S01: Visualization of pathway-related toxicity risks in non-clinical studies. The example of TGF-beta

WuerschKuno Würsch and Christian Pfaff
Novartis AG, Basel, Switzerland

Each nonclinical study generates thousands of new data points, which we analyze to characterize the safety profile of new compounds. While we invest a lot of effort to understand this data and to report the studies, we oftentimes fail to quickly integrate data from different studies for pharmacology comparisons or pathway-related analysis.

In a pilot exercise, we aimed to retrieve TGF-β pathway-related toxicities from our internal database of non-clinical data (ie histopathological findings).

We searched the internal database for compounds hitting the pathway as primary or secondary target. We then used an innovative Relative Risk Ratio (R3) scoring to pick toxicities selectively enriched in compounds targeting the TGF-β pathway. We used the Knime analytics platform to retrieve data, compute scores and generate a risk profile for histopathology findings. By generating R3 scores, we tried to overcome the limited dynamic scale of histopathology data and detect study specific trends. As a next step, the toxicity profile was used to search the eTOX database to find additional compounds or molecules with structural similarities. We also used our internal tool dedicated to visualization of safety and biomarker information.

A pivotal requirement for any approach to integrate data from different sources is a structured database and a sufficient level of standardization. While this is already largely achieved for numerical data such as clinical pathology or body and organ weights, this is more challenging for descriptive data such as clinical signs or pathology findings. With the advent of global initiatives such as CDISC-SEND and INHAND, there has been significant progress to reach a level of standardization that facilitates retrospective or cross-study data analysis of histopathological data. 

Simultaneously, the visualization of such increasingly large datasets becomes a critical business need in order to manage data more efficiently and retrieve key insights (Brown et al 2016).

Pathologists working in toxicologic pathology are clearly trending towards specialists integrating data from different domains and managing more complex datasets. It is no longer sufficient to generate data; we need to be able to efficiently retrieve historical data in order to contribute to hypothesis generation, answering scientific and regulatory questions and to better inform risk-benefit assessments. It also allows to highlight potential target organs and to enhance sampling and analysis.

Literature:

Brown AP (2016) Graphical Display of Histopathology Data From Toxicology Studies for Drug Discovery and Development: An Industry Perspective. Regul Toxicol Pharmacol 82:167-172


S02: Meeting the challenge of reproducible in vivo studies

Kam ThongTony Kam-Thong
Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, Switzerland 

In vivo studies are inherently difficult to perform. Successful operation requires not only highly developed animal handling and logistics skills on the side of the technician, but also a thorough comprehension and stringent application of essential scientific and statistical concepts.

Strikingly, important scientific aspects such as experiment design, sample size estimation and randomization procedures are often not covered by the relevant GLP guidelines and SOPs. Experimental techniques such as (double) blinding – compulsory for any clinical study – are often not or only partially implemented. This may explain why a large number of published preclinical studies either fail to be reproduced completely by peer researchers or turn out to report overstated, i.e. positively biased, effect sizes1.  A recent meta-analysis estimates overall reproducibility of preclinical studies at only 10-50%2. The potential deleterious impact on academic or industrial research is obvious.

Meanwhile, pressure is mounting to reduce animal numbers in accordance with the 3R principles3. This well-justified demand even raises the bar for accurate experimentation and statistical analysis, without which the risks of false negative, false positive and biased results are prone to further increase. 

This talk illustrates some frequently found issues with in vivo studies and corresponding remedies. The link between methodological correctness, reproducibility of findings and responsible animal use is discussed. It is argued that adopting solid scientific practices should be considered a strong imperative of animal welfare and 3R as well.

  1. Ioannidis JPA (2014). PLoS Med 11(10): e1001747
  2. Freedman LP et al. (2015). PLoS Biol 13(6): e1002165
  3. https://www.nc3rs.org.uk/the-3rs

S03: Principal component analysis (PCA) methodology and beyond in toxicological pathology

MaldonadoAna Maldonado1, 2, Christine Ruehl-Fehlert3, Frieke Kuper1
1. TNO, RAPID , 3704 HE Zeist, The Netherlands
2. Qualogy, Data Science, 2288 EC RIJSWIJK (Z-H), The Netherlands
3. Bayer AG, Drug Discovery - Pharmaceuticals, Pathology Wuppertal, Germany

In this presentation, we show an innovative application of data science and more precisely of predictive modelling, to help understand toxic and therapeutic relations between different sets of data involving animal data (rodent and non-rodent) and human data. 

The general idea of predictive modeling (PM) is simple: using available data, predict a future response with as much accuracy as possible. But even though predictive modeling is all about making a practical use of real data to solve real problems, in most cases the data is not clean and the predictive model is not quite perfect.

s03

Using data visualization methods as PCA (Principal Component Analysis), variable reductions can be made in order to find the key factors involved in interesting correlations between the different findings seen in animals and humans. For example, patterns of effects in humans which translate into animal immune findings (reverse translation).

In order to do an extensive comparison between animal and human data, we separated the findings into degenerative and/or immunosuppressive versus inflammatory and stimulatory. We have explored if the findings go in the same direction although they are not identical in different species.

Predictive modelling combined with other data mining and chemometric tools have proven to be a powerful asset in modern molecular and drug discovery, as well as in toxicological research. Using predictive modelling can also help us to understand why a certain response occurs, and which are the key features that govern (most) of its behavior. Finally, relationships between different systems (based on human and animal data) open new possibilities for the experimental testing and safety assessment of novel drugs.


S04: Non-clinical safety evaluation of Antibody-Drug Conjugates: Experience from Non-Human Primate Studies

MecklenburgLars Mecklenburg
Covance Preclinical Science GmbH, Muenster, Germany

Antibody drug conjugates (ADCs) are an emerging class of targeted therapeutics with the potential to revolutionize the field of cancer chemotherapy. ADCs are biopharmaceuticals consisting of a cytotoxic small molecule covalently linked to a targeted protein carrier via a cleavable or non-cleavable linker. This concept allows selective delivery of a cytotoxic drug to cancer cells and is thus able to improve the therapeutic index over traditional chemotherapy.

Critical parameters in ADC development are selection of the target antigen, the antibody against the target, the cytotoxic molecule, and the conjugation chemistry used for the attachment of the cytotoxic molecule to the antibody. The complexity of these molecules requires a science-based approach to safety assessment. The primary objective of this presentation is to review current concepts in the non-clinical safety assessment of ADCs, based on the experience that was gained with more than 20 studies that were conducted with a variety of ADCs in non-human primates.


S05: Antibody-Drug Conjugates: Drug-Induced Renal Toxicity with Secondary Metastatic Calcification in Rats

BodieKaren Bodié, Kirstin Barnhart, Eric Blomme
AbbVie Deutschland GmbH & Co. KG, Global Preclinical Safety, Development Sciences,  Ludwigshafen, Germany

An experimental ADC (monoclonal antibody cross-reactive to rat and monkey) was administered intravenously to male and female Sprague-Dawley rats for three weeks at 10 mg/kg/dose (four weekly doses) and resulted in adverse pathology findings in the kidneys and heart.

In the kidneys, mild to severe degeneration/regeneration of cortical tubules (characterized by basophilic tubular epithelial cells and enlarged nuclei), minimal to moderate tubular epithelial cell necrosis in the cortex and medulla, and small to prominent hyaline casts were noted in all treated rats. The microscopic changes correlated with mild to marked increases in blood urea nitrogen (BUN), serum creatinine, and serum phosphorus.  Additional serum electrolyte changes considered to be secondary to renal injury included mild decreases in sodium, moderate to marked decreases in chloride, and mild increases in calcium. Urinalysis showed mild to moderate increases in urine protein, mild increases in urine glucose, as well as occasional mild decreases in urine pH.  In the heart, minimal to moderate myofiber degeneration (three out of five females) and moderate mineralization of the myocardium and vessel walls (two out of five females) were observed microscopically. Myofiber degeneration and mineralization were considered likely secondary to the kidney findings due to imbalances in the calcium and phosphate serum levels.

Disclosures:

All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.


S06: Pathology Peer Review – regulatory concerns and potential consequences

RomeikeAnnette Romeike
Covance Laboratories Inc., France

Peer review of histopathology data and the pathology narrative has been a widely used, however not mandatory, process to improve the quality and consistency of the pathology interpretation in toxicity studies.

Due to the specific nature of microscopic pathology data, it being an interpretation by a specifically trained expert based on a morphologic evaluation, rather than a “measurement” by a well defined and validated technical instrument, toxicologic pathology evaluations and peer review have been put under special scrutiny by auditors and regulators. Consequently, since the early days of peer review, efforts have been made to clarify and publish specificities of the peer review process, which has ultimately led to the publication of best practices for peer review approved by global societies of toxicologic pathology, widely applied in the profession (“Recommendations for Pathology Peer Review”, Morton et al., Toxicologic Pathology, 2010).

Since the early 2000’s major international societies of toxicologic pathology have tried to inform regulatory bodies and auditors about the benefits, complexities, and limitations of pathology peer review, mainly in order to influence avoiding increasing administrative burden of an overall straightforward, scientifically driven process of quality control. However, regulatory concerns about the potential of undue influence by a peer reviewer have increased with a changing industry environment in which the majority of toxicity studies supporting the safety assessment of new drugs, chemicals or food additives, are conducted in contract research organisations, where the risk of undue influence and pressure on study pathologists is perceived to be higher.

In September 2014, the OECD released Advisory Document No 16 “Guidance on the GLP requirements for Peer Review of Histopathology”, which mainly reflects regulatory expectation for more transparency around the management, planning, conduct and documentation of a Peer review. During its creation, international societies of toxicologic pathology have commented on draft versions of the advisory document twice, in order to provide the OECD working group with more clarity on the currently established best practices of peer review, for example, the important distinctions between contemporaneous and retrospective peer review, and consequences to their documentation. In 2015, a STP SRPC (Scientific  Regulatory & Policy Committee) Working group, in collaboration with international societies of toxicologic pathology, published a review and unified interpretation of this guidance to serve as support for organizations for compliance, as some sections in the guidance appeared unclear and inconsistent (Toxicologic Pathology, 43: 907-914, 2015). More recently (June 2017), the OECD working group published clarifications of two paragraphs on their webpage, mainly around documentation and correspondence between study pathologist and peer review pathologist during the consensus phase (http://www.oecd.org/chemicalsafety/testing/glp-frequently-asked-questions.htm).

While these clarifications still leave some room for interpretation and variable processes, they seem to reflect a major concern of potential undue influence through the peer review pathologist.

Also, an FDA guidance on peer review is currently under internal FDA review. A potential date for issuance of a draft guidance for review and comment is still unknown.

The presentation will offer a review of the historical evolution of peer review in the context of GLP regulation, and provide insight in proposed process adaptions to allow appropriate transparency of the conduct of peer review compliant with regulatory expectations while keeping the general framework of manageable best practices of peer review. In addition, current activities of international societies on this topic, incl. an outlook on the future of international regulations will be given.  After the presentation, sufficient time will be available to allow feedback, questions and comments from the auditorium.


S07: Drug-Induced Neutropenia 

WinterMichael Winter
F. Hoffmann-La Roche Ltd., Pharmaceutical Sciences, pRED Innovation Center Basel, Switzerland


S08: Cytokine release: pre-clinical models and their clinical translation

HintonHeather Hinton
Roche Pharma Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Zurich

Cytokine-mediated infusion-related reactions are a relatively common occurrence upon treatment with monoclonal antibodies. The challenge for pharmaceutical companies is to develop pre-clinical models that accurately assess the clinical risk of these adverse events. Alemtuzumab (CD52 ADCC depleting antibody)1 and TGN1412 (CD28 superagonist antibody)2 both induce cytokine-mediated first infusion reactions in the clinic, although by different mechanisms. In the case of TGN1412 the mechanism is driven by T cells, while for alemtuzumab it is driven via Fc interactions. However, neither molecule induced clinical signs indicative of an infusion reaction in cynomolgus monkey3,4. These examples, combined with others, have led to the perception that cynomolgus are under-predicative for cytokine release and cytokine-mediated infusion reactions.

In recent years there has been an increase in the development of immune-modulating molecules, primarily for oncology but also for infectious indications. These molecules are often Fc inert, stimulating the immune system by agonising activating receptors or blocking inhibitory receptors. Cynomolgus is often the only relevant toxicology species and based on the mechanism of action, cytokines are often considered important safety and pharmocodynamic biomarkers. We have found cytokines secretion in cynomolgus to be useful safety and pharmocodynamic biomarkers that can be used for assessing risk, interpreting findings, guiding first in human doses and as translational pharmacodynamic biomarkers. The relevance of cynomolgus cytokines as biomarkers is highly dependent on the mechanism of action of the drug.  Thus it is important that the cytokine monitored, sampling times and how the data is interpreted is adapted accordingly.

  1. Keating MJ, Flinn I, Jain V, et al: Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: Results of a large international study. Blood (2002) 99:3554-3561.
  2. Cytokine Storm in a Phase 1 Trial of the Anti-CD28 Monoclonal Antibody TGN1412. G. Suntharalingam, M. R. Perry, S. Ward, S. J. Brett, A. Castello-Cortes, M. D. Brunner and N. Panoskaltsis. n engl j med (2006) 355;10, 1018
  3. Concordance of preclinical and clinical pharmacology and toxicology of therapeutic monoclonal antibodies and fusion proteins: cell surface targets. P.J. Bugelski and P.L. Martin. British Journal of Pharmacology (2012) 166 823–846
  4. TGN1412 Investigators Brochure. http://www.circare.org/foia5/tgn1412investigatorbrochure.pdf

S09: Use of Stereology in Animal Model Development and Efficacy Evaluation

Moved to "Joint Interactive Slide Sessions", click here.


S10: Bone histomorphometry in preclinical studies

ChouinardLuc Chouinard
Charles River Laboratories, Montreal ULC

Qualitative histopathology remains the gold standard for hazard identification and safety assessment of potential new therapeutics. In most cases, histopathology evaluation has sufficient sensitivity to detect test article-related effects on decalcified hematoxylin and eosin (H&E) stained bone sections in standard toxicity studies. However, it may lack the sensitivity to detect test articles effects on key processes in bone tissue such as bone formation, mineralization, and resorption. Undecalcified specimens and histomorphometry may be needed to detect low magnitude structural or dynamic changes that cannot be appreciated by qualitative assessment. In addition, long-term dosing could be required for structural changes to manifest as a consequence of test article-related effects on bone cell activity.

While imaging techniques such as dual energy X-ray absorptiometry (DXA), peripheral quantitative computed tomography (pQCT), and micro-computed tomography(µCT) are sensitive methods that can provide excellent information on bone mass and bone structure, these techniques do not explain the tissue-level mechanisms that lead to a bone mass imbalance or altered microstructure.

Bone histomorphometry is an important and indispensable tool for assessing the mechanisms by which therapeutic agents act on the skeleton and for assessing the skeletal safety of therapeutic agents. It can provide mechanistic insight into the effects of a test article on bone and explain structural changes identified by histopathology or imaging. Histomorphometry can also be used prospectively with other methods such as bone markers and imaging modalities to address a potential target- or class-related theoretical bone liability.

This presentation will provide guidance on common concept and techniques for bone histomorphometric analyses including study design, fixation and processing, staining, species and age consideration, fluorochrome labeling techniques, applied to specific example of growth plate, cancellous and cortical bone assessment in preclinical studies.


S11: Predictivity of animal models for human diseases

DoglioniClaudio Doglioni
Scientific Institute San Raffaele,  University Vita-Salute San Raffaele, MILANO, ITALY.

Animal models have been, are, and will be essential in several field of biological and clinical human research. However, the successful translation from animal models to clinical human application is full of hurdles. My main interest in oncology lead me to study different models of spontaneous, chemically induced, or GEM induced   mouse models of carcinogenesis. Human tumor xenotransplant  in immunodeficient mice is another frequently utilized mouse model. Most of these models have been developed with the aim to recapitulate the human neoplastic counterpart.  However  carcinogenesis both in human and mouse is extremely complex. Cancer pathogenesis and the response to different therapeutic regimens are only in part reflected in the available animal models and in particular in the mouse. Factors to be considered are genetic background, genomic landscape of different tumors, immunological response, microbiome. Making a mouse model recapitulating the tumor and the immune response of the single patient in order to identify the more effective treatment is the main goal of several research groups. I will present examples from our experience in developing and investigating mouse models. A special attention to the preanalytical variables, knowledge of molecular/biological properties of the model, a bench to bedside integrated experimental set up and awareness of its limits are prerequisites for the successful translational use of animal models.


S12: Risks of bias in biomedical research – how to identify them and what to do about it

McCannSarah McCann
The University of Edinburgh, Centre for Clinical Brain Sciences, Edinburgh, UK

Biomedical research supports the generation of knowledge in medicine; however, the findings from preclinical studies in animals often do not translate to those in humans in a clinical setting. A plausible hypothesis for this disparity is that animal experiments report exaggerated or falsely positive findings. This theory has been the focus of intense scrutiny in recent years, fuelled largely by the results of systematic reviews of preclinical studies.

Bias can introduce systematic error in the results of a study and can occur due to inadequacies in design, conduct, analysis, or reporting. Frequent findings in systematic reviews of animal studies include exaggerated claims of therapeutic benefit in studies where investigators do not report simple measures to reduce bias in experimental design, or use cohort sizes too small to provide adequate statistical power to answer their research question [1, 2]. Assessing the extent to which experiments are at risk of bias allows us to evaluate the reliability of evidence and the level of confidence in the conclusions drawn. Risks of bias can be assessed by appraising studies critically based on the reporting of experimental design measures to reduce selection, performance, measurement, attrition, and outcome reporting biases.

Measures to reduce risks of bias include random allocation of animals to experimental groups, which helps to ensure that groups are balanced for baseline characteristics. Measured effects are therefore more likely due to the intervention than pre-existing group characteristics. Blinding experimenters to group allocation ensures that biases in the way that animals are handled and outcomes are measured are reduced. To prevent attrition and reporting biases, exclusion criteria and primary and secondary outcomes should be pre-specified. All animals involved in an experiment should be accounted for and all outcomes reported, not only those that are significant or interesting [3].

In publications from different biomedical disciplines, identified in the context of systematic review, the prevalence of reporting measures to reduce selected biases varies considerably, although some improvements have been identified over time. There also appears to be little relationship between the perceived quality of publications or research institutions and the reporting of risks of bias [4].

Establishing the prevalence and impact of risks of bias in the animal literature has in some research disciplines motivated structural changes in the way preclinical research is funded, performed and reported, but there is substantial room for improvement.

  1. Howells DW et al (2014) Nat Rev Neurol 10(1):37–43
  2. Tsilidis KK et al (2013) PLoS Biol 11(7): e1001609
  3. McCann SK et al (2017) Neuroprotective Therapy for Stroke and Ischemic Disease73–93
  4. Macleod MR et al (2015) PLoS Biol 13(11): e1002301.

S13: Animal models of demyelinating diseases, focus on translational medicine

Baumgaertner

The BSTP Chirukandath Gopinath Lecture

Wolfgang Baumgärtner
Department of Pathology, University of Veterinary Medicine, Hannover, Germany

Demyelinating diseases involve the central and peripheral nervous systems (CNS, PNS) and are characterized by inflammatory lesions with a loss of myelin and axonopathy (Waksman, 1999). Depending on the affected region, demyelination causes damage of several nervous functions, including sensitivity, cognition and motor impairments. Further, demyelinating lesions vary in their potential to recover and can be presented as primary or secondary processes (Lempp et al., 2014; Tsunoda and Fujinami, 1996). Primary demyelination damages myelin sheaths or myelin-forming cells, while axons are not affected at early stages. Secondary demyelination results from the damage of neurons or axons followed by myelin breakdown. Similarly, there are two proposed models for the pathogenesis of demyelinating diseases, the inside-out and outside-in models. The outside-in model proposes that the lesion starts from the myelin (outside). Accordingly, the primary target is the myelin or oligodendrocytes. Following the primary myelin destruction, the axon (inside) is damaged. The inside-out model suggests that the lesion develops from the axon (inside) to the myelin (outside), so the primary target is the axon or its cell body (neuron), and its damage triggers secondary demyelination. Inflammation within the CNS mediates myelin loss in most primary demyelinating diseases. The production of mediators by inflammatory cells attracts and stimulates other immune cells and results in disease progression and might trigger secondary by-stander demyelination (collateral damage; Beineke et al., 2009; Lavi and Constantinescu, 2005).

There are several acute and chronic inflammatory primary demyelinating disorders described in humans and in animals. They can be triggered by a variety of causes, including infectious agents, toxins, autoimmune reactions, genetic as well as environmental factors (Baumgärtner et al., 1989; Beineke et al, 2009; Cork et al., 1974; Lavi and Constantinescu, 2005; Love 2006; Martin et al., 1992; Vandevelde et al., 2012; Summers et al., 1995).

Multiple sclerosis (MS) is one of the most frequent CNS demyelinating diseases in humans (Lavi and Constantinescu, 2005; Ebers 2008). The estimated prevalence of MS is more than two million people worldwide, and its distribution is influenced by racial and ethnic differences (Rosati 2001). MS is defined as a chronic demyelinating disease of unknown etiology and multifactorial causes. Genetic disorders, environmental factors and autoimmunity, as well as infectious causes especially virus infections have been suggested as initiators (Bar-Or et al., 1999; Cepok et al., 2005; Love, 2006; Lavi and Constantinescu, 2005; Martin et al., 1992; Trapp and Nave, 2008). Sensory, motor, and cognitive impairments found in MS lead to a wide range of symptoms. MS can be classified in different categories: a benign, a relapsing-remitting, a primary or secondary progressive and a progressive-relapsing form are distinguished (Lublin and Reingold, 1996; Trapp and Nave, 2008).

The pathologic features leading to permanent neurological disability in MS patients are demyelination, inflammation, and axonal damage (Trapp and Nave, 2008). Pathological changes in MS are sub-classified into four groups (I to IV; Lucchinetti et al., 1996) depending upon the major contributing mechanisms: macrophage and T-cell mediated demyelination (I), antibody and complement mediated demyelination (II), peripheral (III) and central (IV) oligodendrogliopathy.

To study myelin disorders, several animal models have been developed and are applied depending on the question to be asked and the mechanism to be investigated (Beineke et al, 2009; Cork et al, 1974; Lempp et al., 2014; Dal Canto et al., 1995; Lavi and Constantinescu, 2005; Mix et al., 2004; Skripuletz et al., 2013; Tsunoda and Fujinami, 1996; Ulrich et al., 2009). They include virus- and toxin-induced as well as experimentally induced autoimmune reactions and gene mutations such as naturally occurring diseases (e.g. canine distemper) and experimentally induced diseases (e.g. murine coronavirus, Semliki forest virus-infection, Theiler’s murine encephalomyelitis, immune-mediated Experimental Autoimmune Encephalomyelitis as well as cuprizone, ethidium bromide and lysolecithin-induced demyelination and genetic mutations [e.g. proteolipid protein mutation (rumpshaker and jimpy mouse), myelin basic protein mutation (shiverer mouse), galactocerebrosidase mutation (twitcher mouse)]. Considering the various disease syndromes causing myelin loss and the fact that none of the models mimics the spontaneously occurring disease perfectly and completely, animal models can only cover certain aspects of the human diseases.

Overall animal models represent an important tool to study pathogenetic mechanisms, develop diagnostic tools and intervention strategies to prevent or cure human diseases. Despite all controversies animal models represent an essential corner stone in the concept of translational medicine (TM; syn., translational science). This is defined by the European Society of Translational Medicine as an interdisciplinary branch supported by three pillars: benchside, beside and community (https://en.wikipdeia,org/wiki/translational_medicine). TM is a highly interdisciplinary field with the goal to improve the global healthcare system significantly. It represents a rapidly growing discipline with the aim to expedite discoveries of new diagnostic tools and treatments by using a collaborative “bench-to-bedside” approach. However, the question whether models really provide substantially new insight into diseases processes and allow meaningful intervention strategies has been discussed controversially. Key words to be mentioned in this context include validity, reproducibility, irreproducibility and robustness of reported data, and statements like “mice lie and monkeys exaggerate” or “mouse models greatly/poorly mimic human inflammatory diseases” have to be taken in account (Seok et al., 2013; Takao and Miyakawa, 2015). Overall researchers have to ensure good scientific practice in their study design and data analysis (e.g. avoid cherry-picking), follow philosophical and ethical fundamental values in their scientific interpretation by performing their studies according to the State of the Art.

 

Selected References

Bar-Or, A., Oliveira, E.M.L., Anderson, D.E., Hafler, D.A., 1999. Molecular pathogenesis of multiple sclerosis. J Neuroimmunol: 100, 252-259.

Baumgärtner, W., Orvell, C., Reinacher, M., 1989. Naturally occurring canine distemper virus encephalitis: distribution and expression of viral polypeptides in nervous tissues. Acta Neuropathol. 78, 504-512.

Beineke, A., Puff, C., Seehusen, F., Baumgartner, W., 2009. Pathogenesis and immunopathology of systemic and nervous canine distemper. Vet Immunol Immunopathol: 127: 1-18, 2009

Cepok, S., Zhou, D., Srivastava, R., Nessler, S., Stei, S., Bussow, K., Sommer, N., Hemmer, B., 2005. Identification of Epstein-Barr virus proteins as putative targets of the immune response in multiple sclerosis. J Clin Invest: 115, 1352-1360.

Cork, L.C., Hadlow, W.J., Gorham, J.R., Piper, R.C., Crawford, T.B., 1974. Pathology of viral leukoencephalomyelitis of goats. Acta Neuropathol: 29, 281-292.

Dal Canto, M.C., Melvold, R.W., Kim, B.S., Miller, S.D., 1995. Two models of multiple sclerosis: experimental allergic encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus (TMEV) infection. A pathological and immunological comparison. Microsc Res Tech: 32, 215-229.

Ebers, G.C., 2008. Environmental factors and multiple sclerosis. Lancet Neurol. 7, 268-277.

Haahr, S., Hollsberg, P., 2006. Multiple sclerosis is linked to Epstein-Barr virus infection. Rev Med Virol: 16, 297-310.

Lavi, E., Constantinescu, C.S. 2005. Experimental models of multiple sclerosis. Springer, New York

Lempp, C., Spitzbarth,I., Puff, C., Cana, A., Kegler, K., Techangamsuwan, S., Baumgärtner  and F. Seehusen, 2014. New aspects of the neuropathogenesis of canine distemper leukoencephalitis. Viruses:6, 2571-2601

Love, S., 2006. Demyelinating diseases. J Clin Pathol: 59, 1151-1159.

Lublin, D.M., Reingold, S.C., 1996. Defining the clinical course of multiple sclerosis: results of an international survey. National Multiple Sclerosis Society (USA) Advisory Committee on Clinical Trials of new agents for multiple sclerosis. Neurology: 46. 907 - 911

Lucchinetti, C.F., Brück, W., Rodriguez, M., Lassmann, H., 1996. Distinct patterns of multiple sclerosis pathology indicates heterogeneity in pathogenesis. Brain Pathol: 6, 259-274.

Martin, R., McFarland, H.F., McFarlin, D.E., 1992. Immunological aspects of demyelinating diseases. Ann Rev Immun: 10, 153-187.

Mix E., Ibrahim S., Pahnke J., Koczan D., Sina C., Bottcher T., Thiesen H.J., Rolfs A., 2004. Gene-expression profiling of the early stages of MOG-induced EAE proves EAE-resistance as an active process. J Neuroimmunol: 151, 158-170.

Rosati, G., 2001. The prevalence of multiple sclerosis in the world: an update. Neurol Sci: 22, 117-139.

Seok, J. et al., 2013. Genomic responses in mouse models greatly mimic human inflammatory diseases. Proc Natl Acad Sci USA: 110. 3507 - 3512

Skripuletz, T., Hackstette, D., Bauer, K., Gudi, V., Pul, R., Voss, E., Berger, K., Kipp, M., Baumgärtner, W. and Martin Stangel. Astrocytes regulate myelin clearance through recruitment of microglia during cuprizone induced demyelination. Brain: 136. 147 – 167, 2013

Summers, B. A., Cummings, J. F., deLahunta, A., 1995. Veterinary neuropathology. Mosby, St. Louis

Takao K. and Miyakawa T, 2015. Genomic responses in mouse models greatly mimic human inflammatory diseases. Proc Natl Acad Sci USA: 122. 1167 - 1172

Trapp, B.D., Nave, K.A., 2008. Multiple sclerosis: an immune or neurodegenerative disorder?. Annu Rev Neurosci: 31, 247-269.

Tsunoda, I., Fujinami, R.S., 1996. Two models for multiple sclerosis: Experimental. allergic encephalomyelitis and Theiler's murine encephalomyelitis virus. J Neuropathol Exp Neurol: 55, 673-686.

Ulrich, R., Kalkuhl, A., Deschl. U. and W. Baumgärtner, 2010. Machine learning approach identifies new pathways associated with demyelination in a viral model of multiple sclerosis. J Cellu Mol Med: 14,  434 – 448

Vandevelde, M., Higgins, R. J., Oevermann, A., 2012. Veterinary neuropathology. Essentials of theory and practice. Wiley-Blackwell, Chichester

Waksman, B.H., 1999. Demyelinating disease: Evolution of a paradigm. Neurochem Res: 24, 491-495.


S14: The human safety of Veterinary Medicinal Products: the importance of regulatory toxicology studies in the assessment of user safety and of residues in foodstuffs

CoussanesEvelyne Coussanes and Audrey Deflandre
Ceva Santé Animale, Libourne, France

Each new Veterinary Medicinal Product (VMP) must be granted a Marketing Authorization (MA) before to be placed on the European Community market.

Among abundant documentation presented in the MA dossier1, safety data coming from studies conducted in laboratory or target animals, allow to guarantee the safety of the VMP for animals but also for human beings. Indeed, humans may be exposed to the VMP during the administration or after consumption of foodstuffs obtained from treated animals.

Pathologists play an important role in assessment of the results of toxicological studies and in the drawing up of conclusions. The impact of conclusions of toxicological studies will be presented through real examples of two marketed VMPs.

The first example is related to the User Safety assessment of a spot-on product, recommended to be used monthly in dogs for the treatment and prevention of flea, ticks and flies infestation.

The assessment of user safety addresses the exposure situations resulting from the normal conditions of use and from foreseeable accidents. This assessment is divided in 4 parts, which will be briefly presented:

-          an appraisal of the inherent toxicity of the VMP,

-          an appraisal of how and when the user may be exposed to the VMP,

-          the risk characterization and

-          the risk management.

The risk characterization is based on the results of toxicology studies conducting for each active ingredient and on potential routes of exposure. It may lead to the identification of some hazardous situations requiring risk management which are mentioned on the leaflet of the VMP. The presentation will be focused on the risk characterization and the resulting measures of risk management for Vectra 3D2.

The second example is related to the safety of residues of VMP or its metabolites in human food. The use of VMP in food-producing animals may result in the presence of residues in foodstuffs of animal origin which might constitute a health hazard for consumer.

A Maximum Residues Limit (MRL) application3 must be submitted before the MA application for each new pharmacologically active substances contained in the VMP intended to food-producing animals.

The MRL application is divided in two parts4. The first part concerns the safety assessment leading to the establishment of a human Acceptable Daily Intake (ADI), which represents the amount of active ingredient or its metabolites that can be safely consumed by a person on a daily basis for a lifetime.  The second part is related to the residues assessment leading to the establishment of MRL for edible tissues (muscle, fat, liver and kidney) and milk coming from treated animals.

For the MA application, residues studies are conducted with the VMP to calculate a Withdrawal Period (WP) which is the period necessary between the last administration of the VMP and the production of foodstuffs from such animals ensuring that such foodstuffs do not contain residues in quantities in excess of the MRL.

The MRL application for an ergot derivative which was developed for a use in dairy cows, will be presented with a focus on the impact of toxicological studies in the establishment of MRL and consequently on the WP which must be applied to the VMP.

 

References:

  1. Commission Directive 2009/9/EC
  2. CVMP assessment report for Vectra 3D (EMEA/V/C/002555/0000)
  3. Regulation (EC) 470/2009 of the European Parliament and of the Council
  4. Volume 8 of the rules governing products in the European Union

S15: Veterinary risk assessments for the target animal, user, consumer and environment, an overview for product approval

HarrimanJay Harriman
Boehringer Ingelheim Animal Health, Duluth, GA, USA 

Successful registration of an Investigational Veterinary Pharmaceutical Product (IVPP) requires the demonstration of product safety under the proposed conditions of use. Margin of safety studies in the target species evaluate health endpoints relative to overdoses. Other target animal safety studies assess injection/dermal application site and breeding safety.  Results are supplemented by safety data obtained in efficacy and field trials. User risk assessments evaluate the hazard of IVPP use in various user exposure scenarios.  Reasonable worst-case exposures are matched with the most conservative health hazard endpoint from similar duration laboratory animal toxicity studies to define a margin of exposure (MOE).   The MOE is regarded as acceptable and the exposure is regarded as safe when the MOE exceeds the uncertainty or safety factor associated with extrapolating laboratory animal data to humans.  Consumer human food safety is assured by using an approach that assesses the need for and defines drug residue exposure mitigation strategies.  Safety is assessed based on the active pharmaceutical ingredient’s (API) toxicologic hazard profile in laboratory animals, its impact on human intestinal flora and evaluated against the IVPP’s residue chemistry profile in edible tissues.    Nationally-defined strategies to ensure consumer safety include drug residue withdrawal periods, milk discard times and product labelling statements.  Environmental impact assessments are also risk-based and employ tiered testing strategies that integrate API environmental fate, exposure and toxicity data. The IVPP’s potential to adversely affect non-target aquatic and terrestrial species is assessed in the context of exposure to confirm safety or define the need for mitigation strategies to protect ecosystems.


S16: Vaccine: Animal models, the necessary link between in vitro experiments and clinical trials

ManuguerraJean-Claude Manuguerra
Environment and Infectious Risks, Laboratory fur Urgent Response to Biological Threats, Institut Pasteur, Paris, France

After antimicrobial drug or vaccine design and the evaluation in cell culture of the inhibitory activity of the new drug or the vaccine induced antibodies and before doing the first phase of clinical trials in human beings, the new products must undergo a series of experiments in vivo, including challenges to show the proof of concept. Generally, a mouse model is the first one to be used and non-human primate the last. Transgenic mice made susceptible to a specific viral infection, for example by adding the cellular receptor for the relevant virus, may be required. Mice bearing a human immune system can also be used.

It is now possible to follow an infection in vivo in mice with and without treatment by new bioluminescent imaging technologies. In this presentation, examples of the use of murine models to study the pathogenesis of viral infections and to evaluate new antivirals and new vaccines will illustrate the value and necessity of animal models in vaccine and drug development.


S17: Nonclinical Development of a viral vectored therapeutic vaccine to treat human cervical cancer

RomeikeAnnette Romeike
Covance Laboratories Inc., France

Cervical cancer is the third most common cancer and second largest cause of cancer deaths in women worldwide. Human papilloma virus (HPV) infection has been recognized as the cause of precancerous cervical lesions (so called CIN2/3) and cervical cancer. HPV infection is the most common sexually transmitted disease, affecting about 400 million women worldwide. Most infections are spontaneously eliminated in less than one year. However, persistent HPV infection can lead to CIN2/3 and progress to cervical cancer. Worldwide, CIN2/3 affects 1,400,000 women each year, while cervical cancer affects 500,000 women each year, resulting in more than 270,000 deaths.

High risk HPVs have the capacity to transform cervical epithelial cells by integrating the open reading frames encoding the viral early proteins E6 and E7 into the host cell genome. This integration may lead to constitutive overexpression of E6 and E7, mediating transformation of the cells to a malignant phenotype. Since the continued production of E6 and E7 is required for the maintenance of the transformed phenotype, E6 and E7 represent tumor-specific antigens in cervical carcinoma and premalignant HPV-transformed cells. As a consequence, E6 and E7 are potential targets for immunotherapeutic intervention strategies involving induction or stimulation of cytotoxic T lymphocyte (CTL) activity against HPV-transformed cells.

ViciniVax, a Dutch biotechnology company and spin-off company from the department of Molecular Virology and the department of Gynaecologic Oncology of the University Medical Center Groningen, Netherlands,  developed SFVeE6,7 (Vvax001), based on an attenuated, recombinant Semliki Forest virus (8rSFV), encoding HVP E6 and E7. SFV represents an excellent platform for generating efficient vector systems for transient high-level gene expression for therapeutic cancer immunization. Vvax001 induces robust HPV-specific cellular immune and memory responses, resulting in excellent therapeutic anti-tumor efficacy in wild type mice. The Vvax001 application is intended as therapeutic immunization of CIN2/3 patients and patients with grade I/II cervical cancer by inducing T helper cells and cytotoxic T cells specific for E6/E7 expressing tumor cells.

The presentation will give an overview of the regulatory environment for nonclinical development of gene therapy medicinal products (GTMP) and specific aspects to be addressed for GTMPs prior to FIH trials and through clinical development using the example of the nonclinical development package of Vvax001.


S18: Nonclinical safety development of Flavivirus vaccines

SyntinPatrick Syntin
Non Clinical Safety Department, Sanofi Pasteur, Marcy l’Etoile, France

Flaviviruses comprise more than 70 different viruses which are transmitted by either mosquitoes or ticks. With respect to medical impact, the most important ones are yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) as well as Zika virus (ZikV) to a lesser extent. Flaviviruses are small enveloped positive stranded RNA viruses which contain three structural proteins only, designed as C (capsid), E (envelope), and M (membrane) (1). Because of its dual role in cell entry – attachment to cellular receptors and membrane fusion – the E protein is the main target of virus neutralizing antibodies which inhibit these two functions and thus prevent the infections. Flaviviruses cause syndromes ranging from febrile illness to acute meningo-encephilitis and hemorrhagic fever, and they are inherently neurotropic, inducing mild meningitis to severe destructive encephalitis associated with mortality (2). ZikV infections have also been associated with microcephalies and Guillain-Barré syndromes (3). Sanofi Pasteur has developed ChimeriVaxTM (CV) vaccines which were constructed by replacing the genes for YF vaccine (YFV 17D 204) pre-membrane (PreM) and E proteins with those of the heterologous flaviviruses (4). Efficient and well tolerated vaccines have been obtained. However, complications - inherent to this particular construct - include for instance, YF-associated viscerotropic disease (YF-AVD) and YF-associated neurotropic disease (YF-AND). Although rare, they represent serious adverse events, and therefore these potential safety issues need to be assessed preclinically. Overall, the nonclinical safety assessment to support a clinical development plan for new CV vaccines should consider a) the systemic toxicity and local tolerance, b) the potential risks related to the parental viruses including viscerotropism and neurotropism, c) the biodistribution, persistence and shedding, and d) the developmental and reproductive toxicity (c and d not covered in this presentation). The NCS evaluation of a CV vaccine requires a species which develops an immune response to the vaccinal antigene(s), and is also pathogenically sensitive to the target virus. Thus, for repeat-dose toxicity, distribution and shedding and neurovirulence studies, the nonhuman primate (i.e. cynomolgus monkey) was selected. In the repeat-dose toxicity study, systemic toxicity and local tolerance are assessed after several administrations of a human dose, following the n+1 paradigm, n being the number of dosing intended in clinical trial. In this study, reactogenicity is carefully evaluated as well as specific parameters including body temperature or CRP (C reactive protein). Viremia and distribution of CV vaccines are used for the risk evaluation for viscerotropism. The clinical symptoms of YF-AVD involved multiple tissue defects such as hepatitis, liver necrosis, spleen damages and renal failure. In the NHP studies, the viremia within these tissues/organs is correlated to histopathology. Viscerotropism effect can also be addressed in an alternative transgenic mouse model deficient for the IFN-α/β receptor (A129) (5). For neurotropism, CV vaccines are evaluated in a safety monkey test (neurovirulence WHO test) following a single intra-cranial administration. A sensitive clinical scoring system and a discriminatory histopathological assessment of inflammation and neuronal damage were implemented (6) promoting this test as being a mandatory test for working and master seed lots within WHO guidelines (7). As an example, this NHP test was carried out as part of the development of a new TBEV vaccine candidate.

The analysis of the spatiotemporal distribution of viral antigens in the CNS of monkeys revealed a prominent neurotropism and higher neurovirulence versus comparators, suggesting insufficient attenuation (8). Alternative methods in transgenic infant mouse or hamster models have been developed but showed limitations and cannot fully replace this NHP test.

The particular design for nonclinical safety assessment of Flavivirus vaccines is pivotal and efficient to bring new vaccines to exposed populations and prevent life-threatening viral diseases.

1- Heinz FX, Stiasny K. Flaviviruses and flavivirus vaccines. Vaccine 2012; 30:4301-06.

2- King NJ and al. Immunopathology of flavivirus infections. Immunol. Cell Biol. 2007; 85 (1):33-42.

3- Aftab A et al. Recent trends in ZikV research: a step away from cure. Biomed. Pharmacother. 2017; 91:1152-59.

4- Guy B et al. Preclinical and clinical development of YFV 17D-based chimeric vaccines against dengue, West Nile and Japanese encephalitis viruses. Vaccine 2010; 28:632-49.

5- Meier KC et al. A mouse model for studying viscerotropic disease caused by yellow fever virus infection. PLOS Pathogens 2009; 5(10):1-10.

6- Levenbook IS et al. The monkey safety test for neurovirulence of yellow fever vaccines : the utility of quantitative clinical evaluation of histological examination. J Biol. Standard. 1987; 15:305-13.

7- World Health Organization. Requirements for Yellow Fever Vaccine. WHO Technical Report Series Report No. 978 _ Annex 5

8- Maximova et al. Comparative neuropathogenesis and neurovirulence of attenuated Flaviviruses in nonhuman primates. J Virology 2008; 82(11):5255-68


S19: Pre-clinical development of gene therapy medicinal products; Comparison with vaccines

FerryNicolas Ferry
NF Consulting SAS,  Boulogne, France

Introduction

Gene therapy is an emerging field of medicine which has now demonstrated its therapeutic efficacy in a number of recent clinical trials. Gene therapy relies on the administration to patients of polynucleotide sequences (mostly DNA sequences), the expression of which will result in a therapeutic effect. There are two broad strategies to achieve this goal. The first one, which is usually referred to as in vivo gene therapy, is the direct administration to patients of therapeutic DNA sequences embedded in the genome of recombinant viral vectors derived from different viruses (adenoviruses, adeno-associated viruses, lentiviruses etc…). The second strategy, also known as ex-vivo gene therapy, requires initial harvest of the target cells from the patient. The cells are then subsequently genetically modified ex vivo and further reimplanted in the patient.

From a regulatory perspective, both gene modified cells and viral vectors are now considered as Gene Therapy Medicinal Products (GTMPs). These products are one category of the Advanced Therapy Medicinal Products (ATMPs) which have been defined and are regulated in Europe by regulation (EC) 1394/2007 amending Directive 2001/83. This regulation also covers the therapeutic use of somatic cell therapy medicinal products.

Because ATMPs have very specific characteristics, the manufacturing, non-clinical and clinical development of ATMPs follow specific guidelines. Regarding the non-clinical requirements, it is considered that ATMPs belongs to biological products and therefore should comply with the requirements of such products. However, it not always clear how to follow these requirements for these very specific products. As an example, the general principles of ICH S6 indicate that preclinical safety testing should be performed in relevant animal species. However, the question as to which animal species are relevant for testing genetically-modified human autologous bone marrow stem cells is still a matter of debate.

ATMPs and vaccines

In many instances, the differences between ATMPs and vaccines appear faint. Recombinant DNA technology is already used for the manufacturing of a number of commercialized vaccines (e.g. some HPV or HBV vaccines …). Ongoing vaccine studies are now based on the direct administration of recombinant viral particles for a vaccination purpose (e.g. rVSV ZEBOV for immunization against Ebola virus). Conversely, some recombinant gene therapy products may match the definition of vaccines. The Committee for Advanced Therapies (CAT) from EMA is in charge of classification of ATMPs. From the published classification it appears that the rationale for classification towards gene therapy or vaccine is not always related to the product characteristics but to the intended use of the product. Indeed, adenoviral vectors encoding proteins from HBV were not classified ATMP because the product was intended for the treatment of chronic HBV infection, hence for the treatment of an infectious disease. In contrast, plasmids encoding antigens from HPV 16 and 18 were classified GTMPs as they were intended for the treatment of high grade squamous lesions of the cervix. This was not considered a vaccination for an infectious disease.

Similarly, active immunization against chronic diseases such as cancer are often referred to as “anti-tumor vaccines”. However, in most instances the corresponding cell or gene therapy products usually match the definition of ATMPs and are regulated as such.

Therefore it appears that in many instances the actual border between vaccine and gene therapy product is very fuzzy.

Consequences for pre-clinical studies

The aim of the presentation is to give an overview of the present requirements for the development of gene therapy products and to compare with the usual requirements for vaccines.

Regarding manufacturing, many provisions applicable to the production of biologics and vaccines also apply to the manufacturing of viral vectors. As discussed above, gene therapy vectors can be directly administered to patients or used to genetically modified autologous cells that will be readministered to patients. The recombinant vectors are thus considered either as a drug substance (for in vivo gene therapy) or a starting material (for ex vivo gene therapy). This classification impacts the pre-clinical requirements.

In vivo gene therapy

For vectors directly administered to patients, primary pharmacodynamic studies and toxicology studies will have to be performed with the drug product following the recommendations of existing guidelines. One of the most important issues is the selection of the relevant animal model to demonstrate the proof of concept for the therapeutic approach. Classical PK and ADME studies are usually not performed. However, a complete biodistribution evaluation is required. Additionally, the duration of expression of the transgene has to be evaluated. A dose finding study (if a pertinent animal model is available) will help to determine the human dose for early clinical phases. Regarding the pre-clinical development program, the toxicity of the vector itself, as well as that of the transgene product should be evaluated. Toxicology studies should comply with GLP and be performed with a 10 fold higher dose that the expected clinical dose. The route of administration which can be very specific (intra-retinal or intra-cerebral administration) should also be taken in consideration. Finally, depending on the integrative capacity of the vector, carcinogenicity and germ line transmission could have to be addressed.

For some anti-cancer application, conditionally replicative vectors can be used (e. g. Imlygic ®). In this specific situation, shedding of viral particles as well as evaluation of viral replication will have to be documented.

Ex vivo gene therapy

In most instances, ex vivo gene therapy is performed with autologous cells, whether for the treatment of genetic diseases or for anti-cancer vaccination purpose. In both situations, preclinical studies are not easy to design. There are no relevant animal models for primary pharmacodynamics except immunodeficient animals. Autologous models may be used, although these are less informative. Regarding toxicology studies, there are no clear regulatory guidance to determine which studies should be performed. One exception may be the evaluation of the tumorigenicity of genetically modified cells in deeply immuno-suppressed animals which can be required by regulatory authorities.

 Conclusion

The preclinical requirements for the development of gene therapy products are specific and more robust than vaccine counterpart, at least when in vivo gene therapy is concerned. Preclinical studies are usually designed on a case by case analysis. According to the current regulation, a risk based approach to determine the extent of preclinical data to be included in the marketing authorization application of ATMPs is possible. Therefore, based on this risk-based analysis, early dialog with regulatory authorities should be strongly encouraged for developers of this type of medicinal products.