Heterogeneous vasomotor responses in segments from Göttingen Minipigs coronary, cerebral, and mesenteric artery: A comparative study.

Göttingen Minipigs (GM) are used as an important preclinical model for cardiovascular safety pharmacology and for evaluation of cardiovascular drug targets. To improve the translational value of the GM model, the current study represents a basic characterization of vascular responses to endothelial regulators and sympathetic, parasympathetic, and sensory neurotransmitters in different anatomical origins. The aim of the current comparative and descriptive study is to use myography to characterize the vasomotor responses of coronary artery isolated from GM and compare the responses to those obtained from parallel studies using cerebral and mesenteric arteries. The selected agonists for sympathetic (norepinephrine), parasympathetic (carbachol), sensory (calcitonin gene-related peptide, CGRP), and endothelial pathways (endothelin-1, ET-1, and bradykinin) were used for comparison. Further, the robust nature of the vasomotor responses was evaluated after 24 h of cold storage of vascular tissue mimicking the situation under which human biopsies are often kept before experiments or grafting is feasible. Results show that bradykinin and CGRP consistently dilated, and endothelin consistently contracted artery segments from coronary, cerebral, and mesenteric origin. By comparison, norepinephrine and carbachol, had responses that varied with the anatomical source of the tissues. To support the basic characterization of GM vasomotor responses, we demonstrated the presence of mRNA encoding selected vascular receptors (CGRP- and ETA-receptors) in fresh artery segments. In conclusion, the vasomotor responses of isolated coronary, cerebral, and mesenteric arteries to selected agonists of endothelial, sympathetic, parasympathetic, and sensory pathways are different and the phenotypes are similar to sporadic human findings.

Species selection for nonclinical safety assessment of drug candidates: Examples of current industry practice.

In drug development, nonclinical safety assessment is pivotal for human risk assessment and support of clinical development. Selecting the relevant/appropriate animal species for toxicity testing increases the likelihood of detecting potential effects in humans, and although recent regulatory guidelines state the need to justify or dis-qualify animal species for toxicity testing, individual companies have developed decision-processes most appropriate for their molecules, experience and 3Rs policies. These generally revolve around similarity of metabolic profiles between toxicology species/humans and relevant pharmacological activity in at least one species for New Chemical Entities (NCEs), whilst for large molecules (biologics) the key aspect is similarity/presence of the intended human target epitope. To explore current industry practice, a questionnaire was developed to capture relevant information around process, documentation and tools/factors used for species selection. Collated results from 14 companies (Contract Research Organisations and pharmaceutical companies) are presented, along with some case-examples or over-riding principles from individual companies. As the process and justification of species selection is expected to be a topic for continued emphasis, this information could be adapted towards a harmonized approach or best practice for industry consideration.

Genomics Integrated Systems Transgenesis (GENISYST) for gain-of-function disease modelling in Göttingen Minipigs.

Göttingen Minipigs show several anatomical, physiological, and pathogenetical similarities to humans and serve an important role in translational studies for example as large animal models of disease. In recent years, the number of transgenic Göttingen Minipigs models has increased, as advanced genetic techniques simplify the generation of animals with precisely tailored modifications. These modifications are designed to replicate genetic alterations responsible for human disease. In addition to serving as valuable large animal disease models, transgenic Göttingen Minipigs are also considered promising donors for xenotransplantation. Current technologies for generation of transgenic minipigs demand a long development and production time of typically 2-3 years. To overcome this limitation and expand the use of Göttingen Minipigs for disease modelling and drug testing, we developed the GENISYST (Genomics Integrated Systems Transgenesis) technology platform for rapid and efficient generation of minipigs based transgenic disease models. As proof of concept, we report the successful generation of transgenic minipigs expressing green fluorescent protein (GFP) in multiple disease-relevant tissues including liver, heart, kidney, lungs, and the central nervous system (CNS). Our data demonstrates the feasibility, efficiency, and utility of GENISYST for rapid one-step generation of transgenic minipigs for human disease modelling in drug discovery and development.

Assessing breed integrity of Göttingen Minipigs.

BACKGROUND: Göttingen Minipigs (GMP) is the smallest commercially available minipig breed under a controlled breeding scheme and is globally bred in five isolated colonies. The genetic isolation harbors the risk of stratification which might compromise the identity of the breed and its usability as an animal model for biomedical and human disease. We conducted whole genome re-sequencing of two DNA-pools per colony to assess genomic differentiation within and between colonies. We added publicly available samples from 13 various pig breeds and discovered overall about 32 M loci, ~ 16 M. thereof variable in GMPs. Individual samples were virtually pooled breed-wise. FST between virtual and DNA pools, a phylogenetic tree, principal component analysis (PCA) and evaluation of functional SNP classes were conducted. An F-test was performed to reveal significantly differentiated allele frequencies between colonies. Variation within a colony was quantified as expected heterozygosity. RESULTS: Phylogeny and PCA showed that the GMP is easily discriminable from all other breads, but that there is also differentiation between the GMP colonies. Dependent on the contrast between GMP colonies, 4 to 8% of all loci had significantly different allele frequencies. Functional annotation revealed that functionally non-neutral loci are less prone to differentiation. Annotation of highly differentiated loci revealed a couple of deleterious mutations in genes with putative effects in the GMPs . CONCLUSION: Differentiation and annotation results suggest that the underlying mechanisms are rather drift events than directed selection and limited to neutral genome regions. Animal exchange seems not yet necessary. The Relliehausen colony appears to be the genetically most unique GMP sub-population and could be a valuable resource if animal exchange is required to maintain uniformity of the GMP.

Perspectives From the 12th Annual Minipig Research Forum: Early Inclusion of the Minipig in Safety Assessment Species Selection Should be the Standard Approach.

Over the last decade, the minipig has been established as a species which can be used in biomedical research, including drug development safety assessment. There are no mandatory regulatory guidelines regarding species selection strategy for safety assessment; hence, choice is at the discretion of companies responsible for drug development. A survey of member companies by IQ DruSafe (2016) highlighted inconsistent and low use of the minipig. At the 12th Annual Minipig Research Forum in 2018, presentations and a workshop examined current practices and considered if the minipig could be utilized more from earliest drug development stages. Despite the agreed utility of scientific data and validity of the minipig, especially for small molecules, each company has its own approach in nonrodent species selection, without consistent rationale. The overall objective should be to ensure the most appropriate species is selected and is scientifically based, with the minipig systematically included from early screening stages.

A shortened study design for embryo-fetal development studies in the minipig.

The minipig is accepted from scientific and regulatory perspectives for the safety evaluation of drug candidates on embryo-fetal development. The relative size and the duration of gestation (112-115 days) in the minipig is, however, considered a drawback compared with routine smaller species. We evaluated if study duration and cost could be optimized without impacting scientific validity by performing all terminal procedures around mid-gestation (60 days). At this stage, minipig fetal size is not too dissimilar to full term rabbit and therefore better suited to fetal processing/examination compared with at the end of gestation. Despite encountering higher than anticipated embryo-fetal death, morphological defects clearly associated with a known teratogen, pyrimethamine, were detected. Although the gonads are poorly differentiated macroscopically at mid-term, a histological examination confirmed that external sexing of the fetuses was accurate. Double staining of the bone and cartilage of the mid-term fetal skeleton allowed a more refined examination.

Impact of blood sampling technique on blood quality and animal welfare in haemophilic mice.

Blood collection in mice can be a challenge, in particular for samples used for coagulation analysis as initiation of coagulation during the procedures can influence the results. Blood collection from the retrobulbar venous plexus is commonly used but the method remains controversial. Several alternatives exist but not all are applicable to mice with a compromised coagulation system because of subsequently excessive bleeding. We therefore wanted to explore whether blood collection by puncture of the submandibular vein could replace blood collected from the retrobulbar venous plexus during pharmacokinetic and pharmacodynamic studies in mice lacking coagulation factor VIII (FVIII). The plasma concentrations of recombinant activated factor VII were independent of the blood collection method in a pharmacokinetic study. The same applied to the thromboelastographic profile of mice with normal coagulation in a pharmacodynamic study. However, excessive haemorrhages were observed in all FVIII knockout mice after a single puncture of the submandibular vein and 60% of the mice were euthanized 2-4 h after the blood collection. In contrast, no or only slight haemorrhage was observed in animals subjected to blood collection from the retrobulbar venous plexus. No signs of distress determined by blood glucose level or clinical abnormalities of the eye were observed after puncture of the retrobulbar venous plexus. In conclusion, blood collected by puncture of the submandibular vein and retrobulbar venous plexus has a quality which allows it to be used in coagulation assays. However, because of excessive bleedings, puncture of the submandibular vein is not recommended in mice lacking FVIII.

New housing conditions: improving the welfare of experimental animals.

As animal experiments and testing are still a necessary part of the discovery and development of new drugs and do not seem likely to be totally replaced in the foreseeable future, it is important that the care and use of these animals are continuously refined. Since the housing facilities are where most experimental animals spend the major part of their lives, this area should be given special attention to ensure optimal welfare for the animals. In a unique collaboration between a pharmaceutical company and an animal welfare organisation, the housing conditions of mice, rats, guinea-pigs, rabbits and dogs, respectively, were reviewed with focus on the basic needs of the animals. Prototypes for new housing systems satisfying the most important of these basic needs of the animals were developed, with valuable input from international experts with special knowledge of the behaviour of experimental animals. These new housing systems and species-specific, newly introduced socialisation programmes contribute to improved animal welfare and a better occupational health of the animal caretakers. Since these new housing systems are more pleasant and appealing, they may also have the added benefit that they contribute to a broader public acceptance of the use of experimental animals.