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Disaggregation of a-Synuclein by way of Isradipine

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1 Disaggregation of a-Synuclein by way of Isradipine
John Chi, Mike DeSalvio, Kevin Ip, Khine Win, Michael Nguyen

2 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

3 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

4 Background & Significance
Isradipine: dihydropyridine Calcium channel blocker Treats high blood pressure and reduces risk of stroke Approved small molecule Multiple trade names Dynacirc Prescal

5 Parkinson’s Disease Neurodegenerative disorder
people in 100,000 Average age of onset 55 to 60 Annual cost approximately $11 billion

6 Parkinson’s Disease Impairs motor skills, cognitive processes, and other functions Tremors Rigidity Bradykinesia Postural instability

7 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

8 Parkinson’s Disease Idiopathic Various hypotheses Viral Genetic
Lewy Bodies

9 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

10 Current Research Improving quality of life Neuroprotection
Therapy and exercise Neuroprotection Drug treatment to slow, stop, reverse progression of PD

11 Neuroprotection Anti-apoptotic drugs Antiglutamatergic agents
Monoamine oxidase-B inhibitors Calcium channel blockers Growth factors

12 Current Treatments Levodopa (L-DOPA) MAO-B inhibitors Amantadine
Anticholinergics Surgery Treatment Standard: L-Dopa

13 Isradipine background
IC uM 60-65% excreted in urine 25-30% excreted through feces 5-15% actually absorbed into the blood Decrease responsiveness Administer intravenously

14 Research Design & Methods
Overview Isradipine as a therapeutic approach Experimental approach Proposed Pre-Clinical trials Prospective Clinical trials

15 Mechanism of α-synuclein cytotoxicity
Figure: Fibrilization pathway Native unfolded monomer Β-sheet rich oligomers or protofibrils Fibrils Lewy body Events in α-synuclein toxicity. The central panel shows the major pathway for protein aggregation. Monomeric α-synuclein is natively unfolded in solution but can also bind to membranes in an α-helical form. It seems likely that these two species exist in equilibrium within the cell, although this is unproven. From in vitro work, it is clear that unfolded monomer can aggregate first into small oligomeric species that can be stabilized by β-sheet-like interactions and then into higher molecular weight insoluble fibrils. α-synuclein can also form annular, pore-like structures that interact with membranes. The addition of ubiquitin (shown as a black spot) to Lewy bodies is probably a secondary process to deposition. Located in cytoplasm, membrane and inside nucleus. It concentrated in presynaptic nerve terminals. α-synuclein protein Reference:

16 Proposed Therapeutic Approach

17 Cav1.3 SNc main ion channels: L-type Calcium channel
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 Dopaminergic Neurons in SNc mainly use Cav1.3 (voltage gated) & NMDA receptor called NMDAR which is Ionotropic glutamate receptor (ligand gated) as ion channel Electrical control system of the heart that co-ordinates heart rate Found in impulse-generating pacemaker tissue of the heart In the central nervous system, it is a modulator of spontaneous pacemaking activity Generate burst of activity through voltage & time dependent ion influx (Ca2+) Activators & inhibitors binding sited on α1D sub unit (pore formation, gating machinery & binding site) 1,4- dihydropyridine (HDPs) binding site Cav 1.2 is late phase long term potentiation in hippocampus neurons Theorized mechanism of action by Isradipine is through changing the conformation of the channel & inhibiting gating mechanism

18 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

19 Comparison with other methods
Rifampicine (Lewy body approach) Antibiotic to treat TB & Leprosy Disintegrate α-syn fibrils Inhibit fibrils formation by stabilization monomer and oligomers No animal models Correlation with PD symptoms not yet tested Might enhance toxicity if protofibril cytotoxicity hypothesis is true Reference: Adphrodite Kapurniotu. Targeting α-synuclein in Parkinson’s Disease. chemibiol 2004;11:

20 Comparison with other methods
Β-synuclein protein (Gene therapy approach) Subfamily of synuclein family Concentrated in presynaptic nerve terminals (133AA) Not found in Lewy body inclusions Studies indicate inhibition of α-syn aggregation Overexpression 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

21 Comparison with other methods
α-synnuclein protofibrils Inhibition by β–synuclein (Green color) Reference: Dr.Igor Tsigelny. Modelling molecular basic of Parkinson’s disease. SDSU review. Winter 2007:52-57

22 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 Research done by implanting interscapular capsule with control release of Isradipine pallets Interscaptular  Pertaining to the upper back, or the part between the shoulders

23 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

24 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

25 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”)

26 Matrix of Mice Groups + - 6-OHDA L-DOPA Isradipine Saline Group 1

27 Pitfalls & Solutions Undue drop in Blood Pressure
Concentration kept low Mouse difference in Metabolic Rate Select for: 4 weeks old, grams Effects not from isradipine Perform Histology to determine

28 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

29 Goal of Phase I Trials Isradipine Effective Improves Quality of Life
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

30 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

31 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

32 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 4 20% $5,333 John Chi $75,000 $7,812 Khine Win $77,000 $6,416 Lab Technician $25/hr 10% $3,000 Animal Technician $41,069

33 Equipment Costs The Bio-Rad Imager is required for the imaging studies
Description Cost Bio-Rad Gel Doc1000 Imager Cabinet w/ Transilluminator $2,000 Labnet International MultiGene Mini Personal Thermal Cycler $2,700 Nuaire Nu 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.

34 Facility Rental Facility Rental: $9,515 Fullerton Business Center #140
1501 E Orangethorpe Ave, Fullerton, CA 92831 Space Size: 1,522 sqft Property Type: Office / R&D Price Per Sqft: $1.25 Rent: $1,903 FS

35 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

36 Research Strategy Run preclinical trials
Determine effect or Ca++ channel blockers on adrenergic neurons in Substantia Nigra

37 Citations

38 Questions


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