John Chi, Mike DeSalvio, Kevin Ip, Khine Win, Michael Nguyen Disaggregation of a-Synuclein by way of...
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Transcript of John Chi, Mike DeSalvio, Kevin Ip, Khine Win, Michael Nguyen Disaggregation of a-Synuclein by way of...
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John Chi, Mike DeSalvio, Kevin Ip, Khine Win, Michael Nguyen
Disaggregation of a-Synuclein by way of Isradipine
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Specific Aims Show the effectiveness of subcutaneous
administration of calcium channel blockers Determine safe dosage Effective concentration
Determine effectiveness of Isradipine against a-synuclein plaques
Restore affected areas by decreasing the intracellular calcium levels
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Objectives
Show spatiotemporal control through subcutaneous injection
Prevent cell death in PD patients Mitigate the effect of Lewy Bodies
Affects Alzheimer's as well as Parkinson’s disease
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Background & Significance
Isradipine: dihydropyridine Calcium channel blocker
○Treats high blood pressure and reduces risk of stroke
Approved small molecule○Multiple trade names
•Dynacirc
•Prescal
http://en.wikipedia.org/wiki/File:Isradipine.png
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Parkinson’s Disease Neurodegenerative disorder 100-200 people in 100,000 Average age of onset 55 to 60 Annual cost approximately $11 billion
http://www.wolframalpha.com/input/?i=parkinsons+disease
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Parkinson’s Disease
Impairs motor skills, cognitive processes, and other functions Tremors Rigidity Bradykinesia Postural instability
http://www.wolframalpha.com/input/?i=parkinsons+disease
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Parkinson’s Disease
Diminished cognitive processes Executive dysfunction
○Planning○Abstract thinking○ Initiating appropriate actions○ Inhibiting inappropriate actions
Eventually develop into dementia Accompanied by behavior and mood
alterations
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Parkinson’s Disease
Idiopathic Various hypotheses
Viral Genetic Lewy Bodies
http://www2.kumc.edu/coa/images/AMED900/Dementia/DementiaSyndromeGraph.gif
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Lewy Bodies
Alpha-Synuclein Eventually accumulate in substantia nigra Accumulate in various parts of the brain
Olfactory bulb Medulla oblongata Pontine tegmentum
Leads to cell death
http://shirley-kwan.com/wp-content/uploads/2007/09/lewy.jpg
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Current Research
Improving quality of life Therapy and exercise
Neuroprotection Drug treatment to slow, stop, reverse
progression of PD
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Neuroprotection
Anti-apoptotic drugs Antiglutamatergic agents Monoamine oxidase-B inhibitors Calcium channel blockers Growth factors
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Current Treatments
Levodopa (L-DOPA) MAO-B inhibitors Amantadine Anticholinergics Surgery
http://www.3dchem.com/imagesofmolecules/Levodopa.jpgc
Treatment Standard: L-Dopa
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Isradipine background
IC50 34+-8 uM 60-65% excreted in urine 25-30% excreted through feces 5-15% actually absorbed into the blood Decrease responsiveness
○ Administer intravenously
http://images.containerstore.com/images/catalog11966/11966.jpg
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Research Design & Methods
Overview Isradipine as a therapeutic approach Experimental approach Proposed Pre-Clinical trials Prospective Clinical trials
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Mechanism of α-synuclein cytotoxicity
Figure: Fibrilization pathway
Native unfolded monomer
•Β-sheet rich oligomers or protofibrils
•Fibrils•Lewy body
α-synuclein proteinReference: http://www.molecularneurodegeneration.com/content/4/1/9
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Proposed Therapeutic Approach
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Cav1.3 SNc main ion channels:
Cav1.3 (voltage gated)NMDAR (iGluR, ligand gated)
L-type Calcium channel Regulate spontaneous pace making activity Modulator of neuronal spiking behavior Binding site on α1D sub unit Isradipine (1,4- dihydropyridine) as inhibitor Theorized mechanism:
Conformational changes by binding Ca2+ influx control approach
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Research Proposal (Intro)
Quantification of effect on targeted drug delivery approach:Behavior study Calcium imaging (striatum & sN)Immunohistochemistry staining by Caspase-
3 antibody to determine apoptotic cells Clinical phase I
Time frame: 4-6 months Potential pitfall: Low blood pressure
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Comparison with other methods Rifampicine (Lewy body approach)
Antibiotic to treat TB & Leprosy Disintegrate α-syn fibrils Inhibit fibrils formation by stabilization monomer and
oligomersNo animal models Correlation with PD symptoms not yet testedMight enhance toxicity if protofibril cytotoxicity hypothesis is
true
Reference: Adphrodite Kapurniotu. Targeting α-synuclein in Parkinson’s Disease. chemibiol 2004;11:1476-1478
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Comparison with other methods Β-synuclein protein (Gene therapy approach)
Subfamily of synuclein familyConcentrated in presynaptic nerve terminals (133AA)Not found in Lewy body inclusionsStudies indicate inhibition of α-syn aggregationOverexpression by upregulating the gene:
○ Human β-syn transgenic mice○ Transfected by lentivector
Major fall back: random insertion of gene might cause cancer and other mutations
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α-synnuclein protofibrils
Inhibition by β–synuclein (Green color)
Comparison with other methods
Reference: Dr.Igor Tsigelny. Modelling molecular basic of Parkinson’s disease. SDSU review. Winter 2007:52-57
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Advantages of our approach No effective inhibitor drug for aS protofibrils
aggregation yet Indirect method to manipulate Ca2+ influx No tendency to cause gene therapy related
implications such as mutation Use of approved drug (Isradipine) Targeted drug delivery Recent studies shows non-targeted delivery
results in reduction in LID Potential to fast-track clinical trial Most beneficial if couple with gene therapy in
future
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Proposed Pre-Clinicals
Administer 6-Hydroxydopamine in mice to induce lesions (40 specimens)
Wait two weeks until lesions mature Administer Isradipine to 20 mice (Dose
2x/day) Administer equivalent volume of saline
to the other 20 mice Sacrifice mice at the end of 18 weeks
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Pre-Clinical
Use B6D2F1/J Mice (Jackson Labs) Transgenic Studies Behavior Studies Bioassays for drugs, pathogens, nutrients
Study Groups: PD, PD + L-DOPA, PD+ Placebo, Wild-Type
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Drug Delivery
Injection of Time Release Pellet Various Concentrations (ex. 0.1 mg/kg/day) 14-day life
Location: Subcutaneously Between shoulder blades (The “scruff”)
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Matrix of Mice Groups
6-OHDA L-DOPA Isradipine Saline
Group 1 + - - -Group 2 + - + -Group 3 + + - -Group 4 + + + -Group 5 - + - -Group 6 - + + -Group 7 - - + -Group 8 - - - +
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Pitfalls & Solutions
Undue drop in Blood Pressure Concentration kept low
Mouse difference in Metabolic Rate Select for: 4 weeks old, 15-20 grams
Effects not from isradipine Perform Histology to determine
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Future Clinical Trials
Phase I 50 Volunteers Early- to Mid-Stage Parkinson’s Blinded Trial
Drug L-DOPA +/- Isradipine Extended Release Formulation
○Packaged in Microspheres
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Goal of Phase I Trials
Isradipine Effective Reduces Adverse Involuntary Movements Did not Drop BP dangerously Low
Improves Quality of Life More control over Movement
Not a replacement for L-DOPA Works in conjunction with L-DOPA
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Prospective Clinical Trials
Phase I: 50 volunteer patients, blind trial Covered by SBIR
Phase II: 200 patients double blind Covered by NIH
Phase III: 2000 patients double blind Investors: VC’s and Industry Sponsors
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Budget Overview
$100,000 for 6 months We plan to borrow equipment
PCR, Ultra Centrifuge, and lab equipment Outsource for DNA sequencing and blots if
necessary Capitalize on help of 4 Interns for lab work Rent small work space in Fullerton, Ca
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Salary Information
Name Base Salary Months Effort Requested Salary
Michael Nguyen$80,000 5 25% $8,333
Michael DeSalvio$82,000 3 35% $7,175
Kevin Ip$80,000 4 20% $5,333
John Chi$75,000 5 25% $7,812
Khine Win$77,000 5 20% $6,416
Lab Technician$25/hr 5 10% $3,000
Animal Technician$25/hr 5 10% $3,000
$41,069
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Equipment CostsDescription Cost
Bio-Rad Gel Doc1000 Imager Cabinet w/ Transilluminator $2,000
Labnet International MultiGene Mini Personal Thermal Cycler $2,700
Nuaire Nu 425-600 Option A-P2 Lab Hood 72" $2,500
Fileserver $1,500
Laboratory laptops x 5 (Apple MacBook Pro 15”) $10,000
2 Experimental PC Workstations $1000
Refrigerator $500
Summit VT 225 Lab Freezer (-30 deg C) $1,600
Liquid Nitrogen System $800
$22,800
•The Bio-Rad Imager is required for the imaging studies•The Thermal Cycler is required to determine •The Nuaire fume hood is necessary for manipulating organic compounds such as fura2 for cell staining and histological studies.
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Facility Rental
Facility Rental: $9,515 Fullerton Business Center #140 1501 E Orangethorpe Ave, Fullerton, C
A 92831 Space Size: 1,522 sqft Property Type: Office / R&D Price Per Sqft: $1.25 Rent: $1,903 FS
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Laboratory Costs Laboratory Supplies: $10,000 Laboratory supplies include items such as
safety equipment gloves oligonucleotides fura2 isradipine
Estimated at approximately $5,000 for startup and $1,000/mo.
• Test tubes• Scales• Slides• Burners• Syringes
• Flasks
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Research Strategy
Run preclinical trials Determine effect or Ca++ channel blockers on
adrenergic neurons in Substantia Nigra
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Citations
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Questions