Disaggregation of a-Synuclein by way of Isradipine John

Disaggregation of a-Synuclein by way of Isradipine John Chi, Mike De. Salvio, Kevin Ip, Khine Win, Michael Nguyen

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 •

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

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

Parkinson’s Disease Neurodegenerative disorder • 100 -200 people in 100, 000 • Average of onset 55 to 60 • Annual cost approximately $11 billion • http: //www. wolframalpha. com/input/? i=parkinsons+disease

Parkinson’s Disease • Impairs motor skills, cognitive processes, and other functions • Tremors • Rigidity • Bradykinesia • Postural instability http: //www. wolframalpha. com/input/? i=parkinsons+disease

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

Parkinson’s Disease Idiopathic • Various hypotheses • • Viral • Genetic • Lewy Bodies http: //www 2. kumc. edu/coa/images/AMED 900/Dementia. Syndrome. Graph. gif

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

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

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

Current Treatments • • • Levodopa (L-DOPA) MAO-B inhibitors Amantadine Anticholinergics Surgery Treatment Standard: L-Dopa http: //www. 3 dchem. com/imagesofmolecules/Levodopa. jpgc

Isradipine background • • • IC 50 34+-8 u. M 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/catalog 11966/11966. jpg

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

Mechanism of α-synuclein cytotoxicity Figure: Fibrilization pathway Native unfolded monomer • Β-sheet rich oligomers or protofibrils • Fibrils • Lewy body α-synuclein protein Reference: http: //www. molecularneurodegeneration. com/content/4/1/9

Proposed Therapeutic Approach

Cav 1. 3 SNc main ion channels: Cav 1. 3 (voltage gated) NMDAR (i. Glu. R, ligand gated) L-type Calcium channel Regulate spontaneous pace making activity Modulator of neuronal spiking behavior Binding site on α 1 D sub unit Isradipine (1, 4 - dihydropyridine) as inhibitor Theorized mechanism: Conformational changes by binding Ca 2+ influx control approach

Research Proposal (Intro) Quantification of effect on targeted drug delivery approach: Behavior study Calcium imaging (striatum & s. N) Immunohistochemistry staining by Caspase- 3 antibody to determine apoptotic cells Clinical phase I Time frame: 4 -6 months Potential pitfall: Low blood pressure

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: 1476 -

Comparison with other methods Β-synuclein protein (Gene therapy approach) Subfamily of synuclein family Concentrated in presynaptic nerve terminals (133 AA) 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

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

Advantages of our approach No effective inhibitor drug for a. S protofibrils aggregation yet Indirect method to manipulate Ca 2+ 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

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 2 x/day) Administer equivalent volume of saline to the other 20 mice Sacrifice mice at the end of 18 weeks

Pre-Clinical • Use B 6 D 2 F 1/J Mice (Jackson Labs) • Transgenic Studies • Behavior Studies • Bioassays for drugs, pathogens, nutrients • Study Groups: PD, PD + L-DOPA, PD+ Placebo, Wild-Type

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”)

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 - - - +

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

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

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

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

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

Salary Information Name Michael Nguyen Michael De. Salvio Kevin Ip John Chi Khine Win Lab Technician Animal Technician Base Salary Months Effort Requested Salary $80, 000 5 25% $8, 333 $82, 000 3 35% $7, 175 $80, 000 4 20% $5, 333 $75, 000 5 25% $7, 812 $77, 000 5 20% $6, 416 $25/hr 5 10% $3, 000 $41, 069

Equipment Costs Description Cost Bio-Rad Gel Doc 1000 Imager Cabinet w/ Transilluminator $2, 000 Labnet International Multi. Gene Mini Personal Thermal Cycler $2, 700 Nuaire Nu 425 -600 Option A-P 2 Lab Hood 72" $2, 500 Fileserver $1, 500 Laboratory laptops x 5 (Apple Mac. Book Pro 15”) 2 Experimental PC Workstations Refrigerator Summit VT 225 Lab Freezer (-30 deg C) Liquid Nitrogen System $10, 000 $1000 $500 $1, 600 $800 $22, 800 • The Bio-Rad Imager is required for the imaging studies • Thermal Cycler is required to determine • The Nuaire fume hood is necessary for manipulating organic compounds such as fura 2 for cell staining and histological studies.

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

Laboratory Costs Laboratory Supplies: $10, 000 Laboratory supplies include items such as safety equipment gloves oligonucleotides fura 2 isradipine • Test tubes Scales Slides Burners Syringes • Flasks • • Estimated at approximately $5, 000 for startup and $1, 000/mo.

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

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