P71 DEXTROMETHORPHAN‐QUINIDINE (DMQ) TREATMENT OF PSEUDOBULBAR AFFECT (PBA) IN AMYOTROPHIC LATERAL SCLEROSIS (ALS)
Brooks BR1, Bradley WG2, Arnold R3, Pope LE3, Berg JE3, Olney RK4, Smith RA5, Study Group AVP‐9233
1University of Wisconsin Hospital and Clinics, Madison, WI, USA, 2University of Miami School of Medicine, Miami, FL, USA, 3Avanir Pharmaceuticals, San Diego, CA, USA, 4University of California San Francisco Medical School, San Francisco, CA, USA, 5Center for Neurologic Study, La Jolla, CA, USA
E‐mail address for correspondence: [email protected]
Background: ALS patients with pseudobulbar affect (PBA) were randomized to dextramethorphan‐quinidine (DMQ) (n = 70), DM (n = 33) or Q (n = 37) as published (1). DMQ effects were measured by the CNS‐Lability Scale which contains self‐rated questions to separately assess threshold and control of crying and laughing lability.
Objective: To determine whether DMQ, an effective treatment for PBA in ALS patients, works through adjusting the threshold, control or both for crying and laughing lability.
Methods: Outcomes for individual questions in DMQ, DM, Q and DM plus Q patient groups were analysed at d1, d15 and d29 after randomization and initiation of treatment. Comparisons were made using one‐way analysis of variance with contrasts.
Results: DMQ significantly decreased, at d15 and d29, CT (−1.21, −1.07 ) (p<0.0001, <0.0001), CC (−1.22, −1.31 ) (p<0.0001, <0.0001) and CCP (−1.38, −1.20) (p<0.0001, <0.0001) compared with Q and CT (−0.93, −0.90 ) (p<0.0001, <0.0001), CC (−0.87, −0.93 ) (p<0.0001, <0.0001) as well as CCP (−0.97, −0.87 ) (p<0.0001, <0.0001) compared with DM and Q. DMQ significantly decreased, at d15, but not d29, LT (−0.61, −0.31) (p = 0.0148, 0.1907), LC (−0.65, −0.36) (p = 0.0096, 0.2328), CLP (−0.63, −0.23 ) (p = 0.0110, 0.3233) compared with Q. Similar effects were seen for DMQ compared with DM and Q, except that DMQ demonstrated a small, but significant decrease in CIAT (−0.35, −0.34) (p = 0.0357, 0.0254) at d15 and d29.
Conclusions: In ALS patients, DMQ had a significantly larger and more persistent effect on threshold, control and control of paroxysms in PBA manifested by crying than threshold, control, control of paroxysms manifested by laughing. A small, but persistent, effect by DMQ on control of intrusive amusing thoughts in PBA was demonstrated.
References
- Neurology 2004; 63:1364–70.
P72 INCIDENCE OF COMMON ADVERSE EVENTS WITH AVP‐923 IN THE TREATMENT OF INVOLUNTARY EMOTIONAL EXPRESSION DISORDER (IEED) PATIENTS OF DIFFERING AGE
BROOKS BR1, POPE LE2, BERG JE2, ARNOLD RA2
1University of Wisconsin, Madison, WI, USA, 2Avanir Pharmaceuticals, San Diego, CA, USA
E‐mail address for correspondence: [email protected]
Background: IEED is associated with various neurological disorders and is characterized by uncontrollable episodes of laughing and/or crying out of proportion, or incongruent with underlying mood. IEED is also commonly known as emotional lability, pathological laughing and crying, emotional incontinence, and pseudobulbar affect. AVP‐923 is an investigational therapy that has been previously shown to be safe and effective in the treatment of IEED in multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) patients, in two phase III clinical trials. A phase III open label safety trial including IEED patients with MS, ALS, traumatic brain injury, stroke, Parkinson's disease, dementias, and other associated conditions is currently ongoing.
Objective: Evaluate the safety profile of investigational therapy AVP‐923 (capsules containing dextromethorphan hydrobromide (DM; 30 mg) and quinidine sulphate (Q; 30 mg), for the treatment of IEED in adults of varying age.
Methods: IEED patients were pooled from two phase III clinical trials, and an open‐label safety study of AVP‐923 (n = 584). Incidence rates of common adverse events (AE) across all trials were analysed for predefined age groups of <45 (n = 185), 45–60 (n = 275), and >60 (n = 124) years of age. All subjects that received study drug or control were included in the analysis.
Results: The observed incidence of common adverse events was similar in each defined age group. Common AEs included dizziness (18% <45 years, 19% 45–60 years, 20% >60 years, 9% placebo), nausea (28% <45 years, 21% 45–60 years, 26% >60 years, 15% placebo), and fatigue (23% <45 years, 18% 45–60 years, 13% >60 years, 20% placebo). There were no significant differences in AE rates across the defined age groups for those patients on AVP‐923 vs. those on placebo.
Discussion and conclusions: AVP‐923 common adverse event incidence rates were similar across all IEED age groups that were studied. The tolerability profile of investigational therapy AVP‐923 is similar in IEED patients of differing adult age groups. AVP‐923 appears well tolerated in multiple studies of IEED patients with differing underlying diseases.
P73 THE COMBINATION OF DEXTROMETHORPHAN AND QUINIDINE IN THE TREATMENT OF LABILE ANGER AND FRUSTRATION IN PATIENTS WITH INVOLUNTARY EMOTIONAL EXPRESSION DISORDER (IEED)
SMITH RA1, ARNOLD RP2, LICHT JM1, POPE LE2
1Center for Neurologic Study, La Jolla, CA, USA, 2Avanir Pharmaceuticals, San Diego, CA, USA
E‐mail address for correspondence: [email protected]
Background: IEED is a condition characterized by the disinhibition of emotional expression secondary to neurological disease or injury. Episodes typically involve involuntary affective displays such as laughing, crying, or related facial features that are incongruent or exaggerated in relation to underlying mood. IEED is associated with neurological diseases such as MS, ALS, Parkinson's disease, dementias; and neurological injuries, such as stroke and traumatic brain injury. Historical terms such as emotional lability, pathological laughing and crying, emotional incontinence, and pseudobulbar affect have also been used to describe the symptomatology of IEED. Dextramethorphan‐quinidine (DM/Q) has been shown in two pivotal trials to reduce the frequency and intensity of episodes of crying and/or laughing associated with IEED, as measured by the validated Center for Neurologic Study‐Lability Scale (CNS‐LS). This study explored treatment effects of DM/Q on anger and frustration, compared to its effects on the hallmark symptoms of crying and/or laughing.
Objectives: Explore the effects of DM/Q (capsules containing dextromethorphan 30 mg and quinidine 75 mg) on labile affective displays of anger and frustration in patients with IEED.
Methods: Analysis of a double‐blind, placebo‐controlled, crossover study of patients with IEED was undertaken to determine the effect of DM/Q on labile anger and frustration. Patients were treated for four weeks with DM/Q and a similar time period with placebo, with a one‐week washout period in between. A questionnaire consisting of 65 questions that focused on episodes of inappropriate tearfulness, laughter, anger, and frustration was administered at baseline and post‐treatment. Responses were scored on a 5 point scale, with 1 = applies never and 5 = applies most of the time. All the questions, related to anger and frustration, were analysed for change from baseline along with the seven validated CNS‐LS questions utilized in clinical trials of IEED.
Results: Patients taking DM/Q reported similar improvement in scores related to anger and frustration as the validated CNS‐LS questions related to laughing and or crying. Patients reported 16% reduction from baseline in episodes of anger, 30% reduction in episodes of frustration, and a 29% reduction in the CNS‐LS questions relating to laughing and/or crying.
Conclusions: DM/Q, previously shown to reduce crying and/or laughing associated with IEED, also appears to reduce anger and frustration. Further studies are needed to explore DM/Q effects on anger, frustration, and other affective disinhibition manifestations.
P74 LEVETIRACETAM TREATMENT FOR SPASTICITY IN MOTOR NEURON DISEASES: A CASE SERIES
Bedlack R1, Bellanich M1, Bromberg M2, Cudkowicz M3
1Duke University, Durham NC, USA, 2University of Utah, Salt Lake City UT, USA, 3Mass General Hospital, Boston MA, USA
E‐mail address: [email protected]
Background: Spasticity is a common problem in patients with motor neuron diseases (MNDs). There are two clinical types: tonic (producing muscle stiffness) and phasic (producing cramps and spasms). Together these can interrupt sleep, exacerbate fatigue, produce pain, impair balance and increase the risk of falls. The pathophysiology of spasticity in MNDs is poorly understood and no treatment is proven to improve it. A number of different agents are being used anecdotally (baclofen, tizanidine, benzodiazepines, dantrolen, gabapentin); side‐effects from these include sedation, exacerbation of fatigue and weakness, and cognitive dysfunction. Levetiracetam (Keppra) is an anti‐epileptic agent that may be useful in the treatment of spasticity. In support, levetiracetam reduces phasic (but not tonic) spasticity in patients with multiple sclerosis.
Objectives: To review a series of patients with MNDs treated with levetiracetam for spasticity.
Methods: We announced our hypotheses regarding levetiracetam and MNDs at the 2005 Northeast ALS Consortium Meeting. Since then, three ALS clinics (Duke University, Massachusetts General Hospital and the University of Utah) submitted case reports of patients with MNDs treated with levetiracetam for spasticity. We are compiling information about patient demographics, dosages and duration, and outcomes.
Results: A total of five patients make up the case series to date. Both ALS and PLS are represented, ages 37–67 years. Most of the patients have had inadequate responses to baclofen. Levetiracetam doses range from 250 to 3000 mg daily, exposures from one to three months. No serious side‐effects have been reported. Three of the five patients report symptomatic improvement starting within 72 h of exposure to the medication. In some cases the symptomatic improvement is dramatic. One patient who had failed baclofen reported “being released from a vice”. In some cases, there is also clear objective benefit – documentation of increased range of motion, improved ability to dress and lengthened walking distances.
Discussion: Given the unpredictable response to anti‐spasticity agents currently being used in MNDs, this small case series suggests that levetiracetam warrants further controlled study. We currently have a Phase II open‐label pilot trial underway that aims to assess safety and efficacy of levetiracetam in 20 patients with MNDs (ALS, PLS or PMA). Eligible patients have cramps with average severity 50/100 points, normal renal function and are on a stable riluzole dose, in addition to meeting other eligibility criteria. After a three‐month baseline period, patients take levetiracetam at increasing doses up to 3000 mg per day over a nine‐month period. Outcome measures include adverse events, tolerability, cramp‐pain‐severity score, cramp‐frequency score, modified Ashworth Spasticity Score, Penn Spasm Score, FVC, ALSFRS‐R and MMT. The results of this study should help determine whether levetiracetam is worth taking into a Phase III trial next year.
P75 BENEFICIAL EFFECTS OF INTRATHECAL IGF‐1 ADMINISTRATION IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS
Murakami T, Nagano I, Nagai M, Shiote M, Ohta Y, Morimoto N, Takehisa Y, Kamiya T, Abe K
Department of Neurology, Okayama University, Okayama, Japan
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder caused by degeneration of the upper and lower motor neurons in the brain and spinal cord resulting in progressive paralysis of voluntary muscles. However, there is currently no effective pharmacological treatment for amyotrophic lateral sclerosis. In a transgenic mouse model of ALS, intrathecal infusion of insulin‐like growth factor (IGF)‐1 showed a promising increase in survival.
Objectives: To examine whether IGF‐1 is effective for the prevention of disease progression, we performed a double‐blind clinical trial to assess the effect of intrathecal administration of IGF‐1 in patients with ALS.
Methods: Nine patients with ALS were randomly assigned to receive either a high dose (3 microg/kg of body weight) or low dose (0.5 microg/kg of body weight) of IGF‐1 every two weeks for 40 weeks. The outcome measurements were the rate of decline of bulbar and limb functions (Norris scales) and forced vital capacity.
Results: The high‐dose treatment slowed a decline of motor functions of the ALS patients in total Norris and limb Norris scales, but not in bulbar Norris or vital capacity. The intrathecal administration of IGF‐1 had a modest but significant beneficial effect in ALS patients without any serious adverse effects.
Discussion and conclusions: Intrathecal IGF‐1 treatment could provide an effective choice for ALS although further studies in more patients are needed to confirm its efficacy and optimize dosages of IGF‐1.
P76 SAFETY AND PHARMACOKINETICS OF REPEATED DOSES OF TRO19622, A DRUG CANDIDATE FOR THE TREATMENT OF AMYOTROPHIC LATERAL SCLEROSIS AND SPINAL MUSCULAR ATROPHY
Abitbol J‐L, Cuvier V, Bordet T, Drouot C, Berna P, Pruss R
TROPHOS, Marseille, France
E‐mail address for correspondence: [email protected]
Background: TRO19622 was selected for its ability to rescue rat motor neurons deprived of trophic factors. In vivo, TRO19622 increases survival, maintains weight and motor function in SOD1G93A mice (3 and 30 mg/kg/day) and increases survival in a mouse model of SMA (NSE‐Cre; F7/F7 – Judith Melki) (30 mg/kg/day, sc). The molecule is orally bioavailable, crosses the blood‐brain barrier and has a large safety margin (NOAEL of 1 g/k/day in four‐week toxicity testing in rat and dog).
Objective: To assess the safety and determine the pharmacokinetic (PK) profile of multiple oral doses of TRO19622 in healthy subjects.
Design: Four oral doses of TRO19622 (50, 150, 250 and 500 mg) were administered for 11 consecutive days in four groups of 12 healthy male Caucasian subjects (nine on TRO19622, three on placebo) in a double‐blind, randomized, sequential, multiple‐dose escalation trial.
Results:
Safety: No SAE as well as no major issues in safety assessment were reported. Sixty‐nine Treatment Emergent Adverse Events (TEAE) were reported: 18 were considered possibly related to the investigated medicinal product (IMP), 22 unlikely related and 27 were considered unrelated to the IMP. Among the possibly related TEAEs, seven occurred on placebo; two on TRO 19622 50 mg dose; none on TRO 19622 150 mg; two on 250 mg dose and seven on TRO 19622 500 mg dose. These TEAEs were mild (48) or moderate (21) in intensity. None was rated as severe. The most common TEAEs (n⩾4 episodes) were diarrhoea (nine episodes), headache (seven episodes), constipation (four episodes), pharyngitis (four episodes) and back pain (four episodes). There was no correlation between occurrence of TEAEs and dosage. No relevant change in vital signs, ECGs parameters, laboratory tests or physical examinations were observed after repeated administration.
Pharmacokinetics. TRO19622 absorption and elimination were slow after oral dosing whatever the dosage: tmax of about 10 h and TRO19622 concentrations measurable up to 19 days after dosing. Mean terminal elimination half‐life was about 120 h and comparable between doses. On day 1 when the dose increased in a ratio of 3, 5 and 10 (from 50 to 150, 250 and 500 mg, respectively), Cmax increased in a ratio of 2.2, 4.4 and 10.2 and AUC0‐t increased in a ratio of 2.1, 4.6 and 10.8. At steady‐state, reached on day 11 whatever the group, Cmax increased with dose in a ratio of 2.1, 7.2 and 12.2 and AUC0‐τ increased in a ratio of 2.0, 6.5 and 11.6. Mean accumulation ratios of Cmax and Ctrough observed between day 1 and day 11 were about 4. Plasma PK profiles were similar between subjects and across doses, the coefficient of variation of Cmax and AUC0‐τ were between 21 and 47% on day 11.
Conclusion: TRO19622 repeated administration up to 500 mg per day was well tolerated in healthy volunteers. PK results suggest that target effective concentrations derived from in vitro and several in vivo pre‐clinical models can be reached at a QD dose of 250 mg and above.
Acknowledgement: The study was supported by Association Française contre les Myopathies.
P77 A MULTICENTRE, DOSE RANGING SAFETY AND PHARMACOKINETICS STUDY OF ARIMOCLOMOL IN ALS
Cudkowicz M1, Shefner J2, Simpson E1, Grasso D1, Sherman A1, Yu H1, Opoliner A1, Balakrishna S1, Schoenfeld D1, Zhang H1, Finkelstein D1, Taft J2, Watson M2, Dibernardo A1, Pulley D1, Verma A3, Koggan D3, Steele J3, Bedlack R4, Grace K4, Heiman‐Patterson T5, Barr C5, Barohn R6, Walsh M6, Herbelin L6, Simmons Z7, Stephens H7, Ensrud E8, Zimmerman M8, David W9, Conn S9, Mozaffar T10, Martin V10, Hung J1, Milan D1, Wieland S11, Barber J11, Brown R1
1Massachusetts General Hospital, Boston, MA, USA, 2SUNY Upstate Medical University, Syracuse, NY, USA, 3University of Miami, Miami, FL, USA, 4Duke University, Durham, NC, USA, 5Drexel University College of Medicine, Philadelphia, PA, USA, 6University of Kansas, Kansas City, KS, USA, 7Penn State University, Hershey, PA, USA, 8University of Texas Health Science Center at San Antonio, San Antonio, TX, USA, 9Hennepin Faculty Associates/Berman Center, Minneapolis, MN, USA, 10University of California Irvine, Irvine, CA, USA, 11CytRx Corp., Los Angeles, CA, USA
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a rare motor neuron disease, usually fatal within three to five years. Although the cause of the disease is unknown, the presence of characteristic protein aggregates in relevant cell types implicates a role for protein misfolding in the disease. Arimoclomol is a small molecule that up‐regulates heat shock proteins (HSP) in cells. HSPs function as protein‐folding chaperones and are critical components of a cell's response to stress. Arimoclomol extends survival when given both pre‐symptomatically and at disease onset in a mutant superoxide dismutase (SOD1) transgenic mouse model of ALS (1). In treated mice, arimoclomol delayed the death of motor neurons and the associated loss of motor unit potentials. It has also been demonstrated to have neuroprotective and neuroregenerative effects in acute models of nerve injury. Therapeutic agents that target protein misfolding, such as arimoclomol, may be helpful in ALS.
Objectives: The primary objective of this study was to assess the safety and tolerability of arimoclomol compared with placebo over 12 weeks of treatment in patients with ALS. The secondary objective of this study was to determine the pharmacokinetic characterization of arimoclomol in blood and cerebrospinal fluid (CSF) penetration in 40 of the 84 total subjects.
Methods: A double‐blind, placebo‐controlled, clinical trial is being conducted. Eighty‐four research subjects with ALS have been randomized to receive oral arimoclomol at one of three dosages (75, 150, and 300 mg/day) or placebo for 12 weeks. Visits and safety testing occur every two weeks. Forty research participants have blood drawn to determine arimoclomol and riluzole levels at the following time points: prior to receiving study dose, and at 0.5, 1, 2, 3, 4, 6, 8, 10, and 12 h after receiving the study dose at the baseline and week 4 study visits. To determine the degree to which arimoclomol enters the CSF, samples are taken by standard lumbar puncture at the week 4 visit in these 40 participants.
Results: Enrollment was completed in March 2006 and the last study visit is expected in July 2006. The full study report will be available by September 2006. All pharmacokinetics visits are complete.
Discussion and conclusions: Should arimoclomol show favorable 12‐week tolerability and safety in patients with ALS, the data from the study would be used to support an efficacy study in ALS. The safety and the pharmacokinetic analyses data will guide selection of dosage for a subsequent efficacy study.
References
- Kieran D, Kalmar D, Dick JR, et al. Treatment with arimoclomol, a coinducer of heat shock proteins, delays disease progression in ALS mice. Nat Med. 2004;10:402–205.
P78 REPEATED ADMINISTRATION OF EDARAVONE, A FREE RADICAL SCAVENGER, FOR PATIENTS WITH ALS
Yoshino H
Yamagata Tokushukai Hospital, Yamagata, Japan
E‐mail address for correspondence: [email protected]
Background: Oxidative stress is presumed to contribute to pathogenesis of ALS.
Objectives: We investigated the efficacy and safety of edaravone, a free radical scavenger previously approved for acute cerebral infarction, in ALS patients.
Methods: Within an open trial design, 20 subjects with ALS received either 30 mg (five subjects) or 60 mg (15 subjects) of edaravone via intravenous drip once per day. Two weeks of administration were followed by a two‐week observation period. This series was repeated six times. A change in the revised ALS functional rating scale (ALSFRS‐R) was used as the primary endpoint, while secondary endpoints of respiratory function (%FVC), and 3‐nitrotyrosine (3NT) levels, a marker of oxidative stress, were assessed.
Results: Efficacy was evaluated in the 60 mg group of patients. Six months following administration, the ALSFRS‐R was inhibited by 2.4±3.5 points (paired t‐test, p = 0.035) compared to the six‐month prior level. The change in %FVC at six months following administration was −4.47±12.30%. In almost all patients, CSF 3NT was markedly reduced to almost undetectable levels at the end of the six‐month treatment period. Stratified analysis showed that in the group pf patients with equal to or greater than 41 points ALSFRS‐R at the commencement of the study (n = 4), %FVC was increased by 7.23±14.77, while in patients with less than 41 points ALSFRS‐R (n = 8), %FVC was reduced by 10.31±5.19.
Conclusion: In the previous placebo‐controlled short term study, efficacy of edaravone was also demonstrated in %FVC and in the change of ALSFRS‐R score in patients with equal to or greater than 41 points ALSFRS‐R. Combined with the present six months open study, edaravone might inhibit the progression of ALS in the early stage. A confirmative placebo‐controlled long‐term study is currently under progress in Japan.
P79 SAFETY CONCERNS IN AMYOTROPHIC LATERAL SCLEROSIS (ALS) PATIENTS WHO ARE ON RILUZOLE AND BU NAO GAO (BNG) CHINESE HERBAL MEDICATION
Brooks BR1, Waclawik AJ1, Peper SM1, Houdek AM2
1University of Wisconsin Hospital and Clinics, Madison, WI, USA, 2William S Middleton Memorial Veterans Affairs Medical Center, Madison, WI, USA
E‐mail address for correspondence: [email protected]
Background: BNG, in Chinese, ‘tincture or solution to revive the brain’, contains 14 herbs: Radix angelica sinensis (DangGui) 29.3%, Ligusticum chuanxiong (ChuanXiong) 7.3%, Hirudo (ShuiZhi) 3.7%, Astragalus membranaceus (HuangQi) 11%, Paeonia rubrae (ChiShao) 7.3%, Lycium chinense mill (GouQiZi) 4.4%, Polygonatum sibiricum (HuangJing) 7.3%, Curculigo orchioides (XianMao) 3.3%, Epimedium grandiflorum (YinYangHuo) 3.3%, Plastrum testudinis (ShengGuiBan) 5.5%, Cornus officinalis (ShanZhuYu) 3.7%, Psoralea corylifolia (BuGuZhi) 4.4%, Leonurus heterophyllus (YiMuCao) 7.3%, Glycyrrhiza uralensis (Gancao) 2.2%. Western literature has no clinical descriptions of observed side effects of BNG alone or in combination with riluzole.
Objective: To report observations of subacute onset of weakness or rapid deteroration of ALS when riluzole and BNG treatments were combined.
Methods: Observational study in patients who experienced ALS worsening following subacute weakness on reinstitution of riluzole when it had been stopped for weakness in combination therapy with BNG.
Results: Four arm onset ALS patients, three males and one female, age 55–62 years, were self‐treated with BNG either before (n = 2) or after (n = 2) initiation of riluzole treatment. On combination therapy, these patients noted asthenia, weakness, loss of appetite, weight loss (10–22 pounds) and decreased ALS FRS‐R. Two patients rechallenged with riluzole worsened and discontinued this medication. One of these patients was rechallenged twice with development of weakness and asthenia each time. One patient added BNG to riluzole with consequential weakness that improved with cessation but returned when rechallenged with BNG. Deep vein thrombosis developed in one of four patients on BNG.
Discussion and conclusions: BNG, a poorly characterized concoction of multiple herbs may be associated with subacute onset of asthenia, worsening of ALS clinimetrics, anorexia and weight loss. ALS patients on riluzole may be at risk when starting BNG. A rapid change in clinical status in an ALS patient should lead to investigations of combination therapy with alternative medicines, particularly BNG.
P80 COMBINATION THERAPY WITH MESENCHYMAL STEM CELL AND ERYTHROPOIETIN IN PATIENTS WITH ALS
Kim H1, Kim K2, Koh S1, Choi M2, Park J2, Cho S1, Kim H1, Kim J1, Kim S1
1Department of Neurology, College of Medicine, Hanyang University, Seoul, South Korea, , 2CoreStem, Seoul, South Korea
E‐mail address for correspondence: [email protected]
Background: The devastating nature of amyotrophic lateral sclerosis (ALS) has led to a major effort to find a treatment capable of arresting the disease. To date, in spite of more than 100 trials in ALS, it has been shown that only one medication, riluzole, has a marginal effect on survival and no effect on muscle strength, quality of life or functional capacity. Although stem cell therapies, such as human umbilical cord cells and adult autologous stem cells, have been tried on a small scale in ALS patients, no positive results have as yet been reported with monotherapy. However, in previous reports, erythropoietin has proved its neuroprotective and regenerative properties in CNS injury.
Objectives: To evaluate the effects of the combination therapy with mesenchymal stem cells and erythropoietin in patients with ALS.
Methods: Eight patients with definite ALS were recruited and treated. Autologous mesenchymal stem cell collection was performed by ex vivo expansion of mesenchymal stem cells following bone marrow aspiration from the patient's own posterior superior iliac spine. Erythropoietin was injected intravenously one day before stem cell therapy. Stem cells were infused intrathecally over a period of minutes using a spinal needle at the level of L3/4. The same procedure was done twice at intervals of 35 days. The patients were monitored by clinical evaluation including forced vital capacity, ALSFRS‐R, Norris score, and Appel score. The neurophysiological index was used as a neurophysiologic assessment.
Results: At the second day after stem cell injection, a mild increase in muscle strength was observed in six patients, and improvement of spasticity was shown in five patients. In addition, two patients experienced improvement in speech and swallowing, and no further functional deficit was observed in five patients during the first two months after first treatment. At the ninth month after the first treatment course, functional decline analyses of ALSFRS‐R showed a less steep slope after treatment. No patient manifested severe adverse events such as death or respiratory insufficiency. Post‐puncture headache, which was transient and disappeared spontaneously, was reported in three patients.
Conclusions: We suggest that this approach including mesenchymal stem cell and erythropoietin has potential therapeutic efficacy and is worthy of further investigation.
P81 ASSESSMENT OF METHODS TO REDUCE MISSING DATA FROM ALS TRIALS
Doorish C, Diamond B, Montes J, Mitsumoto H, Gordon PH
Columbia University, New York, NY, USA
E‐mail address for correspondence: [email protected]
Background: One of the great barriers facing ALS researchers is how best to reduce patient dropout and missing data from clinical trials. Missing data reduce the power of the study and can lead to inconclusive or biased results. Full patient compliance coupled with easy to administer outcome measures is essential for providing high quality data at analysis. Open and frequent dialogue with patients both before enrolling and during the trial might help reduce dropout. It is not known which outcome measures best reduce missing data.
Objective: To describe interventions to reduce dropout rates in a phase II clinical trial, and to determine which clinical outcome measures are associated with the least quantity of missing data.
Methods: We conducted a randomized controlled trial in 30 patients with one baseline and six monthly visits, giving a total of 210 possible patient visits, and assessed the standard outcome measures for data quality. Of the 30 participants, four patients died prior to completion, leaving 13 visits uncompleted and 197 visits for analysis. The ALS Functional Rating Scale‐Revised (ALSFRS‐R), Quality of Life (QOL) questionnaire, manual muscle testing (MMT), and forced vital capacity (FVC) were performed at each clinic visit. Telephone contacts were made when patients could not come to the clinic. At those times, FVC and MMT were not performed. The investigator, coordinator and physical therapist met with each patient individually at each clinic visit. Telephone contacts, for those who could not attend clinic due to weakness, were performed by the coordinator and physical therapist.
Results: There were no dropouts for reasons other than death. Of a possible 197 visits, only one patient missed a full visit. ALSFRS‐R and QOL survey were completed 196 times (99.5% complete data). MMT evaluations were collected on 190 visits (96%). Positions that required patients to lie either prone (75%) or supine (88%) had a higher percentage of missing data than positions in which the patient could sit upright (97%). FVC was performed 187 times (95%) overall. Every patient who performed at least one pulmonary function test, successfully completed three attempts.
Conclusions: Investment of time by the research team was an effective means of reducing patient dropout. Management of clinical trials, therefore, should include a high level of involvement from the investigator and study clinicians beginning with the pre‐screening process and continuing through all study visits. Regular discussions about research and emphasis on good clinical care may help promote retention in clinical trials. Additionally, the use of easily administered outcome measures can further reduce the amount of missing data for analysis. Measures that do not require patients to lie down and that can be administered by phone, such as the ALSFRS‐R, best reduce the amount of missing data.
Acknowledgement: This study was supported by the Wings over Wall Street Fund.
P82 UNIFIED ELECTRONIC DATA CAPTURE AND DATA MANAGEMENT SYSTEM FOR CLINICAL TRIALS AND BIOMARKER STUDIES IN ALS
Sherman AV
1MA General Hospital, Boston, MA, USA
E‐mail address for correspondence: [email protected]
Background: Sharing research data from multiple biomarker studies and clinical trials in amyotrophic lateral sclerosis (ALS) is challenging as different centres use proprietary data storage formats and data dictionaries. Conduct of multiple trials in ALS highlights a growing need for standardization of common data elements (CDE) for the exchange, sharing, integration, analyses, storage and retrieval of data. One of the proposed solutions is creation of the Unified Electronic Data Capture and Data Management system to be used by the entire ALS research community in the US and Canada.
Objectives: Create specifications and develop a single web‐based system for data capture and management to be used by the members of the American ALS Research Group (ALSRG) whose mission is to ‘foster communication, collaboration and research in North America into the cause and treatment of ALS’. Future international collaboration is also feasible.
Methods: With more ALS clinical trials and biomarker studies being conducted by multiple research organizations and individual researchers, there is a clear need to create standards in data acquisition and management. As the collaboration at the methodological and organizational levels proved to be successful with the ALS Biomaterial and Data Banking initiative's commencement in 2006, a larger effort is needed in definition of standard ALS‐specific CDE and requirements specifications for the unified web‐based system. The design of such a system will require flexible architecture to be able to create new studies utilizing library CDE as building blocks. The system will also allow association of new visits and forms with the existing subjects. This feature is necessary when an additional study is being conducted on a subset of subjects participating in the initial study.
Results: Standards for ALS‐specific CDE are being created. Along with this effort, the system specifications must be developed with input from all interested parties. The unified system will be deployed on a central server and securely accessible to all ALSRG members.
Conclusions: The following are the recommendations to the ALS research community for successful specification, design and deployment of the Unified Data Capture and Data Management system in ALS:
Form work groups to develop proposals for the unified system
Form CDE committee to specify ALS‐specific CDE such as outcome measures, disease case report forms (CRFs) and procedures
Develop processes for approval and submission of CDE to the system library of forms
Create specification to the unified system based on the PharmaENGINE™ platform that is already deployed by the NEALS consortium and Neurology Clinical Trials Unit of Massachusetts General Hospital
Develop requirements to the system that will allow maximum flexibility in CDE CRFs creation and utilization through user interface
Define responsibilities of the data management team
Develop compliance to regulatory documents strategies
Find a sponsor to underwrite the effort
P83 THE TIMED GET UP AND GO TEST, A FUNCTIONAL OUTCOME MEASURE FOR CLINICAL TRIALS IN ALS
Montes J, Diamond B, Doorish C, Mitsumoto H, Gordon P
Columbia University, New York, NY, USA
E‐mail address for correspondence: [email protected]
Background: Clinical outcome measures are used as surrogates for survival in clinical trials to reduce sample size and treatment duration. Ideally, outcome measures provide meaningful functional assessments, are easily administered, can be performed in various settings, require minimal equipment or training, and do not burden patients. The Timed Get Up and Go (TGUG) test measures the time it takes an individual to stand up from a chair, walk three meters, turn around and sit down in the same chair. TGUG is used to assess functionally meaningful mobility. It is a reliable test for quantifying mobility in the elderly and is a sensitive measure for identifying elderly individuals prone to falls. In Parkinson's disease it correlates with Unified Parkinson Disease Rating Scale scores, is sensitive to changes associated with levodopa use and correlates with and predicts fatigue.
Objectives: To assess the performance of TGUG in a phase II clinical trial in ALS.
Methods: We conducted a six‐month, prospective, randomized phase II trial of glatiramer acetate in ALS. Thirty subjects were assigned to one of two treatment groups, or a control group, and underwent monthly TGUG, ALS Functional Rating Scale Revised (ALSFRS‐R), forced vital capacity (FVC), manual muscle testing (MMT) and quality of life (QOL) assessments. Pearson correlation coefficients were determined for baseline scores and slopes. T‐tests were used to compare differences in means between those who could and could not perform TGUG. Survival was assessed using Kaplan‐Meier and Cox proportional‐hazards methods.
Results: There were no differences in outcomes between treatment groups, which were therefore combined (n = 30) for analysis. Analyses included imputed values for those unable to perform TGUG (n = 9). The mean TGUG time was 19.3 s (range 6.62–73.8). TGUG time at baseline strongly correlated with leg MMT (r = −0.67, p <0.0001), proximal leg MMT (r = −0.75, p<0.0001), total MMT (r = −0.56, p = 0.0014), and with ALSFRS‐R motor (r = −0.47, p = 0.03) and gait subscores (r = −0.41, p = 0.009). ALSFRS‐R motor (p = 0.0019) and leg (p = 0.0047) subscores, proximal leg MMT (p<0.0001), distal leg MMT (p = 0.0352), and total MMT (p = 0.0043) scores were significantly different in those who could and could not perform TGUG at baseline. The slope of TGUG correlated with the slopes of QOL (r = 0.62, p = 0.0003) and proximal leg MMT (r = 0.42, p = 0.03), and survival was found to be better for those who performed TGUG above the median than for those who performed below the median (p = 0.0561).
Conclusion: TGUG correlates with standard outcome measures, which are different in those who can and cannot perform the test, and may predict survival. TGUG could be a useful addition to the ALSFRS‐R as a simple, less expensive, sensitive, and clinically meaningful outcome in trials of ALS.
P84 PERFORMANCE OF IN‐PERSON VS. PHONE ALSFRS‐R IN A RANDOMIZED, CONTROLLED PHASE III CLINICAL TRIAL OF MINOCYCLINE IN ALS
Flornece JM1, Moore D2, Spitalny GM3, Miller RG2, Gordon PH4
1Washington University School of Medicine, St. Louis, MO, USA, 2California Pacific Medical Center, San Francisco, CA, USA, 3Research Computing & Support Services, Sausalito, CA, USA, 4Columbia University, New York, NY, USA
E‐mail address for correspondence: [email protected]
Background: The ALSFRS‐R has become a principal clinical outcome measure for trials in ALS. It has good reliability, internal consistency, and content validity: declines in tandem with pulmonary function and muscle strength; and predicts survival more strongly than other clinical measures. Telephone administration may be an important way to reduce missing data in ALS trials. Standardized procedures are now used for in‐person and phone administration.
Objective: To assess differences in phone and in‐person ALSFRS‐R scores in a phase III clinical trial that uses the ALSFRS‐R as the primary outcome measure.
Methods: Monthly scores were obtained from 471 subjects in an ongoing phase III clinical trial of minocycline in ALS. One hundred and forty subjects had eight or more monthly scores and at least one evaluation obtained by phone. The number of phone assessments ranged from 1 to 11 with 55 subjects having more than one phone assessment. A locally weighted least squares (lowess) curve was fitted to each subject's data ignoring whether the score was from the clinic or by phone. For each data point a residual, equal to the difference between the observed score and the lowess fit was calculated. The distribution of residuals was compared by assessment type (clinic vs. phone) and a two‐sample t‐test was used to test for differences between types of administration. Analysis of variance was performed to determine whether visit number or total numbers of clinic and phone assessments affected the fits.
Results: The deviations of the phone scores from the lowess fits are similar to those for the clinic scores. The 1302 residuals for clinic (mean 0.08; SD = 1.41) scores are not different from the 346 residuals for phone (mean = −0.06; SD = 1.46) scores (t‐statistic = 1.59, 2‐sided p = 0.11). There are no differences in residuals by visit number (e.g. fifth visit), total number of visits, numbers of clinic visits or numbers of phone visits.
Conclusions: There is no difference between phone and in‐person ALSFRS‐R scores in the large phase III clinical trial where a standard format was used to obtain data. The use of ALSFRS‐R as a primary outcome measure is further strengthened because of its ability to produce valid and complete data from those patients too weak to travel to a research clinic.
P85 ORAL ADMINISTRATION OF MEMANTINE PROLONGS SURVIVAL IN A TRANSGENIC MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
Joo I‐S1, Whang D‐H2, Seok J‐I3, Shin S‐K1, Choi Y‐C4, Kim S‐U5
1Department of Neurology, Ajou University School of Medicine, Suwon, South Korea, 2Brain Disease Research Centre, Ajou University School of Medicine, Suwon, South Korea, 3Department of Neurology, Catholic University School of Medicine, Daegu, South Korea, 4Department of Neurology, Yonsei University School of Medicine, Seoul, South Korea, 5Department of Medicine, University of British Columbia, Vancouver, Canada
E‐mail address for correspondence: [email protected]
Background and objectives: N‐methyl‐D‐aspartate (NMDA)‐mediated neurotoxicity and oxidative stress have been implicated in the aetiology of motor neuron disease. Memantine is a low‐ to moderate‐affinity NMDA receptor antagonist and it may have a potential to protect against motor neuron degeneration.
Methods: Thirty transgenic mice expressing the G93A SOD1 mutation were randomly divided into control, low dose (30 mg/kg), and high dose (90 mg/kg) memantine groups, and mematine was supplied daily in the drinking water, beginning at 75 days of age. Body weight, survival, and behavioural performance including rotarod test, paw grip endurance (PaGE), and hindlimb extension reflex were assessed in control and memantine diet groups.
Results: Clinical disease was evident in the G93A transgenic mice by 11 weeks of age. Memantine was tolerated well. However, although statistically not significant, its treatment showed a tendency toward a protective effect on rotarod performance or on hindlimb extension reflex. Moreover, the low dose memantine group significantly prolonged survival of transgenic mice compared with controls (141 days vs. 134 days, p<0.05).
Conclusions: These findings suggest that memantine, even if administrated at symptom onset, may have some beneficial effect on patients with amyotrophic lateral sclerosis (ALS).
P86 NEUROPROTECTIVE EFFECTS OF TACROLIMUS ON THREE MODELS OF SPINAL MOTONEURON DAMAGE
Ikeda KI1, Aoyagi JA2, Baba BS2, Iwasaki YI2
1PL Tokyo Health Care Center, Tokyo, Japan, 2Toho University Medical Center, Omori Hospital, Tokyo, Japan
E‐mail address for correspondence: [email protected]
Background: Tacrolimus, a neuroimmunophilin ligand, possesses pleiofunctional effects on several kinds of neurons.
Objectives: The purpose of this study is to examine whether this drug can expand survival of motor neurons in three experimental models of damaged spinal motor neurons.
Methods:Experiment 1. Organotypic spinal cord cultures were made from 10‐day‐old Sprague‐Dawley rats. Cultures were exposed to glutamate (10−5 M) or glutamate (10−5 M) + tacrolimus (10−7 to 10−5 M) for 14 days. The number of motor neurons (size⩾25 µm) and choline acetyltransferase activity (ChAT) were determined in the non‐treated control, the glutamate and the glutamate + tacrolimus groups (n = 20 slices/group).
Experiment 2. The left sciatic nerve was cut near the obturator tendon in one‐day‐old Sprague‐Dawley rats. After injury, rats received intraperitoneal injection of tacrolimus (0.1, 1.0, 5.0 mg/kg) or vehicle daily for 14 days. The number of large sized motor neurons (⩾25 µm) with prominent nucleoli was counted in both sides and the survival of motor neurons (%) was calculated as the number of motor neurons in the injured side /that of non‐injured ×100.
Experiment 3. Wobbler mice were given an intraperitoneal injection of tacrolimus (1.0, 5.0 mg/kg) or vehicle from disease onset (three to four weeks of age) daily for four weeks. Motor symptoms of the forelimbs were evaluated every week. After the treatment, neuropathological changes of the biceps muscles and the C5–6 cord were compared among tacrolimus‐ and vehicle‐treated wobbler mice (n = 10 mice/group).
Results:
Experiment 1. Compared to control culture, the survival of motor neurons and the ChAT activities were decreased by 30–40% in glutamate‐exposed culture. Three doses of tacrolimus‐treated cultures significantly inhibited neuronal death (p<0.05) and loss of ChAT activity (p<0.05).
Experiment 2. After axotomy, the survival of motor neurons was approximately 50% in vehicle‐treated rats. High dosage tacrolimus (5 mg/kg) treatment significantly saved axotomized motor neurons by 20% (p<0.05) compared with vehicle.
Experiment 3. Tacrolimus treatment did not delay the progression of motor deficits, denervation muscle atrophy and spinal motor neuron degeneration.
Discussion and conclusions: We studied the neuroprotective effects of tacrolimus on spinal motor neurons in in vivo and in vitro experimental models. This drug has no clinical effects in mutant superoxide dismutase (G93A)‐transgenic mice (1). The present and previous studies indicate that tacrolimus treatment protects spinal motor neurons against acute models of glutamate‐neurotoxicity and axotomy but has no benefits for chronic disease models of wobbler mice and mutant superoxide dismutase‐transgenic mice.
References
- Anneser JMH, Gmerek A, Gerkrath J, et al. NeuroReport 2001;12:2663–5.
P87 CHIP OVEREXPRESSION REDUCES THE MUTANT AR PROTEIN AND AMELIORATES PHENOTYPES OF THE SBMA TRANSGENIC MOUSE MODEL
Adachi H, Waza M, Katsuno M, Minamiyama M, Tokui K, Tanaka F, Doyu M, Sobue G
Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
E‐mail address for correspondence: [email protected]
Background: Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR). The pathologic features of SBMA are motor neuron loss in the spinal cord and brainstem, and diffuse nuclear accumulation and nuclear inclusions of the mutant AR in the residual motor neurons and certain visceral organs. Molecular chaperones are sequestered to inclusions, suggesting that molecular chaperones may be actively engaged in an attempt to degrade or refold components of the inclusions. CHIP (carboxyl terminus of Hsc70‐interacting protein) has three tetratrico peptide repeat (TPR) domains, which interact with the molecular chaperones Hsp70 and Hsp90, and a U‐box domain that interacts with the proteasome and confers E3 ubiquitin ligase activity on CHIP. CHIP has been shown to interact with ubiquitinated unfolded proteins trapped by molecular chaperones and degrade them, thus acting as a ‘quality control E3’.
Objectives: CHIP overexpression has been shown to suppress inclusion formation and cellular toxicity in cellular and zebrafish polyQ disease models. In this study, we examine the effects of CHIP overexpression on a cultured cell model and the transgenic mouse model of SBMA to explore a potential strategy for SBMA therapy.
Methods: The full‐length human CHIP fragment was subcloned into pCAGGS vector. The final plasmids were digested to remove the transgene. We generated CHIP overexpression mice by microinjection into BDF1 fertilized eggs. We crossed the mice expressing full‐length human AR with 24 or 97‐polyQ tract (AR‐97Q mice, 7–8 line) with CHIP overexpression mice.
Results: We demonstrated in a neuronal cell model that transient overexpression of CHIP reduced the monomeric mutant AR more than the wild‐type, suggesting that the mutant AR is more sensitive to CHIP than is the wild‐type. We also demonstrated high expression of CHIP inhibited neuronal nuclear accumulation of the mutant AR and ameliorated motor impairments in the SBMA transgenic mouse model. Western blot analysis showed that both the high‐molecular‐weight form of mutant AR protein complexes retained in the stacking gel and a band of monomeric mutant AR monomer in the spinal cord and muscle of the transgenic mice were diminished in the double transgenic mouse.
Discussion and conclusions: We reported that overexpression of CHIP markedly ameliorated clinical and pathological phenotypes, and that this amelioration was correlated with the reduction of mutant AR protein complexes in the mouse model of SBMA. Furthermore, the amount of monomeric mutant AR was also significantly reduced in the double transgenic mice, suggesting that degradation of mutant AR may have been accelerated by overexpression of CHIP. Thus, CHIP overexpression would provide a potential therapeutic avenue for SBMA.
P88 OLFACTORY ENSHEATHING CELL TRANSPLANTATION IN ALS MODEL MICE
Watanabe Y1, Morita E1, Ishimoto M1, Michio K1, Yasui K1, Fukada Y1, Doi K1, Nakano T1, Murrell WG2, Mackay‐Sim A2, Nakashima K1
1Division of Neurology, Institute of Neurological Sciences, Tottori University, Yonago, Japan, 2School of Biomolecular and Biomedical Science, Griffith University, Nathan, Australia
E‐mail address for correspondence: [email protected]
Background: An effective treatment has still not been established for amyotrophic lateral sclerosis (ALS). In recent years, ever‐greater hopes have been placed in the ability of stem cells to cure a damaged or degenerated central nervous system (CNS). However, motor neurons are widely distributed throughout the CNS. For the treatment of ALS, replenishing the stem cells or the alternatives by focal injection would be impractical for clinical application. To overcome this problem, we developed a transplantation method for mice that allows even diffusion of donor cells throughout the CNS.
Objectives: We chose the olfactory ensheathing cell (OEC) as a suitable donor cell. The OEC reportedly promotes regeneration and remyelination of injured motor pathways and enhances motor function recovery in experimental animal models. As a recipient we used transgenic mice with the mutated Cu/Zn superoxide dismutase (SOD1) gene (ALS‐TgM) (1). First we confirmed the safety of the transplantation procedure, then we transplanted OECs via the fourth ventricle in ALS‐TgM.
Methods: The OECs were extracted from mice which ubiquitously expressed green fluorescent protein (GFP), and then purified and expanded. The transplantation was performed on 90‐day‐old mice (wild‐type mice and ALS‐TgM) according to the report for rats (2), albeit with major modifications. Beginning one week prior to the operation, weekly clinical evaluations were made of body weight, hind limb extension score, and footprints, up to 200 days for wild‐type and until death for ALS‐TgM. Pathological observations were also performed with immunofluorescent staining in frozen sections.
Results: There were no differences between the OEC transplanted and non‐transplanted wild‐type mice with regard to perioperative side‐effects as well as long‐term motor functions. With regard to ALS‐TgM, after clinical evaluation, no significant differences could be found between OEC‐treated and non‐treated ALS‐TgM groups, nor were any differences noted with regard to age at onset, age at death, or disease duration. In the pathological observation, OEC survival in spinal cord was confirmed as long as 30 days after the transplantation.
Discussion and conclusions: We have established a novel cell transplantation method that can be used on mice. In the present experiment, however, we were unable to detect any beneficial effects of OEC transplantation on ALS‐TgM. Considering that OECs survive about 30 days in the recipient spinal cord, modulation of the appropriate transplant time would appear necessary. Furthermore, since it has previously been reported that neural or mesenchymal stem cells have shown promising results in the treatment of ALS, it would seem sensible to examine stem cells other than OEC in greater detail.
References
- Watanabe Y, et al. Brain Res Mol Brain Res. 2005;135:12–20.
- Wu S, et al. Neuroscience Letters 2002;318:81–4.
P89 TOWARDS GENE THERAPY USING GENETICALLY ENGINEERED CELLS AND STEM CELLS IN A MOUSE MODEL OF ALS
Karunaratne A1, Watanabe Y2, Cameron N1, Mackay‐Sim A1, Murrell W1
1Griffith University, Brisbane, Queensland, Australia, 2Tottori University, Yonago, Japan
E‐mail address for correspondence: [email protected]
Background: We have shown olfactory stem cells (OSCs) to be multipotent (1). Olfactory ensheathing cells (OECs) have been therapeutic in a rat model of paraplegia (2). These cells are a glial type and are intrinsically supportive to neurons. They surround the olfactory nerve and assist its continual regeneration. Both these cell types have been labelled with reporter genes under constitutive and tissue‐specific promoters introduced via retroviral vectors enabling stable integration. This result suggests potential to undertake gene therapy using these cells. Watanabe has created a mouse model of ALS by introducing a mutation to SOD1 (3). These mice mimic the symptoms and disease progression seen in human ALS patients, therefore allowing behavioural assessment and histological analysis of any potential gene therapy and cell transfer strategies.
Objectives: We propose to replace damaged motor neurons with the seeding of neural stem cells and protect and nurture new and damaged neurons with olfactory ensheathing glia expressing genetically introduced protective factors.
Initial aims. 1) To generate lentiviral constructs expressing GDNF and BDNF under various tissue specific promoters, a constitutive promoter and an inducible promoter. 2) To transduce these viral constructs into Rosa‐GFP OECs and OSCs. 3)To test these cells in vitro for genuine delivery of growth factors. 4) To establish cell transplant delivery and detection in SOD1 mutant mice. 5) To test various combinations of OEC‐growth factor delivery cells and stem cells in these mice. 6) To assess behavioural parameters to indicate any reduction in disease progression. 7) To analyse post‐mortem tissue.
Methods: Gene sequences were obtained by PCR from genomic and cDNA sources and assembled in commercial plasmids. Inserts were confirmed using restriction analysis and sequencing. Lentivirus particles were generated by transfection in Virapower 293FT producer cell line and components of the Virapower Lentiviral Expression System (Invitrogen). OECs from Rosa‐GFP (BL 6 background) were transduced with various lentiviral vectors, propagated in vitro and assayed for production of GDNF using antibody detection. OSCs from these mice were propagated as described and in some cases transduced with viral vectors. Methods of grafting were initiated and OECs were transplanted into the fourth ventricle along the midline 6 mm backward from Bregma suture at 3.75 mm in depth. Immunosuppression utilized cyclosporin (Cs)10 mg/kg/day, i.p. and tacrolimus (FK506) 3.2 mg/kg/day, p.o. dissolved in drinking water. Transplanted OECs were detected using fluorescence microscopy for GFP signal.
Results: To date the constructs CMV‐GDNF, NF‐GDNF, GFAP‐GDNF and Tet‐CMV‐GDNF have been constructed, packaged and transduced into target cells. In vitro expression of GDNF has been assessed. Transplantation of GFP‐labelled OECs has been successful with detection 30 days after transplant.
Conclusion: Feasibility for this strategy has been demonstrated.
References
- Murrell, et al. Dev Dyn. 2005;233:496–515.
- Lu, et al. Brain 2002;125:14–21.
- Watanabe Y, Yasui K, et al. Brain Res Mol Brain Res. 2005;135:12–20.
P90 HUMAN NEURAL PROGENITOR CELLS SECRETING GLIAL CELL LINE‐DERIVED NEUROTROPHIC FACTOR (GDNF) PROTECT LARGE MOTOR NEURONS IN THE SPINAL CORD OF ALS RATS
Suzuki mM1, McHugh J1, Shelley B1, Wallace K1, Klein SM1, Aebischer P2, Svendsen CN1
1University of Wisconsin‐Madison, Madison, WI, USA, 2Swiss Federal Institute of Technology, Lausanne, Switzerland
E‐mail address for correspondence: [email protected]
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease causing the progressive loss of brain and spinal cord motor neurons. Mutations in the superoxide dismutase 1 (SOD1) gene, which are associated with 1–2% of human patient cases, are thought to induce ALS in a gain of function effect through an unknown pathway suggested to include protein accumulation and aberrant enzymatic activity leading to increased reactive oxygen/nitrogen species. Recent studies suggested the involvement of neuronal‐glial interactions in ALS pathogenesis (1). Motor neuron degeneration in ALS may be due to dysfunction in the surrounding astrocyte populations.
For both drug delivery and astrocyte replacement, human neural progenitor/stem cells (hNPC) can be used as these cells can migrate and integrate into the damaged nervous system (2). Furthermore, recent studies have shown that in animal models of ALS, growth factors such as glial cell line‐derived neurotrophic factor (GDNF) can have significant neuroprotective effects. As stem cells migrate and integrate into the damaged nervous system, produce astrocytes and can be genetically modified to release these growth factors, they represent an ideal source of tissue for delivery. Most recently we prepared hNPC from developing cortex secreting glial cell line‐derived growth factor (hNPC‐GDNF) and transplanted these cells into the lumber spinal cord of rats overexpressing the G93A SOD1 mutation (SODG93A) and observed cell survival, migration, and maintenance of GDNF release in the end‐stage disease animal (3).
In the current study, we showed that hNPC‐GDNF could survive in the spinal cord of ALS rats without tumour formation and spread up to 6.7 mm from the injection sites. We also confirmed high expression (up to 820 pg/mg tissue protein) of GDNF in the transplanted area using ELISA even at the disease end‐stage. To determine whether hNPC‐GDNF transplantation affected motor neuron number and cell size, hNPC‐GDNF was unilaterally transplanted into the lumbar spinal cord of pre‐symptomatic ALS rats (70 days of age). The spinal cords were collected at the mid‐stage of disease (six weeks after surgery). There was a significant protection of large motor neurons (>700 µm2) in the region of GDNF release compared to the non‐grafted side.
These results indicate that hNPC‐GDNF can survive, maintain GDNF release, and protect motor neuron loss at the mid‐stage of disease in a rat model of ALS.
Acknowledgement: This study was supported by the ALS Association.
References
- Clement AM, Nguyen MD, Roberts EA, et al. Wild-type non-neuronal cells extend survival of SOD1 mutant motor neurons in ALS mice. Science 2003;302:113–7.
- Svendsen CN, Langston J. Stem cells for Parkinson’s disease and ALS: replacement or protection? Nature Medicine 2004;10:224–5.
- Klein SM, Behrstock S, McHugh J, et al. GDNF delivery using human neural progenitor cells in a rat model of ALS. Human Gene Therapy 2005;16:509–21.
P91 MUSCLE IS NOT A PRIMARY TARGET IN ALS AS REVEALED BY GENE THERAPY IN MUSCLE OF ALS MICE
Miller TM2, Kim S1, Hester M1, Umapathi P1, Arnson H1, Rizo L1, Mendell JR1, Gage FH3, Cleveland DW2, Kaspar BK1
1Columbus Children's Research Institute, Columbus, OH, USA, 2Ludwig Institute for Cancer Research, La Jolla, CA, USA, 3The Salk Institute for Biological Studies, La Jolla, CA, USA
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a fatal, progressive paralysis arising from premature death of motor neurons. An inherited form is caused by dominant mutation in the ubiquitously expressed enzyme superoxide dismutase (SOD1), whose non‐cell autonomous toxicity to motor neurons requires mutant mediated damage to as yet undefined non‐neuronal cell types.
Objective: Given the severe denervation induced muscle atrophy, two gene therapy approaches were tested in SOD1G93A mice to evaluate the role of muscle in ALS.
Methods: To directly assess whether increased muscle proliferation affected disease course in a mouse model of inherited ALS caused by mutation in SOD1, AAV1‐follistatin or AAV‐GFP (1×1010 viral genomes per injection) was injected bilaterally via intramuscular delivery into the hindlimb quadriceps and tibialis anterior muscles of sixteen 40‐day‐old (equal distribution of male and female) animals. To determine the extent of hyperplasia, hypertrophy and/or muscle sparing in the ALS animals, myofiber numbers within the gastrocnemius muscle were counted in serial sections of AAV‐follistatin‐ or AAV‐GFP‐ treated groups (n = 8 animals).
To test whether SOD1G93A damage directly within muscle contributes to toxicity, a lentivirus was constructed that encodes an siRNA directed against SOD1. Since lentivirus pseudotyped with VSV‐G is not retrogradely transported, intramuscular injection of this virus produced siRNA exclusively within the muscles.
Results: Muscle mass was enhanced with AAV encoded expression in muscle of follistatin, a potent stimulant of muscle growth. Despite increase in muscle mass, myofiber number, fiber diameter and overall strength, survival was not significantly affected. Suppression of mutant SOD1 accumulation by viral delivery to muscles of transcription mediated short interfering RNA against SOD1 did not maintain grip strength. Thus, in SOD1G93A mice mutant damage to muscles is not a significant contributor to pathogenesis in ALS.
Discussion: These data demonstrate that muscle is not a significant contributor to toxicity in ALS mice. While muscle is not a direct therapeutic target, it may nevertheless be an attractive target for synthesis of factors, such as IGF‐1, whose delivery to spinal motor neurons can enhance motor neuron survival.