Regulatory acceptance of animal models of disease to support clinical trials of medicines and advanced therapy medicinal products.

European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Regulatory acceptance of animal models of disease to support clinical trials of medicines and advanced therapy medicinal products Joy Cavagnaro a,n, Beatriz Silva Lima b a b

Access BIO, PO Box 254, Boyce, VA, USA iMED.ULisboa, Universidade de Lisboa, Portugal

art ic l e i nf o

a b s t r a c t

Article history: Received 18 February 2015 Received in revised form 26 February 2015 Accepted 12 March 2015

The utility of animal models of disease for assessing the safety of novel therapeutic modalities has become an increasingly important topic of discussion as research and development efforts focus on improving the predictive value of animal studies to support accelerated clinical development. Medicines are approved for marketing based upon a determination that their benefits outweigh foreseeable risks in specific indications, specific populations, and at specific dosages and regimens. No medicine is 100% safe. A medicine is less safe if the actual risks are greater than the predicted risks. The purpose of preclinical safety assessment is to understand the potential risks to aid clinical decision-making. Ideally preclinical studies should identify potential adverse effects and design clinical studies that will minimize their occurrence. Most regulatory documents delineate the utilization of conventional “normal” animal species to evaluate the safety risk of new medicines (i.e., new chemical entities and new biological entities). Animal models of human disease are commonly utilized to gain insight into the pathogenesis of disease and to evaluate efficacy but less frequently utilized in preclinical safety assessment. An understanding of the limitations of the animal disease models together with a better understanding of the disease and how toxicity may be impacted by the disease condition should allow for a better prediction of risk in the intended patient population. Importantly, regulatory authorities are becoming more willing to accept and even recommend data from experimental animal disease models that combine efficacy and safety to support clinical development. & 2015 Published by Elsevier B.V.

Key words: In vitro In vivo Animal model Species selection Disease model 3Rs Predictive value Preclinical safety assessment Regulatory guidance Guidelines ICH Critical Path Initiative Innovative Medicines Initiative Induced pluripotent stem cells

1. Introduction Regulatory authorities play a major role in the interpretation of results from animal studies conducted to support clinical applications of novel therapeutic modalities. Regulators are tasked with simultaneously promoting innovations that can improve health while implementing policies that ensure that the benefits of new products will outweigh their risks. However, the regulatory environment is also increasingly challenged with a rapid growth in knowledge and technologies. In addition, when a product is withdrawn from the marketplace due to serious safety concerns; the regulatory authorities are under heightened public scrutiny and even criticized for approving an “unsafe” product. Most developers and regulators concerned with assessing the safety of new medicines currently recognize the importance of applying the principles of the 3Rs (Replacement, Reduction and Refinement) for protecting animals used for scientific purposes

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Corresponding author. Tel.: þ 1 540 837 9002. E-mail addresses: [email protected] (J. Cavagnaro), [email protected] (B. Silva Lima).

(Directive 2010/63/EU). However, they also acknowledge that in most cases there are no established alternatives to testing in animals. More importantly, they are also challenged with an increasing imperative to enhance the predictability of the data from animal studies to ensure the safety in humans. While the current methods of safety assessment, mostly animal-based, have been successful in screening out compounds that might cause toxicity in a substantial proportion of patients, they have been less so at predicting serious adverse effects that occur only in a relatively small minority of patients. Some reasons given for why animal studies fail to detect these effects is that animal studies are not powered to detect rare events, and as they are mostly conducted in healthy animals, the impact of the disease on the biological activity of test compounds is not assessed. Arguably, patients enrolled in clinical trials also do not reflect the full range of the population or treatment situations that occur in practice. As a result, new safety issues are often identified only after medicines enter the market (Woodcock and Woosley, 2008). Predictions of safety between species (e.g. rat, dog, monkey and human) are good but not perfect. Retrospective analyses conducted by industry has demonstrated that toxicity evaluation in healthy rodent and non-rodent species results in prediction of

http://dx.doi.org/10.1016/j.ejphar.2015.03.048 0014-2999/& 2015 Published by Elsevier B.V.

Please cite this article as: Cavagnaro, J., Silva Lima, B., Regulatory acceptance of animal models of disease to support clinical trials of medicines and advanced therapy medicinal products. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.03.048i

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J. Cavagnaro, B. Silva Lima / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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human risk in approximately 71% instances (Olson et al., 2000). The present inability to show good concordance between some animal efficacy studies and human clinical outcomes is believed to be due in part to shortcomings in experimental design and conduct, as well as reporting results (Everitt, 2015). The various regulatory agencies house large repositories of in vitro and in vivo animal results that are linked with actual human outcomes data. Data mining efforts which effectively protect proprietary data provide the scientific basis for better predictive preclinical safety models. The decision to utilize an animal model of human disease as part of a preclinical safety submission/dossier has historically been driven by a need to test a specific hypothesis typically generated after target organs have been identified in standard toxicity studies in healthy animals. Animal models of disease have not been used initially based on their inherent limitations e.g. inability to accurately recapitulate all the key aspects of the corresponding human disease and the limited historical data on general health and spontaneous disease pathology. This article specifically highlights current regulatory guidance published by US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), and where appropriate, the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), encouraging the use of animal models of disease to support clinical development. It is acknowledged that other countries may have similar guidance.

2. Modernizing preclinical and clinical development

safety testing are decades old. In addition, although traditional animal toxicology has a good track record for ensuring the safety of clinical trial volunteers, it is resource intensive, time-consuming, requires large quantities of product, and may fail to predict the specific safety problem that ultimately halts development. Clinical testing, even if extensive, often fails to detect important safety problems, either because they are uncommon or because the tested population was not representative of eventual recipients (FDA CPI, 2004). Propelled by CPI, the FDA agreed to form a partnership, together with the University of Arizona and SRI International, to create the Critical Path Institute (C-Path). Because of neutral funding and its mission to focus on process, not products, FDA is able to actively participate in the work of C-Path without concerns about conflicts of interest. The first consortium formed by C-Path was the Predictive Safety Testing Consortium. The goal of C-Path projects is to integrate new and advanced technologies into medical product development, especially those that accelerate pathways for innovative diagnostic tests and therapies. Currently most of the projects are focused on identification of translational biomarkers (e.g. nephrotoxicity, hepatoxicity, cardiotoxicity, vascular injury and muscle injury) (Woodcock and Woosley, 2008). Recognizing the value of biomarkers, FDA's Center of Drug Evaluation and Research (CDER) has issued Letters of Support to submitters, briefly describing CDER's thoughts on the potential value of a biomarker thereby encouraging further evaluation. Although the letter does not connote qualification of a biomarker it is meant to enhance the visibility of the biomarker, encourage data sharing, and stimulate additional studies (Table 1)

2.1. US initiatives

2.2. EU initiatives

In 2004, the FDA launched the Critical Path Initiative (CPI), a project intended to improve the drug and medical device development processes, the quality of evidence generated during product development, and the outcomes of clinical use of these products (FDA CPI, 2004). In FDA's view, the applied sciences that are needed for medical product development have not kept pace with the tremendous advances in the basic sciences. The new science is not being used to guide the technology development process in the same way that it is accelerating the technology discovery process. Specifically, the sophisticated scientific tools used in drug discovery and lead optimization are not being used in the preclinical and clinical development stages. More importantly, insufficient applied scientific work has been done to create new tools to get fundamentally better answers about how the safety and effectiveness of new products can be demonstrated, in faster time frames, with more certainty, and at lower costs. The “tools” identified for safety assessments include product testing, in vitro and animal toxicology studies, and human exposure studies. FDA acknowledged that despite efforts to develop better methods, most of the tools used for toxicology and human

In Europe, an extensive long term consultation with stakeholders in the biomedical research and development process also commenced in October of 2004, organized by the European Commission (EC) in Brussels to address the causes of delay or bottlenecks associated with late attrition of investigational products as well as post marketing, safety – associated withdrawals. The research and development bottlenecks identified were (i) predicting safety, (ii) predicting efficacy, (iii) bridging gaps in knowledge management and (iv) bridging gaps in education and training. A Strategic Research Agenda was prepared describing the recommendations to address those bottlenecks and a plan for their implementation (The Innovative Medicines Initiative Research Agenda, 2008). It was concluded that, for improving the prediction of efficacy and safety of medicines, increased basic knowledge on several areas was needed including a better understanding of basic mechanisms of disease and involved targets, target biology and associated pathways, target cross talk and pathway interconnection would need to be explored. Furthermore, additional and/or alternative preclinical models beyond animal models would be needed. To address these concerns a partnership emerged, similar

Table 1 Example of current letters of support for biomarker development submitted to FDA. Submitter

Biomarkers

Area(s) for further evaluation

Critical Path Institute's (CPI) Predictive Safety Testing Consortium (PSTC), Nephrotoxicity Working Group (NWG) CPI, PSTC, Skeletal Muscle Working Group (SMWG)

Urinary Biomarkers: Osteopontin and Neutrophil Gelatinase-associated Lipocalin (NGAL) Early Clinical Drug Development Serum and Plasma Biomarkers: Myosin Light Chain 3 (Myl3), Skeletal Muscle Troponin I Early Clinical Drug (sTNI), Fatty Acid Binding Protein 3 (FABP3), Creatine Kinase [Muscle Type (CK-M), Development Homodimer (CK-MM)]

adapted from http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DrugDevelopmentToolsQualificationProgram/ucm412833.htm.


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to that established in the US, amongst academia, industry and regulatory scientists, to explore innovative ideas which will hopefully lead to novel solutions. This Public Private Partnership, referred to as the Innovative Medicines Initiative (IMI), is sponsored by the European Commission and the European Federation of Pharmaceutical Industry Associations, to sponsor research aimed at generating a knowledge base and strategies towards faster discovery and development of better medicines for patients. Important outcomes are being reached with the initial 5-year grants, which have the potential to lead to innovative strategies for drug development. Current areas of research include searches for predictive animal and human safety biomarkers, (including rodent carcinogenicity), exploration of in vitro systems with human cells, identification of disease targets for e.g. central nervous system, diabetes and other diseases, and creation of animal models for specific diseases (e.g. autism). The IMI project will continue through IMI2 launching new grants up to 2024, with expanded areas, aimed at the creation of a full “medicines advanced pathway to patients” (www.imi,europa.eu). 2.3. Global harmonization initiatives It is exciting that many unique as well as parallel initiatives are ongoing internationally to facilitate clinical development and the safety of new medicines. Since clinical development often extends across countries, stakeholders will need to determine how best to leverage and complement resources to develop and qualify novel assays and animal models to facilitate worldwide regulatory use.

3. The preclinical safety evaluation paradigm for supporting clinical development 3.1. Historical perspective Over the past three decades differences in emphasis have emerged in the preclinical safety evaluation of new medicines. For new chemical entities, the general approach has provided common ground for evaluation across different product classes. For new biological entities, a more product specific approach evolved where classical toxicology is often less relevant and animal and other preclinical tests have varied according to the specific nature of the agent involved, including its pathological characteristics (Safer Medicines, 2005). In the early 1980s neither industry toxicologists nor regulatory scientists were sure of what constituted an appropriate toxicological assessment program for a new class of NBEs, biotechnology-derived pharmaceuticals or biopharmaceuticals (Hayes and Cavagnaro, 1992). During this time as the first products were transitioning into clinical development, Professor Gerhard Zbinden challenged pharmaceutical toxicologists to ‘refrain from following the beaten track of routine toxicity testing’ (Zbinden, 1987). Departing from traditional practices has more recently endorsed and expanded by the Preclinical Toxicology Working Group of the Academy of Medical Sciences in the Safe Medicines Report. The members of the working group presented a similar challenge for the preclinical safety evaluation extending to all types of medicinal products i.e. ‘to change the present culture of thinking about regulatory toxicity and replace it with the concept of science-driven toxicology’(Safer Medicines, 2005). 3.2. ICHS6 For biotechnology-derived pharmaceuticals, the principles of safety evaluation are not different than for NCEs but the practices have differed based on product attributes (Cavagnaro, 2002). In 1995, under the auspices of ICH, a concept paper was proposed by

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the FDA, for a new safety topic specifically related to the preclinical safety evaluation of biotechnology-derived products. In February 1997 the Thirteenth Centre for Medicines Research International Workshop provided an opportunity for international experts to discuss experiences and difficulties in designing studies for this new class of NBEs. Recommendations arising from this workshop were taken into consideration by the ICH Expert Working Group. The final drafting and agreement on the ICHS6 guideline “Preclinical Safety Evaluation of Biotechnology-derived Pharmaceuticals” was reached at the 4th ICH meeting in Brussels in July 1997 (ICH S6, 1997). At this same meeting another guidance was also finalized, ICH M3 “Nonclinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals” (ICH M3, 1997). This guidance acknowledged that existing paradigms for safety evaluation might not always be appropriate or relevant for biotechnologyderived pharmaceuticals. As such a, science-based, “case-by-case” approach should be considered as described in ICH S6 (Cavagnaro, 2008). 3.2.1. Species selection An essential provision of ICHS6 is the selection of a relevant animal species before beginning a study. The guidance emphasizes that “toxicity testing in non-relevant species may be misleading and are discouraged.” For biotech products, selection of the test species is usually accomplished by an in vitro comparison of binding affinity or functional activity of the product in human and animal cells, followed by in vivo demonstrations of pharmacological activity or cross reactivity in that test species. If there are two pharmacologically relevant species for the clinical candidate (one rodent and one non-rodent) then both species are used for short-term (up to 1 month in duration) toxicology studies (ICHS6 ICHS6(R1)). 3.2.2. Alternative approaches The guidance acknowledges that in some cases, alternative approaches to evaluating safety in a pharmacologically-relevant animal species should be used. Recognizing the progress in development of animal models of disease including induced and spontaneous models of disease, gene knockout(s), and transgenic animals the guidance acknowledges that these models may provide further insight, not only in determining the pharmacological action of the product, pharmacokinetics, and dosimetry, but may also be useful in the determination of safety (e.g., evaluation of undesirable promotion of disease progression). As such in certain cases, the guidance explicitly states that studies performed in animal models of disease may be used as an acceptable alternative to toxicity studies in normal animals (e.g., in cases where the target is only present in the disease state). In all cases the scientific justification for the use of animal models of disease to support safety should be provided (ICHS6: ICHS6(R1)).

4. Considerations for incorporating animal models of disease into the preclinical safety evaluation paradigm While animal models of disease may result in improved safety assessment of human relevance, in many instances, even the optimal use of animal models of disease in preclinical testing will not result in an absolute predictability or understanding of toxicities that may be encountered in a clinical setting, in large part because of limitations in any animal model (Morgan et al., 2013). Table 2 provides a summary of advantages and limitations of using animal models of disease for assessing safety. Important questions to ask when considering using animal disease models to assess safety include: Are there questions that cannot adequately be answered with a conventional (healthy) animal model? Is there


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Table 2 Advantages and limitations of animal disease models in for safety assessment. Advantages

Limitations

Opportunity for leveraging model used in proof-of-concept studies Increased prediction of exposure, activity, toxicity mimicking patient population Direct estimation of therapeutic index possible Identification of potential biomarkers for use in clinical trials Evaluation of potential increased sensitivity in disease state More efficient clinical protocol Opportunity to assess mechanism of toxicity following adverse event in clinic

Lack of historical control data: general health, spontaneous disease Inherent variability; may not mimic all aspects of human disease It may be difficult to distinguish toxicity of test article from disease effects Companion reagents may not be relevant or available Increased sensitivity may not be relevant; lack of appropriate “feedback” controls Model may not be available to meet clinical development timeline Model may be otherwise too prohibitive (e.g. cost, too resource intensive, low fecundity)

Adapted from Cavagnaro (2002).

an appropriate animal model of disease? Do the challenges/ limitations of the animal model of disease support its use in safety testing? Will incorporation of safety endpoints in proof-of-concept studies help to better predict the safety aspects of the medicine in the disease condition? (Morgan et al., 2013).

endpoints in the pharmacological models is addressed in several EU guidelines driving the (clinical or the nonclinical and clinical development of multiple therapeutic classes) (Table 3).

4.1. Robustness of model

5.1. General guidance

An understanding of the limitations of the model is needed in order to determine whether the disease model is suitable, e.g. including evaluation of incidence, severity, and homogeneity of background changes. In addition a determination must be made as to whether the animals can be maintained over the intended duration of the study. For instance, if selecting a chemically or surgically modified model to mimic a human disease, additional issues should be taken into account. Sufficient animal numbers must undergo chemical or surgical modification in order to guarantee animals have been successfully modified to provide enough statistical power to mitigate the impact of individual differences in response to creation of the disease model.

5.1.1. The US “Animal Rule” This guidance provides information and recommendations on product development when human efficacy studies are not ethical or feasible. The use of the FDA Draft Guidance “Product Development Under the Animal Rule” (FDA Product Development Under the Animal Rule, 2014) as a regulatory pathway is not confined to the development of medical countermeasures for chemical, biological, or nuclear threat agents. Medicines intended to ameliorate or prevent serious or life-threatening conditions due to other toxic chemical, biological, radiological, or nuclear substances (e.g., emerging virus, snake venom, industrial chemicals) may be eligible for development under the Animal Rule when it is not ethical to conduct human challenge studies and when field trials to study effectiveness are not feasible. The Animal Rule specifies that the choice of species for the adequate and well-controlled efficacy studies must be appropriate with regard to the disease or condition of interest and the investigational drug. There is no requirement for the use of a specific species. With respect to each animal species selected by sponsors, the sponsors should provide scientific justification that the animal species exhibits key characteristics of the human disease or condition when the animal is exposed to the challenge agent. In addition, the species should be selected based on an understanding of the test article's mechanism of action, such that the effect in the animal species is expected to be predictive of its effect in humans, and the ability to select an effective dose and regimen for humans. The guidance identifies essential elements that should be considered in the development and/or the selection of an animal model for efficacy but anticipates that that the preclinical safety development program is conducted in a manner similar to the traditional regulatory pathways. The guidance acknowledges that animal models used to demonstrate efficacy may not predict specific interactions of the agent-induced disease or condition and the investigational drug in humans and that adverse interactions in humans may not be observed until the drug is used for the disease or condition, reinforcing the critical need for post marketing studies. The guidance recommends that if adverse findings occur only when the investigational product is tested in challenge agent-affected animals, that further investigation may be warranted to determine the pathophysiological mechanism for the unexpected toxicity and its relevance to the risk assessment for the intended human population.

4.2. Interpretation of results From a pathology perspective, the evaluation of animal disease models is challenging. In part because animal disease models are in species or strains that are generally poorly characterized and that lack historical information on spontaneous background findings. In addition, the potential for significant inter-individual variation in primary or secondary disease-associated pathology poses additional challenge sin interpretation. Thus, discerning whether clinical and anatomic pathology findings are attributable to incidental age-related or background changes, anticipate primary or secondary disease manifestations, and reconcile test article-related adverse vs. non adverse effects, will require additional experience and data accumulation specifically with each model system (Morgan et al., 2013).

5. US and EU regulatory guidance recommending use of animal models of disease There are currently several guidance documents in the US that describe the use and development of animal models of disease in preclinical safety assessment. Similarly in the EU, reference to the use of pharmacologically relevant animal models, specifying or not those as disease models are also incorporated in several guidelines, mostly those addressing the development of disease-specific therapies. The use of relevant animal models for proof of concept and preclinical assessment of developing products, as well as their use for safety assessment, mostly by incorporating safely


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Table 3 International and national guidance documents addressing to the use of animal models for the development of medicinal products and ATMPs in specific areas. Indication/area

Guideline title

Reference (internet link)

General

ICHS6 Guideline on the “Preclinical Safety Evaluation of Biotechnologyderived Pharmaceuticals, 1997

General

ICHS6(R1)Addendum Preclinical Safety Evaluation of BiotechnologyDerived Pharmaceuticals (2012) Guidance on nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals ICH M3(R2) Preclinical Assessment of Investigational Cellular and Gene Therapy Products (November 2013)

http://www.fda.gov/downloads/Drugs/ GuidanceComplianceRegulatoryInformation/Guidances/ucm074957. pdf http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/ Guidelines/Safety/S6_R1/Step4/S6_R1_Guideline.pdf http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/ Guidelines/Multidisciplinary/M3_R2/Step4/M3_R2__Guideline.pdf

General

General ATMPs General ATMPs General ATMPs General ATMPs Osteoporosis/ multidisciplinary (US) Osteoporosis (EU)

Rheumatoid Arthritis (US) Red Blood Substitute/ multidisciplinary (US) Epilepsy (EU)

Diabetes Mellitus/ clinical (EU) Muscular Dystrophies (EU) Hypertension/ clinical (EU) Lipid disorders/ clinical (EU) Weight control/ clinical (EU) Depdendence Potential (EU)l

http://www.fda.gov/BiologicsBloodVaccines/ GuidanceComplianceRegulatoryInformation/Guidances/ CellularandGeneTherapy/ucm376136.htm Guideline on human cell based medicinal products http://www.ema.europa.eu/docs/en_GB/document_library/ EMEA/CHMP/410869/2006 Scientific_guideline/2009/09/WC500003898 Guideline on the nonclinical studies required before first clinical use of gene http://www.ema.europa.eu/docs/en_GB/document_library/ therapy medicinal products Scientific_guideline/2009/09/WC500003743 EMEA/CHMP/GTWP/125459/2006 Guideline on the risk-based approach according to annex I, part IV of http://www.ema.europa.eu/docs/en_GB/document_library/ Directive 2001/83/EC applied to Advanced therapy medicinal products Scientific_guideline/2013/03/WC500139748 EMA/CAT/CPWP/686637/2011 FDA Guidelines for Preclinical and Clinical Evaluation of Agents Used in the http://www.fda.gov/OHRMS/DOCKETS/98fr/04d-0035-gdl0001. Prevention or Treatment of Postmenopausal Osteoporosis (April 1994) Guideline on the evaluation of the medicinal products in the treatment of primary osteoporosis CPMP/EWP/552/95 Rev. 2 Guidance on Clinical Development Programs for Drugs, Devices, and Biological Products for the Treatment of Rheumatoid Arthritis (RA) (February 1999) Draft FDA Guidance for Industry Criteria for Safety and Efficacy Evaluation of Oxygen Therapeutics as Red Blood Cell Substitutes (October 2004)

http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2009/09/WC500003406

Guideline on clinical investigation of medicinal products in the treatment of epileptic disorders CHMP/EWP/566/98 Rev.2/Corr Guideline on clinical investigation of medicinal products in the treatment or prevention of diabetes mellitus CPMP/EWP/1080/00 Rev. 1 Guideline on the clinical investigation of medicinal products for the treatment of Duchenne and Becker muscular dystrophy EMA/CHMP/236981/2011, 2013, Draft Guideline on clinical investigation of medicinal products in 7 the treatment of hypertension EMA/CHMP/29947/2013/Rev. 4 Guideline on clinical investigation of medicinal products in the treatment of lipid disorders EMA/CHMP/748108/2013 Guideline on clinical evaluation of medicinal products used 4 in weight control EMA/CHMP/311805/2014 Guideline on the nonclinical investigation of the dependence potential of medicinal products EMEA/CHMP/SWP/94227/2004

http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2010/01/WC500070043

5.1.2. EU Directive 2010/63/EU revising Directive 86/609/EEC The Directive addresses the protection of animals used for the Three Rs, to replace, reduce and refine the use of animals used for scientific purposes. The scope is now wider and includes fetuses of mammalian species in their last trimester of development and cephalopods, as well as animals used for the purposes of basic research, higher education and training. It lays down minimum standards for housing and care, regulates the use of animals through a systematic project evaluation requiring inter alia assessment of pain, suffering distress and lasting harm caused to the animals. The Directive refers to the use of animal models of disease, described as e.g. genetically modified animals, mostly in what concerns the classification based on the level of suffering that could be associated to those conditions. Severe conditions are to be well justified or avoided. The replacement of animals in

http://www.fda.gov/downloads/Drugs/ GuidanceComplianceRegulatoryInformation/Guidances/UCM071579. pdf http://www.fda.gov/BiologicsBloodVaccines/ GuidanceComplianceRegulatoryInformation/Guidances/Blood/ ucm074920.htm

http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2012/06/WC500129256 http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2013/03/WC500139508 http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2013/07/WC500146993 http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2014/01/WC500159540 http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2014/07/WC500170278 http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2009/09/WC500003360

research and development is highly recommended and encouraged, through the implementation of alternative methods, without compromising human health.

5.1.3. Preclinical assessment of investigational cellular and gene therapy products/advanced therapy medicinal products 5.1.3.1. US. The US guidance is consistent with ICH S6 with respect to the importance of using a relevant species stating that the animal species selected for assessment of bioactivity and safety should demonstrate a biological response to the investigational cellular and gene therapy (CGT) product similar to that expected in humans in order to generate data to guide clinical trial design (FDA Guidance Preclinical Assessment of Investigational Cellular and Gene Therapy Products, 2012). The guidance acknowledges that although healthy animals


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represent the standard model test system employed to conduct traditional toxicology studies, due to features of CGT products (e.g., potentially prolonged duration of intended product effect, product persistence in vivo, complex mechanism of action involving interaction between CGT product and the disease environment, invasive ROA), study designs using animal models of disease/injury are frequently modified to incorporate important safety parameters that allow for assessment of the potential toxicology of an investigational CGT product (i.e., hybrid pharmacology-toxicology study design). Thus animal models of disease/injury may be preferable to healthy animals to assess the activity and safety of these products. Every animal model has inherent strengths and weaknesses; thus, no single model will predict with complete accuracy the efficacy and safety outcome of the investigational CGT product in the patient population. Safety data in animal models of disease at the very least can supplement and at best be used in lieu of toxicology studies in healthy animals. Therefore, preclinical studies in disease/injury models are encouraged to better define the risk-benefit ratio associated with investigational CGT products. In addition, use of disease/injury models provides the opportunity for possible identification of activity-risk biomarkers that may be applicable for monitoring in clinical trials. The activity and safety profile of the CGT product may be influenced by the timing of administration relative to the onset of disease state at the initiation of product administration and thus should be characterized and documented. Information describing limitations of potential animal models should be provided e.g. inherent variability of the model, limited historical/ baseline data, technical limitations with the physiological and anatomical constraints of the model, animal care issues, and limited fidelity in modeling human pathophysiology of the disease/injury of interest (FDA Guidance Preclinical Assessment of Investigational Cellular and Gene Therapy Products, 2012). 5.1.3.2. EU. The EU/EMA guideline on human cell-based medicinal products (EMEA/CHMP/410869/2006) while less specific that the US guidelines, clearly considers and encourages the selection, use and justification of relevant models for preclinical testing of these products, including safety studies when considered needed. The animal model may include immunocompromised, knockout or transgenic animals. Homologous models may be advantageous, since the in vivo behavior of the applied cells or tissue in heterologous models could be altered due to species-specific mismatches. Homologous models should be considered for the study of stem cell differentiation. In vitro studies, addressing cell and tissue morphology, proliferation, phenotype, heterogeneity and the level of differentiation may be part of the primary pharmacodynamic analyses. If relevant animal models cannot be developed, in vitro studies may replace animal studies. Expression level of biologically active molecules, the route of administration and the dosages tested should reflect the intended clinical use in humans. When toxicity studies are considered as needed those should be performed in relevant animal models (which will obviously include animal models of disease if considered the most appropriate model). If the human cells are not immediately rejected, the studies may be combined with safety pharmacology, local tolerance, or proof of concept and efficacy studies. Sufficiently characterized analogous animal-derived cells may also be used to support allogeneic products. Also for gene therapy medicinal products the EMA guideline on the preclinical studies required before first clinical trial use, clearly states as general principles that the relevance of the animal model(s), including developmental stages according to intended clinical use, shall be justified by the applicant taking into account the model used to explore the pharmacological effects and the therapeutic function of the expressed gene (EMEA/CHMP/GTWP/125459/2006). The animal model(s) chosen should allow assessment of the pharmacological effects expected in humans as far as possible. Studies should be

designed and carried out aiming at establishing: (i) pharmacodynamic “proof of concept” in preclinical model(s), (ii) bio-distribution; (iii) recommendation on initial dose and dose escalation scheme to be used in the proposed clinical trial; (iv) identification of potential target organs of toxicity; (v) identification of potential target organs of biological activity; (vi) identification of indices to be monitored in the proposed clinical trial and (vii) identification of specific patient eligibility criteria. It is obvious that in many cases this information, when addressing both the pharmacology/proof-of-concept and the safety can only be appropriately conducted if animal model of the disease is used. Furthermore, with regard to the integrated development of advanced therapy medicinal products (ATMPs), the EMA Committee for Advanced Therapies (CAT) has created guidance incorporating the “risk-based approach”, where the quality, know preclinical attributes as well as any experience with the ATMP, its mode of action or that from associated entities shall be used to anticipate the spectrum of risks to be tested in appropriate models for safety assessment (EMA/CAT/CPWP/686637/2011, 2013). 5.2. Disease specific regulatory guidance 5.2.1. Preclinical and clinical evaluation of agents used in the prevention or treatment of postmenopausal osteoporosis 5.2.1.1. US. Preclinical rodent and non-rodent models have shown a strong predictive value to assess the effect of new agents on bone quality during chronic therapy. In addition to the toxicity studies required for all new medicines, preclinical studies of bone quality should be performed for medicines to be used in the prevention and intervention of osteoporosis. For these guidelines, bone quality is considered to be comprised of the architecture, mass and strength of bone. These studies are warranted by instances in which bone density was not positively correlated with architecture and strength. The primary objective of the preclinical studies are to demonstrate that long term treatment with a specific agent will not lead to deleterious effects on bone quality. Animal models for osteoporosis may be classified as either modeling (rats) or remodeling (examples include dogs, ewes, and primates). Modeling of bone is the method by which bone grows and is shaped. In remodeling species, including adult humans, bone undergoes a continuous coordinated process of bone resorption, followed by formation of new bone. The modeling and remodeling species have been further classified as models of accelerated bone loss and of decreased bone formation. Examples of models of accelerated bone loss include castrated male rats and acute post-ovariectomized female rats, lactating pigs and ovariectomized primates. Models of decreased bone formation include aged rats or mice and glucocorticoid-treated rats, and aged canines or primates and glucocorticoid-treated pigs. Additionally, the literature contains reports of transgenic mice and congenitally osteoporotic mice as potential new models. At the present time, an experimental model that precisely mimics the pathophysiology of postmenopausal osteoporosis is unavailable. Although several risk factors for osteoporosis have been identified, there is a predominant association with estrogendeficiency. Hence, ovariectomized animals are the preferred animal models to provide insight into the clinical outcome of an antiosteoporotic drug. Preclinical studies of bone quality in two species are required in the US. One of these studies must be performed in the ovariectomized rat model (modeling species). Although differences in bone metabolism exist between rats and humans, the rat model has been reported to be an appropriate model for cancellous bone changes in humans. The second study should be performed in a larger, remodeling species. The animal model used in the second study is at the discretion of the sponsor (FDA Guidelines for Preclinical and Clinical


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Evaluation of Agents Used in the Prevention or Treatment of Postmenopausal Osteoporosis, April 1994). The proposed animal studies should permit early identification of medicines that result in abnormal architecture or in product of bone in which strength is not positively correlated with density and architecture. Because no single animal species duplicates all the characteristics of human osteoporosis, it is felt that an examination of bone quality in two species is necessary to adequately investigate the effectiveness and safety of drugs for this indication. The time of initiation of treatment in preclinical studies should be reflective of the clinical indication. Specifically, time of initiation of treatment will be different for studies designed for the prevention of osteoporosis versus intervention for the treatment of osteoproosis. Treatment schedule to be used in the preclinical studies should be the same as that intended for clinical use (continuous vs. intermittent). The animals should be treated with two doses; one that is optimally effective in that species, and one that is approximately 5 times greater to give and indication of a safety margin. The duration of preclinical studies should be based primarily on bone turnover (number of complete resorption and formation cycle/year) of a species and should consist of a number of cycles equivalent to 4 years of human exposure. For example if bone turnover in humans is 100-200 days or 2-4 cycles/year, and in rats, baboons and cynomolgus monkeys is approximately 40 days or 9 cycles/year, then 16-month studies in rats and primates are comparable to 4 years in humans. Because of the relatively short life-span in rats, the treatment duration for this species may be limited to 12 months. 5.2.1.2. EU. There is also European Guideline on the evaluation of medicinal products in the treatment of primary osteoporosis (CPMP/EWP/552/95 Rev. 1) which recognizes that while there are no completely satisfactory models of human disease a number of useful models exist. Similarly to the US requirements, for medicines that are aimed for use in the treatment of postmenopausal osteoporosis in women, an evaluation of bone quality should be performed in two species, one of which should be the adult ovariectomized rat and the other an animal with estrogen deficiency induced by ovariectomy and characterized by evaluable cortical bone remodeling. The primate, sheep, adult rabbit or pigs are possible suggestions. The EU guideline also requires as a prerequisite to their clinical development, new chemical entities considered for the treatment of osteoporosis in men should be extensively investigated in the relevant animal models to identify potential gender-specific skeletal toxicity and efficacy. In addition, it is mandatory for stimulators of bone formation to have a preclinical package demonstrating safety of the tested drug in terms of bone biomechanics at the exposure selected for Phase III clinical trials. This information should be made available at the time of the file submission. These studies in the animal models of osteoporosis are therefore tailored to address the efficacy of the clinical candidate and the bone quality emerging from the treatment incorporating efficacy and (bone) safety components. 5.2.2. Clinical development programs for drugs, devices, and biological products for the treatment of rheumatoid arthritis The FDA guidance includes discussions of approaches to address preclinical issues for anti-rheumatic therapies. The biological activity of a potential anti-rheumatic therapy should be established using multiple preclinical model systems. Animals, either healthy, with rheumatic disease (spontaneous or induced), or genetically modified, are subsequently used to determine whether the biological effect can be demonstrated in vivo. It is acknowledged that while the in vivo system used should mimic one or more aspects of

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rheumatoid arthritis or its etiology, it is expected that each model will have limitations. Selection of animal models should be made on the basis of pharmacodynamic responses, similarity of animal disease etiology to clinical disease, and/or to define mechanismbased toxicity. Ideally, products that are targeted for a subset of arthritic patients should be developed in an experimental model (s) that is most relevant to that subset. A variety of animal models of disease are provided for consideration. Sponsors are encouraged to identify and use animal models that assist in selecting drug candidates that selectively inhibit cells and processes responsible for rheumatoid arthritis. Preclinical toxicity studies that evaluate the use of combined agents may be helpful in predicting clinical safety hazards. The guidance encourages the development and validation of in vitro or whole animal models to address concerns regarding short- or long-term toxicity and to identify surrogate markers for patient immunocompetence (FDA Guidance for IndustryClinical Development Programs for Drugs, Devices, and Biological Products for the Treatment of Rheumatoid Arthritis, February 1999)

5.2.3. Criteria for safety and efficacy evaluation of oxygen therapeutics as red blood cell substitutes Recommended points to consider are outlined in this FDA guidance when planning and executing safety studies of oxygen therapeutics in animals. The guidance acknowledges that volume overload and exchange transfusion studies in normal animals can contribute to determination of the dose/toxicity relationship but they may not adequately reflect the circumstances in other clinical or clinical transfusion situations. Thus, the guidance recommends that special animal models will be needed to obtain a complete safety profile. Specifically, the animal model, should be fully instrumented to measure cardiac and pulmonary function, should be stressed so as to resemble the clinical use of the oxygen therapeutic (e.g., volume depleted for resuscitative indication; ischemic model for percutaneous transluminal coronary angioplasty (PTCA); repetitive administration for sickle cell disease; septic shock model for Systemic Inflammatory Response Syndrome; cardiopulmonary bypass model for cardiac surgery). The guidance recommends that concomitant medications and other agents also be included, e.g. contrast agents with hemoglobinbased oxygen carriers for PTCA indications. Controls should include the use of approved oxygen carriers and plasma expanders. Sponsors should also consider a model designed to produce reperfusion injury. Such a model would be a relevant test of clinical situations that involved ischemia (Draft FDA Guidance for Industry Criteria for Safety and Efficacy Evaluation of Oxygen Therapeutics as Red Blood Cell Substitutes, October 2004).

5.2.4. Clinical investigation of medicinal products in the treatment of epileptic disorders This EU guidance recommends that study of the efficacy profile of new medicines should be done in several experimental models, including models of generalized epilepsies with absences. It is important to know if the drug in development displays anti-seizure activity only or if it has a potential for anti-epileptogenesis as well. The neurobiological mode of action of the candidate antiepileptic drug may be important, since it may indicate in which seizure types and epilepsy syndromes the drug will be efficacious. It may be also predictive for the risk of certain adverse events. For instance some drugs have been specifically designed around a given mechanism: promoting γ-aminobutyric acid inhibition; others constitute the extension of a pre-existing family, with a more or less well-known preclinical profile. Other candidates which are the result of systematic screening may need identification of their mode(s) of action (CHMP/EWP/566/98 Rev.2/Corr).


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5.2.5. Clinical investigation of medicinal products in the treatment or prevention of diabetes mellitus development of anti-diabetic drugs Preclinical data are in relevant animal models evaluating the potential effect of the test drug on different safety aspects, including cardiovascular risk, should be conducted and provided as an instrumental element of the safety evaluation are recommended in this EU guideline. Animal studies should focus on athero-thrombotic findings, fluid retention, blood pressure, renal function, electrolyte homeostasis, cardiac functionality, repolarization and conduction abnormalities (pro-arrhythmic effects), etc. as outlined in ICH guidelines. If the drug is developed in the pediatric population the guideline on the need for testing in juvenile animals of pharmaceuticals for pediatric indications should be considered (CPMP/EWP/1080/00 Rev. 1) 5.2.6. Clinical investigation of medicinal products for the treatment of Duchenne and Becker muscular dystrophy The proposed mechanism of action of a new product should be described and discussed in relation to possible testing in available animal models which are currently limited. (e.g. the mdx mouse is limited with respect to the Duchenne Muscular Dystrophy phenotype, while the predictive value of results in the golden retriever muscular dystrophy dog is still unknown). In addition, the changes in biological parameters that are observed in patients or healthy volunteers (if appropriate) should be addressed. It should be explored, whether the pharmacodynamic effect is similar in different stages of the disease (e.g., restoration of dystrophin in early and advanced stages of the disease) (EMA/CHMP/236981/ 2011, 2013, Draft ) 5.2.7. Clinical investigation of medicinal products in the treatment of hypertension Preclinical data in relevant animal models evaluating the potential effect of the new medicine on different safety aspects, including cardiovascular risk, should be conducted and provided as an instrumental element of the safety evaluation. Animal studies should focus, amongst others, on athero-thrombotic findings, fluid retention, blood pressure, renal function, electrolytes homeostasis, cardiac functionality, repolarization and conduction abnormalities (pro-arrhythmic effects), as outlined in ICH Guidelines (EMA/CHMP/29947/2013/Rev). 5.2.8. Clinical investigation of medicinal products in the treatment of lipid disorders Preclinical data in relevant animal models evaluating the potential effect of the new medicine on different safety aspects, including cardiovascular risk, should be conducted. Animal studies should particularly focus on athero-thrombotic findings, fluid retention, blood pressure, renal function, electrolytes homeostasis, cardiac functionality, repolarization and conduction abnormalities (pro-arrhythmic effects), liver, muscle etc., as outlined in specific ICH guidelines. For certain agents, reactions relating to muscle and liver toxicity are of particular significance as are local tolerance and immunogenicity depending on the nature of the medicinal product (EMA/CHMP/748108/2013). 5.2.9. Clinical evaluation of medicinal products used for weight control Special efforts should be made to assess potential adverse reactions that are characteristic of the class of drug being investigated. Preclinical data in relevant animal models evaluating the potential effect of the test drug on different safety aspects should be conducted and provided as an instrumental element of the safety evaluation as outlined in ICH guidelines.

In the clinical studies, an overall plan for the detection and evaluation of potential adverse events including justification of the size and duration of the studies with respect to the possibility of detecting safety signals, should be prospectively designed early during the clinical development, optimally by the time of phase II studies. This program should take into consideration key elements of the primary and secondary pharmacology, as well as key toxicological findings from preclinical studies (EMA/CHMP/ 311805/2014).

6. Regulatory expectations for use of animal models of disease without specific guidance While most guidelines in the US, EU or ICH do not explicitly request preclinical safety studies to be performed in animal models of disease, in general guidance documents do recommend that relevant animal models should be used, which, in many situations, may correspond to animal models of disease as better representing the human condition to be treated rather than the healthy animal models. There are several classes of drugs and biologics where animal models of disease are routinely used to inform clinical trials despite the lack of explicit regulatory guidance/requirements. 6.1. Antithrombolytic and thrombolytic therapies The development and application of animal models of thrombosis have played a critical role in the discovery and validation of novel drug targets and the selection of new agents for clinical evaluation, and have informed dosing and safety information for clinical trials. The development of antithrombotic agents requires preclinical assessment of the biochemical and pharmacologic effects. It is important to note that second-and third-generation antithrombotic products are devoid of in vitro anticoagulant effects, yet in vivo, by virtue of endogenous interactions, they produce protein antithrombotic actions. The initial belief that antithrombotic medicines must exhibit in vitro anticoagulant activity is thus no longer valid. This important scientific observation has been possible only because of the availability of animal models. Numerous models have now been developed to mimic a variety of clinical conditions where anti-platelet and anti-thrombotic drugs are used, including myocardial infarction, stroke, cardiopulmonary bypass, trauma, peripheral vascular diseases, and restenosis. The primate models in particular have been extremely useful, as the hemostatic pathways in these species are comparable to those in humans. The development of such agents as the specific glycoprotein IIb/IIa inhibitor antibodies relies largely on these models. These models are of pivotal value in development of antithrombotic drugs and provide extremely useful data on the safety and efficacy of new medicines developed for human use. (Mousa, 2010) 6.2. Enzyme replacement therapies The two following examples illustrate the expectations from animal models of disease enzyme replacement therapy. 6.2.1. Recombinant human acid sphingomyelinase (rhASM) rhASM is being developed as an enzyme replacement therapy for patients with acid sphingomyelinase deficiency (Niemann–Pick disease types A and B), which causes sphingomyelin to accumulate in lysosomes. In the acid sphingomyelinase knock-out (ASMKO) mouse, intravenously administered rhASM reduces tissue sphingomyelin levels in a dose-dependent manner.


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The only animal with notable findings in preclinical toxicological studies of rhASM is the ASMKO mouse, the animal model of human ASMD. The ASMKO mouse has been critical in characterizing the toxicity of rhASM prior to dosing human patients. Normal animal species e.g. Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys, represent appropriate animals for evaluating the safety of rhASM as each species is pharmacologically relevant due to the presence of mannose and mannos-6-phosphage receptors that allow rhASM to be taken up by cells and transported to lysosomes. However, due to the presence of endogenous ASM in normal animals, there is no sphingomyelin accumulation that mimics the human disease. Thus, studies conducted in normal animals do not provide a complete toxicological assessment that accurately predicts the risk to patients. The ASMKO mouse represents the most sensitive animal for the preclinical safety evaluation of rhASM due to the presence and accumulation of substrate in relevant tissue similar to the human form of the disease. However, the ASMKO mouse does not completely recapitulate Niemann–Pick disease type B pathophysiology. The complete lack of ASM activity in ASMKO mice could represent a worst case scenario as evidenced by the progressive neurological involvement and death by 6–8 months of age that is more reminiscent of Niemann–Pick disease types A. On the other hand, ASMKO mice lack hematological involvement and organomegaly, both of which are hallmarks of ASM-deficient patients. If the toxicity of rhASM is related to the amount of sphingomyelin storage and its rate of breakdown then patients with different extents of sphingomyelin storage may differ in their ability to tolerate rhASM (Murray et al., 2015).

6.2.2. Recombinant human N-acetylgalactosamine 4-sulfatase (rhASB) rhASB is indicated for the long-term enzyme replacement therapy in patients with mucopolysaccharidosis VI (MPS VI), also referred to as Maroteaux–Lamy Syndrome. Galsulfase has been developed and approved in the US and EU for long-term enzyme replacement therapy in patients with a confirmed diagnosis of MPS VI. MPS VI is a rare lysosomal storage disease resulting from a deficiency in the enzyme N-acetylgalactosamine 4-sulfatase. The accumulation causes a progressive disorder with multiple organ and tissue involvement. MPS VI is an inherited autosomalrecessive disorder, and carriers do not exhibit any biochemical or clinical evidence of disease. A feline model of MPS VI is available, which has similar etiology and morphological characteristics/ disease pathology to the human disease and has been used for in vivo pharmacology studies. Similarities to the human disease include facial dysmorphia, corneal clouding, reduced body weight, bone abnormalities and reduced cervical spine flexibility, mild hepatosplenomegaly, thickened cardiac valves and the absence of central nervous system lesions. Differences from the human disease include the lack of respiratory effects in cats, and differences in patterns of urinary glycosaminoglycan excretion. The main breeding colony of MPS VI cats in the world is at the Lysosomal Storage Disease Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, Australia. There is no commercial supplier of these cats, and the pharmacology studies for this product were conducted under the supervision of Dr. J. Hopwood, who maintained the colony at the Unit in Adelaide. The non-GLP studies were initially designed as research studies. While the animal models were not conducted under GLPs they were accepted as conducted in a proper scientific manner, and despite the low numbers of animals the results appeared consistent across studies. Some of the studies, which ranged from 5 weeks to 20 months in duration, included

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evaluation of safety and/or tissue distribution of rhASB which have been considered as contributing to the safety characterization of galsulfase in the disease condition (Naglazyme European Public Assessment Report, 2008). 6.3. Anti-sepsis therapies The complexity of sepsis makes the clinical study of sepsis and sepsis therapeutics difficult. The development and progression of sepsis is multi-factorial, and affects the cardiovascular, immunological and endocrine systems of the body. Animal models have been developed in an effort to create reproducible systems for studying sepsis pathogenesis and preliminary testing of potential therapeutic agents. However, demonstrated benefit from a therapeutic agent in animal models has rarely been translated into success in human clinical trials (Buras et al. 2005). The fact that clinical sepsis is influenced by underlying diseases may help explain why simple animal models of sepsis do not mimic human sepsis and do not predict human response to therapeutics. Animal models of sepsis differ from human sepsis because of age, co-morbidity and use of supportive therapy as well as baseline conditions typically present in septic patients, such as advanced age or chronic disease. Thus complex animal models of human sepsis may be more pharmacologically relevant than simple animal models for the testing of therapeutics as they may ultimately predict human drug responsiveness more accurately (Doi et al., 2009). Animal models of sepsis need to reproduce the complexity of human sepsis and its treatment in the intensive care unit. Ideally, animal models should mimic the pace and severity of human sepsis; reproduce key hemodynamic and immunologic stages; mimic histology findings in key organs and- perhaps even counter intuitively exhibit variability among animals (Fink and Warren, 2014). 6.4. Anti-viral therapies 6.4.1. Respiratory syncytial virus (RSV) RSV is the major cause of serious lower respiratory tract disease in children, manifested as bronchiolitis and pneumonia. The peak incidence of hospitalization of RSV-associated illness is in infants 2–5 months old. Primary RSV infection occurs before one year of age, with 95% of the children displaying serologic evidence of infection by 2 years, and 100% by adulthood. RSV infection occurs in seasonal outbreaks, peaking during the winter in temperate climates and during the rainy season in warmer climates. Repeated infections are common, but development of neutralizing antibodies results in significant protection. MEDI-493 (Synagiss) is specific for the antigenic site A on the highly conserved F protein on the surface of RSV, has potent neutralizing and fusioninhibiting activity, and is cross reactive to strains from both A and B subtypes of the virus. The monoclonal antibody is pharmacologically effective in vitro at blocking RSV infection and syncytia formation. MEDI-493 did not cross react in an immunohistochemical tissue cross reactivity assay with normal human adult tissues or human neonatal tissues. Preclinical safety assessment of MEDI-493 was conducted in normal rat, rabbit and monkeys. No toxic effects were noted. The most relevant safety study was IV prophylaxis of RSV infection in cotton rats. The rats were challenged intranasally with RSV. Lung tissue was collected and pulmonary viral titers determined by plague titration using HEp-2 cells (limit of detection¼ 100 pfu/g tissue). MEDI-493 did not enhance infection or lung pathology upon primary or secondary RSV infection (FDA Pharmacologist Review, 1998).


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6.4.2. Chronic Hepatitis B virus (HBV) infection Fialuridine (FIAU) is a pyrimidine nucleoside analog with antiviral activity against herpes viruses and HBV. During the summer of 1993, patients with chronic HBV infection that had been treated with FIAU for 9.5 to 13 weeks developed signs of severe and in some cases, fatal hepatotoxicity associated with lactic acidosis. The observed toxicity had not been anticipated on the basis of earlier 2- and 4-week clinical trials of FIAU in human patients or on the basis of preclinical toxicological studies using conventional laboratory animals (Tennant et al., 1998). The toxicological studies that preceded the FIAU clinical trials and the studies that followed used healthy animals with normal hepatic morphology and function. To investigate the question on whether HBV-induced liver disease is an essential factor in the pathogenesis of the observed human drug toxicity, woodchucks chronically infected with the woodchuck hepatitis virus were used as a model of HBV. In both humans and woodchucks, the most severe signs of FIAU toxicity including hepatic failure and lactic acidosis were observed after treatment for more than 9 weeks. While not yet in regulatory guidance it is of interest to note that in the report entitled “Nonclinical Assessment of Potential Hepatoxicity in Man” (November 2000) it was acknowledged that some drugs necessitate additional preclinical testing in special animal models when these animal models more appropriately mimic the sensitivity of man to certain classes of hepatotoxins, e.g., woodchuck for nucleoside analogs. Currently the woodchuck is routinely used to support clinical development of this class of new medicines.

7. Animal models recommended for detection of specific toxicities For the detection of specific toxicities, the use of specific animal models is also considered in some guidelines. 7.1. Animal models of cancer Animal models of cancer are routinely used for the characterization of the mode of action of new oncology products (Cheon and Orsulic, 2011). These models are most commonly developed in mice, and correspond to tumor-bearing animal models, like mammary gland tumor models, or immunossupressed animals with human tumors or tumor cells (xenograft models). Depending on the model used, the value of incorporating safety endpoints in the proof of concept studies should be considered by the investigators at the start of their research, as disease condition by itself may well affect the biological response. For instance, if a product is intended to act on a tumor target which also exists in non tumor cells, it might be expected that healthy animals will not respond similarly to the diseased ones in terms of targetrelated adverse effects, given the much higher population of the target receptors in the tumor as compared to normal cells. The tumor tropism of the product may then determine the adverse event pattern in diseased vs. healthy animals, as would be also expected in humans. 7.2. Animal models for assessment of carcinogenic risk Several animal models for human cancer prediction have been developed and are being used and accepted by regulatory authorities to test the carcinogenic potential of new medicinal products, as alternative to the two years mouse carcinogenicity study. The objectives of the various models are to more closely predict human carcinogens and discriminate between genotoxic and nongenotoxic mechanisms. Currently there are three models which have been discussed and considered by US and EU regulators as

valuable models for cancer risk prediction (European Medicines Agency, 2004). CPMP SWP Conclusions and Recommendations on the use of genetically modified animal models for carcinogenicity assessment (CPMP/SWP/2592/02). The C5BL p53 þ/ transgenic mouse model, in general responds positively to genotoxic carcinogens. The wild type p53 protein suppresses tumors in humans and rodents. In this model one Trp53 allelle is replaced with one null TRp53 allelle, making the strain more susceptible to genotoxic hits silencing the functional Trp53 allele. The TG-rasH2 (CB6F1-TG-rasH2) transgenic mouse carries the human prototype c-Ha-ras gene. The mice are hemzygous carrying 3–6 copies of the human prototype gene with its own promoter integrated into the genome in a tandem array. The total amount of the gene product p21 is reported to be 2–3 times higher in the Tg than in the nonTg mice. The model is accepted by regulatory authorities as useful for studying the carcinogenic potential, responding to genotoxic or non-genotoxic compounds. The TG-AC transgenic mouse model carries the human prototype c-Ha-ras gene. It is created in FVB/N mouse strain by pronuclear injection of a v-Ha-ras oncogene flanked 5´by a mouse zeta-globin promoter and 30 by an SV-40 polyadenylation signal sequence. The v-Ha-ras oncogene confers on the TG.AC mouse the property of ‘genetically initiated skin’. This model is able to identify tumor promoters as well as complete carcinogens when topically applied in the skin. 7.3. Animal models of tolerance and dependence Specific preclinical animals models of behavioral disturbances have been developed for investigating dependence potential (Baster and Bigelow, 2003). The choice of the model used should be justified based on the biochemical, pharmacological and clinical information already available. Pharmaceutical characteristics of the product may also influence the choice of the model. Specific aspects of dependence potential may require different models to study them. Examples include the self-administration paradigm and conditioned place preference models (Panillio and Goldberg, 2007). 7.4. In vitro preclinical safety assessment using human cells and tissues The main challenge at developing proof-of-concept data for advancingto a clinical trial is the lack of an animal model which has similar functional and morphologic features to the intended disease. Reprogramming of somatic cells (obtained through phlebotomy) represents a novel approach to obtaining patient-specific stem cells harboring individual disease mutations. Because of the unlimited replicative capacity and clonability, induced pluripotent stem cells (iPSCs) can provide adequate material for understanding the disease pathology and developing sustained treatment approaches. The advantages of iPSCs include the fact that they can be developed from a wide variety of tissue sources, including white blood cells obtained through phlebotomy or from cells that had already been banked, thus minimizing repeated invasive procedures. Importantly the cells do not undergo senescence and they have an unlimited lifespan. Further, they can be differentiated in a tissue-specific fashion to generate in vitro tissue models. A recent example of the use of iPSCs was for the preclinical development was a gene therapy product for choroideremia (CHM). This is an X-linked retinal degeneration that is symptomatic in the 1st or 2nd decade of life causing nyctalopia and loss of peripheral vision. An animal model that accurately reflects the human condition is not available. Cells from patients were efficiently transduced with AAV2. Transduction of CHM cells with a


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wild type hCHM cDNA results in functional restoration of REP1mediated enzymatic activity and protein trafficking. To screen for short-term toxicity resulting from infection with AAV2. hCHM, CHO cells were evaluated for apoptosis by TUNEL staining following infections. Neither dose of AAV2.hCHM resulted in cell death (Vasireddy et al., 2013). The scientific advances which are currently occurring through iPSC technology allow differentiated cells to be obtained from healthy or diseased humans, in multiple conditions, and the possibility for the construction of in vitro, human-based systems of multiple diseases, which can be used for the understanding of the mechanisms of diseases and the study of molecules aimed at correcting the pathological disturbances in the specific conditions. Several large research initiatives funded by public and private funds are ongoing in multiple regions, including the US and EU. A number iPSC specific projects under TOx21 in the US designed at producing cell specific and/or disease specific cells lines for in vitro screening include Cellular Dynamics and Molecular Devices; QPS, PhoneixSongsBiologicas and the Hamner Insititues; XCell; the Buck Institution, Primorigen Biosciences and Vala Sciences. The IMI initiative previouslydescribed above is also funding several projects using human cell-based systems, of which, due to its transversal applicability, StemBANCC is a good example. StemBANCC aims at establishing and optimizing the processes for obtaining iPSC from healthy and disease subjects with several types of diseases like diabetes, neurodegenerative, etc. The field of mechanistic safety, where specific questions need to be posed to understand how certain effects emerged is benefiting from in vitro cell-based technology. For example, human cardiomyocyte – based systems, hepatocyte-based and neural cell line systems. These systems are hoped someday to result in combined in efforts towards obtaining comprehensive in vitro human cell based microphysiological systems (organs on chips; human on a chip) to screen and mechanistically scrutinize specific safety aspects most relevant for humans (http://www.ncats.nih.gov/research/ reengineering/tissue-chip/tissue-chip.html). These in vitro models, if and when accepted by the Regulatory Authorities have the potential to reshape the preclinical safety paradigm to support clinical development (Silva Lima, 2014).

8. Conclusion There are many complimentary efforts in the US and the EU as well as international collaborations aimed at developing better “tools”, specifically, preclinical in vitro and in vivo models that can more reliably and more efficiently predict the clinical benefits and risks of new medicines. All models will have limitations. Disease models include various tissues and cells derived from patients; spontaneous animal disease models, induced animal disease/ injury models (e.g. chemical, immunological, surgical), genetically modified animal models and humanized mouse models. While in vivo animal models of disease have played a critical role in drug discovery and lead candidate selection to confirm activity and proof-of-concept, they have not been generally used to support their safety. As an increasing number of innovative medicines and ATMPs are emerging from academic research, it is especially important for research scientists to be aware of the value of extracting relevant safety information from proof-of-concept studies but at the very least understand the full dose response of the therapeutic modality. Importantly, in order to improve extrapolation, the similarities and differences between the pathophysiology of the disease/injury animal model and the pathophysiology of the disease/injury of humans should be understood. Specifically, the effect of the disease/injury status of the animal on the pharmacology/

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Risk of discontinuation of Advanced Therapy Medicinal Products clinical trials.

Whither advanced therapy medicinal products?

Advanced therapy medicinal products - a multiple challenge.

Regulatory and clinical aspects of psychotropic medicinal products bioequivalence.

New European regulation for clinical trials of medicinal products.

Animal cadaveric models for advanced trauma life support training.

Advanced Therapy Medicinal Products for Rare Diseases: State of Play of Incentives Supporting Development in Europe.

Advanced Therapy Medicinal Products: How to Bring Cell-Based Medicinal Products Successfully to the Market - Report from the CAT-DGTI-GSCN Workshop at the DGTI Annual Meeting 2014.

The treatment of Alzheimer's disease using Chinese medicinal plants: from disease models to potential clinical applications.

Disease modification in epilepsy: from animal models to clinical applications.

Bottlenecks in the Efficient Use of Advanced Therapy Medicinal Products Based on Mesenchymal Stromal Cells.

Animal models of Alzheimer's disease.

Ovarian autoimmune disease: clinical concepts and animal models.

Clinical trials with herbal medicinal products in children: a literature analysis.

Translating dosages from animal models to human clinical trials--revisiting body surface area scaling.

Advancing hospital pharmacy practice through new competences in advanced therapy medicinal products.

The regulatory sciences for stem cell-based medicinal products.

[Use of animal models of clinical pain].

Use of Simulation to Study Nurses' Acceptance and Nonacceptance of Clinical Decision Support Suggestions.

A review of disease progression models of Parkinson's disease and applications in clinical trials.

Acceptance of surrogate end points in clinical trials supporting approval of drugs for cancer treatment by the Japanese regulatory agency.

Anesthetic neurotoxicity--clinical implications of animal models.

Contribution of the spanish agency for medicines and healthcare products to the European committee for the evaluation of medicinal products for human use.

Transgenic animal models for study of the pathogenesis of Huntington's disease and therapy.