P1 THE COCHRANE COLLABORATION – A DECADE OF EVIDENCE-BASED REVIEWS IN ALS/MND
MILLER RG1, MITCHELL JD2, MOORE DH1, BENATAR MG3, JEWITT K4, HUGHES RA4
1California Pacific Medical Center, San Francisco, CA, United States,2Royal Preston Hospital, Preston, United Kingdom,3Emory University, Atlanta, GA, United States,4National Hospital for Neurology and Neurosurgery, London, United Kingdom
Email address for correspondence: [email protected]
Keywords: cochrane collaboration, evidence-based medicine, ALS management
Background: The Cochrane Collaboration is a global voluntary scientific network dedicated to making up-to-date, accurate information about the effects of therapeutics readily available worldwide.
Objectives: To review the progress of the Cochrane Neuromuscular Research Group (NMRG) that evaluates various therapies for ALS/MND.
Methods: Each Cochrane systematic review utilizes the standard methodology of the Cochrane Collaboration including a software program for statistical analyses of the reported data from relevant randomized-controlled clinical trials. Interested reviewers meet annually at the International ALS/MND meeting to share progress and plans. The editorial office in London, UK, provides support for literature searches and editorial assistance.
Results: To date there have been 11 systematic reviews and 10 protocols published in the Cochrane library (issue 1, 2009). The systematic review of riluzole has been recently revised to include recently published data from large registries. The review of insulin-like growth factor-1 examined the evidence in two published trials, pointing out methodological concerns, soon to include the third recently completed IGF-1 trial. Earlier reviews of antioxidant treatment and ciliary neurotrophic factor were published.
Within the past year, three systematic reviews appeared: clinical trials in familial ALS, exercise in ALS/MND and treatment for pain in the terminal phase of ALS/MND. In the familial ALS review the authors found no difference between familial and sporadic patients in clinical trial responses, but point to the need for inclusion of familial cases, for trials to document whether or not ALS is familial and for sharing of data once trials are completed. The review of exercise, based on two small studies, found no harmful effects of exercise and some modest benefits, but larger studies are needed. The review of pain treatment pointed to the lack of controlled studies in ALS and the need to move forward with trials in this important area.
Two reviews on management issues were recently published, one for enteral feeding and the other for spasticity. In the review of spasticity, patients who were performing twice daily exercise had significantly less spasticity after three months than those who were not.
Protocols that have been published with reviews in progress include: breaking the news; treating sialorrhea; mechanical ventilation; multidisciplinary clinics and clinical trials of minocycline, gabapentin and creatine.
Discussion: Since the first systematic review of riluzole in ALS/MND in 1999, there has been a steady increase in the number of reviews. A substantial number of new reviews have appeared or are close to publication. The quality of clinical trials has improved, but reviews of management highlight the paucity of strong evidence in ALS therapeutics and point the way toward future research in this field.
Conclusion: More high quality clinical studies are needed to guide management in ALS.
P2 ONLINE SURVEYS OF PALS REVEAL THEIR OPINIONS ON ALS CLINICS AND RESEARCH STUDIES
BEDLACK R
Duke University, Durham, NC, United States
Email address for correspondence: [email protected]
Keywords: clinics, trials, enrolment
Background: Less than 50% of North American patients with ALS (PALS) get care in a specialized ALS clinic. Only 10–25% of PALS in these clinics participate in research studies. Meta-analyses suggest that trial design factors do not account for low and variable enrolment in ALS research studies; rather, "doctor factors" and "patient factors" may be responsible.
Purpose: To gain a better understanding of the opinions of PALS toward specialized ALS clinics and the research offered there and ultimately to develop educational interventions that improve attendance in ALS clinics and enrolment in ALS research studies. Similar surveys have been used in cancer patients and led to interventions that improved enrolment in cancer studies.
Methods: An online survey directed toward PALS is underway, hosted by the website Survey Monkey. This consists of questions related to clinics, research studies and demographics and has been advertised on a large number of individual North American ALS Clinic websites (for example www.dukealsclinic.com) and on MDA and ALSA websites.Data from the survey will be analyzed using simple descriptive statistics.
Results: The survey has been open and advertised since 29 December 2008. To date, 77 PALS have participated in this survey.
Participants thus far were white (100%) males (55%), mean age 57, with college or higher education (76%). A surprising percentage did not know that specialized ALS Clinics existed (15%). Those who did, usually learnt about them from their first neurologist (64%). Those who came to specialized ALS Clinics did so primarily for the multidisciplinary team (77%). The biggest complaints about the clinics were the amount of driving (50%) and time required to attend (30%). Information about research was obtained primarily from ALSA and MDA websites (59%) and support groups (48%). Only half the responders had ever been asked to be in a research study and of those asked 80% agreed to participate. Most common reasons for participating were beliefs that the study would help others (85%) and that it would help the participant themselves (73%). The majority of those who participated in a research study stated that they were never asked to participate again (74%). Most participants had not participated in online research studies, but said that the results of an online study would not affect their interest in a clinic based study.
Discussion and Conclusion: Even this preliminary data suggests that changes can be made to optimize enrolment in clinics (better advertising and geographic distribution, shorter clinic visit times) and enrolment in research studies (asking more often and creating a registry of past participants).
P3 UPDATE ON ALSUNTANGLED; A GLOBAL SCIENTIFIC EFFORT TO INVESTIGATE ALTERNATIVE AND OFF-LABEL TREATMENT OPTIONS FOR PATIENTS WITH ALS
BEDLACK R1, HARDIMAN O2
1Duke University, Durham, NC, United States,2Trinity College, Dublin, Ireland, United Kingdom
Email address for correspondence: [email protected]
Keywords: ALSUntangled, internet, alternative therapies
Background: Patients with ALS (PALS) often consider alternative and off-label treatments (AOTs) they read about on the internet. These are high stake decisions, as AOTs are can be expensive and harmful; pursuit of an AOT is often undertaken in place of participation in a clinical trial (potentially delaying the search for better ALS therapeutics). Unfortunately, the evidence presented for the efficacy and safety of these typically ranges from scant to frankly inaccurate.
In April 2009, we initiated a system by which the community of ALS clinician scientists might work more closely and regularly together to systematically assess AOTs, ultimately helping PALS make more informed decisions about their care. This system, collectively referred to as ALSUntangled (ALSU) operates using newer internet tools. There are three parts to ALSU: learning about the AOTs that PALS are considering, a "war room" in which these AOTs are investigated by ALS Clinician Scientists and finally, public release of the results of the investigations. The first part utilizes Twitter; the second part utilizes an invitation-only site called NING and the third part utilizes free access publications via the journal Amyotrophic Lateral Sclerosis.
Objectives: To remind people of the availability of ALSUntangled and review early experiences with ALSUntangled.
Methods: Components of ALSUntangled were reviewed, including how PALS and ALS clinicians and scientists can access it. The number of "tweets" received, the amount of activity on the NING and the number of free access publications via the journal Amyotrophic Lateral Sclerosis will be reported.
Results: Thus far we have received 4 ALSUntangled tweets, all from members of the ALSRG with requests to evaluate Lyme Disease treatment, the X-Cell Center, the Hickey Wellness Center and Dr. Zannos Grekos' Stem Cell Clinic in Dominican Republic. The NING has attracted 20 members, with 4 forum topics being actively discussed (Lyme Disease treatment, the X-Cell Center, the Hickey Wellness Center and Dr. Zannos Grekos' Stem Cell Clinic in Dominican Republic). One free access publication from the program is scheduled for June 2009 (an introduction to ALSUntangled).
Discussion and Conclusions: ALSUntangled fills an important need: to facilitate organized scientific investigation of AOTs, which in turn can help PALS make more informed decisions about their care. Early experience is promising. In the ensuing months, we will begin to advertise the program and hope to see increased numbers of tweets and increased activity on the NING, which will result in more free access publications to help PALS and their providers.
P4 ENROLMENT IN THE STAR TRIAL
BEDLACK R1, HEPNER A2, KAY R2
1Duke University, Durham, NC, United States,2Avanir Pharmaceuticals, Aliso Viejo, CA, United States
Email address for correspondence: [email protected]
Keywords: zenvia, enrolment, trials
Background: Surveys suggest that only 10–25% of PALS enrol in clinical trials; a meta-analysis revealed an average trial enrolment rate of only 2 subjects/site/month. These studies suggest tremendous variability in enrolment across sites (range 0–75%) and across trials (0.1–7.5 subjects/site/month). This variability is not explained by trial design factors, for example presence of placebo, or invasiveness of the intervention under study. “Doctor factors” and “patient factors” may be responsible.
We reviewed enrolment data from the STAR Trial. There were several differences between this trial and the previous trials reviewed in the above retrospective analyses. STAR was a trial of symptomatic (not disease-modifying) therapy and had more preliminary human data. STAR enrolled both PALS and patients with MS and utilized sites across North and Latin America. Finally, awareness initiatives for STAR were more extensive than previous trials, including the use of web-based advertising through Patients-Like-Me (PLM).
Methods: Enrolment data from the STAR trial were obtained from Avanir. Simple descriptive statistics were used to analyze these data and compare them to similar data from prior published ALS trials.
Results: The percentage of patients screened for this trial was not available, since the number of patients being followed at each clinic was not recorded. The percentage of screened patients that were enrolled was 79.7% (326 enrolled/409 screened). Overall the enrolment rate was 0.33 subjects/site/month (326 subjects/65 sites/15 months). Enrolment rate did not differ between North American sites (0.32 subjects/site/month; 224 subjects/47 sites/15 months) and Latin American sites (0.38 subjects/site/month; 102 subjects/18 sites/15 months). Enrolment rate did not differ between PALS and patients with MS, either. ALS enrolment rate was 0.24 subjects/site/month (197 subjects with ALS/55 sites enrolling either ALS or both/15 months) and MS enrolment rate was 0.34 subjects/site/month (129 subjects with ALS/25 sites enrolling either MS or both/15 months).Variability in enrolment across sites was more impressive, ranging from 0 to 1.6 subjects/month.
Discussion and Conclusions: As seen in previous studies, the percentage of screened patients that enrolled in STAR was high; some "pre-screening" may have occurred to explain this (for example, not offering the study to patients who seem unlikely to qualify at first glance). Enrolment rate in the STAR trial was within the range seen in previously published trials of disease modifying therapies; also, the variability in enrolment across sites was much higher than the variability in enrolment across countries or across disease status. These findings support the previous hypothesis that trial design factors are not as important as "patient factors" and "doctor factors" in influencing enrolment. Surveys are underway to better understand patient and doctor factors, with the hope of developing strategies that enhance enrolment in future trials.
P5 A COMPARISON BETWEEN ALS PATIENTS PARTICIPATING IN CLINICAL TRIALS AND THOSE WHO DO NOT IN A LARGE MULTICENTER ALS CLINIC
WATSON M-L, SHEFNER J
SUNY Upstate Medical University, Syracuse, NY, United States
Email address for correspondence: [email protected]
Keywords: clinical trials, recruitment strategies, disease progression
Background: Advances in basic science have led to an increase in the number of ALS clinical trials either currently being conducted or in planning stages. Within any clinical trial site, however, only a minority of patients are ultimately enrolled in trials. Understanding the differences between patients who are ultimately enrolled in trials and those who are not is critical for maximizing trial enrolment. In addition, any significant difference between these groups may have implications for how research findings may be generalized to the entire ALS patient population.
Objectives: To compare patient attributes between patients ultimately enrolled in clinical trials and those who are not, in a large multispecialty ALS clinic.
Methods: All patients seen at a large multispecialty ALS clinic were consecutively evaluated from June 2005 until May 2009. Baseline characteristics and measures of rate of progression (ALS-FRS-R) were obtained at clinic visits and differences were assessed between those patients who never participated in a clinical trial (Group 1) and those who did (Group 2).
Results: A total of 238 new patients were seen during the above time period. Of these, 72 (30%) participated in clinical trials. Average age at presentation was 60.7 (SD = 15.9) years for those not in trials and 56.7 (SD = 9) for those who did. Average ALSFRS-R at presentation for Group 1 was 34.2 (SD = 8.2) and 38.7 (SD = 5.1) for Group 2 and average vital capacity was 72.3% (SD = 26.4) for Group 1 and 84% (SD = 20.1) for Group 2. Rate of progression was 1.01 (SD = 0.96) ALSFRS-R units per month for Group 1 and 1.09 (SD = 0.75) for group 2, while change in vital capacity was 2.6% (SD = 3.9) per month for Group 1 and 2.7% (SD = 2.76) for Group 2.
Discussion and Conclusions: In the last 4 years, 30% of patients seen were ultimately enrolled in clinical trial. This is a markedly higher rate than previously reported and is likely due in part to the large number of trials enrolling at the center. Subjects who enrolled in trials were somewhat younger than those who did not, but rate of progression as measured by pulmonary function and ALSFRS-R was similar in the two groups. Of note, there was approximately 35% more variability in progression rate in those patients not in trials. One possible explanation for this is that patients either progressing faster or slower than average are less likely to be able to access trials.
P6 MINIMIZING ADVERSE EVENT REPORTING BIAS IN A HISTORICALLY-CONTROLLED SCREENING TRIAL
CHAMPION S, FORSHEW D, KUSHNER G, MILLER R, SPITALNY M, KATZ J
California Pacific Medical Center, San Francisco, CA, United States
Email address for correspondence: c[email protected]
Keywords: clinical trial, historical controls, bias
Background: A new Phase II screening trial paradigm using historical controls has been designed in order to more efficiently identify promising candidate therapies in ALS. The first such screening trial of lithium carbonate is underway, using the placebo group from the recent minocycline trial as the control group. Determining safety by comparing the lithium carbonate arm to a historical control group raises special challenges.
Objectives: To report on the empirical analysis of potential biases surrounding the comparison of safety data from non-concurrent clinical trials.
Methods: Since the lithium trial began, ongoing analyses of safety data have been performed. When designing the trial, measures were taken to ensure that safety reporting would parallel the previous trial for a fair comparison. These included using similar inclusion and exclusion criteria, creating safety forms that were similar to the original trial, tracking adverse events (AE) and serious adverse events (SAE) at each visit over the same timeframe and instructing centers on how to collect safety data. The demographics and markers of disease severity were evaluated in order to form a comparative picture of both patient populations and to compare AE, SAE and mortality rates. Differences in safety reporting procedures were separated between trials in order to determine what variables can and cannot be controlled.
Results: Several possible issues that could lead to inconsistencies in AE reporting were identified. There were slight differences in AE forms because different types of side effects were expected in each trial. As some of the study sites and coordinators differed between trials, AE interview styles may differ. There may have been slight differences in the frequency or manner that the coordinating center reminded sites to enquire about AEs. The lithium patient population know they are on drug versus minocycline where there was a possibility of placebo. These biases seem to have less of an effect on the more severe markers of safety such as SAEs, unexpected increases in rate of progression and death where comparison to the historical placebo data appears robust.
Conclusions: Several potential biases were identified; however, many can be corrected. These results highlight the importance of standardizing AE reporting methods in order to ensure that safety comparisons to placebo are accurate. Known side effects of the agent must also be considered to obtain an accurate estimation of whether the side effects are those that are expected. Finally, safety data must be viewed within the context of the risk benefit analysis of the study as a whole.
P7 SELECTION TRIAL DESIGN FOR PROMISING THERAPEUTICS IN AMYOTROPHIC LATERAL SCLEROSIS (ALS)
AGGARWAL SI1, SCHOENFELD D1, CUDKOWICZ M1, KATZ J2, MILLER R2, MOORE D2
1Massachusetts General Hospital, Boston, MA, United States,2California Pacific Medical Center, San Francisco, United States
Email address for correspondence: [email protected]
Keywords: selection design, Phase 2, drug development
Background: The process of developing new drugs for ALS is particularly challenging because of the relative rarity of this disorder and the resulting limited number of patients eligible for pharmaceutical trials. At least 32 unique compounds have been tested in phase II/III clinical trials with largely negative results. Given the poor success rate, new initiatives are necessary to optimize early clinical trial design to optimize the limited pool of resources.
Objectives: To use a selection trial as a means of choosing a treatment that indicates superiority to other treatments. This is fundamentally different from a conventional phase II trial that uses a formal hypothesis testing approach. The hypothesis approach requires data to prove significant difference in efficacy between the two treatments before a selection can be made. In contrast, a selection trial is designed to choose a treatment that indicates superiority over other treatments. If one in ten ALS drugs are effective and each drug is tested against placebo at 80% power, an average of 1/(0.8*0.1) = 12.5 clinical trials would be necessary to find an effective drug. A multi-drug selection design can considerably reduce this time.
Since selection is made between active treatments, the selection procedure provides no information as to whether the selected treatment is beneficial, harmful, or neither. Therefore, a secondary non-superiority test against placebo should be conducted.
Methods: In the proposed selection design phase II schema, efficacy of multiple active drugs shown to be efficacious in models (in vitro and in vivo) of ALS will be compared. Primary efficacy will be measured by analyzing the rate of decline in the revised ALS functional rating scale (ALSFRS-R) slope in the different treatment arms. For the three-arm selection trial, there will be 30 participants in each arm. With a total sample size of 90 participants, there would be an 83% chance that the best of the three treatments would be chosen if one of the three treatment groups has a 30% decrease in the rate of ALS-FRS (or equivalently, a 0.3 decrease in slope) when compared to the other two treatments after six months.
Results:Assuming a six-month (26 weeks) three arm selection trial is conducted, with 30 participants in each of the three treatment arms and ALSFRS-R measurements at 0, 1, 2, 4 and 6 months; the trial would take only 65% of the time as a conventional Phase II study (consisting of a placebo and one treatment arm) would take to find an effective treatment, with a comparable sample size of 228 patients in the conventional Phase II trial to achieve similar power.
Conclusion: Selection design can speed the search for effective drugs in situations where there are several new treatments to be tested.
P8 AN HISTORICAL DATABASE OF PLACEBO CONTROLS FOR PLANNING CLINICAL TRIALS IN ALS
MOORE D, KATZ J, MILLER R
Forbes-Norris ALS Center, San Francisco, CA, United States
Email address for correspondence: [email protected]
Keywords: database, trials, model
Background: Many large phase III clinical trials have been carried out since riluzole was approved for ALS and all have been negative. With many compounds to evaluate, more efficient trial designs, including some that will utilize historical controls, are needed to find drugs that are promising, prior to launching large, expensive phase III trials.
Objectives: To develop a database of historical placebo controls to compare with patients receiving treatment in phase II ALS trials. To examine the controls for systematic changes over time and differences among different treatment centers. To use this database to plan phase III trials and confirm results of computer simulations.
Methods: With IRB approval, we have begun to assemble a large database of placebo patients from completed ALS clinical trials. This database can be used to design clinical trials in ALS and is currently being used as a source of historical controls for a phase II trial of lithium. Patient characteristics at enrolment included in the database are: age, first symptom (bulbar or limb), time since first symptom, time since diagnosis, FVC and ALSFRS. Periodic follow-up data include: ALSFRS or ALSFRS-R, FVC, muscle strength, weight loss and vital status at last follow-up. Computer models for power calculations were based on hypothesized distributions of drug efficacies and multi-stage testing.
Results: The database currently includes de-identified data from 748 placebo patients enrolled in 7 randomized clinical trials of treatments for ALS. Results to date show: rates of decline as measured by ALSFRS and ALSFRS-R have not changed over the 10-year period 1997-2007. ALSFRS/ALSFRS-R slopes vary with symptom duration and initial values of ALSFRS/ALSFRS-R and FVC, but are independent of age, sex and site of onset. Survival time depends on patient age, initial FVC, initial ALSFRS/ALSFRS-R and symptom duration. There are large, statistically significant differences in survival but much smaller differences in ALSFRS slopes among the different trials. The differences in slopes can be explained by differences in initial patient values, but differences in survival are not so readily explained. Through computer modelling, the number of patients required to discover an effective drug for ALS can be reduced by a factor of 2 or more using historical, in place of concurrent, controls in phase II screening trials prior to definitive phase III trials.
Discussion and Conclusions: A well-defined database of patient data from clinical trials has already proven to be useful in planning clinical trials for ALS. we look forward to expanding the database, which we will make broadly available, to include patients from additional clinical trials so that we can continue to examine the variability of commonly used measures such as ALSFRS-R over time and among different trial centers.
P9 CENTRAL VENOUS CATHETER STUDY DRUG SELF ADMINISTRATION IS SAFE IN ALS
PULLEY D1, CUDKOWICZ M1, TINDALL K2, GROSSO M2, RADAKE T3, HOLMES S4, GUINGRICH S5, JOHNSTON-CREWS T6, POLAK M7, YU H1, BELLANICH M1
1Massachusetts General Hospital, Charlestown, Massachusetts, United States,2State University of New York, Syracuse, NY, United States,3Washington University, St. Louis, MO, United States,4Carolinas Medical Center, Charlotte, NC, United States,5Indiana University, Indianapolis, IN, United States,6Wake Forest University, Winston-Salem, NC, United States,7Emory University, Atlanta, GA, United States
Email address for correspondence: [email protected]
Keywords: Hickman, safety, care
Background: In the multicenter, adaptive design clinical research trial of ceftriaxone in ALS, study participants and caregivers were taught central venous catheter (CVC) care. Each site is comprised of an ALS nurse; physician's assistant; and infectious disease, interventional and ALS physicians. Teaching included standard protocol for clean and sterile technique, how to self administer study medication twice daily via CVC via mechanical timed pump. Study participants and caregivers were taught signs and symptoms of infection, possible complications related to CVC and to report findings to study staff immediately. A questionnaire was administered at the end of training to establish study participant's understanding of CVC care.
Objectives: To demonstrate that through proper education by licensed personnel, CVCs are a safe route of "self" drug administration in clinical research. To make neurology researchers aware of the feasibility of CVC usage in future research studies broadening the scope of potential investigational drugs.
Methods: Hickman CVC safety data were collected for 66 participants. Safety was assessed by adverse events deemed by the site investigators to be related or possibly related to the CVC.
Results: The 66 participants cumulatively had 24,826 days of intravenous treatment. There were 10 serious adverse events related or possibly related to CVC, an incidence of 0.4/1000 days. These included 8 reports of bacteremia incidence of 0.3/1000 days and one pulmonary embolism incidence of 0.04/1000 days. There were 8 exit site infections (non-serious); incidence of 0.3/1000 days. There were 11 line breaks or displacements; incidence of 0.44/1000 days.
Discussion: It is possible to safely administer study drug twice a day by teaching caregivers to perform the administration. The risk of bacteremia from a CVC is related to the length of time that the catheter remains in place. The safety of home administration of study drug by trained caregivers was excellent; catheter related infections occurred at a rate of 0.4 infection per 1000 days of catheter use. This is lower than infection rates of 2.9 to 11.3 per 1000 catheter days reported by Center for Disease Control in their guidelines for prevention of intravascular catheter-related infections. In a review of the literature, only one study had a lower rate of 0.34 per 1000 catheter days in a cohort 50,470 patients treated in the home setting. Additional education to minimize line breaks and tears is in place for the next phase of the study.
Conclusion: CVCs are safe if study subjects and/or caregivers are educated for CVC care and followed by licensed personnel. Future research should incorporate these findings when designing clinical research trials.
P10 SAFETY, DOSAGE, TOLERABILITY AND PHARMACOKINETICS OF TETRAHYDROCANNABINOL (THC) IN ALS-PATIENTS
FAGAGNINI S1, BALDINGER R1, BRENNEISEN R2, HARTMANN S3, GOLDMAN B1, SCHNEIDER U1, WEBER M1
1Muskelzentrum/ALS Clinic, Kantonsspital, St. Gallen, Switzerland,2Department of Clinical Research, University of Bern, Bern, Switzerland,3Palliative Care Unit, Kantonsspital, St. Gallen, Switzerland
Email address for correspondence: [email protected]
Keywords: cramps, tetrahydrocannabinol, pharmacokinetics
Background: About 10% of ALS patients use Cannabinoids for control of symptoms such as muscle cramps, spasticity and dyssomnia (1). Furthermore, Cannabinoids may also be an important disease modifying medication, as they are neuroprotective in mice (2).
Objectives: To evaluate the safety, dosage and side effects of 5mg and 10mg THC in ALS patients.
Methods: A subgroup of 7 of 24 ALS-patients (enrolled in a randomized double-blind placebo-controlled crossover study to evaluate the efficacy of THC for cramps) were evaluated. 5mg of THC (Dronabinol®) were orally ingested on the first and 10mg on the second day, respectively. THC and its metabolites were measured before and at 30, 60, 120, 240 and 480 minutes after ingestion by using gas chromatography-mass spectrometry (GC-MS) as previously published (3). At the same interval, pulse and blood pressure were measured. At 480 minutes, a standardized questionnaire concerning side effects was answered (VAS; 0 to 10 points).
Results: Mean THC-plasma levels rose from 0 to 0.7ng/mL. Mean pulse rose from 81 bpm to 90 bpm with 5mg and from 79 bpm to 90 bpm with 10mg THC, respectively. Only one patient experienced tachycardia of 105 bpm. Three patients had an exacerbation of their (already at baseline elevated) systolic and diastolic blood pressure. Concerning side effects, 5mg were well tolerated by all patients. At 10mg almost all patients had side effects. Sleepiness, euphoria and drowsiness were the most common complaints.
Discussion and Conclusions: Single doses of 5mg were well tolerated, but unlike in healthy controls, doses of 10mg were much less well tolerated. This suggests that affinity to Cannabinoid receptors (CB1 and CB2) is different in ALS patients. THC is known to have central sympathomimetic properties and at 10mg, but not 5mg, blood pressure has to be controlled carefully in patients with arterial hypertension. The potential use of THC in ALS patients will be discussed.
Reference:
- Amtmann D, Weydt P, Johnson KL, et al. Am J Hosp Palliat Care. 2004;21(2):95–104
- Raman C, McAllister SD, Rizvi G, et al. Amyotroph Lateral Scler Other Motor Neuron Disord. 2004;5(1):33–9
- Naef M, Curatolo M, Petersen-Felix S, et al. Pain 2003; 105(1–2):79–88
P11 A DOUBLE BLIND, MULTI-NATIONAL, PLACEBO CONTROLLED TRIAL OF TALAMPANEL IN AMYOTROPHIC LATERAL SCLEROSIS: STUDY DESIGN AND SUBJECT BASELINE CHARACTERISTICS
SHEFNER J1, MEININGER V2, ROTHSTEIN J3, CHIO A4, LUDOLPH A5, GENGE A6, MORA J-S7, VAN DEN BERG L8, DRORY V9, ROBBERECHT W10
1SUNY Upstate Medical University, Syracuse, NY, United States,2l'Hopital de la Petie-Salpetriere, Paris, France,3Johns Hopkins University, Baltimore, MD, United States,4University of Torino, Italy,5University of Ulm, Germany,6McGill University, Montreal, Canada,7Hospital Carlos III, Madrid, Spain,8University Medical Center Utrecht, Netherlands,9Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel,10University of Leuven, Belgium
Email address for correspondence: [email protected]
Keywords: clinical trial, glutamate, Talampanel
Background: Glutamate mediated excitoxicity plays an important pathogenic role in the progression of patients with ALS. Both glutamate receptor antagonists and drugs that up-regulate glial glutamate transport proteins prolong survival and improve function in the SOD1 transgenic mouse models. Talampanel, an orally available selective AMPA antagonist with CNS penetration, shows efficacy in multiple models of epilepsy and neuroprotection. A pilot study of Talampanel in ALS patients showed promising results in reduction rate of progression as measured by ALSFRS.
Objectives: To study the safety, tolerability and efficacy of orally administered Talampanel in subjects with ALS as compared to placebo, in a 12 month global study run concurrently in North America, Europe and Israel.
Methods: Subjects were recruited from 25 clinical trial sites in Belgium, France, Germany, Israel, Italy, Spain, Netherlands, Hungary, USA and Canada. Eligible subjects with definite, probable or laboratory probable ALS and a slow vital capacity of >70% of predicted were randomized into 3 groups: placebo, talampanel 25mg three times a day by mouth (PO TID), or talampanel 50mg PO TID. The primary outcome measure is the rate of change of ALSFRS-R as compared to placebo. Secondary outcome measures include survival, change in strength as measured by manual muscle testing, vital capacity and quality of life. Safety testing includes serial ECGs, clinical laboratory assessments, adverse events and routine medical monitoring. Last visit for the last patient is expected in April 2010.
Results: From 29September 2008 to 31March 2009, 559 subjects were enrolled (196 (35%) females; 363 (65%) males). The average number of subjects/site/month (enrolment rate) ranged between 2.28 to 7.77 across countries. Average age was 55.9 years (range: 18.9-80.8). Subjects experienced their first ALS symptom 18.7±8.4 months prior to study entry and had a diagnosis of ALS for 8.1±6.7 months prior to study entry. Mean Body Mass Index (BMI) at entry was 25.6±3.4 for males and 25.1±4.9 for females. Mean ALSFRS-R at entrance was 38.4±5.1 (range; 20-47), mean strength across 22 muscle groups was 91.0±14.4% and mean vital capacity at entrance was 93.1% (range; 68-164%).
The mean number of patients per North America (NA) site was 13 (range: 5-25). The mean per Rest of the World (ROW) site was 29 (range: 11-52). There were no apparent clinically significant variations in subject characteristics between NA and ROW (e.g. time from first symptom to diagnosis, BMI, ALSFRS-R and gender).
Discussion and Conclusions: The rapid enrolment of this study demonstrates the feasibility of performing international clinical trials in ALS. The subject's entry characteristics across countries will be discussed and will be compared to other recent ALS clinical trials.
P12 TOLERANCE AND SAFETY OF CDP-CHOLINE (CERAXON) IN MND
RAJI A, WINKLER G
Center of Neurology, Hamburg, Germany
Email address for correspondence: [email protected]
Keywords: therapy, CDP-coline, neuroprotection
Background: There is evidence that in vitro CDP-choline protects motor neurons against apoptotic changes of chronic glutamate exitotoxicity. In MND this effect could be synergistic to riluzole, a proven therapy in MND. CDP-choline clinical trials are ongoing in both ischemic and hemorrhagic stroke. Safety and possible efficacy trials had been promising in clinical stroke. Up to now there are no data concerning CDP-choline in MND.
Objectives: To prove the hypothesis that CDP-choline in patients with MND is safe and well-tolerated even in combination with riluzole. This is the assumption for a CDP-choline efficacy trial.
Methods: After giving their informed consent 10 consecutive patients with sporadic MND were included in this study. Three patients were primarily bulbar. Seven patients were continuously treated with 100mg/day riluzole. Patients with the necessity of tube feeding were excluded because of missing data about bioavailability of tube fed CDP-choline. At baseline all patients received a complete clinical examination, a blood sample and a questionaire covering all potential side effects.
Patients received oral CDP-choline in a dosage of 1500mg/day corresponding to that for the treatment of stroke. All previous medication including riluzole remained unchanged. Any new medication in the observed time interval was avoided.
The second assessment as performed at baseline followed after 6 weeks of treatment with CDP-choline.
Both assessments were compared intra-individually. Negative progression of any of the assessed parameters was listed and interpreted. The potential worsening of the neurological findings was refered to MND, if they could be classified as MND specific.
Results: The clinical examination revealed no side effects of CDP-choline. There were no cutaneous or mucosal reactions. The blood sample showed no negative changes of blood count, liver or kidney function or signs of inflammation. The questionaire revealed no symptoms related to the CDP-choline treatment. There was no measurable interaction in the patients with combined CDP-cholone and riluzole therapy. One patient complained of a headache but showed progressive sleep related hypoxic periods. The headache disappeared after ventilatory assistance. One patient had a gastrointestinal infection in the observed time interval, but tolerated CDP-choline well before and after the infection. All but the one patient with the headache summarized the treatment with CDP-choline as positive and wished to be included in the following efficacy pilot study.
Discussion and Conclusions: CDP-choline was safe and well-tolerated in a small number of patients with MND in a short time period of observation. This result is in accordance with the data from CDP-choline studies in acute stroke. Furthermore a dual glutamate exitotoxicity inhibition with CDP-choline and riluzole is well-tolerated in MND. As a consequence a CDP-choline efficacy pilot trial has started.
P13 THE POSITIVE EFFECTS OF MEMANTINE ON TAU ASSOCIATED NEURONAL DEGENERATION IN ALS: AN OPEN LABEL PILOT TRIAL
LEVINE T1, BOWSER R2, HANK N1, SAPERSTEIN D1
1Phoenix Neurological Associates, Phoenix, AZ, United States,2University of Pittsburgh, PA, United States
Email address for correspondence: [email protected]
Keywords: Rilutek, memantine, biomarkers
Background: There are several lines of reasoning to support the potential benefit of memantine in patients with ALS: 1) Memantine is an uncompetitive NMDA receptor antagonist that may reduce the effects of glutamate mediated excitotoxicity; 2) Memantine can inhibit and reverse the abnormal hyperphosphorylation of tau; 3) Memantine has been shown to prolong survival in a mutant SOD1 transgenic mouse model of ALS.
Objectives: 1) To see if ALS patients treated with a combination of rilutek and memantine progress more slowly than historical controls. 2) To follow levels of CSF biomarkers during the course of memantine treatment to explore whether changes correlate with response to treatment.
Methods: Twenty patients with ALS were enrolled in this open label pilot trial. Patients were treated with a combination of rilutek and memantine for 18 months. Lumbar punctures were performed at baseline, 6 and 12 months. CSF was analyzed in a blinded manner to assess specific biomarkers. ALSFRS was performed every three months.
Results: Compared to historical controls from other placebo controlled trials that lost an average of −0.90 points per month, the patients followed in this study for 18 months treated with memantine and rilutek lost on average −0.56 points per month. The levels of tau and neurofilament subunit (NF-H) at baseline were significantly higher in our patients with ALS compared to healthy controls. In addition, patients who progressed faster than −0.5 points per month had a higher levels of CSF tau and NF-H at baseline than those who lost less than −0.5 points per month.
Preliminary data have indicated that throughout the disease patients exhibit increased levels of CSF tau. In contrast patients in this trial showed a 27% decline in CSF tau after 12 months treatment. Further, patients whose CSF tau levels corrected back to that observed in healthy controls exhibited the slowest decline in ALS disease progression, reducing the rate of decline to -0.42 ALSFRS points per month.
Discussion: These data suggest that combination treatment with rilutek and memantine may slow down the disease course in patients with ALS. Further to this, higher levels of CSF tau and NF-H predict a faster rate of progression of the disease. Patients treated with memantine and rilutek who exhibit decreased levels of CSF tau over 12 months showed the most significant slowing of their disease progression.
Conclusion: The results of this open label pilot trial suggest that a double blind placebo controlled trial of rilutek and memantine is indicated. It is suggested that CSF tau and NF-H may be valid biomarkers for disease progression.
P14 RESULTS OF A RANDOMIZED CONTROLLED PHASE II TRIAL OF MEMANTINE FOR ALS.
DE CARVALHO M1,2, PINTO S2, ALVES M2, FERREIRA J1, EVANGELISTA T1,2, OHANA B2, COSTA J2, PINTO A2
1Department of Neurosciences. Centro Hospitalar Lisboa Norte. Hospital de Santa Maria, Lisbon, Portugal,2Neuromuscular Unit. Instituto de Medicina Molecular, Lisbon, Portugal
Email address for correspondence: [email protected]
Keywords: memantine, clinical trial, neuroprotection
Background: There is sound evidence that glutamatergic excitoxicity is associated with motor neurons demise in ALS. Memantine is a novel class of Alzheimer′s disease medication acting on the glutamatergic system by blocking NMDA glutamate receptors. This drug prolongs survival in an ALS mouse model (G93A) but there are no reports of clinical trials in human ALS.
Objectives: To report the detailed results of the clinical trial as an academic-funded, Phase II, double-blind, single-centre randomized, clinical trial of memantine 20mg/day and Riluzole vs placebo and Riluzole.
Methods: Sixty-three patients who were treated for 12 months were studied. Inclusion criteria were 18-75years of age, disease duration <36 months, probable or definite ALS (revised El Escorial criteria) and ALS-FRS score 25-38. Exclusion criteria were the presence of other medical or neurological diseases, FVC <60% predicted, ADM strength > 2 bilaterally. Primary outcome was ALS-FRS change. Secondary outcomes were FVC, muscle force as evaluated by manual testing, analogue scale for disease progression, MUNE and NI of the ADM muscles. A questionnaire regarding side-effects was applied. The patients were evaluated at diagnosis, 1 month later, 3 months after entry and every 3 months thereafter for 12 months, Full evaluation was performed at entry, at 6 months and at one year. Number of patients needed to treat was calculated taking into account a 50% change in the ALS-FRS score (power 80%, P < 0.05). Intention to treat and per-protocol analyses were performed.
Results: Both treatment and placebo groups were well balanced concerning age at onset, disease duration, gender, region of onset, ALS-FRS, FVC and body mass index. Drop-outs (8) and deaths (7) were equally distributed between groups. There was no difference between treatment groups in the primary or secondary outcomes measures at 6 months and after 12 months, with intention to treat or per-protocol analysis. Side-effects were comparable between groups.
Discussion: The effectiveness of Riluzole, an anti-glutamatergic drug, has raised the expectation that other drugs acting in the same pathway could prove efficacious. Memantine is a licensed drug with neuroprotective action in Alzheimer′s disease. This exploratory clinical trial in ALS was negative and does indicate that a larger Phase III is not promising.
Conclusions: The negativity if this trial suggests that modulating NMDA receptors by blocking glutamate transmission does not change the rate of motor neuron drop-out in ALS, in particular lower motor neurons.
P15 OXIDATIVE STRESS AND LITHIUM THERAPY IN AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
CARLESI C1, PIAZZA S1, LO GERFO A1, PASQUALI L1, FULCERI F2, FORNAI F2, SICILIANO G1
1Department of Neuroscience,2Deparment of Human Morphology and Applied Biology, University of Pisa, Italy
Email address for correspondence: [email protected]
Keywords: lithium, oxidative stress
Background: The causes of motorneuron loss in Amyotrophic Lateral Sclerosis (ALS) are still unknown, but accumulating evidence indicates that oxidative stress is involved in the pathogenesis of this disease. The only treatment approved for this disease is Riluzole, an antiglutamatergic drug, that prolongs survival in ALS patients by about two months on average. Recently, a possible role of lithium salts for the treatment of this disease has been proposed. Lithium, a well known mood-stabilizing drug used for the treatment of bipolar affective disorders, has recently been recognized as a neuroprotective factor, mainly by its effects on cellular mechanisms linked to autophagy.
Objectives: To test the effect of lithium carbonate on clinical and oxidative-stress-related laboratory parameters of 30 sporadic ALS patients (mean±SD age: 64.3±7.0 years, mean±SD duration of the disease 15.3±9.4 months) through an open label therapeutic trial,. The daily dosage of lithium has been targeted to the plasma levels ranging from 0.4 to 0.8 mEq/litre.
Methods: In the enrolled patients (20% bulbar, 30% cervical and 50% pseudopolyneuritic form), we assessed blood levels of advanced oxidation protein products (AOPP), ferric reducing ability of plasma (FRAP), total glutathione, as well as disease-related ALSFRS-r and MRC scales at baseline and at 6 months after the targeted plasma lithium level was achieved (T1 evaluation).
Results:A significant reduction of AOPP values (mean±SD: 432.4±279.8 vs 211.3±163.1 µmol/L, P: 0.005) and increase of total glutathione (mean±SD: 0.28± 0.13 vs 0.66±0.33 nmol/µl of cells, P: 0.002) was observed, as compared to the values obtained before treatment. FRAP level was increased but not significantly (Mean±SD: 0.52±0.17 vs 0.83±0.63 mmol/L Fe2 +-TPTZ, P: 0.08). Clinical evaluations at 6 months after the targeted plasma lithium level was achieved, showed a decrease of ALS-FRS and MRC scale scores compared to pre-treatment's scores.
Conclusion: Lithium therapy is able to reduce circulating levels of blood oxidative stress markers in ALS. Whether or not the modification of oxidative stress markers and anti-oxidant defence is related to a direct effect of this drug on the pathogenic mechanism of the disease is still an open question, to be addressed with long term studies in conjunction with the assessment of the clinical effects.
P16 SAFETY OF LITHIUM IN ALS PATIENTS
ANDREWS J1, CHEN IA1, PAGEOT N2, JUNTAS-MORALES R2, DALTON K1, HARRINGTON-MORONEY G1, BELL D1, RABKIN J3, MITSUMOTO H1, CAMU W2
1The Neurological Institute, Columbia University, New York, NY, United States,2CHU de Montpellier, University Montpellier, France,3Department of Psychiatry, Columbia University, New York, NY, United States
Email address for correspondence: [email protected]
Keywords: lithium carbonate, safety
Background: ALS is a fatal neurodegenerative motor neurone disorder leading to death within 3 years after onset. Recently, an open-randomized therapeutic trial suggested that ALS could be significantly slowed by lithium carbonate administered orally (1). This study generated enormous interest and many ALS patients requested lithium therapy despite its unknown efficacy and unclear safety in this population.
Objectives: To describe the clinical profile and outcome of ALS patients treated prospectively with lithium carbonate.
Methods: A total of 55 patients with ALS according to the El-Escorial criteria were treated with lithium at two centers. Group 1 included 28 patients treated in the US with IRB approval. Lithium was titrated on a weekly basis for the first month and then monthly, following the American Psychiatric Association practice guidelines. Group 2 included 27 patients in France where it is acceptable to treat ALS patients, providing the patients understand the risks and benefits of therapy. Contraindications were excluded, patients were followed quarterly and blood levels were monitored.
Results : In Group 1, 28 patients were treated with lithium. Five stopped prematurely (less than 3 months after lithium initiation) due to intolerance. Of 23 patients treated for a mean time of 6 months, 54% were male, mean age of onset was 56±11 years, mean disease duration prior to lithium was 25±16 months. Two patients died during treatment: One had a tracheostomy at 3 months and one died at 8 months. In Group 2, 27 patients were treated with lithium. Six stopped prematurely: two had intolerance; three perceived rapid worsening; and one suddenly died. Of 21 patients treated for a mean time of 6 months, 48% were male, mean age of symptom onset was 54±12 years and mean disease duration prior to lithium was 36±20 months. Four patients died during treatment within 7 months.
The rate of weight loss on lithium was greater (Group 1: 0.49 kg/month; Group 2: 0.46 kg/month) compared to the rate of weight loss prior to lithium (Group 1: 0.01 kg/month, P = 0.04; Group 2: 0.04 kg/month,P = 0.02). The rate of ALSFRS decline after lithium treatment was also greater (Group 1: 1.5 points (pts)/month; Group 2: 0.95 pts/month) compared to prior to lithium (Group 1: 1.1pts/month; Group 2: 0.64pts/month).
Conclusion: The experience of lithium therapy among ALS patients in the centers was consistent and an increased weight loss on lithium therapy was noted. Weight loss cannot be excluded for being responsible for global worsening. Additionally, patients died while on lithium therapy which is different from that published previously. Worsening with lithium therapy should be considered, as it has already been observed in prior drugs that were thought to be promising. These data underline the need to strictly monitor lithium therapy. Only a well-designed randomized controlled trial can determine true efficacy and side effect profile.
References:
- Fornai F, et al. PNAS 2008; 105(6):2052–2057
Acknowledgements: MDA, Wings Over Wall Street
P17 CHRONIC LITHIUM DOSING IN A SIBLING-MATCHED, GENDER BALANCED, INVESTIGATOR-BLINDED TRIAL USING A STANDARD MOUSE MODEL OF FAMILIAL ALS HAS NO THERAPEUTIC BENEFIT
VIEIRA F, KIDD J, THOMPSON K, PERRIN S, LINCECUM J, GILL A
ALS Therapy Development Institute, Cambridge, MA, United States
Email address for correspondence: [email protected]
Keywords: SOD1 G93A, therapy, lithium
In any animal model of human disease a positive control therapy that demonstrates efficacy in both the animal model and the human disease can validate the application of that animal model to the discovery of new therapeutics. Such a therapy has recently been reported using chronic lithium carbonate treatment(1) and demonstrated therapeutic efficacy in both the high-copy SOD1 G93A mouse model of familial ALS and in human patients. However, recent work by the Bendotti group did not identify any therapeutic or neuroprotective effect of lithium in SOD1G93A female mice (2)
As part of an independent effort to identify a positive control therapy, we tested chronic lithium dosing in a sibling-matched, gender balanced, investigator-blinded trial using the high-copy (average 23 copies) SOD1 G93A mouse (n = 27-28/group). Lithium-treated mice received single daily 36.9 mg/kg i.p. injections from 50 days of age to death. This dose delivered 1 mEq/kg (6.94 mg/kg/day lithium ions). 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. This study did not show lithium efficacy by any measure.
Reference:
- Fornai et al. Proc Natl Acad Sci U S A. 2008 12;105(6): 2052–7
- Pizzasegola C, et al. Amyotroph Lateral Scler 2009;23:1–8
P18 CHRONIC APOCYNIN DOSING IN A SIBLING-MATCHED, GENDER BALANCED, INVESTIGATOR-BLINDED TRIAL USING A STANDARD MOUSE MODEL OF FAMILIAL ALS PROVIDES NO THERAPEUTIC BENEFIT
GILL A, MORENO A, KIDD J, VIEIRA F, LINCECUM J, PERRIN S
ALS Therapy Development Institute, Cambridge, MA, United States
Email address for correspondence: [email protected]
Keywords: SOD1 G93A, therapy, apocynin
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to motor neuron degeneration. Encouraging recent studies employing NOX knockout mice or treatment with a NOX inhibitor in the SOD1 G93A preclinical mouse model of ALS, concluded that dysregulated redox stress caused by NADPH oxidases Nox1 and Nox2 significantly influences the progression of motor neuron disease. The NOX inhibitor apocynin is a prodrug that is used to inhibit all members of the NOX family. Treatment of SOD1 G93A mice with the NOX inhibitor apocynin was shown to significantly prolong their average life span by approximately 4 months (1). Seeking to verify this as a positive control therapy in the SOD1 G93A mouse model, chronic apocynin dosing was tested at 150 mg/kg/day formulated in drinking water (an efficacious dosage level in the referenced study). Two sibling matched, gender balanced, investigator blinded trials were executed with two start ages, 30 days (n = 27/group) and 50 days (n = 26/group). The drug was determined to have been stable in its formulation throughout the study by LC/MS. Water consumption was measured twice weekly throughout the course of each study to confirm dosing estimates and rule out taste aversion to apocynin. 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 paresis onset and age at death were compared using Kaplan-Meier and Cox proportional hazards analyses.
Although the intended 150 mg/kg/day apocynin consumption occurred throughout the treatment period, neither treatment regimen showed apocynin efficacy by any measure in these rigorously powered, investigator-blinded, gender-balanced, sibling-matched survival studies.
References:
- Harraz et al. J. Clin. Invest 2008;118(2):659–70
P19 A THERAPEUTIC AGENT (NDDPX08) DELAYS DISEASE PROGRESSION, IMPROVES MOTOR FUNCTION AND PROLONGS SURVIVAL IN A MOUSE MODEL OF ALS
TANAKA K1, KANNO T1,2, YANAGISAWA Y1,2, AOKI M3, HADANO S2, ITOYAMA Y3, OGINO M4, IWASAKI Y5, YOSHII F6, IKEDA J-E1,2
1Neugen Pharma Inc., Kanagawa, Japan,2Department of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, Japan,3Department of Neurology, Tohoku University Graduate School of Medicine, Miyagi, Japan,4Department of Neurology, Kitasato University East Hospital, Kanagawa, Japan,5Department of Neurology, Toho University Omori Hospital, Tokyo, Japan,6Department of Neurology, Tokai University School of Medicine, Kanagawa, Japan
Email address for correspondence: [email protected]
Keywords: oxidative stress, transgenic mouse, post-onset administration
Background: ALS is a devastating neurodegenerative disorder characterized by the selective loss of upper and lower motor neurons. Unfortunately, our understanding of the molecular pathogenesis of ALS is still limited. There is no perfectly validated molecular target in ALS. However, advancements in motor neuron disease research provide compelling evidence that oxidative stress is heavily implicated in neurodegenerative diseases including ALS. Neuronal apoptosis inhibitory protein (NAIP, aka BIRC1) selectively suppresses oxidative stress-induced cell death and the up-regulation and ectopic expression of NAIP exerts the neuroprotective activity in in vivo model of human cerebral ischemia. Based on the converging findings, NAIP is a potent molecular target in ALS drug discovery. A neurotropic compound library (1,000 chemicals) containing medical drugs was screened in NAIP-based drug screening. Among hit compounds, this study's interest converged on NDDPX08, which is being used in another neurodegenerative disease and has shown to selectively protect neuronal cell against oxidative insults in vitro.
Objectives: To conduct an efficacy test of NDDPX08 from disease onset in a congenic SOD1H46R mouse model of ALS, including neuropathological analysis.
Methods: Intraperitoneal injection of NDDPX08 (0, 1 and 10 mg/5 mL/kg) in 0.5% CMC-Na was conducted once a day starting at the day at which SOD1H46R mice exhibited signs of disease onset (post-onset administration). NDDPX08 efficacy in SOD1H46R mice was analyzed as follows; 1) behavioral testing (balance beam test, vertical pole test and footprint analysis) was performed once a week to assess motor function; 2) neuropathological analysis by immunohistochemical staining was performed by using the mice at 22 weeks of age.
Results: The post-onset administration of NDDPX08 extended survival interval of SOD1H46R mice after the sign of the onset compared to vehicle control, 39.0±7.6 days (1mg/kg, n = 33) and 39.4±7.1 days (10mg/kg, n = 38) vs. 35.2±6.7 days (vehicle, n = 31), P < 0.05 by log-rank test. Behavioral analyses showed the preservation of motor function in the NDDPX08-treated mice when compared with the vehicle-treated mice. The treatment of NDDPX08 reduced the immunoreactivity for GFAP and Iba-1 in spinal cord of SOD1H46R mice. Furthermore, the delayed loss of ChAT-positive neurons in the anterior horn was observed in the NDDPX08-treated mice as compared with vehicle-treated mice.
Discussion and Conclusions: The post-onset treatment of NDDPX08 preserved the motor function and delayed the disease progression in a SOD1H46R mouse model. Thus, NDDPX08 is a highly promising candidate drug to the treatment of ALS. The clinical research of NDDPX08 as add-on to riluzole, a double-blind placebo-controlled trial with ALS patients, is being conducted.
Acknowledgements: This work was supported by the National Institute of Biomedical Innovation and the Ministry of Health, Labour and Welfare.
P20 PROOF-OF-CONCEPT FOR A NOVEL SMALL MOLECULE HSP90 INHIBITOR FOR SBMA
PANDEY U1,2, JAFFER Z3, CHEN R3, TAYLOR JP1, WINSSINGER N4
1St. Jude Children's Research Hospital, Memphis, TN, United States,3NexGenix Pharmaceuticals, Inc., New York, NY, United States,4Universite Louis Pasteur, Strasbourg, France,2Louisiana State University Health Sciences Center, New Orleans, LA, United States
Email address for correspondence: [email protected]
Keywords: Hsp90, SBMA, neuroprotection
Background: A hallmark of motor neuron diseases (MNDs) and other neurodegenerative diseases is the misfolding and aggregation of mutant or otherwise mis-regulated proteins. In the case of spinal bulbar muscular atrophy (SBMA), a mutant poly-glutamine (poly-Q) expanded androgen receptor (AR) becomes mislocalized and aggregates to form inclusions. It has been shown that inhibition of the heat shock protein 90 (Hsp90) promotes the upregulation of multiple HSPs, the clearance of misfolded proteins and is neuroprotective in models for MNDs, including SBMA and other neurodegenerative diseases. Furthermore, the fact that AR is a client protein of Hsp90 provides additional rationale for Hsp90 inhibition as a therapeutic strategy for SBMA. NexGenix Pharmaceuticals is developing a series of fully synthetic small molecule Hsp90 inhibitors based on the natural radicicol scaffold. The lead compound, NXD30001, is a potent and relatively safe Hsp90 inhibitor which demonstrates in vivo efficacy in murine cancer models. In addition, NXD30001 readily penetrates the blood-brain barrier and accumulates in the brain, making it a viable therapeutic candidate for MNDs. This study hypothesized that NXD30001 will clear poly-Q AR aggregates in diseased motor neurons and will be efficacious in a drosophila model for SBMA.
Objectives: To develop an oral therapy for SBMA and MNDs based on the novel Hsp90 inhibitor series. This study's short-term objectives are to demonstrate efficacy for NXD30001 in a drosophila model for SBMA and to obtain data elucidating the mechanism(s) of protection by Hsp90 inhibition.
Methods: The efficacy of NXD30001 was evaluated in a drosophila model for SBMA that recapitulates many key features of human disease. The SBMA model also exhibits poly-Q length dependent impairment of ubiquitin proteasome function (1). In addition, the effects of NXD30001 on poly-Q AR localization and solubility as well as the validility of Hsp90 inhibition in the SBMA model both genetically and by assaying for biomarkers for inhibition was examined. The effects of NXD30001 in a conditional model for proteasome impairment was also investigated.
Results: NXD30001 treatment strongly suppressed neurodegeneration caused by expression of expanded poly-Q AR in a drosophila model of SBMA. Interestingly, NXD30001 rescued the degenerative phenotype in a dose dependent manner. NXD30001 treatment also rescued lethality caused by ubiquitous proteasome impairment in a drosophila model.
Discussion and Conclusions: These data provide evidence supporting the rationale for Hsp90 inhibition with NXD30001 in motor neuron degeneration associated with SBMA. Furthermore, the data presented here indicate a role for Hsp90 inhibition in suppressing neurodegeneration in the absence of a functional proteasome. Based on these results, pharmacologic inhibition of Hsp90 with NXD30001 is an attractive therapeutic strategy for SBMA and potentially other MNDs.
References:
- Pandey UB, Batlevi Y, Baehrecke EH, et al. Autophagy 2007;3:643–5
P21 EVALUATING THE THERAPEUTIC POTENTIAL OF A NOVEL SMALL MOLECULE HSP90 INHIBITOR FOR MOTOR NEURON DISEASES
DURHAM H1, CHA J1, JAFFER Z2, CHEN R2, WINSSINGER N3, RUBENSTEIN A2
1Montreal Neurological Institute, McGill University, Montreal, Qc, Canada,2NexGenix Pharmaceuticals, Inc.,, New York, NY, United States,3Université Louis Pasteur, Strasbourg, France
Email address for correspondence: [email protected]
Keywords: Hsp90 inhibitor, neuroprotection, heat shock proteins
Background: Heat shock proteins (HSPs) facilitate proper folding or clearance of misfolded proteins. Upregulation of multiple HSPs is protective in cell culture and transgenic mouse models of motor neuron diseases. Such treatments include the Hsp90 inhibitors, geldanamycin (GA) or 17-AAG, which increase expression of HSPs ostensibly through disrupting complexes of Hsp90 with factors involved in signaling the heat shock response and/or facilitating clearance. Toxicity and poor penetrability of the blood-brain barrier limit therapeutic use of these agents in neurological diseases. In order to identify analogues with more favourable pharmacokinetic profiles, NexGenix Pharmaceuticals is developing a series of fully synthetic small molecule Hsp90 inhibitors based on the natural radicicol scaffold. The lead compound, NXD30001, is effective in the nanomolar range, exhibits greater safety factor in terms of induction of HSPs relative to cytotoxicity and accumulates in nervous tissue.
Objectives: To evaluate NXD30001 for its ability to induce HSPs, promote degradation of Hsp90 client proteins and confer neuroprotection in models of motor neuron disease.
Methods: Effects of NXD30001 on induction of HSPs (Hsp70, Hsp40 and Hsp90), turnover of Hsp90 client proteins and cytotoxicity were evaluated in BT474 breast cancer cells and in dissociated cultures of E13 mouse spinal cord-DRG, in comparison to other Hsp90 inhibitors (geldanamycin, 17-AAG and radicicol). Expression of each HSP was evaluated by Western blotting. Expression in neural cell types, motor neurons in particular, was evaluated by immunocytochemistry. Activation of the major heat shock transcription factor Hsf1 was determined by gel shift assay. Neuroprotective properties of NXD30001 are being investigated in primary culture models of familial ALS (cultured motor neurons expressing ALS-causing mutant proteins-including mutant SOD1) using multiple endpoints of toxicity (formation of inclusions, mitochondrial rounding, mitochondrial and cytosolic calcium and viability).
Results: NXD30001 depleted Hsp90 client proteins at lower concentrations than inducing Hsp70 expression in BT474 cells. In spinal cord-DRG cultures, NXD30001, GA and 17-AAG induced dose-dependent expression of Hsp70 and Hsp40, but not Hsp90 at non-cytotoxic concentrations. NXD30001induced expression of Hsp70 predominantly in motor neurons, whereas Hsp40 was upregulated in a larger number of cells. Hsf1 hyperphosphorylation, indicative of activation, was increased in a dose-responsive manner.
Discussion and Conclusions: Radicicol, the parent compound of NXD30001 transiently induced HSPs in cultured motor neurons, but only at cytotoxic concentrations. NXD30001 induced more sustained expression of multiple HSPs and was less cytotoxic. Hsp90 inhibitors show promise in therapy of disorders of protein misfolding including ALS-GA is highly neuroprotective in this primary culture model of fALS1. Due to its more favourable pharmacological profile and ability to accumulate in nervous tissue, NXD30001 is an attractive candidate for testing in animal models and for therapeutic development.
P22 SOD1G93A MICE TREATED WITH A SOLUBLE FORM OF THE ACTIVIN RECEPTOR TYPE IIB (ACTRIIB) HAVE INCREASED LEAN MUSCLE MASS AND IMPROVED GRIP STRENGTH
LACHEY J, WONG V, PULLEN A, UCRAN J, SEEHRA J
Acceleron Pharma, Cambridge, MA, United States
Email address for correspondence: [email protected]
Keywords: myostatin, muscle hypertrophy, activin receptor type IIB
Background: Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of muscle mass, strength and mobility due to the deterioration of motor neurons. SOD1G93A mice express a mutant form of the human Cu/Zn superoxide dismutase (SOD1) gene and display progressive muscle weakness, muscle atrophy and paralysis similar to ALS. Activin receptor type IIB (ActRIIB) mediates the effects of multiple ligands of the TGF-beta superfamily which act as negative regulators of muscle mass. Administering RAP-031, a fusion protein derived from a form of the extracellular region of ActRIIB fused to an IgG Fc domain, prevents signalling of these ligands resulting in dramatic increases of muscle mass and strength.
Objectives:To evaluate the effects of RAP-031 on muscle mass and strength in SOD1G93A mice with advanced disease.
Methods: 125-day old B6.Cg-Tg(SOD1G93A)1Gur/J mice, a SOD1G93A strain maintained on the C57BL/6 background with a life expectancy of approximately 160 days, were divided into male and female groups that received either control vehicle or RAP-031 twice-a-week for 3 weeks. NMR scans were performed at baseline (125 days of age) and study end to assess body composition. Forelimb grip strength was measured at baseline and 143 days of age.
Results: At study end, control group male mice lost 8.3% of body weight (day 125: 25.4±1.3g, day 146: 23.3±1.2g P < 0.001); however, the body weight of RAP-031-treated males did not change significantly compared to baseline (day 125: 25.7±1.2g, day 146: 26.2±1.7g P = not significant (ns)). In female mice, body weight of control animals did not change significantly (day 125: 18.8±1.0g, day 146: 18.7±0.9g P = ns); however, RAP-031 treatment resulted in a 12% increase in body weight (day 125: 19.1±1.0g, day 146: 21.4±1.2g P < 0.001). The different effects on body weight of RAP-031 and control animals were attributable to changes in lean mass, as assessed by NMR scans. At 146 days of age, RAP-031-treated male and female mice had 11.5% and 14.5% more lean tissue mass, respectively, compared to vehicle-treated mice (P < 0.01). Treatment with RAP-031 also mitigates loss of motor function, as assessed by forelimb grip strength. Male control animals lost significantly more grip strength (-39.6%) than RAP-031 treated animals (-18.6%) in the day 143 trial compared to the baseline grip strength trial. Similarly, female control animals lost significantly more grip strength (-34.0%) compared to RAP-031 treated animals (-21.6%) across the two trials.
Discussion and Conclusions: These data demonstrate that RAP-031 increases body weight, lean tissue mass and improves muscle function in late-stage SOD1G93A mice. Therefore, treatment with ACE-031, the fully human analogue of RAP-031, could provide clinical benefit for ALS patients with advanced disease. A Phase I clinical study of ACE-031 is currently underway.
P23 NEUROPROTECTIVE ACTIVITY OF NOVEL MULTIMODAL IRON CHELATING DRUGS IN MOTOR NEURON-LIKE NSC-34 CELLS AND TRANSGENIC MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
KUPERSHMIDT L1, WEINREB O1, MANDEL S1, AMIT T1, CARRI MT2,3, YOUDIM MBH1
1Technion, Haifa, Israel,2University of Rome "Tor Vergata", Rome, Italy,3Laboratory of Neurochemistry, Fondazione Santa Lucia, Rome, Italy
Email address for correspondence: [email protected]
Keywords: multimodal iron chelating drugs, hypoxia-inducible factor, G93A-SOD1 mouse model
Background: Amyotrophic Lateral Sclerosis (ALS) is a multifactorial disease where several pathological mechanisms have been suggested to contribute to neurodegeneration. Novel therapeutic approaches for the treatment of ALS may, therefore, consider drug candidates designed specifically to act on multiple central nervous system targets. A multifunctional, non-toxic, brain-permeable iron chelating drugs, M30 and HLA20, possessing the N-propargylamine neuroprotective moiety of the anti-Parkinson's drug, rasagiline (Azilect) and the iron-chelating moiety of VK28 have recently been synthesized.
Objectives: To examine the neuroprotective activities of our multifunctional drugs in the motor neuron-like cell line NSC-34 and transgenic mouse model of ALS.
Methods: The protective action of M30 and HLA20 were investigated in NSC-34 naïve and NSC-34 expressing mutant G93A-SOD1 protein cell lines. The effects of M30 and HLA20 on protein and mRNA levels of apoptosis/survival-related markers and signaling pathways were examined by Western immunoblotting, RT-PCR and immunohistochemical analyses. To investigate the effect of M30 in vivo, G93A-SOD1 transgenic mice were randomly assigned to vehicle or M30. M30 (1mg/kg) and vehicle were administered by oral gavage method, four times a week, starting at 70 days of age. Various behavioral tests were performed to assess the effects of the drugs on the onset and extent of neurological deficits.
Results: M30 and HLA20 possess a wide range of pharmacological activities in mouse NSC-34 motor neuron cells, including neuroprotective effects against hydrogen peroxide- and 3-morpholinosydnonimine (SIN-1)-induced neurotoxicity; induction of differentiation and up-regulation of hypoxia-inducible factor (HIF)-1α and HIF-target genes (enolase1 and vascular endothelial growth factor). Both compounds induced NSC-34 neuritogenesis, accompanied by a marked increase in the expression of brain derived neurotrophic factor (BDNF) and growth associated protein (GAP)-43, which was inhibited by PD98059 and GF109203X, indicating the involvement of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) and protein kinase C (PKC) pathways, respectively. A major finding was the ability of M30 to significantly extend the survival of G93A-SOD1 ALS mice and delay the onset of the disease.
Discussion: The neuroprotective/neuro-rescue potential of the novel compounds may result from their multifunctional activities: a) similar to rasagiline and other propargyl-containing molecules they activate the canonical survival pathways, MEK and PKC, associated with elevation of BDNF; b) the promotion of neurite sprouting may result from activation of the above pathways, combined with the ability of iron-complexing molecules to interfere with cell cycle progression via deactivating cell cycle regulators, thus triggering differentiation through various iron-associated biological events; c) activation of HIF-1 and induction of its pro-survival/ neuroprotective target genes, an action that has been ascribed to iron chelation and inhibiton of HIF prolyl 4-hydroxylases (PHDs).
Conclusions: These data indicate that these new multifunctional molecules may have the potential to modify the progression of ALS.
P24 OVEREXPRESSION OF G-CSF PROTECTS MOTONEURONS AGAINST APOPTOTIC DEATH IN PERIPHERAL NERVE AXOTOMY OF NEONATAL MICE
HENRIQUES A, PITZER C, SCHNEIDER A
SYGNIS Bioscience GmbH & Co. KG, Heidelberg, Germany
Email address for correspondence: [email protected]
Keywords: G-CSF, axotomy, neonatal
Granulocyte colony stimulating factor (G-CSF) is known as a hematopoietic growth factor that is essential for generation of neutrophilic granulocytes. Potent neuroprotective and regenerative properties of G-CSF in the CNS have recently been uncovered. The G-CSF receptor and G-CSF itself are expressed in large motoneurons, G-CSF protects motoneuronal cell lines and improves outcome in the SOD1 (G93A)-transgenic mouse model for amyotrophic lateral sclerosis (ALS). Due to the pleiotrophic effects of G-CSF it is not fully clear at present if the effects seen in ALS models are solely due to motoneuron-specific effects, or if other components are also important.
This study sought to further delineate the anti-apoptotic potential of G-CSF on motoneurons in vivo in a different model, sciatic nerve axotomy in newborn mice. The sciatic nerve axotomy model was used in neonatal mice overexpressing G-CSF in the CNS. This is a well established experimental paradigm of motoneuron death. This study found that G-CSF transgenic animals displayed higher numbers of surviving lumbar motoneurons 4 days following axotomy.
In this model of pure apoptotic cell death, the protective effects of G-CSF indicate direct actions of G-CSF on motoneurons in vivo. This strengthens the argument for activity of this direct mechanism of action also in complex motoneuron disease models like the SOD1 (G93A) transgenic mouse.
P25 RESCUE OF MOTOR NEURONS IN ALS BY TARGETING THE BNIP3 CELL DEATH PATHWAY
KONG J, WENG J, ZHANG S, YANG X, MA X
University of Manitoba, Winnipeg, Manitoba, Canada
Email address for correspondence: [email protected]
Keywords: mitochondria, oxidative stress, BNIP3
Oxidative stress, mitochondrial dysfunction and morphologically necrotic-like motor neuron death are major features in ALS. Previously it has been shown that oxidative stress provides a redox signal to activate hypoxia-inducible factor 1α (HIF-1α), which is the primary, if not the only, transcriptional factor for the death-inducing gene BNIP3 (1). Expression of BNIP3 caused a caspase-independent form of neuronal cell death in vitro and in vivo (2).
Here it is shown that BNIP3 expression was inducted at the onset of the disease in transgenic mice expressing the G93A and the G37R mutations of SOD1. BNIP3 was not detectable in the brain of control animals nor in the G93A or G37R mice before the onset of disease. Levels of BNIP3 expression increased with disease progression as determined by immunohistochemistry, Western blotting and RT-PCR analyses. The expressed BNIP3 was found to be primarily localized to motor neurons. BNIP3 was not detectable in the liver, kidney and lung tissues from the same groups of G93A and G37R animals that showed high levels of BNIP3 in the spinal cord. BNIP3 was detected in the mitochondrial membranes after alkaline extraction, indicating that the expressed BNIP3 was active because inactive BNIP3 is known to be dissociated from mitochondria after alkaline treatment.
To further determine the role of BNIP3 in mutant SOD1-induced neuronal death, a lentiviral shRNA vector targeting the nucleotides 167-188 of the BNIP3 mRNA, which was able to almost completely inhibit BNIP3 expression (2), was injected into the lumbar spinal cord of the G93A mice at the age of 8 weeks. Animals injected with a scramble shRNA vector were used as controls. Inhibition of BNIP3 by RNAi significantly increased the number of axons in the L5 ventral roots (p = 0.015). Analysis of axon size distribution showed clearly the protection of middle to large (larger than 6 mm in inner diameter) axons by the lentiviral BNIP3 shRNA vector. The BNIP3 pathway was further analysed and found that BNIP3 interacted with the ion channel VDAC to induce mitochondrial release of endonuclease G leading to a caspase-independent apoptosis. To look for an inhibitor for the BNIP3 pathway, the small chemical necrostatin-1 was identified that was able to inhibit BNIP3 cell death pathway by preventing integration of BNIP3 to the outer membrane of mitochondria. The results demonstrate that BNIP3 plays a role in mediating mutant SOD1-induced motor neuron death. The BNIP3-induced cell death pathway provides a molecular linkage for mitochondrial degeneration, oxidative stress and caspase-independent neuronal death. Necrostatin-1 appears to be a potent inhibitor for the BNIP3 pathway and may be a new therapy for ALS.
Reference:
- Zhang S et al. Brain Res 2007;1138:221–230
- Zhang Z et al. Stroke 2007;38:1606–1613
P26 LIF DECREASES AMPA RECEPTORS IN THE BRAINSTEM AND SENSORIMOTOR CORTEX OF SOD1G93A(G1H) MOUSE MODEL OF ALS
MA C1, LIU J1,2, ZANG D1
1Department of Neurology, Tianjin First Center Hospital, Tianjin, China,2Department of Laboratory Medicine, Tianjin First Center Hospital, Tanjin, China
Email address for correspondence: [email protected]
Keywords: AMPA, LIF
Background: Motor neurons degenerate in patients with amyotrophic lateral sclerosis (ALS) and the mechanism of this disease is still unclear. Endogenous neural stem cells (ENSCs) are considered to be a promising way of treating ALS in the future.
Objectives: To observe the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the brainstem and sensorimotor cortex of the SOD1G93A (SOD1) transgenic mouse model of ALS and identify whether leukemia inhibitory factor (LIF) can reduce the expression and inhibit the neurotoxicity of AMPA receptors.
Methods: Fourty-eight transgenic mice resourced from B6SJL-TgN (SOD1-G93A) which expressed a human mutation in the superoxide dismutase 1 gene were used in this study. These mice were separated into 3 groups; ALS control (ALSC); LIF treated (LIF) and normal control (CON) group each group contained 16 mice (8 males and 8 females). Radioligand binding assays were carried out in this experiment to estimate the expression of AMPA receptors in brainstem and sensorimotor cortex in each of the three groups. The number of AMPA receptors was calculated using the confocal counting system.
Results: A total of 48 mice were involved in the result analysis. 1) Compared with the CON, at the time points of postnatal 90-day and 120-day, the number of AMPA receptors on the motor neurons of brainstem increased to 1.86 and 3.30 times in the ALSC group (both P < 0.01), 1.16 times (P > 0.05) and 2.48 times (P < 0.01) in the LIF group, respectively. Compared with the ALSC group, the expression of AMPA receptors in brainstem and sensorimotor cortex in the LIF group decreased significantly (P < 0.01). 2) The number of AMPA receptors on the motor neurons of sensorimotor cortex increased to 1.71 times and 4.95 times in the ALSC group (both P < 0.01), while 1.18 times (P > 0.05) and 3.35 times (P < 0.01) in the LIF group, respectively at days 90 and 120 after birth. The expression of AMPA receptors decreased in the sensorimotor cortex in the LIF group compared with the ALSC group (P < 0.01).
Discussion: AMPA is a glutamate receptor found on the postsynaptic membrane and is related to the activity of excitotoxic glutamate. In this study the number of AMPA receptors in an ALS model increased at the late stage of disease, which may contribute to the loss of neurobehavior function. Further to this, the number of AMPA receptors was also found to be decreased in the LIF treated animal model, this mechanism needs to be further investigated.
Conclusion: The number of AMPA receptors in the brainstem and sensorimotor cortex of ALS mice significantly enhances at the middle and late stages of this disease, which is associated with glutamate-mediated toxicity of motor neurons.
P27 MULTIPLE ADMINISTRATIONS OF HUMAN MARROW STROMAL CELLS THROUGH CEREBROSPINAL FLUID MAY PROLONG SURVIVAL OF SOD1-G93A MICE
ZHANG C1, ZHOU C2, ZHAO C1, GU R1, LIU Z1
1Department of Neurology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China,2Department of Neurology, Affiliated Hospital of Medical College, Qingdao University, Qingdao, Shandong, China
Email address for correspondence: [email protected]
Keywords: SOD1 G93A mice, marrow stromal cells, cerebrospinal fluid
Background: The blood-brain barrier (BBB) is the main obstacle for cell therapy in neurological disorders such as amyotrophic lateral sclerosis (ALS). Intrathecal injection is a potential method for cell transplantation because it would bypass the BBB. we investigated the effects of human marrow stromal cells (hMSC) delivered through cerebrospinal fluid (CSF) in SOD1-G93A mice, a transgenic mouse model of ALS.
Methods: 5 x 105 hMSC were delivered into the CSF of SOD1-G93A transgenic mice at the age of 8 weeks (single transplantation group) or 8, 10 and 12 weeks (multiple transplantation group). Clinical features, weight, hanging wire test and motor neuron count were observed to assess disease progression in the SOD1-G93A mice. Immunohistochemistry was performed with a human-specific antibody against HuNu to measure the distribution of hMSC in the lumbar spinal cord parenchyma of SOD1-G93A mice at the age of 15 weeks.
Results: A single transplantation of hMSC did not have a beneficial effect in SOD1-G93A mice. Multiple transplantations of hMSC attenuated weight loss, enhanced motor performance, decreased motor neuron loss and, importantly, increased survival in SOD1 transgenic mice. However, only a few hMSC delivered through the CSF migrated into the lumbar spinal cord parenchyma of SOD1 mice.
Conclusions: Multiple administrations of hMSC through CSF may have a therapeutic effect in SOD1-G93A mice, although limited numbers of cells migrate into the lumbar spinal cord parenchyma. It is likely that the hMSC remaining in CSF are responsible for the effect in SOD1-G93A mice.
P28 CELLULAR FATES OF SYSTEMICALLY DELIVERED NEURAL STEM CELLS IN A RAT MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
MITRECIC D, NICAISE C, POCHET R
Universite Libre de Bruxelles, Bruxelles, Belgium
Email address for correspondence: [email protected]
Keywords: neural stem cells, cell fates, cell transplantation therapy
Background: One of the common drawbacks of focal injections of stem cells in ALS experimental models is the limited number of regions which can be targeted by applying such procedure.
Objectives: As ALS is defined by widely spread pathogenetic processes affecting the motor cortex and the spinal cord, it was of high interest to test whether blood delivery route could yield satisfactory level of cell survival and differentiation. Herewith, we report our work with neural stem cell (NSC) therapy of ALS rat animal model (G93A).
Methods: In order to obtain an insight into cellular fates after blood stream injection, we developed a specific protocol: it included isolation of NSC from the cortex of 16 day old rat embryos ubiquitously expressing GFP, cultivation of neurospheres and obtaining of uniform 100% nestin/GFP positive population of NSC.
Results: Injection of GFP positive NSC into the blood stream of affected animals resulted in the presence of NSC within the affected CNS. The cell amount found within their wild type counterparts was significantly lower. NSC remained in an undifferentiated state for the first day, but after 3 days started to differentiate: approximately 35% were GFAP positive (astrocytes) and 25% were Map2 positive (neurons). The remaining 40% cells were undifferentiated (30%) or expressed other markers including O4 (oligodendrocytes) and Iba1 (microglia). Analyses of programmed cell death using a caspase specific assay revealed that approximately 35% of Map2+ cells and less than 15% of GFAP+ cells died within 7 days after injection possibly caused by failure of cell integration within the host tissue. However, after 14 days, the remaining Map2+ and GFAP+ cells continue to be present in the motor cortex.
Conclusions: Prolonged survival of injected NSC cells and the presence of synaptic marker synaptotagmin reveal that transplanted cells can survive and integrate within disease affected regions. Thus, these results suggest that systemic route for delivery of NSC in ALS affected CNS represents a promising strategy for cell transplantation therapy of ALS.
P29 EVALUATION OF THE THERAPEUTIC POTENTIAL OF INTRASPINAL TRANSPLANTATION OF HUMAN UMBILICAL CORD BLOOD CELLS IN G93A ALS MICE
KNIPPENBERG S1,2, WILLENBROCK S3, MURUA EH3, MEIER M4, HASS R5, DENGLER R1,2, PETRI S1,2
1Department of Neurology, Hannover Medical School, Hannover,2Center for Systems Neuroscience (ZSN), Hannover,3Small Animal Clinic, University of Veterinary Medicine, Hannover,4Department of Cardiology, Hannover Medical School, Hannover,5Department of Gynecology and Obstetrics, Hannover Medical School, Hannover, Germany
Email address for correspondence: [email protected]
Keywords: adult stem cells, G93A mice, neuroprotection
Background: Cellular therapies appear to be a promising option for both familial and sporadic ALS even though there are still major limitations to overcome. The focus of cell therapies in ALS currently lies in the generation of a neuroprotective environment for degenerating motor neurons by transplantation of non-neuronal cells. Adult stem cells from umbilical cord blood present an option worth further investigations as they are barely immunogenic, ethically inoffensive, non-tumorigenic and easily available. Despite their hematopoetic origin, human umbilical cord blood cells (hUCBC) were shown to differentiate in vitro along neuronal or glial lineages.
Methods: G93A SOD1 transgenic mice received intraspinal injections of hUCBCs positive for CD34+ and expanded in presence of stem cell growth factors for 8 days before (d40) or after symptom onset (d90). Lentiviral eGFP-labeling was used for post mortem detection of transplanted cells by fluorescence microscopy. For in vivo detection by MRI, cells were labeled with iron oxide nanoparticles. Survival time of G93A-ALS transgenic mice transplanted with either hUBCB or vehicle before or after symptom onset was assessed. The locomotor activity was determined weekly via rotarod and footprint analyses. In addition, immunohistological and molecular biological evaluation of spinal cord tissue was performed.
Results: With the aid of a stereotactic frame a reproducible and defined administration of cells into the affected region was found to be possible and the blood brain barrier can be overcome. Detection of transplanted cells was also possible both post mortem and in vivo.
Discussion: For the most efficient administration of stem cells for the treatment of ALS, a local injection of the cells appears to be plausible. Intraspinal transplantation into the lumbar spinal cord does not cause persisting motor impairment or major tissue lesions. Before translation into clinical trials can be considered, basic questions need to be clarified, such as migration, functional state and interaction with the environment of the transplanted cells in vivo.
P30 ACETYLCHOLINESTERASE IS OVEREXPRESSED IN ALS MICE AND ANTISENSE INHIBITION PROLONGS SURVIVAL
GOTKINE M, ROZENSTEIN L, EVRON T, EINSTEIN O, ABRAMSKY O, ARGOV Z, SOREQ H, ROSENMANN H
Hadassah University Hospital, Jerusalem, Israel
Email address for correspondence: [email protected]
Keywords: antisense oligonucleotides, acetylcholinesterase, mouse model
Background:Acetylcholinesterase (AChE) may be involved in ALS through various mechanisms, including exacerbation of excitotoxicity and accelerated apoptosis. Most deleterious effects of AChE are unrelated to its enzymic activity. AChE enzyme inhibitors such as neostigmine are not candidate treatments in ALS as they upregulate a deleterious form of AChE known as "read-through" AChE (AChE-R). AChE antisense oligonucleotides (EN101) decrease AChE-R levels in muscle and spinal-cord neurons and alleviate symptoms of myasthenia gravis in humans. The beneficial clinical effects of EN101 in ALS mice have previously been described.
Objective: To further investigate the role of AChE-R in ALS by checking AChE-R levels in ALS mice. To investigate the clinical, pathological and biochemical effect of EN101 on ALS mice when compared to treatment with neostigmine.
Design and Methods: 1) Untreated G93A ALS mice and wildtype(WT) mice were sacrificed for comparison of AChE-R levels in spinal neurons (3 mice per group). 2) ALS mice received either saline (NS, n = 35), EN101( n= 35), inverse-EN101(n = 15) or neostigmine (n = 15). Treatments were administered daily from 5-weeks (oligonucleotide dose = 200 µg/kg, neostigmine dose = 1mg/kg). 3) disease-onset and survival were recorded. Additional mice (7 from the EN101 group and 3 from the NS group) were sacrificed for motor-neuron count and AChE-R level measurement (quantitative ELISA).
Results: AChE-R staining density was significantly higher in G93A mice compared to WT( 80µ vs 140µ, P < 0.01). Compared to NS, EN101 resulted in a non-significant 5-day delay in disease onset and a 9-day delay in death (P < 0.001). Neostigmine treatment resulted in earlier onset (5 days, P < 0.05) and death (6 days, non-significant). No significant clinical effects were observed with inverse EN101. Mice treated with EN101 had significantly higher cell-counts at 15 weeks of age compared to untreated mice. AChE-R staining density was significantly lower in EN101 treated ALS mice compared to untreated ALS mice. (110µ vs 140µ, P < 0.01).
Conclusions and Relevance: High AChE-R levels in ALS mice supports the role of AChE-R in ALS.Treatment with EN101 reduces AChE-R expression in ALS mice to levels found in WT mice.The beneficial effects of EN101 coupled with the detrimental effects of neostigmine (which raises AChE-R) further supports the role of AChE-R in ALS.
P31 POTENTIAL NANOTECHNOLOGY PLATFORM FOR ALS: EFFICACY IN THE MSOD1G93A MOUSE MODEL
WILEY N, MITCHELL R, MADHANKUMAR AB, SIMMONS Z, CONNOR J
Pennsylvania State University, Hershey, PA, United States
Email address for correspondence: [email protected]
Keywords: nanotechnology, animal model, inflammation
Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease hallmarked by the selective death of motor neurons accompanied by inflammation and microgliosis, the latter of which has been shown to have a neurotoxic role in the pathogenesis of the disease. To this end, several therapeutic agents have aimed to curb microglia activation, albeit with little success. To increase site-specific concentration and thus drug efficacy, a liposomal drug delivery system with targeting moieties specific to microglial cell surface markers was utilized to deliver the anti-inflammatory compound minocycline.
Objectives: To develop a targeted nanotechnology platform that will improve treatment options for individuals suffering from ALS. To address this objective we provide a proof of concept study using minocycline contained in liposomes and delivered via intracerebroventricular (ICV) pump to mice carrying the Cu/Zn superoxide dismutase-1 (SOD1) mutation.
Methods: To test these hypotheses, the liposomes were conjugated with an LPS targeting moiety that is recognized by the Toll4 receptors on microglia. The drug was delivered continuously via an ICV osmotic pump that was implanted at 70 days of age. Control animals had a pump inserted that delivered only a buffer. Efficacy was determined by the time to disease onset in treatment versus control groups, as well as the time to endpoint stages. Motor performance was measured with a rotarod apparatus with disease onset defined as the point in which a mouse can no longer stay on the rotating rod for >1 s.e.m. of the number of times it fell during the pre-symptomatic phase.
Results: Control mice have an average disease onset at 107 days of age, whereas mice receiving targeted and non-targeted minocycline liposomes have an average disease onset at 113.4 (P < 0.05) and 118.3 days (P < 0.001), respectively. Targeting the nanovesicles to microglia appears to significantly slow later disease progression.
Discussion and Conclusions: In the present study, both age of disease onset and lifespan are significantly increased with minocycline liposomal treatments. These effects were obtained with concentrations that were lower than those given in previous studies using intraperitoneal or oral routes of delivery. This is a consistent advantage of delivering therapeutic agents with a nanotechnology platform. Minocycline has not been successful in treating ALS in humans. However, this study serves as a proof of concept for a drug delivery platform in which the "payload" of the nanovesicles can be easily modified to other compounds, including cDNA or siRNA, which could be delivered via an intraventricular route with the goal of altering the disease course of ALS.
P32 INVESTIGATING NOVEL ROUTES OF DELIVERY FOR GENE THERAPY APPROACHES TO ALS
BARNES L1, NING KE2, MITROPHANOUS K1, AZZOUZ M2, RALPH S1
1Oxford BioMedica Ltd, Oxford, United Kingdom,2University of Sheffield, United Kingdom
Email address for correspondence: [email protected]
Keywords: gene therapy, lentivector, VEGF
Background: It has previously been shown that delivery of vascular endothelial growth factor (VEGF) using a retrogradely transported EIAV lentiviral vector pseudotyped with a rabies-G envelope protein (MoNuDin®) prolongs survival and delays motor neuron degeneration in a mouse model of ALS (SOD1G93A) following intramuscular delivery. Clinical advancement of this approach has been limited by the necessity to administer large amounts of vector to many muscle groups. For these reasons alternative routes of administration are currently being investigated with the aim of maximising delivery of VEGF to motor neurons and minimising the dose of vector required.
Objective: Identification of novel routes of delivery for therapeutic application of MoNuDin.
Methods: A bicistronic EIAV vector encoding the human VEGF and the reporter gene GFP (EIAV-VEGF-GFP) was constructed to allow visualisation of transduced cells. The vector was administered using several routes of delivery including intraspinal, intraventricular and direct injection into the deep cerebellar nucleus (DCN). Transgene expression was evaluated in target tissues at one month post vector delivery.
Results: Intraventricular delivery of EIAV-VEGF-GFP vector pseudotyped with the VSV-G envelope protein resulted in some transduction of the ependymal cell layer lining the ventricular system as assessed by GFP immunohistochemistry. Direct delivery of an EIAV vector pseudotyped with a rabies-G envelope protein to the DCN resulted in efficient retrograde transport of vector to several brain stem nuclei, including the ventral tegmental nucleus, inferior olive and medial nuclei. Assessment of vector transduction and transgene expression following direct intraspinal delivery is under investigation.
Discussion and Conclusions: The intraventricular delivery route provides an interesting alternative site of delivery for MoNuDin. Although modest transduction efficiency was achieved in this study, improvement may be made by altering the pseudotype of the EIAV vector to confer a higher tropism for the ependyma and by increasing vector dose.
Although the brain stem nuclei transduced by DCN delivery are not necessarily those most relevant for ALS therapy, the secreted VEGF protein would be likely to spread from the sites of transduction to the motor neuron populations of the brain stem.
The results of the intraspinal delivery of EIAV vectors will also be presented at the meeting.
P33 GENE THERAPY APPROACHES TO NEUROPROTECTION IN EXPERIMENTAL MODELS OF AMYOTROPHIC LATERAL SCLEROSIS
NANOU A, HIGGINBOTTOM A, VALORI C, WYLES M, NING K, SHAW P, AZZOUZ M
The university of Sheffield, United Kingdom
Email address for correspondence: [email protected]
Keywords: gene therapy, lentiviral vectors, anti-oxidant genes
Background: Amyotrophic lateral sclerosis (ALS), is a progressive neurodegenerative disorder characterized by the loss of upper and lower motor neurons with no effective treatment to date. Although it has a multi-factorial aetiology, oxidative stress is hypothesized to be one of the key pathogenic mechanisms since in cellular models of ALS decreased expression of anti-oxidant genes and disrupted anti-oxidant defence in mitochondria has been observed.
Objectives: It has been previously found that three specific anti-oxidant genes, namely peroxiredoxin 3, leukotriene B4, 12-hydroxydehydrogenase and nuclear factor erythroid 2-related factor 2 (Prdx3, LTB412HD and NRF2), are down-regulated in the presence of mutant SOD1 (mutated in approximately 5% of ALS cases). The main aim of this project is to alter the expression of one or more target genes using viral vectors and study the effect on the vulnerability of cells expressing mutant SOD1 both in vitro and in vivo.
Methods: Two different cellular models (NSC34 cell line or NSC34 carrying a common mutation in SOD1 – G93A mutation and mouse primary motor neurons) were used in order to achieve the over-expression of the target genes using a lentiviral delivery system. After verification of the target gene over-expression through western blotting and Q-PCR, the oxidation stress levels were measured using 2’,7’-dichlorofluorescein (DCF) dye. Cell survival of NSC34 cells was assessed by 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay.
Results: The cells transduced with the lentiviral vectors expressing the anti-oxidant genes achieved stable over-expression of the target proteins and preliminary data showed promising neuroprotection properties of most of the genes. In particular, wild type NSC34 cells transduced with lentiviral vectors expressing LTB412HD showed no neuroprotection after inducing oxidative stress and this gene was not studied any further. When NSC34 cells were transduced with viral vectors expressing NRF2, over-expression of NRF2 was not stable over time possibly due to tight regulation with maximum over-expression seen only 2 days post transduction. However, when NSC34 G93A cells were transduced, the over-expression remained stable for at least 10 days and a 60% reduction in oxidative stress was seen (N = 3, P < 0.001). Finally, NSC34 cells transduced with lentiviral vectors expressing Prdx3 exhibited a consistent 30% protection against induced oxidative stress (serum withdrawal or menadione, N = 5, P < 0.01) and 40% reduction of oxidative stress in NSC34 cells carrying the G93A SOD1 mutation at the basal level (N = 5, P < 0.001). The neuroprotective properties of Prdx3 are currently being verified by cell viability assays in mouse primary neurons.
Discussion and Conclusions: The lentiviral delivery of anti-oxidant genes is proven to be an attractive strategy to protect motor neurons long term. Thus, the most promising gene (Prdx3) will be taken forward to the animal model of ALS, with ultimate aim the translation of this strategy to the clinic.
P34 INTRAVENOUS OPTIMIZED SCAAV9 VECTORS FOR TRANSGENE DELIVERY TO ADULT MOTOR NEURONS.
DUQUE S, MARAIS T, JACOB A, ASTORD S, BARKATS M
INSERM UMR 974, Paris, France
Email address for correspondence: [email protected]
Keywords: adeno-associated vector, gene therapy, motor neuron
Motor neuron diseases (MNDs) such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are neurodegenerative disorders of the central nervous system (CNS) characterized by motor neuron (MN) degeneration. To date, there is no treatment for these disorders mainly due to the blood brain barrier (BBB) which prevents drug diffusion into the CNS. Gene transfer to the spinal cord based on intramuscular injections of viral vectors has been successfully used in laboratory animals to produce therapeutic proteins within MNs. However, this strategy is clinically difficult because of the size and the number of muscles that should be targeted in the whole human body.
Here the potential for MN transduction of an alternative gene therapy strategy was analysed, based on the intravenous administration of recombinant vectors derived from the adeno-associated-virus of serotype 9 (scAAV9). Single stranded (ss) and double-stranded self-complementary (sc) AAV1 and 9 vectors were injected intraperiteonaly, intramuscularly or intravenously in neonatal or adult mice and analyzed transgene expression in the CNS. These vectors encoded either the murine secreted alkaline phosphatase (mSEAP) or the green fluorescent protein (GFP) under control of the cytomegalovirus (CMV) promoter. These results showed, for the first time, that intravenous scAAV9 enabled widespread, non-invasive and long-term MN transduction in adult mice, without pharmacological disruption of the BBB.
As the scAAV9 vectors used in this study expressed the reporter gene under control of the CMV promoter, systemic vector-mediated transduction was not restricted to MNs. This could yield severe side effects due to undesired transgene expression (for example, trophic factors) in tissues such as the heart or the liver. We thus proposed to optimize the scAAV9 vectors in order to restrict transgene expression to neurons following intravenous injection, thereby reducing possible side effects due to ectopic transgene expression. In this aim, the study used and compared two strategies: (i) the use of the Synapsin neurospecific promoter (ii) the use of Neuron-Restrictive Silencer Elements (NRSEs) placed upstream from the ubiquitous phosphoglycerate promoter. The potential of these new scAAV9 vectors for achieving high and specific transgene expression in the CNS was assessed both in culture and in mouse models of MND.
P35 FRAGMENT C OF TETANUS TOXIN, MORE THAN A CARRIER: NOVEL PERSPECTIVES IN NON-VIRAL ALS GENE THERAPY
CALVO A1, MORENO M1, PENAS C2, MANZANO R1, OLIVÁN S1, MUÑOZ MJ1, ZARAGOZA P1, AGUILERA J3, NAVARRO X2, OSTA R1
1LAGENBIO-I3A, University of Zaragoza, Miguel Servet, Zaragoza (Spain), Spain,2Group of Neuroplasticity and Regeneration, Institut de Neurosciènces, Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain and CIBERNED, Spain,3Institut de Neurosciènces, Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
Email address for correspondence: [email protected]
Keywords: SOD1 (G93A) mice, fragment C of tetanus toxin, non-viral gene therapy.
Background: The non-toxic carboxy-terminal fragment of tetanus toxin heavy chain (TTC) has been implicated in the activation of cascades responsible for trophic actions and neuroprotection by inhibition of apoptosis (1,2). In particular, in Amyotrophic Lateral Sclerosis (ALS), a progressive neurodegenerative disorder involving the loss of cortex, brainstem and spinal cord motor neurons, TTC may act as a potential therapeutic agent. TTC is sufficient for neuron binding, internalization, retrograde and transynaptic transport (3), thus becoming an attractive and promising therapeutic agent.
Objectives: To investigate the effects of intramuscular delivery of a TTC-encoding plasmid in a neurodegenerative mouse model. To analyze clinical features and focus on gene expression and protein assays to detect anti-apoptotic processes in the spinal cord of one of the best characterized ALS mouse models SOD1G93A mice.
Methods: Naked DNA encoding for TTC was injected intramuscularly into the quadriceps and triceps muscles in SOD1G93A mouse model, neuromuscular function and clinical behaviours were monitored until end stage. Gene expression variations in spinal cord and muscle tissues due to TTC treatment were assayed by real-time PCR. Protein expression variations in spinal cord due to TTC treatment were assayed by Western blot analysis. Statistical significance was evaluated by one- or two-factor ANOVA and posterior Post-hoc analysis.
Results: These results indicate that TTC treatment ameliorated the decline of hindlimb muscle innervation, significantly delayed the onset of symptoms and functional deficits, improved spinal motor neuron survival and prolonged lifespan by 12.8 days in the TTC-treated group compared to the vehicle-plasmid group. Furthermore, caspase-1 and caspase-3 proapoptotic genes were down-regulated in the spinal cord of treated mice. Western blot analysis showed that the active form of caspase-3 was also down-regulated. Survival signals, such as Akt phosphorylation, were also up-regulated after TTC treatment compared to vehicle-plasmid mice. TTC treatment revealed variations in calcium-related gene expression (Ncs1 and Rrad) in spinal cords of TTC-treated SOD1G93A animals.
Discussion and Conclusions: These results suggest that fragment C of tetanus toxin, TTC, provides a potential therapy for neurodegenerative diseases. Antiapoptotic pathways and survival signal activation were increased in spinal cords of TTC-treated mice. These results also indicate that the delivery of this gene by means of intramuscular injection of naked-DNA is successful, plays a significant role in neuroprotection and delays disease progression.
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