WO2005016229A2 - Use of lipophilic diesters of chelating agent for the treatment of amyloidosis and atherosclerosis - Google Patents

Abstract

The present invention relates to the use of lipophilic diesters of the chelating agent 1,2-bis(2 aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) for preventing, treating or managing atherosclerosis, amyloidosis-related diseases and disorders and other pathologies associated with proteins aggregations. In particular, these compounds are useful in treating cardiac and vascular amyloidosis, neurodegenerative diseases and disorders and cerebral amyloid angiopathy (CAA).

Description

USE OF LIPOPHILIC DIESTERS OF CHELATING AGENT FOR THE TREATMENT OF AMYLOIDOSIS AND ATHEROSCLEROSIS

FIELD OF THE INVENTION The present invention relates to the use of lipophilic diesters of the chelating agent l,2-bis(2 aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) for the treatment of atherosclerosis, amyloidosis and other pathologies related to protein aggregation.

BACKGROUND OF THE INVENTION Amyloidosis refers to a pathological condition characterized by extracellular deposition of various proteins in a characteristic insoluble fibrillar form (amyloid fibrils). The amyloid fibrils characteristic of amyloidoses are straight, unbranching structures, about 7-12 nanometers in diameter, formed by self-assembly of a diverse group of normally soluble proteins. Some natural wild-type proteins, which are inherently amyloidogenic, form fibrils and cause amyloidosis in old age or if present for long periods at abnormally high concentrations. Other amyloidogenic proteins are acquired or inherited variants, containing amino-acid substitutions that render them unstable so that they populate partly unfolded states under physiological conditions, and these intermediates then aggregate into the stable amyloid fold. Many of the protein aggregation processes involved in the amyloidosis cascade and other pathologies of protein aggregation are metal dependent. Examples include: (i) the copper dependent deposition of beta-2-microglobulin as amyloid plaques in the joint space is a debilitating complication of long-term hemodialysis [Eakin et al. (2002) Biochemistry 41: 10646-56]; (ii) copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide PrP106- 126 [Jobling et al. (2001) Biochemistry 40: 8073-84] and (iii) evidence has been gathered to suggest that amyloid-beta (Aβ) precipitation and toxicity in Alzheimer's disease are caused by abnormal interactions with neocortical metal ions, especially Zn, Cu and Fe [Bush, AL (2003) Trends Neurosci. 26: 207-14]. Alzheimer's disease (AD) is the most frequent type of amyloidosis in humans and the commonest form of dementia. The disease is pathologically characterized by over-accumulation of amyloid plaques and neurofibrillary tangles in the brain. Polymers of amyloid-beta, the 39-43 amino acid peptide product of the transmembrane protein, amyloid protein precursor (APP), are the main components extracted from the amyloid of Alzheimer's disease brains [Masters et al. (1985) Proc Natl Acad Sci USA 82: 4245-4249]. In most AD cases, Aβ peptides also form some deposits in the cerebrovasculature, leading to cerebral amyloid angiopathy (CAA) and hemorrhagic stroke. CAA [Revesz et al. (2002) Brain Pathol.12: 343-57] is the term used to describe deposition of amyloid in the walls of arteries, arterioles and, less often, capillaries and veins of the central nervous system. CAAs are an important cause of cerebral hemorrhage and may also result in ischemic lesions and dementia. Amyloid may be clinically classified into primary and secondary amyloid. Primary amyloid, is amyloid appearing de novo, without any preceding disorder. For example, primary amyloid has been shown as antecedent of plasma cell dysfunction such as the development of multiple myeloma or other B-cell type malignancies. In this case the amyloid deposits are made up of immunoglobulin light chain proteins created in the bone marrow by malfunctioning plasma cells. Here the amyloid appears before rather than after the overt malignancy. Secondary amyloid, on the other hand, appeared as a complication of a previously existing disorder. Patients with rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, can develop secondary amyloidosis as patients with tuberculosis, lung abscesses and osteomyelitis. Amyloidoses and pathologies related to protein aggregation in general, where specific amyloid proteins are deposited, have also been associated with chronic inflammation and various forms of malignancy (e.g. multiple myeloma, endocrine tumors, Hodgkin's disease, Non-Hodgkin's disease, renal cell carcinoma), as well as with prion diseases, long-term hemodialysis and type II diabetes. Thus, amyloid deposits and proteins aggregations (both extracellular and intracellular) are associated with a number of different diseases, including diseases in vital organs such as kidney, liver, spleen, gastrointestinal tract, skin, pancreas, adrenal glands, brain, and heart. These pathologies leads to organ dysfunction, organ failure, and eventually death [Pepys M,Hawkins P. Amyloidosis. In:Warrell D, Cox T, Firth J, Benz EJ, eds. Oxford Textbook of Medicine (4th ed.) Oxford:Oxford University Press , 2003:162-73.]. Another family of diseases and disorders which is associated with arterial wall deposits includes lipid /cholesterol based substance. Atherosclerosis is the name of the process in which deposits of fatty substances, cholesterol, cellular waste products, extra-cellular matrix, calcium and other substances build up plaques in the inner lining of an artery. These plaques, usually affect large and medium-sized arteries, and their calcification was found to increase with age. Plaques can grow large enough to significantly reduce the blood's flow through an artery. In addition, damage may occur when plaques become fragile and rupture, thus generating blood clots that can block blood flow at various parts of the body. For example, if the blood clot blocks a blood vessel that feeds the heart, it may cause a heart attack, and if it blocks a blood vessel that feeds the brain, it causes a stroke. Calcium antagonists were tested in animal models and the data suggest an important pathogenetic role of Ca and pronounced anti-atherosclerotic potencies of Ca antagonists in Ca-dominated types of experimental arteriosclerosis [Fleckenstein-Gran et al. (1994) Basic Res. Cardiol. 89 Suppl 1 :145-59]. Moreover, there is compelling mechanistic evidence for the potential role of iron in atherosclerosis; iron is involved in oxidizing low- density lipoprotein (LDL) causing endothelial cell damage, iron chelators have been found to prevent endothelial cell dysfunction and vascular smooth muscle proliferation [Shah and Alam (2003) Am J Kidney Dis. 41(3 Suppl 2): S80-3]. Metal chelation therapy for coronary artery disease by ethylene diamine tetraacetic acid (EDTA) is practiced in the U.S. but its benefits are still debated by the medical establishment [Villarruz et al. (2002) Cochrane Database Syst. Rev. (4):CD002785]. There is no available therapy capable of either preventing or halting the atherosclerotic process. Similarly, for most of the amyloidoses, there is no apparent cure or effective treatment. The consequences of proteins deposition, either as extracellular deposits or as aggregations at intracellular sites, can be detrimental to the patient. Treatments may involve correcting organ failure and treating any underlying illness (such as myeloma, infection, or inflammation). However, the disease itself is difficult to reverse since it is so frequently discovered after significant organ damage has already occurred. To date there is no approved treatment for preventing cerebral amyloid angiopathy (CAA). Acute management of CAA-associated lobar hemorrhage consists of aggressive control of associated hypertension and supportive care. Surgical removal of the hemorrhage has not been shown to improve survival. Furthermore, antiplatelet and anticoagulant therapy should be avoided in elderly patients with known CAA [Feldmann and Tornabene (1991) Clin. Geriatr. Med. 7:617-630]. U.S. Patent No. 6,329,356, issued to Neurochem Inc. and Queen's University at Kingston, discloses the use of phosphonocarboxylate compounds for inhibiting the deposition of toxic amyloid fibrils that cause amyloidosis. These compounds are currently in Phase II/III clinical trial. The use of the chelating agent clioquinol for the therapy of Alzheimer's disease has been suggested by Gerolymatos (U.S. Patent No. 6,001,852) and by Bush et al. (in U.S. Patent Application Publication No. 2002/0025944). However, oral treatment with clioquinol was stopped in Japan and it was withdrawn from the market in most other countries as it was associated with the neurological side effect subacute myelo-optic neuritis (SMON) and was reported to induce amnesia in humans. Thus, there is an unmet need for development of a new effective, yet safe therapeutic agent for the treatment of diseases and disorders associated with deposition of harmful substances, such as in atherosclerosis and certain amyloidoses, and CAA in particular.

BAPTA-diesters Stable lipophilic diesters of the divalent metal ion chelator l,2-bis(2 aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) have been disclosed, by applicants of the present application, in U.S. Patent No. 6,458,837 and in the corresponding International Patent Publication No. WO 99/16741. Also disclosed in these publications is the use of the compounds in treating diseases and disorders related to excess of divalent metal ions. Among these diseases and disorders are brain and cardiac ischemia, stroke, myocardial infarction, epilepsy and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. At that time, however, the mechanism by which these chelating agents exert their neuroprotective effects had not been elucidated. Furthermore, no indication or suggestion has been made as to the potential beneficial effect of these molecules in treating amyloidoses in general and amyloid angiopathy in particular.

SUMMARY OF THE INVENTION It has now been found, in accordance with the present invention, that certain diesters of the chelating agent l,2-bis(2 aminophenoxy)ethane-N,N,N',N'- tetraacetic acid (hereinafter denoted as "DP-BAPTAs") are capable of markedly reducing the burden of amyloid plaques and the degree of cerebral amyloid angiopathy (CAA) in brains. Accordingly, the present invention provides, in one aspect, a method of attenuating or inhibiting metal ion-dependent amyloidosis, pathologies of protein aggregation, or atherosclerosis, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the general formula (I): (I)

wherein

R is saturated or unsaturated alkyl, cycloalkyl, arylalkyl or cycloalkyl-alkyl radical having from 1 to 28 carbon atoms which may be interrupted by any combination of 1-6 oxygen and/or nitrogen atoms, provided that no two oxygen atoms or an oxygen and a nitrogen atom are directly connected to each other; and M and M' independently represent a hydrogen or a physiologically acceptable cation. According to currently preferred embodiments of the invention, the useful compounds for treating amyloidosis and/or atherosclerosis are l,2-bis(2- aminophenoxy)ethane, N,N'-di(2-octodecyloxyethyl acetate), N,N'-diacetic acid (denoted herein DP- 109) and 1 ,2-bis(2-aminophenoxy)ethane, N,N'-di(2- octoxyethyl acetate), N,N'-diacetic acid (denoted herein DP-b99), and physiologically acceptable salts thereof. Currently most preferred compound is DP- 109 or a physiologically acceptable salt thereof. In another aspect of the invention, there are provided methods for preventing, treating or managing amyloidopathy-related diseases, disorders and conditions and other pathologies of proteins aggregations in mammals. Said methods comprise administering to a mammal in need thereof, a pharmaceutical composition containing, as an active ingredient, a therapeutically effective amount of a compound of the above-mentioned general formula (I). It should be understood that the diseases, disorders, conditions and pathologies that may be treated by the compounds of the general formula (I) relate to protein deposits which are metal-ion dependent. The detrimental protein aggregates may include deposits associated with amyloidoses and amyloidopathies, namely mainly extracellular deposits and plaques, as well as other protein aggregates that are intracellular inclusions at intracellular sites and organelles of various tissues. Both cases of extracellular and intracellular pathological depositions are meant to be covered within the scope of the present invention. The amyloidopathy-related diseases, disorders and conditions and pathologies of proteins aggregations, may be selected from, but not limited to, the group consisting of neurodegenerative diseases and disorders (e.g. Alzheimer's disease, Parkinson's disease, Lewy body diseases, synucleinopathies), dementias, chronic inflammation (e.g. tuberculosis, lung abscesses, osteomyelitis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis), cardiac amyloidosis, malignancies (e.g. multiple myeloma, endocrine tumors, Hodgkin's disease, Non-Hodgkin's disease, renal cell carcinoma), prion diseases, cataract, long-term hemodialysis and type II diabetes. In yet another aspect of the invention, there are provided methods for preventing, treating or managing atherosclerosis-related diseases, disorders and conditions in mammals. Said methods comprise administering to a mammal in need thereof, a pharmaceutical composition containing, as an active ingredient, a therapeutically effective amount of a compound of the above-mentioned general formula (I). The atherosclerosis-related diseases, disorders or conditions, may be selected from, but not limited to, the group consisting of peripheral vascular disease (PVD), ischemic heart disease (IHD), cerebro-vascular accidents (CVA), renal artery stenosis, carotid artery stenosis, mesenteric arterial thromboembolism and vascular dementias. It should be noted that the methods of the invention can be used therapeutically to treat pathologies of protein aggregations, including amyloidosis, as well as atherosclerosis-related diseases, disorders and conditions, or can be used as prophylactic treatment in a subject susceptible to intracellular or extracellular aggregation of proteins or atherosclerotic precipitates. The methods of treatment in accordance with the invention may further comprise treating the patient with additional therapeutic means which may be carried out concurrently with, preceding or subsequent to the administration of the pharmaceutical composition comprising a compound of the general Formula (I). In still another aspect of the invention, there is provided the use of a compound of the general formula (I) for the preparation of a medicament for the treatment of pathologies of proteins aggregations, amyloidopathy- and atherosclerosis-related diseases or disorders.

Further objects of the present invention will become apparent to those skilled in the art upon further review of the following disclosure, including the detailed descriptions of specific embodiments of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1A-C depict the effect of DP-109 treatment on amyloid plaque burden in the cerebrum. Brains of Tg2576 mice, treated daily for 3 months with either DP- 109 (black bars) or vehicle (gray bars), were assayed for amyloid plaques. Fig. 1A- bars denote total number of congophilic plaques in whole cerebral sections 200 μm lateral from the midline.

Fig. IB- bars denote plaque density (per mm²) in the cerebral sections. Fig. 1C- area of plaques presented as percentage of total cortical area in cerebral cortex. Figs. 2A-C depict the effect of DP-109 treatment on the total amounts of soluble and insoluble Aβ40+ Aβ42 in the cerebrum of Tg2576 mice. Mice were treated daily for 3 months with either DP-109 or vehicle as indicated, after which soluble and insoluble levels of Aβ were determined in the cerebrum (pmol/g of wet tissue). Fig. 2A and Fig. 2B show, respectively, insoluble and soluble Aβl-40 (light bars) and Aβl-42 (dark bars) levels (pO.OOl, different from vehicle treatment). Fig. 2C shows the total level of Aβ (soluble and insoluble, AB1-40 and AB1-42) in the vehicle- and DP-109-treated mice ( ρ<0.005). Figs. 3A-B depict the effect of DP-BAPTA on cerebral amyloid angiopathy (CAA). Tg2576 mice were orally administered with DP-109 (B) or vehicle (A) daily for 3 months, after which brains were removed and assayed for amyloid deposition around blood vessels (arrows) and parenchyma (arrowheads). Sections show Congo red-stained cortex. Scale bar, 200 μm.

DETAILED DESCRIPTION OF THE INVENTION The synthesis and some utilization of stable lipophilic diesters of BAPTA

(DP-BAPTAs) have been disclosed in U.S. Patent No. 6,458,837 and the corresponding International Patent Publication No. WO 99/16741 of the same applicants, the teaching and disclosure of which are expressly incorporated herein in their entirety by reference. In these publications, the neuroprotective effects of DP- BAPTAs were demonstrated in neuronal cell cultures in- vitro, and in ischemia model systems in- vivo. However, the effect of the DP-BAPTA molecules on specific cellular targets or biochemical processes have not been taught or suggested.

Accordingly, it was neither taught, recognized or suspected that these compounds could be effectively use for the treatment of atherosclerosis and of amyloidosis and other pathologies of protein aggregation, and in particular amyloid angiopathy, as disclosed in the present application. It is now demonstrated, for the first time, that certain diesters of the chelating agent BAPTA are capable of attenuating or inhibiting amyloidosis and amyloid angiopathy. Likewise, it is suggested that these compounds will be useful also in inhibiting other metal-ion dependent aggregation processes of fatty substances or proteins, thus may be helpful in treating atherosclerosis-related diseases and disorders and pathologies involving protein aggregation. The useful compounds in accordance with the invention are of the general formula (I) as described above. It is to be understood that within the scope of the invention are included also salts of addition and hydrates as well as other active forms of the compounds of the general formula (I).

Currently preferred useful compounds are diesters of BAPTA with alkyl chains comprising from around 8 to 20 carbon atoms. The alkyl chains may be saturated or unsaturated alkyls including one or more double bonds and/or a triple bond. According to preferred embodiments of the invention, the alkyl chain is interrupted by from 1 to 3 oxygen atoms. According to most preferred embodiments, the R moiety of a compound of the general formula (I) includes a monoalkyl ether of ethylene glycols, preferably mono- ,di- or tri-ethylene glycols.

Currently most preferred DP-BAPTA molecule for use in treating amyloidosis, in accordance with the invention, is l,2-bis(2-aminophenoxy) ethane, N,N'-di(2- octodecyloxy ethyl acetate), N,N'-diacetic acid, also referred to as DP-109, and physiologically acceptable salts thereof. Without wishing to be bound by theory, it is believed that the DP-BAPTA compounds act as chelating agents and thus are capable of removing the metal ions that are required for protein or lipid/ cholesterol aggregation, such as in pathologies involving intracellular protein-, amyloid- or lipidic substance deposition. Furthermore, it is believed that the useful effect of the DP-BAPTAs is not only in preventing, inhibiting or disrupting protein aggregation, but these chelators may also be effective in dissolution of formed amyloid and lipidic plaques and aggregates, hence further contributing to reduction of amyloid and atherosclerotic burden. It was shown that metal ions, in particular calcium, copper, zinc and iron, play a pivotal role in regulating protein structures. For example, it was shown in many age-related diseases that metal ions are involved in the process of peptides and proteins aggregation. Examples include cataracts in which proteins aggregate in the lens, aggregation of prion peptides, and the formation of Lewy bodies which are composed of filamentous aggregates of α-synuclein [Nielsen et al. (2001) J. Biol. Chem. 276: 22680-84]. The useful DP-BAPTA compounds in accordance with the invention may be used in the treatment of a whole range of indications which involve deleterious proteins precipitation. These indications include, but are not limited to, diseases, disorders and conditions such as neurodegenerative diseases and disorders (e.g. Alzheimer's disease, Parkinson's disease, Lewy body diseases, synucleinopathies), dementias, chronic inflammation (e.g. tuberculosis, lung abscesses, osteomyelitis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis), cardiac amyloidosis, malignancies (e.g. multiple myeloma, endocrine tumors, Hodgkin's disease, Non- Hodgkin's disease, renal cell carcinoma), prion diseases, cataract, long-term hemodialysis and type II diabetes. The DP-BAPTA compounds may be useful also in the treatment of atherosclerosis-related diseases, disorders and conditions that may include, but are not limited to, peripheral vascular disease (PVD), ischemic heart disease (IHD), cerebro- vascular accidents (CVA), renal artery stenosis, carotid artery stenosis, mesenteric arterial thromboembolism and vascular dementias. The term "treatment" as used herein means to include therapeutic procedures aiming at preventing, ameliorating, palliating, inhibiting or delaying the onset and/or development and/or progression of a pathological condition associated or related to metal ion-dependent protein or lipid/ cholesterol aggregation, or improving its manifestations. The term "protein" as used throughout the specification and claims is intended to include a diverse group of molecules of proteinaceous nature including proteins, peptides and polypeptides. As used in the specification and claims of this application, the term "pathogenic aggregation of proteins" should be understood to encompass a variety of different diseases, disorders and pathological conditions characterized by extracellular or intracellular deposition of various proteins (including peptides or polypeptides) in a characteristic insoluble forms such as plaques aggregates, deposits, inclusions, precipitates and the like. The terms "plaque", "aggregate", "deposit", "deposition", "inclusion" and

"precipitate" are known in the art, and are used interchangeably throughout the specification and claims. In accordance with the methods of the invention, a pharmaceutical composition comprising a therapeutically effective amount of a compound of the general Formula (I) is administered to a patient in need thereof.

The administered pharmaceutical composition may include a compound of the general Formula (I) either as a sole active ingredient, or in combination with one or more additional agents known to be effective in the treatment of a particular disease or disorder. The compound(s) of the general Formula (I) and the additional therapeutically active agent(s) may be included in the same pharmaceutical composition or may be administered in separate compositions. Furthermore, the use of DP-BAPTA in combination with another therapeutically active agent (or another therapeutic means) may be concurrently or not. The methods of the invention include administration of the DP-BAPTA compound(s) either at the same time, preceding or following exposure to the additional therapeutic agent or procedure. The additional agents that may be used in combination with the DP-BAPTA compounds may be therapeutic and prophylactic drugs, hormones, immuno- modifying agents, anti-proliferative agents etc. and may include other chelating agents, proteins, peptides, carbohydrates, lipidic molecules, DNA and RNA sequences etc. It will be readily apparent to those of ordinary skill in the art that a large number of other beneficial drugs, reagents, means or procedures may be useful in the treatment of particular pathological conditions. Pharmaceutical compositions including these therapeutically effective agents and methods applying them or other medical procedures are also included within the scope of the invention as compositions and methods useful in combination with the compounds of the general formula (I). The pharmaceutical compositions comprising the compound of the general formula (I) may be in a liquid, aerosol or solid dosage form, and may be formulated into any suitable formulation including, but not limited to, solutions, suspensions, micelles, emulsions, microemulsions, aerosols, ointments, gels, suppositories, capsules, tablets, and the like, as will be required for the appropriate route of administration. Any suitable route of administration is encompassed by the invention including, but not being limited to, oral, intravenous, intraperitoneal, intramuscular, subcutaneous, inhalation, intranasal, topical, rectal or other known routes. In preferred embodiments, the useful pharmaceutical compositions are orally, intravenously or subcutaneously administered. The dose ranges are those large enough to produce the desired attenuation or inhibitory effect on the pathological protein aggregation, amyloidosis or atherosclerosis. The dosing range varies with the specific DP-BAPTA used, the treated pathological condition and the route of administration and is dependent on the additional treatment procedure, if such additional treatment is applied. The dosage administered will also be dependent upon the age, sex, health, weight of the recipient, concurrent treatment, if any, frequency of treatment and the nature of the effect desired. The specific dosage, regimen and means of administration will be determined by the attending physician or other person skilled in the art.

The Invention will now be illustrated by the following non-limiting examples. EXAMPLES

EXAMPLE 1: Synthesis of BAPTA diesters of alkyl or alkylaryl and salts thereof Synthesis of disodium or calcium salts of alkyl or alkylaryl diesters of 1,2- bis(2-aminophenoxy)ethane-N,N,N',N' -tetraacetic acid (DP-BAPTA) was carried out in three stages as follows: Step 1. Preparation of BAPTA anhydride BAPTA (24 gr.,0.05 mol), pyridine (8 gr., 0.1 mol) and acetic anhydride (95 ml, 1.0 mol) are introduced into a round-bottom single-neck flask (500 ml), equipped with a reverse condenser (water cooling) and magnetic stirrer. The reaction mixture is heated at 90°C for 5 hours with vigorous stirring by magnetic stirrer. The temperature is then decreased to 50°C and heating is continued at this temperature for 10 hours longer. At the end of the 10-hour period the reaction mixture is cooled to room temperature and the precipitate is extracted by filtration. The precipitate is then washed four times with ethyl acetate (50 ml each wash) and twice with ether (60 ml each wash). The precipitate is dried under vacuum at 50°C for 6-8 hours. The product is a BAPTA anhydride. Yield 80% (17.6g.). White solid, m.p. 148-149°C. Analyses: TLC . The compound decomposed in the course of analysis. 1H NMR (C₆D₅N0₂), δ (ppm): 4.40 (s, 8H), 4.47 (s, 4H) and 6.85-7.01 (m, 8H). IR: 1762.9 cm^-1 (s), 1820.7 cm^-1 (s).

Elemental. C₂₂H₂o0₈N₂. Calculated: C 60.00%, H 4.54%, N 6.36%. Found: C 59.60%, H 4.66%, N 6.20%.

Step 2. Preparation of alkyl or aryl diester of BAPTA The BAPTA anhydride of step 1 (10 g, 0.023 Mol) and corresponding absolute alcohol (300 ml) are introduced, under argon atmosphere, into a round-bottom single-neck flask, equipped with reverse condenser and magnetic stirrer. The mixture is heated in an oil bath at 90°C (for methyl and ethyl diesters at 70°C) with vigorous stirring. After 6 hours about half of the alcohol is distilled from the reaction mixture (high molecular alcohols are distilled under vacuum). The obtained mixture is cooled to 0°C and kept at this temperature for 5-8 hours. The precipitate is separated from the solution by filtration (glass filter N4) under vacuum and is washed 3-4 times with about 40 ml of ethanol, followed by three washes (100 ml each) of ethyl acetate and finally with three washes (150 ml each) of diethyl ether. The product is dried under vacuum for 8 hours. For example, the synthesis scheme of DP-109 (acid) is shown below.

+2 C₁₈H₃₇OCH₂CH₂OH (C₃H₇)CHCOONa

Step 3. Preparation of disodium salt of the diester of BAPTA

In the third stage, the final product which is, in this exemplified case DP-109 sodium salt, is obtained by NaOH solution titration of DP-109 acid.

Elemental analysis C₆₂H₁o₂N₂0₁₂Na₂. Calculated (for M^'2H₂0): C 64.78%, H 9.29%, N 2.43%, Na 4.00%. Found: C 64.59%, H 9.41%, N 2.39%, Na 3.81%. 1H NMR data: 0.86-0.92 (t, 6 H); 1.27 (broad, 60H); 1.52-1.54 (m., 4H); 3.39-3.44 (t, 4H); 3.50-3.53 (m., 4H); 3.65 (s., 4 H); 3.84 (s., 4 H); 4.01-4.05 (m, 4H); 4.33 (s, 4 H); 6.88-7. l l (m., 8H). Other DP-BABTAs compounds useful in accordance with the invention, can be prepared by following synthesis procedures analogous to the procedure described above for DP-109, while using the corresponding R moiety of the respective compound of the general formula (I) and the desired M cation. Syntheses of additional diesters of BAPTA are described in International Patent Publication no. WO 99/16741, the disclosure of which is herein incorporated by reference.

EXAMPLE 2: Lipophilicity and dissociation constant measurements of BAPTA diesters salts Octanol/saline Distribution Coefficient D The lipophilicity values for BAPTA diesters (DP-BAPTAs) were studied by comparing the solubility of these compounds in organic versus aqueous solutions. Octanol and physiological saline were used, respectively, as the organic and aqueous solutions. The octanol/saline distribution coefficient was measured by the shake-flask technique. 10 mg of DP-BAPTA compound were dissolved in 5 ml of octanol. Saline (5 ml) was added to the octanol solution. The octanol/saline mixture was shaken by a Stuart Scientific reciprocating shaker (model 02) overnight at 25°C. Then, the octanol and saline phases were separated. A sample of each phase (1 ml) was dissolved in ethanol and the DP-BAPTA concentration in each phase was determined by UV-Vis spectroscopy. Distribution coefficients were calculated. The logarithm of octanol/saline distribution coefficients, logD (25 °C) values for DP-109 and DP-b99 were found to be 2.8±0.1 and 1.4-tO. l, respectively. Dissociation Constants Dissociation constants of BAPTA-diesters calcium (Ca) and copper (Cu) salts were determined by potentiometric titration with ion-selective electrodes. The Zinc (Zn) salt dissociation constants were determined by spectrophotometric method. According to the potentiometric titration method, the dissociation constant (K_D) is determined as the slope of a Scatchard plot: [Cation]_b/[Cation]_f vs. [Cation]_b (where [Cation]_f is the free cation concentration and [Cation]_b is the concentration of the cations of calcium salts of the BAPTA-diester). [Cation]_b is calculated as the difference between total cation concentration and free cation concentration. [Cation]_f is determined by an ion-selective electrode. The dissociation constants of DP-BAPTA-Zn salts were determined by spectrophotometric method using Zn⁺² specific indicator: 4-(2-Pyridylazo)resorcinol (PAR). The method is based on competition between the indicator and the tested molecule for Zn chelation. The K_D values for Ca⁺⁺, Zn⁺⁺ and Cu⁺⁺ salts of some DP-BAPTA molecules in water solution containing 2.5-10%) ethanol are presented in Table 1.

Table 1 : Dissociation constant (K_D) of calcium, zinc and copper salts of BAPTA-diesters.

The dissociation constant (K_D) of metal salt of DP-BAPTA is a measure of the capability of the compound to solvate (chelate) a cation. Thus, as it follows from the data shown in Table 1, the tested DP-BAPTA di-esters have much higher chelating capability to zinc (about 10³ fold) and copper (about 10⁴ fold) ions than to calcium ions.

EXAMPLE 3: DP-109 effect on brain amyloid plaque formation The effect of the DP-109 on amyloid plaque formation was studied in the animal model system of human Swedish mutant

Tg2576 mice [Hsiao et al. (1996) Science 274: 99-102]. These transgenic mice over-express the 695-amino acid isoform of human Alzheimer beta-amyloid precursor protein, and accordingly show increased levels of Aβ (1-40) and Aβ (1-42/43). It was shown that numerous amyloid-beta plaques were present in cortical and limbic structures of the mice with elevated amounts of Aβ. The correlative appearance of behavioral, biochemical, and pathological abnormalities reminiscent of Alzheimer's disease in these transgenic mice suggests them as an in-vivo model system for exploring this disease. Female human Swedish mutant _4EE,_{5 5}-transgenic Tg2576 mice, 18 months of age, were randomly separated into two treatment groups (17 mice in each group). One group was administered with 5 mg/kg/day (0.1 ml/10 g body weight) of DP- 109. The other group was given an equivalent volume of vehicle (control group). The DP-109 or vehicle was orally administered on a daily basis for 3 months using a syringe-linked intubation tube (total of 90 treatments per mouse). There were no differences in body weight or clinical appearances between the drug-treated and control mice at the end of the 3 months treatment. In addition, there was no significant difference in mortality between DP-109-treated (23.5%; 4/17 mice) and control mice (11.8%, 2/17 mice) (p = 0.386).

The next day after the final drug treatment, mice were sacrificed and brains removed for analysis. Each brain was dissected into two hemispheres and snap frozen in liquid nitrogen. Sagittal sections (12 μm thickness) of the right hemisphere (for histological evaluation) were obtained using a cryostat (Leica, Nussloch, Germany) and mounted on poly-L-lysine-coated glass slides. The left hemisphere was weighed and then stored at -80°C for Aβ measurement. Congo red staining To identify amyloid plaques, brain sections, 200 μm lateral from the midline, were stained with Congo red dye (Sigma, St. Louis, MO). After staining in Gill's hematoxylin solution (Sigma), sections were incubated in alkaline sodium chloride solution and then stained with alkaline Congo red solution (0.2% in 80% ethanol saturated with sodium chloride). The total number of congophilic plaques in a sagittal section was determined by eye using a light microscope (magnification ^χ400; Olympus). The percent area loaded with plaques in the whole cerebrum was calculated using a computer-assisted image analysis program (Image-Pro). All measurements were performed in an evaluator- blinded manner.

Statistical analysis of the data was performed using one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls test. P value less than 0.05 was considered significant. The counted congophilic amyloid plaques in the cerebrum are depicted in Figure 1. As shown in Fig. 1, the DP-109 treatment markedly reduced both the total number of plaques (Fig. 1A) and the number of plaques per area (Fig. IB) compared to the vehicle-treated mice (21.6 ± 15.1 vs. 126.1 ± 39.5 plaques/section, p< 0.001). Moreover, the cerebrum area loaded with amyloid plaques was decreased by 81.1% in the DP-109-treated mice compared to control mice (0.11 ± 0.08% vs. 0.58 ± 0.23%, p < 0.001) (Fig. 1C).

EXAMPLE 4: Effect of DP-109 on insoluble and soluble amyloid beta (Aβ) The in-vivo model system of human Swedish mutant

Tg2576 mice as described above in Example 3, was used for assessing the effect of the DP-109 treatment on the levels of soluble and insoluble Aβ 40/42 in the cerebrum. For the purpose of Aβ measurement, the cerebellum was removed before preparing the cerebrum protein extract, and the sandwich enzyme linked immunosorbent assay (ELISA) was applied. Brain protein was extracted following the procedure as described by Kawarabayashi et al. [J. Neurosci. (2001) 21 : 372- 381]. Briefly, cerebrum left hemispheres were homogenized in Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.6; 1 ml/150 mg wet weight) supplemented with proteinase inhibitors (Complete™ mini-protease inhibitor cocktail, 2 tablets/50 ml, Rosche Diagnostics, Mannheim, Germany). The suspension was centrifuged at 100,000g for 1 hour at 4°C and the supernatant containing soluble Aβ was collected for assay. The pellet was dissolved in 70% formic acid, resonicated and centrifuged again, and the supernatant containing insoluble Aβ collected for assay. Soluble and insoluble Aβ fractions were diluted (x 5-20) immediately prior to being subjected to ELISA. The Aβ_40/42 concentrations were determined by ELISA using a monoclonal antibody specific for the NH₂ terminus of human Aβ as a capture antibody, and a biotinylated rabbit polyclonal antibody specific for the human Aβl-40 or 1-42 sequence as a detection antibody (Biosource, Camarillo, CA). The results are shown in Figure 2. It was found that compared to control mice, DP-109 treatment significantly decreased the insoluble Aβ40/42 in the cerebrum (p< 0.01) and increased the soluble Aβ40/42 (p< 0.01). As summarized in Fig. 2C, when taken together, there is a net reduction in total amyloid beta, i.e. soluble and insoluble, Aβl-40 and Aβl-42, in the DP-109-treated mice ( ρ<0.005). The increase in the soluble/insoluble Aβ40+42 ratio in DP-109- treated animals in the present study suggests metal chelation by the drug facilitates disaggregation of Aβ from plaques into soluble forms.

EXAMPLE 5: DP-109 effect on cerebral amyloid angiopathy (CAA) The in-vivo model system of human Swedish mutant -4EP_<j₉₅-transgenic

Tg2576 mice as described above in Example 3, was used for evaluating the effect of DP-109 on cerebral amyloid angiopathy (CAA).

Typical sections of Congo red-stained cortex of DP-109-treated and untreated mice are depicted in Figure 3. As can be seen in Fig. 3, CAA was markedly reduced in the brains of the DP-

109 treated mice (Fig. 3B) compared to control mice (Fig. 3 A). These results demonstrating the effect of DP-109 in attenuating cerebral amyloid angiopathy in the brain of old female Tg2576 mice, which resemble the Alzheimer's disease in human, suggest that DP-BAPTA metal chelators may be useful as therapeutic agents against CAA also in human.

EXAMPLE 6: Effect of DP-BAPTAs on intracellular Zn²⁺ levels The effects of the DP-BAPTA compounds on intracellular zinc levels and chelation kinetics were studied in depolarization-induced Zn²⁺-loaded Pancreatic Min-6 cells. The Min-6 cells were chosen for this experiment since these cells lack the ZnTl zinc (extruding) pump and zinc levels remain relatively stable for a period after the loading. Pancreatic Min-6 cells were loaded with a fluorescent zinc indicator (ZnAF- 2F DA, K_d~400nM -final concentration lμM), in calcium-free Ringer's buffer. An influx of Zinc was stimulated by potassium-induced depolarization (50mM), as demonstrated by increased fluorescence. Experiments were performed using epifluorescence microscopy and constant perfusion so as to easily exchange buffers. Data is represented as %/- /₀ //„ where /„ is the initial average fluorescence at the start of the run. / represents the fluorescence at time t. /- /„ is represented as Δ/. After 100 sec Zn^+ (lOOμM) was added. After another 100 sec, a Zn2+ intracellular load was induced by increasing extracellular K+ (50mM) to induce depolarization. This Zn -load was monitored by the increased fluorescence of the zinc indicator. After a further approximately 200 sec (or longer, until the fluorescence increase reaches a plateau), the cells were washed with Ringer's buffer (Zn2+ free, normal K+). After a total of 500 sec DP- 109 (20μM) or DP-b99 (20μM) was added. In the case of pre-incubation, DP-109 was added to the cells 20 min before the addition of Zn and the rate and extent of increase in fluorescence was examined concurrent with the depolarization step. The average fluorescence value of each cell was taken and the mean of the fluorescence values of all the cells were calculated. In order to minimize errors, multiple recordings were made at a high frequency from several different sites. Results of these experiments show that both DP-b99 and DP-109 can attenuate depolarization-induced zinc influx. It was further shown that both DP-b99 and DP-109 exert their biological effect in a dose dependent fashion. It was found that DP-109 chelates intracellular zinc at a much slower rate than DP-b99 when added to zinc-loaded cells. The slow rate of chelation most likely is due to DP-109 not entering freely into the cytosol (the log_p~2.5) once it has partitioned into the plasma membrane. Thus, it probably interacts with zinc in the membrane vicinity 94- and expected to modulate Zn -dependent functions in the vicinity of the membrane. It is important to note that, in contrast to the DP-109 and DP-b99 effects, the addition of a zinc chelator that is not membrane permeable, such as EGTA, does not affect intracellular fluorescence.

EXAMPLE 7: Zinc chelation in the brain bv DP-109 In order to further explore the chelating effect of DP-109 on endogenous metal ions, the vesicular free zinc in the brains of DP-109-treated and vehicle- treated Tg2576 mice was determined. Mice were given DP-109 or vehicle daily following the procedure as described above in Example 3. Brains were then removed, sagittal sections prepared and the zinc-specific fluorescence intensity in the mossy fiber region of the hippocampus was determined using histofluorescent staining with TSQ dye followed by quantification of vesicular zinc. Unfixed brain sections were stained with the zinc-specific fluorescent dye N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ; 4.5 μM; Molecular Probes, Eugene, OR), in 140 mM sodium barbital and 140 mM sodium acetate buffer (pH 10.0) for 90 seconds [Frederickson et al. (1987) J. Neurosci. Methods 20: 91-103; Lee et al (1999). J. Neurosci. 19(RC10): 1-5]. After washing with saline, TSQ- stained sections were examined under a fluorescence microscope (dichroic mirror, 400 nm; excitation filter, 330-385 nm; barrier filter, 420 nm) (BX60; Olympus, Tokyo, Japan), and photographed with a digital camera (Camedia C2000-Z; Olympus). The quantification of vesicular zinc was performed as previously described by Lee et al. [Proc Natl Acad Sci USA (2002) 99: 7705-7710]. Fluorescence intensity in the mossy fiber region of the hippocampus was determined using a computer-assisted image analysis program (Image-Pro, Media Cybernetics, Silver Spring, MD). After subtracting background fluorescence (determined as fluorescence occurring in the thalamus that lacks vesicular zinc), all values were normalized to the mean fluorescence of vehicle-treated mice sections, which was given a value of 100%). The TSQ-fluorescence intensity (mean ± SEM, n = 13) in the mossy fiber region for the DP-109-treated animals was about 75% of the fluorescence measured in the vehicle-treated mice sections (p < 0.05). The significant attenuation of the Zn/TSQ fluorescence in the mossy fiber region of the hippocampus by the DP-109 treatment compared to vehicle treatment, confirmed the zinc chelating effect of DP-109 in the brain.

EXAMPLE 8: DP-109 is a safe chelating agent As the therapeutic use of potent divalent metal ion chelators could be problematic due to their effect on circulating ions and on basal ion-dependent processes and enzymes, the DP-BAPTA compounds were assayed for possible side effects. The toxicity of DP-109 was examined in Wistar rats following oral and intravenous multiple administration for 5 days. Male Wistar rats (220-290 g) were treated with the tested DP-BAPTA compound by either oral (PO) or intravenous (IV) route of administration, as follows:

Oral administration: DP-109 (500 mg/kg) or vehicle (10 ml/kg) was orally administered to Wistar rats (5 animals in each group), once a day for five consecutive days.

IV administration: for intravenous toxicity, DP-109 (50 mg/kg) and vehicle (5 ml/kg) were administered, respectively, to 5 and 3 Wistar rats via the tail lateral vein once a day for five consecutive days.

Toxicity was evaluated by monitoring the following parameters: a) Monitoring of animal weight, motor function, general sedative effect and overall well-being. b) Evaluation of biochemical parameters in serum collected on days 1 and 5, four hours after drug administration. c) Histological and pathological analysis of body toxic-prone organs, i.e. lungs, heart, spleen, liver, kidneys and adrenals, brain, stomach and small intestine.

In addition, following oral administration, stomach was also visually-assessed for ulcers. Results: Well- being of the animals treated with DP-109 was fine throughout the experiment. No mortality or general morbidity and no signs of discomfort or sedation effect were observed. The biochemical analysis of the serum shows an acute slight increase of some liver enzymes (Alkaline phosphatase, Aspartate aminotransferase and Alanine aminotransferase). However, these increases (about 2 fold) were all transient and returned to normal levels after 5 days of sub-chronic treatment. No pathological changes were observed in all the organs taken for histological analysis at the end of the treatment. None of the animals, which were subjected to oral administration of vehicle and DP-109, exhibited any signs of stomach ulcers. Conclusions: The above results indicate that DP-109 is a safe drug which does not have toxic effects in animals treated with oral dose which is 100-fold of the effective oral dose. Moreover, the DP-BAPTA compounds were found to be non- toxic whether being administered via oral or intravenous routes.

Claims

CLAIMS;

1. A method of attenuating or inhibiting metal ion-dependent amyloidosis, pathologies of protein aggregation, or atherosclerosis, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the general formula (I):

(I)

wherein

R is saturated or unsaturated alkyl, cycloalkyl, arylalkyl or cycloalkyl-alkyl radical having from 1 to 28 carbon atoms which may be interrupted by any combination of 1-6 oxygen and/or nitrogen atoms, provided that no two oxygen atoms or an oxygen and a nitrogen atom are directly connected to each other; and M and M' independently represent a hydrogen or a physiologically acceptable cation.

2. The method according to claim 1 wherein said metal ion is Zn 2+

3. The method according to claim 1 wherein said metal ion is Cu' 2+

4. The method according to claim 1 wherein said amyloidosis is amyloid angiopathy.

5. The method according to claim 1 wherein said amyloidosis is selected from cerebral, cardiac and vascular amyloidosis.

6. The method according to claim 1 wherein said amyloidosis is cerebral amyloid angiopathy (CAA).

7. The method according to claim 1 wherein R is a monoalkyl ether of mono-, di-, or tri-ethylene glycol.

8. The method according to claim 1 wherein R is Cι₈H₃₇θCH₂CH₂ .

9. The method according to claim 1 wherein R is C₈Hi₇θCH₂CH₂.

10. A method for preventing, treating or managing pathogenic aggregation of proteins in a mammal comprising administering to a mammal in need thereof, a pharmaceutical composition comprising a therapeutically effective amount of a compound of the general Formula (I).

11. The method according to claim 10, wherein said pathogenic aggregation of proteins is selected from the group consisting of neurodegenerative diseases and disorders, dementias, chronic inflammation, malignancies, prion diseases, cataract, long-term hemodialysis and type II diabetes.

12. The method according to claim 10, wherein said pathogenic aggregation of proteins is a neurodegenerative disease.

13. A method for preventing, treating or managing atherosclerosis-related disease or disorder in a mammal comprising administering to a mammal in need thereof, a pharmaceutical composition comprising a therapeutically effective amount of a compound of the general Formula (I).

14. The method according to claim 13 wherein said atherosclerosis-related disease or disorder is selected from peripheral vascular disease (PVD), ischemic heart disease (IHD), cerebro-vascular accidents (CVA), renal artery stenosis, carotid artery stenosis, mesenteric arterial thromboembolism and vascular dementia.

15. The method according to any one of claims 10 to 14, wherein said mammal is a human.

16. The method according to any one of claims 10 to 14, wherein said compound of the general formula (I) is selected from l,2-bis(2-aminophenoxy)ethane, N,N'- di(2-octodecyloxy ethyl acetate), N,N'-diacetic acid (DP-109) and l,2-bis(2- aminophenoxy)ethane, N,N'-di(2-octoxyethyl acetate), N,N'-diacetic acid (DP-b99).

17. The method according to claim 10 or 13, wherein said compound of the general formula (I) is orally, intravenously or subcutaneously administered.

18. Use of a compound of the general formula (I): (I)

wherein

R is saturated or unsaturated alkyl, cycloalkyl, arylalkyl or cycloalkyl-alkyl radical having from 1 to 28 carbon atoms which may be interrupted by any combination of 1-6 oxygen and/or nitrogen atoms, provided that no two oxygen atoms or an oxygen and a nitrogen atom are directly connected to each other; and M and M' independently represent a hydrogen or a physiologically acceptable cation, for the preparation of a medicament for the treatment of amyloidopathy-related disease or disorder or pathogenic aggregation of proteins. O 2005/016 28

19. Use of a compound of the general formula (I) :

(I)

wherein

R is saturated or unsaturated alkyl, cycloalkyl, arylalkyl or cycloalkyl-alkyl radical having from 1 to 28 carbon atoms which may be interrupted by any combination of 1-6 oxygen and/or nitrogen atoms, provided that no two oxygen atoms or an oxygen and a nitrogen atom are directly connected to each other; and M and M' independently represent a hydrogen or a physiologically acceptable cation, for the preparation of a medicament for the treatment of atherosclerosis-related disease and disorder.

20. The use according to claim 18 or 19, wherein said compound of the general formula (I) is selected from l,2-bis(2-aminophenoxy)ethane, N,N'-di(2- octodecyloxyethyl acetate), N,N'-diacetic acid (DP-109) and l,2-bis(2- aminophenoxy)ethane, N,N'-di(2-octoxy ethyl acetate), N,N'-diacetic acid (DP-b99).

21. The use according to claim 18, wherein the medicament is for the treatment of amyloidopathy-related disease or disorder or pathogenic aggregation of proteins selected from the group consisting of neurodegenerative diseases and disorders, dementias, chronic inflammation, malignancies, prion diseases, cataract, long-term hemodialysis and type II diabetes.

22. The use according to claim 19, wherein the medicament is for the treatment of atherosclerosis-related disease or disorder selected from the group consisting of peripheral vascular disease (PVD), ischemic heart disease (IHD), cerebro-vascular accidents (CVA), renal artery stenosis, carotid artery stenosis, mesenteric arterial thromboembolism and vascular dementia.