Prevention of Alzheimer's Disease by Control of Beta-Amyloid

Prevention of Alzheimer’s disease by control of beta-amyloid production in the periphery

Authors: Abstract

J. Gregor Sutcliffe1,2 Using a novel approach for identification of

Brian S. Hilbush1 disease modifier genes, it was demonstrated that the level of expression of the Presenilin2 gene by

the liver regulates the accumulation of pathogenic 1 ModGene Pharma, LLC, PO Box concentrations of Alzheimer’s disease (AD)- 757, Cardiff-by-the-Sea, CA 92007, initiating beta-amyloid within the brain. The anti- USA. leukemia therapeutic imatinib (trade name E-mail: [email protected] Gleevec), which does not cross the blood-brain barrier, reduced liver production of beta-amyloid 2 The Scripps Research Institute, and lowered its accumulation in the brain below La Jolla, CA, USA pathogenic levels. These observations suggest E-mail: [email protected] that Alzheimer’s disease is preventable. The

imatinib-related compound, imatinib para- diaminomethylbenzene trihydrochloride, is more

than three-fold more potent in inhibiting beta- amyloid production than imatinib and exhibits th only 1/16 of the activity of imatinib in the inhibition of Abl kinase (the imatinib target in

leukemia), resulting in a selectivity ratio of nearly 60 for the AD indication. These studies suggest that prophylactic reduction of beta-amyloid at the

site of its production in the livers of aging humans has the potential to lower the incidence

AD and point to the identity of a drug that can accomplish that goal.

Keywords: Alzheimer's disease; beta-amyloid; imatinib

Medical Research Archives, Vol. 4, Issue 8, December 2016 Prevention of Alzheimer’s disease by control of beta-amyloid production in the periphery

1. Background 1.1 AD Genetics

Alzheimer’s disease (AD) is Some of the genes whose protein characterized by the age-dependent products affect AD risk have been deposition of beta-amyloid within identified. For example, certain variations vulnerable regions of the brain, (mutations) in the gene that encodes the particularly the frontal cortex and Amyloid Precursor Protein (APP; Tanzi hippocampus (Terry 2006). Beta-amyloid 1989), a cell membrane protein produced aggregates have a pathogenic effect, in all body tissues, predispose individuals leading to progressive neuronal loss that to early-onset AD. APP is a substrate for causes deterioration of the ability of those proteolysis by the endogenous proteases brain regions to orchestrate both higher beta and gamma secretase, liberating beta- order and basic neural processes. As the amyloid proteolytic fragments ranging deterioration worsens, the affected from 37 to 43 amino acid residues. The individual faces dementia and a worsening 42-residue species is thought to be the quality of life, and eventually the most pathogenic (Wolfe 2006), and forms condition is fatal. Age is the greatest aggregates, which, in addition to known risk factor for AD with an contributing to the plaques that deposit in incidence of 30–50% in people 85 years or the AD-affected brain, are thought to older. For a given individual, the time at initiate processes that lead to cognitive which AD manifests is the consequence of deficits (Barten & Albright 2008). AD- an additional series of risk factors, some predisposing variations in APP cluster in of which might be due to environmental the vicinity of the cleavage sites, affecting causes, but many of which are attributable the rate at which pathogenic beta-amyloid to that individual’s genetic endowment. fragments are generated, their stability,

and their ability to form aggregates

(Selcoe 2001). Individuals inheriting such

APP variations usually show signs of AD

Medical Research Archives, Vol. 4, Issue 8, December 2016 Prevention of Alzheimer’s disease by control of beta-amyloid production in the periphery in their 50s, whereas sporadic AD is not Brain Barrier (BBB) so as to reduce local common until individuals reach their 70s generation of the beta-amyloid. The BBB,

(Waring & Rosenberg 2008). Rare which protects the mostly non- variations in two other genes, Presenilin 1 regenerating cells of the brain by isolating and Presenilin 2, also confer high risk to it, is normally impermeable to small polar early-onset AD. These two genes encode molecules. Efforts to develop BBB- independent proteins of similar structures penetrating AD therapeutics have thus far that function as part of the gamma been disappointing. Recent research secretase protein complex (Wolfe 2006). (Sutcliffe et al. 2011) has now shown that

As a consequence of these genetic cerebral levels of beta-amyloid are observations and considerable regulated by peripheral tissues. One experimentation, the so-called “amyloid implication of this discovery is that cascade” model that has emerged holds therapeutic penetration of the BBB is that biochemical events that increase the unnecessary. This represents a production and accumulation of beta- fundamental change in concepts amyloid, particularly Aβ-42, accelerate concerning AD pathogenesis. the onset and progression of AD. 2. Alzheimer’s Modifier Gene 1.2 The Blood-Brain Barrier and Although mice do not normally the Implications of Regulation of beta- develop Alzheimer’s-like disease, they amyloid in Peripheral Tissues can be “engineered” to get Alzheimer’s

The tacit assumption of the disease by the introduction of an APP researchers in this field has been that local transgene that contains the so-called over-production of pathogenic beta- Swedish mutations: two mutations that in amyloid within the brain is the problem humans predispose individuals to early and that the likely solution would involve onset Alzheimer’s (Kulnane & Lamb development of beta and gamma secretase 2001). In some genetically pure strains of inhibitors designed to penetrate the Blood-

Medical Research Archives, Vol. 4, Issue 8, December 2016 Prevention of Alzheimer’s disease by control of beta-amyloid production in the periphery mice (such as B6) the APP transgene database that contained the activities of causes high levels of brain beta-amyloid every one of the mouse’s approximately leading to cognitive deficits as measured 25,000 genes (how much mRNA product in maze tests (Hock et al. 2009) and each gene made) in each of 10 tissues deposition of beta-amyloid in plaques (including liver and several areas of the

(Lehman et al. 2003). Other pure strains of brain) in all 40 pure mouse stains was mice (such as D2) accumulate less beta- constructed. The database was queried as amyloid in the brain, do not develop to whether any genes had activities that cognitive deficits, nor do they get plaques. were inherited in the 40 strains in exactly

The genetic differences in the mouse the same manner in which susceptibility to genome responsible for the difference in Alzheimer’s disease was inherited. A gene susceptibility to Alzheimer’s disease have was discovered in each of the three been mapped to three broad chromosomal chromosomal intervals: chromosome 1, regions by Bruce Lamb and colleagues Psen2; chromosome 2, Zfhx 1b;

(Ryman et al. 2008), but the identities of chromosome 7, Cib1 (Sutcliffe et al. the responsible genes were not known. 2011).

Forty pure strains of mice that had It was expected that their heritable been generated by breeding B6 mice with differences in activities would be observed

D2 mice were studied (Sutcliffe at al. within the brain, but that was not what

2011). These 40 recombinant inbred (RI) was found. Instead, unexpectedly, it was strains had each been made “pure” by discovered that the activity level of each brother-sister matings over more than 20 of the three genes (how much mRNA generations. Each of the 40 RI strains product it made) was higher in the liver of inherited some (approximately half) of its all strains of mice that were susceptible to genes from B6 and the remaining Alzheimer’s disease than in any of those approximately half from D2, but each RI that were resistant to the disease. strain had a different mix of genes. A

Medical Research Archives, Vol. 4, Issue 8, December 2016 Prevention of Alzheimer’s disease by control of beta-amyloid production in the periphery

For example, trait correlation overlapping distributions of mRNA regression analysis was performed expression levels between lines carrying between the genotype of the Psen2 the alternative genotypes at the Psen2 interval on chromosome 1 and the amount locus. Thus, Psen2 expression in the brain of Psen2 mRNA that accumulates in each is not a modifier of brain beta-amyloid of the 10 tissues in the RI mice, accumulation. However, in the liver, the calculating the Pearson’s product-moment amount of Psen2 mRNA was highly correlation values (Sutcliffe et al. 2011). correlated (p = 4.99 e-41) with the

The values are shown in Table 1. None of genotype at the Psen2 locus. Furthermore, the tissue samples derived from brain mice inheriting the B6 genotype express shows high heritability of Psen2 2-to-8-fold more Psen2 mRNA in the liver expression, as was evident by the than do D2-genotype mice.

Table 1: Heritability of Psen2 mRNA accumulation in various tissues*

r r2

brain │r│<0.05 <0.0025 cerebellum r = 0.6344 0.4025 eye │r│<0.35 <0.1225 hippocampus │r│<0.36 <0.1296 kidney r = -0.4733 0.2240 liver r = -0.9402 0.8840 nucleus accumbens r = 0.7260 0.5271 neocortex r = 0.5500 0.3025 prefrontal cortex │r│<0.51 <0.2601 striatum r = 0.5329 0.2840

* Pearson’s product-moment correlation value (r), negative correlation values indicate expression of Psen2 mRNA in B6 genotypes is higher than in D2 genotypes. │r│< 0.8 represent incomplete heritabilities unlikely to account for mapped QTLs because of overlapping distributions.

Although each of the three genes correlation between the activity level of

(Psen2, Zfhx 1b and Cib1) was also the genes in the brain and the inheritance expressed in the brain, there was no of Alzheimer’s disease resistance. That

Medical Research Archives, Vol. 4, Issue 8, December 2016 Prevention of Alzheimer’s disease by control of beta-amyloid production in the periphery suggested that the genes were acting in the hypothesis correct, then one ought to be liver to modify the susceptibility of the able to reduce the amount of beta-amyloid mice to Alzheimer’s disease. in the brain by reducing how much is

Strengthening this argument is that the made by the liver. The leukemia drug chromosome 7 gene, Cib1, encodes imatinib (Gleevec) was known to inhibit calmyrin, a protein discovered because of the APP clipping rate by interfering with its preferential interaction with Presenilin the interaction between gamma-secretase

2 (the Psen2 product) in a two-hybrid and its activator, GSAP (He et al. 2010). screen (Stabler et al. 1999). Imatinib does not enter the brain;

therefore, it had not been tested to 3. Pathogenic Amyloid Comes determine whether it could reduce From Peripheral Tissues accumulation of beta-amyloid in the brain. The Psen2 gene encodes the 448 The test was conducted by injecting amino acid trans-membrane protein mice with imatinib for 1 week. Even Presenilin2. In humans, some Presenilin2 though the drug cannot get to the brain, it mutations confer risk for early-onset reduced the amount of brain beta-amyloid Alzheimer’s disease. The product of the by about 50% (Sutcliffe et al. 2011). That Presenilin2 gene is the probable catalytic implied that a considerable portion of the component of the gamma secretase brain amyloid was produced outside of the complex, responsible for cleavage of beta- brain but rapidly entered the brain. Liver amyloid from APP. It was hypothesized is a major source of beta-amyloid origin, that higher activity of Presenilin2 in the but there might be other non-brain sources liver increases the clipping rate leading to in addition to liver that have not been more beta-amyloid released by the liver tested. The take-away message is that the into the blood, where it can form small amount of cerebral amyloid, hence the risk aggregates and migrate to the brain and for Alzheimer’s disease, is regulated by eventually initiate pathology. Were this peripheral tissues. This concept has been

Medical Research Archives, Vol. 4, Issue 8, December 2016 Prevention of Alzheimer’s disease by control of beta-amyloid production in the periphery confirmed and the results replicated in in the human body and byproducts of its studies (Weintraub et al. 2013) which organic synthesis, were studied to assess show that hippocampal beta-amyloid and compare their activities in inhibiting accumulation accompanied by cognitive beta-amyloid production and tyrosine deficits initiated by endotoxin-induced kinase inhibitor function. Ideal peripheral inflammation is eliminated by Alzheimer’s medications would have imatinib administration, and (Cancino et higher potency in reducing beta-amyloid al. 2008) that imatinib administration to but lower potency in inhibiting tyrosine amyloid plaque-bearing APP transgenic kinase activity, and thus their long-term mice led to plaque reduction and cognitive maintenance use would be less cause for improvement. Furthermore, it has been concern. Two compounds were identified shown (Eisele et al. 2014) that that are more potent than imatinib in intraperitoneal injection of beta-amyloid reducing beta-amyloid production by aggregates leads to brain amyloidosis, cultured cells transfected to produce demonstrating directly that peripheral human APP and which are more selective beta-amyloid can enter the brain to initiate than imatinib in sparing tyrosine kinase disease. activity in an in vitro assay for Abl kinase,

the imatinib target enzyme in leukemia. 4. More Selective Amyloid- The better of the two, imatinib para- lowering Compounds diaminomethylbenzene trihydrochloride,

Any treatment that lowered the is more than three-fold more potent in concentration of blood beta-amyloid to an inhibiting beta-amyloid production than acceptable level would be a candidate for imatinib and exhibits only 1/16th of the chronic use for individuals wishing to activity of imatinib in the inhibiting Abl lower their risk for this frightening disease. kinase, thus represents a 60-fold

Compounds closely related in structure to improvement over imatinib in its imatinib, including its natural metabolites selectivity for the Alzheimer’s indication

Medical Research Archives, Vol. 4, Issue 8, December 2016 Prevention of Alzheimer’s disease by control of beta-amyloid production in the periphery

(Sutcliffe & Hilbush 2016), positioning it (specifically Aβ-42) in mutation carriers as the potential drug for disease was shown to be stable for decades prevention over decades of use. preceding age of onset (Fagan et al. 2014),

suggesting a prolonged period of 5. Further Considerations progressive accumulation that can be

These studies identify a peripheral halted by drugs lowering its production in source of beta-amyloid as a prime target the periphery. for disease prevention in the therapeutic There is a general scientific ‘window of opportunity’ preceding onset agreement that non-aggregated beta- of AD. The unexpected discovery that amyloid has beneficial effects within the lowering the production of beta-amyloid brain and probably on many types of cells peripherally leads to a reduction in brain in the body. After all, its structure and amyloid implies that elevated beta- activity has been selected through amyloid in the circulation is potentially a evolution. It is known that in humans, hallmark (or clinical biomarker) of the some variations in the APP gene that preclinical stage preceding AD onset. encodes beta-amyloid confer risk for Longitudinal studies have shown early-onset Alzheimer's, and individuals unequivocally that plasma beta-amyloid with Down syndrome who carry an extra levels are consistently higher in copy of this gene develop Alzheimer's individuals destined to develop very early; so beta-amyloid can have Alzheimer’s as compared to healthy negative effects as well. Aggregated beta- controls both in autosomal dominant amyloid is toxic to neurons and initiates carriers (APP, PSEN1 and PSEN2) the cellular pathologies we recognize as (Bateman et al. 2012; Fagan et al. 2014) Alzheimer's disease. The studies reviewed and in Down syndrome patients (Zigman here suggest that, at least in mice, the & Lott 2007; Head et al. 2011). The beta-amyloid produced locally within the elevation of plasma beta-amyloid brain is benign as far as Alzheimer's.

Medical Research Archives, Vol. 4, Issue 8, December 2016 Prevention of Alzheimer’s disease by control of beta-amyloid production in the periphery

Beta-amyloid produced by the liver pills ought to be found to prevent or aggregates in the blood and some of it greatly delay the pathogenic effects of enters the brain. When added to the brain- beta-amyloid in the brain – a future world made beta-amyloid, the toxic effect over with lower Alzheimer’s incidence. Patent prolonged decades initiates the disease applications were filed on the dual process. As larger aggregates form, they discoveries that pathogenic beta-amyloid are no longer soluble and deposit as has a primary source in peripheral tissues plaques. The plaques themselves are outside of the brain (particularly the liver) probably benign, but demonstrate the sites and that the FDA-approved anti-leukemia within the brain where toxic aggregates drug imatinib is effective in reducing have initiated pathology. amyloid accumulation in the brain, even

though it does not cross the blood-brain 6. Conclusions and Future barrier (BBB), and additional applications Directions on the discoveries of compounds that are

The mouse studies, both genetic and more potent and selective for the with imatinib, suggest that lowering blood Alzheimer’s indication. Both patents have

(liver-derived) beta-amyloid by as little as been approved and issued in Europe

20% can reduce the brain beta-amyloid (Sutcliffe et al. 2015; Sutcliffe & Hilbush burden to a level below that which is 2016) and are pending in other pathogenic. It is probably a bad idea to get jurisdictions. The patent portfolio has rid of all beta-amyloid, as for example it been assigned to ModGene Pharma, LLC, would be a bad idea to completely rid the which is seeking investors and blood of cholesterol: you need some, but pharmaceutical partners to run clinical too much takes a toll over time. Imatinib trials that would allow development of and drugs with similar actions taken as AD-preventing medications.

Medical Research Archives, Vol. 4, Issue 8, December 2016 Prevention of Alzheimer’s disease by control of beta-amyloid production in the periphery

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Tanzi RE Molecular genetics of Alzheimer's disease and the amyloid beta