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Regulation of human pharmaceuticals in Europe was not generally established until the thalidomide disaster around 1960, more than 50 years ago. The political willingness to have harmonized criteria on a more European level arose already soon, resulting in a first Directive 65/65. Harmonized pharmacological and toxicological criteria were established 10 years later in Directive 75/318. It took another 10-15 years before the Committee on Proprietary Medicinal Products started as an advisory committee to the European Commission, and its member states.
The tasting of harmonization did also result in an initiative to start a global process with Japan and the United States in 1989, with the first International Conference on Harmonisation in Brussels in November 1991.
The starting point for assessment of new human pharmaceuticals is always to understand the clinical relevancy of the toxicity data, instead of focusing on full characterization of the toxicity of a substance up to the lethal level. This can be illustrated in the choice for dose selection.
Restriction of use of animals. The discussion about the protection of animals for scientific purposes is always important in the EU. A recent directive (2010/63) renewed the previous one after 25 years, further emphasizing that animals should only be used when clearly needed. In the legislation around cosmetics animal data are no longer allowed for regulatory purposes. Stimulation of in vitro approaches is therefore important in the research activities. Reproduction toxicity, carcinogenicity and sensitization are actual topics with promising perspectives to reduce animal use.

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Preclinical assessments of a drug’s abuse liability typically involve studies that assess the capacity of a drug to produce physiological dependence and those that assess the reinforcing effects of drugs with self-administration procedures. Most often, studies that examine physiological dependence do so by assessing whether a drug reverses the withdrawal signs that follow treatment with a standard drug. Withdrawal also occurs from drugs that are used therapeutically, but is typically referred to as rebound. Problems of abuse stem therefore, not from physiological dependence per se, but from the reinforcing effects of abused drugs, though dependence may influence reinforcing effects of drugs. Simple results from self-administration procedures are predictive of whether a drug will be abused in human populations. Nonetheless, there is documentation that the pharmacological history of a subject may predispose or counter predispose to reinforcing effects of other agents. Self-administration procedures are also used in searches for medications to treat drug abuse. This approach is the logical extension of abuse liability assessments, but has not been validated, the key concern of translation. Critical to assessments of the effects of potential drug-abuse medications is the shape of the self-administration dose-effect curve and how it is changed by treatment with the potential medication. Two types of effects of treatments have been discussed in the clinical literature – antagonism (e.g. naltrexone for opioid abuse) and substitution (e.g. methadone). How these types of treatments relate to the change in the self-administration dose-effect curve requires substantial focus to fully realize translational potential for laboratory studies.

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Many neurological diseases develop by unknown mechanisms. A common feature in many of these is the appearance of so-called “neuroinflammatory changes”. This refers to the widespread activation of microglial cells in the areas where the nervous system is being damaged. It is unclear whether such changes are part of the pathogenesis, or merely a reaction to damage that has occurred. Animal models of such diseases provide evidence that microglial activation may be a key step in the disorder’s development. If this is true, then it might be possible to delay or halt the disease with therapeutic agents specifically targeting the microglial cells. A major impediment in this quest is identifying compounds effectively targeting microglia in CNS tissue. Whole animal testing of each compound would be impossibly expensive and time consuming. In view of this our lab has developed a strategy that can select promising compounds by testing for cellular effects in vitro, then applying a sensitive CNS tissue slice method to detect agents that have anti-inflammatory effects at the tissue level. By using RT-PCR to select agents that inhibit the production of inflammatory mediators such as TNF and IL-1, we can demonstrate the effects of known anti-inflammatory agents and also detect novel compounds with similar effects. The presentation will discuss the method and show how it can be used to test agents for their ability to inhibit neuroinflammatory changes.

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ICH M3(R2) Guidance has helped clarify the timing of reproductive toxicology studies in drug development. However, the guidance remains general and may not apply to all drug candidates for all circumstances. Therefore, in addition to the guidance document, other factors such as pharmacological, pharmacokinetic (PK), and toxicological characteristics of the drug candidate, and clinical trial design should be considered when planning the timing of these studies. In embryo-fetal development studies, exposure to drug could be indicated by the presence of maternal toxicity, and ICH did not require collection of toxicokinetic (TK) data in these studies. This results in the absence of TK data in some studies, and assessment of risk to pregnant mothers is done using the conversion of animal dose to human equivalent dose based on body surface area. This approach is less precise and occasionally not accepted by regulatory agencies. In rabbit embryo-fetal development study, the absence of PK/ADME data, coupled with the occasional absence of TK data, has led many to question the relevance of this species in risk assessment. A survey has been conducted to address this question. Under the EMA regulation, planning for the juvenile animal study should be submitted early in development. Two approaches are generally considered when designing the juvenile animal study. In the targeted design approach, the study will evaluate target organs of concern in the target population. In the absence of target organs, the modified general toxicity screening approach may be used to identify hazards in the young and developing animals.

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Toxicogenomics is a mature field which provides invaluable information on the molecular events preceding or accompanying toxicity. Most traditional uses of transcriptional profiling and other –omics data in toxicology are: mode-of-action analysis, classification/prediction, and biomarker discovery. While these applications are very informative for the regulatory decisions, they do not contribute to quantitative evaluation of the margin of safety and/or characterization of the uncertainties in species-to-species extrapolation and population variability. Studies that explore the shape of dose-response relationships at the molecular pathway level, and the impact that inter-individual differences in gene expression patterns may have on both efficacy and safety outcomes, represent some of the novel frontiers in toxicogenomics. Dose-response toxicogenomic data may be used for calculating the point-of-departure at which either adverse or adaptive “molecular pathology” phenotypes are observed after exposure to chemicals. Aggregation of the dose-response genes into pathways further improves the linkages between mechanistic understanding and estimating the margin of safety. In addition, the challenge of elucidating the genetic determinants of inter-individual differences in toxicity may be met through a combined analysis of the toxicity phenotypes, genetic polymorphisms and gene expression data from the population-based experimental in vivo and in vitro model systems. The intricate interplay between genetic polymorphisms and gene expression requires careful consideration of the genetic background-dependent and –independent toxicity pathways. Collectively, as the number of toxicogenomic studies which incorporate dose-response and/or population-based study designs is on the rise, the applicability of such data to next-generation human health risk assessments will also increase.

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The promise of RNA-targeting therapeutics (antisense, siRNA and RNA-splice modulation) is that it is possible to design therapeutic entities based solely on the sequence of the RNA target, taking advantage of Watson-Crick base-pairing rules for rational drug design. The concept is simple, however full realization of the potential has not yet been achieved. Recent advancements are turning the promise into reality. Since the first description of antisense acitivty and the subsequent discovery of siRNA, significant progress has been made in the understanding of mechanisms of action and how these processes exploit naturally occurring phenomena. In addition to a better understanding of the enzymologic basis for these technologies, there have been remarkable advancements in understanding of mechanisms associated with the adverse effects of RNA-targeted therapies. How administering oligonucleotides produces adverse effects and how to avoid adverse effects has been the subject of intensive research in bioinformatics, innate immunity, pharmacokinetics and pharmaceutics. Delivery of oligonucleotide-based therapeutics remains a challenge. Nature has solved the delivery problem using exosomes and self-assembling oligonucleotide containing nanoparticles (viruses). To solve the delivery problem it might be useful to learn from nature. We are in an important time for oligonucleotide technology and there are many new advancements and new clinical results each week. This talk will highlight some of the key activities ongoing in the field.

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Phospholipdosis is an unusual intracellular accumulation of phospholipids associated with a variety of diseases such as inborn errors of metabolism and exposure to toxic xenobiotics. Characterized by intracellular inclusions called lamellar bodies, phospholipidosis is also a common finding in nonclinical toxicology studies designed to enable clinical trials and marketing applications for new drugs. When observed, there is uncertainty concerning the safety implications of the finding. Phospholipidosis is a finding usually associated with what are generally referred to as cationic amphiphilic drugs (CADS). CADs have a distinct structure: they contain both hydrophobic and hydrophilic domains, which enables transit through cellular membranes and intracellular concentration. This chemical property may be desirable for drugs that act on intracellular targets and in some circumstances phospholipidosis could be related to pharmacodynamic activity and drug efficacy. However, there are many examples of phospholipidosis associated with pathology. Thus, there is a dilemma in assessing the safety implication of phospholipidosis. Many factors should be considered in evaluating phospholipidosis: structural relationship to chemicals known to cause pathology, tissues affected, and histopathology associated with lamellar bodies. This is a very complex subject and requires assessment of many parameters in order to determine if demonstration of phospholipidosis is a cause for concern.

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Questions are often raised about the safety of vaccine adjuvants, particularly in relation to autoimmunity or autoimmune disease(s)/disorder(s) (AID). The International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) formed a scientific committee, which included technical experts from academia, government regulatory agencies and industry to consider adjuvant safety and conducted a wide literature review and a two-day workshop. This large topic was broken into subtopics and focused on oil-in-water emulsions and Toll-like Receptor (TLR) agonists adjuvants, use of animal models and biomarkers for the evaluation and prediction of AID and addressed key issues including: the value of animal models of autoimmunity for studying novel vaccine adjuvants; whether there is scientific evidence indicating an intrinsic risk of autoimmunity with adjuvants, or a higher risk resulting from the mechanism of action; and if there is compelling clinical data linking adjuvants and AID. The tripartite group of experts concluded that there is no compelling evidence supporting the association of vaccine adjuvants with autoimmunity signals. Additionally, it recommended that if future research focused on the potential effects of adjuvanted vaccines on AID, it should consider carefully the experimental design in animal models particularly if they are to be used in any risk assessment, as depending on the study design, the results could be misleading. In addition, studies on the mechanistic aspects and potential biomarkers related to adjuvants and autoimmunity phenomena could be developed.

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The WHO took the initiative of writing a guideline on the nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines. The EU guideline on adjuvants became in force in 2005, and several adjuvants came on the market since then. With this experience and new insights on the impact of Toll-like receptors on the immune responses, an update of the regulatory testing was needed, preferably from a global perspective. After a consultation workshop in 2011, a drafting group consisting of experts on nonclinical testing from various regulatory areas in the world (Indonesia, Canada, United States, Europe) was established. Important topics being discussed were the testing of the adjuvant independent from the antigen, the design of reproductive toxicity studies and a possible relation with the induction of autoimmunity.
It became clear that testing of the adjuvant alone is of limited value, and combination with an antigen in a single relevant species is to be recommended. With respect to reproductive toxicity it is clear that vaccination during pregnancy might be important to protect the mother as well as the child, but an adjuvant might be a risk especially very early in pregnancy. Recommendations for an adapted study design have been given. Autoimmunity has been suggested to be a specific risk for adjuvanted vaccines, and this has been discussed thoroughly. There is no compelling evidence, however, that there is an association between adjuvants and autoimmunity disease symptoms. Animal models are not present to support such releationships.

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Protection of animals used for scientific purposes in the European Union is afforded by Directive 2010/63/EU, which being adopted on 22 September 2010 and coming into force on January 1^st 2013, superseded Directive 86/609/EEC and set out revised measures for the protection and welfare of animals by raising minimum animal welfare standards in line with latest scientific developments. By placing emphasis upon the 3Rs of replacement, reduction and refinement, the Directive not only addresses fundamental requirements for animal care, such as housing and environmental enrichment, but also introduced a number of new elements including a fourth ‘R’ of rehoming. It also establishes the need for Animal Welfare Bodies, assurance of competency of researchers and animal carers, and increased statistics including reporting of actual severity of procedures.

This presentation will provide an overview of the scope, structure and general provisions of the EU Directive in relation to conventional and genetically altered animals, with discussion of specific elements that have been introduced to improve animal welfare. There will also be reference to statistics for animal use in the EU, EU guidance on care of animals, discussion of how a European contract research facility has implemented? new requirements, and comparison to the animal welfare requirements of Switzerland, which is not a member of the European Union.

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