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Guidelines

Guidelines for preclinical animal research in ALS/MND: A consensus meeting

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Pages 38-45 | Published online: 26 Feb 2010

Abstract

The development of therapeutics for ALS/MND is largely based on work in experimental animals carrying human SOD mutations. However, translation of apparent therapeutic successes from in vivo to the human disease has proven difficult and a considerable amount of financial resources has been apparently wasted. Standard operating procedures (SOPs) for preclinical animal research in ALS/MND are urgently required. Such SOPs will help to establish SOPs for translational research for other neurological diseases within the next few years. To identify the challenges and to improve the research methodology, the European ALS/MND group held a meeting in 2006 and published guidelines in 2007 (1). A second international conference to improve the guidelines was held in 2009. These second and improved guidelines are dedicated to the memory of Sean F. Scott.

Introduction

As the elderly population within society grows, it is widely recognized that the incidence of neurodegenerative diseases will significantly increase. The imperative for the development of effective therapeutic strategies for this group of disorders has therefore never been greater. Among these diseases, amyotrophic lateral sclerosis (ALS) is rarely given a high priority as a target disease since it is considered as a rare condition, or a so-called ‘orphan disease’. The importance of ALS is usually underestimated since in the industrialized world more than one in 500 people will die of the disease. Furthermore, the scientific and methodological basis for development of therapeutics for ALS is comparatively strong. This conclusion is justified since three relevant, major causes of the disease are known – mutations in the SOD1, TDP-43, and FUS genes Citation[2–6]. Well characterized rodent models already exist or are currently being established Citation[7–9]. In addition, the development of relatively fast in vivo screening models is underway, such as zebrafish and Drosophila models. By contrast, translation of apparent therapeutic successes from in vivo models of ALS to the human disease has proven difficult Citation[10], Citation[11]. A large number of successful pharmaceutical interventions in ALS mouse models have been reported, none of which could be prospectively translated into human therapeutics, and a considerable amount of financial resources has been apparently wasted.

Therefore, the European ALS/MND group decided in 2005 to initiate an international discussion on the principles of preclinical research and therapy development in ALS/MND. This was undertaken with the support of the North American ALS Therapy Development Foundation (ALS-TDF) and the personal commitment of Sean Forrester Scott and Fernando Vieira Citation[12]. A first document was developed during a European Neuromuscular Centre Conference in Naarden, The Netherlands, in 2006 and published in 2007 Citation[1]. In the meantime, one of the main driving forces behind the discussion, Sean F. Scott, had died of ALS. The group decided to organize a second conference to improve the guidelines, held in Reisensburg Castle, southern Germany, 11–13 June 2009. This second set of guidelines is the outcome of these discussions and is dedicated to the memory of Sean F. Scott. They may eventually result in standard operating procedures (SOPs) for preclinical animal research in ALS/MND within the next few years, but are not intended to weaken novel approaches or innovation by over-regulation. Accordingly, we have produced a set of guidelines, but no rules.

Preclinical studies: clinical needs

The development of preclinical models in ALS has raised great expectations in the ALS clinical community, also with patients and their families. However, the failure to translate the positive results of drug testing in animals into successful trials in humans has led to a loss of confidence in the relevance of these models for ALS patients. It is now clear that this failure may, at least in part, be related more to methodological pitfalls than to problems inherent in the models themselves. There is now a greater imperative towards improving the validity of animal models and the design of preclinical experimental therapeutic approaches. Moreover, it remains the case that preclinical studies are an important step towards increasing the probability of identifying drugs that can be effective also in the clinical setting.

The clinician who initiates a clinical trial, and who has no direct knowledge of the preclinical drug testing must be confident that the pre-clinical methodology is sound. In the clinical setting this has been obtained with the implementation of recommendations for report of clinical trials (the CONSORT guidelines), which are now accepted by the major clinical journals as a requirement for the publication of the trial (www.consort-statement.org).

It is important to easily identify whether an experimental study is a ‘proof of concept’ or a ‘preclinical drug testing’ trial. In the first case, the study may use a drug as an investigational tool, but its primary goal is to decipher a mechanism of the disease, without an immediate application to therapeutic approaches in humans. In the case of a preclinical drug testing study, the primary goal is to evaluate a drug for use in humans. While acknowledging the importance of proof of concept studies, the clinicians are primarily interested in assessing the preclinical drug testing studies. Therefore, we suggest the inclusion of a clear statement describing the type of research and its methodological foundation in both the abstract and in the body of the paper.

It is also important to know whether the experimental drug has been administered at the presymptomatic or at the symptomatic phase. It is clear that the treatment of mice after the onset of symptoms is more relevant for the clinical setting (‘disease modification’), even though treatment during the presymptomatic phase can give important information concerning the potential ‘preventive’ effects of the experimental drug.

If a study reveals a positive effect of the experimental drug on a biomarker, the procedure must be clearly described to determine its suitability as a possible surrogate measure for clinical studies. This could be the case with new neuroimaging techniques such as PET/CT and high-field MRI. We also suggest the use of biomarkers with a known direct relevance to the human disease course and its pathophysiology as a primary endpoint.

The strength of a new finding is related to the confirmation of the observation by an independent study. We propose that positive findings in experimental animals should be replicated by another laboratory. Most published studies report positive results Citation[11]. This is likely to be a function of publication bias. Non-positive (negative) studies are also important for understanding the real impact of a drug on the disease model and to determine pathogenesis of its specific pattern of damage. We encourage the publication of these non-positive preclinical drug-testing studies in specialized journals, as well as the meta-analysis of published studies.

Any paper reporting the results of a preclinical drug testing study should clearly indicate the strengths and weaknesses of the experimental design and should not inflate the relevance of the findings for the patient. Therefore, overstating the efficacy of a drug and the possible rapid translation to therapy in humans should be avoided, in particular when the experimental drug is available on the market. The consequences of such non-critical facile interpretations for patients and their families are potentially devastating, giving false hope, and may put them in danger of possible adverse events by uncontrolled (self) administration of the drug.

The reporting of conflict of interest is now mandatory in clinical research. We suggest that preclinical studies should also include a sentence regarding any possible conflict, including the sources of financing and whether the drug was provided by a pharmaceutical company.

From the clinical viewpoint, the following prerequisites for the conduct of a preclinical animal study with a therapeutic aim must be explained or – ideally – be present:

  • Recognition of the validity of the model: this relates to the ‘genetic validity’ of hereditary/familial form of the condition, and the importance of genetic background. For most studies that are likely to progress to a clinical phase, at least two species should be considered in the pre-clinical phase.

  • Validity of the characterization of such a model: comprehensiveness phenotyping should be undertaken.

  • Distinction between ‘proof of concept’ (to show that a pathogenetic mechanism is therapeutically relevant) and a ‘preclinical drug testing’ trial (which may include biomarker and dose-finding studies).

Table I.  Proposals to improve clinical understanding and impact of a preclinical study.

Methodological aspects of preclinical studies

Systematic review articles on preclinical drug testing in SOD animals Citation[1], Citation[10], Citation[11], Citation[13] clearly show the failure of translating preclinical research in effective therapies in patients. However, the problem is not only the discordance between animal and human studies, but there is also growing evidence showing the lack of reproducible effects in studies of one drug in the same animal model (Citation[13], Citation[15]). Therefore, there is an urgent need to standardize the protocols in selected animal models to improve the comparison of results obtained in different laboratories. Today, experience with experimental drug testing for ALS/MND is largely limited to the use of the high-copy G93A mice. Therefore, some of the recommendations are broader (not limited to the model) and some are based on the experience with this specific model. In future, the latter need to be adjusted for other or related models. gives the minimum requirements for any proof of concept or preclinical study and the additional requirements specific to preclinical studies (see also Ludolph et al., 2007). We strongly recommend that reviewers use this table as a checklist to assess studies.

Table II.  Table II gives the minimum requirements for any proof of concept or preclinical study and the additional requirements specific to preclinical studies. We suggest this table be used as a check list for both investigators and reviewers.

Those measures not previously outlined in detail (Ludolph et al., 2007) are discussed below.

Disease onset

Implementation of measures of onset varies widely between laboratories, with most using first signs of tremor and hind-limb splay defects or a reduction in rotarod performance. Frequent (e.g. twice weekly), blinded monitoring for onset is needed and clear criteria for defining onset determined prior to study commencement.

The reporting of onset data also varies with studies published without these data. We recommend that a robust measure of onset is developed by individual groups and that these onset data are always reported in publications, particularly where treatment was initiated prior to onset of disease. This is particularly important in view of the fact that the mechanisms driving preclinical and clinical phases of the disease may be different.

Disease progression

For any preclinical experiment the measurement of disease progression is critical for full assessment of the therapeutic effects of an agent. With respect to translation, therapies that extend survival without slowing progression may be less attractive than those that do both. Typically the measures most commonly used involve weight loss, grip strength, wire hanging, gait analysis and neurological scoring. Quantitative measures are preferable and since it is a simple measurement, weight change data are expected as an absolute minimum in all published studies.

End stage

Time to reach end stage of disease is the most consistently measured and reported parameter in the mutant SOD1 mouse models. Virtually all studies use loss of righting reflex with the time taken to assess this parameter varying from 10 to 30 s. Institutional or national ethics committees or regulations can dictate practice. We question whether there is any real difference between using 10 or 30 s as an endpoint – both are likely to give the same results. The focus on extension in survival may be misguided, particularly as little work has been conducted on the changes occurring during the rapidly progressive final few days of disease. Statistically significant effects on quantitative measures of disease progression or physiology may merit equal or greater weight.

Histology

The reporting of histology is critical in interpretation of positive data showing effects on survival or progression. Consideration must be given to the entire neuromuscular unit where previous studies have shown preferential effects on preservation of motor neuron number or neuromuscular junction integrity. Ideally, histological analysis of cell bodies, axons and neuromuscular junctions (NMJs) should be considered. At the very least a separate cohort of animals (n of at least five per group with each group representing the same proportion of each gender) to assess motor neuron numbers within a significant portion of the spinal cord should be conducted. Since motor neurons in the lumbar spinal cord are preferentially affected in mutant SOD1 mice, analysis of motor neuron survival must include an assessment of the number of motor neurons present throughout the lumbar region of the spinal cord. This will involve analysis of spinal cord sections between L3 and L5 region of the spinal cord. Strategies should be employed and thoroughly described to ensure that the same motor neurons are not counted twice in consecutive sections. For example, if the spinal cord is sectioned at 20 um, motor neuron counts should only be performed on every third section.

External validity and reproducibility

The concept of external validity expressed in the CONSORT statement for clinical studies should be applied to the interpretation of therapeutic studies in the mutant SOD1 mouse models. Data for the control group should be consistent with data from other studies for the same strain/transgene/copy number combination. There are examples in the literature where control group data for survival are inconsistent with the experience of most groups running these models Citation[14], Citation[15].

Equally, experimental variability across all measures should be considered within research groups and publication of historical analyses of control group data showing variability in measured outcomes is to be encouraged. Such data could form the basis for justification of a particular testing paradigm and selection of the ‘n’ for studies from that group.

Selection of numbers and statistical analysis

The previous guidelines (Ludolph et al., 2007) recommended 48 mice in a single preclinical study (12 males and females per group). This was based on analysis of survival and potential confounding factors in the SJLB6 G93A-SOD1 model at the ALS Therapy Development Institute (ALSTDI) (Scott et al., 2008). While this recommendation holds for this model and endpoint, we recognize that other models and read-outs may justify alternative n numbers based on power calculations. As an absolute minimum for any study of survival (time to loss of righting reflex), 12 animals of a single sex must be used unless justified by power analysis with data shown in supplemental figures or tables.

Additional requirements for preclinical studies

In order to make claims about the possible therapeutic utility of a treatment, further data are required as detailed in . Of particular importance is replication of the therapeutic effect in an alternative mammalian model or in a different laboratory. Post-symptom onset studies are also strongly recommended and efficacy in these paradigms would greatly enhance confidence.

Exposure (pharmacokinetic) and drug action (pharmacodynamic) read-outs

A critical aspect in relation to 1) confirming the mechanism of action of the therapeutic agent, and 2) translating results to the clinic, is the development of assays for measuring drug action in the target tissue.

Such assays should have the potential to be applied in human subjects and should ideally use easily accessible peripheral tissues. Linking efficacy in the preclinical model to these markers of drug action provides an initial target for clinical studies. At the very least, the target tissue concentrations of the therapeutic agent should be determined and correlated with peripheral (plasma) levels. This will inform initial pharmacokinetic studies in humans. Such data are particularly valuable if a dose-response relationship between drug level (or action) and therapeutic effect is developed. Such assays can also accelerate development of related drugs, particularly if they become validated in the clinic.

Additional quantitative measures of disease progression and physiology

To further define the therapeutic action, additional measures are recommended. Of particular interest are measures that can be applied in the clinical setting such as MRI and estimates of motor unit number. In addition, further comprehensive histopathology, gene profiling and proteomic analysis may give greater understanding of the therapeutic effect and also inform the development of pharmacodynamic read-outs as well as preclinical biomarkers as described above.

Other recommendations include the following:

  • the development of functional scales for rodents, in particular for motor assessment (Eumorphia Project)

  • considerations regarding the use of inbred versus outbred strains

New models (rodent and non-rodent)

SOD1 rodent models

There is a clear need to identify a cheap and convenient screening tool that could identify drugs that are promising for rodent studies. Most researchers strongly suspect that the lack of success, including translational efficacy, of preclinical studies in ALS/MND is due to the use of one single rodent model, the transgenic SOD model carrying a high number of G93A mutations Citation[16]. Although its use remains justified as results are rapidly achieved, there are some major concerns:

  • the extent of the toxic effect of the overexpressed gene, which might make the pathogenesis different from the one found in humans carrying only one copy

  • the fact that the pathogenesis of SOD mutation carriers does not represent one important signature of the disease, i.e. TDP-43 accumulation Citation[17]

Four recommendations are made regarding rodent models:
  1. Comparative use of transgenic mice carrying SOD mutations other than the G93A

  2. Comparative use of mice which carry mutations that produce toxicity to a lesser extent than the high-copy G93A mouse. This includes the low-copy G93A animals Citation[18].

  3. Development of a high-copy G59S transgenic dynactin mouse to compare it with other models Citation[9]

  4. Development of transgenic animals carrying Fus and TDP-43 mutations and comparison of pathogenesis with SOD models Citation[17]

Other rodent models

The current rat models overexpressing SOD mutations show variability with respect to site of onset and individual life span, independently from the levels of mutant SOD. This recapitulates the variable phenotype of the human disease. The larger size is advantageous for the availability of tissues to be analyzed for biomarkers. On the other hand, the size of the animals and their variable phenotype represent a disadvantage for drug-testing trials, with regard to both space and costs.

The wobbler mouse displays TDP-43 neuropathology and could be used to dissect the pathogenetic principles specific for the death of motor neurons. However, as cervical motor neurons are selectively affected and systemic developmental abnormalities are present, the reliability of this model for preclinical drug trials remains to be established.

The current SMA (spinal muscular atrophy) animal models are not currently useful for drug development; although their aetiological principles seem to be close to the TDP-43 and Fus mutations. The development of biomarkers specific and representative of a drug effect should be part of each animal study to improve translation from mice to man.

New screening tools

The group also saw the need for new and innovative drug screening tools: major advances have been made in the use of zebrafish (which will be discussed below); unfortunately, a reliable Drosophila model is still not available.

The use of the Zebrafish, Danio rerio, in biomedical research is rapidly increasing, because of its major advantages. It produces 250 offspring per week, making large numbers of animals available, its embryonic development is external, rendering it amenable to genetic manipulation and both overexpression (injection of RNA) and knockdown (through injection of a morpholino, a stabilized antisense oligonucleotide) of genes in the embryo are possible. The embryo is transparent during the first days of development, making visual inspection easy and, in addition, transgenic fish expressing fluorescent dyes in a tissue-specific way have been raised, visualizing certain organs directly using fluorescence microscopy. Finally, large libraries of morpholinos are available that can be used for genetic screening.

In developmental studies, the zebrafish embryo has been used extensively, mostly through the study of the effect of morpholinos to knockdown gene expression during the initial stages of development. Disease models in zebrafish have been generated only recently. Huntington's disease, spinomuscular atrophy and amyotrophic lateral sclerosis are some of these. Although most of the models have been generated by transient overexpression or knockdown of disease causing genes, truly transgenic fish are now being generated.

In the field of ALS (with or without FTD), zebrafish research is at an early stage. Models based on mutant SOD1-induced axonal abnormalities, alsin knockdown and progranulin knockdown have been reported and a tauopathy model that can be used for drug screening is available. Several research groups are working on TDP-43 and Fus/TLS models.

These models have advantages and limitations. Disease models in zebrafish (embryos) are useful for experiments that are not possible in other animals. Genetic screening of a morpholino library, compound screening and the study of the interaction of multiple gene products, all difficult or expensive in rodent models, are feasible in zebrafish. It is obvious that the results obtained in zebrafish need confirmation in larger animal models.

Table III.  Recommendations for the use of animals.

Pharmacology and toxicology

The following recommendations mainly concern studies for ‘preclinical drug testing’ (in contrast to ‘proof of concept’ studies).

If a given species (e.g. mice and rats, dogs or non-human primates) is used for preclinical drug testing and an attempt is made by an academic laboratory to use this drug for a clinical trial in humans, the following pharmacological and toxicological testing is necessary before or after testing in experimental animals is carried out.

If nothing is known about pharmacokinetics and toxicology of the drug, then, in a first step, qualitative understanding of the drug metabolism of the compound is mandatory. In most cases PK studies will not be performed by an academic laboratory but rather by an industrial counterpart. The group feels that in the year 2009 the necessary know-how is present in the setting of industry, less so in academia. ADME (absorption, distribution, metabolism, and excretion) testing should be performed in the relevant species. If unknown, analysis of pharmacokinetics may also include tissue distribution (such as the central nervous system). Further quantitative assessment of drug metabolism and distribution may be necessary to explain drug effects in experimental animals and facilitate dose-finding in mice and man. At later stages of development, PK parameters of the effective dose in experimental animals should be sought in humans.

In addition to determining PK data, preliminary toxicological parameters should be tested. Again, this should in many cases be performed by an experienced industrial laboratory. The NOAEL (no adverse effect level) and MTD (maximal tolerated dose) should be compared to the dose in which efficacy was obtained.

The choice of the route of application of a given drug may depend on its absorption from the gastrointestinal tract, its hepatic metabolism and its ability to cross the blood-brain barrier.

If oral administration via food or drinking water is chosen, then measurements of daily intake are necessary (including weekends). To ensure reliable dosing of a critical compound (e.g. with a taste not preferred by the animal), oral gavage is advisable. Often this procedure needs to be justified for an application to an animal care committee; however, we strongly recommend to researchers to initiate and conduct these discussions forcibly, if necessary, in order to prevent animal studies that are not useful.

For the choice of the route of administration, in general the preference for i.p., s.c., or oral application or even by Alzet pumps is dependent upon the pharmacological and pharmacokinetic properties of the drug. Pharmacokinetic data are also necessary if a drug is invasively administered. For example, intraventricular application does not guarantee sufficient spinal cord concentrations.

In a ‘proof of principle study’ a single dose may be sufficient; however, (hepatic) metabolism, route of application and – if uncertain – tissue distribution must be considered for a meaningful study. This can be done in advance or after efficacy data are obtained. If these studies are not performed, a meaningful positive result may be missed and these limitations of a study must be thoroughly explained and discussed. In all other studies, dose-response curves must be obtained. It is often useful to demonstrate a biological effect on a disease marker to facilitate translation of a result from animals to man.

Table IV.  Recommendations for pharmacological and toxicological studies in a preclinical drug testing study. We recommend that most of the information will be obtained by industrial partners.

How can new technologies be integrated into guidelines for preclinical work in ALS?

In general, preclinical research focusing on SOD animals should adhere to all considerations for a better standardization of studies as outlined above. By this approach, feasibility, resources, and time should be optimized. A better standardization would have the following advantages:

  • Facilitate and improve non-hypothesis driven and unbiased research by using comprehensive screening programs and automated analysis

  • Improve throughput in rodents (for example, by continuous parallel recordings in multiple cages), but also other species

  • Improve sensitivity. This can be done by extracting minimal essential read-outs derived from large scale screening programs.

  • Improve interlaboratory comparisons to obtain higher reliability

  • Permit multivariate datamining

The organization and integration of such approaches is often undertaken by a not-for-profit organization or multinational research collaboration. This has been successful in other conditions including Huntington's disease and muscular dystrophy. In the field of ALS, the ALS-TDF supports the research community ().

Table V.  Summary of new technologies providing a high likelihood of fulfilling these needs.

Summary and conclusions

The need for improvement in preclinical animal research to facilitate drug development for neurodegenerative diseases is accepted and the challenges have been identified Citation[12]. The field of ALS/MND has the advantage of having one of the first reliable and reproducible animal models (Gurney et al., 1994) for a disease in which therapeutic advances are in the early stages Citation[19]. Consequently, the field has experienced foreseeable and unforeseeable challenges that, however, can be resolved in a stepwise fashion. The goal is a better predictability and translatability of preclinical research results to humans; the general approach is based on both comprehensiveness and caution and consequent improvement and implementation of research methodology. During the meeting, the group was certain that this goal can be achieved; we wish to point out that – in the entire framework – the development of biomarkers in models (mice) and man is a central need. A second critical need – the publication of negative studies – has been accepted as editorial policy by the ALS Journal. The danger that rigorous standardization may have the disadvantage of discouraging innovation is acknowledged. However, the ultimate goal is to develop robust criteria and guidelines for preclinical animal studies to justify investments into a clinical drug study. This in turn will help to develop therapeutics for diseases of central epidemiological importance in our societies.

Acknowledgements

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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