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 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. Little attention has however been focussed on degradation of b-amyloid. In this respect recent data emerging from the Mayo Clinic (Jacksonville) is groundbreaking. Dr Eckman's group has demonstrated convincingly that endothelin converting enzyme (ECE) plays a fundamental role in b-amyloid metabolic pathways. The development of strategies able to stimulate or over-express ECE could therefore offer a novel therapy for Alzheimer's disease. This group has developed many of the assays necessary for exploiting this target including HTS compatible screens and therefore represents a powerful research partner for industry. The present dossier has been produced to highlight the proof of concept, commercial potential and development strategy of this target.

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 (see inset; source, AHAF). 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. The appearance of plaques in the hippocampus occurs long before the appearance of neurofibrillary tangles. 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.

Aß42 is invariably elevated with all mutations that cause Alzheimer's disease and this form of ß-amyloid is considerably more neurotoxic that 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) have been suggested. 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.

Increasing Aß42 degradation may be a better therapeutic strategy than preventing its accumulation - 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. 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) or small molecules.

Endothelin converting enzyme represents a target for boosting Aß42 degradation - A number of extracellular protease 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). ECE-1 is expressed as 4 isoforms, ECE-1a, ECE-1b, ECE-1c and ECE-1d each with different localization patterns. ECE-1a is localized predominantly at the plasma membrane and also maybe in intracellular vesicles (Parnot et al, 1977) thus matching the cycling pattern of APP and its Aß products. In contrast, ECE-1b is present only within the cell. ECE-1c and ECE-1d are located both within the cell and at the plasma membrane (Schweizer et al, 1997; Valdenaire et al, 1999). Specific promoter regions determine the differential expression of these isoforms (Funke-Kaiser et al, 2000; Valdeniare et 1999).

ECE-1 as a candidate regulator of Aß accumulation: A groundbreaking study has been performed to determine the role of ECE-1 in Aß accumulation. For experimental detail the reader is referred to the original publication describing this study (Eckman et al, 2001).

• Culturing H4 neuroglioma cells with phosphoramidon, which is a broad spectrum inhibitor of peptidases including ECE, caused an increase in Aß accumulation supporting a role for ECE-1 in the endogenous modulation of Aß accumulation. ECE appears to exert its effect on intracellular Aß.

• Over-expression of ECE-1a or ECE-1b in CHO cells evoked a dramatic drop in extracellular accumulation of Aß40 and Aß42. Levels were reduced by up to 90%, and effect reversed by phosphoramidon. The production of sAPP was unaltered suggesting specificity with respect to APP processing.

• Over-expression of ECE-1a, the isoform that is expressed at the plasma membrane caused a modest metabolism of extracellular Aß40. ECE-1b, which is only present within the cell, did not share this property suggesting ECE works to degrade intracellular Aß primarily but can degrade extracellular Aß when the enzyme expression is appropriate

• A recombinant soluble fragment of ECE-1 was able to degrade Aß40 and Aß42 with favorable kinetics.

• The production of Aß40 and Aß42 occurs both intracellularly and at the plasma membrane. This study concluded that both ECE-1a and ECE-1b activation reduces the intracellular generation of Aß40 and Aß42, and furthermore ECE-1a may also be able to degrade protein preexisting in or escaping into the extra-cellular matrix.

• ECE-1 plays an important role in the generation of vasoactive endothelin and there is a risk that up-regulation of ECE-1 could cause cardiovascular side effects. This does not appear to be a problem since mice over-expressing ECE-1 did not display an increase in circulating endothelin suggesting that ECE is not the rate-limiting step in endothelin productions. Thus targeting ECE-1 remains a viable therapeutic option.

Patent position: US and PCT patents have been filed or are in the process of being filed in order to establish a proprietary position on the targeting of ECE as a strategy for treating Altzheimer's disease.

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 ECE 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). A number of Aß modulators are in development however ECE modulators offer a unique approach. Instead of blocking the amyloid pathway, which may have detrimental side effects, they are able to remove Aß thereby directly targeting a postulated basis of disease. This is likely to have considerable advantages.

Strategic analysis and suggested further studies: The results of the study presented in this dossier demonstrate clearly the potential of targeting ECE-1 for a novel treatment of Alzheimer's. The deposition of Aß in amyloid plaques is considered by many to represent an early step in the disease's path. Although this has not been shown incontrovertibly the evidence is strong. Hippocampal amyloid plaques are exclusive to Alzheimer's and this precedes many other hallmarks of the disease. Further more Aß is cytotoxic and can also impair cholinergic transmission both of which lead defective neuroregulation, the causative factor responsible for the symptomology of Alzheimer's. Successful Aß modulators are expected to occupy a place alongside conventional Alzheimer's therapies in the market and the commercial potential of such drugs is immense. Aß modulation can be conceivably be achieved through a number of approaches, none of which have been significantly exploited by current drug development activities. Targeting ECE-1 is particularly attractive since it takes away "bad" products of the amyloid pathway without affecting those that may occupy a physiological role. Furthermore, the ability to metabolize Aß as it is being formed and also after it has been deposited could be of dramatic importance in terms of treatment. The potential for ECE-1 modulators is therefore immense. Two key questions need to be addressed before a pharmaceutical development should start:

• Is ECE able to prevent Aß accumulating in vivo with an acceptable safety margin? If so is there a preferred target isoform of ECE-1, or mixture of isoforms, or indeed what would be the role of ECE-2?

• How can modulation of ECE be achieved?

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 exploiting his research. Dr Eckman would be able to set up assays for HTS studies performed by interested parties. In addition, Dr Eckman would be able to take responsibility for cell-based assays and animal models. This input would be offered in return for financial support and/or a percentage of income generated from candidate therapeutics. 

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 screen in order to exploit the commercial potential from proprietary research. Focussing on the biochemistry and genetics of Altzheimer's disease Dr Eckman has become a field-leader in the identification of genetic determinants of FAD allowing him to co-develop one of the first transgenic model of this disease.