Strategic focus on 3R principles reveals major reductions in the use of animals in pharmaceutical toxicity testing.

The principles of the 3Rs, Replacement, Reduction and Refinement, are being increasingly incorporated into legislations, guidelines and practice of animal experiments in order to safeguard animal welfare. In the present study we have studied the systematic application of 3R principles to toxicological research in the pharmaceutical industry, with particular focus on achieving reductions in animal numbers used in regulatory and investigatory in vivo studies. The work also details major factors influencing these reductions including the conception of ideas, cross-departmental working and acceptance into the work process. Data from 36 reduction projects were collected retrospectively from work between 2006 and 2010. Substantial reduction in animal use was achieved by different strategies, including improved study design, method development and project coordination. Major animal savings were shown in both regulatory and investigative safety studies. If a similar (i.e. 53%) reduction had been achieved simultaneously within the twelve largest pharmaceutical companies, the equivalent reduction world-wide would be about 150,000 rats annually. The results point at the importance of a strong 3R culture, with scientific engagement, collaboration and a responsive management being vital components. A strong commitment in leadership for the 3R is recommended to be translated into cross-department and inter-profession involvement in projects for innovation, validation and implementation. Synergies between all the three Rs are observed and conclude that in silico-, in vitro- and in vivo-methods all hold the potential for applying the reduction R and should be consequently coordinated at a strategic level.

A triple-transgenic immunotolerant mouse model.

Avoiding unwanted immunogenicity is of key importance in the development of therapeutic drug proteins. Animal models are of less predictive value because most of the drug proteins are recognized as foreign proteins. However, different methods have been developed to obtain immunotolerant animal models. So far, the immunotolerant animal models have been developed to assess one protein at a time and are not suitable for the assessment of combination products. Our aim was to develop an animal model for evaluating the impact of manufacturing and formulation changes on immunogenicity, suitable for both single protein and combination products. We constructed two lines of transgenic mice expressing the three human coagulation factors, II, VII, and X, by inserting a single vector containing the three coagulation factors encoding sequences separated by insulator sequences derived from the chicken beta-globin locus into the mouse genome. Immunization of transgenic mice from the two lines and their wild-type littermates showed that transgenic mice from both lines were immunotolerant to the expressed human coagulation factors. We conclude that transgenic mice immunotolerant to multiple proteins can be obtained, and that these mice are potentially useful as animal models in the assessment of immunogenicity in response to manufacturing changes.

Refined analysis of antigen-specific antibody responses--a new one-step tool in immunogenicity studies.

Immunogenicity is a continuous efficacy and safety issue of biopharmaceuticals. Pre-clinical models for prediction of immunogenicity itself as well as biomarkers to reveal potential mechanisms behind an already existing antibody response are still needed. A sensitive, robust and specific immunogenicity assay has therefore been developed that can detect and measure antibodies of five classes against an administered recombinant human protein drug. Additionally, a validation was performed to evaluate the reproducibility and specificity of this newly developed assay. The production of drug-induced antibodies in mice injected with a recombinant human protein drug has been measured by using a modified version of a multi-parametric bead analysis technique. Competitive binding was used to verify drug-specificity of the antibodies. Results showed that the mouse response against the recombinant human protein was IgG1- and IgG2b-specific, suggesting that the drug-induced response was driven by both Th1/Th2 cells; a finding confirmed by measurement of the cytokine profile. With this assay, anti-drug antibody class and subclass screening may be executed in one step.