CEB-2001 a therapeutic candidate for Alzheimer's disease identified using AMYScreen an HTS screen for specific inhibitors of Aß42 accumulation

Project number 100101

This dossier has been prepared for Dr Eckman of the Mayo clinic by Lead Discovery

Aim: LeadDiscovery is a company of industrial scientists dedicated to identifying areas of research with pharmaceutical/biotech potential. Using our two key services, TherapeuticAdvances and DiscoveryDossiers, we use our experience to help academic scientists or biotech companies highlight this potential. Equally we provide an impartial and non-commission based service to industry identifying field leaders and by suggesting how their research areas can be adapted to product development.

Abstract Accumulation of extracellular b-amyloid, and in particular Aß42, in the CNS is a hallmark feature of Alzheimer's disease, improved treatment of which represents one of the highest priorities of the pharmaceutical industry. Considerable attention is being focussed on preventing production of b-amyloid synthesis and consequently several synthesis inhibitors are in various stages of development. In a previous dossier we analyzed the ground breaking work of Mayo Clinic (Jacksonville) researchers highlighting endothelin converting enzyme (ECE) as a candidate drug discovery target whose exploitation could reduce the accumulation of b-amyloid. Through the development of AMYScreen, an improved Alzheimer's assay able to screen at high throughput levels for molecules that specifically reduce the accumulation of b-amyloid 42, Dr Chris Eckman has now made a further breakthrough. Representing perhaps one of this years highlights in Alzheimer's research, this group has identified a proprietary product, CBE-2001 which is able to specifically reduce the accumulation of Aß42. Even more impressive is the observation that this product is able to reduce levels of Aß42 when administered into the mouse brain while leaving levels of Aß40 unaffected. This product confers significant advantages over preceding Alzheimer's leads which have targeted synthesis pathways and carry a reduced specificity and increased risk of adverse effects. AMYScreen is now being offered as a tool to companies wishing to out-source studies involving the screening of libraries. Of equal if not greater importance, CBE-2001 is available as a licensing/co-development option to companies interested in supporting the optimization of this molecule towards an effective Alzheimer's treatment.

Background: Approximately 15 percent of people who live to the age of 65 will develop some form of dementia; by age 85, that proportion increases to at least 35 percent. The most common of all the dementias is Alzheimer's disease. Existing in two forms, early onset familial disease (FAD) and late onset disease. Four million Americans currently suffer from the condition, and experts estimate that 22 million people around the world will be so afflicted by 2025. Until recently, researchers had almost no understanding of the disorder's causes, and it still lacks preventive or curative therapies. The symptoms of Alzheimer's disease parallel a gradual death and shrinkage of brain tissue. The grooves or furrows in the brain, called sulci (plural of sulcus), are noticeably widened and there is shrinkage of the gyri (plural of gyrus), the well-developed folds of the brain's outer layer. In addition, the ventricles, or chambers within the brain that contain cerebrospinal fluid, are noticeably enlarged. In the early stages of Alzheimer's disease, short-term memory begins to decline when the cells in the hippocampus, which is part of the limbic system, degenerate. The ability to perform routine tasks also declines. As Alzheimer's disease spreads through the cerebral cortex, judgement declines, emotional outbursts may occur and language is impaired. Progression of the disease leads to the death of more nerve cells and subsequent behavior changes, such as wandering and agitation. The ability to recognize faces and to communicate is completely lost in the final stages. Patients lose bowel and bladder control, and eventually need constant care. This stage of complete dependency may last for years before the patient dies. The average length of time from diagnosis to death is 4 to 8 years, although it can take 20 years or more for the disease to run its course.

Plaques comprising ß-amyloid are a key early feature of Alzheimers - Much of our early understanding of Alzheimer's disease has been due to histological studies in which the shrinking of the brain was related to a loss of neurons including cholinergic fibers in the hippocampus and the cerebral cortex. This also led to the development of first generation therapies. The other directly observable hallmarks of Alzheimer's disease are clusters of proteins in the brain. These accumulations occur inside nerve cells in the form of neurofibrillary tangles. Analyses performed in the 1980s at several laboratories made it clear that these tangles consist of a protein called tau. Tau is significant because it binds to a protein named tubulin, which in turn forms microtubules which are responsible for cell structure and also the movement of various molecules within the cell. The tau tangles disrupt the microtubule structures in the nerve cell, impairing the transport of nutrients as well as the transmission of neuronal messages. Tangles of tau, however, are not unique to Alzheimer's disease. For that reason, even though the high density of neurofibrillary tangles in Alzheimer's patients is distinctive and strongly correlates with the severity of dementia, many investigators have not considered disruptions of tau to be as important as the second kind of protein deposits identified in amyloid plaques found in patients. Plaques contain a wide variety of proteins and general debris however of these proteins, the 40 or 42 amino acid ß-amyloid peptides first reported in 1984 by Glenner & Wong are now generally accepted markers and possibly etiological factors of Alzheimer's disease. Glenner & Wong's study was quickly followed by the sequencing of the gene encoding the precursor of ß-amyloid, APP and the location of this gene to chromosome 21. Three isoforms APP have been identified. APP695 is the major isoform and is expressed exclusively in neurons, while APP751 and APP770 are expressed in both neural and non-neural cells. The primary structure of APP has a signal sequence, a large extramembranous N-terminal region, a single transmembrane domain, and a small 47 aa residue cytoplasmic C-terminal tail. The APP proteins mature in the endoplasmic reticulum and Golgi apparatus and exhibit post-translational modifications, including phosphorylation, glycosylation and sulfation.

Accumulation of Aß42 contributes to Alzheimer's - Evidence that ß-amyloid plays an important role in Alzheimer's came from the study of Down's disease and also FAD patients. Individuals with Down's disease invariably develop Alzheimer's disease at an early age if they live past the age of 50. These patients as well as those with FAD were found to have genetic abnormalities related to chromosome 21. Down's disease patients carry an extra copy of chromosome 21, while FAD patients display mutations flanking the ß-amyloid sequence of APP. As a result increased levels of ß-amyloid are deposited in both patient groups and this led to the hypothesis that altered accumulation of this protein may be an early and critical event in the pathogenesis of Alzheimer's (see Goate et al, 1991; Chartier-Harlin, 1991; Mullan et al, 1992). Mutations in two other genes, presenilin 1 on chromosome 14 and presenilin 2 on chromosome 1 have also now been identified in FAD and this has given insight into the pathogenesis of Alzheimer's disease. Although the incidence of FAD is low compared with late onset Alzheimer's disease, patients develop clinical and neuropathological characteristics that are essentially identical and ß-amyloid is therefore generally accepted to play a role in both diseases.

Three proteases, a-, ß- and g-secretases, are involved in APP processing. At the cell surface, APP undergoes proteolysis by a-secretase releasing a large, soluble ectodomain (a-APP; sAPP). The C-terminal fragment is retained within the cell membrane. This fragment can then be cleaved by g-secretase releasing the p3 peptide. In an alternate pathway, ß-secretase cleaves APP releasing a large secreted derivative sAPPß and a C-terminal fragment CTFß that can be further cleaved by g-secretase to form Aß which is released into the extracellular milieu. The predominant peptide secreted is Aß40 with about 10-15% being Aß42.

Aß42 is invariably elevated with all mutations that cause Alzheimer's disease and this form of ß-amyloid forms toxic fibrils much faster than Aß40, its shorter homologue. Neurotoxicity is thought to be due to altered calcium regulation, mitochondrial damage and/or immune stimulation. Exactly what causes the increase in neurotoxic Aß42 is unclear however mutations in APP (Citron et al, 1992; Suzuki et al, 1994) and in presenilin 1 or 2 (Sherrington et al, 1995; Levy-Lahad et al, 1995; Rogaev et al, 1995) are known to contribute to this phenomenon. The presenilins are involved in protein modification and may in fact be g-secretase. Mutations in APP and the presenilins may account for about 50% of familial Alzheimer's and about 5% of the total cases. Other causes of Aß42 build up are unclear and moreover the discrepancy between plaque density and disease severity is also challenging. Consequently some researchers dispute the suggestion that ß-amyloid plays a major role in Alzheimer's however a more conciliatory stance has been that ß-amyloid accumulation is important for initiating a cascade of events leading to disease progression. Despite this doubt considerable efforts have focussed on developing inhibitors of the amyloid cascade.

 

• Specific targeting of Aß42 accumulation offers a desirable therapeutic approach to Alheimer's disease- Blocking APP expression may be of therapeutic interest however this is countered by studies showing that APP is required to promote neurite outgrowth (Araki et al, 1991) and cell adhesion (Breen et al, 1991) and therefore neural plasticity (Sisodia & Gallagher, 1998). Inhibitors of ß-secretase represent an alternative target being a key enzyme in the production of ß-amyloid from APP. Therapeutic development has however been hampered because the enzyme was only identified in 1999 when Vassar et al and later Sinha et al, Yan et al and Hussain et al, identified the gene coding ß-secretase (also termed BACE). Furthermore, although ß-secretase is an attractive target its physiological role is not yet clear and thus possible side effects are yet to be established. Likewise, although targeting g-secretase may prevent the accumulation of Aß42, inhibitors of this enzyme have also been shown to seriously compromise the immune system (Hadland et al, 2001) as well as causing a build up of CTFs which may themselves be toxic. An alternative approach has been through the increased clearance of Aß42 and Aß40, for example by antibodies (Bard et al, 2000; Thatte, 2001) or small molecules. A number of extracellular proteases have been suggested to play a role in Aß degradation including insulin-degrading enzyme, neprilysin MMP-9, thimet oligopeptidase and a-macroglobulin (Kurochkin & Goto, 1994; Qiu et al, 1998; Vekrellis et al, 2000; Perez et al, 2000; Chesneau et al, 2000; Howell et al, 1995; Iwata et al, 2000; Takaki et al, 2000, Backstrom et al, 1996; Yamin et al, 1999; Qiu et al, 1996). Recent evidence also points to endothelin-converting enzyme-1 (ECE-1) as a candidate Aß degrading enzyme. ECE-1 and ECE-2 are responsible for the hydrolysis of several peptides including bradykinin, neurotensin and substance P. ECE-1 is widely expressed by vascular endothelial cells where it degrades big endothelin-1 to produce the potent vasoconstrictor, endothelin-1. ECE-1 is also expressed by non-endothelial cells of the lung, pancreas, testis, ovary, adrenal gland, and of relevance to Alzheimer's, cells of the cortex and the hippocampus (Xu et al, 1994; Davenport et al, 1998; Korth et al, 1999; Barnes et al, 1997).  In a previous dossier (Click here for access) we overviewed research emerging from the Mayo Clinic establishing ECE-1 as a therapeutic target for Alzheimer's disease. The current dossier describes further research emerging from the Mayo Clinic identifying CBE-2001 as a novel inhibitor of Aß42 accumulation.

AMYScreen bioassay for Aß42: The first step in the identification of CBE-2001 was the development of AMYScreen, an improved screen for inhibitors of Aß42 accumulation. AMYScreen was then used to screen for therapeutic candidates in a large library of proprietary natural extracts. The screen used by Dr Eckman's group is based on the use of the H4 human glioma cell-line obtained from the ATCC following stable transfection with ßAPP695 the predominant form of Aß to be found in neural cells. Screening was performed in a 96-well format, full characterization of which is reported by Haugabook et al, 2001. Screens were performed at high throughput. Fresh culture medium containing extracts diluted to a final concentration of 0.05% and 0.5% (v/v) were added and allowed to incubate on the cells for 18 hours. Each extract was analyzed in sextuplet for each dose (0.05% and 0.5% v/v). Positive and negative negative controls were included on each plate. Positive controls for toxicity were also included. Aß40 and Aß42 was detected using the human antibodies, BAN50/BA27 and BAN50/BC05 respectively human and measured by sandwich ELISA as previously described (Suzuki et al, 1994). AMYScreen represents an improvement over other HTS compatible screens that have been used in the past (see Dovey et al, 2001), since unlike it's predecessors it is able to assay both Aß40 and the more amyloidogenic Aß42. Furthermore this assay is based on cells originating from the human CNS. Please note that this assay is available to companies wishing to screen molecules for activity against Aß40 and Aß42 accumulation. This can either be done on a service contract basis or alternatively Dr Eckman would be able to supervise in house assay installation. Further details are given in the strategic analysis section of this dossier.

The identification of CBE-2001, a novel inhibitor of Ab 42 accumulation: AMYScreen was used to screen a proprietary collection of natural product extracts for their ability to alter Aß accumulation. Each extract was tested in sextuplet at 0.05% and 0.5% (v/v) with an exposure time of 18 hours, and compared against positive and negative controls. From the starting library, 10 extracts were identified that reduced total Aß accumulation. One extract, CBE-2001 was identified that selectively reduced Aß42 accumulation (seen inset), while no significant effect on Aß42 accumulation was observed up to the maximum concentration investigated.

Further study showed that CBE-2001 was non-toxic to cells and that, it had no demonstrable effect on levels of ßAPP, CTFa or CTFß. This is important since as mentioned above, CTFs may be toxic. The active component of CTF-2001 was a small heat stable molecule soluble in ethanol, DMSO, ethyl acetate, and acetonitrile. Perhaps of greatest excitement was the observation that within 3 hours of icv administration, CBE-2001 was able to reduce Aß42 by over 30% (see inset).

Patent position: A patent has been filed to protect the use and development of CBE-2001.

Market size: Analysts predict that the US Alzheimer's market alone will be worth $2.3 billion by 2003.

Market competition: Many drugs are commonly used to treat behavioral symptoms associated with Alzheimer's such as agitation, aggression, paranoia, delusions, or depression. In addition to symptomologic treatment a large number of products are available or are in advanced development to slow the course of disease progression. A search of drug development databases has been performed to identify products in development or on the market for Alzheimer's. A total of 219 products were identified. Well over 50 molecules are in advanced development or on the market (see table 1). Most of these are aimed at slowing disease progression by increasing endogenous transmitter release, prolonging the half-life of released neurotransmitter or complementing the level of endogenous transmitter. And furthermore the majority focus on the cholinergic pathway. Notable exceptions are PTI-00703 and cerivastatin, both of which modulate the amyloid pathway. Of particular interest cerivastatin and Lovastatin demonstrate the general ability of cholesterol lowering agents to reduce Aß deposition (Kojro et al, 2001). Likewise there is growing evidence to support the concept that modulating the cholinergic pathway alters Aß accumulation.

Table 1: Treatments for Alzheimer's on the market or in advanced stages of development. In each phase, for clarity, cholinergic drugs have been separated from non-cholinergic products

Table 2: Molecules in development targeted towards reduced production or increased degradation of Aß

Comparison of AMYScreen with previous Ab bioscreens: prior screens have focused on either total Ab or Ab 40 which would likely miss any compounds that specifically influence Ab42. Furthermore AMYScreen employs human cells of human origin again differing from earlier studies and therefore of presenting the opportunity for improved clinical predictability.

Comparison of Aß inhibitors with other Alzheimer's targets: Most current therapies or molecules in advanced development compensate for altered neurotransmission. Agents able to block the amyloid pathway are unlikely to compete with these molecules - on the contrary the dual use of both classes of drug is likely to optimize therapeutic options. This is particularly the case as significant overlap between amyloid toxicity and cholinergic signaling has been reported. On one hand, Aß reduces hippocampal cholinergic release in cognitively impaired rats (Vaucher et al, 2001), binds strongly to nicotinic receptor blocking the conductance of their channel sub-units (Pettit et al, 2001), reduces choline uptake and inhibits acetylcholine release (Kar et al, 1998). On the other hand cholinesterase inhibitors can block Aß accumulation (Lahiri et al, 2000). Perhaps the closest competitor to CBE-2001 that has been described to date is a family of g -secretase inhibitors from Elan & Lilly. These inhibitors block the total production of Aß and therefore CBE-2001 presents improved specificity. Furthermore, inhibitors of g-secretase have been shown to seriously compromise the immune system (Hadland et al, 2001) as well as causing a build up of CTFs which may themselves be toxic. To our knowledge therefore, CBE-2001 represents a unique advance in the potential treatment of Alzheimer's disease. 

Strategic analysis and suggested further studies: Dr Eckman's group has developed AMYScreen, a convenient model for the high throughput screening for specific inhibitors of Aß42 accumulation. Although this was considered insufficiently novel to be the subject of a patent application, it represents a major advance in Alzheimer's research all the same. Dr Eckman would be willing to screen molecules for interested parties using this tool. In addition, using this model, CBE-2001 has been identified as a class leader. This naturally derived extract represents, to our knowledge, the first product that specifically targets the accumulation of Aß42. Since AMYScreen employs cells derived from the human CNS, the demonstrated activity of CBE-2001 is predicted to be relevant to Alzheimer's disease. This will of course only be the case if CBE-2001 is active in vivo and secondly if it can be delivered to the CNS at therapeutically active doses. The first question has already been addressed in part since icv administration of CBE-2001 reduced Aß42 levels in the murine brain. This study should be repeated in animal models of Alzheimer's disease, preferably employing both local and systemic routes of administration.

Up until recently useful animal models of Alzheimer's have not been available. However thanks to the generation of transgenic mice with mutated genes and clinical features similar to those found in FAD (see Hsiao et al, 1996; Sommer et al, 2000; van Leuven, 2000; Bornemann & Staufenbiel, 2001) the first key question can now be addressed.

Dr Eckman is seeking companies interested in the further development of CBE-2001. Dr Eckman would be able to take responsibility for screening and optimization studies. Partners would be expected to offer chemistry and compound purification support. Optimally any agreement would involve licensing of the use and/or development of CBE-2001 and its derivative to partner companies. The financial terms of such an agreement are available upon request. Partners are invited to contact Dr Eckman directly or through LeadDiscovery. In addition Dr Eckman would be willing to screen drug candidate libraries. Financial details will be negotiated on the basis of the size of chemical libraries to be tested. Alternatively Dr Eckman would be able to assist companies in establishing the AMYScreen in house for interested parties

About the study authors: Dr Eckman received his PhD in Neuroscience from Case Western Reserve University and is currently a faculty member at the Mayo clinic in Jacksonville, Florida. Within a relatively short period of time Dr Eckman has emerged as a prolific researcher in the field of Alzheimer's disease with over 40 publications since 1994. This work has led to lead authorship on two pending US patent applications and perhaps of even greater importance Dr Eckman has developed several high throughput screens to exploit the commercial potential from proprietary research. Focusing on the biochemistry and genetics of Alzheimer's disease Dr Eckman has become a field-leader in the identification of genetic determinants of FAD.