Alzheimer’s/Mild Cognitive Impairment Program

Alzheimer’s/Mild Cognitive Impairment Program – Introduction –

Non-‐Confidential Summary

AD SWITCHING PROGRAM INTRODUCTION

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The Buck Institute For Research On Aging

TABLE OF CONTENTS

1 EXECUTIVE SUMMARY .......................................................................................... 2

2 ALZHEIMER’S DISEASE: PROBLEM STATEMENT ..................................................... 4

2.1 THE MAGNITUDE OF THE PROBLEM 4

2.2 PROBLEMS WITH CURRENT ALZHEIMER’S TREATMENTS 4

3 THE TARGET .......................................................................................................... 4

3.1 APP AS A DEPENDENCE RECEPTOR 4

4 NETRIN-‐1 & MIMETICS .......................................................................................... 6

4.1 HISTORY 6

4.2 THE SCIENCE 6

4.3 NETRIN-‐1 AND NETRIN-‐1 MIMETIC DEVELOPMENT 7

5 SMALL MOLECULES -‐ F03 & ANALOGS, ASBIS ........................................................ 7

5.1 SMALL MOLECULE SCREENING 7

5.2 F03 & ANALOGS 7

5.3 F03 SUMMARY 8

5.4 APP-‐SPECIFIC BASE INHIBITORS (ASBIS) 9

6 INTELLECTUAL PROPERTY ................................................................................... 10

1 EXECUTIVE SUMMARY The Buck Institute for Research on Aging (“Buck Institute” and “Buck”) is the United States’ only independent, nonprofit research institute dedicated to the study of aging and age-related disease. The Buck Institute’s mission is to understand aging in all its aspects and through that knowledge increase both longevity and healthspan - the healthy years of life. From opening its doors in 1999, the Buck Institute now has twenty Principal Investigators with diverse expertise directing over 130 scientists toward an understanding of the biology of aging and age-related disease.

Since the Buck’s inception, the laboratory of Dale Bredesen, MD, has been conducting pioneering research to identify the critical mechanisms underlying the development of Alzheimer’s disease (AD). Dr. Bredesen, a world renowned expert in the field of programmed cell death and neurodegeneration, has created the Alzheimer’s Drug Discovery Network (ADDN) at the Buck. The ADDN team is composed of internationally recognized scientists and product development experts with complementary skills who work together to translate the discoveries from the Bredesen

AD SWITCHING PROGRAM INTRODUCTION 3

The Buck Institute for Research On Aging

lab into novel therapeutic candidates and advance these candidates toward optimal clinical trials.

The Bredesen lab views AD as a disease of biological signal imbalances, with the amyloid precursor protein (APP) molecule as a central switch. This work has been published extensively in scientific journals including Nature, Science, and Proceedings of the National Academy of Sciences.

In this well-characterized model:

• AD results from an imbalance between trophic (i.e., pro-growth, survival, and connectivity) and anti-trophic (i.e., anti-growth, survival, and connectivity) influences that govern key neuronal functions in the brain, including cellular connectivity, synaptic efficacy and maintenance, neurite retraction, and ultimately, programmed cell death. Thus AD is not simply a β-amyloid disease or a tau disease, but instead a network imbalance.

• APP is a dependence receptor that functions as a “molecular switch”, integrating disparate biochemical signals (trophic, hormonal, extracellular matrix-derived, transmitter-mediated, etc.) to mediate plasticity via both connective and de-connective effects (see Section 3.1).

Using a novel screening approach for drug discovery, the ADDN team has identified and developed compound “hits” that interact with APP itself, as well as other members of the underlying AD plasticity network. These APP interactors modulate APP processing, switching it toward trophic signaling (as well as effecting downstream signals including tau phosphorylation). A number of drug candidates have emerged from this screening approach. Three classes of candidates are being researched in the Bredesen lab, all of which display disease-modifying properties in AD animal models.

1. An endogenous biologic, netrin-1, whose function in adult physiology was previously unknown. Netrin-1 has been shown by the Bredesen lab to stabilize APP, reducing aberrant processing in multiple in vitro and in vivo AD models.

2. Several small molecule compounds that have shown good potency in cell-based assays and efficacy in validated animal models of AD. The lead candidate identified from the screen, a small molecule with oral availability designated “F03”.

3. SAR and medicinal chemistry performed on F03 and other key hits in the above mentioned screen have led to novel compositions of matter exhibiting greater potency and BBB permeability than the original compounds.

The Buck Institute is pursuing clinical development of F03, a repurposed generic molecule, which is moving towards a pilot Phase 2a clinical study. Additional drug development as well as preclinical and clinical studies are being designed to support its full development in the United States and worldwide. Concurrently, Buck AD researchers have developed a pipeline of promising compounds, with unique mechanisms of action and novel compositions of matter, that have produced very encouraging results in both discovery and preclinical assessments. It is the Buck’s intent to move quickly towards identifying additional suitable drug candidates and take them to clinical development in collaboration with corporate partners. To that end, the Buck is seeking partners who can collaboratively develop these novel drug candidates into potential AD therapeutics.


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2 ALZHEIMER’S DISEASE: PROBLEM STATEMENT

2.1 THE MAGNITUDE OF THE PROBLEM Alzheimer’s disease (AD) represents one of the largest unmet medical needs of the 21st century. There are currently 5.4 million Americans with AD, and this number will reach 16 million by 2050 if no preventative or curative treatments are developed (Alzheimer’s Association, 2012). AD’s current annual economic cost in the US of $183 billion (over $600 billion worldwide) does not begin to explain the heartache and disruption to the millions of families affected. Clearly there is a critical need for effective preventive and ultimately curative therapies for this debilitating and increasingly common disease.

2.2 PROBLEMS WITH CURRENT ALZHEIMER’S TREATMENTS It is well established that the currently approved drugs for the treatment of AD (e.g., Aricept and Namenda) show minimal efficacy at best, with modest symptomatic but no disease-modifying effects. Current therapeutic candidates focusing on amyloid deposition do not ameliorate progressive cognitive dysfunction and in fact, in one case, have shown to hasten it. Furthermore, all recent clinical trials with novel therapeutics whose discovery was based on the prevailing theories of Alzheimer’s have ended in failure (Flurizan, Rember, Dimebon, Semagacestat, Alzhemed, Bapineuzemab, Solanezumab etc.). AD clinical trial failures are likely due to multiple factors:

• There is a lack of understanding of the fundamental mechanisms that drive Alzheimer’s disease, and therefore the developed treatments have failed to target critical features of the disease process. This stems from the misconceived postulate that AD is primarily a disease of β-amyloid toxicity.

• Many of the trials have been carried out with patients who have advanced disease, when anatomical and pathophysiological changes are too advanced for any individual or combination treatment to effectively attenuate the disease. It is likely that earlier treatment will be more effective, just as for cancer and many other diseases. With improved biomarkers and earlier diagnosis, it is now the right time for a clinical trial with an optimized therapeutic system to address the signalling imbalance that we postulate to be the root of Alzheimer’s disease.

3 THE TARGET

3.1 APP AS A DEPENDENCE RECEPTOR Dependence receptors function as molecular switches, mediating contrasting cellular signals depending on trophic ligand availability (Rabizadeh et al., 1993; Mehlen et al., 1998; Bredesen et al., 2004; Bredesen, 2009); APP is one of more than 20 such receptors identified to date. General consensus has settled upon Alzheimer’s disease (AD) being caused by APP-derived amyloid peptides inducing neuronal cell death, however there are many other factors involved in APP processing.



Figure 1. The APP “switch”. APP may be cleaved by proteases to yield either four pro-apoptotic, pro-AD peptides, or two anti-apoptotic, anti-AD peptides. Just as for the HDL:LDL ratio, the ratio of these peptides is critical. The pro-AD peptides induce retraction of synapses, while the anti-AD peptides support synaptic stabilization.

APP is a transmembrane protein localized primarily at synapses. Differential cleavage of APP results in alternative plasticity-related fates: cleavage at the α-secretase site mediates neurite outgrowth and synaptic maintenance, inhibiting programmed cell death; conversely, cleavage at β-secretase, γ-secretase, and caspase sites initiates synaptic reorganization, neurite retraction, and caspase activation (Figure 1).

The neurodegenerative (anti-trophic) cleavage of APP produces peptides sAPPβ, Aβ, Jcasp and C31, which mediate neurite retraction, synaptic reorganization and programmed cell death. Considerable evidence from multiple laboratories exists for the detrimental synaptic and neurodegenerative effects of these anti-trophic APP fragments.

In contrast, APP α-site cleavage produces the trophic peptides sAPPα and α-CTF (the latter inhibits γ-site cleavage and mediates neurite extension). Studies have shown that this “anti-AD” fragmentation pattern leads to a discernible effect on synaptic formation and neurite extension, as well as neuronal health and synaptic maintenance.

APP acts as a dependence receptor as its processing fate is determined by its environment. Ligand binding to APP has been shown in the Bredesen lab to be a key directional mediator of APP processing. For example, netrin-1 and the Aβ peptide promote differential α and non-α (i.e., β, γ, and caspase) cleavage, respectively. These alternative cleavage pathways can initiate positive feedback loops, and so we coined the term “AD molecular switch” to describe the state in AD wherein the trophic pathway is switched off and neurodegenerative response pathway is switched on. This signaling behavior explains, in part, the true character of the Aβ peptide as a physiological anti-trophin and also the confounding existence of the “high pathology control” subpopulation of elderly deceased who present considerable plaque formation yet no signs of dementia. The plaques, in these cases, act as a repository for Aβ and prevent it from interacting with APP.

Understanding APP’s role as a dependence receptor also reveals a valuable new target for the development of AD therapeutics. The Buck Institute’s ADDN is focused on APP “switching” compounds that flip the molecular switch in AD back to the trophic pathway and is working to translate these novel therapeutic candidates from the lab to the clinic, advancing basic research findings toward clinical trials.


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4 NETRIN-‐1 & MIMETICS

4.1 HISTORY The Bredesen lab, in collaboration with the University of Lyon (France), has identified the netrin-1 protein as being capable of switching APP signaling toward synaptic and neuronal maintenance with significant efficacy. In addition to studying the anti-AD effects of netrin-1 itself, the Buck team has developed mimetic peptides with characteristics that improve delivery and biodistribution.

4.2 THE SCIENCE In preclinical pharmacology studies, the Bredesen lab have shown that netrin-1 functions as a ligand for APP, in that it modulates APP signaling and regulates anti-trophic APP fragment production in AD transgenic mouse models. Furthermore, netrin-1 appears to ameliorate the cognitive AD phenotype in a mouse model of AD when delivered intracerebroventricularly (ICV) (Figure 2).

Netrin-1 displays a unique combination of characteristics that renders it highly attractive as both a new therapeutic and a therapeutic template for Alzheimer’s disease:

• Netrin-1 is a diffusible ligand that reduces Aβ peptide markedly (over 80%) in organotypic brain cultures, primary neuronal cultures, and in vivo.

• Netrin-1 reduces the generation of C31, a neurotoxic peptide derived from the C-terminus of APP.

• Netrin-1 reduces the activity of caspases, cell death proteases activated in AD. • Netrin-1 alters the processing of APP, favoring the trophic peptides (sAPPα and

CTFα), and inhibiting the neurite-retractive and cell death peptides (sAPPβ, Aβ, Jcasp, and C31).

• Netrin-1 alters the intracellular signaling mediated by APP, favoring synaptic maintenance.

• Netrin-1 has shown relevance in both AD and MCI (mild cognitive impairment) • The delivery of netrin-1 to multiple transgenic mouse models improves memory

and AD biomarkers, and therefore represents a novel therapeutic agent for AD. • Netrin-1 interaction with APP can be used as a structural template for the

development of small molecule netrin mimetics.

Figure 2. Netrin-1 restores memory in a transgenic AD mouse model. Netrin 1-treated (blue) J20 and non-transgenic mice show significantly greater preference for a novel object over a familiar one as compared to vehicle-treated J20 mice after only two weeks of treatment.



4.3 NETRIN-‐1 AND NETRIN-‐1 MIMETIC DEVELOPMENT The ADDN team has completed various animal POC studies with netrin-1. Pathophysiology and cognitive function improve markedly with netrin-1 administration. Multiple AD animal models have been utilized in this research including several transgenic models that were specifically created to support the study of netrin-1. In addition, potent peptide mimetics of netrin-1 have been developed that have improved brain tissue penetration and distribution, and therefore could be delivered therapeutically to the brain using conventional drug delivery approaches. Netrin-1 is expected to move towards advanced preclinical studies upon completion of fundraising.

5 SMALL MOLECULES -‐ F03 & ANALOGS, ASBIs

5.1 SMALL MOLECULE SCREENING The ADDN small molecule portfolio emerged as part of our discovery efforts to screen and identify drug candidates that enable APP switching. These molecules have multiple anti-AD functions, including: reducing pro-apoptotic and increase trophic peptides derived from APP; reducing hyperphosphorylated tau; and improving cognitive function.

To facilitate small molecule development the Buck has developed a hierarchical screening method wherein the levels of specific APP cleavage products (biomarkers) are assessed following treatment with drug candidates, and improved potency in vitro and efficacy in vivo through medicinal chemistry. The Buck has developed novel, sensitive biomarker assays identifying the production of APP cleavage products at α and non-α (β, γ, and caspase) cleavage sites, which have been fully validated for, among other things, peptide biomarkers sAPPα, sAPPβ, Aβ40, Aβ42.

Potential drug candidates originating from extensive compound libraries are identified by parallel and sequential testing using a number of screening methods utilizing the following:

1) Immortalized cell lines expressing APP; 2) Primary neurons dissected from AD mouse model brains; 3) Assessment of in vivo brain penetration and pharmacokinetics; 4) Ex vivo tissue extracts derived from AD mouse model brains; 5) In vivo analysis in validated AD mouse models; and 6) Drugability screens.

5.2 F03 & ANALOGS Using the unique discovery approach detailed above, the ADDN has developed a number of drug candidates, the most advanced of which is a repurposed compound, designated “F03”. F03, and a suite of novel compounds built from the F03 pharmacophore, constitute a new paradigm in the development of therapeutics for AD. Lead compound F03 is currently under development for treatment of AD and mild


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cognitive impairment (MCI). F03 is a compound approved in more than 40 countries and for which there are over 15 years of human safety data. It is well tolerated, can be administered orally, shows good BBB permeability and is efficacious at a low dose. It is currently registered for an acute indication in a hospital setting.

Considerable research has been performed to assess F03 as a potential clinical candidate. This compound has shown to have remarkable efficacy in AD models in biochemical and cognitive assays (Figure 3A and 3B). Studies suggest F03 and some of its analogs have disease-modifying properties in MCI and early to moderate AD.

The Buck Institute has already received initial funding to test F03 in a human clinical trial. The Buck will commence a Phase 2a clinical trial to test F03 in patients with mild cognitive impairment in 2013. F03 clinical testing will provide clinical POC for F03 as an AD therapeutic and for APP as a dependence receptor and novel target. Optimized novel F03 analogs are currently under preclinical development following F03.

5.3 F03 SUMMARY F03 represents a traditional small molecule approach to treating MCI and AD with APP as the target. F03 and the suite of analogs that have been identified and synthesized are

Figure 3B. Memory effects of F03 in an AD mouse model. F03 treatment (0.5 mpk) improved both short-term working memory (left panel, novel object recognition test, 4 week-treatment) and spatial memory (right panel, Morris water maze, 8-week treatment) in J20 AD transgenic mice.

Figure 3A. The sAPPα /Aβ1-42 ratio improves with F03. F03 reproducibly (n = 56) increases sAPPα and decreases Aβ1-42 in J20 AD model mice, increasing the slope of the line representing “switching” of APP processing to a trophic balance by almost 50%.



all orally bioavailable, and appear to act via the key putative mechanism: switching APP processing towards trophic factors and away from AD-promoting peptides.

In summary:

• F03 is a small molecule drug that is both gut and BBB permeable; • F03 reduces the activity of caspases, decreasing C31, and other cell death

proteases that are activated in AD; • F03 markedly reduces Aβ peptide levels; • F03 lowers the amount of sAPPβ; • F03 alters the trophic processing of APP, increasing peptides sAPPα and CTFα; • F03 alters the intracellular signaling mediated by APP, favoring synaptic

maintenance; • F03’s base motif has been used to develop second generation compositions with

enhanced BBB penetration; and • F03 has shown relevance in both AD and MCI.

5.4 APP-‐SELECTIVE BASE INHIBITORS (ASBIs) While there is a large drug discovery effort focused on development of direct BACE inhibitors, none so far have advanced significantly in clinical testing. The Bredesen laboratory at the Buck Institute has identified a chemical family of APP-selective BACE1 inhibitors (ASBI drugs and prodrugs). These inhibit APP pro-AD cleavage without being associated with off-target BACE1 inhibition effects.

Studies in the Bredesen Laboratory have demonstrated that their ASBI drug was able to increase sAPPα levels without changing APP levels in the SHSY-5Y neuroblastoma cell line. Subcutaneous administration of an ASBI drug to an AD mouse model showed that the drug could be detected in the brain (Fig 2A) and effectively decreased Aβ1-42 levels (Fig 2B).

These results in AD transgenic mice indicate that the ASBI drug was able to switch APP processing from the pro-AD pathway to the anti-AD, pro-trophic pathway. The Bredesen laboratory has additionally developed a prodrug with the objective of further increasing ASBI drug brain levels. This prodrug is currently being evaluated.

Furthermore, other ASBIs that are novel compounds with high brain permeability and in vivo alterations of biomarkers are currently under development.

Figure 4. The ASBI drug crosses the BBB and has in vivo effects. The ASBI drug has good blood brain barrier penetrance after subcutaneous injection at 10 mg/kg (left panel). ASBI treatment (40 mkd) decreases Aβ1-42 (middle panel) and slightly increases sAPPα (right panel) in brain tissue from AD model mice .


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6 INTELLECTUAL PROPERTY The Buck has sought to capture the broadest IP base around switching drugs and the attenuation of aberrant pro-AD APP processing. The following patent applications have been filed. The first patent application has been granted in multiple countries, including the US.

Additional filings are currently being contemplated.

Patent File Date

Compositions and Methods for Suppression of Amyloid Plaque Formation Associated with Neurodegenerative Disorders

Apr 07

Small molecule APP switches Aug 10

F03 & Related Compounds to Promote Normal Processing of APP

Aug 11

Netrin Loop Peptides Nov 11

Formulations & methods for treatment or prophylaxis of pre-MCI and/or pre-AD conditions

Feb 12

APP-specific BACE inhibitors (ASBI’s) and uses thereof Mar 12

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Alzheimer’s/Mild Cognitive Impairment Program