Development of anti tuberculosis therapy using nanoparticles enclosing antibiotics in zebrafish embryos
The purpose of the experiment is to use a well-established tuberculosis model in zebrafish embryos involving Mycobacterium marinum to test different nanoparticles (NP) encapsulating anti TB antibiotics.
The use of embryos up to 6 days post-fertilisation are not regulated but when feeding starts around day 6 they are considered animals and subject to regulation. This application concerns permission to use the embryos up until day 12 post-fertilisation.
The level of distress expected is moderate.
Twenty embryos per experimental condition is calculated to be needed (e.g NP with drug versus without).
The rationale for using zebrafish embryos is that these transparent animals allow detailed imaging of fluorescent NP and bacteria, as well as host cells to be visualized in live animals. This is a technical limitation of mouse models, the preferred preclinical model. By allowing rapid screening of NP the use of zebrafish embryos can select for the most promising NP to be tested in mouse models of TB, both for therapy and for potential undesirable toxicity of the NP. 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 and NP needed for testing.
The use of embryos up to 6 days post-fertilisation are not regulated but when feeding starts around day 6 they are considered animals and subject to regulation. This application concerns permission to use the embryos up until day 12 post-fertilisation.
The level of distress expected is moderate.
Twenty embryos per experimental condition is calculated to be needed (e.g NP with drug versus without).
The rationale for using zebrafish embryos is that these transparent animals allow detailed imaging of fluorescent NP and bacteria, as well as host cells to be visualized in live animals. This is a technical limitation of mouse models, the preferred preclinical model. By allowing rapid screening of NP the use of zebrafish embryos can select for the most promising NP to be tested in mouse models of TB, both for therapy and for potential undesirable toxicity of the NP. 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 and NP needed for testing.
Etterevaluering
Forsøket er klassifisert som betydelig belastende.
Begrunnelse for etterevalueringen
This project has established and validated the zebrafish TB (tuberculosis) model for the evaluation of nanoparticle-enclosed antibiotics. New non-toxic and highly effective derivatives of the anti-TB drug pretomanid were identified. All the aims of this study have thus been achieved.
Based on these experiments, the most promising candidate drugs for subsequent evaluation in mouse models of TB have been selected. In addition, by exploiting the accessibility of the zebrafish larvae for in vivo high-resolution imaging, a better understanding has been gained of the mechanisms governing the interaction of drug-loaded nanoparticles with the infected tissue.
The use of 7800 zebrafish was approved and in total 6000 zebrafish larvae older than 6 dpf were used. 5 849 were categorized with the severity level severe (betydelig belastning), due to disease development (TB) and/or death. 28 were given moderate severity and 123 mild. Experimental groups, subjected to procedures considered to cause only moderate stress, were typically non-infected control groups or non-infected groups used in imaging experiments. Fewer larvae than anticipated were used due to efforts to coordinate experiments for example by sharing the same control groups in different but simultaneously conducted experiments. This is within the limits of the decision letter and as expected.
Using zebrafish embryos and larvae in the evaluation of new nanoparticle-enclosed antibiotics satisfies the 3R principles as it can reduce the number of mammalian animals needed in subsequent experiments. Before evaluating new drugs or drug-nanoparticle formulations, both anti-mycobacterial activity as well as toxicity were routinely tested in cell culture systems to eliminate compounds with poor activity and/or significant toxicity. As of now, there does not exist an in vitro cell culture system that could replace the in vivo model of the zebrafish embryos and larvae.
By coordinating experiments, using multiple assays in the same experiment, the number of zebrafish embryos and larvae needed was reduced. Limiting the duration of experiments, where possible, to 6 dpf, the pain load was reduced (refinement). Using the humane endpoints as defined in the application combined with tight monitoring during critical phases of experiments, prevented unnecessary suffering in the zebrafish larva. The euthanasia method worked satisfactorily.
Based on these experiments, the most promising candidate drugs for subsequent evaluation in mouse models of TB have been selected. In addition, by exploiting the accessibility of the zebrafish larvae for in vivo high-resolution imaging, a better understanding has been gained of the mechanisms governing the interaction of drug-loaded nanoparticles with the infected tissue.
The use of 7800 zebrafish was approved and in total 6000 zebrafish larvae older than 6 dpf were used. 5 849 were categorized with the severity level severe (betydelig belastning), due to disease development (TB) and/or death. 28 were given moderate severity and 123 mild. Experimental groups, subjected to procedures considered to cause only moderate stress, were typically non-infected control groups or non-infected groups used in imaging experiments. Fewer larvae than anticipated were used due to efforts to coordinate experiments for example by sharing the same control groups in different but simultaneously conducted experiments. This is within the limits of the decision letter and as expected.
Using zebrafish embryos and larvae in the evaluation of new nanoparticle-enclosed antibiotics satisfies the 3R principles as it can reduce the number of mammalian animals needed in subsequent experiments. Before evaluating new drugs or drug-nanoparticle formulations, both anti-mycobacterial activity as well as toxicity were routinely tested in cell culture systems to eliminate compounds with poor activity and/or significant toxicity. As of now, there does not exist an in vitro cell culture system that could replace the in vivo model of the zebrafish embryos and larvae.
By coordinating experiments, using multiple assays in the same experiment, the number of zebrafish embryos and larvae needed was reduced. Limiting the duration of experiments, where possible, to 6 dpf, the pain load was reduced (refinement). Using the humane endpoints as defined in the application combined with tight monitoring during critical phases of experiments, prevented unnecessary suffering in the zebrafish larva. The euthanasia method worked satisfactorily.