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Candidate Alzheimer’s disease (AD) drugs that increase the sAPP a /A b 42 or sAPP a /sAPP b ratio improve working memory Patricia Spilman, Jesus Campagna, Olivia Gorostiza, Karen Poksay, Alex Matalis, Olivier Descamps, Varghese John, Dale Bredesen

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Candidate Alzheimer’s disease (AD) drugs that increase the sAPPa/Ab42 or sAPPa/sAPPb ratio improve working memory

Patricia Spilman, Jesus Campagna, Olivia Gorostiza, Karen Poksay, Alex Matalis, Olivier Descamps, Varghese John, Dale Bredesen

Alzheimer’s Drug Discovery Network (ADDN), Buck Institute for Research on Aging, Novato, CA, 94945 USA

ABSTRACT. Using a hierarchical chemical-genetics approach, we have identified compounds that increase the sAPPa/Ab42 and/or the sAPPa/sAPPb ratio. Compounds from a clinical compound library underwent high-throughput screening (HTS) in CHO-7W cells stably transfected with huAPPwt, those that increased sAPPa were re-tested in primary neurons from PDAPPSwe/Ind (J20) mice. Candidate therapeutics were then subjected to a battery of in vivo tests using J20 mice. In addition to biochemical readouts for efficacy, improvements in working memory as reflected by performance in the Novel Object Recognition (NOR) task were ascertained. The lead AD therapeutic candidate “F03” was identified, and we confirmed that working memory correlates to the sAPPa/Ab1-42,sAPPb ratio(s), whether the increase is driven by sAPPaincreases, Ab42 or sAPPb decreases, or both.

The Lead Candidate: F03

F03 vs. Donepezil (Aricept)

A B

C D

A B

C D

E F

G H

I

Working Object Memory Testing using Novel Object Recognition. Working memory uses pre-existing neuronal pathways. A mouse is acclimated to the empty arena, a 30 X 39 cm opaque black banker’s box with bedding for 15 min. one day before testing. On the test day, the mouse is placed in the arena with two identical objects for 10 min.. After a 1-hour interval, the mouse is returned to the arena, wherein one of the objects has been changed (above). Healthy, normal mice are curious, and if the mouse remembers the “familiar” object, will spend more time investigating the novel object. Noldus Ethovision software tracks a non-transgenic (NTg) mouse interacting more with the novel object below (A). The mice are scored by counting the number of interactions with each object. An interaction is the nose coming within 2 cm of the object. Novelty preference NP) is calculated by the percentage increase (or decrease) in interactions with the novel object as compared to the familiar. Most NTg mice show NP; J20 mice as a group tend to interact with novel and familiar objects more equally (B). Successfully treated J20 mice show increased NP (C). Some untreated J20 mice do show NP and others neophobia. Other aspects of behavioral phenotype such as activity level can be determined in NOR by distance traveled or total object interaction.

Both F03 (0.5 mkd) and donepezil (1 mkd) increased sAPPa, but only F03 decreased Ab42 after 28-day treatment (data not shown). Only F03 increased novelty preference (A). The NP improvement correlated weakly with sAPPa (B) and Ab42 (C), but had good correlation to the sAPPa/Ab42 ratio.(sAPPb data was not available)

Older J20 Mice

A B

C

BACKGROUND – Amyloid precursor protein (APP) Processing. In AD, there is a shift in the balance of APP processing towards an anti-trophic state, which may in part be due to aberrant upregulation of the Ab pathway, leading to a lowering of the sAPPa/Ab ratio. We identify “switching” drugs that restore the trophic balance by increasing the ratio. The trophic pathway comprises ADAM10 (a-secretase) cleavage of APP and generates sAPPa and aCTF (C80). In the anti-trophic pathway, BACE (b-secretase) cleaves APP, followed by g-secretase, and caspase cleavages generating sAPPb, Ab, and C31. It is our hypothesis that it is the ratio, rather than increases in Ab alone, that influences cognitive function and disease progression. A decrease in the sAPPa/sAPPb ratio can similarly reflect an anti-trophic state.

A B C

In most pilot studies, 5 m.o. J20s are used. These mice are pre-plaque. In the studies shown in (A) and (B), older (6+ m.o.) mice in which plaques were starting to form were used. In these mice, NP is most closely correlated to sAPPa . High sAPPa outliers (circled) typically had the best NOR performance.

In 1+ year-old mice (C), NP is almost exactly correlated to sAPPa.

Validating NOR: Protein Netrin-1

A B

C D

E

HTS

CHO-7W cells

(huAPPwt)

sAPPa

Primary

Hippocampal neuronal culture

sAPPa

Ab 42

Pharmacokinetics

10 mkd sub-Q

Plasma:Brain

levels

CONCLUSIONS.In pre-plaque J20 mice, there is some correlation between novelty preference and the sAPPa/Ab42 ratio; but as sAPPbmay better reflect de novo Ab42 production (an additionalg-secretase cleavage is necessary to form Ab), there appears to be better correlation between the sAPPa/sAPPb ratio and working memory. Most J20 mice will have Ab plaques by 6-7 months of age, but younger individuals may still have significant insoluble Ab before they enter a study, and this may confound the correlation between cognition and the sAPPa/Ab42 ratio.

In older mice with Ab plaques, sAPPa correlates well with working memory. This phenomenon may explain the existence of non-symptomatic humans with significant plaque load – these individuals may have high levels of sAPPa.

F03 and compounds like it that improve the ratio(s) by lowering sAPPb and therefore Ab42 production, and/or by increasing sAPPa, are promising AD therapeutics.

Large n# studies

Morris Water Maze

sAPPa, Ab40, Ab42, sAPPb, C83, C99

I5 huAPPwt mice

sAPPa, Ab42

(Hip)

Small n# studies

J20 huAPPSwe/Ind

sAPPa,, Ab 42

Ab 40, sAPPb

F03 increases sAPPa, lowers Ab42, and increases NP; NP improvement correlates well with the sAPPa/sAPPb*ratio.

As a result of 28-day treatment with F03 at the human-equivalent dose of 0.5 mkd by subcutaneous Alzet pump, (A) sAPPa was slightly increased in the entorhinal cortex (EC), and novelty preference (NP) was increased (B) in J20 mice. Ab42 was decreased in both hippocampus (Hip) and EC of F03-treated J20s (C), and therefore the sAPPa/Ab42 ratio increased (D). While sAPPb didn’t apparently decrease in Hip or EC in group analysis (E), the sAPPa/sAPPb ratio (F) increased in individual F03-treated mice as compare to controls. Correlation between the sAPPa/Ab42 ratio and NP was weak (G), but NP correlated well with the sAPPa/sAPPb ratio (H). When data from further F03 studies are combined in meta analysis, the correlation between the sAPPa/sAPPb ratio and NP remains strong. Behavior and biochemical ratios are individual and sAPPb production may better reflect de novo Ab42 production than Ab42 itself as mice can enter the study with pre-existing insoluble Ab42 at varying levels.

*The sAPPb AlphaLISA is new and data was not available for some studies.

Combine with compounds with complementary mechanisms

Compare with current AD therapeutics

Expanded analysis IHC, p-tau, calbindin, c-Fos, ARC, NPY, ACh levels

Treatment with protein netrin-1 was used to validate NOR.

After 2-week ICV administration of netrin-1 (R&D systems), sAPPa was similar between treated and control groups (A), but Ab42 was lower in netrin-treated mice (B). Therefore, the sAPPa/Ab1-42 ratio was increased in treated mice (C) as was novelty preference (D). There was good correlation between increases in the sAPPa/Ab42 ratio and novelty preference. The NOR protocol was based on that described in Bevins & Besheer (Nat. Prot. Vol. 1 3:1306 (2006).

Hierarchical Assay Flow Scheme. Libraries are screened in CHO-7W cells (huAPPwt) with sAPPa(AlphaLISA, Perkin-Elmer) as a readout. Compounds that increase sAPPaundergo testing in primary neuronal culture (J20, huAPPSwe/Ind) with sAPPaand Ab42as the readouts. Brain penetrance and the brain/plasma ratio is then determined in non-transgenic (NTg) mice (IAS, Berkeley, CA). Candidates are tested in pilot studies in both J20 and I5 (huAPPwt) mice; sAPPa, Ab42, Ab40(Invitrogen ELISA) and sAPPb (AlphaLISA, Perkin-Elmer) are determined. In some pilot studies, mice undergo Novel Object Recognition (NOR) memory testing. Successful candidates are tested in larger n# studies, and test mice undergo both NOR and spatial memory (Morris Water Maze, Barnes Maze) testing. Promising candidates are then compared with current AD therapeutics such as memantine (Namenda) and donepezil (Aricept). Finally, expanded pathological analysis is performed including immunohistochemistry and additional biochemistry.

Acknowledgements: This work was supported by the Rosenberg Challenge and Eureka 1200704 Grants

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