Poster Abstracts 11 to 21
5th Annual Meeting
Poster Abstracts
11
THE DOPAMINE-1 RECEPTOR PROMOTER CONFERS CELL-TYPE SPECIFICITY IN CULTURED BASAL GANGLIA NEURONS
Zhang, S., Yool, A.J.
Depts. Of Neurology and Physiology, The University of Arizona, Tucson, Arizona 85724.
According to the classical theory of basal ganglia function, there are two distinct GABA-ergic output pathways originating from neurons of the putamen. The “direct” pathway bears dopamine-1 receptors (D1), uses substance P as a co-transmitter, and facilitates movement. The “indirect” pathways bear dopamine-2 (D2) receptors, use enkephalin as a co-transmitter, and suppresses motor activity. Since many movement disorders are characterized by an imbalance between the activities of these two pathways. We propose that a gene therapy based on their selective control could provide a new therapeutic avenue. For example, cell-type specific promoters coupled to voltage-gated ion channels could selectively modulate basal ganglia activity. In order to test the feasibility of this approach, we have: 1) developed a primary culture model of basal ganglia suitable for gene transduction and electrophysiological experiments; 2) shown that these cultures contain the same neurochemical markers found in vivo; and 3) demonstrated that cell-type specific promoters can differentially drive gene expression in neuronal subpopulations found in these cultures. Immunocytochemical methods demonstrated population of neurons positive for the D1 and D2 receptors, substance P, enkephalin, and other markers. The D1-receptor promoter was placed in an expression vector to drive a green fluorescent protein reporter gene. The D1-receptor promoter showed neuronal selectivity in cell lines and cell-type specificity in basal ganglia cultures. Neurons that were strongly immunoreactive for the D1 receptor demonstrated high reporter-gene expression when transduced with the D1-receptor-promoter construct. We conclude that cultured basal ganglia neurons can be used as a model for gene transfer experiments; and that the D1-receptor promoter confers cell-type specificity in this system.
12
POTENTIAL THERAPEUTIC USES OF NEURAL STEM CELLS IN A RAT MODEL OF ALS
Klein, S., Hoffman, K., Wallace, K., Behrstock, S., Svendsen, C.N.
Waisman Center, University of Wisconsin-Madison, Madison, WI 53703.
Amyotrophic Lateral Sclerosis is a fatal disease resulting from the degeneration of upper and lower motor neurons. Mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene lead to motor neuron loss in a rat model of ALS. Part of the mechanism for this cell death may be dysfunctional astrocytes leading to a lack of glutamate uptake. In this study we further characterize the model of ALS with regard to overall survival times, rotorod behavioral testing and anatomical changes in the brain. We hypothesized that transplantation of human neural stem cells genetically modified to release glial derived neurotrophic factor (GDNF) may result in new astrocytes which would be neuroprotective. The initial phase of this study, establishing anatomical integration of transplanted stem cells, is currently underway and will be presented. Support by ALS Association to CNS.
13
REGULATED GLIAL DERIVED NEUROTROPHIC FACTOR PRODUCTION BY NEUROSPHERES PROTECTS DOPAMINE NEURONS
Behrstock, S., Klein, K., Hoffman, K., Delong, N., Aebischer, P., Svendsen, C.N.
Waisman Center, University of Wisconsin-Madison, Madison, WI;
Swiss Federal Institute of Technology, Lausanne, Switzerland.
Human neurospheres (hNS), which contain a mixture of stem and progenitor cells, can be grown indefinitely in vitro and can survive transplantation. We assessed whether such cells can be manipulated to produce therapeutic molecules, specifically glial derived neurotrophic factor (GDNF) that protects dopamine neurons in several models of Parkinson’s disease. It is vital that GDNF production is regulatable for an eventual clinical setting. Therefore, we co-infected hNS with a lentivirus carrying either the GDNF or green fluorescent protein (GFP) gene under control of a tetracycline promoter and a lentivirus carrying the tetracycline transactivator (tTA) gene). We show infected hNS produce GFP, which is rapidly and tightly regulated by doxycycline. Furthermore, we show infected hNS produce high levels of GDNF, which is regulated in a time-dependent manner by doxycycline. When transplanted, infected hNS can survive and express foreign proteins for up to two weeks. Infected human neurospheres released GDNF into the media at levels high enough to increase dopamine neurite outgrowth and cell body size. As hNS survive transplantation, regulatable GDNF-producing hNS may have therapeutic potential in Parkinson’s disease. Support by Northwestern Mutual Foundation to CNS.
14
ETHYL-EICOSAPENTAENOATE (ETHYL-EPA) IN HUNTINGTON DISEASE: A RANDOMIZED, PLACEBO-CONTROLLED TRIAL
Leavitt, B.R., et al, Vancouver, BC, Canada.
This trial was designed to test the effect of Ethyl-EPA versus placebo on motor signs in HD. 135 patients with early stage HD entered a double-blind study at three US centers and one center each in Canada, the UK and Australia. Patients were randomized to receive 2g/d Ethyl-EPA or matching placebo for one year. Ethyl-EPA was generally well tolerated with similar adverse events to placebo. The primary end-point of the trial was the total motor score-4 (TMS4) sub-scale of the Unified Huntington’s Disease Rating Scale (UHDRS). Secondary end-points were other UHDRS sub-scales and the total UHDRS. Pre-planned analyses were carried out on both an intent-to-treat (ITT) group that included all 135 randomized patients, and a per protocol (PP) group that excluded 38 patients for non-compliance with protocol. Pre-planned analyses on both groups were: ANCOVA with center, baseline TMS4 (disease severity), CAG size and treatment as covariates; and chi square with a binary division into patients who improved or deteriorated from baseline. On the primary ITT analysis, there was no significant difference between Ethyl-EPA and placebo. In the PP analysis, Ethyl-EPA was significantly better than placebo on the chi square (p<0.05) but just missed significance on ANCOVA (p=0.06). Because non-compliant patients tended to be more severely affected, the PP group was significantly less symptomatic at baseline than the ITT group. No significant effects of Ethyl-EPA were seen on total UHDRS or other sub-scales. Sub-group analysis revealed no effect of center, but baseline TMS4 score and CAG size appeared to influence outcome in both the ITT and PP Groups. Ethyl-EPA may have a modest beneficial effect in HD. Further trials are indicated to confirm these findings, and determine whether the effect is immediate or represents a slowing of progression.
15
ONSET OF ACTION OF OLANZAPINE/FLUOXETINE COMBINATION IN BIPOLAR DEPRESSION
Dube’, S., Tollefson, G., Briggs, S., Van Campen, L., Case, M., Tohen, M., Eli Lilly & Co., Indianapolis, IN 46285. Thase, M., Western Psych. Inst. & Clinic, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213.
The present study investigated the onset of action of olanzapine/fluoxetine combination (OFC) in a study (n-833) designed to compare OFC with olanzapine and placebo for treating bipolar depression. The primary efficacy was the Montgomery-Asberg Depression Rating Scale (MADRS). Additional analyses were conducted for a reduced MADRS total score excluding sleep and appetite items. Onset of action was analyzed using traditional and pattern analyses, mixed-effects curvilinear regression, and survival analysis of sustained response. An area under the curve analysis was also conducted to assess overall effectiveness. Traditional analysis revealed significantly greater improvement in MADRS scores at week 1 for OFC versus placebo (-9.55 vs. -5.08, p<.001) and for olanzapine versus placebo (-8.31 vs. -5.08, p<.001). Significant differences were maintained for the reduced MADRS total score. Pattern analysis revealed that OFC had a significantly greater percentage of early persistent responders (defined as response within two weeks and not followed by a relapse) than the OLZ or PLA groups (32.4% vs. 18.3%, p<.05; and 12.7%, p<.001, respectively). Survival analysis of sustained response revealed a significantly shorter time to sustained response for OFC versus placebo (p<.001), for OFC versus olanzapine (p<.05), and for olanzapine versus placebo (p<.05). Mixed-effects curvilinear regression analysis revealed a significant therapy by time interaction (p<.001). Area under the curve analysis revealed a significantly greater percentage of total possible improvement (48.2%) for OFC vs. olanzapine (38.5%, p<.01) or placebo (27.8%, p<.001). OFC and olanzapine demonstrated significantly faster onset of action compared to placebo as measured by traditional, pattern, and survival analyses. OFC showed significantly faster onset of action than olanzapine as measured by pattern and survival analyses.
Funding provided by Eli Lilly and Company.
16
OLANZAPINE ALONE AND OLANZAPINE IN COMBINATION WITH FLUOXETINE IMPROVE HEALTH-RELATED QUALITY OF LIFE IN PATIENTS WITH BIPOLAR DEPRESSION
Shi, L., Namjoshi, M., Baker, R., Dube, S., Tohen, M., Breier, A., Eli Lilly and Company, Indianapolis, IN 46285; Yu, X., MedFocus Inc., Chicago, IL 60018.
Health-related quality of life (HRQL) was compared for olanzapine monotherapy, olanzapine/fluoxetine combination (OFC), and placebo in a pooled sample from two double blind randomized clinical trials. Patients with bipolar depression as defined is DSM IV and baseline MADRS rating of e” 20 were randomized to either olanzapine (5-20 mg/day, n=370), combination of olanzapine (6 or 12 mg/day) plus fluoxetine (25 or 50 mg/day) (n=86), or placebo (n-377) for 8 weeks. Mean changes from baseline to week 8 in the Medical Outcomes Study Short Form-36 (SF-36) were used as the measure of HRQL improvement. As compared with placebo, OFC treatment exhibited significantly greater improvement in 5 domains [general health perception (p<.001), mental health (p<.001), role limitations due to mental health problems (p<.001), social functioning (p<.001) and vitality (p=.002)] and in the mental health summary score (p<.001). The improvement in the same 5 domains and mental health summary score was also significantly greater among patients on OFC than olanzapine alone. Olanzapine compared with placebo exhibited significantly greater improvement in 3 of 8 SF-36 domains [mental health (p=.015), role limitations due to mental health problems (p=.046) and social functioning (p=.006)], and in the mental health summary score (p=.002). Compared to placebo, both olanzapine alone and OFC were associated with improvement in HRQL outcomes in patients with bipolar depression. In addition, OFC treatment produced greater improvement in HRQL outcomes than olanzapine monotherapy.
Funding provided by Eli Lilly and Company.
17
PDA-ENHANCED SPEECH TREATEMENT FOR INDIVIDUALS WITH PARKINSON'S DISEASE
Halpern, A.E.1, Matos, C.E.2, Ramig, L.O.1,2, Spielman, J.L.1, Bennett, J.K.2
National Center for Voice and Speech1, Denver, CO 80204
University of Colorado-Boulder2, Boulder, CO 80309.
At least 89% of individuals with Idiopathic Parkinson Disease (IPD) have disordered speech. Previous studies utilizing LSVT® have generated the first short and long-term experimental efficacy data for voice treatment for individuals with IPD (Ramig LO et al, JNNP 2001: 71:493-8). A preliminary PET study has documented changes in neural functioning following LSVT® (Liotti MM et al, Neurology 2003; 60:432), thus indicating the potential impact of early intervention for speech treatment. Despite these advancements in establishing the efficacy of speech treatment for individuals with IPD, barriers such as physical limitations and lack of access to a qualified therapist prohibit many individuals from receiving speech treatment. The purpose of this study is to describe the development and use of a personal digital assistant (PDA), as an enhancement tool for the LSVT®. This device, named the LSVT companion or LSVTC, was developed in response to the need for a tool that could make treatment accessible to more individuals and help to sustain treatment effects. Pilot data from two subjects (Ss) with IPD who trained with the LSVTC demonstrate the feasibility of utilizing a specially programmed, but otherwise conventional PDA for these clinical treatment and research purposes. Both Ss made substantial gains in vocal loudness (up to 6db SPL increase), and demonstrated a significant improvement in psychosocial functioning on a self-administered voice rating scale. The goal of the LSVTC is to make speech treatment a reality for more individuals with IPD and allow them to reap the benefits of functional communication and expression. This study will demonstrate the progress and commitment toward this goal.
This work is funded by NIH/NIDCD and NINDS R21-DC05583.
18
A2A RECEPTOR ANTAGONIST PREVENTS DOPAMINE AGONIST-INDUCED MOTOR COMPLICATIONS IN PRIMATE AND RODENT MODELS OF PARKNISON’S DISEASE
Bibbiani, F.1, Oh, J.D.2, Chase, T.N.1, 1ETB, NINDS, National Institutes of Health, Bethesda, MD; 2Psychology Dept., Central Michigan University, Mount Pleasant, MI.
Adenosine A2A receptors are highly expressed in the GABAergic output neurons of the striatum and their blockade has been shown to exert antiparkinsonian and neuroprotective effects in experimental models of Parkinson’s disease (PD). The present study investigated whether the selective adenosine A2A receptor antagonist KW-6002 has a prophylactic effect on motor response complications in primate and rodent parkinsonian models. In 6-OHDA hemiparkinsonian rats, oral administration of KW-6002 (2.5 mg/kg, p.o.) in combination with levodopa treatment once daily for three weeks significantly reversed the shortened motor response to acute levodopa challenge (p<0.01). In monkeys, co-administration of KW-6002 (75 mg/kg, p.o.) apomorphine (0.2 mg/kg, s.c.) once daily completely prevented the development of aopmorphine-induced dyskinesia (p<0.01). Control animals showed dyskinesia in about ten days of apomorphine treatment. The KW-6002-treated animals also manifested apomorphine-induced dyskinesia in about ten days after the cessation of KW-6002 co-administration. In both rats and monkeys, KW-6002 did not exacerbate parkinsonian signs when given with dopamine agonists. These results implicate adenosine A2A receptor-mediated mechanisms in the pathogenesis of dopamine agonist-associated dyskinesia in PD and suggest that drugs acting to block these receptors might confer therapeutic benefits to these patients.
19
LONG-TERM SAFETY OF ZYDIS® SELEGILINE IN PARKINSON’S DISEASE: INTERIM RESULTS
Wess, M., Scott, D., Medical Affairs, Amarin Pharmaceuticals, Inc., Mill Valley, CA.
The long-term safety of Zydis selegiline (ZS) when used as an adjunct with levodopa was assessed in an open-label extension study involving 26 US sites. ZS is a rapidly dissolving formulation of selegiline. All patients received 2.5 mg ZS/day for up to 4 years. Doses of levodopa could be modified as needed. Visits were scheduled every 6 months. Safety was assessed by adverse events (AEs), vital signs, physical examinations and laboratory results. A total of 254 patients were enrolled; including 83 patients previously treated with placebo (P). Patient demographics were: 172 male/82 female, mean age 67 years, mean duration of PD- 7 years, and 94% Caucasian. The durations of treatment and numbers of patients are: N=180 – 6 months, N=156 – 12 months, N=132 – 18 months, N=91 – 24 months, N=6 – 36 months. Drug related AEs included: dizziness, dyskinesias, insomnia, dry mouth and nausea. AE discontinuations occurred in 39 patients (15%) with a slightly higher proportion in patients previously treated with P – 22% (18/83) compared to ZS – 12% (21/171). No other differences were observed. The most frequent AEs resulting in discontinuation were: depression (5), hallucinations (4), dizziness (4) and hypotension (4). No notable changes from baseline were noted in other safety parameters. Since prior P patients were started immediately on 2.5 mg without an initial dose of 1.25 mg, this may have led to the higher discontinuation rate observed with prior P patients. These results are consistent with previous ZS studies. Overall, ZS was generally well tolerated and this study supports the long-term use.
This study was sponsored by Elan Pharmaceuticals, Inc.
20
THE PHARMACODYNAMICS OF ZYDIS® SELEGILINE: CONFIRMATION OF MAO-B SELECTIVITY
Wess, M., Scott, D., Medical Affairs, Amarin Pharmaceuticals, Inc., Mill Valley, CA 94941.
To confirm monoamine oxidase type B (MAO-B) selectivity of Zydis selegiline (ZS), an open-labeled, randomized, parallel group study of 3 doses of ZS (1.25 mg, 2.5mg, 5 mg), compared to 5 mg BID selegiline (S) was performed in 61 healthy male volunteers. MAO-A degrades serotonin to 5-hydroxyindoleacetic acid (5-HIAA) and metabolizes norepinephrine to 3-methoxy-4-hydroxyphenylglycol (MHPG). MAO-B primarily degrades dopamine and phenylethylamine (PEA). Decreases in cumulative urinary excretion of 5-HIAA and plasma MHPG are indirect markers indicative of MAO-A inhibition whereas increased urinary PEA is an indirect marker of MAO-B inhibition. Urine and plasma samples were obtained at Baseline and on Day 10 (Steady state). Mean cumulative amounts excreted of 5-HIAA increased with all 3 ZS dosages in comparison to S, which decreased slightly. The mean percentage changes from baseline for 5-HIAA were: 26%, 11.3%, 4.0% and -0.04% for the 1.25, 2.5, 5.0 mg ZS and S treatments respectively. The mean percentage changes from baseline in MHPG were: -10.7% (1.25 mg ZS), -2.8% (2.5 mg ZS), -14.5% (5 mg ZS), and -9.1% (S). None of the MHPG decreases represent clinically significant MAO-A inhibition. Typically non-selective MAO inhibitors produce decreases exceeding 70% from baseline (Pickar, D et al, Psychopharmacology 1981;74:4-7). In contrast, mean cumulative amounts excreted of PEA increased 20- to 40-fold across all treatment groups. The increase in PEA excretion were dose related with ZS. In conclusion, ZS is a selective MAO-B inhibitor and this selectivity is maintained at all doses studied.
This study was sponsored by Elan Pharmaceuticals.
21
MODELING PARKINSON’S DISEASE IN ZEBRAFISH
Kobayashi, K., Guo, S.,
Department of Biopharmaceutical Sciences, University of California, San Francisco, CA 94143-0446.
Parkinson’s Disease (PD), the 2nd most common neurogdegenerative disorder, is characterized by an age-dependent loss of dopaminergic neurons leading to disabling motor impairments including difficulty initiating movements, resting tremors, and slowness of movement. We hope to understand the molecular basis of PD pathogenesis and progression through a zebrafish model of PD. We have generated a transgenic zebrafish model of PD that express human alpha-synuclein (á-syn), a protein believed to be central to PD, in neurons of the fish. So far, ten different lines that express either the wildtype of the mutant forms (A53T or A30P) of á-synuclein have been created. We have begun to characterize these lines at the cellular and molecular level by Western blot analysis to determine the expression level of á-syn and the presence of ligomerized á-syn, which is believed to have toxic properties. We will further determine the neurotoxicity of á-syn expression by examining the states of dopamine neurons and the locomotor behavior of zebrafish. Later, we plan to utilize the strength of the zebrafish model to conduct genetic screens to further elucidate the process of dopaminergic neuron degeneration seen in PD. Furthermore, these fish can be used to screen for small molecule compounds that can halt dopamine neuron degeneration.
Supported by NIH/NINDS.