HK1201736B - Compositions and methods for reduction of amyloid-beta load - Google Patents

Description

Compositions and methods for reducing beta amyloid loading

Priority is claimed for U.S. provisional application serial No. 61/554,375 filed on 1/11/2011 and U.S. provisional application serial No. 61/682,031 filed on 10/8/2012, each of which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to methods and compositions for modulating the level of beta amyloid peptide (a β) displayed by non-neural (i.e., peripheral) cells, fluids, or tissues. The invention also relates to the modulation of brain a β levels via selective modulation (e.g., inhibition) of γ -secretase activity in peripheral tissues. The invention further relates to methods of preventing, treating, or ameliorating the symptoms of disorders, including but not limited to neurological a β -related disorders, by peripherally administering compounds that result in the modulation, directly or indirectly, of γ -secretase. The invention also relates to the use of modulators of gamma-secretase activity via peripheral administration for preventing, treating or ameliorating the symptoms of alzheimer's disease. The invention further relates to the use of inhibitors of a β production with reduced kinase inhibitory activity.

Background

Amyloid beta (a β) peptide is a metabolite of the alzheimer's disease-associated precursor protein, β Amyloid Precursor Protein (APP), and is believed to be a major pathological determinant of Alzheimer's Disease (AD). AD is a neurodegenerative disorder characterized by age-dependent deposition of A β in vulnerable areas of the brain, particularly in the frontal cortex and hippocampus (Terry RD. J Geriatr PsychiatryNeurol19:125-128, 2006). A β has a pathogenic effect, resulting in progressive neuronal loss, which causes a deterioration in the ability of those brain regions to control higher order and essential neurological processes. When the degeneration worsens, the affected individual faces dementia and a worsening quality of life, and the final condition is fatal (Brookmeyer R, Johnson E, Ziegler-Graham K, Arrighi HM Alzheimer' S comment 3:186-191, 2007; Power JM. neurobiol Aging18: S53-S54,1997).

It is believed that the development of AD is caused by natural biochemical processes associated with aging, and that each individual, whether alive for a sufficient period of time or not, almost eventually manifests symptoms of the disease. Age is the largest known risk factor for AD and the incidence of disease is 25-50% in people 85 years of age or older (Giacobini e.ann NY Acad Sci920: 321-. For a given individual, the time at which the condition manifests is the result of a series of additional risk factors, some of which can be attributed to environmental causes, but many of which are attributed to the individual's genetic endowments: natural variation in the structure and activity of individual genes produces a collection of proteins whose complex interaction network makes individuals more or less susceptible to AD. Several genes whose protein products influence the risk of AD have been identified. For example, there are three common variants of the gene encoding serum apolipoprotein E, designated E2, E3, and E4. Individuals who inherit the e 4-encoding allele are at higher risk for AD than the average and are predisposed to develop disease at an earlier time than individuals without the e4 allele. Those who inherit the e4 allele from both parents are at higher risk of early onset of AD, while individuals with the e2 allele are at much lower risk, if at all, of disease at a later time in life compared to the average level (Cedazo-Minguez A.J Cell Mol med.11:1227-38, 2007). Traumatic brain injury and repeated brain trauma have also been found to accelerate brain a β deposition and cognitive disorders. Uryu et al J.Neurosci.22(2):446 (2002).

Most, if not all, AD is considered to have some genetic components associated with each individual's risk threshold. However, some forms of human AD are particularly highly heritable. These heritable forms result from rare mutations in a single gene encoding a protein that is associated with this neurodegenerative disorder and that plays a central role in the initiation of the disease process. Mutations in these genes may be inherited or may occasionally occur.

One of these genes encodes Amyloid Precursor Protein (APP) (Tanzi RE. Ann Med.21:91-94,1989). APP is a membrane protein, the biochemical function of which is now unknown. APP is known to be a proteolytic substrate for a variety of endogenous proteases, and proteolysis releases fragments with different structures. Two of these protease activities are called β -secretase and γ -secretase. APP produces fragments by proteolysis of β -secretase, which fragments can be subsequently cleaved by γ -secretase at multiple sites to produce a β peptides. Gamma-secretase is a complex of proteins (including presenilin 1 and presenilin 2), and cleavage of APP by gamma-secretase produces isoforms of a β that range from 37 to 43 amino acid residues (see, e.g., Steiner H, Fluhrer R, Haass c., J Biol chem.2008jul 23). A.beta.in the form of a 42-residue was considered to be the most pathogenic (Wolfe MS. biochemistry45:7931-7939, 2006). The 42-residue A β fragments form oligomeric structures that are thought to cause cognitive deficits in addition to the formation of plaques that deposit in AD-affected brain (Barten DM, Albright CF. mol Neurobiol37: 171-.

AD-inducing APP mutations aggregate near the proteolytic site, affecting the rate of production of pathogenic A β fragments, their stability and their ability to form oligomers (Selkoe DJ. physiol Rev81:741 and 766, 2001). Individuals who inherit this type of APP variation often show signs of AD at their fifties, while sporadic AD does not become common until the individual reaches their seventies (Waring SC, Rosenberg RN. Arch neuron.65: 329-34, 2008).

The complete molecular identity of the gamma-secretase enzyme is still unknown. Presenilin 1 or closely related presenilin 2 is essential for gamma-secretase activity. In cultured cells obtained from embryos genetically deleted for presenilin 1, γ -secretase activity was reduced by 80%. In cells without presenilin 1 and presenilin 2, the γ -secretase activity is totally lost. Peptidomimetic inhibitors of gamma-secretase activity can crosslink with presenilin 1 and 2, suggesting that these proteins are catalytic subunits for cleavage. However, the gamma-secretase activity isolated from the cells was analyzed by chromatography as larger complexes of >1M daltons. Recent genetic studies have identified three additional proteins required for gamma-secretase activity; dull protein, aph-1 and pen-1. (Francis et al 2002, development cell3(1): 85-97; Steiner et al 2002, J.biol. chemistry:277(42): 3906239065; and Li et al 2002, J.neurochem.82(6): 1540) 1548). In cells without these proteins, the accumulation of presenilins as high molecular weight complexes is altered. Rare variations in the genes encoding the presenilin 1 and presenilin 2 components of gamma-secretase also confer a high risk of premature AD (Waring SC, Rosenberg RN. Arch neuron.65: 329-34, 2008).

The third enzyme, α -secretase, cleaves the precursor protein between the β -and γ -cleavage sites, thereby precluding a β production and the release of an approximately 3kDa peptide called P3, which is non-pathological. Cleavage by β -and α -secretases also produces soluble, secretory terminal fragments of APP, which are referred to as sAPP β and sAPP α, respectively. The sAPP α fragment has been proposed to have neuroprotective effects.

Due to these genetic observations and a large number of biochemical and neuroanatomical experiments, the following models have emerged: biochemical events that increase the production and accumulation of a β, particularly a β -42, accelerate the onset and progression of AD. Thus, therapeutic and prophylactic procedures aim to reduce the production of a β or to reduce its accumulation.

The current focus of AD treatment is to reduce a β production and/or accumulation in the brain. Several methods are currently under investigation (Rojas-Fernandez CH, Chen M, Fernandez HL. Pharmacotherapy22: 1547. cndot. 1563, 2002; Hardy J, Selkoe DJ. science.297: 353. cndot. 356, 2002). Mice that are transgenic for AD-inducing APP and additionally carry an inactivating knockout mutation of the β -secretase gene exhibit an almost complete reduction of A β in the brain (Luo Y, Bolon B, Kahn S, Bennett BD, Babu-Khan S, Denis P, Fan W, Kha H, Zhang J, Gong Y, Martin L, Louis JC, Yan Q, Richards WG, Citron M, Vassar R. nat Neurosci4: 231: -232, 2001). However, it has been shown that such mice still exhibit cognitive deficits, premature death and low myelination (Ohno M, Chang L, TsengW, Oakley H, Citron M, Klein WL, Vassar, Disterhoft JF Eur J Neurosci23:251-, hicks CW, He W, Wong P, Macklin WB, Trapp BD, Yan R. nat Neurosci9:1520-1525, 2006). This leads to the following conclusions: beta-secretase activity in the brain is required for healthy neurological function, and therapeutic agents that reduce the brain activity of beta-secretase may have adverse side effects. In addition, it is difficult to design effective, brain penetrating β -secretase inhibitors (Barten DM, Albright CF. MolNeurobiol37: 171-.

The effect of gamma-secretase inhibitors in reducing brain a β has also been investigated. Brain penetrating gamma-secretase inhibitors have been shown to reduce A β synthesis and to reduce cognitive deficits in mouse models of AD (Barten DM, Meredith JEJr, Zaczek R, Houston JG, Albright CF. drugs R D7:87-97,2006). However, gamma-secretase has targets other than APP (Pollack SJ, Lewis H. Curr Opin Investig Drugs6:35-47,2005), one of which is a transmembrane receptor of the Notch family. Inhibition of Notch signaling by chronic administration of gamma-secretase inhibitors results in changes in the gastrointestinal tract, spleen and thymus, thereby limiting the degree of A β inhibition achievable in vivo using the compounds studied (Searfoss GH, Jordan WH, Calligoro, Galbreeth EJ, Schirtzinger LM, Berridge BR, Gao H, Higgins MA, May PC, Ryan TP. J Biol Chem278:46107-46116, 2003; Wong GT, Manfra D, Poulet FM, Zhang Q, Josien H, Bara T, Engstrom L, Pinzon-Ortiz M, Fine JS, Lee MilHJ, Zhang L, Higgins GA, Parker EM. J Biol Chem279: 12876-82, 2004; Cano J, Kay J, Daaiys C, Brown-Brown, Jagns-Brown J, Jagnson L, Jagnson J-J biochem R341, J-D, Jacq J-E J-D, J-E J-O-D, J-E-D, J-E J-D, C, J-E J-D, E-D, J-E J-.

Us patent application 20020128319a1 states that certain nonsteroidal anti-inflammatory drugs (NSAIDS) reduce the production and/or level of a β 42 in cell cultures expressing a β 40 and a β 42 obtained by cleaving APP. Because there is ample evidence that high a β 42 levels are a major risk factor for AD, such drugs may be useful in preventing, slowing or reversing the progression of AD. However, the disadvantages of using such drugs are the large dose of NSAIDS required to significantly reduce A β 42, and the significant gastrointestinal side effects, including bleeding ulcers, associated with the prolonged use of high doses of NSAIDS (Langman et al 1994, Lancet343: 1075-1078). In addition, there is still an unknown risk of alzheimer's disease due to amyloid formation by a β 40 and other forms unaffected by a β 42-lowering agents. Accordingly, there is a need in the art to develop therapies for diseases or disorders associated with modulation of a β production.

One class of compounds has been found to reduce a β production without affecting Notch signaling. Such compounds include the tyrosine kinase inhibitor imatinib mesylate (STI-571, trade name GLEEVEC) and related compounds, 6- (2, 6-dichlorophenyl) -8-methyl-2- (methylthiophenyl-amino) -8H-pyrido [2,3-d ] pyrimidin-7-one, which is known as inhibitor 2(Netzer WJ et al Proc Natl Acad Sci U S A.100: 12444-. See also U.S. patent publication 2004/0028673 and PCT patent publication WO2004/032925, each of which is incorporated herein by reference. STI-571 is currently approved for the treatment of myeloid leukemia and gastrointestinal stromal tumors. In APP-transfected neuroblastoma cells and cell-free extracts of transfected cells, STI-571 effectively reduces the production of A β via a mechanism that does not require the Abl tyrosine kinase, an important target of this drug in leukemia cells (Netzer, supra). STI-571 and related compounds called "inhibitor 2" were found to reduce a β production in cultures of primary neurons prepared from the cerebral cortex of embryonic day 18 rats (Netzer, supra), indicating that these drugs affect proteolytic processing of proteins from endogenous and transfected APP genes.

According to the product data of GLEEVEC, STI-571 is administered orally. This drug has been studied for its effect on a β accumulation in the brain and has been shown to have poor blood brain barrier permeability. In STI-571 treated leukemia patients receiving this drug, the cerebrospinal fluid (CSF) level of the drug is 92-fold lower than the blood level (Takayama N, Sato N, O' Brien SG, Ikeda Y, Okamoto S.Br J Haematol.119:106-108, 2002). Thus, its use as a potential therapeutic agent for AD in unmodified form has not been considered (Netzer, supra).

In view of poor blood brain barrier permeability, researchers investigating the effect of STI-571 on brain a β used implanted osmotic micropumps to deliver either STI-571 or inhibitor 2 intrathecally to guinea pig brain (Netzer, supra). Although Netzer et al observed a reduction in ap accumulation in the brain, they still concluded that "in the case of Gleevec and related drugs, the ability to obtain high blood brain barrier permeability is necessary for the possibility of improved therapeutic benefit" (Netzer, supra).

In the development of small molecule therapeutics for most diseases, compounds that inhibit protein kinases or block the ATP-binding domain of any enzyme are generally less desirable than compounds that exert the same therapeutic effect via alternative mechanisms. Protein kinases regulate many essential cellular processes including cell cycle progression, DNA damage response, cell proliferation, metabolism and cell death, differentiation and survival. In fact, the human genome contains at least 500 different genes encoding protein kinases. Kinase inhibitor drugs, such as imatinib, have known off-target interactions that alter their toxicity and side effect profiles (see, e.g., Force, T. & Kolaja, k.l. cardio-oxidation of kinase inhibitors: the prediction and transmission of pre-clinical models to clinical output. nat. rev. drug discovery. 10,111-126 (2011)). Imatinib inhibits kinases Abl, ARG (Abl-related gene proteins), PDGF-Ra/B and KIT. The tyrosine kinase inhibitor sunitinib (see, e.g., Chu, t.f. et al, Cardiotoxicity associated with tyrosine kinase inhibitor subsitinib. Lancet 370,2011-2019(2007)) and other kinase inhibitors exhibit Cardiotoxicity (see also Cheng, H. & Force, t.molecular mechanisms of Cardiotoxicity associated with targeted cancer therapy, circ.res.106,21-34 (2010)). Thus, there is concern that the use of kinase inhibitory drugs such as imatinib to prevent alzheimer's disease in long-term treatment regimens may have negative consequences that are not observed in relatively transient chemotherapy regimens. Although the reported side effects of imatinib are considered to be limited for chemotherapeutic agents used in cancer therapy, if tens of millions of people take this drug for maintenance, it is expected that new side effects associated with protein kinase inhibitory activity will be observed.

There remains a need for therapies that effectively reduce a β levels in the brain, and in addition there remains a need for therapies that effectively reduce a β levels and result in less inhibition of Abl kinase activity.

Summary of The Invention

The present invention relates to methods of treating, preventing or monitoring brain a β disorders by testing and/or treating peripheral (non-brain, non-CNS) tissues. In some preferred embodiments, the peripheral tissue comprises liver, while in other embodiments, the peripheral tissue comprises blood and/or serum. In some embodiments, the invention includes evaluating a subject for the presence of AD or predisposition to AD by peripherally administering a compound that modulates the accumulation or production of a β and evaluating the subject for AD or progression of AD.

The present invention provides methods, compositions, and processes relating to treating or preventing AD by treating the liver of a subject. In particular, the invention relates to altering a β production, processing, accumulation or trafficking in the liver of a subject by directly inhibiting production (e.g., by inhibiting APP expression) or by modulating a factor, thereby modulating a β production, processing, accumulation or trafficking in the liver. Such factors include, but are not limited to, gamma-secretase, presenilin 1, presenilin 2, ApoE, cadherin, synaptic growth related proteins, inositol 1,4, 5-triphosphate receptor (instp 3R) or Smad interacting protein-1 (SIP1, encoded by Zfhx1 b), clusterin (encoded by CLU, also known as ApoJ), phosphoinositide binding clathrin assembly protein (encoded by PICALM), complement component receptor 1 (encoded by CR 1), Insulin Degrading Enzyme (IDE), gamma secretase-activating protein (GSAP), and modulators thereof. The invention encompasses the treatment or prevention of AD by modulating any factor that, when modulated, affects the production of a β in the liver of a subject either directly (e.g., by acting on APP production or processing) or indirectly (e.g., by acting on a factor that in turn acts on a factor that acts on APP). The present invention is not limited by the nature of the modulation, or the identity or number of factors that operate to modulate a β in the liver of a subject.

In some embodiments, the invention provides methods of treating a subject diagnosed with or susceptible to a brain a β disorder, comprising peripherally administering a compound that modulates production of a β in peripheral tissues. In some preferred embodiments, the compounds inhibit the production of a β. In a particularly preferred embodiment, the peripherally administered compound has a partition coefficient of less than 2.0, more preferably less than 1.5 and still more preferably less than about 1.0. In a particularly preferred embodiment, the compound does not substantially cross the blood brain barrier.

In some embodiments, the invention provides a method of treating a subject for a brain a β disorder or predisposition to a brain a β disorder in a subject, comprising peripherally administering a compound that modulates expression of a gene in a peripheral tissue of the subject. In a preferred embodiment, modulating said expression of said gene results in modulation of a β production or accumulation in said peripheral tissue. In certain preferred embodiments, the peripheral tissue is the liver of the subject.

The present invention encompasses any method of affecting a β production in the liver, including but not limited to altering APP expression and/or processing. In some embodiments, the invention provides methods comprising peripherally administering a compound that modulates the expression of one or more of Psen1, Apo E, inst 3R, Psen2, APP, Cib1, Ngrn, Zfhx1b, CLU (also known as ApoJ), PICALM, IDE, GSAP, and CR1 genes. In some embodiments, the methods of the invention comprise peripherally administering a compound that modulates the activity of one or more of: presenilin 2, calintegrin binding protein, synaptic growth related protein, Zfhx1b, clusterin, phosphoinositide-binding clathrin assembly protein, complement component receptor 1, insulin degrading enzyme, GSAP, or APP expression or activity. In some embodiments, one or more of these genes or activities are modulated in the liver of the subject. In some embodiments, modulating comprises inhibiting expression or activity, and in some embodiments, modulating comprises stimulating expression or activity.

In some embodiments, the invention includes, for example, a method of treating a brain a β disorder, the method comprising the steps of: the subject is assessed for the presence of a brain a β disorder or predisposition to a brain a β disorder, a compound that modulates production of a β is administered peripherally, wherein the compound does not substantially penetrate the blood brain barrier, and the subject is assessed for the brain a β disorder or progression of the brain a β disorder. It is further contemplated that in some embodiments, the results of the assessment before and after treatment are compared to determine, for example, the effect of the treatment on the brain a β disorder state (e.g., to determine the effect on the onset or rate of disease progression or remission). Modulation of production of a β is not limited to any particular means or pathway of modulation. Modulation of production may include, for example, altering (e.g., decreasing) expression of APP, or altering the processing of APP into a β.

In some embodiments, the present invention comprises the steps of: the subject is assessed for the presence of a brain a β disorder or predisposition to a brain a β disorder, the compound that modulates the accumulation of a β is administered peripherally, wherein the compound does not substantially penetrate the blood brain barrier, and the subject is assessed for the brain a β disorder or for the progression of the brain a β disorder. The regulation of the accumulation of a β is not limited to any particular means. Modulation of accumulation may include, for example, decreasing production of a β and/or increasing degradation or clearance of a β, or altering a β to produce an improvement (e.g., a non-pathogenic form) with a different property.

It is contemplated that in some embodiments of the invention, the modulation of production and/or accumulation of a β, the administered compound comprises a modulator of γ -secretase activity, while in some preferred embodiments, the compound comprises an inhibitor of γ -secretase activity.

It is further contemplated that in some embodiments of the invention, the administered compound comprises a modulator of presenilin 2, modulation of production and/or accumulation of a β. In some preferred embodiments, the compound comprises an inhibitor of presenilin 2. In some embodiments, the compound comprises a modulator that cleaves amyloid precursor protein, and in some embodiments, the compound comprises an inhibitor that cleaves amyloid precursor protein.

In some embodiments, the compound comprises a composition selected from the group consisting of: STI-571, imatinib p-diaminomethylbenzene (e.g., trihydrochloride), N-desmethyl imatinib, compound 1, compound 2, LY450139, GSI-953, levoflurbiprofen, and E2012(Eisei) compounds, or blood-brain barrier impermeable variants thereof. In particularly preferred embodiments, the composition has a partition coefficient (e.g., in an octanol/water system) of less than 2.0, more preferably less than 1.5, and still more preferably less than about 1.0. In a particularly preferred embodiment, the compound does not substantially cross the blood brain barrier.

In some embodiments, the compound comprises an interfering oligonucleotide, while in preferred embodiments, the compound comprises an interfering RNA. In still more preferred embodiments, the interfering RNA is selected from the group consisting of: siRNA, shRNA and miRNA. In some embodiments, the interfering RNA comprises interfering RNA against amyloid precursor protein RNA, while in other embodiments, the interfering RNA comprises interfering RNA against presenilin 2 RNA. In other embodiments, the interfering RNA is directed against Psen1, ApoE, instp 3R, Cib1, Ngrn, Zfhx1b, CLU (also known as ApoJ), PICALM, IDE, GSAP, or CR1 RNA.

It is contemplated that in some embodiments, the compounds further include known therapeutic agents that treat, ameliorate, or reduce the risk or severity of a brain a β -related disorder. In certain preferred embodiments, the known therapeutic agent is selected from the group consisting of: cannabinoids, dimebom, prednisone, ibuprofen, naproxen, indomethacin; statins, selective estrogen receptor molecules, antihypertensive drugs, alpha-blockers, beta-blockers, alpha-beta blockers, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers, diuretics, and antioxidants.

The peripheral administration of the compounds in the methods of the invention is not limited to any particular route. Routes of administration include, but are not limited to, via the eye (ophthalmic), oral (oral), skin (transdermal), nose (nasal), lung (inhalant), oral mucosa (buccal), ear, injection (e.g., intravenous, subcutaneous, intraperitoneal, etc.), and the like. In certain preferred embodiments, peripheral administration comprises oral administration.

In some embodiments of the methods of the invention, the assessing comprises assessing the mental state. In some preferred embodiments, the assessment comprises one or more of neuropsychological testing and brain imaging.

It is contemplated that in some embodiments, the present invention provides methods of assessing the risk or presence of a brain a β disorder in a subject, comprising determining a level of a β in a peripheral tissue of the subject. In some other embodiments, the present invention provides a method of monitoring a brain a β disorder in a subject, comprising determining a level of a β in a peripheral tissue of the subject. In some embodiments, the peripheral tissue is blood, and in some embodiments, the peripheral tissue is serum. In some particularly preferred embodiments, monitoring comprises measuring a β of said peripheral tissue at a plurality of time points.

In a preferred embodiment of the method disclosed hereinabove, the brain a β disorder is alzheimer's disease.

In some embodiments, the present invention provides a method of monitoring a brain a β disorder in a subject, comprising analyzing expression or activity of a gene product in a peripheral tissue of the subject. In certain preferred embodiments, the gene product is from a gene selected from the group consisting of: psen2, APP, Cib1, Ngrn, and Zfhx1 b.

In some embodiments, the present invention provides methods comprising the steps of: a subject is assessed for the presence of a brain a β disorder or a predisposition to a brain a β disorder, and a compound that inhibits peripheral a β transport across the blood brain barrier is administered peripherally, wherein the compound is not an anti-a β antibody. In a preferred embodiment, further comprising assessing the subject for a brain a β disorder or progression of a brain a β disorder. In a particularly preferred embodiment, the brain a β disorder is alzheimer's disease.

In some embodiments, the present invention provides a method of identifying a genetic target for treating a brain a β disorder, comprising comparing the hepatic gene expression profiles of offspring from a first parent having or susceptible to the a β disorder and a second parent having a reduced susceptibility to the a β disorder, identifying a heritable genetic marker having a level of expression in the liver, wherein an increase or decrease in the expression of the heritable genetic marker in the liver of the offspring relative to the level of expression in the liver of the first parent correlates with the inheritance of the genetic marker from the second parent.

In some embodiments, the invention includes a compound selected from the group consisting of: STI-571, imatinib para-diaminomethylbenzene, N-desmethyl imatinib, compound 1, compound 2, LY450139, GSI-953, levoflurbiprofen, and E2012 compounds, or blood brain barrier impermeable variants thereof, for use in modulating production of a β in peripheral tissues of a subject having or susceptible to a β disorder. In some embodiments, the a β disorder is a brain a β disorder. In a particularly preferred embodiment, the compound has a partition coefficient of less than 2.0, more preferably less than 1.5 and still more preferably less than about 1.0. In a particularly preferred embodiment, the compound does not substantially cross the blood brain barrier.

In some embodiments, the present invention provides a compound selected from the group consisting of: STI-571, imatinib para-diaminomethylbenzene, N-desmethyl imatinib, compound 1, compound 2, LY450139, GSI-953, levoflurbiprofen, and E2012 compounds, or blood brain barrier impermeable variants thereof, for use in modulating (e.g., inhibiting) production of a β in the liver of a subject having or susceptible to an a β disorder. In some embodiments, the a β disorder is a brain a β disorder. In a particularly preferred embodiment, the compound has a partition coefficient of less than 2.0, more preferably less than 1.5 and still more preferably less than about 1.0. In a particularly preferred embodiment, the compound does not substantially cross the blood brain barrier.

In some embodiments, the present invention relates to the use of a compound selected from the group consisting of: imatinib (STI-571), imatinib p-diaminomethylbenzene, N-desmethyl imatinib, WGB-BC-15, compound 1, compound 2, LY450139, GSI-953, levoflurbiprofen, and E2012 compounds, blood brain barrier impermeable variants thereof, and/or pharmaceutically acceptable salts thereof, for use in the manufacture of a medicament for modulating production of a β in peripheral tissues of a subject suffering from or susceptible to a brain a β disorder. In a preferred embodiment, the medicament is formulated for oral administration. In a particularly preferred embodiment, the peripheral tissue comprises liver. In a particularly preferred embodiment, the compound has a partition coefficient of less than 2.0, preferably less than 1.5 and still more preferably less than about 1.0. In a particularly preferred embodiment, the compound does not substantially cross the blood brain barrier. In some preferred embodiments, the present invention relates to the use of imatinib or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for inhibiting a β production in the liver of a subject suffering from or susceptible to a brain a β disorder.

The invention also provides the use of a compound as described above for the manufacture of a medicament comprising a second therapeutic agent for the treatment of a brain a β disorder. In some embodiments, the second therapeutic agent is selected from imatinib (STI-571), imatinib p-diaminomethylbenzene, N-desmethyl imatinib, WGB-BC-15, compound 1, compound 2, LY450139, GSI-953, levoflurbiprofen, and the E2012 compound, blood-brain barrier impermeable variants thereof, and/or pharmaceutically acceptable salts thereof. In certain preferred embodiments, the second therapeutic agent comprises one or more agents selected from the group consisting of: cannabinoids, dimebom, prednisone, ibuprofen, naproxen, indomethacin; statins, selective estrogen receptor molecules, antihypertensive drugs, alpha-blockers, beta-blockers, alpha-beta blockers, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers, diuretics, and antioxidants. In certain particularly preferred embodiments of the methods and compositions described above, the compound comprises imatinib para-diaminomethylbenzene and/or N-desmethyl imatinib or a pharmaceutically acceptable salt thereof.

Brief Description of Drawings

Figure 1A shows a graph comparing the amount of Psen2mRNA in liver samples from subject mice, compared to the genotype of mice at the Psen2 locus.

FIG. 1B shows a graph plotting Psen2 locus genotype (B6/B6 or D2/D2) versus Psen2mRNA concentration in6 tissues (arbitrary units) from up to 89 Recombinant Inbred (RI) lineages. Parental C57 and DBA values were plotted around those from the RI lineage. Some tissues have data from a single RI lineage that is heterozygous at the Psen2 locus: these are indicated by B6/D2. Data were obtained from genenetwork.org (j.wang, r.w.williams, k.f. manly KF, Neuroinformatics 1,299 (2003)). For the liver, expression data was initially expressed as the ratio of the liver fluorescence signal to the signal generated by the reference mRNA sample for each probe. The data is normalized using a robust LOWESS smoothing method that adjusts for signal non-linearity in both channels. The log base 2 (median) of these ratios is then calculated. A value of-1 indicates that expression in the liver is about 1/2 of expression in the control; a value of-2 indicates that expression in the liver is approximately 1/4, etc., of expression in the control. Conversely, a value of +2 indicates that expression is 4-fold greater in the liver than in the liver. A liver dataset from 40 recombinant inbred lines is described by d.gatti et al Hepatology 46,548 (2007). For other tissues, expression values and alternative normalization methods such as indicated (Wang, supra).

FIG. 2 is STI-571, mesylate GLEEVEC^TM) STI-571 variant ("WGB-BC-15"), Compound 1(PD173955, Mosser et al 1999, Cancer Research 59: 6145-; wisniwski et al cancer research2002,62(15):4244-55) and Compound 2(PD 166326; chemical structure of cancer research2002,62(15):4244-55 of Wisniwski et al。

Figures 3A-3F show the effect of peripheral administration of STI-571 on a β levels in plasma and whole brain. Wild type B6 and D2 mice (8-12 weeks of age [ A-F ] or 15-18 months of age [ G, H ]) were administered the drug or vehicle twice daily by intraperitoneal injection for 7 days. Figure 3A shows a western blot showing a β hexamer levels in plasma of young D2 mice treated with saline vehicle (lanes 1,2, 9 and 10) or three doses of STI-571: lanes 3, 4, 11 and 12 show the results using 1 mg/kg; lanes 5, 6, 13 and 14 show the results using 10 mg/kg; and lanes 7, 8, 15 and 16 show the results using 100 mg/kg; n is 4/group. Fig. 3B shows bar chart quantification of the western blot image in fig. 3A. Figure 3C shows a western blot showing Α β hexamer levels in brain extracts of young B6 mice treated with saline vehicle or STI-571 at 20mg/kg (total n 10/group; only n5 is shown in western blot). Fig. 3D shows bar chart quantification of the western blot image in fig. 3C. Fig. 3E and 3F show bar graphs indicating a β hexamer levels in brain extracts (E) or plasma (F) of aged B6 mice (n 4/group) treated with saline vehicle or STI-571 at 20 mg/kg.

Fig. 4 shows a graph comparing the amount of Ngrn mRNA in liver samples from subject mice, compared to the genotype of the mice at the Ngrn locus.

FIG. 5 shows a graph plotting Cib1 (FIG. 5A) or Zfhx1B (FIG. 5B) genotypes (B6/B6, B6/D2, or D2/D2) compared to calnexin-binding protein (FIG. 5A) or Zfhx1B (FIG. 5B) mRNA concentrations in the livers (arbitrary units) of 40 recombinant inbred lines, as in FIG. 1B. Data obtained from genenetwork.org (Wang, supra); the liver dataset is described by Gatti above.

Figure 6 shows a graph comparing the effect of imatinib and desmethyl imatinib on a β concentration in treated cells.

Figure 7 shows a graph comparing the effect of imatinib, imatinib p-diaminomethylbenzene 3HCl, imatinib (pyridine) -N-oxide and imatinib (piperidine) -N-oxide on the concentration of a β in treated cells.

Figure 8 shows a graph comparing the effect of imatinib, desmethyl imatinib, and imatinib on diaminomethylbenzene on Abl kinase activity in a cell-free assay system.

Figure 9 shows a selectivity graph showing the ratio of the fold difference in Α β -lowering activity (compared to imatinib) for each compound to the fold difference in kinase inhibitor activity for this compound at each of the three concentrations shown.

FIGS. 10A-D show the structures of N-desmethyl imatinib, imatinib p-diaminomethylbenzene 3HCl, imatinib (pyridine) -N-oxide, and imatinib (piperidine) -N-oxide, respectively.

Definition of

As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the terms "subject" and "subject" relate to animals, preferably mammals including non-primates (e.g., cows, pigs, horses, donkeys, goats, camels, cats, dogs, guinea pigs, rats, mice, sheep) and primates (e.g., monkeys such as cynomolgus monkeys, gorillas, chimpanzees, and humans), preferably humans. In one embodiment, the subject is a subject with Alzheimer's Disease (AD).

As used herein, the term "a β -related disorder" or "a β disorder" is a disease (e.g., alzheimer's disease) or condition (e.g., senile dementia) that involves abnormal or deregulated a β levels. A β -related disorders include, but are not limited to, AD, brain trauma-related amyloid disorders, down's syndrome, and inclusion body myositis.

As used herein, the term "at risk for disease" refers to a subject (e.g., a human) susceptible to a particular disease. This predisposition may be genetic (e.g., a particular genetic predisposition to experience a disease such as a heritable disorder), or due to other factors (e.g., age, weight, environmental conditions, exposure to harmful compounds present in the environment, etc.). Thus, it is not intended that the present invention be limited to any particular risk, nor that the present invention be limited to any particular disease.

As used herein, the term "suffering from a disease" refers to a subject (e.g., a human) experiencing a particular disease or diagnosed with a particular disease. The present invention is not intended to be limited to any particular sign or symptom, nor to disease. Thus, it is contemplated that the invention encompasses subjects experiencing any range of disease (e.g., from subclinical manifestations to general illness), wherein the subject exhibits at least some signs (e.g., signs and symptoms) associated with a particular disease.

As used herein, the terms "disease" and "pathological condition" are used interchangeably to describe a state, sign and/or symptom associated with any disorder of the normal state of a living animal or any of its organs or tissues that interrupts or defines the performance of normal function, and may be a reaction to environmental factors (such as emotional trauma, physical trauma, nutritional disorders, industrial hazards, or climate), specific infectious agents (such as worms, bacteria, or viruses), inherent defects of an organism (such as various genetic abnormalities, or a combination of these and other factors.

As used herein, the term "subject with AD" or "subject displaying signs or symptoms or condition indicative of AD" or "subject suspected of displaying signs or symptoms or condition indicative of AD" refers to a subject identified or diagnosed as having or likely to have AD based on known AD signs, symptoms and condition.

As used herein, the terms "subject at risk of displaying a pathology indicative of AD" and "subject at risk of AD" refer to a subject identified as at risk of developing AD.

As used herein, the term "AD therapeutic agent" refers to an agent used to treat or prevent AD. Such agents include, but are not limited to, small molecules, drugs, antibodies, pharmaceuticals, and the like.

As used herein, the term "cognitive function" refers generally to the ability to think, reason, concentrate, or remember. Thus, the term "cognitive decline" refers to a diminished ability to think, reason, concentrate, or remember.

As used herein, the terms "modulate", "modulating", "modulated" or "modulation" shall have their ordinary meaning and encompass the meaning of the words "enhance", "promote", "increase", "promotion", "inhibition", "decrease" or "antagonism". For example, a modulator of an enzymatic activity, such as gamma-secretase activity, may act directly, i.e., by interacting directly with an enzyme having an activity to be modulated, or it may act indirectly, i.e., without interacting directly with the enzyme, but via a pathway that results in modulation of the activity.

As used herein, the term "assessing a subject for AD" refers to performing one or more tests to determine, for example, the presence or progression of AD in a subject, or the risk of developing AD in a subject. Assessing a subject for AD and/or distinguishing alzheimer's disease from other causes of memory loss may include assessing one or more of:

1. medical history, including assessment of general health and past medical problems of a subject, problems that a subject may have while performing daily activities

2. Basic medical tests, including, for example, blood tests to rule out other potential causes of dementia, such as thyroid disorders or vitamin deficiencies.

3. Mental state assessment, such as screen memory, problem solving ability, attention span, computational skills and language.

4. Neuropsychological testing, including broader assessment of memory, problem solving ability, attention span, computational skills, and language.

5. Brain scanning or imaging using, for example, computed tomography (CT Magnetic Resonance Imaging (MRI); and Positron Emission Tomography (PET)) to detect visible abnormalities.

As used herein, an "agonist" is any compound that acts directly or indirectly on a molecule to produce a pharmacological effect, while an "antagonist" is any compound that acts directly or indirectly on a molecule to reduce a pharmacological effect.

The terms "sample" and "specimen" are used in their broadest sense and encompass samples or specimens obtained from any source. As used herein, the term "sample" is used to refer to a biological sample obtained from an animal (including a human) and encompasses fluids, solids, tissues and gases. In some embodiments of the invention, the biological sample comprises neural tissue (e.g., brain tissue) cerebrospinal fluid (CSF), serous fluid, urine, saliva, blood, and blood products such as plasma, serum, and the like. However, these examples should not be construed as limiting the type of sample that is suitable for use in the present invention.

As used herein, the term "blood brain barrier" refers to a structure in the Central Nervous System (CNS) that limits the transport of various chemicals and microscopic objects (e.g., bacteria) between the blood and neural tissue. The directional indications of "inside" and "outside" the blood-brain barrier refer to objects that are on the brain/nerve tissue side of the blood-brain barrier, or the non-brain/nerve side of the blood-brain barrier, respectively.

As used herein, the term "blood brain barrier impermeable variant" as used in reference to a material or compound (e.g., a drug) refers to a variant of a compound that has a reduced ability to penetrate the blood brain barrier when administered peripherally to a subject, as compared to the permeability of a parent or reference compound, such that, for example, the variant does not substantially penetrate the blood brain barrier of the subject to which it is administered. As discussed below, the ability of a compound to cross the blood-brain barrier can be characterized by any of a number of methods known in the art, such as by in vivo or in vitro testing, by computational modeling, or by characterizing the compound relative to a feature associated with blood-brain barrier permeability, such as size, charge, and the like (e.g., by physical testing or computational modeling).

Methods to determine or assess brain/CNS uptake of drugs include in vivo methods (e.g., intravenous or carotid injection followed by brain sampling or imaging), in vitro methods using, for example, isolated brain microvasculature or cell culture models, and computational (on-silicon) predictive methods that are generally based on factors such as molecular weight and lipophilicity. See, e.g., u.bickel, neurorx.2005, month 1, incorporated by reference herein; 15-26 to obtain an overview and comparison of methods for measuring drug transport across the blood-brain barrier.

The lipophilicity/hydrophilicity of a compound is generally related to the rate and extent of entry of the compound into the brain. The lipophilicity/hydrophilicity of a drug is often expressed in terms of a partition coefficient, which represents the property of a drug when dissolved in an immiscible organic/aqueous solvent system. The 1-octanol/water partition system has been widely used to assess the ability of compounds to cross the blood-brain barrier. The 1-octanol/water partition coefficient "log P" has long been used as a descriptor of lipophilicity, and computer algorithms that provide calculated log P values, such as Clog P and Mlog P, often closely match experimental measurements (within about 0.3 log units; Bickel, supra). For ionizable molecules, a partition coefficient, i.e. log P value at a defined pH (typically physiological plasma pH of 7.4) is used. If logP and pKa are known, then log D (log partition coefficient) can be obtained using the Henderson-Hasselbalch equation. Log D at pH7.4 is often cited to give an indication of the lipophilicity of a drug at plasma pH.

Hansch and colleagues have determined that drugs with a log P of about 2 are generally accessible to the central nervous system (Hansch et al 1987, J.Pharm.Sci.76(9):663-687, which is incorporated herein by reference) and are more hydrophilic such that drugs with low log P values (e.g., about 1) generally have a reduced ability to access the CNS. This observation has been applied to improving drugs to reduce CNS permeability as a means of controlling, for example, CNS toxicity or side effects. Such as CNS permeability of cardiac drug ARL-57. This drug is considered an excellent cardiotonic, but is not useful for patients because it results in a "bright color vision" in humans. ARL-57 has log P of 2.59 at pH8. A more hydrophilic variant of this material was generated, ARL115, (sumazol); log P1.17 at pH 8; calculated value 1.82) and was found to be free of CNS side effects, demonstrating that altering lipophilicity/hydrophilicity could be used as a means to alter, e.g., reduce, the permeability of drugs to the blood-brain barrier (Hansch, et al, supra).

The partition coefficient (log P) of imatinib mesylate was calculated as 1.198 and 1.267 at 25 ℃ and 37 ℃ respectively (Velpandian et al, Journal of Chromatography B,804(2):431-434 (2004)). This log P value is consistent with the data, showing that imatinib does not substantially penetrate the blood brain barrier.

The terms "peripheral" and "outer periphery" as used in relation to a location in or on a subject's tissue relate to all locations and tissues of the subject outside the blood-brain barrier.

As used herein, the phrase "does not substantially cross the blood brain barrier" or "does not substantially penetrate the blood brain barrier" refers to materials or compounds, such as GLEEVEC imatinib mesylate (STI-571), which, if administered to a peripheral tissue or orally, are either completely absent from a CNS sample (e.g., brain tissue, cerebrospinal fluid) or present in a CNS sample at a lesser percentage of the concentration found in the peripheral tissue, e.g., less than about 10%, preferably less than about 5%, and more preferably less than about 2% of the concentration found in the peripheral tissue. For example, GLEEVEC/STI-571 has poor permeability of the blood brain barrier as demonstrated in STI-571-treated leukemia patients where the cerebrospinal fluid (CSF) level of the drug is 92-fold lower than the level in blood (Takayama N, Sato N, O' Brien SG, Ikeda Y, Okamoto S.Br JHaematol.119: 106-. Thus, GLEEVEC/STI-571 imatinib mesylate does not substantially penetrate the blood brain barrier.

As used herein, the term "effective amount" refers to an amount sufficient to produce a selected effect (e.g., an amount of a composition comprising a modulator of gamma-secretase activity of the invention). An effective amount may be administered in one or more administrations, applications or dosages and is not intended to be limited to a specific formulation or route of administration.

As used herein, an "amount of a compound" is an amount that is at least the minimum amount needed to achieve a predetermined result. These amounts can be routinely determined by one skilled in the art based on data from the study using analytical methods such as those disclosed herein.

As used herein, the term "about" means within 10 to 15%, preferably within 5 to 10%.

As used herein, the terms "control", "controlling" and "management" refer to the beneficial effect that a subject obtains from a compound that does not cause a cure of disease, such as a compound that reduces the level of a β displayed by a cell or tissue. In certain embodiments, one or more such agents are administered to a subject to "control" a disorder in order to prevent or slow the progression or worsening of the disorder.

As used herein, the terms "prevent", "preventing" and "prevention" refer to hindering the recurrence or onset of an a β -related disorder or one or more symptoms of an a β -related disorder in a subject.

As used herein, "protocol" includes dosing schedules and dosing regimens. The protocols herein are methods of use and include prophylactic and therapeutic protocols.

As used herein, the terms "administration" and "administering" refer to the act of administering a drug, prodrug, or other agent, or therapeutic therapy (e.g., a composition of the invention) to a subject (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs). Exemplary routes of administration to the human body can be via the eye (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lung (inhalant), oral mucosa (oral), ear, rectum, vagina, injection (e.g., intravenous, subcutaneous, intratumoral, intraperitoneal, etc.), and the like. By "peripheral administration" is meant any route of administration that is given outside the blood-brain barrier.

As used herein, the terms "co-administration" and "co-administration" refer to the administration of at least two agents (e.g., a composition comprising STI-571, N-desmethyl imatinib, imatinib para-diaminomethylbenzene, and one or more other agents, such as a β -related disease therapeutic agents) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies occurs simultaneously. In other embodiments, the first agent/therapy is administered before the second agent/therapy. One skilled in the art will appreciate that the formulation and/or route of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at a lower dose than the appropriate dose for their separate administration. Thus, co-administration is particularly desirable in embodiments where co-administration of an agent or therapy reduces the necessary dose of a potentially harmful (e.g., toxic) agent, and/or co-administration of two or more agents results in sensitization of a subject to a beneficial effect of one agent via co-administration of another agent.

As used herein, the terms "treat" and "treating" include administering a therapy to prevent, cure, or reduce/prevent symptoms associated with a particular disorder, disease, injury, or condition.

As used herein, the term "treat," "treating," or grammatical equivalents encompasses ameliorating and/or reversing the symptoms of a disease (e.g., an a β -related disease, such as alzheimer's disease). Thus, a compound that results in an improvement in any parameter associated with a disease may be identified as a therapeutic compound when used in the screening methods of the present invention. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. For example, those who may benefit from treatment with the compositions and methods of the invention include those who already have a disease and/or disorder (e.g., an a β -related disease, or a symptom or condition consistent with an a β -related disease) as well as those who the disease and/or disorder is to be prevented (e.g., using the prophylactic therapies of the invention).

The term "compound" refers to any chemical entity, drug, pharmaceutical, etc., that can be used to treat or prevent a disease, condition, illness, or functional condition of the body. As used herein, a compound can be a single composition (e.g., a pure preparation of a chemical) or it can be a composition comprising multiple chemicals (e.g., one or more effective pharmaceutical agents and one or more inert agents). The compounds may include known and potential therapeutic compositions. Compounds can be screened for identification as therapeutic agents using the screening methods of the invention.

However, known therapeutic compounds are not limited to compounds having a particular level of effectiveness in The treatment or prevention of a disease (e.g., a β -related disease) and include, for example, compounds whose data suggest The presence of some beneficial effects and few negative effects (e.g., generally recognized as safe compounds such as food extracts and nutraceutical compounds). examples of known therapeutic agents for treating, ameliorating or reducing The risk or severity of a β -related diseases (e.g., alzheimer's disease) include, but are not limited to, cannabinoids (see, for example, Ramirez et al, The Journal of Neuroscience, february23,2005,25(8): 1904-one 1913), dimebomom (see, for example, RS doedy et al, The atorvastatin Lancet, 372: 207; 2008; anti-inflammatory agents such as prednisone (2008), and statins, including, anti-inflammatory drugs such as ibuprofen, and anti-inflammatory drugs (NSAIDs), such as ibuprofen, and/or anti-inflammatory drugs such as heart protective drugsAnd/or cerivastatinA mixture of fluvastatin (for example,) Mevastatin, pitavastatin (e.g. mevastatin)) And a pharmaceutically acceptable carrier, pravastatin (e.g.,) Rosuvastatin (e.g.,) And simvastatin (for example,) (ii) a Selective Estrogen Receptor Molecules (SERMs), e.g. raloxifeneAntihypertensive agents including α -blockers, β -blockers, α - β blockers, angiotensin converting enzyme inhibitors, angiotensin receptor blockers (ARBs, such as valsartan (e.g.,) Calcium channel blockers and diuretics (see, e.g., I Hajjar et al The journal of Geronology Series A: Biological Sciences and Medical Sciences60:67-73 (2005)); and antioxidants such as garlic extract, curcumin, melatonin, resveratrol, ginkgo biloba extract, green tea, vitamin C and vitamin E (see, e.g., B Frank et al Ann Clin Psychiatry17(4):269-86 (2005)).

As used herein, the term "small molecule" generally refers to a molecule of less than about 10kDa molecular weight, including, but not limited to, natural or synthetic organic or inorganic compounds, peptides, (poly) nucleotides, (oligo) sugars, and the like. Small molecules include, inter alia, smaller non-polymeric (i.e., not peptides or polypeptides) organic and inorganic molecules.

As used herein, the term "extract" and similar terms refer to a process of isolating and/or purifying one or more components from their natural source, or when used as a noun, to a composition produced by such a process.

As used herein, the term "kit" refers to any delivery system for delivering a material. In the case of kinase activity or inhibition assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents and/or support materials (e.g., buffers, written instructions for performing the assay, etc.) from one location to another. For example, a kit includes one or more covers (e.g., cassettes) containing the relevant reaction reagents and/or support materials. As used herein, the term "discrete kit" refers to a delivery system that includes two or more separate containers, each container containing a sub-portion of the total kit components. These containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for the assay, while a second container contains a standard for comparison with the test compound. The term "sporadic kit" is intended to encompass a kit containing an analyte-specific Agent (ASR) administered according to part 520(e) of the Federal Food, Drug, and Cosmetic Act, but is not so limited. In fact, any delivery system comprising two or more separate containers each containing a sub-portion of the total kit components is encompassed by the term "discrete kit". In contrast, a "combination kit" refers to a delivery system that contains all of the components of a reaction assay in a single container (e.g., a single cartridge containing each of the desired components). The term "kit" includes both discrete and combination kits.

As used herein, the term "toxicity" refers to any adverse or detrimental effect on a subject, cell, or tissue as compared to the same cell or tissue prior to administration of the toxicant.

As used herein, the term "pharmaceutically purified" refers to a composition of sufficient purity or quality to be prepared for pharmaceutical use.

As used herein, the term "purifying" refers to treating a starting composition to remove at least one other component (e.g., another component, contaminant, synthetic precursor or byproduct, etc. from the starting composition (e.g., plant or animal tissue, environmental sample, etc.) such that the ratio of purified component to removed component is greater than the ratio in the starting composition.

As used herein, the term "pharmaceutical composition" refers to a combination of an active agent (e.g., a composition comprising a modulator of gamma-secretase activity) and an inert or active carrier, such that the composition is particularly suitable for in vitro, in vivo, or ex vivo diagnostic or therapeutic use.

As used herein, the term "pharmaceutically acceptable" or "pharmacologically acceptable" refers to a composition that does not substantially produce an adverse reaction, such as a toxic, allergic, or immune reaction, when administered to a subject.

As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as oil/water or water/oil emulsions), and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption retardants, disintegrants (e.g., potato starch or sodium starch glycolate), and the like. The composition may also include stabilizers and preservatives. Such as carriers, stabilizers and adjuvants. (see, e.g., Martin, Remington's Pharmaceutical Sciences,15th Ed., Mack Publ. Co., Easton, Pa. (1975), incorporated herein by reference).

As used herein, the term "pharmaceutically acceptable salt" refers to any salt of a compound of the present invention (e.g., obtained by reaction with an acid or base) that is physiologically tolerated in a subject of interest (e.g., a mammalian subject, and/or in vivo or ex vivo, a cell, a tissue, or an organ). "salts" of the compounds of the present invention may be obtained from inorganic or organic acids and bases. Example acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, p-toluenesulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic, and the like. Other acids, such as oxalic, while not per se pharmaceutically acceptable, may be used to prepare salts which may be used as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and NW₄ ⁺A compound wherein W is C_1-4Alkyl groups, and the like.

Examples of salts include, but are not limited to: acetates, adipates, alginates, aspartates, benzoates, benzenesulfonates, bisulfates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, chlorides, bromides, iodides, 2-hydroxyethanesulfonates, lactates, maleates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, oxalates, palmitates, pectinates, persulfates, phenylpropionates, picrates, pivalates, propionates, succinates, tartrates, thiocyanates, tosylates, undecanoates, and the like. Other examples of salts include salts with suitable cations such as Na⁺、NH₄ ⁺And NW₄ ⁺(wherein W is C_1-4Alkyl) and the like. For therapeutic use, salts of the compounds of the present invention are expected to be pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable acids and bases may also be suitable, for example, for the preparation or purification of pharmaceutically acceptable compounds.

For therapeutic use, salts of the compounds of the present invention are expected to be pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable acids and bases may also be suitable, for example, for the preparation or purification of pharmaceutically acceptable compounds. In some embodiments of the invention, the pharmaceutical composition comprises a form selected from the group consisting of: powder, solution, emulsion, micelle, liposome, gel, and paste forms. In some embodiments, the pharmaceutical composition comprises a tablet or a filled capsule, wherein the tablet or filled capsule optionally comprises an enteric coating material.

As used herein, the term "excipient" refers to an inactive ingredient (i.e., not pharmaceutically active) added to the active ingredient formulation.

The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences required for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). A polypeptide can be encoded by a full-length coding sequence or any portion of a coding sequence, so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length or fragment are retained. The term also encompasses the coding region of the structural gene as well as sequences located adjacent to the coding region on the 5 'and 3' ends, up to a distance of about 1kb or more on either end, such that the gene corresponds to the length of the full-length mRNA. Sequences located 5 'to the coding region and present on the mRNA are referred to as 5' untranslated sequences. Sequences located 3 'or downstream of the coding region and present on the mRNA are referred to as 3' untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. Genomic forms or clones of a gene contain coding regions interrupted by non-coding sequences called "introns" or "insertion regions" or "insertion sequences". Introns are gene segments transcribed into nuclear rna (hnrna); introns may contain regulatory elements such as enhancers. Introns are removed or "clipped" from the nucleus or original transcript; thus, introns are not present in messenger rna (mrna) transcripts. The mRNA functions during translation to specify the sequence or order of amino acids in the nascent polypeptide.

As used herein, the terms "gene expression" and "expression" refer to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) via gene "transcription" (i.e., via the enzymatic action of an RNA polymerase), and for protein-encoding genes, into protein via mRNA "translation". Gene expression can be regulated at many stages in the process. "Up-regulation" or "activation" refers to regulation that increases and/or enhances production of a gene expression product (e.g., RNA or protein), while "down-regulation" or "inhibition" refers to regulation that decreases production. Molecules involved in up-regulation or down-regulation (e.g., transcription factors) are commonly referred to as "activators" and "inhibitors", respectively.

In addition to containing introns, genomic forms of a gene may also include sequences located on the 5 'and 3' terminal sequences present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5 'or 3' to the untranslated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct transcription termination, post-transcriptional cleavage, and polyadenylation.

The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. Wild-type genes are the genes most frequently observed in a population, and are therefore arbitrarily designed as "normal" or "wild-type" versions of the gene. Conversely, the term "modification" or "mutant" refers to a gene or gene product that exhibits a change in sequence and or functional properties (i.e., altered characteristics) as compared to the wild-type gene or gene product. It should be noted that naturally occurring mutants can be isolated; these mutants are identified by the fact that: it has altered properties (including altered nucleic acid sequence) compared to the wild-type gene or gene product.

As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the amino acid sequence along the polypeptide (protein) chain. Thus, the DNA sequence encodes an amino acid sequence.

The term "eukaryote" as used refers to an organism that is distinguishable from "prokaryotes". This term is intended to encompass all organisms whose cells exhibit the usual characteristics of eukaryotes, such as the presence of a true nucleus defined by the nuclear membrane within which the chromosomes are disposed, the presence of membrane-bound organelles, and other characteristics commonly observed in eukaryotic organisms. Thus, this term includes, but is not limited to, organisms such as fungi, protozoa, and animals (e.g., humans).

As used herein, the term "in vitro" refers to an artificial environment and processes or reactions occurring within an artificial environment. The in vitro environment may consist of, but is not limited to, test tubes and cell cultures. The term "in vivo" refers to the natural environment (e.g., an animal or cell) and processes or reactions occurring within the natural environment.

The terms "test compound" and "candidate compound" refer to any chemical entity, drug, pharmaceutical, etc., that is candidate for use in treating or preventing a disease, condition, disease, or a functional disorder of the body (e.g., cognitive function, amyloid-related disorders, circulation, hypertension, heart disease, etc.). Test compounds include known and potential therapeutic compounds. Test compounds can be screened using the screening methods of the invention for identification as therapeutic agents.

As used herein, a "functional" molecule is a molecule in a form in which it exhibits the property it characterizes. For example, a functional enzyme is an enzyme that exhibits a characteristic catalytic activity by which the enzyme is characterized.

As used herein, the term "antisense oligonucleotide" refers to a nucleic acid, e.g., an RNA or DNA segment, that is complementary to a sequence of a target RNA (or fragment thereof). Typically, the target RNA is mRNA expressed by the cell.

As used herein, the term "interfering oligonucleotide" refers to an oligonucleotide capable of inhibiting the function of a target gene product, regardless of the mechanism of inhibition. As used herein, interfering oligonucleotides, including but not limited to antisense oligonucleotides, aptamers, small RNA molecules (mirnas), short interfering RNAs (sirnas), and short hairpin RNAs (shrnas), short interfering RNAs generally consist of double stranded RNA molecules of 19-22nt in total, while short hairpin RNAs consist of palindromic sequences linked by a circulating sequence of 19-29nt in total. Methods for generating interfering oligonucleotides are well known to those skilled in the art and include, but are not limited to, chemical synthesis, recombinant DNA techniques, or cleavage using enzymes, e.g., by Dicer enzymes, from larger precursor molecules.

As used herein, the term "antibody" refers to an immunoglobulin or immunoglobulin-derived protein that includes an antigen recognition site. Antibodies include, but are not limited to, natural or recombinant immunoglobulins comprising two heavy chains and two light chains, as well as modified forms comprising different combinations of a portion of a heavy chain and a light chain, including, for example, fragment antibodies and single chain antibodies. This term includes polyclonal and monoclonal antibodies.

As used herein, the term "kinase-inhibiting imatinib derivative is intended to refer to imatinib-related compounds having reduced protein kinase activity as compared to imatinib, e.g., imatinib para-diaminomethylbenzene and N-desmethyl imatinib. These imatinib derivatives are not necessarily obtained from imatinib as a starting material, and this term encompasses variants of imatinib produced, for example, by chemical synthesis.

Detailed Description

Specific embodiments of the present invention are described in this detailed description and summary of the invention, which is incorporated herein by reference. While the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. For example, the methods and compositions of the invention are described in connection with specific modulators of gamma-secretase activity, such as GLEEVEC (STI-571) imatinib mesylate, and specific cerebral amyloid disorders, such as alzheimer's disease. It is to be understood that the present invention is not limited to methods or compositions using or including imatinib mesylate, or AD. The present invention relates to the use of a kinase-inhibiting imatinib derivative for the treatment of an a β -related disorder.

The present invention is based, in part, on applicants' unexpected discovery: modulation of a β expression or accumulation in peripheral tissues such as the liver provides therapeutic effects in a β -related diseases of the brain such as alzheimer's disease. Accordingly, the present invention relates generally to methods and compositions for preventing or treating brain a β -related disorders, such as AD, via administration of compounds that modulate a β production and/or accumulation in non-neural (i.e., peripheral) cells, fluids, and/or tissues.

As discussed above, amyloid beta (a β) peptide is a metabolite of Amyloid Precursor Protein (APP) and is considered to be a major pathological determinant of Alzheimer's Disease (AD). APP is proteolyzed by beta and gamma-secretases to produce a β peptides, with a β in the form of 42 residues being considered the most pathogenic. Beta-secretase is required for healthy brain function and is therefore a poor candidate for inhibition in order to reduce a β. Many brain permeable gamma-secretase inhibitors exhibit undesirable side effects due to interference with gamma-secretase action on other targets, particularly Notch family transmembrane receptors. One class of compounds has been found to reduce a β production without affecting Notch signaling. Such compounds include the tyrosine kinase inhibitor imatinib mesylate (STI-571, trade name GLEEVEC) and related compounds, 6- (2, 6-dichlorophenyl) -8-methyl-2- (methylthiophenyl-amino) -8H-pyrido [2,3-d ] pyrimidin-7-one, which is known as inhibitor 2(Netzer WJ et al ProcNatl Acad Sci U S A.100: 12444-. However, such compounds are no longer considered as a therapy for brain Α β disorders because they do not cross the blood brain barrier and are therefore very difficult to deliver to brain tissue.

As mentioned above, we have found that modulating a β production or accumulation in peripheral tissues such as the liver provides therapeutic effects in a β -related diseases of the brain such as alzheimer's disease. The present invention provides methods, compositions, and processes relating to treating or preventing AD by treating the liver of a subject. In particular, the invention relates to altering a β production, processing, accumulation or trafficking in the liver of a subject by directly inhibiting production (e.g., by inhibiting APP expression) or by modulating a factor, thereby modulating a β production, processing, accumulation or trafficking in the liver. In a preferred embodiment, inhibition is achieved via the use of a compound that does not substantially cross the blood brain barrier. In a particularly preferred embodiment, the compositions and methods for treatment comprise the use of STI-571, or a pharmaceutically acceptable salt thereof, administered peripherally, e.g., orally. In a further especially preferred embodiment, the compositions and methods for treatment comprise the use of a reduced kinase inhibition imatinib derivative or a pharmaceutically acceptable salt thereof administered peripherally, e.g., orally. In a still further preferred embodiment, the imatinib derivative is selected from the group consisting of: n-demethyl imatinib and imatinib p-diaminobenzene compositions such as the trihydrochloride salt.

Use of a composition in the manufacture of a medicament

Imatinib is the general name [ international non-proprietary name ] for the compound 4- (4-methylpiperazin-1-ylmethyl) -N- [ 4-methyl-3- (4-pyridin-3-yl) pyrimidin-2-ylamino) phenyl ] -benzamide of formula I:

STI-571 refers generally to imatinib mesylate and has been approved for the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors. The use of imatinib for the treatment of breast cancer is described in WO 2004/032925. Imatinib, its manufacture, pharmaceutically acceptable salts such as acid addition salts, and its protein kinase inhibitory properties are described in U.S. patent No. 5,521,184, which is incorporated herein by reference. "imatinib" corresponds to 4- (4-methylpiperazin-1-ylmethyl) -N [ 4-methyl-3- (4-pyridin-3-yl) pyrimidin-2-ylamino) phenyl ] -benzamide in the form of the free base or the mesylate salt. The preparation of imatinib and its use are described in example 21 of European patent application EP-A-O564409, which is incorporated herein by reference.

N-demethyl imatinib, also known as an N-demethylated piperazine derivative of imatinib, is an active metabolite of imatinib having the structure shown in FIG. 10A.

Imatinib p-diaminomethylbenzene is a variant having the structure shown in figure 10B.

Although peripheral administration is not limited to any particular route of administration, in some preferred embodiments administration is oral. Thus, in some preferred embodiments, the invention includes the use of STI-571 and/or a kinase inhibitory imatinib derivative for the preparation of an orally administered medicament for the treatment or prevention of a brain Α β disorder. In some embodiments, the oral administration form comprises a tablet, and in some embodiments, the oral administration form comprises a capsule.

In a preferred embodiment, the invention comprises preparing a tablet or capsule comprising an effective amount of imatinib and/or a kinase inhibitory imatinib derivative to reduce a β levels in the brain. For example, a capsule or tablet may include 100 to 1000mg of active agent (e.g., imatinib or a derivative thereof). For example, a tablet or capsule may include 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000mg, or any convenient dose therebetween (e.g., 125mg, 150mg, 175mg, 225mg, 250mg.. 975mg, etc.). In some embodiments, the tablet or capsule is configured to contain a smaller effective dose of imatinib or a kinase inhibitory imatinib derivative, e.g., 1 to 5mg (e.g., 1,2, 3, 4, or 5mg, or a convenient fractional amount thereof), 6 to 10mg, 11 to 15mg, and the like.

Compositions and formulations for oral administration include, for example, powders or granules, suspensions or solutions in water or non-aqueous media, capsules, wafers in sachets, dissolvable strips and tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In preferred embodiments, the tablet or capsule (or other form of peripheral administration) is configured to deliver a dose or equivalent amount of any whole integer mg amount between 1 and 1000mg (e.g., 1,2, 3, 4,5, etc.), or any fractional mg amount between 1 and 1000 mg. In certain embodiments, the formulation may include, for example, a capsule filled with a mixture of the compositions.

In some embodiments, the capsule or tablet comprises an enteric coating. "enteric" refers to the small intestine, and thus "enteric coating" generally refers to a coating that substantially prevents release of the drug until it reaches the small intestine. While not limiting the present invention to any particular mechanism of action, it is understood that most enteric coatings function by presenting a surface that is stable at acidic pH but rapidly decomposes at higher pH.

Compositions and formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical formulations of the invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of associating the active ingredient with a pharmaceutical carrier or excipient. In general, the process for preparing the formulations is carried out by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The pharmacokinetics of imatinib mesylate (GLEEVEC) have been evaluated in studies with healthy subjects as well as in population pharmacokinetic studies. Imatinib is completely absorbed after oral administration, C_{Maximum of}Obtained 2-4 hours after administration. The average absolute bioavailability was 98%. After oral administration in healthy volunteers, the elimination half-lives of imatinib and its major active metabolite N-demethyl derivative are approximately 18 and 40 hours, respectively. The mean imatinib AUC (area under the plasma drug concentration versus time curve) increases proportionally with increasing dose in the range of 25mg to 1000 mg. The pharmacokinetics of imatinib did not change significantly after repeated dosing and accumulated 1.5-2.5 fold at steady state upon once daily dosing. At clinically relevant concentrations of imatinib, in vivoBinding to plasma proteins in the in vitro experiments was approximately 95%, binding primarily to albumin and α 1-acid glycoprotein see, e.g., "Gleevec Prescription Information"2003revision T2003-09; Printed in U.S. A.89019001(Novartis).

CYP3a4 is the major enzyme responsible for metabolizing imatinib. Other cytochrome P450 enzymes, such as CYP1a2, CYP2D6, CYP2C9 and CYP2C19, play secondary roles in their metabolism. The major circulating active metabolite in the human body is the N-demethylated piperazine derivative, N-demethylated imatinib, formed primarily by CYP3a 4. It exhibits in vitro potency similar to the parent imatinib. The plasma AUC for this metabolite is about 15% of the AUC for imatinib.

Elimination is mainly in the feces, mostly in the form of metabolites. Based on compound recovery after oral administration of the 14C-labeled dose of imatinib, approximately 81% of the dose was eliminated in feces (68% dose) and urine (13% dose) within 7 days. Unchanged imatinib accounted for 25% of the dose (5% urine, 20% feces), with the remainder being metabolites.

Typically, the clearance of imatinib in 50-year-old patients weighing 50kg is expected to be 8L/h, whereas for 50-year-old patients weighing 100kg, the clearance increases to 14L/h. However, the interpatient variability of clearance of 40% does not justify the need to adjust the initial dose based on body weight and/or age, but indicates the need to closely monitor treatment-related toxicity.

As in adult patients, imatinib is reported to be rapidly absorbed following oral administration in pediatric patients, and C_{Maximum of}For 2-4 hours. Apparent oral clearance was similar to adult values (11.0L/hr/m 2 in children versus 10.0L/hr/m2 in adults), as was half-life (14.8 hours in children versus 17.1 hours in adults). Pediatric administration of 260mg/m2 and 340mg/m2 achieved AUC similar to 400-mg doses in adults. Comparison of AUC (0-24) at the dose levels of 260mg/m2 and 340mg/m2 at day 8 compared to day 1 after repeated once daily dosing showed 1.5 and 2.2 fold drug accumulation, respectively. The mean imatinib AUC did not scale up with dose escalationAnd (4) adding. "Gleevec Presscrinbinginformation" 2003 review T2003-09; printed in U.S. A.89019001(Novartis).

Although modulation of a β production in the liver by treatment with imatinib is used as an example above, the invention is not limited to treatment of the liver with this compound and provides a general method of treating a subject for a brain a β disorder or a predisposition to a brain a β disorder in a subject comprising peripherally administering a compound that modulates expression of a gene in a peripheral tissue of the subject. In a preferred embodiment, modulating said expression of said gene results in modulation of a β production or accumulation in said peripheral tissue. In certain preferred embodiments, the peripheral tissue is the liver of the subject.

In a particularly preferred embodiment, modulation of a β production comprises the use of a composition having reduced protein kinase inhibitory activity compared to, for example, imatinib.

As described in example 3 below, the compositions provided herein inhibit the formation of a β while exhibiting substantially reduced protein kinase inhibition as compared to imatinib. In particular, the present invention provides formulations of imatinib para-diaminomethylbenzene and/or N-desmethyl imatinib for use in reducing a β loading in treated cells and subjects.

The present invention encompasses any method of affecting a β production in the liver, including but not limited to altering APP expression and/or processing. In some embodiments, the invention provides methods comprising peripherally administering a compound that modulates the expression of one or more of Psen1, Apo E, inst 3R, Psen2, APP, Cib1, Ngrn, Zfhx1b, CLU (also known as ApoJ), PICALM, IDE, GSAP, and CR1 genes. In some embodiments, the methods of the invention comprise peripherally administering a compound that modulates the activity of one or more of: presenilin 2, calintegrin binding protein, synaptic growth related protein, Zfhx1b, clusterin, phosphoinositide-binding clathrin assembly protein, complement component receptor 1, insulin degrading enzyme, GSAP, or APP expression or activity. In some embodiments, one or more of these genes or activities are modulated in the liver of the subject. In some embodiments, modulating comprises inhibiting expression or activity, and in some embodiments, modulating comprises stimulating expression or activity.

Evaluation and monitoring of brain a β disorders during peripheral treatment

The present invention relates to testing and treating AD and AD risk by testing and administering to peripheral (i.e., non-brain) tissue of a subject. As discussed below, the present study demonstrates that presenilin 2 expression in the liver and/or one or more peripheral tissues can alter a β accumulation, and that a decrease in peripheral a β is sufficient to alter its deposition in the brain. Thus, despite extensive counter-teaching in the literature, effective therapeutic or prophylactic treatment for AD that reduces a β accumulation does not require crossing the blood-brain barrier and entering the brain. Inhibition of Psen2 or gamma-secretase activity outside the central nervous system (i.e., outside the blood-brain barrier), or by other means to reduce a β production or accumulation is useful in preventing the brain from developing a β -related conditions. Treating peripheral tissues has the added benefit of protecting the brain from any adverse side effects that may occur if a therapeutic agent enters the brain.

In some embodiments, the present invention provides methods of adapting a therapy to a biochemical state of a subject or patient. It is contemplated that the characteristics of the effective dose of one or more compounds selected for modulation of a β in peripheral tissues may be influenced by the particular biochemical condition of the subject or patient, including but not limited to the presence of other drugs or agents (e.g., for treating a β disorders or unrelated conditions), or biochemical changes resulting from other conditions. The methods provided herein comprise monitoring a subject for assessment of a brain a β disorder or progression of a brain a β disorder before and after administration of a compound that modulates a β production, e.g., in the liver. In some embodiments, a therapy for a brain a β disorder is selected, adjusted, or altered accordingly.

Experimental examples

The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting the scope thereof.

Example 1

Identifying modified genes that are afflicted with an AD-like pathology

Transgenic mouse models have been developed that recapitulate key features of human alzheimer's disease. The APP gene carrying some of the mutations that induce AD in humans is linked to various transcriptional promoters and introduced into the mouse germline (Games D, Adams D, Alessandrini R, Barbour R, Berthelette P, Blackwell C, Carr T, Clemens J, Donaldson T, Gillespie F, et al Nature373: 523-527; Hsia AY, Masliah E, Mclogue L, Yu GQ, Tatsuno G, Hu K, Kholodenko D, Malenka RC, Nicoll RA, Mucke L. Proc Natl Acad Sci US A.96: 8-3233, 1999; Hsiao K, Chapman P, Nilsen S, Kkman C, Hariglayya Y, Ynhakus S, Yang F, Style G.32233, Calkuki K, Junkuki D.92, Junkuki D.D, Geckhol K, Gecko K, Calkuki D.83, Junkuki D, Gecko K D, Geckii K, Gecko K21, Geckii K, Gecko K21K, Gecko K D80, Gecko K, dewachter I, LorentK, Revers WeD, Baekeladt V, Naidu A, Tesseur I, Spittarels K, Haute CV, Checler F, Godaux E, Cordell B, Van Leuven F.J Biol chem.274: 6483-; richardson JC, KendalCE, Anderson R, Priest F, Gower E, Soden P, Gray R, Topps S, Howlett DR, Lavender D, Clarke NJ, Barnes JC, Haworth R, Stewart MG, Rupniak HT. neuroscience122:213-228, 2003; buttini M, Yu GQ, Shockley K, Huang Y, Jones B, Masliah E, Mallory M, Yeo T, Longo FM, Mucke L.J Neurosci.22: 10539-. The resulting transgenic mice developed a β deposits, but the time varied between 3 and 15 months of age. Variables contributing to these age differences include the particular transcriptional promoter selected, the particular AD-inducing mutation in the APP gene, the chromosomal site of transgene integration, and the background strain of mice in which the transgene is maintained (reviewed in Bloom FE, Reilly JF, Redwine JM, Wu CC, Young WG, Morrison JH. Arch Neurol.62:185-187, 2005).

A report (Kulnane LS, Lamb BT. Neurobiol Dis.8:982-992,2001) introduced R1.40, a human APP transgene carrying a so-called Swedish mutation (K670N, M671L, making it easier for those who inherit this mutant gene to develop early-onset AD variations), into the mixed C57Bl/6x129/Sv mouse genetic background. Expression of the R1.40 transgene is driven by the native human APP promoter. A β deposits were first detectable in the brains of these mice at months 14-16. Subsequently, the R1.40 transgene was individually crossed from its original background into the C57Bl/6(B6), DBA/2(D2) and 129/Sv backgrounds. Each of these 3 strains was then propagated to homogeneity: 10 or more strains were backcrossed into the same background to allow the generation of 3 transgenic lines with uniform but different backgrounds (Lehman EJ, Kulnane LS, Gao Y, Petriello MC, Pimpis KM, Younkin L, Dolios G, Wang R, Younkin SG, Lamb BT. hum MolGenet.12: 2949-. Although all three transgenic lines produced the same amount of APP precursor (indicating that the transgene was equally expressed in the 3 line background), B6 accumulated more a β (pathogenic fragment of APP) on days 21 and 60 compared to the other 2 lines as measured by ELISA for brain homogenate and plasma, and at 13.5 months amyloid deposits characteristic of human AD appeared, while D2 was protected (no deposition within 2 years). Thus, this indicates the presence of genes that distinguish B6 from D2 mice, and that these genes alter the development of AD-like pathology, and that it is likely that these genes are involved in the accumulation of the causative agent, Abeta (Lehman EJ, Kulnane LS, Gao Y, Petriello MC, Pimpis KM, Younkin L, Dolios G, Wang R, Younkin SG, Lamb BT. hum Mol Genet.12: 2949-. The identity of the modifier gene may suggest therapeutic or prophylactic patterns that mimic the effect of the modifier gene and delay or prevent the development of AD pathology.

To assign the modifier genes to the chromosomal interval, Ryman and colleagues (Ryman D, Gao Y, Lambtb. neurobiol Aging29: 1190. sup. 1198,2008) crossed female B6R1.40 mice (homozygous for the transgene) with male D2R1.40 mice (also homozygous for the transgene) and then crossed their F1 progeny (all having 2 copies of the R1.40 transgene) with non-transgenic B6x D2F1 progeny, resulting in 516F2 mice, each carrying a single transgene. These transgenes were genotyped with 909 SNP. In brain homogenates from 516 mice, a β was measured by ELISA. Regression analysis correlating Α β accumulation amounts to genotype of 516 mice allowed assignment of 3 modifying loci to broad regions concentrated at the following positions: chromosome 1, 182.049374 megabases (Mb); chromosome 2, 41.216315 Mb; chromosome 7, 63.680922 Mb.

Determination of modified genes

The mouse gene encoding presenilin 2, Psen2, is located at 182.06371 megabases on chromosome 1, centered between trait loci, suggesting that it is a candidate gene for altering a β accumulation and deposition. This is consistent with its function as a component of gamma-secretase. In order for Psen2 to represent the actual modified gene located on chromosome 1 by Ryman and colleagues, its activity must be genetically variable (in mendelian fashion) between the B6 and D2 mouse strains, and Psen2 activity must be greater in B6 mice compared to D2 mice, since lower γ -secretase activity is expected to be protective in AD. We investigated this problem by determining the amount of mRNA accumulated by the Psen2 gene in various tissues of B6 and D2 mouse strains and up to 89 strains of recombinant inbred mice, which were generated by crossing B6 and D2 mice and feeding the progeny to homogeneity. The concentration of each of over 20,000 mRNAs in 10 tissues (brain, cerebellum, liver, striatum, kidney, hippocampus, eye, prefrontal cortex, nucleus accumbens and neocortex) of B6 and D2 mouse strains and 89 recombinant inbred mouse strains was obtained in a public database compiled with http:// www.GeneNetwork.org. For each of the 89 recombinant inbred mouse strains, it has been determined by genotyping whether this strain inherits every interval of its genome from the B6 or D2 parents.

Probe rs13476267 is located at 182.120454Mb of chromosome 1. Using software on the world wide web public site of genenetwork.org/webqtl.py, we performed a trait association between genotypes at the rs13476267 interval and the amount of Psen2mRNA accumulated in each of 10 tissues in up to 89 recombinant inbred mice by calculating Pearson's product-moment. These values are:

none of the tissue samples obtained from the brain exhibited high inheritance of Psen2 expression (| r | >0.9), and for the two brain regions, cerebellum and nucleus accumbens, exhibiting limited inheritance of Psen2mRNA expression, more Psen2mRNA was associated with the D2 genotype than with the B6 genotype. Thus, Psen2 expression in the brain is not an altering factor for a β accumulation. However, in the liver, the amount of Psen2mRNA was highly correlated with the genotype of the Psen2 locus (fig. 1A). In addition, B6 mice expressed more Psen2mRNA than D2 mice.

The data demonstrate that expression of Psen2 in the liver and/or one or more peripheral tissues can alter a β accumulation, and that a decrease in peripheral a β is sufficient to alter its deposition in the brain. Thus, despite the broad contrary teachings in the literature, effective therapeutic or prophylactic treatment for AD that reduces a β accumulation does not require crossing the blood-brain barrier and entering the brain, based at least in part on the natural hypothesis that brain disease is caused by events occurring in the brain. Inhibition of Psen2 or gamma-secretase activity outside the central nervous system, or by other means to reduce a β production or accumulation is sufficient to prevent the brain from developing a β deposits, while preventing the brain from any adverse side effects that may occur if therapeutic agents enter the brain. Treatment of peripheral a β accumulation may be accomplished using drug delivery routes that do not include direct application to the CNS (e.g., via CSF delivery), such as via oral administration.

Example 2

Peripheral administration of STI-571 imatinib mesylate to reduce A β in brain

Data from mapping studies and our further ideas suggest novel therapeutic approaches to the treatment of AD (its onset, progression or severity) based on modulating Α β production in the liver. The basis of the new therapeutic strategy is that agents that reduce the steady-state level of a β in the blood (by inhibiting the production of a β in the liver) will reduce the a β concentration in the brain.

Experiments were designed to test the effect of STI-571 imatinib mesylate administration on the a β protein levels of brain and blood tissues of 2 mouse strains. STI-571 imatinib mesylate was administered to mice over the course of one week by IP injection, and brain and tissue samples were removed and Α β protein levels were measured by ELISA or western blot.

Male wild-type C57Bl/6 and DBA/2J mice (8-12 weeks old) were administered the drug or vehicle by intraperitoneal injection twice daily for 7 days. The vehicle group (each strain, n ═ 4 animals) was injected with 100ul saline and the pharmacotherapy group (n ═ 4) received 1, 10 or 100mg/kg of sti-571(GLEEVEC imatinib mesylate, catalog No. I-5508, lclaborides, Woburn, MA). The prescribed dose of STI-571 for human cancer patients is between 100mg and 1000 mg. See, e.g., Gleevec descriptive Information2003 review T2003-09; printed inu.s.a.89019001(Novartis), which is incorporated herein by reference.

Animals were sacrificed 12 hours after the last injection. Individual mice were anesthetized with isoflurane and blood samples (100-. The samples were placed on ice in the presence of EDTA for 30 minutes and then centrifuged at 16,000Xg for 20 minutes at 4 ℃. The plasma fractions were removed and stored at-80 ℃. The brain was removed and snap frozen on dry ice and stored at-80 ℃.

Mouse A β in blood and brain samples_1-40Detection of (D) Using a commercially available immunoassay kit (Biosource mouse A β)_1-40Catalog number KMB3481, Invitrogen, Carlsbad, CA) or western blot. Mouse brain samples are prepared by homogenizing brain tissue in the presence of 5M guanidine HCl and 50mM Tris HCl, pH8.0 in a polylaurone (polytron), e.g.(see, e.g., Masliah, E. et al, (2001) β analog peptide expression α -synuclein accumulation and neural derivatives in a transgenic animal linking the animal's disease and Parkinson's disease PNAS 98: 12245-12250; Johnson-Wood, K, et al (1997) analog peptide expression processing and Abeta42 expression in a transgenic animal model of animal disease PNAS94: 1550-1555; and Chishiti, M.A. 2001; Early-analog precipitation and chemical peptide expression 21562. J. for example Biochemical expression 215276

For the assay, brain homogenates were diluted 1:10 in reaction buffer containing Dulbecco's phosphate buffered saline with 5% BSA and 0.03% Tween-20 supplemented with a protease inhibitor cocktail (Cat. No. 539131, EMD Biosciences, La Jolla, Calif.). Blood samples were diluted 1:5 in standard diluent buffer. The sample was assayed in duplicate and OD450 was measured on a Tecan infinite2000 microplate reader.

Oligomeric a β was extracted in SDS fractions, essentially as described (t. kawarabayashi et al, Neurosci 21,372 (2001)). For western blotting, samples were subjected to PAGE analysis, transferred to PVDF membrane and A β hexamers were observed using monoclonal antibody 4G8(1:1,000; Covance) against mouse A β using the manufacturer's suggested protocol. The blot was scanned by densitometry and then probed again with an antibody to histone H3 (1:50,000; Abcam) as a loading and transfer control. Data are depicted as normalized optical density.

The A β levels in brain and blood were different between the two mouse strains tested (C57Bl/6 and DBA/2J). In the vehicle-treated control group, a β levels were higher in brain and blood samples from C57Bl/6 mice compared to DBA/2J, as previously shown.

Figure 3 shows the effect of peripherally administered STI-571 on a β levels in plasma and brain. Figure 3A shows a western blot showing a β hexamer levels in plasma of young D2 mice treated with saline vehicle (lanes 1,2, 9 and 10) or three doses of STI-571: lanes 3, 4, 11 and 12 show the results using 1 mg/kg; lanes 5, 6, 13 and 14 show the results using 10 mg/kg; and lanes 7, 8, 15 and 16 show the results using 100 mg/kg; n is 4/group. Fig. 3B shows bar chart quantification of the western blot image in fig. 3A. Figure 3C shows a western blot showing Α β hexamer levels in brain extracts of young B6 mice treated with saline vehicle or STI-571 at 20mg/kg (total n 10/group; only n5 is shown in western blot). Fig. 3D shows bar chart quantification of the western blot image in fig. 3C. Fig. 3E and 3F show bar graphs indicating a β hexamer levels in brain extracts (E) or plasma (F) of aged B6 mice (n 4/group) treated with saline vehicle or STI-571 at 20 mg/kg.

A dose-dependent reduction in plasma a β was observed (fig. 3A-B), and the highest dose reduced circulating a β by approximately 75%. The middle dose, 20mg/kg, was chosen to study brain effects. In young and old B mice, this dose reduced brain and plasma levels of a β by approximately 50% (fig. 3B and 3C). These levels of a β were observed to be protective in the R1.40 mouse model (e.j. lehman et al, Hum Mol genet12,2949 (2003)).

These results demonstrate that short-term (one week) treatment with STI-571 imatinib mesylate significantly reduced a β levels in the blood and brain. Furthermore, since the drug did not significantly cross the blood brain barrier at the concentrations used in this study, the results indicate that STI-571 imatinib mesylate may indirectly alter brain Α β levels by modulating Α β production peripherally.

Example 3

Identification of candidate chromosome 2 and 7 modifier genes

The studies described above demonstrate that pathogenic a β may be from the liver. Using the same database and method as described above, we also searched for genes that mapped the interval between chromosomes 2 and 7, and whose activity in the liver was genetically variable between the B6 and D2 mouse strains.

Marker rs4226715 is located at 80.138616Mb of chromosome 7, within the modifying locus of said chromosome. Two genes from this interval show very high inheritance of intrahepatic expression: the Ngrn gene, and the Cib1 gene. The Ngrn gene encodes a synaptogenesis-associated protein, a widely expressed protein of unknown function, whose expression is increased in some cancers and associated with neuroblastoma differentiation (S.Ishigaki et al, Biochem Biophys Res Commun279,526(2002), S.R.Hustinx et al, Cancer Biol Ther 3,1254(2004)), and the Cib1 gene encodes a calcium integrin-binding protein, which is a myristoylation calcium and integrin-binding membrane-associated protein, which was originally found due to its preferential interaction with presenilin 2 in HeLa cells (S.M.Stabler et al, J Cell Biol14,145,1277 (1999)). These genes showed the highest correlation: the corresponding pearson value r is 0.945 and r is-0.913, p <4.99e-39, (fig. 5 and 4, respectively). Ngrn is located at 80.138736Mb of chromosome 7 and Cib1 is located at 80.101507, which is consistent with the enantiomodified locus.

As mentioned above, the calcium integrin binding protein has a proven interaction with presenilin 2. However, since the distribution of calnexin-binding proteins in the brain is not fully correlated with the distribution of brain presenilins or the areas most susceptible to AD pathology, previous studies suggest that they have potential effects in contributing to a β production in the forebrain, but judging such effects to be impossible (m.blazejczyk et al, Biochim biophysis Acta1762,66 (2006)). However, calintegrin binding protein is highly expressed by the liver (s.m. stabler, supra). One suggested activity of the calintegrin-binding protein is as a protein ligand for the inositol 1,4, 5-triphosphate receptor Ca (2+) release channel (c.white et al, J Biol chem281,20825 (2006)), which is abnormal in chick cells whose gating activity is transfected with a mutant presenilin gene (k.h. cheung et al, neuron58,871 (2008)).

The heritability of hepatic calcium integrin binding protein mRNA expression is very high. In each line that inherited its Cib1 gene from the B6 parent, the amount of calintegrin binding protein mRNA was higher compared to that observed in the line that inherited its Cib1 gene from the D2 parent (fig. 5A). One line (lineage 73) appeared to be heterozygous at the probe, but expressed the same amount of calnexin-binding protein mRNA as D2. This suggests that low-calintegrin binding protein expression in the liver reduces a β accumulation in the brain and protects mice from its adverse effects.

Treatment with compounds that reduce the abeta potentiating activity of the calnexin-binding protein mimics the low expression of the D2 genotype and is therefore protective.

Synaptic growth related proteins have an inverse correlation (FIG. 4). The abundance of synaptic growth related proteins in the liver is associated with lower a β accumulation, suggesting that treatment with compounds that increase synaptic growth related proteins should be protective.

Marker rs3669981 is located at 44.943029Mb of chromosome 2, within a rather broadly modified locus of said chromosome. The Zfhx1b gene (44.810557Mb) encoding the zinc finger homeobox 1b protein showed the highest association: r-0.919, p-4.99 e-39 (fig. 5B). The Zfhxb1 protein is a Smad-interacting transcription co-repressor involved in Wnt and hedgehog signaling (G.Bassez et al, Neurobiol Dis15,240 (2004); G.Verstappen et al, Hum Mol Genet17,1175 (2008); N.Isohata et al, Int J cancer125,1212 (2009)). Deleterious variants of the gene cause the developmental disorder Mowat-Wilson syndrome, causing a number of congenital defects, including mental retardation (c.zweier et al, Am JMed Genet 108,177 (2002)). Although Zfhx1b mRNA is widely expressed during adult mouse development, particularly within the nervous system, it is most highly expressed in the liver (g.basez, supra). The Zfhx1b gene is located at 44.810557Mb on chromosome 2, consistent with an enantiomodified locus. For this gene, the heritability of hepatic mRNA expression is very high. In almost every line that inherited its Zfhx1B gene from the B6 parent, the amount of Zfhx1B mRNA was greater compared to the line that inherited its Zfhx1B gene from the D2 parent (fig. 5B). Under probing, lines 12 and 36 differed in genotype but had similar mRNA levels. These data suggest that low Zfhx1b expression in the liver reduces a β accumulation in the brain and protects mice from its adverse effects. Treatment with compounds that inhibit the activity of Zfhx1b mimics low expression of the D2 genotype and is therefore protective.

Example 3

Measurement of A beta inhibition of Imatinib derivative compositions

Protocol

1. SY5Y-APP cells were thawed and added to warm high glucose DMEM with 10% serum, pen-strep in t-75 flasks. On day 2, cultures were divided into 4 flasks. Cells from 3 flasks were collected and frozen in liquid N2. The rest of the cultures were used for the experiments.

2. The 24-well plates were seeded with cells in the same medium. Growing until fusion.

3. Preparation of stock solutions of imatinib and desmethyl imatinib:

500ug in 100ul is a 10mM stock

1mM stock was also prepared

4. Medium was changed (1ml) and inhibitors (in DMSO vehicle) or vehicle only were added as follows:

1. vehicle only

2. 3ul Imatinib-3 uM final concentration from 1mM stock

3. 3ul Demethyl Imatinib from 1mM stock (Santa Cruz Biotechnology Cat. No. SC-208027; Toronto Research Chemicals, Cat. No. D292045) ═ 3uM Final concentration

4. 10ul imatinib to 10uM final concentration from 1mM stock

5. 10ul final concentration of demethyl imatinib ═ 10uM from 1mM stock

6. Final concentration of 3ul imatinib to 30uM from 10mM stock

7. 3ul final concentration of demethyl imatinib from 10mM stock 30uM

8. 10ul final concentration of demethyl imatinib ═ 100uM from 10mM stock

After 5.16 hours incubation, the medium was isolated, 10ul protease inhibitor was added and cells and debris (3000xg) were spun out;

4. measurement of A β in 100 μ L aliquots using a Covance ELISA kit SIG-38952 luminometer

The results are shown in figure 6. These data indicate that the metabolite desmethyl imatinib (shown in fig. 10A) produced a more effective reduction of Α β when administered at the same range of concentrations compared to imatinib (Gleevec).

In addition to the above, the effect of three variants of imatinib on a β concentration was tested as described above except that a β was measured in 150 μ L of culture supernatant instead of 100 uL.

A. Imatinib (Gleevec)3, 10 and 30. mu.M;

B. imatinib p-diaminomethylbenzene 3HCl (shown in fig. 10B, Toronto Research Chemicals, catalog No. I267995)3, 10, 30, and 100 μ M;

C. imatinib (pyridine) -N-oxide (shown in fig. 10C, Toronto Research Chemicals, catalog No. I268010); and

D. imatinib (piperidine) -N-oxide (shown in fig. 10D, Toronto Research Chemicals, catalog No. I268000).

The results are shown in figure 7. These data indicate that the active metabolite imatinib produces a stronger inhibition of a β to diaminomethylbenzene 3HCl (shown in fig. 10B) than imatinib (Gleevec) when administered over the same range of concentrations. These data also indicate that imatinib (pyridine) -N-oxide and imatinib (piperidine) -N-oxide have little effect on a β concentrations.

Example 4

Measuring inhibition of Abl kinase by imatinib related compositions

The following are combined in order:

10 uL2.5Xkinase assay buffer

2.5 μ L of Abl kinase (Millipore, Temecula, CA)

1 μ L of DMSO vehicle or inhibitor in DMSO on ice

10μLγ³²P-ATP

2.5 μ L Abltide synthetic peptide substrate, Biotin Label (Millipore, Temecula, CA)

Incubation at 30 ℃ for 10 min;

termination was by vortexing by adding 12.5 μ L7.5M guanidine HCL to each reaction.

Coating 12.5. mu.L on SAM2 Biotin Capture Membrane (Promega Corp., Madison, Wis.)

The membrane was rinsed (4x2M NaCl; 4x2M NaCl, 1% H)₃PO₄，2x H₂O, at room temperature)

Kinase activity was determined by scintillation counting.

And (3) scintillation counting: display of final drug concentration

Each assay was performed in duplicate. The counts per minute measured are as follows, and the average of the two determinations is shown in the right-hand column:

the data is shown in figures 8 and 9. Figure 8 shows a semilogarithmic graph of Abl kinase activity measured in the presence of each drug at concentrations from 0 to 100 μ M. Imatinib substantially inhibited the Abl kinase even at the lowest concentration tested, 10 μ M. N-desmethyl imatinib inhibits the Abl kinase less than imatinib and treatment with imatinib p-diaminomethylbenzotrihydrochloride shows significantly lower levels of Abl kinase inhibition even at the highest concentration tested, 100 μ M.

Figure 9 shows a selectivity graph showing the ratio of the fold difference in Α β -lowering activity (compared to imatinib) for each compound to the fold difference in kinase inhibitor activity for this compound at each of the three concentrations shown. Imatinib is the reference compound, so the ratio value for this drug was set to 1.

In terms of selectivity, N-desmethyl imatinib demonstrated a 3.8 to 4.8 fold improvement over imatinib. Imatinib exhibits the greatest selectivity for p-diaminomethylbenzotrihydrochloride. At a concentration of 30 μ M, the p-diaminobenzene composition showed about 3.7-fold greater activity in reducing A β, and only 1/16 of the imatinib activity in the Abl kinase assay, resulting in a selectivity ratio of almost 60.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described compositions and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the invention.

Claims (16)

1. Use of an imatinib derivative exhibiting reduced protein kinase inhibition as compared to imatinib for the manufacture of a medicament for modulating a β production in a peripheral tissue of a subject suffering from or susceptible to a brain a β disorder, wherein the imatinib derivative is imatinib para-diaminomethylbenzene or a pharmaceutically acceptable salt thereof.

2. The use of claim 1, wherein the brain a β disorder is alzheimer's disease.

3. The use of claim 1, wherein said modulation comprises a reduction in production of a β in said peripheral tissue.

4. The use of claim 1, wherein the peripheral tissue is liver.

5. The use of claim 1, wherein the imatinib para-diaminomethylbenzene is in the mesylate salt form.

6. The use of claim 1, wherein the medicament further comprises a known therapeutic agent for treating, ameliorating, or reducing the risk or severity of a brain a β -related disorder.

7. The use of claim 6, wherein the known therapeutic agent is selected from the group consisting of: cannabinoids, statins, selective estrogen receptor molecules, antihypertensive drugs, NSAIDS and antioxidants.

8. The use of claim 6, wherein the known therapeutic agent is selected from the group consisting of: imatinib, dimebom, prednisone, ibuprofen, naproxen, and indomethacin.

9. The use of claim 6, wherein the known therapeutic agent is selected from the group consisting of: alpha-blockers, beta-blockers, alpha-beta blockers, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers, and diuretics.

10. The use of claim 1, wherein the medicament is formulated for oral administration.

11. A composition comprising imatinib para-diaminomethylbenzene or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

12. The composition of claim 11, wherein the composition further comprises a known therapeutic agent for treating, ameliorating, or reducing the risk or severity of a brain a β -related disorder.

13. The composition of claim 12, wherein the known therapeutic agent is selected from the group consisting of: cannabinoids, statins, estrogen receptor molecules of choice, antihypertensive drugs, NSAIDS and antioxidants.

14. The composition of claim 12, wherein the known therapeutic agent is selected from the group consisting of: imatinib, dimebom, prednisone, ibuprofen, naproxen, and indomethacin.

15. The composition of claim 12, wherein the known therapeutic agent is selected from the group consisting of: alpha-blockers, beta-blockers, alpha-beta blockers, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers, and diuretics.

16. The composition of any one of claims 11-15, wherein the composition is configured for oral administration.