Neuro Group: Discovery of new secretase inhibitor drug leads for

neurodegeneration and Alzheimer's disease. Mission Statement & Timeline

Dorothy Du, Cathy Hutchinson and Melissa McCoy

Advisor: Gilbert Rishton, PhD

Mission Statement

• To address the unmet medical need in Alzheimer's disease by the discovery and development of safe and effective drug leads and, ultimately, to help bring new mechanism-based medicines to the Alzheimer's disease patient population.

Discovery• Alzheimer’s disease introduction

– Disease history and outlook, market analysis of current treatments on the market and in development (i.e. gamma-secretase inhibitors, Amyloid specific mAb, ect.), pro’s/con’s of current treatments and need for more drug development

• Neurodegeneration• What causes it in AD? What is the hypothesized disease pathway and

what factors are still not well understood?• APP, β-secretase, and A plaques

• Secretase inhibitor design from APP structure– Gil’s example of small molecule drug design

• Biochemical screening– Model Systems for testing efficacy (i.e. transfected cell lines, transgenic

mouse model of AD, ELISA for detecting Beta Amyloid levels)– Methods for screening blood-brain barrier permeability (computational,

in vivo, and in vitro methods)

Figure 1 – Actual and estimated number of new AD cases in the US through 2050

Figure 2- Actual and estimated costs of AD in the US. Purple bars =Medicare costs, Yellowbars=Medicaid cost

Mount, Claire & Downton, Christian. Nature Medicine. 12, 780-784 (2006)

Disease Outlook

Drug Company Mechanism IndicationAricept (donepezil)

Eisai/ Pfizer AChE inhibitor Mild to moderate AD

Exelon (rivastigmine)

Novartis AchE inhibitor Mild to moderate AD

Razadyne (galantamine)

Johnson and Johnson

AchE inhibitor Mild to moderate AD

Namenda/ Ebixa (memantine)

Lundbeck/ Forest NMDA receptor antagonist

Moderate to severe AD

Current Treatment Market

Figure 3- Existing and forecasted Alzheimer’s disease treatment marketMount, Claire & Downton, Christian. Nature Medicine. 12, 780-784 (2006)

APP and β-Secretase• APP= Amyloid Precursor Protein

– Genetic link in Alzheimer’s Disease

• Protein is cleaved by enzymes known as secretases

– 3 types: α-secretase, β-secretase, and γ-secretase– Cleave the protein at different positions

Familial Alzheimer’s Disease Sporadic Alzheimer’s Disease

Mutations in APP/Presenilin genes

Genetic risk factors:ApoE4, other genes? Aging & environmental

Life-long increase in Aβ42 production Failure of Aβ clearance, gradual increasing levels

Aβ accumulation and oligomerization

Aβ oligomers subtly effect synapses

Aβ oligomers form plaques

Immune cell activation/ inflammation

Oxidative stress/ altered ion flow

Neuron dysfunction/transmitter deficits

Dementia

Amyloid Cascade Hypothesis

Blennhow, Kaj et al. The Lancet. 368 (2006)

Normal Tau Protein

Abnormally hyperphosphorylated tau

Kinases Phosphatases

Sequestration of MAPs and tau by hyperphosphorylated tau

Tau polymerization

NFT formation

Microtubule disassembly

Disturbed axonal flow/transport

Dementia

Neuronal dysfunction

Neuronal Death

Tau Protein in Alzheimer’s Disease

Blennhow, Kaj et al. The Lancet. 368 (2006)

Potential Drug Targets of AD

Journal of Pharmacology And Experimental Therapeutics Fast Forward First published on November 25, 2002; DOI: 10.1124/jpet.102.035840

Link Between Amyloid Hypothesis and Tau Protein

Journal of Pharmacology And Experimental Therapeutics Fast Forward First published on November 25, 2002; DOI: 10.1124/jpet.102.035840

Competitive LandscapeDrug Target/Mechanism Company Stage of Development

β-secretase inhibitor Amgen, Bristol-Meyer Squibbs, Elan Pharmaceuticals, Scios Inc, GlaxoSmithKline

Pre-clinical

γ-secretase inhibitors or modulators

Eli Lilly, Myriad Pharmaceuticals

Phase II/III

Aβ immunotherapy Elan Pharmaceuticals, Baxter Healthcare, Wyeth

Phase I, II and III

Aβ fibrillization inhibitors Neurochem, Prana Biotechnology

Phase II/III

CDK5/GSK-3β inhibitors UCSB Pre-clinical

Anti-inflammatory drugs Proctor & Gamble, Roche Phase III

Cholesterol lowering drugs Pfizer Phase II

http://www.alzforum.org

Why Target β-secretase?

• Plays a crucial role in the first step of the amyloid cascade

• Mice missing the β-secrectase gene are normal and cannot produce Aβ

• The enzyme is an aspartic protease similar to the HIV protease for which successful inhibitors have already been produced

Biochemical Screening

• Biochemical Assay– Kinetic measurements-

determines how potent the inhibitor is

– β-Secretase FRET Assay ( Fluorescence Resonance

Energy Transfer) Inhibition of β-secretase by a Statine-derived Inhibitor.

http://www.invitrogen.com/content/sfs/manuals/L0724.pdf

Biochemical Screening• Functional Activity Assays

– Utilize in vivo modelsystem:

• Transfected cell line• Transgenic mouse• Both produce excess Aβ

– Detect levels of Aβ in cell lysate or brain tissue

• Utilize immuno-assays• Western Blot, ELISA,

Immunoprecipitation

Rational Methods

• Medicinal Chemistry• X-ray Crystallography, Co-crystallization• Assay Development

– Enzyme Assay Development to measure pharmacokinetics of drug leads

– Need to measure initial velocity of reaction, Km and Vmax with selected substrates

– Determine IC50 and Ki for inhibitors (used to determine how good the inhibitor is at inhibiting the enzyme and what type of inhibition is occuring)

– Assay would be an In vitro system that measures the ability of the drug lead to inhibit secretase

Assay Development

• Biochemical Screening• Blood Brain Barrier Permeability (BBB)

– In vivo (transgenic mice) and in vitro (artificial membranes) approaches

– Computational approaches

• Other important Factors to Consider:– Pharmacokinetics-metabolic stability, metabolic liability,

permeability, protein binding– ADME- Absorption, Distribution, Metabolism and Excretion– Toxicology- will need to be outsourced

Literature

• Patent search– Lead molecules, composition-of-matter patents

• Novelty, new intellectual property• Building cost at CSUCI

Patent• Provisional patent

– Specific claims, composition-of-matter, use, function

– Structure, structure-activity-relationship– New drug lead, secretase inhibitor

• IP: Develop intellectual property position– Provisional patents– PCT patents

Development

• Facilitate development by attention to quality in discovery stage– Leadlike, druglike, low MW, oral bioavailability,

blood-brain barrier permeability• Facilitate development by partnering

– Use contract development labs for in vivo work.– Partner with a drug company for clinical

development

Characteristics of a Good Lead

Pajouhesh, Hassan et al. NeuroRx. 2, 541-553. (2005)