John Chi, Mike DeSalvio, Kevin Ip, Khine Win, Michael Nguyen

Disaggregation of a-Synuclein by way of Isradipine

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 age 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://www2.kumc.edu/coa/images/AMED900/Dementia/DementiaSyndromeGraph.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

http://www.3dchem.com/imagesofmolecules/Levodopa.jpgc

Treatment Standard: L-Dopa

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

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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 proteinReference: http://www.molecularneurodegeneration.com/content/4/1/9

Proposed Therapeutic Approach

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

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

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

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

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

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

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

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

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 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

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.

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

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

Research Strategy

Run preclinical trials Determine effect or Ca++ channel blockers on

adrenergic neurons in Substantia Nigra

Citations

Questions