P1 ANALYSIS OF MOTOR SKILL ACQUISITION AMONG CHILDREN WITH TYPE I SPINAL MUSCULAR ATROPHY SUBMITTED TO MEDICATION WITH VALPROIC ACID
CONCEIÇÃO E1, SILVA T2, UMBERTINA R1, MARIA JOAQUINA D1
1Universidade de São Paulo, São Paulo , Brazil, 2Universidade Federal de São Paulo, São Paulo, Brazil
E-mail address for correspondence: [email protected]
Keywords: Type I Spinal Muscular Atrophy, valproic acid, motor acquisition
Background: Spinal Muscular Atrophy (SMA) is the most frequent of the neuromuscular diseases responsible for hypotonia (floppy infant syndrome) caused by the degeneration of cells in the anterior horn of the spinal cord. Symptoms consist of progressive, symmetrical motor deficit of the affected limbs and the condition sometimes affects the musculature innervated by the cranial nerves. Valproic acid has proven effective in avoiding the deletion of exon 7, which is thought to prolong the survival of neurons. Based on this new treatment possibility, a physical-functional assessment protocol was developed for monitoring this group of patients.
Objective: The aim of the present study was to analyze and describe possible motor acquisitions among patients with SMA-I submitted to a medication with valproic acid and assess whether observational analysis using filming is a resource of easy application and adequate reproducibility as a form of evaluating and following up such patients.
Methods: Between November 2005 and December 2007, five infants with a confirmed diagnosis of SMA-I between four and 18 months of age were evaluated. The inclusion criteria were SMA-1 (definition based on the European Neuromuscular Centre Workshop), difficulty breathing on the first evaluation and no need for assisted ventilation. Data collection was carried out following a previously structured protocol involving the determination of motor acquisition using filming with the patient in dorsal decubitus.
Results: Fifteen patients with a confirmed diagnosis of SMA-I submitted to treatment with valproic acid between November 2005 and December 2007 were evaluated. Three patients died; six became dependent on mechanical ventilation during the evaluation period, with the evaluations and medication subsequently interrupted; and one patient did not undergo the proposed physiotherapy because the parents were not available to participate. At the end of the data collection, only patients who underwent the five evaluation sessions were considered eligible; thus, the results analyzed were on only five patients, all of whom were Caucasian. The results demonstrated that none of the children exhibited a worsening of the motor skills, which would be expected in the natural progression of the disease.
Conclusion: Based on the results of the present study, valproic acid may be considered a medication that assists in the maintenance of motor skills as well as new acquisitions, as none of the children who participated in the study exhibited any worsening in motor ability, which would be expected in the natural progression of the disease.
P2 UNPROVED CELLULAR TREATMENTS FOR ALS PATIENTS: AN OBSERVATIONAL STUDY
GAMEZ J
ALS Unit, Neurology Department, Hospital Universitari Vall d'Hebron, Barcelona, Spain, Autonomous University of Barcelona, Barcelona, Spain
E-mail address for correspondence: [email protected]
Keywords: stem cells, ensheathing cells, cellular therapy
Background: No curative therapy for ALS is yet available, and the drug treatment used has an almost imperceptible effect on the disease's clinical course. Cellular therapies have recently become a promising strategic approach in the treatment of neurodegenerative diseases, as they may replace dysfunctional or dying neurons. Cell transplantation may provide other benefits, including neuronal growth factors for the host cells, stimulation of axonal growth or glial function, secretion of neurotransmitters deficient in the host, differentiation into oligodendrocytes and myelination of host axons and differentiation into neurons. Many patients are using the Internet to seek these innovative procedures at clinics offering a variety of cellular treatments for ALS before their effectiveness and safety is confirmed. The increasing number of clinics offering ‘cure-all’ therapies for financial gain, taking advantage of patients’ understandable desperation to find a cure, has prompted considerable ‘stem cell tourism’.
Objectives: To analyze the effectiveness of cellular therapy (TX) in a series of 12 Spanish ALS patients undergoing cytotherapy in clinics found on the Internet on their own account.
Methods: Twelve ALS patients with a mean age of 48.6 years old (SD 12.8) received cytotherapy 26.9 months (SD 15.8) after clinical onset. ALSFRS-R and FVC at TX were 32.3 (SD 6.8) and 63.4% (SD 15.3) respectively. TX involved transplants of olfactory ensheathing cells in three patients, and autologous mesenchymal stem cells in the remainder.
Results: One patient died 33 months post-TX, after surviving for 49 months. Five required mechanical non-invasive home ventilation 7.4 months post-TX. Two required invasive ventilation (13.0 months post-TX). Five patients needed gastrostomy feeding (23.3 months post-TX). Survival between clinical onset and the study end date was 50.0 months (SD 17.2). No significant adverse events or changes in the decline of FVC and ALSFRS-R compared to the disease's natural history were observed.
Discussion: The ever-increasing availability of stem cells in many countries, often offered by “for profit” clinics, together with straightforward laws of supply and demand, raises ethical implications. These clinics provide therapies that are not proven which may cause serious psychological and economic distress to the patients concerned, and endanger the legitimate progress made by scientists in the stem cells field.
Conclusions: Our observations and those of other groups suggest that cell therapy in ALS is not yet sufficiently effective in curing or halting the disease, and its presumed ability to slow down the disease's progress is not yet proven. Cytotherapy cannot yet be considered a curative treatment for ALS. Patients’ associations and experts treating the disease must strongly dissuade their patients from undertaking the worrying new type of medical tourism arising from their desperate search for effective treatment.
Acknowledgements: JG was supported by a Spanish Fondo de Investigaciones Sanitarias grant (IP 10/01070).
P3 STRENGTHS AND WEAKNESS OF THE HISTORICAL CONTROL DESIGN FOR SCREENING TRIALS IN ALS: AN ANALYSIS OF THE WALS MULTICENTER LITHIUM TRIAL
KATZ J, MILLER R, MOORE D
California Pacific Med Center, San Francisco, CA, United States
E-mail address for correspondence: [email protected]
Keywords: clinical trials, historical controls, clinical progression
Background: The WALS group recently completed a multicenter study of Lithium. Because of circumstances related to the positive Lithium study from Italy, we used Minocycline historical placebo controls (MP) and compared them with actively treated Lithium patients. Our work was supplemented by additional data from 748 placebos from 7 earlier trials. Our methodology maximized efficiency, acknowledged Lithium was available by prescription, and avoided ethical conflicts in case the agent turned out effective. The year-long trial enrolled 109 patients, and concluded Lithium was not a candidate for further development.
Now, we wish to focus specifically on our methodology to discuss implications for future trials. Since our trial is complete, we have a unique opportunity to study biases and efficiency tradeoffs, especially since other trials were performed simultaneously.
Methods: We tested four rules regarding the validity of historically controlled trials: 1) There should be no drift in the primary endpoint over time. We compared the change in ALSFRS-R of placebo groups from different epochs; 2) There should be no differences in types of patients that enrolled in the separate trials. This might relate to the open design, or to variations in timing or screening strategies. Here, we compare baseline demographics between this study and our historic control group; 3) No factors should lead to variations in drop outs that could secondarily bias conclusions. We compare dropout rates in our cohort and the historical control group; 4) Simultaneous trials should reach similar conclusions. This is a way to check for other potential biases such as a lack of blinding. We discuss two other Lithium trials, one using randomization and blinding, done concurrently to ours.
Results: 1) There is no change in the 6-month FRS slopes between 1997 and 2007, with mean FRS declines of near 1.0/mo. for 7 trials; 2) There were no differences in baseline features (Lithium, MP): FVC (94.7, 94.3); FRS-R (37.1, 37.9); months since disease onset (18.5, 16.8); age, gender, bulbar involvement or riluzole use; 3) Dropouts due to death or other causes were 19% for MP and were 18% for Lithium; 4) Two concurrent trials reported Lithium was ineffective, one with concurrent controls. All found trends suggesting treated subjects fared worse than controls and clearly refuted the original positive study. All found similar trends in secondary outcome measures (FVC slopes).
Conclusions: There were no clear differences in types of patients enrolled or rates of progression between distinct trials. This lends credibility to the historical-control design for screening new agents. As long as these trials are performed under conditions that parallel earlier work, the risk of introducing bias seems small and given the savings in cost and acceptance, the trade-off may be of benefit to the field.
P4 LESSONS LEARNED FROM LITHIUM TRIALS IN ALS
MILLER RG, KATZ J, MOORE D
California Pacific Medical Center, San Francisco, CA, United States
E-mail address for correspondence: [email protected]
Keywords: clinical trials, trial design, therapy
Background: In 2008, Fornai et al, reported in PNAS that lithium dramatically slowed disease progress in both a mouse model and a small trial with 16 subjects with ALS (1). Supported by scientific background related to autophagy, the trial generated an unprecedented reaction with many patients taking lithium and a groundswell of activity to test the agent further. Eventually, over 700 patients were enrolled in eight clinical trials organized in five countries, using a variety of methods to re-test the result.
These events give us a unique opportunity to inspect different approaches for screening compounds and to examine the most effective ways to utilize resources.
Methods and objective: We evaluated designs (inclusion criteria, sample size, treatment duration, outcome measures, controls) and findings (efficacy and safety) of these trials with two specific goals: 1) to determine which were most efficient as screening trials; and 2) to learn how to develop a more optimal response for new agents.
Results: There were four open-label case series (2–5) (three US, one French), one dose ranging study (6) (Italy) and three placebo-controlled trials (7–9) (US/Canada, ongoing UK, ongoing Italian). The trials differed in sample size from 27 to 220.
All but the English (8) and Italian (9) trials are complete and all except the initial small trial (1) have reported negative results. None were terminated based on reports of an earlier trial being negative. All four case series reported frequent adverse events with one stopped for safety (2–5). Two compared outcomes to historical values and concluded that lithium was not effective (4,6). One placebo controlled trial using a novel ‘time-to-event’ endpoint was terminated at the first interim analysis due to futility (7). The three completed controlled trials found no therapeutic benefit from lithium (4,5,7). Two randomized controlled trials are ongoing (8,9).
Conclusions: Despite differences, these trials have thus far, consistently refuted the initial Italian study. This suggests a variety of designs might be practical, especially when the goal is to screen for phase III or test the possibility of a very large beneficial effect. The findings point to the need for a more efficient overall approach to screening since the current atmosphere sets the stage for committing too many resources to a single agent. These lithium trials lend a considerable opportunity to ask why any given evidence leads to such a large number of trials and how to organize research across borders.
References and principal investigators:
- Fornai 2008.
- Barohn 2009.
- Andrews 2010.
- Miller 2010.
- Camu 2010.
- Chio 2010.
- Aggarwal 2010.
- Leigh 2010.
- Silani 2010.
P5 AUTOMATED DATA CHECKS PROCESSES: IMPROVING DATA QUALITY IN CLINICAL TRIALS AND BIOMARKER STUDIES
CHAN M, SINANI E, SHERMAN A, YU H, CUDKOWICZ M
Massachusetts General Hospital, Charlestown, MA, United States
E-mail address for correspondence: [email protected]
Keywords: data management, neurology clinical trials unit, EDC
Introduction: The Neurology Clinical Trials Unit at Massachusetts General Hospital serves as a data management center for multi-site clinical trials and biomarker studies. It utilizes PharmaENGINE™ software platform for data capture and data management. Clinical Data Management is the process of ensuring that data collected during the course of a clinical trial are accurate, complete, logical and consistent. The amount of data accumulated during a clinical trial may be significant and depends on a trial's duration, the number of subjects, number of sites and number of procedures performed. Manual data validation in most cases is not a feasible alternative as it is an inefficient process that most likely results in invalid data and, as a result, incorrect analysis and results interpretation. An automated data checks functionality built into PharmaENGINE™ is used in both data entry and data verification to detect incorrect or incomplete data.
Methods: The PharmaENGINE™ system has been designed to check for data errors and data consistency through two methods: automated queries at the point of entry and logic checks.
Automated Queries detects data discrepancies within a form such as missing fields, out-of-window visits, out-of-range values, and unknown values with no comments associated with them.
Logic checks functionality that is built into the system allows detection of data discrepancies within a form or across multiple forms and visits. The module allows entering SQL based scripts that will check the data, specify the field for which an automatic query will be created if the script returns positive results, and provide the query message.
A query is automatically created in a field or a form that has a discrepancy. The trial personnel is able to check for any outstanding queries.
Results: In early 2010, the Logic Checks functionality was introduced for the Clinical Trial of Ceftriaxone in subjects with amyotrophic lateral sclerosis. This multi-center trial has 58 participating sites and a target enrollment of 600 subjects. There were a total of 2216 queries opened in the month of February alone; 45% of these queries were created by the automated queries and logic checks combined.
Conclusions: Utilizing Automated Queries and Logic Checks is a must for any data management effort in a multi-site clinical trial. The alternative is time-consuming and costly, not only in human resources utilization, but also from a high risk of missing important data entry errors that may lead to invalid results and their interpretations. Running Logic Checks takes minutes versus weeks and months for the manual alternative. Hence, these tools should be viewed as an important asset in service of data management professionals.
P6 EFFECTS OF TREATMENT WITH DEXPRAMIPEXOLE ON ALSFRS-R SUB-DOMAIN SCORES AND CROSS-VALIDATION OF CHANGE IN SUB-DOMAIN SCORES WITH PREVIOUSLY PUBLISHED DATA
INGERSOLL E1, CUDKOWICZ M2, MITSUMOTO H3, MATHER J1, ARCHIBALD D1, GRIBKOFF V1, BOZIK M1
1Knopp Neurosciences Inc., Pittsburgh, PA, United States, 2Massachusetts General Hospital, Boston, MA, United States, 3Columbia University, New York, NY, United States
E-mail address for correspondence: [email protected]
Keywords: dexpramipexole, clinical trial, ALSFRS-R
Background and objectives: Dexpramipexole (KNS-760704; [6R]-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine), a compound that may be cytoprotective by ameliorating mitochondrial dysfunction, is being developed to treat ALS. The objective of this study was to evaluate changes in the 4 sub-domains of the ALS Functional Rating Scale, Revised (ALSFRS-R) in Study KNS-760704-CL201 (CL201) for treatment effects and, within the placebo group, for concordance with previously published patterns of sub-domain dynamics (1). A treatment effect in an ALS clinical trial would be most likely observed within sub-domains that exhibit the greatest change in the reference group over the treatment period.
Methods: Results of safety and clinical effects in study CL201 have been reported previously (2). This was a randomized, multicenter, double-blind, placebo-controlled study in ALS subjects. A total of 102 subjects were randomized to receive 50, 150, or 300 mg/day dexpramipexole or placebo for 12 weeks. Clinical effects were, in part, measured by the ALSFRS-R, which has 4 sub-domains (fine motor, gross motor, bulbar, and respiratory), each assessed according to 3 questions directed to specific activities of daily living. Cedarbaum and colleagues previously showed that the greatest decline in any of the ALSFRS-R sub-domains at 3 months was in the fine motor sub-domain (estimated as −0.9 absolute/40% of total score change), followed, in order, by the gross motor (−0.65/29%), bulbar (−0.4/18%) and respiratory (0.3/13%) sub-domains. In study CL201, the patterns of change in the ALSFRS-R sub-domains were compared for treatment differences, and the placebo group was compared with those reported by Cedarbaum et al over a comparable time period.
Results: The patterns of change in ALSFRS-R sub-domain scores observed by Cedarbaum et al over a 3 month period were replicated in the placebo cohort of this study, with the greatest mean decline observed in the fine motor sub-domain (−1.4/38% of total score change), followed by gross motor (−0.9/24%), bulbar (−0.8/22%), and respiratory (−0.6/16%) sub-domains. In the 300 mg dexpramipexole group, mean changes were −0.6 (fine), −0.9 (gross), −0.3 (bulbar), and −0.5 (respiratory). The largest magnitude nominal treatment effect was in the fine motor domain.
Discussion and conclusions: The concordance between the placebo cohort in this study and the population studied by Cedarbaum et al provides external validation for the ALSFRS-R as a measure of functional decline in a randomized clinical trial. The greater magnitude of decline observed in CL201 may be a function of differences in inclusion criteria, but the pattern and relative percentages of decline were similar across the sub-domains. The sub-domain with the greatest decline over 12 weeks in the placebo group was fine motor, in which domain the largest magnitude treatment effect with 300 mg dexpramipexole was also seen.
References:
- Cedarbaum, et al. J Neurol Sci 1999;169:13–21.
- Bozik, et al. ALS (Supplement 1) 2009;10:28–29.
P7 MECHANISMS OF NEUROPROTECTION BY HSP90 INHIBITORS IN MODELS OF FALS
DURHAM H1, CHA J1, TRADEWELL M1, ZINKIE S1, JAFFER Z2, CHEN R2, RUBINSTEIN A2
1McGill University, Montreal, QC, Canada, 2NexGenix Pharmaceuticals, New York, NY, United States
E-mail address for correspondence: [email protected]
Keywords: Hsp90 inhibitor, stress response, culture model
Background: Many therapeutic strategies for ALS are designed to reduce cellular stress or upregulate endogenous stress responses to compensate for the high vulnerability of motor neurons to the disease. Relative to astrocytes, motor neurons have a high threshold for transactivation of stress response genes, including heat shock and phase II antioxidant genes. Evidence that these properties compromise maintenance of protein quality control in ALS includes: chaperoning activity is decreased in lumbar spinal cord of presymptomatic SOD1G93A mice; specific proteasomal enzyme activities are reduced in lumbar cord of P45+ SOD1G93A mice; chymotrypsin-like activity is decreased in sporadic ALS spinal cord, but not in cerebellum (unpublished data).
Objectives: The Hsp90 inhibitors, geldanamycin and NXD 30001 (a novel radicicol-based Hsp90 inhibitor with improved in vivo pharmacodynamic properties from NexGenix Pharmaceuticals), were highly neuroprotective in a primary culture model of fALS1, preventing aggregation of mutant SOD1 into inclusions and prolonging viability of motor neurons. The objectives of this study are to determine: 1) If Hsp90 inhibitors prevent other manifestations of mutant SOD1 toxicity, including calcium dysregulation and mitochondrial abnormalities; 2) If Hsp90 inhibitors exert neuroprotection through reducing cellular burden of mutant protein either through upregulation of HSPs or by disrupting association of Hsp90 with mutant SOD1 or other client proteins.
Methods: Mutant or wildtype SOD1 (+/− eGFP tag) are expressed in motor neurons of dissociated spinal cord-DRG cultures by intranuclear microinjection of plasmid construct. Microfluorometric techniques are used to measure levels of Ca2+ in mitochondria, ER and cytoplasm; mitochondrial shape, fission/fusion and axonal transport; formation of inclusions, and viability in individual motor neurons. To estimate levels of plasmid-derived eGFP-tagged protein in motor neurons and background cells, microscopic images are captured using a Hamamatsu cooled CCD camera (500 msec exposures). MetaFluor software (Universal Imaging) is used to calculate pixel density of fluorescence in defined regions.
Results and conclusion: NXD30001, as geldanamycin, induced HSPs, ie Hsp70 and Hsp40, but not Hsp90. Also upregulated was clusterin, a chaperone expressed in motor neurons and astrocytes and well known for its secretion and chaperoning function in the extracellular space. NXD30001 reduced accumulation of SOD1-eGFPG93A in motor neurons following gene transfer. 40 μM NXD30001 significantly reduced accumulation of SOD1-eGFPG93A on both day 1 and 2 following microinjection, but had minimal effect on accumulation of SOD1-eGFPWT. Significant reduction of SOD1-eGFPG93A was measured on day 2, but not on day 1 following treatment with 5 μM NXD30001, demonstrating a dose-response effect. These results provide evidence that Hsp90 inhibitors are protective at least in part by reducing levels of mutant protein. Studies on mitochondrial manifestations of toxicity are in progress and NXD30001 is under further testing and therapeutic development for ALS.
P8 ADMINISTRATION OF RECOMBINANT METALLOTHIONEIN-I STARTING AT ONSET PROLONGS SURVIVAL IN A MOUSE MODEL OF MUTANT SOD1-LINKED FAMILIAL ALS
TOKUDA E1,2, ONO S-I1,3
1Nihon University, Funabashi, Japan, 2Umea University, Umea, Sweden, 3Akiru Municipal Clinical Center, Tokyo, Japan
E-mail address for correspondence: [email protected]
Keywords: metallothionein, non-neuronal cell, transgenic mouse
Background: Mutations in the gene of copper/zinc superoxide dismutase (SOD1) are the most common known cause of amyotrophic lateral sclerosis (ALS). Non-neuronal cells, including glial cells, influence motor neuron survival in ALS and are a potential target to prevent motor neuron degeneration.
Metallothionein-I (Mt-I), a metal binding protein, is primarily located within glial cells. When neurons are injured, Mt-I is released by glial cells toward the injured neurons where it acts to promote neuronal survival and regeneration. Overexpression of Mt-I prolongs survival of SOD1G93A mice (1), whereas, absence of Mt-I shortens survival (2,3). Despite the fact that Mt-I plays a critical role in protecting motor neurons, it is not known whether administration of Mt-I ameliorates mutant SOD1 toxicity.
Objectives: The aim of the present study was to elucidate the effects of intraperitoneal administration of recombinant Mt-I (rMt-I) starting at disease onset on the disease course in SOD1G93A mice.
Methods: rMt-I was purchased from Alexa Biochemicals. Since rMt-I contains 5∼7% zinc ions, we used two types of control groups: a zinc sulfate (ZnSO4) group and a phosphate buffered-saline (PBS) group. SOD1G93A mice (B6SJL, high copy number) were assigned to receive a daily intraperitoneal administration of rMt-I (0.5 mg/kg: n = 24), ZnSO4 (0.025 mg/kg: n = 24), or PBS (n = 24). Treatment was started at 13 weeks of age, when the SOD1G93A began to exhibit ALS-like symptoms.
The clinical onset of the disease was evaluated by examining mice to identify shaking of the limbs when suspended in the air by the tail. The end-point was defined as inability of a mouse to right itself within 30 s after being pushed onto its side. For analysis of the survival of the motor neurons, lumbar sections of the spinal cord were immunostained using anti-NeuN antibody. The number of NeuN-positive motor neurons in the ventral horn was counted.
Results: Treatment of recombinant Mt-I prolonged the survival of the mice by 17% (rMt-I: 146±2.8, ZnSO4: 124±3.2, PBS: 122±1.9 days). The progression of the disease was slowed by 78% (rMt-I: 52±7.1, ZnSO4: 29±2.6, PBS: 27±2.0 days).
At 17 weeks of age, SOD1G93A mice had only 50% surviving motor neurons, whereas 77% remained in mice treated with rMt-I.
Conclusions: rMt-I might be a potential therapeutic target for familial ALS patients with SOD1 mutations.
References:
- Tokuda E, Ono S. ALS (Suppl. 1) 2009;10:85.
- Nagano S, Satoh M, Sumi H, et al. Eur J Neurosci 2001;13:1363–70.
- Puttaparthi K, Gitomer WL, Krishnan U, et al. J Neurosci 2002; 22:8790–96.
P9 INTRAMUSCULAR ADMINISTRATION OF METALLOTHIONEIN-IIA IMPROVES SURVIVAL OF G93A SOD1 MOUSE
FOSTER S, LEWIS K, WEST A, CHUNG R, CHUAH MI
University of Tasmania, Hobart, Tasmania, Australia
E-mail address for correspondence: [email protected]
Keywords: metallothionein, G93A SOD1 mouse, intramuscular injection
Background: Mutations to superoxide dismutase 1 (SOD1) enzyme have been linked to familial Amyotrophic Lateral Sclerosis. These mutations typically cause a toxic gain-of-function resulting in tyrosine nitration and protein aggregation and excitotoxicity (1). Mutant SOD1 also has a higher affinity to aggregate when it is zinc deficient (2). The neuroprotective protein metallotheionein-IIA (MT-IIA) is involved in metal homeostasis, particularly that of zinc, and can act as an antioxidant (3).
Objectives: (i) To determine whether MT-IIA can delay neurodegenerative decline and improve survival in the G93A SOD1 transgenic mouse; (ii) To investigate a possible route through which MT-IIA can access the CNS.
Methods: At 10 weeks of age, MT-IIA or a saline control was injected intramuscularly into the left hindlimb of litter pairs comprising a wild type and a mutant G93A SOD1 mouse twice a week until the mice reached endstage (loss of 20% maximum body weight). At each injection time point, weight and disease symptoms (muscle wastage, hindlimb mobility and tremors) were assessed. In addition, mice deficient in MT (MTKO) were used to investigate whether there is any difference in tissue access when MT-IIA is administered intramuscularly or intraperitoneally.
Results: Symptom analysis showed that from 147 days of age, the MT-IIA treated group showed significantly less severe symptoms compared to the control group. There was also improved survival rate in the MT-IIA treated group compared to the control group, beginning at 145 days of age. Survival of the control and MT-IIA treatment group was 15% and 40% respectively at 150 days of age. Histological analysis of MTKO showed that MT-IIA was present in blood vessels as soon as 15 minutes after intramuscular injection. Intraperitoneal injection resulted in detection of MT-IIA in the proximal convoluted tubules of the kidney 15 minutes later, suggesting that the MT-IIA may have been taken up rapidly in the vascular circulation and formed a component of the glomerular filtrate.
Discussion and conclusions: These results suggest that MT-IIA can delay neurodegenerative decline and prolong survival in the G93A SOD1 mouse. The evidence suggests that both intramuscular and intraperitoneal injection can rapidly deliver MT-IIA to the blood circulation. Recent studies show that the blood brain barrier in the G93A SOD1 transgenic mouse is altered (4). An important question is whether the MT-IIA in blood circulation can access the CNS of the G93A SOD1 mouse readily and is therefore able to act on the neurons.
References:
- Gurney ME, Pu H, Chiu AY, et al. Science 1994;264:1772–5.
- Banci L, Bertini I, Boca M, et al. PLoS ONE 2008; 3:e1677.
- Taylor DM, Minotti S, Agar JN, et al. Neurotoxicol 2004;25:779–92.
- Zhong D, Deane R, Ali Z, et al. Nat Neurosci 2008;11420–2.
P10 NO BENEFIT FROM CHRONIC DIMEBOLIN DOSING IN A SIBLING-MATCHED, GENDER BALANCED, INVESTIGATOR-BLINDED TRIAL USING A STANDARD MOUSE MODEL OF FAMILIAL ALS
VIEIRA F, MORENO A, KIDD J, THOMPSON K, DHILLON H, GILL A
ALS TDI, Cambridge, MA, United States
E-mail address for correspondence: [email protected]
Keywords: SOD1-G93A, dimebolin, therapy
Background: Dimebolin is a non-selective anti-histamine drug approved in Russia for treatment of allergy. The drug has been shown to be protective in a beta-amyloid fragment Abeta 25-35 cerebellar granule cell model that may mimic some neurodegenerative aspects of Alzheimer's Disease and has been demonstrated as neuroprotective in glutamate-induced apoptosis in striatal neuronal cultures derived from a Huntington's disease mouse model. Dimebolin preserves learning and memory after chronic administration in a preclinical AF64A cholinergic-lesion in rats. Dimebolin has not been tested for efficacy in any preclinical models of motor neuron degeneration.
Objectives: The aim of the current study was to test chronic dimebolin dosing for efficacy in a sibling-matched, gender balanced, investigator-blinded trial using the high-copy (average 23 copies) SOD1G93A mouse (n= 30 per group).
Methods: Pharmacokinetic parameters in high-copy SOD1G93A mice of dimebolin were determined after single 30 mg/kg intraperitoneal injection of dimebolin dihydrochloride in 1X phosphate buffered saline. The parameters were used to establish a dosing regimen for chronic delivery of dimebolin in SOD1G93A mice which could result in constant exposure of drug to the spinal cord. In the survival efficacy study, treatment commenced when mice were approximately 55 days old. Dimebolin-treated mice received a single loading dose of 1.75 mg/kg on the first day of treatment and were subsequently implanted with Alzet osmotic mini-pumps which delivered 384 μg of dimebolin free-base per day to each mouse in 6 μL of 1X PBS. Exhausted pumps were removed and replaced after 30 days for the duration of the study as necessary. Neurological disease severity score and body weight were determined daily during the dosing period. Age at onset of definitive disease and survival duration were recorded. Summary measures from individual body weight changes and neurological score progression, age at disease onset, and age at death were compared using Kaplan-Meier and Cox proportional hazards analysis.
Results and conclusions: Rigorous survival study design that includes sibling matching, gender balancing, investigator blinding, and transgene copy number verification for each experimental subject minimized the likelihood of attaining a false positive therapeutic effect in this standard animal model of familial ALS. Results from this study do not support taking dimebolin into human clinical trials for ALS.
P11 TREATMENT WITH THE UPA INHIBITOR WX-340 DELAYS ONSET OF CLINICAL SYMPTOMS IN G93A ALS MICE
HENSLER M1, LORENZL S1, SCHMALIX W1
1Department of Neurology, LMU, Munich, Germany, 2Wilex AG, Company, Munich, Germany
E-mail address for correspondence: [email protected]
Keywords: uPA, uPA inhibitor WX-340, G93A mice
Purpose: We have recently shown that treatment with the uPA inhibitor WX-340 (Wilex AG, Germany) significantly prolonged survival of G93A mice. The objective of this study was to investigate whether delayed start of treatment with WX-340 alters disease progression.
Methods: 150 transgenic mice were used. Mice were grouped as follows: vehicle group d60 (start of treatment), WX-340 10 mg/kg d30, WX-340 1 mg/kg d60, WX-340 10 mg/kg d60, Riluzole 30 mg/kg/day d60, and WX-340 10 mg/kg + Riluzole 30 mg/kg/day d60 (administered in drinking water). Clinical scoring and body weight was performed once-a-week, open field test measurement clinical onset of disease (score = 4) and survival data were recorded.
Results: There were no differences in the body weight between the treatment groups. Number of rearings was significantly decreased on age week 15 in WX-340 1 mg/kg d60 and WX-340 10 mg/kg d60 plus Riluzole treatment groups compared to vehicle group. Clinical score was significantly improved on age week 11 in WX-340 1 mg/kg d60 treatment group compared to vehicle group. The WX-340 10 mg/kg d60 group had a significantly delayed onset of clinical symptoms. There were no significant differences in the survival between the treatment groups.
Conclusions: WX-340 administered at dose of 10 mg/kg starting at the age of 60 days significantly delayed the onset of clinical symptoms. Chronic treatment with 1 or 10 mg/kg of WX-340 at the age of 60 days has no significant effects on survival, body weight, or open field activity.
P12 COPPER BIS(THIOSEMICARBAZONATO) COMPLEX ADMINISTRATION REDUCES TDP-43 PATHOLOGY, DELAYS DISEASE ONSET AND EXTENDS SURVIVAL IN A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
SOON C1,2, DONNELLY P4, TURNER B3, CROUCH P1,2, SHERRATT N1,2, TAN J-L1, LIM N1,2, LAM L2, BICA L1, LIM S4, HICKEY J4, CULVENOR J1, DUCE J1,2, MASTERS C1,2, WHITE A1,2, BARNHAM K1,2, LI Q-X1,2
1The University of Melbourne, Department of Pathology, Parkville, Australia, 2The Mental Health Research Institute, Parkville, Australia, 3Florey Neuroscience Institutes, Parkville, Australia, 4The University of Melbourne, School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, Parkville, Australia
E-mail address for correspondence: [email protected]
Keywords: SOD1G93A, CuII(atsm), TDP-43
Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by deterioration of motor neurons with no effective treatment. Diacetylbis(N(4) methylthiosemicarbazonato)copper(II) (CuII(atsm)) is a coordination complex that crosses the blood-brain barrier and has antioxidant properties.
Objective: This study examined the effects of systemic CuII(atsm) treatment on disease onset and progression in the SOD1G93A mouse model of ALS.
Methods: TgSOD1G93A mice in C57B6 background with delayed phenotype due to low copy of transgene were characterized previously (1) and administered CuII(atsm) orally at 30 mg/kg at both pre-symptomatic and symptomatic age. Clinical assessment and motor function tests including rotarod and stride length, as well as biochemical and histological analysis were performed.
Results: CuII(atsm) pre-symptomatic treatment led to 70% increase in survival after motor impairment onset and 14% increase in overall lifespan. Importantly, treatment beginning at symptom onset also increased survival after motor impairment onset by 59%, and prolonged overall lifespan by 10%. These effects were accompanied by rescue of motor neurons, attenuation of oxidative damage, suppression of astrocyte and microglial activation and prevention of pathologic up-regulation of metalloproteinase 9 in spinal cords, compared to vehicle-treated control mice. Furthermore, CuII(atsm) treatment reduced abnormal phosphorylation and cleavage of TDP-43.
Discussion and conclusion: This reduction in pathological changes may contribute to the neuroprotective effect of CuII(atsm) in this mouse model. The study also provides a link between disease progression and TDP-43 pathology, which can be pharmacologically modulated. This study indicates that CuII(atsm) may be a promising therapeutic drug candidate for ALS.
Reference:
- Soon CPW, Crouch PJ, Turner BJ, et al. Neuromuscul Disord 2010;20:260–6.
P13 ACTIVATION OF THE TRANSCRIPTION FACTORS CREB AND NRF2: MECHANISM OF ACTION STUDIES TO DEFINE THE POSITIVE THERAPEUTIC OUTCOMES FOR CU(ATSM) IN ALS MODEL MICE
CROUCH P1,2, PATTERSON B1, LIM S1, SOON C1,2, MASTERS C1,2, LI Q-X1,2, DONNELLY P1, BARNHAM K1,2, WHITE A1,2
1University of Melbourne, Melbourne, Victoria, Australia, 2Mental Health Research Institute of Victoria, Melbourne, Victoria, Australia
E-mail address for correspondence: [email protected]
Keywords: therapeutic, mechanism of action, transcription factor
Background: The paucity of effective therapeutic treatments for ALS is highly disproportional to the devastating impact ALS has on the people affected. The development and testing of new and more effective therapeutic strategies is therefore an important research goal. CuII(atsm) is a metal-ligand complex that crosses the blood-brain barrier after oral administration. We have shown that treating a SOD1G93A mouse model of ALS with CuII(atsm) improves locomotor function and delays paralysis when administered pre- or post-symptom onset.
Objective: This study examined CuII(atsm) at a cellular level in order to elucidate the mechanisms through which CuII(atsm) improves locomotor function in ALS model mice.
Methods: Neuronal- and glial-like cells were grown in culture and treated with CuII(atsm) or its vehicle control. Treatments were performed under normal growing conditions, or under conditions to induce a specific cellular stress (hypoxia, glucose deprivation, glutathione depletion etc).
Results: Treating neuronal-like SH-SY5Y cells with CuII(atsm) under normal growing conditions showed that although the compound is taken up by these cells it is relatively inert under normal conditions. Stress conditions such as hypoxia and glutathione depletion however induced CuII(atsm)-mediated responses including activating phosphorylation of the signalling kinase Akt, inhibitory phosphorylation of GSK3β, and activating phosphorylation of the transcription factor CREB. In contrast to neuronal like SH-SY5Y cells, we found that glial-like U87-MG cells responded to CuII(atsm) in the absence of any specific cellular stress. U87-MG responses to CuII(atsm) included activation of Akt and up-regulation of Nrf2, a transcription factor that regulates levels of cellular antioxidants.
Discussion and conclusions: These mechanistic studies indicate the therapeutic outcomes for CuII(atsm) in ALS model mice involve the activation of transcription factors with the potential to increase synaptic activity (CREB activation in neuronal cells) and protect against oxidative insult (Nrf2 activation in glial cells). Our on-going work aims to define the mechanism of action for CuII(atsm) in greater detail in order to better understand its potential as a therapy for ALS and to define the relevance of CREB and Nrf2 activation as valid therapeutic targets.
P14 NEUROPROTECTION OF RAPAMYCIN AND COMBINED TREATMENT OF RAPAMYCIN AND GSK-3 INHIBITOR IN AN ALS MOUSE MODEL
AHN S-W, YOUN Y-C, PARK K-Y, SHIN H-W, KWON O-S, JANG J-W, KIM K-E
Chung-Ang University Hospital, Seoul, Republic of Korea
E-mail address for correspondence: [email protected]
Keywords: autophagy, rapamycin, GSK-3 inhibitor
Background: In previous studies of neurodegenerative disorders, autophagy is the major route for lysosomal degradation of misfolded protein aggregates and oxidative cell components. Thus, the study of the autophagy pathway and its therapeutic effect is important in ALS. In addition, it is well known that GSK-3 plays significant roles in intracellular apoptotic pathways, and the suppression of GSK-3 activity could be one of the potential pathogenic mechanisms of ALS.
Objective: Given that apoptosis and autophagy play a central role in motor neuron degeneration and can contribute to neuronal death through distinctive routes in ALS, we hypothesize that up-regulation of autophagy would prolong the survival of motor neurons and suppress the disease progression in ALS. Moreover, the concurrent administration with both down-regulation of apoptosis and up-regulation of autophagy would probably have additive effects on neuronal survival and motor function.
Methods: A total of 24 transgenic mice harboring the human G93A mutated SOD1 gene and 6 wild type mice were used following confirmation of their genotype. The 24 transgenic mice were equally divided into 4 groups; transgenic control, GSK-3 inhibitor treatment, rapamycin treatment and concurrent administration group. The clinical status, rotarod test and survival of the ALS mice model was evaluated beginning 60 days after birth. Cumulative probabilities of symptom onset, rotarod failure, and endpoint were analyzed with the Kaplan-Meier survival analysis.
Results: The onset of symptoms was significantly delayed in both GSK-3 inhibitor treatment group and rapamycin treatment group, and non-significantly delayed in concurrent administration group relative to the control group. In addition, motor activity using rotarod test was significantly improved in both rapamycin treatment group and concurrent administration group in incipient stage, and non-significantly improved in GSK-3 inhibitor treatment group relative to the control group. The time of rotarod failure and endpoint were non-significantly delayed in GSK-3 inhibitor group relative to the control group. However, unexpectedly, the time of rotarod failure and end point were shortened in both rapamycin treatment group and concurrent administration group relative to the control group, although the results were not statistically significant.
Conclusion: The present study suggest that: GSK-3 inhibitor has a neuroprotective effect which could delay disease progression in the ALS mouse model consistent with previous reports; treatment with rapamycin in incipient stage of ALS mouse model delayed motor function deficit and onset of symptoms; excessive dose or prolonged treatment of rapamycin exacerbated the disease progression in the ALS mouse model. Thus we suggest that an appropriate dose of rapamycin would be a novel promising therapeutic strategy in ALS, although this is a preliminary study with a few limitations.
P15 CHRONIC TREATMENT WITH THE MACROLIDE IMMUNOSUPPRESSANT RAPAMYCIN IN A SIBLING-MATCHED, GENDER BALANCED, INVESTIGATOR-BLINDED TRIAL ACCELERATES NEUROLOGICAL DISEASE PROGRESSION IN A STANDARD MOUSE MODEL OF FAMILIAL ALS
VIEIRA F, MORENO A, KIDD J, THOMPSON K, GILL A
ALS Therapy Development Institute, Cambridge, MA, United States
E-mail address for correspondence: [email protected]
Keywords: G93A-SOD1, rapamycin, immunomodulation
Background: Rapamycin is an immunosuppressive macrolide antibiotic that binds to the cytosolic protein FKBP12. The rapamycin-FKBP12 complex inhibits the mammalian target of rapamycin (mTOR) pathway by directly binding the mTOR Complex1 (mTORC1). Immunomodulatory treatment with an anti-Cd40lg monoclonal antibody delays onset of paresis and extends survival in the high copy G93A-SOD1 mouse model, but its mechanism of action remains unclear. Like anti-Cd40lg monoclonal antibodies, macrolide immunosuppressants have been used in preclinical models of organ transplant to improve likelihood of engraftment. It is possible that macrolide immunosuppressants like rapamycin and fk506 could have downstream mechanistic overlap with anti-Cd40lg therapy that would be efficaceous in the G93A-SOD1 mouse model of ALS.
Objectives: The aim of the current study was to test chronic rapamycin dosing for efficacy in a sibling-matched, gender balanced, investigator-blinded trial using the high-copy (average 23 copies) G93A -SOD1 mouse (n=30 per group).
Methods: Pharmacokinetic parameters in high-copy G93A-SOD1 mice of rapamycin were determined after single 5 mg/kg intraperitoneal injection of rapamycin in 1% DMSO in 1x phosphate buffered saline confirming peripheral and central nervous system exposure of the drug in mice. A sibling-matched, gender balanced, investigator-blinded efficacy study was initiated in G93A-SOD1 mice. Treatment commenced when mice were approximately 55 days old. Treatment cohort mice received daily injections of 1 mg/kg rapamycin in 1% DMSO in phosphate buffered saline. Neurological disease severity score and body weight were determined daily during the dosing period. Age at onset of definitive disease and survival duration were recorded. Summary measures from individual body weight changes and neurological score progression, age at disease onset, and age at death were compared using Kaplan-Meier and Cox proportional hazards analysis.
Results and conclusions: In this initial test of early (55 days of age) immunomodulation using daily 1 mg/kg IP rapamycin treatment, body weight was less well maintained, neurological disease showed an earlier onset, and neurological symptoms progressed more rapidly than in vehicle control animals. There was no significant change in survival compared to vehicle treated control mice.
P16 GRANULOCYTE COLONY STIMULATING FACTOR REDUCES INFLAMMATION IN A MOUSE MODEL OF ALS
POLLARI E1, KANNINEN K1, GINIATULLINA R1, AHTONIEMI T2, MALM T1, GINIATULLIN R1, KOISTINAHO J1, MAGGA J1
1A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland, 2Medeia Therapeutics Ltd, Kuopio, Finland
E-mail address for correspondence: [email protected]
Keywords: G-CSF, G93A-SOD1 mice, inflammation
Background: Amyotrophic lateral sclerosis (ALS) is a lethal motoneuron affecting neurodegenerative disease and currently has no known cure. We propose that granylocyte colony stimulating factor (G-CSF), a neuroprotective agent, is a promising drug candidate for treatment of ALS. G-CSF is clinically used to mobilize stem cells from bone marrow and to treat neutropenia. It promotes angiogenesis and neurogenesis, has anti-apoptotic and anti-inflammatory effects and is neuroprotective in models of several acute and chronic diseases.
Objectives: Our aim was to assess G-CSF mediated protection to the central nervous system (CNS) and to the inflammatory status in mouse model of ALS.
Methods: Transgenic mice overexpressing mutant Cu, Zn superoxide dismutase, G93A-SOD1, received weekly subcutaneous injections of G-CSF starting at pre-symptomatic stage. At symptomatic stage and at end-stage disease we analyzed an array of inflammatory markers and determined reactive oxygen species (ROS) in CNS. Immunohistochemical antibody stainings were performed to evaluate gliosis and neuronal survival in lumbar spinal cord (SC) at end-stage disease. Additionally, disease progression and survival were monitored. Neuroprotective effect of G-CSF was confirmed in primary SC cultures.
Results: G-CSF prolonged survival and slowed down the disease progression from the onset to death in transgenic G93A-SOD1 mice. Production of proinflammatory cytokines was reduced in G-CSF treated transgenic mice in comparison to untreated transgenic mice both at symptomatic and end-stage disease. In addition, G-CSF treatment reduced gliosis and promoted neuronal survival in the ventral horn of lumbar SC and diminished ROS levels in the CNS at the end-stage. Furthermore, in primary neuronal cultures we were able to verify neuroprotective properties of G-CSF.
Discussion and conclusions: Our data reveal new anti-inflammatory properties of G-CSF in a mouse model of ALS. We were able to demonstrate that G-CSF prolongs survival of G93A-SOD1 mice and protects neurons in culture. These results validate beneficial properties of G-CSF as a neuroprotective factor for the treatment of ALS.
P17 THE PLEOTROPHIC EFFECTS OF IGF-I ON HUMAN SPINAL CORD NEURAL PROGENITOR CELLS
LUNN S1, PACUT C1, BACKUS C1, HONG Y1, JOHE K3, HEFFERAN M2, MARSALA M2, FELDMAN E1
1University of Michigan, Ann Arbor, MI, United States, 2University of California San Diego, La Jolla, CA, United States, 3Neuralstem, Inc., Rockville, MD, United States
E-mail address for correspondence: [email protected]
Keywords: stem cell, IGF-I, therapeutic
Background: Amyotrophic lateral sclerosis (ALS) is a lethal neurological disorder that leads to progressive degeneration and loss of motor neurons (MN). Neural progenitor cells derived from the spinal cord have the potential to differentiate into various cell types, including both neurons and glia, within the disease microenvironment. Combining growth factors with Schwann cells, glia, and stem cells delivers both cellular and neurotrophic support. Insulin-like growth factor-I (IGF-I) is a growth factor with neuroprotective properties highly considered for treatment of ALS. The combination of human spinal cord stem cells (HSSC) with IGF-I treatment may provide a systems approach to the treatment of ALS as well as other motor neuropathies
Objectives: Our hypothesis is that IGF-I in combination with HSSC will enhance HSSC integration into the host tissue and offer greater neuroprotection in neurodegenerative diseases. Our goal is to understand the role of IGF-I in stem cell biology and how IGF-I may interact with stem cells to increase their neuroprotective effects.
Methods: HSSC were prepared and provided by Neuralstem, Inc. Briefly, cells were prepared from a cervical–upper thoracic region of spinal cord tissue obtained from a single 8-week human fetus after an elective abortion. The fetal tissue was donated by the mother in a manner fully compliant with the guidelines of NIH and FDA and approved by an outside independent review board. The direct effects of IGF-I on the differentiation of HSSC were assessed either by western blotting or immunocytochemistry. Cell death was assessed using TUNEL.
Results: Our findings demonstrate that IGF-I is produced early in HSSC differentiation. Direct treatment with IGF-I does not change the expression profile of HSSC as they are differentiating, suggesting IGF-I does not affect lineage selection, but rather enhances neural development and neurite outgrowth. Signaling via AKT, but not MAPK mediates IGF-I-stimulated neurite outgrowth. HSSC are more resistant to glutamate excitotoxicity than mature MNs. As such, HSSC may be less susceptible to pathogenic factors while they mature; however, IGF-I remains a potent neuroprotective factor for excitotoxic stress in HSSC. Finally, IGF-I does not promote proliferation of HSSC.
Discussion and conclusions: We show that IGF-I increases the proportion of HSSC producing neurites and increases neurite length. Furthermore we show that IGF-I mediated AKT signaling is required for neurite extension. IGF-I does not change the proliferation profile of differentiating HSSC, or the terminal fate decisions. We go on to show that IGF-I protects HSSC against glutamate-induced excitotoxicity. Our data support the idea that upregulation of IGF-I production in HSSC may offer additional therapeutic benefits when HSSC engineered to overexpress IGF-I are transplanted into the nervous system of animal models and humans with neurological disorders.