The RETHINK project on minipigs in the toxicity testing of new medicines and chemicals: conclusions and recommendations.

The objective of the RETHINK project was to evaluate the potential impact of toxicity testing in the minipig as an alternative approach in regulatory toxicity testing that can contribute to the replacement, refinement and reduction of animal testing (3Rs). Expert study groups (Working Groups) were assembled to review five different areas relating to the use of minipigs in regulatory safety testing: ethical issues, welfare and animal care, development of new medicines and chemicals, safety testing issues and emerging technologies in safety testing. The conclusions and recommendations of the projects are presented in this article. It is concluded that there are no specific areas where restrictions to the use of minipigs in toxicology are required for welfare reasons. The minipig model is generally acceptable to regulatory authorities, provided it is adequately justified. The minipig is an interesting model for safety testing since there are numerous anatomical, physiological, genetic and biochemical similarities to humans. In addition many features of the minipig make it a practical and flexible model for safety testing. The use of the minipig in development of products does not bring any financial penalty in terms of the cost of testing. Benefits in terms of 3Rs can be identified in terms of life-cycle analysis of the use of minipigs compared to dogs and non-human primates. Finally the minipig (unlike the dog) is well positioned to take advantage of genomics and gene manipulation technologies. Specific recommendations for further research are made, which could bring 3Rs benefits. To deploy the minipig to the best advantage, clear information is needed about the predictivity of the minipig for human toxicities, and focussed action to define the potential role of the minipig in testing of biologics.

The utility of the minipig as an animal model in regulatory toxicology.

In this article we review the value and utility of the minipig as an animal model in regulatory toxicity testing. Our review is based on detailed consideration of the comparative biology of the minipig, and of the practical features of toxicity testing in the minipig. The minipig presents a favourable profile as a non-rodent toxicology model, in terms of the similarity to man and also in terms of applicability to different study types. Studies of general toxicology can be performed in the minipig by oral, cutaneous, parenteral and inhalation routes. For reproductive toxicology studies the minipig offers numerous advantages as a non-rodent model although the lack of placental transfer of macromolecules may limit the role of the minipig in reproductive testing of biotechnology products. For safety pharmacology studies the minipig is an advantageous model, particularly as regards the cardiovascular system. The immune system of the pig is better characterized than that of the dog, making the pig an interesting alternative model to the nonhuman primate for therapeutic approaches based on manipulation of the immune system. Overall, this review leads us to believe that the minipig might be a better non-rodent toxicology model than the dog. At the present time, however, insufficient comparative data is available to permit a rigorous evaluation of the predictivity of the minipig for human drug-induced toxicities and research is urgently needed to provide experimental data for evaluation of the hypothesis that minipig studies may better reflect human drug-induced toxicities than studies performed in traditional non-rodent toxicology models. It would be of particular value to gain a better vision of the potential utility of the minipig as a model for the safety testing of new biologics, where the minipig could potentially replace the use of non-human primates in the testing of some new products.

The RETHINK project--minipigs as models for the toxicity testing of new medicines and chemicals: an impact assessment.

The objective of the RETHINK project was to evaluate the potential impact of toxicity testing in the minipig as an alternative approach in regulatory toxicity testing that can contribute to the replacement, refinement and reduction of animal testing (3Rs). Minipigs are strains of domestic pigs that are markedly smaller than farmyard varieties, and thus better adapted to laboratory housing. The pig closely resembles man in many features of its anatomy, physiology, biochemistry and lifestyle. In particular, the cardio-vascular system, skin and digestive tract are considered to be very good models for man. Because of these similarities the toxic effects of chemicals and drugs in pigs may resemble the effects in man more closely than do some other commonly used laboratory animals. The pig also has some features that make it a very practical model for laboratory studies. Finally, being a food animal, testing in the minipig may be more acceptable to the public than animals such as dogs or monkeys. Expert study groups (Working Groups) were assembled to review five different areas relating to the use of minipigs in regulatory safety testing: ethical issues, welfare and animal care, development of new medicines and chemicals, safety testing issues and emerging technologies in safety testing. Their conclusions are presented in the articles of this special issue. The RETHINK project was funded as a Specific Support Action under the European Community 6th Framework Programme.

Short-term increase of serum troponin I and serum heart-type fatty acid-binding protein (H-FABP) in dogs following administration of formoterol.

In this paper, changes in serum levels of the cardiac biomarkers troponin I and the heart-type fatty acid-binding protein (H-FABP) following administration of a long-acting beta(2)-sympathicomimeticum (long-acting beta-agonist, LABA) to dogs were measured. We measured troponin I in dogs in a 4-week repeated-dose study with inhalative administration of formoterol (13microg/kgd) and a glucocorticoid/formoterol combination (143/16microg/kgd). The medians of troponin I increased within 3 days in both groups, far beyond the cut-off level (0.1microg/L), but returned to baseline levels on study day 9. The increase was more pronounced in the formoterol-only group (3.29microg/L) compared to the glucocorticoid/formoterol combination group (1.32microg/L). In a second study, we measured serum troponin I as well as serum H-FABP levels in several samples over 7 days in dogs, receiving a single inhalative dose of a glucocorticoid/formoterol combination (120/12mug/kgd). The median of the troponin I concentration increased above the cut-off level within 2h and that of H-FABP within 4h. The medians of both parameters were temporarily above the cut-off levels even on study day 7. Both studies were conducted according to national animal welfare guidelines. To our knowledge, this is the first report that shows a corresponding increase of troponin I and H-FABP in dogs treated with formoterol. Both parameters are more sensitive in detecting a drug-induced cardiac injury compared to total LDH, total CK as well as CK MB activity. However, it is recommended to take at least three blood samples per day to assess a temporary increase of troponin I.

Teratogenic effects of nitroimidazopyridazine on the CNS in fetal Wistar (WU) rats.

Teratogenic effects caused by a new nitroimidazopyridazine were examined in Wistar (WU) rats after repeated oral administration of 0, 2.5, 10, and 40 mg/kg, given on days 6-17 post coitum (p.c.) (Day of mating = Day 0) in a regular study on embryo-fetal development according to ICH S5A. At day 20 p.c., fetuses were removed and carefully examined under a dissecting microscope for external, visceral and skeletal malformations. The exposure to the high dose of the test compound during the organogenesis and early histogenesis periods of prenatal development induced prominent CNS malformations (exencephaly, neural tube defects (NTD)) associated with external malformations (hyperflexion of the forelimbs). To support the data from this study additional histological evaluation of the brains was performed with the following results: disorganization of the cerebral cortex associated with ectopic subcommissural organs. Additionally, an in vitro test (whole embryo culture, WEC) showed alterations of the developing neural tube after the incubation of rat embryos with the test compound on gestation days 9.5-11.5. Our data demonstrated that nitroimidazopyridazine caused NTDs and limb malformations during organogenesis. Based on these data the further development of the test compound was stopped.

Mesenteritis precedes vasculitis in the rat mesentery after subacute administration of a phosphodiesterase type 4 inhibitor.

Inhibitors of phosphodiesterase type 4 (PDE4) are currently exploited as potent drugs for pulmonary diseases. Some PDE4 inhibitors induce necrotizing panarteritis in the mesentery of rats, comparable to spontaneous polyarteritis nodosa in rats and vascular alterations that are induced by various vasoactive compounds, such as fenoldopam and inhibitors of PDE3. The mechanism of toxicity is unknown. In order to investigate the development of arteritis in the splanchnic vasculature of rats, a time-course study was performed with high doses of a compound (BYK169171), specifically inhibiting PDE4. Rats were treated orally for 1-28 days, and alterations in the mesentery were evaluated by histology, morphometry, and immunohistology. As early as 3 days after the onset of treatment, a mesenteritis was found, characterized by macrophage infiltration, fibroblast proliferation, neovascularization, and loss of adipocytes. Incidence and severity of the mesenteritis were low during the first 2 weeks of treatment, but increased with duration of treatment, finally affecting 2/3 of all animals. A segmental necrotizing panarteritis was detected in some rats treated for 21 or 28 days, but always followed a mesenteritis, whereas many animals with mesenteric inflammation did not have vascular lesions. We postulate that PDE4 inhibitors do not cause a primary vasculitis/arteritis in rats, but induce a non-purulent inflammation as the predominant initial toxic effect in the mesentery. This renders their toxic effect distinct from that of PDE3 inhibitors.

Integration of safety pharmacology endpoints into toxicology studies.

In the ILSI Human Toxicity Program, human toxicity was identified with 94% in studies of 1 month or less duration. Safety pharmacology studies and 1 month toxicity studies are prerequisites of INDs. These studies contributed in 69% to the predictivity of human toxicity. Correlating data from pharmacology and toxicology data will therefore enhance the predictivity of human toxicity. The ILSI Human Toxicity Program also showed that non-rodent toxicology studies were more predictive of human toxicity than rodents. Consequently, the usage of non-rodents, especially dogs, produces data more relevant to the safety of humans. Integration of data from safety pharmacology and pharmacological endpoints from integrated toxicology studies, which cover a wide dose range, allow data interpretation also concerning chronic effects. Differences in relation to chronic exposure and species specific pharmacodynamic effects can be taken into consideration. In vivo data in pharmacology are taken from a larger number of species. Usually, pharmacokinetic data and histopathology are often lacking in pharmacology (e.g. guinea pig). Studies in a smaller number of species, which are incorporating pharmacology, pharmacodynamic and toxicology, allow also crossinterpretation with data from clinical chemistry, haematology and histopathology. Endpoints relating to behaviour (functional observation battery/FOB) and cardiovascular toxicity can be integrated into regulatory toxicology. Technical progress in non-invasive methodology and refined measurements for pharmacological parameters and standardization of study design allow the incorporation into regulatory toxicity studies today. The limitations of conducting pharmacological measurements in regulatory toxicology studies are acknowledged. Safety pharmacology studies should complement toxicity studies in terms of choice of species and dose regimen. Ethical usage of animals, especially dogs or monkeys, can only be justified in the future, when more clinically relevant data can be gained from fewer in vivo studies. Multidisciplinary co-operation between pharmacology, pharmacokinetics and toxicology will lead to refinements and reduction of in-vivo studies when functional parameters are integrated into regulatory studies.

ICH topic: the draft ICH S7B step 2: note for guidance on safety pharmacology studies for human pharmaceuticals.

During the last ICH Expert Working Group meeting for Safety Pharmacology in February 2002 in Brussels, the appended step 2 S7B document on QT Prolongation was signed off. This paper will now be published by the three agencies. The comment period will be 6 months. Nevertheless, the EU and EFPIA would be very appreciative of comments received before the end of May 2002, in order to start the discussion on how this step 2 document should be modified. In addition, everybody is requested to share data that could support the evaluation process of all methods addressed in this document. The EWG appreciates every support.