P01 NEW APPROACHES TO THE ANALYSIS OF OBSERVATIONAL DATA ON THE USE OF RILUZOLE IN ALS
MITCHELL D1, TAYLOR S2, CALLAGHER P1, DODD S2
1Preston MND Care & Research Centre, Preston, Lancashire, United Kingdom, 2Centre for Medical Statistics and Health Evaluation, Liverpool, United Kingdom
E-mail address for correspondence: [email protected]
Keywords: Riluzole, effectiveness, analysis
Background: Although further study of the efficacy of riluzole was suggested by NICE in 2001 all new data since then has come from observational studies rather than RCT's.
Objective: To apply more rigorous methodology for analysing observational data through auditing the use of riluzole in a large MND Centre.
Methods: Data on survival from first symptom were recorded in 548 ALS patients. Date of diagnosis was available in 450 of these along with data on gender, onset site and onset age. We used Kaplan-Meier (KM) analyses and log-rank tests to assess categorical variables (including treatment) for univariable association with survival, and univariable Cox regression to assess significance of continuous variables. Variables with a univariable p-value of ≤0.2 were considered for inclusion in the multivariable Cox regression model. Riluzole and PEG treatment were fitted as time varying covariates. We examined survival from onset and diagnosis as two separate outcomes.
Results: Univariable KM analyses showed that onset site, riluzole and PEG status were significant predictors of survival from onset. Multivariable Cox regression showed that patients on riluzole had a 67% increased hazard of death (HR 1.67, 95%CI: 1.38–2.02) compared to patients who had not taken riluzole.
For analysis of survival from diagnosis, we only included patients who started riluzole within 95 days of diagnosis. In this model, only PEG was fitted as a time-varying covariate and age at diagnosis was used in place of age at onset. Multivariable Cox regression showed that age at diagnosis (p = 0.001), riluzole group (p = 0.018) and PEG group (p < 0.001) were significant predictors of survival from diagnosis. This model showed an advantage for riluzole (HR 0.78, 95%CI: 0.64–0.96). A patient receiving PEG has more than three times the hazard of death (HR 3.40, 95%CI: 2.71–4.27) compared to a patient not receiving PEG at that time.
Discussion: When analysing survival from onset using riluzole as a time-dependent covariate, only that proportion of the patient's journey while actually on riluzole was attributed to the treatment. The remainder of the patient's survival experience contributed to the non-treatment effect. The apparent disadvantage for riluzole in this analysis arose because of the time between first symptom and starting riluzole. Patients on riluzole were inevitably more advanced in the disease journey and thus at greater risk of death.
Patients generally start riluzole soon after diagnosis. The analysis based on survival from diagnosis showed a survival advantage broadly consistent with RCT data. We suggest that survival from diagnosis gives a more balanced comparison than survival from first symptom in interpreting the results of observational studies of riluzole.
P02 A PATIENT-LED TRIAL OF LITHIUM IN ALS USING THE INTERNET
WICKS P1, MASSAGLI M1, FROST J1, MACEDO H2, FELZER K3, HEYWOOD J1
1PatientsLikeMe, Cambridge, Mass, United States, 2Patient, Distrito Federal, Brazil, 3Caregiver, Pasadena, Ca, United States
E-mail address for correspondence: [email protected]
Keywords: Lithium, internet, trial
Background: In November 2007, a patient relayed an Italian news report about a promising result of a human trial to an online ALS community, PatientsLikeMe. The report described the apparent slowing of symptoms of ALS in patients taking lithium carbonate. In advance of formal publication of the study, a number of patients were prescribed lithium by their doctors off-label. A group of these patients began completing self-report ALSFRS-R scales and combining their data in a shared, online spreadsheet. To eliminate duplicate effort and to encourage standardised data collection, PatientsLikeMe enhanced its treatment recording and community report tools, with the result that over 160 ALS patients are tracking their use of lithium and reporting on disease progress and side-effects. This is alongside the remainder of over 1,500 ALS PatientsLikeMe users who are not using lithium, creating a unique opportunity to study the impact of lithium on a patient community.
Objectives: To study the evolution of a patient-lead open-label trial and examine how estimated effect sizes and other data in a non-experimental setting might inform the design of formal trials.
Methods: Patients who had obtained lithium carbonate through their own doctors were invited to enter self-report ALSFRS-R data on a regular basis, along with their lithium blood levels and any side effects they experienced. Participants were provided with a graphing tool that allowed them to compare, at a glance, the progression of all other patients taking lithium. Optional filters allowed users to customise the data displayed on the graph. All available data are being processed to develop statistical models of (a) the decision to use lithium, (b) the impact of lithium on ALS patients.
Results: At the time of writing, 162 patients with ALS were tracking their use of lithium, 10 times larger than the group reported in the Italian study. Before commencing lithium, the average rate of onset was 0.57 (S.D. 0.05) ALSFRS-R points per month, indicating a relatively slowly progressive sample relative to the known literature. Contrary to the Italian study, several users in our sample have died since starting lithium, and side effects have been reported including perceived worsening of respiratory function. Results for any detectable drug effects will be presented at the Symposium.
Discussion: The high level of internet use by ALS patients, the rapid flow of scientific information, and the ability to prescribe drugs off-label for compassionate use means that many patients will not wait patiently in line for a placebo-controlled trial. With sufficient improvements to data quality and safety monitoring, the use of a site such as PatientsLikeMe for a distributed clinical trial in ALS could prove time-efficient and cost effective.
P03 GRANULOCYTE COLONY-STIMULATING FACTOR ADMINISTRATION IN AMYOTROPHIC LATERAL SCLEROSIS: A PHASE I/IIA, DOUBLE-BLIND, RANDOMIZED, PLACEBO-CONTROLLED CLINICAL TRIAL
NEFUSSY B1, ARTAMONOV I1, NAGLER A2, DEUTSCH V1, NAPARSTEK E1, DRORY V1
1Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel, 2Sheba Medical Center, Tel-Hashomer, Israel
E-mail address for correspondence: [email protected]
Keywords: granulocyte colony-stimulating factor, GCSF, clinical trial
Background: Recombinant human granulocyte colony-stimulating factor-GCSF (Neupogen®) is an approved drug used to mobilize CD34+ hematopoietic stem cells from the bone marrow to the peripheral blood. In previous studies it was shown that mobilized cells are able to cross the blood-brain barrier and can be found after treatment in brain tissue.
Objective: We hypothesized that autologous peripheral blood progenitor cells, mobilized from the bone marrow with GCSF, would slow down disease progression in patients with amyotrophic lateral sclerosis (ALS).
Methods: Patients with clinically definite or probable ALS of less than six years duration, with a forced vital capacity of at least 50% of predicted and limitation of motor function in at least one limb were randomly assigned in a double-blind fashion to receive either GCSF or placebo. GCSF was injected subcutaneously at a dose of 5µg/Kg once a day, for four consecutive days. Each treatment cycle was repeated every three months for a total of four cycles. The primary outcome measure was the decline over time of the revised ALS Functional Rating Scale score (ALSFRS-R). Secondary outcome measures were forced vital capacity, a manual muscle strength megascore derived from all limbs, compound muscle action potential amplitudes, neurophysiological index, a visual analogue scale for quality of life and tracheostomy-free survival.
Results: 39 patients (25 males) were enrolled, of them 19 received the active drug. 17 patients completed the one-year trial. The decline over time of ALSFRS-R, as well as of the secondary outcome measures, was not significantly different between placebo and active treatment groups. GCSF treatment did not alter survival of ALS patients in this study.
Conclusion: The presented treatment protocol had no beneficial effect on disease progression in patients with ALS.
P04 RECOMBINANT HUMAN ERYTHROPOIETIN THERAPY IN AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
KIM SH1, KIM HY1, KOH SH1, CHO GW1, KIM HJ1, OH SC1, KANG BY1, KIM KS2, CHOI MR2, LEE KW3
1Department of Neurology, Hanyang University Hospital, Seoul, 2Bioengineering Institute, CoreStem Inc., Seoul, 3Department of Neurology, Seoul National University Hospital, Seoul, Republic of Korea
E-mail address for correspondence: [email protected]
Keywords: Erythropoietin, intravenous, ALS functional rating scale-revised
Background: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative condition characterized by loss of upper and lower motor neurons in the brain and spinal cord. In spite of numerous researches, riluzole remains the only effective therapy, but with only minimal effects on survival. In previous reports, erythropoietin (EPO) has been shown to have neuroprotective effects, including anti-apoptotic and anti-inflammatory actions on central nervous system in vivo as well as in vitro.
Objectives: To evaluate the effect of EPO on the modification of disease progression and to ascertain most appropriate candidates for EPO therapy.
Methods: Fifty-seven patients with probable and definite ALS on the basis of El Escorial criteria were recruited for this study. After the lead-in period for 3 months to assess the progression of disease, recombinant human erythropoietin (rhEPO) was injected intravenously (35,000 IU/30min/50ml) four times at an interval of one month (0, +1, +2, +3 month) to rhEPO group. Patients were monitored by clinical evaluation including ALS Functional Rating Scale-Revised (ALSFRS-R), subjective symptoms and signs by 6 months follow up period. As a primary outcome measure, the change of ALSFRS-R score was compared between rhEPO (n = 21) and control (n = 20) groups. Additionally, the demographic and clinical characteristics in patients showing beneficial effects on EPO therapy were evaluated as a secondary outcome analysis. To assess the safety, patients were closely monitored for adverse events including thrombocytosis, malignant hypertension, and myocardial ischemia. Besides hemodynamic monitoring, each patient had complete blood cell count, blood urea nitrogen, creatinine, electrolytes, glucose and liver enzyme.
Results: When compared with the control group, the rhEPO treatment could reduce the change of ALSFRS-R during the first half follow up period. In addition, these disease modifying effects were enough to improve the functional deficits during 2 to 3 weeks after each rhEPO injection in 10 patients and more prominent in patients with short duration and rapid progression among the rhEPO group. The intravenous administration of rhEPO also had modest but significant beneficial effects including improvement of muscle power, decreased drooling, mood stabilization, and decreased spasticity. No significant adverse effects were noted. The hematocrit, hemoglobin, red blood cell counts, liver enzyme, and blood pressure remained stable.
Conclusions: Intravenous rhEPO in ALS is both safe and well tolerated. Moreover, this approach may be worthy of further investigation with various treatment interval and dosage.
P05 A PHASE II CLINICAL TRIAL USING THALIDOMIDE FOR THE TREATMENT OF AMYOTROPHIC LATERAL SCLEROSIS IS NOT EFFECTIVE
STOMMEL E1, COHEN J1, FADUL C2, COGBILL C4, GRABER D4, KINGMAN L2, MACKENZIE T3, HARRIS B4
1Section of Neurology 2 Section of Oncology, Department of Medicine, 3 Department of Medicine, Dartmouth Medical School, 4Department of Pathology, Dartmouth Medical School, Lebanon, NH, United States
E-mail address for correspondence: [email protected]
Keywords: tumor necrosis factor-alpha, thalidomide, clinical trial
Background: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by degeneration of upper and lower motor neurons. No treatment halts disease progression, which inevitably results in paralysis. Pathogenesis of motor neuron loss is likely initiated by genetic and/or environmental factors, but the mechanisms ultimately remain unclear. Immune activation in the central nervous system (CNS) is postulated to play an important role. Information gathered from transgenic rodent models of ALS and human post-mortem studies implicates that the cytokine tumor necrosis factor – alpha (TNF-α) is involved and may be detrimental to motor neurons. In ALS patients and transgenic models serum levels and TNF-α soluble receptors are elevated compared to healthy controls. Increased TNF-α levels may induce apoptosis or disrupt axonal transport.
Thalidomide is a small molecular-weight pharmacological agent that penetrates the blood brain barrier. It has been shown to selectively reduce levels of TNF-α pre-transcriptionally and post-transcriptionally. It also inhibits angiogenesis by targeting basic fibroblast growth factor (bFGF). Thalidomide and other TNF-α antagonists have prolonged disease duration and extended the lifespan of transgenic animal models of ALS.
Objectives: An open-label, single-institution clinical phase II trial tested the efficacy of thalidomide in patients diagnosed with ALS. The primary endpoint in the trial was the ALS Functional Rating Scale. Secondary endpoints were, pulmonary function testing, survival stratified for newly diagnosed and progressive disease, toxicity, quality of life, and cytokine measurements. Controls from an existing ALS data base at another institution were used for statistical analysis.
Results: Twenty-three patients enrolled in the trial and were evaluated every 3 months for up to 9 months and in 2 cases longer. The major side effects of thalidomide included deep-vein thrombosis and peripheral neuropathy. Minor side effects included constipation, rash, sedation, bradycardia and elevated liver functions. Despite promising evidence from in vitro and in animal model studies, the trial was terminated early when we found that thalidomide had no statistically significant benefit on functional assessment scoring, survival, or pulmonary function. Serum cytokine analysis and post-mortem pathology are reported and discussed.
Discussion: The lack of efficacy may relate to any number of issues including the lack of appropriate receptor and intracellular targeting with thalidomide, a lack of efficacy related to an insufficient dosing, and the late initiation of therapy because of the inherent late diagnosis of the disease. Further clinical trials targeting TNF-α might be warranted, but this small trial does not provide support that reducing TNF-α levels improves ALS disease progression.
P06 CEFTRIAXONE TREATMENT IS ABLE TO REDUCE OXIDATIVE STRESS IN ALS PATIENTS
CARLESI C1, ANNALISA LG1, PIAZZA S1, MOLESTI E1, SERRADORI M2, SICILIANO G1
1Department of Neuroscience, Neurology, Pisa, Italy, 5Pulmonary Unit, Cardio-Thoracic Department, Pisa, Italy.
E-mail address for correspondence: [email protected]
Keywords: Oxidative stress, Glutamate, Ceftriaxone
Introduction: Glutamate-mediated neurotoxicity has been suggested as a mechanism of motor neuron death. Ceftriaxone has been considered putative therapeutic agent for ALS, by a modulation of astroglial GLT1 glutamate transporter expression.
Objectives: To report laboratory results of blood oxidative stress markers in ALS patients treated with a first course of ceftriaxone (Rocefin, 2g/day, for 14 days) within a planned one year study with bimonthly drug cycles.
Methods: The study has included 38 patients, 28 males and 10 females (mean age 62.2±SD 12.1-years). 14 patients presented with the bulbar form of disease, 10 the classic form and the remaining cases a predominant lower motorneuron onset form. Disease was evaluated by ALS-FRS-r, MRC scale, dynamometric test and respiratory functionally parameters as FVC, MIP and MEP. Blood levels of oxidative stress markers as glutathione (GSH), the advanced oxidation protein products (AOPP) and the ferric reducing ability of plasma (FRAP) were evaluated with colorimetric and spectrometric methods.
Results: Before therapy no correlation was found between oxidative stress marker blood levels, the duration and the severity of the disease. After ceftriaxone therapy AOPP levels decrement significantly (p < 0.001), while GSH and FRAP showed a slight not significant decrement. Respiratory functionally parameters, in particular FVC, were unchanged function.
Discussion: Based on the results, short term therapy with ceftriaxone is able to reduce circulating levels of some markers of oxidative stress in ALS. Whether or not this finding is antibacterial related to a direct effect of this drug on pathogenic mechanism of the disease or to an indirect effect mediated by its antibacteric action is still an open question, this is to be addressed with long term studies in conjunction with the assessment of the clinical effects.
P07 COMBINATION HIGH-THROUGHPUT SCREENING TO IDENTIFY EFFECTIVE THERAPEUTIC CHEMICAL COMBINATIONS TO UPREGULATE ASTROGLIAL PROTEIN EXPRESSION
LI Y, ROTHSTEIN J, SATTLER R
Johns Hopkins University, Baltimore, MD, United States
E-mail address for correspondence: [email protected]
Keywords: astroglia, combination, EAAT2
Background: Glutamate is the predominant excitatory amino acid neurotransmitter in the mammalian CNS. Glutamate transporter, EAAT2 (or GLT1), is the physiological dominant astroglial protein to inactive synaptic glutamate. Animal studies show that EAAT2 dysfunction leads to excessive extracellular glutamate, and may contribute to various neurological disorders including amyotrophic lateral sclerosis (ALS), Huntington's disease, and growth of glioblastomas. Modulation of transporters may also be valuable in behavioral disorders such as depression. Through a recent screen of 1040 FDA approved drugs, we had revealed that beta lactam antibiotics protect against neural injury and delay disease in ALS mice through increasing GLT1 /EAAT2 expression. Thus, agents that upregulate the EAAT2 transporter might be beneficial in ALS and other neurological/psychiatric disorders by augmenting astrocytic uptake of glutamate. However, the beta lactam antibiotics had poor efficacy in increasing transporter protein.
Objectives: We proposed to employ a new drug discovery approach for identifying effective and synergistic combinations of compounds. Multi-component therapies through deliberate mixing of drugs have a successful history in a number of medical areas including HIV and oncology. Here we report our effort to develop a novel combinational high-throughput screening (cHTS) paradigm for identifying effective combinations of compounds to upregulate EAAT2 expression.
Methods: We generated fetal derived-human immortalized astroglial cells that are stably expressing a firefly luciferase reporter under the control of the human EAAT2 promoter (2.5 kb). Through cHTS of a library of up to 2000 FDA approved compounds and natural products, we discovered a serious of candidate drug-pairs to show synergistic effect to turn on the EAAT2 promoter. We have quantified synergy in our screening experiments and identified combinations in which one drug enhances the potency or the intrinsic activity of the other drug. Follow-up analysis employed EAAT2 BAC luciferase reporter mice for whole animal validation of in vivo efficacy
Conclusions: One of our lead compounds, JR01, acts not only by increasing the intrinsic activity of its pair, but also mediating a shift in potency. We are further testing our leading candidates to see if they increase endogenous EAAT2/GLT1 expression in our in vitro and in vivo systems. Our studies provide potential neurotherapeutics by modulating the activity of astroglial proteins via gene activation.
P08 TREAT ALS PLATFORM: BRINGING STANDARDS AND EFFICIENCIES TO CLINICAL RESEARCH IN ALS
SHERMAN A, WALLACE K, BADAYAN I, BELOUIN F, GRASSO D, ROYCE-NAGEL G, SWARTZ A, SWEET B, THORNELL B, ANDRES P, CUDKOWICZ M
Massachusetts General Hospital, Charlestown, MA, United States
E-mail address for correspondence: [email protected]
Keywords: Clinical Trials Management System, TREAT ALS, Clinical Trial
Background: The Northeast ALS consortium (NEALS) was awarded a grant through The ALS Association's (ALSA) Translational Research Advancing Therapy for ALS (TREAT ALS) initiative to form the TREAT ALS/NEALS Clinical Trials Network with the goal to “translate research advances rapidly into clinical trials for patients with ALS”.
Objective: To build a distributed Web-based clinical trial management platform to support the goals of the TREAT ALS initiative that will bring efficiency, scalability, regulatory compliance and standardization in conducting clinical trials and biomarker studies to ALS.
Methods: The TREAT ALS platform has been built on top of the existing PharmaContent system utilizing the “state machine” model for workflow configuration and customization; it is comprised of several modules that serve the following purposes: 1) To acquire and maintain the sites’ information including contact data, sites’ experiences with the TREAT ALS network, sites’ IRB information and a list of equipment at each site. 2) To track the site and site members’ regulatory documentation including professional licenses as well as compliance documents. 3) To study-specific workflow applications that help to expedite, regulate and standardize processes in clinical trials/studies management through using the following methods, i) Protocol module to track study protocols and protocol amendments at all participating institutions ii) Supply chain management module that allows sites and coordination centres to create orders of study supplies and track their status. iii) Financial module for automatic invoice generation for participating institutions based on either procedure- or visit-specific model. 4) Creation and maintenance of a bio-specimens inventory that includes samples from 15 clinical trials/studies. Samples may be searched upon by multiple criteria. 5) Standardization of data acquired from multiple NEALS studies and trials, which enables data analyses from multiple data sources.
Results: The TREAT ALS platform is currently deployed at the Neurology Clinical Trials Unit of MGH and is available to all member sites of the NEALS consortium. Four new investigators use this platform for their studies. Site and member information has been collected and is being maintained. The system issues regular notifications to the sites to update and upload expired and next-to-expiration regulatory documents. It became possible to track financial information for each study in real time knowing site payments and study supplies expenses.
Conclusion: Several new clinical research initiatives utilize the TREAT ALS platform. Developing a clinical trials management system for ALS studies adds efficiency and regulatory compliance to trials’ conduct.
Acknowledgements: We thank The ALS Association for its continuing support.
P09 EXPLORING STOPPING RULES FOR ALS SCREENING TRIALS USING A BAYESIAN MODEL AND HISTORICAL PLACEBO DATABASE
KATZ JS1, AGOSTA JM2, MOORE DH1, MILLER RG1
1California Pacific Medical Center, San Francisco, CA, 2Intel Research, Santa Clara, CA, United States
E-mail address for correspondence: [email protected]
Keywords: futility, trial design, historical controls
Background: The vast majority of large clinical trials in ALS have failed, raising questions about how best to allocate resources for drug discovery. One option is to perform screening trials that avoid placing too many patients on an ineffective drug. Futility designs can play a role, but they allow only the most deleterious treatments to be stopped. Another way is to consider that most drug trials in ALS have failed and then to account for the likelihood that the chance that any given agent in the ALS pipeline will be effective is small. Under these constraints, even after a relatively small number of patients are tested, a response that looks similar to placebo would suggest a trial can be stopped.
Objective: To develop a Bayesian mathematical model, and use a database of historical placebo controls from three prior ALS trials, to explore the effects of different stopping rules for clinical trials. Understand how to predict likelihoods of drug efficacy after treating fixed numbers of patients in open label trials in ALS.
Methods: We created a Bayesian mathematical model that assumes a theoretical distribution of efficacies for potential agents in a pipeline of ALS drugs. Only a percentage of drugs will turn out to be effective and the prior probabilities of effect sizes of all drugs on the ALSFRS-R slope decline form a normal distribution with a mean of zero. We then calculated the posterior probability that a drug is effective, varying the number of patients treated (n, ranging from 30–100) and using means and standard deviations from a pooled distribution of 409 actual placebo patients from three previous phase III ALS trials, based on any observed outcome. We defined “effective” as a drug that reduces the slope by 20% or more. For the purpose of this study, we report results when the outcome is equivalent to historical placebo controls (decline in ALS-FRS of 0.83 units per month).
Results: We assumed a range of prior distributions where 5% of drugs tested would reduce ALSFRS-R slopes by 20% or greater; and a distribution where 20% of drugs reduce the slope by 20% or greater (the former probability roughly reflects outcomes of past ALS trials have been effective. The latter is a generous assumption that could reflect an upper limit for sensitivity analysis). Under the 5% assumption, an observed slope identical to historical placebo yielded a 2.5% probability that a drug will be effective with n = 30 patients. For n = 60 the probability was 1.3% and n = 100 was 0.5%. Using the 20% assumption, n=(30, 60, 100), corresponding probabilities are 8.8%, 4.2% and 1.7%.
Conclusions: Considering the efficacy of drugs in the pipeline can help design stopping algorithms in ALS clinical trials. Bayesian modelling is a rational approach to trial design and can show the small chance a drug will be effective after a small number of patients are treated. Decisions to stop trials are ultimately based on cost and understanding these probabilities. This can be contrasted with traditional statistics that avoid assumptions about the pipeline, and primarily focus on avoiding type II errors.
P10 PASSIVE IMMUNIZATION USING MONOCLONAL ANTIBODY SPECIFIC FOR MUTANT SOD1 DELAYED MORTALITY IN ALS G93A MUTANT SOD1 MICE
GROS-LOUIS F, JULIEN JP
CHUL Research Centre, Quebec, Canada
E-mail address for correspondence: [email protected]
Keywords: ALS, Passive immunization, monoclonal antibodies
Amyotrophic lateral sclerosis (ALS) is a fatal, yet untreatable, neurodegenerative disorder leading to motor neuron degenerartion, muscle atrophy, limb paralysis and death. There is emerging evidence for the existence of secretory pathways for superoxide dismutase (SOD1) mutants-linked ALS and for neurotoxicity of extracellular mutant SOD1. Active immunization paradigm has illustrated the potential to delay disease onset and mortality of ALS mice models. However, we believe that passive immunization approaches would be safer in future human ALS clinical tests. Thus, we tested a passive immunization approach through intraventricular infusion with miniosmotic pump, using conformation-specific antibodies against misfolded SOD1 mutant that we generated. The data show that it was also effective in alleviating disease symptoms and delaying mortality of ALS mice. From these results, we propose that immunization strategies, especially passive immunization, should be considered as potential avenues for treatment of familial ALS caused by SOD1 mutations.
P11 TARGETED LIPOSOMES FOR TREATMENT OF ALS IN MUTANT SOD1 MOUSE MODEL
WILEY N, MITCHELL R, MADHANKUMAR A, SIMMONS Z, CONNOR J
Pennsylvania State University, Hershey, PA, United States
E-mail address for correspondence: [email protected]
Keywords: liposome, targeted, minocycline
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, we utilized a liposomal drug delivery system with targeting moieties specific to microglial cell surface markers to delivery the anti-inflammatory compound minocycline.
Objectives: The broad, long-term objective for our research is to develop a targeted nanotechnology platform that will improve treatment options for individuals suffering from ALS. Our central hypothesis is that minocycline contained in liposomes and delivered via intracerebroventricular (ICV) pump will delay disease onset and increase lifespan in mice carrying the Cu/Zn superoxide dismutase-1 (SOD1) mutation as compared to SOD1 mice receiving no treatment or sham pumps.
Methods: To test these hypotheses, we conjugated our liposomes with an LPS targeting moiety that is recognized by the Toll4 receptors on microglia. The drug is delivered continuously via an ICV osmotic pump that is implanted at 70 days of age. Efficacy is determined by the time to disease onset in treatment versus control groups, as well as the time to endpoint stages. Motor performance is 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 (n = 7) have an average disease onset at 107 days of age, whereas mice receiving targeted (n = 7) and non-targeted (n = 6) minocycline liposomes have an average disease onset at 113.4 (p < 0.05) and 118.3 days (p < 0.001), respectively. In addition, treatment groups experienced a similar increase in the time to endpoint stages (experiment ongoing).
Discussion and Conclusions: In the present study, we have shown that both disease onset and lifespan are significantly increased with our minocycline liposomal treatments. There appears to be little advantage to targeting the nanovesicles through an LPS peptide. Whether these data suggest minocycline is less effective if taken up by microglia will be pursued in future studies. Finally, this study serves as a proof of concept study for a drug delivery platform in which the “payload” of the nanovesicles can be easily modified to other compounds.
P12 EVALUATING THE EFFICACY OF SIMULTANEOUSLY DELIVERING IGF-1 AND VEGF EXPRESSING VIRAL VECTORS TO THE VENTRICULAR SYSTEM OF ALS MICE
DODGE J, TRELEAVEN C, FIDLER J, YANG W, PASSINI M, HAIDET A, RAO M, EAGLE A, CHENG S, KASPAR B, SHIHABUDDIN L
1Genzyme Corporation, Framingham, MA, United States, 2The Research Institute at Nationwide Children's Hospital, Columbus, OH, United States
E-mail address for correspondence: [email protected]
Keywords: neuroprotection, adeno-associated virus, trophic factors
Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by a selective loss of motor neurones in the motor cortex, brain stem and spinal cord. Although a number of studies have demonstrated that trophic factors have potent effects on motor neuron survival their delivery to the CNS remains to be a challenge. Recently we showed that intraventricular delivery of trophic factor (i.e., IGF-1 and VEGF) expressing viral vectors resulted in trophic factor delivery to the CNS and improved survival in ALS mice.
Objective: In our current study we wished to determine if delivering IGF-1 and VEGF expressing viral vectors in combination to the ventricular system would lead to either an additive or synergistic improvement in efficacy.
Methods: Starting at 80 days of age (and every 10 days thereafter), SOD1G93A mice underwent behavioral testing (rotarod, hindlimb and forelimb grip strength) to assess motor function. At 90 days of age mice received either intraventricular injection of AAV4-IGF-I or AAV4-VEGF or a combination of both vectors. Control mice were injected with AAV4-GFP.
Results: We found that delivery of trophic factor (either IGF-1 or VEGF alone) expressing AAV4 vectors to the ventricular system significantly extended lifespan in ALS mice. Treatment with AAV4-GFP provided no survival benefit. Although a trend toward improved survival was observed in mice simultaneously treated with both IGF-1 and VEGF vectors, median survival between different trophic factor treatment groups was not significantly different.
Conclusion: Our results indicate that intraventricular injection of trophic factor expressing AAV4 vectors is an effective approach for slowing disease progression in a mouse model of ALS. Our findings also suggest IGF-1 and VEGF may be modifying disease progression in ALS mice by acting on similar biological pathways as co-administration of these trophic factors did not lead to a statistically significant enhancement in survival over treatment with either tropic factor alone.
P13 TREATMENT WITH A SOLUBLE ACTIVIN RECEPTOR TYPE LLB PROMOTES LEAN TISSUE GAIN AND IMPROVES GRIP STRENGTH IN SOD1G93A MICE
LACHEY J, WONG V, PULLEN A, CADENA S, PEARSALL RS, SEEHRA J
Acceleron Pharma, Cambridge, MA, United States
E-mail address for correspondence: [email protected]
Keywords: Myostatin, ActRllB, Muscle
Background: In amyotrophic lateral sclerosis (ALS), motor neurons responsible for innervating muscle progressively deteriorate. This muscle denervation results in muscle weakness and eventual atrophy. SOD1G93A mice express the mutant human Cu/Zn superoxide dismutase gene and therefore, serve as a rodent model of familial ALS. As in ALS patients, SOD1 mice display progressive muscle weakness and as the disease progresses, muscle atrophy and paralysis. Activin receptor type llB acts as the receptor for myostatin, a TGF beta family member which negatively regulates muscle mass, as well as other unidentified muscle mass inhibitors. RAP-031, a fusion protein comprised of the ActRllB extracellular region and an IgG domain, acts as a soluble receptor and inhibits endogenous ActRllB signalling, leading to significantly increased muscle mass. Previous work demonstrated that blocking myostatin lead to an improvement in the early stages of disease in SOD1G93A mice. This study evaluates the potential beneficial effects of RAP-031 treatment on muscle mass and strength in disease progressed SOD1G93A mice.
Objective: The aim of this study was to determine if treatment with a soluble activin receptor type llB can increase muscle mass and strength in SOD1G93A mice.
Methods: SOD1G93A mice already exhibiting signs of disease were divided by gender and further into treated vehicle or RAP-031 (once a week for 4 weeks) treated groups. Mice were assessed for grip strength and body composition at baseline and study end. At study completion, individual muscle groups were dissected and weighed.
Results: RAP-031 treatment increased body weight in both genders compared to vehicle treated groups. Both the male and female vehicle groups exhibited significantly decreased forelimb grip strength between study days 0 and 21 (male: −34.9%; female: −30.8%). At day 21, the male RAP-031 grip strength was also lower than its baseline values although to a significantly lesser extent (Male RAP-031: -12.9%). The RAP-031 treated female group maintained their baseline strength over the 21 days. The male vehicle group also exhibited a significant 6% lean tissue loss between the baseline and study day 28 measurements. In contrast, the RAP-031 cohort gained 9% of its baseline lean tissue mass. The female vehicle mice did not lose lean mass, but RAP-031 females increased total lean mass by 11%. At the end of the study, RAP-031 mice had significantly greater gastrocnemius, femoris and pectoralis weights compared to vehicle groups (male: 22.2%, 25% and 50%, respectively; female: 30.8%, 11.1% and 50%, respectively).
Discussion and Conclusions: These data demonstrate that RAP-031 increases body weight, lean tissue mass, individual muscle weights and improves grip strength in late-stage SOD1G93A mice and therefore could provide clinical benefit to ALS patients.
P14 METALLOTHIONEIN-IIA TREATMENT INCREASES SURVIVAL IN THE G93A SOD1 MOUSE.
FOSTER S, WEST A, KIRKCALDIE M, THOMSON R, VICKERS J, CHUAH MI, CHUNG R
Menzies Research Institute, University of Tasmania, Hobart, Tasmania, Australia
E-mail address for correspondence: [email protected]
Keywords: Metallothionein, G93A SOD1 mice, therapeutic
Background: Mutations to superoxide dismutase 1 (SOD1) enzyme have been linked to familial amyotrophic lateral sclerosis (ALS). These mutations typically cause a toxic gain-of-function resulting in tyrosine nitration and protein aggregation Citation[1], mitochondrial dysfunction Citation[2] and excitotoxicity Citation[3]. Mutant SOD1 also has a higher affinity to aggregate when zinc deficient Citation[4]. The neuroprotective protein metallothionein-IIA (MT-IIA) is involved in metal homeostasis Citation[5], particularly that of zinc, and can act as an antioxidant Citation[6].
Objectives: To test our predictions that MT-IIA may delay neurodegenerative decline and thus increase survival in the G93A SOD1 transgenic mouse.
Methods: At 10 weeks of age, litter pairs comprising a wild type and a mutant G93A SOD1 mouse, were injected intramuscularly into the left hindlimb with either MT-IIA or a saline control twice a week until the mice reached endstage (defined as a loss of 20% maximum body weight). At each injection time point, weight and disease symptoms (muscle wastage, hindlimb mobility and tremors) were assessed. Histopathological techniques are currently being used to assess the effect of MT-IIA treatment upon spinal motor neuron survival and integrity of the sciatic nerve.
Results: Symptom analysis showed a delay in disease development in the MT-IIA treatment group beginning at approximately 147 days of age. This finding can be attributed to the increase in survival seen in the MT-IIA treated group, which began at 145 days of age. Survival of the saline control group declined to 15% at 150 days of age while the MT-IIA treatment group still had 50% survival.
Conclusion: These results suggest that MT-IIA can prolong survival in the G93A SOD1 mouse. However, further research is required to determine the mechanism of neuroprotection by MT-IIA and its site of action.
P15 AMMONIUM TETRATHIOMOLYBDATE, A COPPER-CHELATING DRUG, SUPPRESSES SOD1 AGGREGATION AND HAS THERAPEUTIC EFFECTS IN A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
TOKUDA E1, OKAWA E1, SUZUKI T2, ONO SI3
1College of Pharmacy, Nihon University, Funabashi, Chiba, Japan, 2Department of Pediatrics, Nihon University School of Medicine, Itabashi, Tokyo, Japan, 3Division of Neurology, Akiru Municipal Medical Center, Akiruno, Tokyo, Japan
E-mail address for correspondence: [email protected]
Keywords: SOD1 oligomer, Copper, Ammonium tetrathiomolybdate
Background: Amyotrophic lateral sclerosis (ALS) is an adult-onset fatal motor neuron disease. Toxic gain-of-function mutations in superoxide dismutase1 (SOD1) are responsible for a subset of cases with familial ALS. Mutant SOD1 proteins form high-molecular-weight oligomers which tend to aggregate within the affected cells, such as the spinal motor neurons. A shift of the redox status of mutant SOD1 from the reduced form to the oxidized form is probably a key step in the aggregation process.
Objectives: To address the involvement of copper ions, which are known to possess redox properties, in the aggregation of mutant SOD1, we evaluated the effects of ammonium tetrathiomolybdate (TTM), a selective copper chelator, in a mouse model of ALS (G93A strain).
Methods: G93A mice were randomly assigned to receive a daily intraperitoneal administration with TTM (5 mg/kg) or phosphate buffered saline. Treatment was started at 4 weeks of age, before the G93A mice began to exhibit ALS-like symptoms. The clinical onset of the disease was evaluated by examining the mouse for shaking of its limbs when suspended in the air by its tails. The end-point was defined as the inability of the mouse to right itself within 30 seconds after being pushed onto it's side. The progression of the disease, duration of disease or survival after onset, was calculated as the period between the onset and the endpoint of the disease. For analysis of SOD1 oligomers, detergent insoluble fractions from spinal cords were suspended in Laemmli sample buffer without 2-mercaptoethanol. The fractions were electrophoresed, transferred onto a membrane, and immunoreacted with human SOD1 antibody.
Results: Immunoblotting showed that the removal of copper ions by TTM inhibited oligomer formation in the spinal cord. TTM also showed substantial therapeutic effects; it ameliorated ALS-like symptoms, including delaying of the symptom onset, slowing the progression and limiting the duration of the disease. Especially, survival was prolonged by approximately twofold as compared with that following treatment with riluzole, the sole approved drug for clinical use against ALS (11% for riluzole vs. 24% for TTM).
Conclusions: Copper ions are likely to contribute to oligomerization and aggregation of mutant SOD1. TTM may be a promising drug for familial ALS.
p16 SIGNALS IN PATHOLOGICAL MICROENVIRONMENT OF ALS MICE PROMOTE HMSCS NEUROGENIC DIFFERENTIATION IN VITRO
ZHANG C1, ZHAO C2
1Department of Neurology, First Affiliated Hospital,Sun Yat-sen University, Guangzhou, China, 2Department of Neurology, Second Affiliated Hospital, Shandong University, Shandong, China
E-mail address for correspondence: [email protected]
Keywords: Differentiation; mesenchymal stem cells; amyotrophic lateral sclerosis
Background: Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disease, currently without any effective therapy. Multiple advantages make mesenchymal stem cells (hMSCs) a good candidate for cellular therapy in many intractable CNS diseases. But some studies have cast doubt on neuronal differentiation of the MSCs using non-physiological chemical inducing agents in vitro which are apparently different from physiological or pathological state in vivo.
Objectives: This study was designed to use conditioned medium to investigate whether signals from pathological state of ALS were competent to induce a program of neurogenic differentiation in expanded cultures of hMSCs and to assess their therapeutic potency in ALS.
Methods: hMSCs were isolated from iliac crest aspirates from healthy donors and kept in cell cultures. Conditioned medium of SOD1-G93A mice was made. The morphological change of hMSCs and the expression of neuronal markers were analyzed.
Results: We observed that incubation of hMSC with conditioned medium prepared from CNS of SOD1-G93A mice resulted in a time-dependent morphological change from fibroblast-like into neuron-like cells, concomitant with increase in the expression of nestin and subsequent β-TubIII, NSE and Gap43. Moreover, signals in pathological environment of ALS mice were more effective in promoting the neurogenic differentiation of hMSCs than in the physiological environment of CNS in adult mice.
Conclusion: These results show that pathological microenvironment of ALS is endowed with capacity to induce neurogenic differentiation of hMSCs and hMSCs have shown a potential candidate in cellular therapy for ALS.
P17 OPTIMAL MESENCHYMAL STEM CELLS FOR CLINICAL USE IN AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
KIM HY1, KIM SH1, KOH SH1, CHO GW1, KIM HJ1, OH SC1, KANG BY1, KIM KS2, CHOI MR2, LEE KW3
1Department of Neurology, Hanyang Univeristy Hospital, Seoul, Korea, Republic of, 2Bioengineering Institute, CoreStem Inc., Seoul, 3Department of Neurology, Seoul National University Hospital, Seoul, Republic of Korea
E-mail address for correspondence: [email protected]
Keywords: Mesenchymal stem cell, cytokine, growth factor
Background: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal, neurodegenerative condition characterized by loss of upper and lower motor neurons in brain and spinal cord. At present, riluzole, a glutamate antagonist, remains the only effective therapy, but with only minimal effects on survival. Therefore, stem cell research has highlighted ALS as a target disease for stem cell treatment. As a source of stem cell therapy, it has been reported that Mesenchymal Stem cells (MSCs) display neuroprotective effects and could influence on disease progression in ALS. Considering the low density (0.01% to 0.001%) in bone marrow, ex vivo expansion of MSCs is necessary prior to clinical use. However, the stability and characterization of MSCs during long-term in vitro expansion have not been evaluated yet.
Objectives: To evaluate the optimal passage for autologous MSCs therapy in ALS patients.
Methods: MSCs from bone marrow of ALS patients were isolated and expanded. The cumulative population doublings, specific antigen expressions, karyotype, differentiation potential, and the expression of various cytokines and growth factors in successive passages were evaluated.
Results: During long-term culture, the size and the morphology of the MSCs transferred from small and spindle-like cells to large and polygonal types although the specific antigenic expressions for MSCs were shown in late passages. Population doublings were gradually decreased when passages went on. MSCs at fourth passage were induced to mesenchymal lineage cells, including adipocytes, osteocytes, and chondrocytes. When we analyzed the cultured media of MSCs at third, fifth, seventh, and ninth passages, IL-6, VEGF, and IL-8 showed high expression of more than 50 pg/10,000 cells in all these passages but their expression rate was continuously decreased. Additionally, secretion of IL-15, GM-CSF, IL-10, PDGF bb, G-CSF, IL-1β, FGF basic, and IFN-gamma was gradually decreased as culture time passed on.
Conclusions: It is clear from our studies that MSCs gradually become large and shows the decrease of growth rate, differentiation potential, and cytokine release during long-term culture. Therefore, we might suggest that MSCs at earlier passage would be suitable for stem cell therapy considering its stability and neuroprotective roles associated with various cytokines and growth factors.
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