Screening of nanoparticle-drug formulations against tuberculosis in the zebrafish embryo model
1. The purpose of the experiment is to use a well-established tuberculosis model in zebrafish embryos involving Mycobacterium marinum to test the efficacy and toxicity of new anti-TB antibiotics formulated in nanoparticles (NP). The use of embryos up to 5 days post-fertilisation (dpf) are not regulated by Norwegian Law (“Forskrift om bruk av dyr i forsøk (FOR-2015-06-18-761)"), but when feeding starts around day 5 they are considered animals (larvae) and subject to regulation. This application concerns permission to use embryos up until day 10 post fertilization (dpf).
2. The level of distress in infected/treated zebrafish larvae is expected to be severe according to the “forskrift om bruk av dyr i forsøk vedlegg B as the experiment procedures strongly affect the general condition and the well-being of the animals in a negative manner.
3.The rationale for using zebrafish embryos/larvae is, that it is a quicker, less expensive and more sensitive animal model than the mouse models, the preferred preclinical model. By allowing rapid screening of free and NP-formulated antibiotics the use of zebrafish embryos/larvae can select for the most promising drugs and NP-formulations to be tested in mouse models of TB, both for therapy and for potential undesirable toxicity.
4. Twenty-five embryos for each experimental condition, i.e. free and NP-formulated drug, each at three different concentrations and untreated control, are calculated to be needed. In total 10500 zebrafish embryos/larvae are needed.
5. Collectively, these zebrafish screens contribute greatly to reduce both the number of mice needed in the next step (in support of the 3R principle) and in the volume of drugs needed for testing. Based on our experience from previous projects (ID13580;ID13653), we are able to apply several different assays in the same animals (f.ex. imaging and survival assays), providing maximal information from every single experiments, thus reducing the number of zebrafish embryos/larvae necessary. We also define - based on our previous experience - stringent humane endpoints, which ensure that larvae are euthanized before they succumb to infection or treatment. Thus, we are able to keep distress induced in the animals to a minimum.
2. The level of distress in infected/treated zebrafish larvae is expected to be severe according to the “forskrift om bruk av dyr i forsøk vedlegg B as the experiment procedures strongly affect the general condition and the well-being of the animals in a negative manner.
3.The rationale for using zebrafish embryos/larvae is, that it is a quicker, less expensive and more sensitive animal model than the mouse models, the preferred preclinical model. By allowing rapid screening of free and NP-formulated antibiotics the use of zebrafish embryos/larvae can select for the most promising drugs and NP-formulations to be tested in mouse models of TB, both for therapy and for potential undesirable toxicity.
4. Twenty-five embryos for each experimental condition, i.e. free and NP-formulated drug, each at three different concentrations and untreated control, are calculated to be needed. In total 10500 zebrafish embryos/larvae are needed.
5. Collectively, these zebrafish screens contribute greatly to reduce both the number of mice needed in the next step (in support of the 3R principle) and in the volume of drugs needed for testing. Based on our experience from previous projects (ID13580;ID13653), we are able to apply several different assays in the same animals (f.ex. imaging and survival assays), providing maximal information from every single experiments, thus reducing the number of zebrafish embryos/larvae necessary. We also define - based on our previous experience - stringent humane endpoints, which ensure that larvae are euthanized before they succumb to infection or treatment. Thus, we are able to keep distress induced in the animals to a minimum.