblasts, in Linköping. This 3D model makes it possible to study effects on mechanical strength of the tissue after for example drug treatment or changes in loading situations. This is in contrast to conventional cell culturing. Studies on mechanical strength is an important tool in orthopedic research and this has previously only been done in animal models.

Moreover, this in vitro model also allows me to study mechanotransduction. Mechanotransduction is the way a cell converts mechanical stimulus into a biochemical response. I will use this model to study the effect of loading without micro-damage associated inflammation (which is present in animal models) to investigate the mechanotransduction component during loading. I have previously observed that tendon fibroblasts from some patients lack the ability to form tendon tissue, in vitro. I will study the differences between cells that can form tendon tissue with those that can´t. This might help to identify factors that are vital for tendon formation.
I have also started a project where I study some aspects on why tendon injuries appear. We will perform epidemiological methods to study associations between statin use and tendon injuries in different part of the body. I will also use my in vitro model to study the mechanisms why tendons are affected by statins and high cholesterol levels.
The goal with my studies is to understand some factors which might contribute to the appearance of different tendon injuries, and to gain information to increase the understanding on what happens after a tendon injury, so that treatment and rehabilitation protocols can improve.

Martin Hallbeck, Linköping university: Development of a modell to test future drugs to treat Alzheimer’s disease
The goal of the project is to develop a model for testing drugs against Alzheimer’s disease. It is a progressive disease and the patient suffers constant deterioration. Our knowledge of how the disease spreads in the brain is incomplete but there is a growing understanding that it is due to the spread of small protein collections. Our hope is to impede this spread.
It is difficult to study the human brain directly, good models are needed. We have developed models to study how Alzheimer’s and other neurodegenerative diseases can spread between nerve cells. Through previous funding, we have further developed the model using human induced pluripotent stem cells developed by programming skin cells from donors. If we can show that this model can be used by the pharmaceutical industry and in research on dementia and result in better results than with animal research, many animal tests can be replaced and at the same time research can take important steps forward.

Maria Karlgren, Inst f pharmacy, Uppsala university: Human cell based models for accurate predictions of brain drug uptake
Poor predictions of CNS drug exposure is a major problem in CNS drug development and is primarily the result of relying on animal-based models although major species differences are well-known for the blood-brain barrier (BBB) and the expression of drug transporters. Here, we will solve this problem by developing predictive models taking the drug transporters expressed in human BBB into account and thereby accurately quantify drug delivery to human brain.
This will be done by development of mechanistic transporter models. Thereafter in vitro kinetics from mechanistic transporter models and human BBB in vitro models will be combined with quantitative proteomics and physiological parameters to predict the human in vivo situation. The project will be performed at Uppsala University in close collaboration with UDOPP (part of DDDp/SciLife Lab) giving a unique opportunity for validation in real-life drug development projects.
With our model drug candidates with predictable and favorable human BBB properties will be identified and selected early in drug discovery/development. As a result an initial reduction of animal PK experiments with 50-60%, with further reduction potential, is estimated. Thereby providing the refined tools needed to substantially reduce and, in a longer perspective, replace animal BBB studies throughout the drug discovery/development process.

Pekka Kohonen, IMM, Karolinska institute: Deepened mechanistic validation of toxicity pathway functionality in a patented analysis tool for toxicity prediction
Our aim is to apply a systems biology-based approach and innovative bioinformatics tools to evaluate the dose-dependent activation of a patented first-ever mechanistically comprehensive human cell-based toxicogenomics space generated in our laboratory. Specifically, we are now to evaluate in depth its capacity to predict activation of p53-mediated signaling, as it is possibly the most central cellular mechanism activated in response to diverse toxic insults. The experiments set out are to assess cellular dose–response thresholds related to adverse outcomes. In order to adapt the analysis tool for regulatory toxicology use, the current project shall deepen the mechanistic validation of the toxicity pathway functionality. The work effort shall enhance it with entirely new methods that permit quantitative dose-dependent Bench Mark Dose (BMD) modeling of the transcriptomic response captured by the toxicogenomics space. “Forska utan Djurförsök” funding will specifically enable elaboration and more targeted validation of research being carried out under the Swedish Academy of Sciences “3R” call and in the EU H2020 / Eurostars “ToxHQ”project (http://www.toxhq.net/). This project is aimed at generating a proof-of-concept for the novel solutions towards toxicity prediction based on in vitro data only. This goal covers also creating suggested standard operating procedures that form a part of a real product-based applicable testing tool and provide deepened proof of the applicability of the toxicogenomics space concept for regulatory practice.

Johan Lundqvist, The Swedish University of Agricultural Sciences, Uppsala: Establishment of animal-free cell models for studies of aquatic toxicity
This research program aims to develop a system for animal-free testing to assess aquatic toxicity, based on the principle of toxicity pathways. Today, European regulations require that a large number of chemicals should undergo assessment of aquatic toxicity, which often include animal experiments. Assessment of the aquatic toxicity in toxicity pathway-based cell culture models instead of in animal experiments, will allow analysis of a large number of chemicals to a moderate cost and will significantly reduce the number of animals used for aquatic toxicity assessment of chemicals. The European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) has recently proposed development of cell cultured based assays as a key strategy to reduce and replace the use of fish in toxicity testing.
The aims of this project are

• To establish a panel of 10-15 robust toxicity pathway-based assays that are measuring toxic effects of high relevance for fish toxicity assessment

• To use the established toxicity pathway-based assays to study the toxicity of approx. 25 chemicals that has already underwent classical animal-testing for aquatic toxicity, and to compare the results from the in vitro-assays with the results from the already performed animal experiments

• Based on the results from this project, we will suggest a panel of toxicity pathway-based assays that can be used as an initial screening step for aquatic toxicity and provide information to prioritize which substances that needs to undergo further toxicological investigations

Pär Matsson, Inst f pharmacy, Uppsala university
Methods for predicting intracellular drug exposure
Current methods for predicting the effect of new drugs do not adequately address that drug concentrations can differ substantially between the circulating blood and the cells where the drug target is expressed. We have developed a rapid method for measuring unbound drug concentrations in the interior of cultured cells. The specific aims of this project is to 1) further develop the method and validate it for prediction of unbound drug exposure in different cell and tissue types, 2) to use the method to explore how transport proteins affect intracellular drug concentrations, 3) to study if the method can be used to improve predictions of modulation of intracellular drug targets such as protein kinases, and 4) to develop computational models that can predict intracellular drug exposure from the chemical structure alone.

Results so far: An experimental method for intracellular concentration measurements was developed and applied to the human cell line HEK293, and to primary human hepatocytes. The method was shown to give comparable results to previously described methods that use tissues from laboratory animals, and we also show that cell binding measurements in the HEK293 cell line can be used to predict binding in several different human tissues, including the liver and the brain. Studies of the link between drug transport proteins and intracellular concentrations (Aim 2) and of how unbound drug concentrations affect intracellular drug effects are ongoing and expected to be finalized during the coming funding period. Using the data obtained so far, computational models were developed that accurately predict cellular binding for new drug compounds, without the need for experiments.

Stina Oredsson, Lund university: Novel 3D Cell Culturing Methods in Cancer Research -For Better Prediction, Efficacy, and Less Animal
Millions of animals are used in cancer research to develop new drugs for treatment. Many times initial testing of potential drugs is done in 2D cancer cell line systems and when positive results are achieved, the compounds are injected into tumour bearing animals without investigation of toxic side effects. Unfortunately, cell culture work with cancer and normal cells involves animal-derived products such as fetal calf serum, collagen, and matrigel, the latter from tumour bearing mice. Our aim is to develop a 3D cell culturing system for co-culturing of cancer cells and stromal cells found in a tumour to replace and reduce animal experiments in cancer drug discovery. The basis is a unique collagen mimicking polycaprolactone fibrous structure made by electrospinning. Cancer cells, fibroblasts (normal or cancer-associated), and immune cells are co-cultured in medium without products derived in animal-ethics questionable ways. Using phase contrast and holographic time laps imaging of cultures incubated in normoxia and hypoxia we can follow cell dynamics such as cell movement. After fixation and staining with non-animal-derived antibodies we use confocal microscopy to investigate cell-cell and cell-matrix interactions and calibrate/compare to in vivo systems. Using a unique library of potential cancer drugs we investigate effects on cancer cell sub-populations as well as on the stromal cells. We anticipate that this system can replace many animal experiments in cancer research.

Lena Palmberg, IMM, Karolinska institute: New lung models with primary cells and exposure to components in air pollutants
The primary aim is to develop and evaluate unique and relevant normal and chronic bronchitis-like airway wall models with primary human bronchial cells from healthy subjects and smokers with and without COPD. The sophisticated airway wall models will be co-cultured with innate effector cells (neutrophils and alveolar macrophages) from different patient groups and mimic the in vivo-situation better than animal models where species differences always is an issue. These models with multiple cell types enable us to study cell-to cell interactions and cross-talk between the cells. By using our advanced exposure systems we can expose cell cultures to airborne particles, only consuming minimal amounts of test substance if the access is limited. Our chosen test substances of different ambient air pollutants like biodiesel particles, nanoparticles and aldehydes contribute to the development of asthma, chronic bronchitis, COPD and cardiovascular diseases and trigger symptoms in subjects suffering from those diseases. Development and validation of these models enable us to develop an in vitro testing strategy in order to reduce the requirement for animal inhalation studies. The applicant team has long experience of studies of in vivo-effects induced by exposure in an exposure chambers of the above mentioned agents in healthy subjects and subjects with respiratory diseases. Therefore the clinical relevance of the models will be well evaluated.

Peter Sartipy, Systems Biology Research Center, School of Bioscience, University of Skövde: Stem cell derived human cardiomyocytes as in vitro model for toxicity testing
Our research aims towards the development of an in vitro model based on cardiomyocytes derived from human pluripotent stem cells. Such a model is expected to be very valuable in the future, in order to reduce the number of test animals used for drug development.
The specific focus for this project is to evaluate human stem cell-derived cardiomyocytes, as a relevant alternative to animal studies, to detect and in detail study drug-induced cardiotoxicity using doxorubicin. Doxorubicin is an effective chemotherapeutic agent that is associated with severe cardiac side effects. The model systems available today are mostly based on the use of animal models, with a limited value for the study of human toxicity. The use of primary human cardiac tissue is problematic due to a very low availability of donated material, which often also is associated with a history of disease and drug treatment.
The cardiomyocytes are studied during treatment with doxorubicin for up to 48 hours. The global protein expression is measured using quantitative proteomics. Bioinformatic algorithms are applied to the data in order to identify differentially expression proteins in the doxorubicin exposed cells. The proteomics data is also integrated with already available transcriptomics data (global mRNA and microRNA expression data) in order to identify affected signaling pathways and mechanisms linked to the toxicity.
The results show a clear effect of doxorubicin on the cells protein expression. A number of vital functions are affected within the cells, such as proteins linked to the cardiomyocytes contractility, apoptotic signaling, energi metabolism, and synthesis and modulation of proteins. Interesting differential expression patterns that show a linkage between the proteome, transcriptome, and the regulatory microRNA network, were identified. Findings that help to increase the understanding of the mechanisms behind and suggest putative biomarkers for anthracycline-induced cardiotoxicity.

Lena Svensson, Lund university: Replacing animal models by microfluidic vascular models to study cell migration
This project aims to adopt and adapt microfluidic vascular models and employ these to study leukocyte transmigration and cancer cell extravasation, the process in which cells migrate through the endothelial cell lining in blood vessels in order to invade the surrounding tissue. For leukocytes, this process is key to reaching sites of infections and for cancer cells it is part of the overall process of metastasis, when cancer cells leave the original tumour site and spread throughout the body, the major cause of death among cancer patients. The microfluidic model we aim to use will reduce the need for animal trials while simultaneously improving experimental control and microscopy imaging conditions. It will also add a layer of refinement to existing in vitro techniques by increasing physiological relevance by using a three dimensional co-culture system with an extracellular matrix model, fluid flow and the possibility for chemotaxis. Using this system we will study mechanical effects (such as sheer forces and device geometry) and chemical effects (signalling molecules, antibodies, and drugs) on transmigration and extravasation to learn more about this all-important processes.

 

 

Senast uppdaterad: 20 april 2017