SESSION 6A USE OF THE G93A SOD1 MOUSE IN THERAPEUTIC TESTING

C33 THE SOD1 MOUSE MODEL OF ALS?

GREENSMITH L

Institute of Neurology, University College London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: G93A mice, SOD1, model

For the past 10 years or more, the SOD1 mouse model has been one of the most important research tools for scientists working in the field of ALS. However, in recent months the validity of this mouse model has been widely questioned. This concern stems primarily from the failure of several successful preclinical trials in the SOD1 mouse to translate into effective therapies for human ALS. The most recent example of this failure was the publication in Lancet Neurology of negative results from a large phase III randomised trial of minocycline in ALS patients [1]. This trial was initiated by positive findings that show that minocycline delays disease progression in the SOD1 mouse and the results of the trial led the authors to conclude that “either the current approach to translational neuroscience is unsatisfactory or the transgenic mouse model is a poor representation of sporadic ALS”. Thus, opinion now varies as to whether the SOD1 mouse is simply a model of motoneuron degeneration or specifically SOD1-ALS rather than sporadic ALS.

In January 2008, a paper from Sean Scott [2] and colleagues added to the growing unease about the SOD1 mouse. These authors presented data indicating that most of the preclinical trials that have been published to date in the SOD1 mouse are likely to be a measure of noise in the distribution of survival means in this model rather than actual drug effect. Thus, for example, genetic variation (gene copy number) will be a significant determinant of lifespan.

However, before we discard the SOD1 mouse as a good model for ALS, we must ask ourselves whether the "problem" with the SOD1 mouse lies more in how we have used this model than in the model itself? For example, it is clear that human and mouse trials are performed very differently. Perhaps one of the most significant differences is that most preclinical trials have been based on presymptomatic treatment of mice. Is it really surprising therefore, that such trails may never be predictive of the outcome of treatment of human ALS patients, who are clearly well beyond symptom onset when they are enrolled into clinical trials? In addition, it is clear that we also need to improve our understanding of mouse pharmacokinetics and how similar they are to humans, in order to determine appropriate doses for use in human trials.

Although we look forward to the development of new models of ALS, it is clear that it will take several years for us to become as familiar with the pros and cons of these new models as has been the case for the SOD1 mouse. With this in mind it is likely that SOD1 mice will continue to be used to screen new ALS drugs. The need is therefore to use the SOD1 model as effectively as possible, bearing in mind the variables that we now know are associated with both the model as well as the researchers that work with it! The process towards standardization has been started by a consortium of European ALS researchers with the publication of a set of guidelines for preclinical testing of potential therapeutic agents in the SOD1 mouse [3]. These guidelines attempt to address the issues of genetic background, pre-and post-symptomatic treatment regimes, as well as the need for dose-response curves and pharmacokinetic studies.

The emphasis should now therefore lie with implementing these guidelines to ensure that we produce results that are robust and use the SOD1 mouse to its full potential, whilst bearing in mind its shortcomings. The responsibility to get this right lies with all of us involved in the ALS research community, not only the scientists, but the funding agencies and journal reviewers and editors.

C34 DESIGN, POWER, AND INTERPRETATION OF STUDIES IN THE STANDARD MURINE MODEL OF ALS

SCOTT S, RAMASUBBU J, BOSTROM A, LINCECUM J, KRANZ J, COLE J, VIEIRA F, THOMPSON K, THEODOSS J, AL-NAKHALA B, KELLY N

ALS Therapy Development Institute, Cambridge, MA, United States

E-mail address for correspondence: [email protected]

Keywords: G93A mice, pre-clinical, SOD1 FALS

Identification of SOD1 as the mutated protein in a significant subset of familial amyotrophic lateral sclerosis (FALS) cases has led to the generation of transgenic rodent models of autosomal dominant SOD1 FALS. Mice carrying 23 copies of the human SOD1G93A transgene are considered the standard model for FALS and ALS therapeutic studies. To date, there have been at least 50 publications describing therapeutic agents that extend the lifespan of this mouse. However, no therapeutic agent besides riluzole has shown corresponding clinical efficacy. We used computer modeling and statistical analysis of 5429 SOD1G93A mice from our efficacy studies to quantify the impact of several critical confounding biological variables that must be appreciated and should be controlled for when designing and interpreting efficacy studies. Having identified the most critical of these biological variables, we subsequently instituted parameters for optimal study design in the SOD1G93A mouse model. We retested several compounds reported in major animal studies (minocycline, creatine, celecoxib, sodium phenylbutyrate, ceftriaxone, WHI-P131, thalidomide, and riluzole) using this optimal study design and found no survival benefit in the SOD1G93A mouse for any compounds (including riluzole) administered by their previously reported routes and doses. The presence of these uncontrolled confounding variables in the screening system, and the failure of these several drugs to demonstrate efficacy in adequately designed and powered repeat studies, leads us to conclude that the majority of published effects are most likely measurements of noise in the distribution of survival means as opposed to actual drug effect. We recommend a minimum study design for this mouse model to best address and manage this inherent noise and to facilitate more significant and reproducible results among all laboratories employing the SOD1G93A mouse.

C35 REFINEMENT OF MOUSE MODELS OF MOTOR NEURONE DISEASE AND GENERATION OF NOVEL READOUTS FOR THERAPEUTIC ASSESSMENT.

MEAD R, KASHER P, COX L, GRIERSON A, SHAW P

University of Sheffield, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: method, mouse, models

Background: Motor neurone disease (MND) is a devastating neurodegenerative disorder where loss of upper and lower motor neurones leads to gradual loss of motor function and death typically within three years. Mutations in SOD1 are responsible for a significant proportion of inherited MND cases. Mouse models exist which rely on the systemic transgenic expression of mutant human SOD1 (G93A SOD1). These mice show stereotypical loss of motor neurones leading to mild tremor and gait abnormalities obvious from about 75 days of age, progressing to significant paralysis, with humane intervention at about 140 days of age (‘survival’ time). Testing of new therapeutic approaches in this model is time-consuming and resource intensive and carries a significant burden of disease and distress for the mice, as studies are typically designed to measure extension in survival time.

Objectives: The primary aim was to describe the disease course in a pure genetic background, as opposed to the standard mixed genetic background and validate our standardised protocol for therapeutic testing. The second aim was to determine whether early behavioural or biochemical readouts could be identified and used as a rapid ‘pharmacodynamic’ readout for the efficacy of new therapeutic agents.

Methods: Multiple trials 6 were conducted in the C57Bl/6 G93A mouse model using standard methods of measuring disease progression (rotarod scoring, neurological state scoring, weight) over a period of two and a half years. At the outset we applied principles of good experimental design based on ‘The Design of Animal Experiments’. In addition, systems for measuring mitochondrial function and oxidative stress in situ in spinal motor neurones were investigated.

Results: Analysis of this large data set lead to two main conclusions. Firstly, the disease course is remarkably consistent and significantly less variable in the pure genetic background. Power analysis indicated that the reduced variability would reduce n numbers by at least 60% when looking at effects on disease onset or survival time. Secondly, motor deficits are detectable very early and correlate with published patterns of synaptic remodelling at motor end plates. We identified early changes in motor function at about 40 days of age, long before classical onset of disease.

Discussion and Conclusions: This finding supports recent data indicating that the neuromuscular junction is lost first, followed by dying back of the axon itself. Our methods to quantify these early changes may enable early assessment of the neuroprotective potential of new therapeutic agents, increasing throughput and reducing the distress burden for mice on pharmacological studies.

C36 IDENTIFICATION OF THERAPEUTIC COCKTAILS FOR ALS BASED ON: COMBINATIONS OF FDA-APPROVED DRUGS, NUTRACEUTICALS AND METABOLIC PRECURSORS

CROW J, REED R, JOHNSON J, BENNETT P

University of Arkansas Medical School, Little Rock, AR, United States

E-mail address for correspondence: [email protected]

Keywords: symptomatic administration, G93A mice, drug cocktail

Background: Many existing therapeutic agents have shown significant survival effects in G93A mice when given at symptom onset – the more relevant approach for extrapolation to human ALS patients. These agents are only now reaching the stage of human clinical trials, and the outcome of these trials will provide much more compelling evidence for the predictive value (or lack thereof) of the G93A mouse in terms of therapeutics testing. However, even if the survival and motor function preservation benefits extrapolate directly to humans, no agent to date has shown the promise of totally arresting disease progression.

Objectives: To systematically test combinations of FDA-approved drugs and nutraceuticals that each extend survival individually when given at onset, to identify a drug “cocktail” which dramatically slows or halts disease progression. The use of approved drugs, and agents derived from foodstuffs (generally regarded as safe), will allow any such combination to be immediately taken to the clinic for small scale human trials. Candidate ALS patients include those who, for a variety of reasons, are unlikely to enroll in clinical trials involving experimental agents.

Methods: G93A SOD1 overexpressing mice, bred and maintained in-house, are tested in groups of 10, relative to vehicle controls. Simultaneous breeding of 50–55 non-transgenic females with littermate transgenic (Tg) males typically yields approximately 220 age-matched Tg mice. Treatment and control groups are randomized to give equal numbers of males/females per group, and treatment is begun at onset (∼ 90 days of age). Established (published) criteria are used to determine the time to sacrifice, and the investigator making the determination is blinded to the treatment status. Survival results are expressed as a ratio of survival after onset, relative to vehicle controls from the same group of pooled littermates, tested simultaneously.

Results: Thus far, over 80 different agents and combinations (2 – 5 agents) have been tested. A number of new therapeutic agents and chemical classes have been identified, and at least three combinations have demonstrated additive effects far exceeding those seen with any individual agent. Most combinations to date have produced results comparable to individual agents, while some have shown sub-additive effects. Overall, the results have tended to validate the proof-of-concept, and have yielded one combination (four agents) of such magnitude to warrant consideration of a small human trial.

Discussion: Combination drug therapy has been a mainstay of cancer and HIV chemotherapy for many years, and is becoming more common for the treatment of dyslipidemias and microbial infections.Until more is known about the molecular pathogenesis of ALS, the best hope for an effective treatment in the short-term is via the use of combinations of agents which are already approved or regarded as safe based on years of human consumption without ill effects.