Strategies to apply 3Rs in preclinical testing.

Animal experimentation has been fundamental in biological and biomedical research. To guarantee the maximum quality, efficacy and/or safety of products intended for the use in humans in vivo testing is necessary; however, for over 60 years, alternative methods have been developed in response to the necessity to reduce the number of animals used in experimentation, to guarantee their welfare; resorting to animal models only when strictly necessary. The three Rs (Replacement, Reduction, and Refinement), seek to ensure the rational and respectful use of laboratory animals and maintain an adequate projection in terms of bioethical considerations. This article describes different approaches to apply 3Rs in preclinical experimentation for either research or regulatory purposes.

3D cell culture models: Drug pharmacokinetics, safety assessment, and regulatory consideration.

Nonclinical testing has served as a foundation for evaluating potential risks and effectiveness of investigational new drugs in humans. However, the current two-dimensional (2D) in vitro cell culture systems cannot accurately depict and simulate the rich environment and complex processes observed in vivo, whereas animal studies present significant drawbacks with inherited species-specific differences and low throughput for increased demands. To improve the nonclinical prediction of drug safety and efficacy, researchers continue to develop novel models to evaluate and promote the use of improved cell- and organ-based assays for more accurate representation of human susceptibility to drug response. Among others, the three-dimensional (3D) cell culture models present physiologically relevant cellular microenvironment and offer great promise for assessing drug disposition and pharmacokinetics (PKs) that influence drug safety and efficacy from an early stage of drug development. Currently, there are numerous different types of 3D culture systems, from simple spheroids to more complicated organoids and organs-on-chips, and from single-cell type static 3D models to cell co-culture 3D models equipped with microfluidic flow control as well as hybrid 3D systems that combine 2D culture with biomedical microelectromechanical systems. This article reviews the current application and challenges of 3D culture systems in drug PKs, safety, and efficacy assessment, and provides a focused discussion and regulatory perspectives on the liver-, intestine-, kidney-, and neuron-based 3D cellular models.

Combination of cassette-dosing and microsampling for reduced animal usage for antibody pharmacokinetics in cynomolgus monkeys, wild-type mice, and human FcRn transgenic mice.

PURPOSE: The aim of this study was to develop a useful antibody PK evaluation tool using a combination of cassette-dosing and microsampling in mice and monkeys in order to reduce the number of animals used. METHODS: Cetuximab, denosumab, infliximab, and a mixture of the three antibodies, i.e., cassette-dosing, were administered intravenously to cynomolgus monkeys, C57BL/6J mice, and homozygous human neonatal Fc-receptor transgenic (Tg32) mice. Mouse blood was collected from one animal continuously via the jugular vein at nine points. RESULTS: In cynomolgus monkeys, infliximab showed faster elimination in the cassette-dosing group than in the single-dose group. Anti-drug antibody production was observed, but the PK parameters of the clearance and distribution volume were similar in both groups. In C57BL/6J and Tg32 mice, each of the plasma concentrations-time profiles after cassette-dosing were similar to those after single dosing. PK evaluation using a combination of cassette-dosing and microsampling in mice may reduce the number of mice used by approximately 90% compared with the conventional method. CONCLUSIONS: The combination of antibody cassette-dosing and microsampling is a promising PK evaluation method as a high-throughput and reliable with reduced numbers of mice and cynomolgus monkeys.

Functional testing of topical skin formulations using an optimised ex vivo skin organ culture model.

A number of equivalent-skin models are available for investigation of the ex vivo effect of topical application of drugs and cosmaceuticals onto skin, however many have their drawbacks. With the March 2013 ban on animal models for cosmetic testing of products or ingredients for sale in the EU, their utility for testing toxicity and effect on skin becomes more relevant. The aim of this study was to demonstrate proof of principle that altered expression of key gene and protein markers could be quantified in an optimised whole tissue biopsy culture model. Topical formulations containing green tea catechins (GTC) were investigated in a skin biopsy culture model (n = 11). Punch biopsies were harvested at 3, 7 and 10 days, and analysed using qRT-PCR, histology and HPLC to determine gene and protein expression, and transdermal delivery of compounds of interest. Reduced gene expression of α-SMA, fibronectin, mast cell tryptase, mast cell chymase, TGF-ß1, CTGF and PAI-1 was observed after 7 and 10 days compared with treated controls (p < 0.05). Histological analysis indicated a reduction in mast cell tryptase and chymase positive cell numbers in treated biopsies compared with untreated controls at day 7 and day 10 (p < 0.05). Determination of transdermal uptake indicated that GTCs were detected in the biopsies. This model could be adapted to study a range of different topical formulations in both normal and diseased skin, negating the requirement for animal models in this context, prior to study in a clinical trial environment.

[Strategic considerations on the design and choice of animal models for non-clinical investigations of cell-based medicinal products]. / Strategische Betrachtungen zur Konzeption und Wahl von Tiermodellen bei nicht-klinischen Prüfungen von zellbasierten Therapeutika.

For the development of medicinal products animal models are still indispensable to demonstrate efficacy and safety prior to first use in humans. Advanced therapy medicinal products (ATMP), which include cell-based medicinal products (CBMP), differ in their pharmacology and toxicology compared to conventional pharmaceuticals, and thus, require an adapted regime for non-clinical development. Developers are, therefore, challenged to develop particular individual concepts and to reconcile these with regulatory agencies. Guidelines issued by the European Medicines Agency (EMA), the U.S. Food and Drug Administration (FDA) and other sources can provide direction.The published approaches for non-clinical testing of efficacy document that homologous animal models where the therapeutic effect is investigated in a disease-relevant animal model utilizing cells derived from the same species are commonly used. The challenge is that the selected model should reflect the human disease in all critical features and that the cells should be comparable to the investigated human medicinal product in terms of quality and biological activity. This is not achievable in all cases. In these cases, alternative methods may provide supplemental information. To demonstrate the scientific proof-of-concept (PoC), small animal models such as mice or rats are preferred. During the subsequent product development phase, large animal models (i.e. sheep, minipigs, dogs) must be considered, as they may better reflect the anatomical or physiological situation in humans. In addition to efficacy, those models may also be suitable to prove some safety aspects of ATMP (e.g. regarding dose finding, local tolerance, or undesired interactions and effects of the administered cells in the target tissue). In contrast, for evaluation of the two prominent endpoints for characterizing the safety of ATMP (i.e. biodistribution, tumorigenicity) heterologous small animal models, especially immunodeficient mouse strains, are favourable due to their tolerance to the human cell therapy product. The execution of non-clinical studies under the principles of good laboratory practice (GLP) increases the acceptance of the results by authorities and the scientific community.

The safety, efficacy and regulatory triangle in drug development: Impact for animal models and the use of animals.

Nonclinical studies in animals are conducted to demonstrate proof-of-concept, mechanism of action and safety of new drugs. For a large part, in particular safety assessment, studies are done in compliance with international regulatory guidance. However, animal models supporting the initiation of clinical trials have their limitations, related to uncertainty regarding the predictive value for a clinical condition. The 3Rs principles (refinement, reduction and replacement) are better applied nowadays, with a more comprehensive application with respect to the original definition. This regards also regulatory guidance, so that opportunities exist to revise or reduce regulatory guidance with the perspective that the optimal balance between scientifically relevant data and animal wellbeing or a reduction in animal use can be achieved. In this manuscript we review the connections in the triangle between nonclinical efficacy/safety studies and regulatory aspects, with focus on in vivo testing of drugs. These connections differ for different drugs (chemistry-based low molecular weight compounds, recombinant proteins, cell therapy or gene therapy products). Regarding animal models and their translational value we focus on regulatory aspects and indications where scientific outcomes warrant changes, reduction or replacement, like for, e.g., biosimilar evaluation and safety testing of monoclonal antibodies. On the other hand, we present applications where translational value has been clearly demonstrated, e.g., immunosuppressives in transplantation. Especially for drugs of more recent date like recombinant proteins, cell therapy products and gene therapy products, a regulatory approach that allows the possibility to conduct combined efficacy/safety testing in validated animal models should strengthen scientific outcomes and improve translational value, while reducing the numbers of animals necessary.

Animal welfare and use of silkworm as a model animal.

Sacrificing model animals is required for developing effective drugs before being used in human beings. In Japan today, at least 4,210,000 mice and other mammals are sacrificed to a total of 6,140,000 per year for the purpose of medical studies. All the animals treated in Japan, including test animals, are managed under control of "Act on Welfare and Management of Animals". Under the principle of this Act, no person shall kill, injure, or inflict cruelty on animals without due cause. "Animal" addressed in the Act can be defined as a "vertebrate animal". If we can make use of invertebrate animals in testing instead of vertebrate ones, that would be a remarkable solution for the issue of animal welfare. Furthermore, there are numerous advantages of using invertebrate animal models: less space and small equipment are enough for taking care of a large number of animals and thus are cost-effective, they can be easily handled, and many biological processes and genes are conserved between mammals and invertebrates. Today, many invertebrates have been used as animal models, but silkworms have many beneficial traits compared to mammals as well as other insects. In a Genome Pharmaceutical Institute's study, we were able to achieve a lot making use of silkworms as model animals. We would like to suggest that pharmaceutical companies and institutes consider the use of the silkworm as a model animal which is efficacious both for financial value by cost cutting and ethical aspects in animals' welfare.

Juan josé Heydeck, un alemán en la corte de Carlos IV: experimentos contra la viruela / No disponible

En los primeros años del sigo XIX, Juan José Heydeck propone la realización de unos experimentos con el pus de cabras en lugar del pus de vacas, para luchar contra las viruelas naturales. Estos experimentos requerían un número indeterminado de niños donde verificarlos. En España, el pus de cabras sería una alternativa al pus de vaca. En estos territorios era difícil encontrar el pus de vacas. Esta iniciativa del pus hircino no tuvo el éxito esperado

First years of the XIX century, Juan José Heydeck proposes the realization of the experiments with the pus of the goats instead of the pus of the cows, for combat the smallpox. This experiments require more childrens, where to be verified them. In Spain, the pus of the goats will be the one alternative to the p us of the cows. In this country is difficult fall in with the pus of the cows. This iniciative haven´t the hope results