RU2461377C2 - Method for improving cognitive function (versions) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely neurology and may be used for improving a cognitive function in a patient with diminished cognitive function being due to protein ADDL neurotoxicity. The methods use non-peptide compounds of molecular weight less than 1000 which can react against the formation of neurotoxic proteins ADDL from monomer peptides Aβ 1-42, characterised by general formula I.

EFFECT: group of inventions provides improving memory, ideation, intellectual abilities and/or amnesia in the patients with such diseases as Alzheimer's disease, Down syndrome, stroke and moderate cognitive defects.

20 cl, 2 dwg, 2 tbl, 3 ex

Description

FIELD OF THE INVENTION

The invention relates to a method for improving cognitive function in a patient with reduced cognitive function due to the neurotoxicity of ADDL proteins by administering a non-peptide compound with a molecular weight of less than 1000.

State of the art

Alzheimer's disease (AD) is a progressively developing dementia with a fatal outcome, incurable today. Although the molecular mechanism of the disease has not been established, a large number of existing data indicate the participation in this mechanism of neurotoxins originating from beta-amyloid (Aβ) peptides, and, in particular, the beta-amyloid peptide, consisting of 42 amino acid residues. Aβ is an amphipathic peptide, the relative content of which increases due to mutations in genes and risk factors associated with AD. Fibrils formed from Aβ make up the rods of amyloid senile plaques, which are a sign of a brain with AD. Similar in vitro fibrils are lethal to cultured brain neurons. This information was the main reason for putting forward the primary hypothesis of the “amyloid cascade," a theory that suggested that memory loss was due to neuronal death caused by fibrillar Aβ (Hardy and Higgins (1992), Science 256: 184-185).

Despite good experimental evidence and intuitive arguments, it has been shown that the initial hypothesis of the amyloid cascade is incompatible with key observations, including a weak correlation of dementia with the mass of amyloid senile plaques (Katzman (1988) Ann. Neural. 23 (2): 138-144). Through the use of a transgenic mouse-based AD model that was treated with monoclonal antibodies, two surprising observations were made regarding Aβ: (1) the process was reversed in memory impaired mice, with an obvious restoration of normalcy within 24 hours; and (2) there was an improvement in cognition during vaccination, although the level of senile plaques remained unchanged (Dodart et al. (2002) Nat. Neurosci 5: 452-457; Kotilinek et al. (2002) J. Neurosci. 22: 6331-6335). Such observations are not compatible with a mechanism in which memory impairment is attributed to neuronal death caused by amyloid fibrils.

The obvious shortcomings of the initial hypothesis of the amyloid cascade were eliminated in the improved hypothesis of the amyloid cascade, in which additional neuroactive molecules formed as a result of self-assembly of Aβ played a key role. These molecules are proteins derived from Aβ-amyloids (hereinafter referred to as ADDL), which are assembled from monomeric beta-amyloid peptides consisting of 42 amino acid residues (hereinafter referred to as Aβ _1-42 ) at low concentrations (Lambert et al. (1998) Proc. Natl. Acad. Sci. USA 95: 6448-6453). Being essentially the missing link in the original theory of the amyloid cascade, ADDL proteins quickly suppress prolonged potentiation (Lambert et al. (1998) Proc. Natl. Acad. Sci. USA 95: 6448-6453; Walsh et al. (2002) Nature 416: 535-539; Wang et al. (2002) Brain Res. 924: 133-140), a classic experimental paradigm in memory and synaptic plasticity. In the improved Aβ cascade hypothesis, memory loss is attributed to the occurrence of a synapse disorder before neuronal death, and the disorder is caused by ADDL rather than fibrils (Hardy and Selkoe (2002) Science 297: 353-356). ADDLs occur in the brain tissue and are strikingly accumulated in the brain tissue of a patient with AD compared with controls of the same age group (Kayed et al. (2002) Science 300: 486-489; Gong et al. (2003) Proc. Natl. Acad. Sci. USA 100: 10417-10422), and also accumulate in models of transgenic mice with AD (Kotilinek et al. (2002) J. Neurosci. 22: 6331-6335; Chang et al. (2003) J. Mol. Neurosci. 20: 305-313).

A simplified mechanistic explanation of this theory can be represented as follows:

where the formation of ADDL is a separate direction, different from the formation of amyloid senile plaques, each of these directions is in equilibrium with the monomeric Aβ _1-42 .

In further experiments, important neurological properties of ADDL were shown. ADDL was shown to have selective toxicity to hippocampal CA1 neurons compared to CA3 neurons, and they completely lack toxicity to cerebellar neurons (Kim et al. (2003) FASEB J . 17: 118-120). Based on the abdominal injection of Aβ _1-42 oligomers to wild rats, fast abnormal behavior patterns were obtained in which complete recovery takes 24 hours (Cleary et al. (2005) Nat. Neurosci. 8: 79-84), and these lesions are attributed to oligomers higher order, in particular, oligomers of 12 units (Lesne et al. (2006) Nature 440: 352-357).

The binding of ADDL to neurons proceeds with high specificity and is localized on postsynaptic receptors from the subgroup of hippocampal neurons (Lacor et al. (2004) J. Neurosci. 24: 10191-10200). This binding initiates a fast and stable stimulating regulation of the line of pre-early gene products, the translation of which depends on the activity of polyribosomes located on the subgroup of dendritic spines (Steward et al. (1998) Neuron 21: 741-751; Guzowski et al. (2000) J. Neurosci . 20: 3993-4001). More recently, ADDL has been considered as activators of phosphorylation of tau proteins, and it has been shown that they affect the behavior of animals at the femtomolar level (Matsubara et al. (2004) Neurobiol. Aging 25: 833-841).

The reversibility of memory loss in mouse models, combined with the neurological properties of ADDL and their content in the brain of a patient with AD, is a good argument for the hypothesis that AD is a disease associated with synapse disorder induced by ADDL (Lambert et al. (1998) Proc. Natl. Acad. Sci. USA 95: 6448-6453; Klein et al. (2001) Trends Neuroscis. 24: 219-220; Selkoe (2002) Science 298: 789-791). The use of antibodies specific for ADDL is a powerful way to change the balance between monomeric Aβ _1-42 and ADDL, which can cure painful disorders mediated by ADDL. However, antibody delivery is usually limited to injectable solutions, which create problems associated with patient compliance with the treatment regimen and regimen, as well as the need for the presence of a physician. Especially useful in this case would be small molecules that change this equilibrium and can be delivered by non-injection means, in particular, by ingestion, transdermal delivery, through the lungs, nasal region, etc.

A number of small molecules, originally developed as inhibitors of amyloid fibrils, are believed to have the properties of inhibiting self-assembly of Aβ _1-42 oligomers. Some of these compounds include Alzhemd ™ (Gervais (2004) Neuirobiol. Aging 25: S11-12), clioquinol (Ritchie et al. (2003) Arch. Neural. 60: 1685-1691), substituted G-cyclodextrins (Yu and et al. (2002) J. Mol. Neurosci. 19: 51-55), trehalose (Lui (2005) Neurobiol. Disease 20: 74-81), simple amino-, carbonyl- and nitro-substituted phenols (De Felice et al. ( 2001) FASEB J. March 20; De Felice et al. (2004) FASEB J. 18: 1366-1372), curcumin (Yang et al. (2005) J. Biol. Chem. 280 (7): 5892-5901) analogs of cyclohexanehexol (McLaurin et al. (2006) Nature Med. 12: 801-808), spirosteroid alcohols (Lecanu et al. (2004) Steroids 69: 1-16) and tricyclic pyrons (Maeqawa et al. (2006) J Neurochem. 98: 57-67). Of the above, two compounds, Alzhemd ™ and clioquinol, have been clinically tested.

Alzhemed ™ (3-amino-1-propanesulfonic acid), the so-called "mimetic analogue of GAG (glucoseaminoglycans)", is believed to lower the level of soluble and insoluble amyloids by binding to Aβ monomer, although there are no obvious experimental details confirming the proposed mechanism of action . Alzhemed ™ has recently passed the II stage of a 20-month open extended trial, and there has been a slow cognitive decline in some patients with mild AD, however, the drug did not show efficacy during the latent phase of the study (Gervais (2004) Neuirobiol. Aging 25: S11- 12).

The second compound, Clioquinol, has been shown to stabilize the cognitive ability of patients compared to untreated patients in stage II of a clinical trial and lower plasma levels of Aβ _1-42 (Ritchie et al. (2003) Arch. Neurol. 60 : 1685-1691). However, the toxic impurity (diiodized form of clioquinol) that appears during the manufacture of the drug caused the study to cease and replace clioquinol with an analog called PBT2 (Blennow et al. (2006) Lancet 368: 387-403).

Finally, it has been reported that an unidentified compound or compounds from ginkgo biloba leaf extract lowers the level of Aβ _1-42 trimmers and tetramers and increases the level of high molecular weight polymers depending on the dose (Yao et al. (2001) Brain Res. 889: 181 -190). Dose-dependent protection of PC-12 cells against toxicity caused by the Aβ oligomer has also been reported.

Apparently, only a few compounds of those that have been reported to inhibit self-assembly of Aβ and binding to Aβ _1-42 monomer have a high therapeutic potential. Given the very simple structure and hydrophilic properties of Alzhemed ™, it is highly unlikely that it would have a high and selective affinity for the Aβ _1-42 monomer. Any effect of Alzhemed ™ on Aβ aggregation or disaggregation is likely due to its interaction with ionic residues near the N-terminus of Aβ _1-42 . Cyclodextrins have neither prototype nor drug properties, so that they can be recommended for development (Orhea et al. (2001) J. Chem. Inf. Comput. Sci. 41: 1308-1315; Vieth et al. (2004) J. Med. Chem. 47: 224-232), and De Felice phenols contain aldehyde and nitro-functional groups that are often considered chemically active and are excluded from screening libraries (Walters and Namchuk (2003) Nat. Rev. 2: 259 -266). A number of molecules containing phenolic functional groups have been reported as “frequently found drugs” in screening libraries (Roche et al. (2002) J. Med. Chem. 45: 137-142). Thus, further evaluation of the activity and selectivity of De Felice phenols is necessary in order to confirm the possibility of their effective use. It was reported that some compounds with a steroid skeleton may act as heterogeneous inhibitors during an unexpected process of auto-aggregation (McGovern et al. (2002) J. Med. Chem. 45: 1712-1722), which can explain the ambiguous results when using spirosteroidal alcohols. Finally, it is still unknown what is the active ingredient in the ginkgo biloba leaf extract. Thus, most of the putative Aβ assembly inhibitors cannot be considered as compounds suitable for development as drugs. Despite the alleged results mentioned above, a binding analysis indicates that these compounds are, at best, mild antagonists of ADDL formation.

Accordingly, the development of small molecules providing improved suppression, regulation, and / or modulation of ADDL formation could be useful for improving cognitive function.

Disclosure of invention

This invention addresses the discovery that it is possible to counteract the formation of the soluble, globular, non-fibrillar, neurotoxic Aβ _1-42 oligomer (ADDL) by using substantially pure non-peptide compounds with a molecular weight of less than 1500, preferably less than 1000, that counteract the formation of ADDL on higher level than what could be achieved earlier. By counteracting the formation of ADDL, cognitive function improves. In addition, this invention is directed to the discovery of a fact that has been established in the study of a large number of structural frameworks exhibiting increased antagonism, that the antagonism does not depend on the structural framework.

One embodiment of the invention relates to a method for improving cognitive function in a patient with cognitive function decreased due to neurotoxicity of ADDL. The method includes administering to the patient a therapeutically effective amount of a substantially pure non-peptide compound, wherein said compound is characterized in that:

(a) has a molecular weight of less than 1000;

(b) is an antagonist of the formation of neurotoxic ADDLs from monomeric Aβ _1-42 ; and

(c) its IC ₅₀ value is about 55 μM or less, which is found in the analysis of Example 1, which determines the formation of neurotoxic ADDL.

In some embodiments, a cognitive impairment in a patient is associated with the patient suffering from a disease or at risk of developing a disease associated with the formation and / or activity of ADDL. In other embodiments, the disease is selected from the group consisting of Alzheimer's disease, Down syndrome, stroke, and mild cognitive impairment.

In other embodiments, a cognitive impairment in a patient is associated with the patient suffering from a disease or having a risk of developing a disease associated with insoluble amyloid fibrils, senile plaques and / or tangles. Alternatively, a patient’s cognitive impairment is associated with the patient suffering from a disease or having a risk of developing a disease associated with overexpression of Aβ _1-42 protein. In some embodiments, the disease is selected from the group consisting of focal ischemia associated with dementia and neuronal dystrophy.

In another embodiment, the invention provides a method for suppressing, regulating and / or modulating the binding of neurotoxic ADDL to spines and / or synapses of nerve cells. The method includes the interaction of the specified nerve cell with an effective amount of a practically pure non-peptide compound, wherein said compound is characterized in that:

(a) has a molecular weight of less than 1000;

(b) is an antagonist of the formation of neurotoxic ADDLs from monomeric Aβ _1-42 ; and

(c) its IC ₅₀ value is about 55 μM or less, which is found in the analysis of Example 1, which determines the formation of neurotoxic ADDL.

In yet another embodiment, the invention provides a method for suppressing, regulating and / or modulating the binding of prolonged potentiation of nerve cells. The method includes the interaction of these cells with an effective amount of a practically pure non-peptide compound, wherein said compound is characterized in that:

(a) has a molecular weight of less than 1000;

(b) is an antagonist of the formation of neurotoxic ADDLs from monomeric Aβ _1-42 ; and

(c) its IC ₅₀ value is about 55 μM or less, which is found in the analysis of Example 1, which determines the formation of neurotoxic ADDL.

In another embodiment, the invention relates to a method for treating a patient with reduced cognitive function, which is a consequence of the patient suffering from a disease or having a risk of developing a disease selected from the group consisting of Alzheimer's disease, Down syndrome, stroke, mild cognitive impairment, focal ischemia associated with dementia, and neuronal dystrophy. The method includes administering to the patient a therapeutically effective amount of a substantially pure non-peptide compound, wherein said compound is characterized in that:

(a) has a molecular weight of less than 1000;

(b) is an antagonist of the formation of neurotoxic ADDLs from monomeric Aβ _1-42 ; and

(c) its IC ₅₀ value is about 55 μM or less, which is found in the analysis of Example 1, which determines the formation of neurotoxic ADDL.

In one embodiment, a substantially pure non-peptide compound is administered in an amount of 0.05 to 1000 mg one or more times per day. In another embodiment, the compound is administered in a pharmaceutical composition, which further comprises a pharmaceutically acceptable excipient.

In some embodiments, the compound inhibits the formation of neurotoxic ADDLs as determined in the analysis of Example 1 by an IC ₅₀ value of about 5 μM, 2 μM, 1 μM or less. In addition, the invention relates to a composition for improving cognitive function in a patient with reduced cognitive function, which is a consequence of neurotoxicity of ADDL, this composition contains a therapeutically effective amount of a practically pure non-peptide compound, wherein said compound is characterized in that:

(a) has a molecular weight of less than 1000;

(b) is an antagonist of the formation of neurotoxic ADDLs from monomeric Aβ _1-42 ; and

(c) its IC ₅₀ value is about 55 μM or less, which is found in the analysis of Example 1, which determines the formation of neurotoxic ADDL.

The composition may further comprise a pharmaceutically acceptable excipient.

In addition, the present invention relates to a method for the manufacture of a medicament comprising the compound mentioned in the embodiments of this section.

Brief Description of the Drawings

Figure 1 shows an exemplary dose-dependent inhibition of assembly by the compounds described herein, which is determined by analysis of RPEF (resonance fluorescence energy transfer). Shows the results of the analysis of test compound A, taken at a concentration of from 0.05 to 3 μm, as well as the results for test compound B, taken at a concentration of 30 μm. Positive and negative controls are also shown. The time in minutes is indicated on the X axis, and the relative fluorescence units (OEF) × 10 ³ determined by the RPEF analysis method in Example 1 are indicated on the Y axis.

Figure 2 shows an exemplary dose-dependent inhibition of assembly by a putative Alzhemed ™ amyloid fibril inhibitor (Neurochem), tested by the RPEF analysis method described in this document. Positive and negative controls are also shown. The time in minutes is indicated on the X axis, and the relative fluorescence units (OEF) × 10 ³ determined by the RPEF analysis method in Example 1 are indicated on the Y axis.

The implementation of the invention

A. Methods of Invention

Before describing the methods, it must be taken into account that the invention is not limited to the individual techniques described, protocols, cell lines, assay methods and reagents, as they may vary. It should also be understood that the terminology used in this document is intended to describe individual embodiments of the present invention and in no way limits the scope of the present invention set forth in the accompanying claims.

It should be noted that the singular objects used in this description and the claims also relate to the plural, unless the context clearly indicates otherwise.

Unless otherwise indicated, all technical and scientific terms used herein have the meanings generally understood by one skilled in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the implementation or testing of the present invention, preferred methods, devices, and materials will now be described. All publications cited here are incorporated herein by reference in their general form. The absence of anything in this document does not give the right to contrast the disclosure of this invention with an earlier invention.

The definitions used in this document are limited to the use of small molecules insofar as they relate to the aggregation or oligomerization of Aβ _1-42 in ADDL and related diseases.

This invention addresses the discovery that it is possible to counteract the formation of soluble, oligomeric, globular, non-fibrillar, neurotoxic Aβ _1-42 peptides (ADDL) by using substantially pure non-peptide compounds with a molecular weight of less than 1500, preferably less than 1000. The compounds counteract the formation of ADDL is at a higher level than what could be achieved earlier. A detailed description of embodiments related to counteracting the formation of ADDL can be found in PCT application No. US2007 / entitled "Methods for Modifying β Amyloid Oligomers with Non-Peptide Compounds" filed July 12, 2007, Attorney Register No. 089265-1400, which is included in this document is by reference in its general form.

Not limited to any theory, it is believed that the administration of a therapeutically effective amount of one or more of the compounds described herein will affect key assembly elements in Aβ _1-42 monomers or important elements in Aβ _1-42 oligomers. This effect, in turn, will prevent the formation of neurotoxic ADDLs or inhibit the activity of such ligands. The destruction of ADDL or the suppression of the activity of such ligands provides prolonged potentiation of nerve cells, eliminating and / or thereby reducing the neurotoxicity associated with ADDL. In addition, this effect will not prevent the formation of senile Aβ plaques.

The invention is directed to improving cognitive function in a patient in whom it is reduced. The term "cognitive function" refers to the cognitive process by which the individual perceives, comprehends or conceptualizes any ideas. Cognitive function includes cognitive characteristics, which include all aspects of perception, recognition, understanding, thinking, reasoning, remembering and imagination.

The term "reduced cognitive function" refers to impaired memory, slowed thinking, impaired mental ability and / or amnesia. Memory impairment may be characterized by difficulty or lack of immediate or delayed memorization. Deterioration of thinking is a complication in processing or completing previously studied topics in a timely manner or in processing new information in a quick manner. Mental impairment is defined as loss of information or inability to use information that an individual previously had or used. Amnesia is a very severe cognitive impairment that leads to a partial or complete inability to remember past knowledge, or a violation or complete loss of the ability to speak or write. Weakening of cognitive function can be caused by a number of painful disorders, which are discussed in more detail below.

Methods for assessing cognitive function include the use of standardized tools, such as a short scale for assessing the mental state according to Volstein, a modified short scale for assessing the mental state, a midsex assessment of the mental state of the elderly, a short portable questionnaire on the mental state, a scale for assessing the severity of Alzheimer's disease, test " clockwork, "clinical scale for assessing dementia, a neuropsychiatric assessment questionnaire, or any other tests similar specified conditions. Using the above assessment methods, a qualified doctor can evaluate the level of cognitive decline in a patient or improve cognitive function after treatment. In addition, to assess cognitive function, you can use informal observations or contacts of individuals with a patient, these individuals include family members, friends, official carers, such as nannies, as well as individuals previously familiar with the patient (but the list is not limited to this).

Mechanical methods for assessing neurons and nerve tissue can also be used to evaluate cognitive function, such methods include computed tomography (CT), axial computed tomography (ACT), magnetic resonance imaging (MRI), functional magnetic resonance imaging (fMRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), diffuse optical imaging (PDI), diffuse optical tomography (DOT) or any other analysis methods developed with ozhim way (and the list is not limited).

The term "non-peptide compound" refers to compounds, examples of which are described herein and do not include peptides and / or proteins. It is believed that the presence of 1-10, 1-5 or 1-2 amino acid residues associated with the structural framework of the compounds disclosed herein does not make such frameworks peptide, provided that the molecular weight of the compound is less than 1500, preferably less than 1000 , and amino acid residues do not have any antigen binding function, and also provided that the framework itself in the absence of amino acid residues is capable of inhibiting the formation of ADDL, as described in Example 1 of this document. In a preferred embodiment, the non-peptide compounds described herein do not contain amino acid residues derived from 1-20 natural amino acids.

The terms “peptides” and “proteins” refer to high molecular weight compounds containing many amino acid residues linked by amide bonds —C (O) —NR—. Amino acid residues, as a rule, come from 1-20 natural amino acids.

The compounds described herein are used in a method of suppressing, regulating and / or modulating the assembly of neurotoxic ADDLs either in vitro or in vivo. The term "ADDL" usually refers to diffusing ligands derived from amyloid beta, which have the following characteristics: they are soluble, oligomeric, globular, non-fibrillar, neurotoxic Aβ _1-42 peptides.

The term "neurotoxicity" refers to the toxic effects of ADDL on nerve cells in vitro and / or in vivo. ADDL binds specific nerve receptors, initiating abnormal transmission of nerve signals, which disrupts prolonged potentiation and causes memory impairment. Thus, ADDLs alter the function of the nerve cell in such a way that, despite its continued viability, neurons cease to function properly. Such altered functionality is referred to in this document as “neuronal dysfunction,” which is a subspecies of neurotoxicity. The prolonged effect of ADDL on signaling leads to improper transcription and the progressive disappearance of synapses, and with a very long exposure and accumulation of structural pathologies, the final death of nerve cells and severe brain dystrophy occur.

The term "soluble" means the ability of a particular substance to dissolve in any solvent (an example of such a substance in the present invention is an Aβ _1-42 oligomer). In the context of the present invention, soluble Aβ oligomers can be fractionated by centrifugation.

The term "oligomeric" refers to a protein complex of a certain number of monomer units. In the context of this invention, oligomers are called trimers, oligomers with a small number of (n) units, dodecamers (containing 12 units), and multimers with a large number of (n) units, consisting of Aβ _1-42 peptides. The term "oligomeric" does not include senile amyloid plaques.

The term “globular” means a large soluble protein complex that can be separated from fibrils and amyloid plaques. Preferably, the globular structure in size is from 4 to 12 nanometers (nm), preferably from 4.7 to 11 nm, as determined by atomic force microscopy (AFM) analysis of the supernatant fractions of the soluble Aβ _1-42 oligomer preparations described in U.S. Patent No. 6,218,506.

The term “non-fibrillar” refers to Aβ _1-42 peptides and oligomeric complexes that do not line up to form morphologically distinct structures, the so-called amyloid protofibrils or amyloid fibrils.

The term "practically pure" means that the substance is practically free of impurities, for example, contains impurities in an amount of less than 20%.

In one embodiment, the compound is considered to be substantially pure if it contains impurities in an amount of less than 10%, and in another embodiment, less than 1%.

As mentioned above, cognitive decline can be caused by a number of diseases. The terms “disease,” “disorder,” and “disorder” are used to include each other and refer to any condition that is at least partially mediated by ADDL.

In the context of this invention, the disease may be associated with insoluble amyloid fibrils, senile plaques, neurofibrillary tangles and / or overexpression of amyloid Aβ _1-42 protein. Examples of such diseases include Alzheimer's disease, Down syndrome, mild cognitive impairment, stroke, focal ischemia associated with dementia, and neuronal dystrophy. Patients susceptible to treatment include individuals with a risk of developing the disease, but who do not manifest their symptoms, as well as patients with symptoms at the moment. Thus, the compounds described in this document can be administered to all segments of the population for the purpose of prevention without the need for any assessment of the patient group.

The term “tangles” refers to neurofibrillary tangles that form inside degenerate neurons upon binding of paired helical fragments that are assembled from

hyperphosphorylated forms of proteins associated with microtubules, called tau.

The term "amyloid fibrils" refers to protein masses with special structural features. In histopathological methods, structures are usually established due to the birefringence of light green light when stained with Congo red dye, followed by viewing under polarized light.

In one aspect of the invention, invented compounds, when administered, can inhibit, regulate and / or alter the prolonged potentiation of nerve cells. The term "prolonged potentiation" means an increase in the strength of the chemical synapse, the duration of which is from several minutes to several days. It is known that potentiation is one of the main mechanisms by which the formation and storage of memory in the brain occurs.

A "nerve cell" or "neuron" is a cell that transmits and processes signals in the brain or other organs of the nervous system. In addition, the nerve cells used in the present invention can be separated from the tissue of an animal or brain tissue or grown in tissue culture. Isolated cells may consist of stable nerve cell lines selected, for example, from MC65; HCN-2; SH-SY5Y; SK-N-AS; SK-N-FI; SK-N-DZ; H19-7 / IGF-IR; QNR / D; QNR / K2; C8-D30; C8-S; C8-D1A; OLGA-PH-J / 92; Daoy; RSC96; SW10; RT4-D6P2T; RN33B; RS-12; DBRTG-05MG; C8-B4; SK-N-SH; B35; R3 [33-10ras3]; Neuro-2A and HCN-IA or any of their genetically, chemically and / or biochemically modified variations (the list may be extended). Isolated cells may also consist of germ and / or astroglial cells of nerve tissues selected, for example, from the hippocampus, cerebellum, cerebral cortex, hypothalamus, midbrain, spinal cord, striatum, frontal, temporal, parietal, occipital lobe of the brain and their any genetically, chemically and / or biochemically modified variations. Isolated, cultured animal cells may consist of nerve stem cells or any mutated, genetically, chemically and / or biochemically modified variations of these cells.

As used herein, the term “nerve tissue” refers to any part of the central nervous system, including but not limited to the brain or spinal cord. Nerve tissue may consist, at least in part, of nerve cells.

Mentioned methods are especially useful for individuals who have a genetic risk of developing Alzheimer's disease. Such individuals include individuals whose relatives were diagnosed with a disease or the risk of developing it was determined by analyzing genetic or biochemical markers. Genetic risk markers for Alzheimer's include mutations in the gene for the amyloid precursor protein (BPA), in particular, mutations at positions 717 and 670 and 671, referred to as Hardy mutations and Swedish mutations, respectively. Other risk markers include mutations in the presenilin genes PS1 and PS2, and ApoE4, a family history of Alzheimer's disease, hypercholesterolemia, or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be recognized by their characteristic dementia, as well as by the presence of the risk factors listed above. In addition, a number of diagnostic tests are available to identify individuals with Alzheimer's disease. Such tests include measuring the content of tau protein and Aβ _1-42 in cerebrospinal fluid. Individuals suffering from Alzheimer's disease can also be diagnosed by the criterion of Alzheimer's disease and related disorders (ADRDA) or by the method disclosed herein.

Patients who do not show symptoms of the disease can begin to undergo treatment at any age (for example, from 10-, 20-, 30 years of age). However, as a rule, it is necessary to begin treatment until the patient reaches 40-, 50-, 60- or 70 years of age. Typically, the course of treatment includes repeated dosing over a period of time. The course of treatment can be observed by analyzing the content of ADDL over time.

The term "patient" refers to animals, including mammals, humans, etc. In certain embodiments, the patient is an animal, in particular, an animal selected from mammals such as rat, rabbit, bull, sheep, pig, dog, cat, mouse, horse and primate, in particular humans.

The term "treating" a disease includes: (1) preventing a disease, i.e. stopping the development of clinical symptoms of the disease in a patient who is at risk of developing the disease or predisposed to the disease, but who is not yet experiencing or exhibiting symptoms of the disease; (2) suppression of the disease, i.e. stopping or reducing the risk of developing a disease or its clinical symptoms; or (3) relieving the disease, i.e. stimulation of relapse of the disease or its clinical symptoms.

The expression “suffers” in the context of the term “treatment” refers to a patient or individual who is diagnosed or predisposed to a disease. They may also say that the patient "has a risk of developing the disease." The indicated patient has not yet shown a characteristic pathology of the disease, however, it is known that he is predisposed to the disease due to a family history, i.e. he is genetically predisposed to the disease or diagnosed with a disease or disorder that predisposes him to the development of a disease that needs to be cured.

In addition to Alzheimer's disease, there are several other diseases associated with the formation of Aβ _1-42 , which include Down syndrome, stroke and mild cognitive impairment (and the list is not limited to this). It can be assumed that, as in the case of Alzheimer's disease, treatment of patients suffering from or at risk of developing these diseases can be carried out through parallel mechanisms of the disease.

The terms “senile plaque” or “senile plaque formation” refer to extracellular deposits of amyloid in the gray matter of the brain. Deposits are associated with degenerate nerve structures. It is understood that senile plaques are different from ADDL or, in other words, differences can be made between them and ADDL.

Similarly, overexpression of Aβ _1-42 is associated with focal ischemia associated with dementia, or is associated with neuronal dystrophy. Overexpression of Aβ _1-42 is believed to be a consequence of the accumulation of ADDL, which cause neurotoxicity. Treatment of a patient suffering from or at risk of developing one of these diseases by administering to him one or more of the described compounds will help to eliminate the neurotoxicity of overexpressed Aβ _1-42 .

For therapeutic purposes, a patient with a suspicion of a disease (or already suffering from a disease) associated with the accumulation of ADDL is administered a pharmaceutical composition containing one or more of the described compounds, while these compounds are administered in an amount sufficient to cure or at least to partially suppress the symptoms of the disease (biochemical, histological and / or behavioral), including when complications or intermediate pathological phenotypes occur during the development of the disease.

For prophylactic purposes, a pharmaceutical composition containing one or more of the described compounds is administered to a patient susceptible to a disease or at risk of developing a disease associated with the accumulation of ADDL, while these compounds are administered in an amount sufficient to eliminate or reduce the risk of the disease, reduce the severity or delay the onset of the disease. This includes biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes that manifest themselves in the development of the disease.

A “therapeutically effective amount” varies depending on the compound used, the type and severity of the disease, as well as the age, weight and other characteristics of the patient being treated, with each of these parameters being within the competence of the attending physician. It is anticipated that a therapeutically effective amount of one or more of the compounds described herein will affect the formation of ADDL (including, suppress or reverse the direction of formation of ADDL) in a patient compared to the process of ADDL binding without treatment. In fact, this suppresses the process of deterioration of long-term potentiation and subsequent memory impairment. A therapeutically effective amount is different from a biologically effective amount (a “biologically effective amount”). The compound of the present invention has one or more biological effects in vitro or even in vivo, for example, to some extent reduces the formation of ADDL. However, the biological effect may not lead to the manifestation of the above therapeutic effect at a clinically noticeable level, which is determined by methods that are within the competence of the attending physician.

In some methods, when the compound is administered, mild cognitive impairment is reduced or eliminated in patients who have not yet developed Alzheimer's characteristic pathology. In certain embodiments, a therapeutically effective amount is an amount of one or more of the described compounds administered or delivered to a patient, in which the patient is most likely to exhibit the desired response to treatment.

An “effective amount” is an amount of one or more of the compounds described herein by which an ADDL-mediated disease is treated. Preferably, the compounds of this invention help to reduce the formation of ADDL either in vitro or in vivo by at least 10%, 25%, 40%, 60%, 80%, 90% or 95% compared to the control.

B. Compounds

The compounds used in the described methods are substantially pure non-peptide compounds with a molecular weight of less than 1000. In one embodiment, the compounds used in the methods of the invention preferably have one or more of the following characteristics: (1) low or sub-micromolar content in the drug when tested in the RPEF analysis described in this document; (2) lack of aggregation; (3) slight or zero neurotoxicity when administered to a patient; (4) convenient solubility in an aqueous medium; (5) amenable to chemical treatment; (6) have dose-dependent characteristics; (7) reversible binding to Aβ protein; (7) the ability to bind β-amyloid monomer (Aβ _1-42 ); (8) the ability to bind soluble β-amyloid oligomers (ADDL).

In one embodiment of the invention, the compounds used to treat patients are suitable for oral administration. In this embodiment, the compounds comply with the Lipinski rule 5, which provides a criterion for evaluating drug properties. This rule generally states that a drug that is active when administered orally has: no more than 5 donors of hydrogen bonds (OH and NH groups); no more than 10 acceptors of hydrogen bonds (in particular, N and O atoms); molecular weight less than 500 g / mol and distribution coefficient log P less than 5.

1. Connection selection

A preferred method for selecting compounds for use in the methods of the invention involves analyzing the compounds for resonance fluorescence energy transfer (RPEF). The RPEF method is used to measure, detect, identify, evaluate, analyze and characterize various interactions and processes in biological systems (see, for example, Mitra et al. (1996) Gene 173: 13-17; De Angelis (1999) Physiol. Genomics . 21: 93-99; Latif and Graves (2000) Thyroid 10 (5): 407-412; Rye (2001) Methods 24 (3): 278-288; Kenworthy (2001) Methods 24 (3): 289-296 ; Periasamy (2001) J. Biomed. Opt. 6 (3): 287-291; Truong and Ikura (2001) Curr. Opin. Struct. Biol. 11 (5): 573-578; Zhang et al. (2002) Nat. Rev. Mol. Cell. Biol. 3 (12): 906-918; Sitte and Freissmuth (2003) Eur. J. Pharmacol. 479: 229-236; Milligan (2004) Eur. J. Pharm. Sci. 21 (4): 397-405; Herman et al. (2004) Methods Mol. Biol. 261: 351-370; Roda et al. (2004) Trends Biotechnol. 22 (6): 295-303; Wallrabe and Periasamy (2005 ) Curr. Opin. Biotechn ol. 16 (1): 19-27; Milligan and Bouvier (2005) FEBS J. 272 (12): 2914-2915; as well as references in any of the above documents, etc.).

The methods, protocols, techniques, evaluations and other similar procedures used in the analysis of the RPEF are described in general and in particular in a number of patents and patent applications, including: US patent No. 6908769; U.S. Patent No. 6,824,990; U.S. Patent No. 6,762,280; U.S. Patent No. 6,698,574; U.S. Patent No. 6,661,909; U.S. Patent No. 664,2001; U.S. Patent No. 6,639,078; U.S. Patent No. 6,472,156; U.S. Patent No. 6,456,734; U.S. Patent No. 6,376,257; U.S. Patent No. 6,348,322; U.S. Patent No. 6,323,039; U.S. Patent No. 6291201; U.S. Patent No. 6,280,981; U.S. Patent No. 5,914,245; U.S. Patent No. 5,661,035; and in the links of any of the listed documents, etc.

Similarly, the method of fluorescence polarization (FP) was used to measure, detect, identify, identify, evaluate, analyze and characterize various interactions and processes in biological systems (see, for example, Lundblad et al. (1996) Mol. Endocrinol. 10 (6 ): 607-612; Nasir and Jolley (1999) Comb. Chem. High Throughput Screen. 2 (4): 177-190; Park and Raines (2004) Methods Mol. Biol. 261: 161-166; as well as references in any of the above documents, etc.).

The methods, protocols, techniques, evaluations and other similar procedures used in the FP method are described in general and in particular in a number of patents and patent applications, including: US patent No. 6794158; U.S. Patent No. 6,632,613; U.S. Patent No. 6,630,295; 6,596,546; U.S. Patent No. 6,569,628; 6,555,326; US patent No. 6511815; U.S. Patent No. 6,448,018; U.S. Patent No. 6,432,632; U.S. Patent No. 6,331,392; U.S. Patent No. 6,326,142; US patent No. 6284544; U.S. Patent No. 6,207,397; U.S. Patent No. 6,171,807; U.S. Patent No. 6,066,505; US patent No. 59976820; US patent No. 5804395; U.S. Patent No. 5,756,292; U.S. Patent No. 5,445,935; U.S. Patent No. 5,427,960; U.S. Patent No. 5,407,834; US patent No. 5391740; U.S. Patent No. 5,315,015; U.S. Patent No. 5,206,179; US patent No. 5070025; U.S. Patent No. 5066426; US patent No. 4952691; U.S. Patent No. 4,863,876; U.S. Patent No. 4,751,190; US patent No. 4681859; U.S. Patent No. 4,666,640; U.S. Patent No. 4,614,823; U.S. Patent No. 4,585,862; U.S. Patent No. 4,510,251; U.S. Patent No. 4,476,229; U.S. Patent No. 4,429,230; U.S. Patent No. 4,420,568; U.S. Patent No. 4,203,670; as well as in the links of any of the listed documents, etc.

RPEF and AF methods are used in the field of amyloid research (see, for example, US patent No. 6927401; US patent No. 6906104; US patent No. 6905827; US patent No. 6881546; US patent No. 6864290; US patent No. 6864103; US patent No. 6858383; US patent No. 6846813; US patent No. 6828106; US patent No. 6803188; US patent No. 6770448; US patent No. 6713276; US patent No. 6600017; US patent No. 6515113; US patent No. 6495664; US patent No. 6323039; US patent No. 6294330; US patent No. 6280981; US patent No. 6197928; US patent No. 5981200; Kim and Lee (2004) Biochem. Biophys. Res. Commun. 316 (2): 393-397; Bacskai et al. (2003) J. Biomed. Opt . 8 (3): 368-375; Gorman, PM et al. (2003) J. Mol. Biol. 325 (4): 743-757; Garzon-Rodrequez et al. . (1997) J. Biol. Chem. 272 (34): 21037-21044; Lindgren et al. (2005) Biophys. J. 88 (6): 4200-4212; Lewis et al. (2004) Neurobiol. Aging 25 (9): 1175-1185; Leissring et al. (2003) J. Biol. Chem. 278 (39): 37314-37320; Taylor et al. (2003) J. Protein Chem. 22 (l): 31-40 ; Allsop et al. (2001) Biochem. Soc. Symp. 67: 1-14; Allsop et al. (2001) Biochem. Biophys. Res. Commun. 285 (l): 58-63; Huang et al. (2000) J. Biol. Chem. 275 (46): 36436-36440; as well as links in the above documents, etc.).

2. Applications of the RPEF method for finding a suitable compound

By the RPEF analysis method in Example 1, various compounds from commercially available libraries were examined for specific binding of ADDL to each of the compounds. Prototype compounds were identified and libraries of compounds were obtained. In addition, the dependence of the activity of prototype compounds on the structure was established, which allowed to further increase their activity.

3. Exemplary Compounds Used in the Methods of the Invention

Specific examples of the compounds used in the invention are presented below in Table 1. It is implied that the illustration of these compounds in no way limits the invention to the compounds listed in the tables, and therefore it is assumed that other compounds are suitable for use in this invention.

The table also shows the IC ₅₀ values of the compounds tested by the analysis method described in Example 1. In each case, these compounds are either commercially available or their synthesis is described in the examples of this application. It should be noted that the following compounds have spatial isomers (i.e., E- and Z-isomers), and the invention contemplates the use of any of the possible isomers or mixtures thereof.

C. Testing and introduction

The effective doses of the compositions of the present invention used to treat the above diseases can vary depending on various factors, including the route of administration, the physiological condition of the patient, other medications administered, whether the patient is a human or an animal, and for what purpose the treatment is carried out: prophylactic or therapeutic. Typically, the patient is a human, but in certain embodiments, the patient is an animal, in particular an animal selected from mammals, including rat, rabbit, bull, sheep, pig, dog, cat, mouse, horse and primate.

Compounds can be administered multiple times, with single dose intervals being days, weeks, months, or years. The intervals can also be uneven, which is determined by measuring the content of Aβ _1-42 protein or ADDL, or ADDL complexes in the patient’s blood. Alternatively, one or more of the compounds of the invention can be administered as a sustained release formulation, in which case the drug is administered at a lower frequency. Dosage and frequency of administration may vary depending on the half-life of the compounds of the invention. For therapeutic purposes, it is sometimes required to administer a relatively high dose for relatively short periods of time until the development of the disease decreases or stops, and preferably until the patient has a partial or complete elimination of the symptoms of the disease. After that, the patient can be transferred to a preventive regimen.

The administration of a pharmaceutical composition containing the compounds described herein can be carried out in various ways, in particular through the mouth, lungs, local, rectal, subcutaneous, intradermal, intranasal, intracranial, intramuscular, intraocular methods, intraarticular injection and other similar methods. The most preferred route of administration is by ingestion, although other methods may be equally effective. One or more of the compounds described herein may optionally be administered in combination with other biological or chemical agents that are at least partially effective in the treatment of diseases associated with Aβ _1-42 . An example of such agents are antibodies against Aβ _1-42 , described in international applications No. WO 2003/253673, WO 2006/014478, US patent No. 2489195, US Patent Publication No. 2007-0048312 and US Patent Application No. 11/571532, which are incorporated herein by reference.

The compounds described herein can be administered to a patient in an amount sufficient to suppress, regulate and / or modulate the formation of neurotoxic ADDLs or the activity of similar ligands in the body of said patient. A qualified physician can readily determine the appropriate amounts of the compounds described herein to effectively suppress, regulate and / or modulate the formation of neurotoxic ADDLs or the activity of similar ligands in the body of said patient. Estimated amounts of the compounds described herein are, for example, from 0.05 to 2000 mg / (m ² / day) for one or more compounds.

As noted above, the compounds described herein can be administered, for example, by mouth, topically, through the lungs, rectum, subcutaneous, intradermal, through the nose, intracranial, intramuscular, intraocular, intraarterial and other similar methods. The carrier or excipient or mixture of excipients in the drug can be a solvent or dispersion medium containing, for example, various polar or non-polar solvents, their suitable mixtures or oils. As used herein, the term “carrier” or “excipient” means a pharmaceutically acceptable carrier or excipient and includes, without exception, all solvents, dispersing agents or media, coatings, antimicrobial agents, iso / hypo / hypertonic substances, absorption modifying substances, etc. P. The use of such substances and agents for pharmaceutically active compounds is well known in the art. Moreover, any other or auxiliary active ingredients may be included in the final composition.

Diseases treated by the methods described herein include Alzheimer's disease, Down syndrome, stroke, mild cognitive impairment, focal ischemia associated with dementia, and neuronal dystrophy.

D. Pharmaceutical Formulations and Methods of Administration

When used as medicines, the compounds of this invention are usually administered in the form of pharmaceutical compositions. These compounds can be administered in various ways, including, by mouth, topically, through the lungs, rectum, subcutaneous, intradermal, intranasal, intracranial, intramuscular, intraocular or intraarticular injection. These compounds are effective in compositions administered both by injection and by mouth. Such compositions are prepared according to a technique well known in the pharmaceutical art and contain at least one active compound. This invention also includes pharmaceutical compositions that contain, as an active ingredient, one or more of the compounds described herein along with pharmaceutically acceptable carriers. In preparing the compositions of the invention, the active ingredient is usually mixed with an excipient, diluted with an excipient, or placed inside a carrier, which may be in the form of a capsule, sachet, paper sheet or some other container. The excipient used is generally suitable for administration to a patient. In the event that the filler acts as a diluent, it can be a solid, semi-solid or liquid material that serves as a carrier, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, starch capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or inside a liquid medium), ointments containing, for example , the active compound in an amount up to 10% by weight of soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

When preparing the composition, it may be necessary to grind the active compound to obtain a suitable particle size in order to mix it with other ingredients. If the active compound is essentially insoluble, then it is usually ground to obtain particles with a size of less than 200 mesh. If the active compound is substantially water soluble, then the particle size is usually controlled by crushing the composition to obtain an almost uniform distribution, for example, obtaining particles of about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, panitylpyrrolidone, cellulose, water, syrup and methyl cellulose. The compositions may also include: lubricants, such as talc, magnesium stearate and mineral oil, moisturizers, emulsifying and suspending agents, preservatives, such as methyl and propyl hydroxybenzoates, sweeteners and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained and sustained release of the active ingredient after administration to the patient according to methods known in the art.

The intravenous administration of therapeutic agents is well known in the pharmaceutical industry. Compositions for intravenous administration should have certain properties, not to mention the fact that the therapeutic agent must be dissolved in them. For example, the composition should contribute to the overall stability of the active ingredient (s), and the production of the composition should be cost-effective. All these factors ultimately determine the overall success of the application and the usefulness of the intravenous composition.

Other adjuvants, such as solvents, may be included in pharmaceutical formulations with the compounds of the present invention:

ethanol, glycerin, propylene glycol; stabilizers: ethylenediaminetetraacetic acid (EDTA), citric acid; antimicrobial preservatives: benzyl alcohol, methyl paraben, propyl paraben; buffers: citric acid / sodium citrate, potassium bitartrate, sodium bitartrate, acetic acid / sodium acetate, maleic acid / sodium maleate, sodium hydrophthalate, phosphoric acid / potassium dihydrogen phosphate, phosphoric acid / disodium hydrogen phosphate; tonicity modifiers: sodium chloride, mannitol, dextrose.

A buffer may be needed to maintain the pH of the aqueous solution in the range from 4 to 8, and more preferably in the range from 4 to 6. The buffer system is usually a mixture of a weak acid and its soluble salt, for example, a mixture of sodium citrate and citric acid; or a mono-cationic or bi-cationic salt of a dibasic acid, for example, potassium bitartrate, sodium bitartrate, a mixture of phosphoric acid and potassium dihydrogen phosphate and a mixture of phosphoric acid and disodium hydrogen phosphate.

The amount of buffer system used depends (1) on the desired pH and (2) on the amount of drug. Usually used in the composition, the amount of buffer in a molar ratio to the drug is from 0.5: 1 to 50: 1 (the amount of buffer is calculated based on the total number of moles of buffer ingredients, for example, sodium citrate and citric acid), to maintain a pH in the range from 4 to 8, the molar ratio of the buffer (total) to the drug should be from 1: 1 to 10: 1.

One of the buffers suitable for the invention is a mixture of sodium citrate and citric acid in an amount of 5-50 mg / ml sodium citrate per 1-15 mg / ml citric acid, sufficient to maintain the pH of the aqueous composition at a level of 4-6.

A buffer substance can also be added to prevent the deposition of the drug during the formation of a soluble metal complex under the action of dissolved metal ions, for example, Ca, Mg, Fe, Al, Ba ions, which can be washed out of glass vessels or rubber stoppers, or can be contained in ordinary tap water. The additive can also act as a complexing agent competing with the drug and give a soluble metal complex, leading to the formation of unwanted particles.

In addition, the presence of any additive, for example, sodium chloride in the amount of 1-8 mg / ml, may be required to regulate the tonicity at the level of the tonicity of human blood, to prevent swelling or compression of red blood cells with the introduction of an intravenous composition, which leads to undesirable side effects such as nausea and diarrhea, and even concomitant blood diseases. In general, the tonicity of the composition corresponds to the tonicity of human blood, which ranges from 282 to 288 mOsm / kg and, as a rule, 285 mOsm / kg, which is equivalent to the osmotic pressure created by a 0.9% sodium chloride solution.

The intravenous composition can be administered by direct intravenous injection, through an intravenous bolus, or by infusion of a suitable infusion solution, for example, a 0.9% sodium chloride solution or other compatible infusion solution.

Compositions may be formulated as an oral dosage form. The term "dosage forms" refers to physically discrete units used as unit doses for a patient, with each dose containing, along with a suitable pharmaceutical excipient, a predetermined amount of active material designed to produce the desired therapeutic effect.

The active compound is effective in a wide dosage range and is usually administered in a pharmaceutically effective amount. However, it is understood that the actual amount of the compound administered will be determined by the physician taking into account the appropriate conditions, including treatment conditions, the chosen route of administration, the type of compound currently being administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, etc.

When preparing solid compositions in the form of tablets, the main active ingredient is mixed with a pharmaceutical excipient to obtain a solid preliminary composition containing a homogeneous mixture of the compounds of the present invention. When mentioning homogeneous pre-formulations, it is understood that the active ingredient is uniformly dispersed throughout the composition so that it can be easily divided into dosage forms with equal efficacy, such as tablets, pills and capsules. Next, the solid preliminary composition is divided into the dosage forms described above, containing, for example, from 0.05 to 2000 mg of the active ingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwise formulated to provide a sustained release dosage form. For example, a tablet or pill may contain an internal or external drug component, the latter being in a shell covering the internal component. These two components can be separated by an enteric layer, which counteracts the grinding process in the stomach and allows the internal component to enter the duodenum in its entirety or to be delayed during release. A variety of materials can be used for such enteric layers or coatings, including a number of polymeric acids and mixtures of polymeric acids with substances such as shellac, cetyl alcohol and cellulose acetate.

Liquid dosage forms which include the new compositions of the present invention and which can be administered by mouth or injection include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions with edible oils such as cottonseed, sesame, coconut or vegetable peanut butter, as well as elixirs and similar pharmaceutical bases.

Compositions administered by inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. Preferably, the compositions are administered by mouth or respiratory tract to provide local or systemic effects. Compositions in preferred pharmaceutically acceptable solvents may be nebulized using inert gases. Spray solutions can be discharged directly from the spray device, or the spray device can be attached to the support of a protective mask or breathing apparatus with positive intermittent pressure.

Dissolved, suspension or powder compositions may preferably be administered by mouth or nose from devices by which proper delivery of the composition is effected.

The following formulation examples illustrate the intended pharmaceutical compositions of the present invention.

Composition Example 1

Hard gelatin capsules are prepared containing the following ingredients:

Ingredient Amount (mg / capsule) Active ingredient 30.0 Starch 305.0 Magnesium stearate 5.0

The above ingredients are mixed and filled with the resulting mixture in hard gelatin capsules in an amount of 340 mg.

Composition Example 2

The composition of the tablets is prepared using the following

ingredients:

Ingredient Amount (mg / tablet) Active ingredient 25.0 Microcrystalline Cellulose 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are mixed and the resulting mixture is compressed to give tablets weighing 240 mg each.

Composition Example 3

The dry powder inhalation composition is prepared using the following components:

Ingredient % by weight Active ingredient 5 Lactose 95

The active ingredient is mixed with lactose, and the resulting mixture

added to the dry powder inhalation device.

Composition Example 4

A tablet, each containing 30 mg of the active ingredient, is prepared as follows:

Ingredient Amount (mg / tablet) Active ingredient 30.0 mg Starch 45.0 mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone (as 10%) solution in sterile water) 4.0 mg Sodium Carboxymethyl Cellulose 4,5 mg Magnesium stearate 0.5 mg Talc 1.0 mg

The active ingredient, starch and cellulose are passed through a 20 mesh sieve. (US standard) and mix thoroughly. The solution of polyvinylpyrrolidone is mixed with the obtained powders, and the resulting mixture is passed through a sieve with a pore size of 16 mesh.

The granules thus obtained are dried at 50-60 ° C. and a sherry with a mesh size of 16 mesh is passed through. Sodium carboxymethyl cellulose, magnesium stearate and talc are passed through a sieve with a pore size of 30 mesh, and then added to the granules, which after mixing are compressed on a tablet machine to obtain tablets weighing 120 mg.

Composition Example 5

Capsules, each of which contains 40 mg of the active ingredient, are prepared as follows:

Ingredient Amount (mg / capsule) Active ingredient 40.0 mg Starch 109.0 mg Magnesium stearate 1.0 mg Total amount 150.0 mg

The active ingredient, starch and magnesium stearate are mixed, the mixture is passed through a sieve with a pore size of 20 mesh. and fill it with hard gelatin capsules in an amount of 150 mg.

Formulation Example 6

Suppositories, each of which contains 25 mg of the active ingredient, are prepared as follows:

Ingredient amount Active ingredient 25 mg Saturated Fatty Acid Glycerides up to 2000 mg

The active ingredient is passed through a 60 mesh sieve. and suspended in glycerides of saturated fatty acids, previously melted with minimal heating. The mixture is then poured into a suppository mold with a nominal capacity of 2.0 g and allowed to cool.

Example composition 7

Suspensions, each of which contains 50 mg of the drug substance per 5.0 ml dose, are prepared as follows:

Ingredient amount Active ingredient 50.0 mg Xanthan gum 4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline Cellulose (89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavoring and coloring agent a sufficient amount Purified water 5.0 ml

The active ingredient, sucrose and xanthan gum are mixed, the mixture is passed through a sieve with a pore size of 10 mesh, and then mixed with a pre-prepared solution of microcrystalline cellulose and sodium carboxymethyl cellulose in water. Sodium benzoate, flavor and color are diluted with a small amount of water and added to the first mixture with stirring. Then bring the volume of the solution to the desired value by adding water.

Composition Example 8

Ingredient Amount (mg / capsule) Active ingredient 15.0 mg Starch 407.0 mg Magnesium stearate 3.0 mg Total amount 425.0 mg

The active ingredient, starch and magnesium stearate are mixed, the mixture is passed through a sieve with a pore size of 20 mesh. and fill it with it

hard gelatin capsules in an amount of 425.0 mg.

Composition Example 9

The subcutaneous composition can be prepared as follows:

Ingredient amount Active ingredient 5.0 mg Corn oil 1.0 ml

Composition Example 10

An intravenous formulation can be prepared as follows:

Ingredient amount Active ingredient 250 mg Isotonic solution 1000 ml

For another formulation used in the methods of the present invention, transdermal delivery devices ("patches") are used. Such transdermal patches can be used to provide a continuous or intermittent infusion of the compounds of the present invention in a given amount. The structure and use of transdermal patches for the delivery of pharmaceuticals is well known in the art. See, for example, US patent No. 5023252, issued June 11, 1991 and incorporated herein by reference. Such patches can be intended for continuous, pulsating delivery of pharmaceuticals or for delivery when necessary.

Often, it is required or necessary to administer the pharmaceutical composition to the brain directly or indirectly. Direct methods typically include placing a catheter to deliver the drug to the patient’s ventricular system while bypassing the blood-brain barrier. One such implantable delivery system used to transfer biological factors to particular anatomical parts of the body is described in US Pat. No. 5,011472, which is incorporated herein by reference.

Indirect methods typically include formulating compositions to give the drug stealth by converting hydrophilic drugs into a lipid-soluble form. Secrecy is usually achieved by blocking the hydroxyl, carbonyl, sulfate and primary amino groups present in the drug in order to keep the drug in a more lipid-soluble and accessible state when transporting it through the blood-brain barrier. Alternatively, the delivery of hydrophilic drugs can be improved by intraarterial infusion of hypertonic solutions, which can briefly open the blood-brain barrier.

Other formulations suitable for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985).

As noted above, the compounds described herein are suitable for use in the various drug delivery systems described above. In addition, in order to increase the in vivo half-life of the administered drug from the blood serum, the compounds can be encapsulated, injected into the liposome cavity, prepared in the form of a colloid, or other methods can be used to increase the half-life of the compounds from blood serum. A variety of liposome preparation methods are available, as described, for example, by Szoka et al. in US patent No. 42235871, 4501728 and 4837028, each of which is incorporated into this document by reference.

For prophylactic purposes, compositions are administered to a patient at risk of developing AD (which is determined, for example, by studying the patient’s genome or hereditary characteristics) in an amount sufficient to suppress the initial symptoms of the disease. An amount satisfying this condition is called a "prophylactically effective dose." Amounts effective for this purpose depend on the prescription of the attending physician, who determines it taking into account factors such as age, weight and general condition of the patient, etc.

As noted above, the compounds administered to the patient are in the form of the pharmaceutical compositions described above. These compositions may be sterilized by conventional sterilization techniques or may be sterilized by filtration. The resulting aqueous solutions can be packaged and used in the form in which they are available, or they can be dried in a vacuum, and the lyophilized preparation is mixed with a sterile aqueous carrier before administration. The pH of the preparations of the compounds is usually in the range from 3 to 11, more preferably from 5 to 9, and most preferably from 7 to 8. It is understood that when using the particular excipients, carriers, or stabilizers listed above, pharmaceutical salts will be formed.

The following synthetic and biological examples are given to illustrate the invention and in no way limit the scope of this invention. Unless otherwise specified, all temperature values are given in degrees Celsius.

Examples

The invention will be better understood with reference to the following examples, which are intended only to illustrate the invention. The scope of the present invention is not limited to these embodiments, which are merely illustrations of certain aspects of the invention. Any functionally equivalent methods are within the scope of the invention. Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications fall within the scope of the appended claims.

In the examples and other places of this document, the abbreviations have the following meanings:

μl = microliter

g = gram

h = hour

Hz = hertz

M = mol per liter

mg = milligram

min = minute

ml = milliliter

mm = millimole per liter

mm = millimeter

mmol = millimol

mol = moth

MS = mass spectroscopy

H = normality

nm = nanometer

μM = micromol per liter

DMSO = dimethyl sulfoxide

Example 1. Analysis RPEF

Forster's resonant fluorescence energy transfer (RPEF) is a non-radiative energy transition dependent on internuclear distances, in which the deexcitation of one fluorophore (donor) is interconnected with the excitation of another fluorophore (acceptor). The RPEF occurs if (1) the energy quantum emitted by the donor fluorophore corresponds to the excitation energy of the acceptor fluorophore, (2) the directions of the dipole moments of the transitions for the donor and acceptor are almost parallel, and (3) the emission spectrum of the donor fluorescence partially coincides with the absorption spectrum of the acceptor.

In this analysis, the formation of the Aβ _1-42 oligomer is monitored by RPEF using the N-terminal fluorescein – Aβ _1-42 conjugates as donor and acceptor fluorophores (for the fluorescein-fluorescein conjugate R _O ~ 45 Å). The assembly of Aβ _1-42 monomers into oligomeric particles leads to a decrease in fluorescence, since Aβ _1-42 peptides labeled with fluorescein approach each other, as well as to an increase in the efficiency of RPEF. Inhibition of the assembly of Aβ _1-42 is manifested by the absence or attenuation of quenching of fluorescence. RPEF and AF analyzes are performed on a 384-well black, opaque microtiter plate of calcium borophosphate glass with a non-binding surface, and the analysis buffer consists of MOPS-Tris (3- (N-morpholine) propanesulfonate acid - N-tris (hydroxymethyl) - aminomethane) with a concentration of 50 mM (pH 8.0) and MgCl ₂ with a concentration of 100 mM. The analyzed volume containing FITC-Aβ ( _1-42 ) (fluorescein-isothiocyanate - Aβ ( _1-42 ), concentration 0.2 μM) and Aβ ( _1-42 ) (0.8 μM) is 50 μl, and the temperature - 37 ° C. The DLPA assembly is observed on a Tecan GENios Pro plate reader with an excitation wavelength of 485 nm, measuring emission at a wavelength of 515 nm. The conclusion about the kinetic mechanisms is made by the measured fluorescence intensity and by reading the polarization readings every five minutes for 3-6 hours. In the reactions of the negative control, in which there is no noticeable assembly in DLPA for the indicated period of time, MgCl _{2 is} not involved, but all the other buffer and peptide components are included. In the reactions of the positive control, all buffer components are present, while there are no additional reagents based on small molecules and antibodies. To test for the suppression of ADDL assembly, the non-peptide compound was incubated with a mixture of peptides, preparing solutions with six different concentrations, which were gradually reduced, ranging from 30 μM to 0.05 μM.

Using the above analysis method with the following conditions: the total concentration of Aβ _1-42 monomer is 1 mM, MgCl ₂ is 100 mM, the concentration of test compound A varies from 0.05 to 3 mM, it was found that test compound A is characteristic dose-dependent inhibition of Aβ _1- oligomer assembly 42 (ADDL), as shown in Figure 1. Test compound A is compound 7 of Table 1. At the same time, test compound B analyzed at a concentration of 30 μM was characterized only by partial suppression of assembly at very high concentrations (Figure 1). Other examples of compounds with a molecular weight of less than 1000, having the necessary activity, as established by this method of analysis, are presented in Table 1.

Example 2. Comparative example of a putative Aβ assembly inhibitor

Using the above-described RPEF analysis method, several compounds originally developed as amyloid fibril inhibitors are tested to inhibit the assembly of Aβ _1-42 monomer into Aβ oligomers. Test compounds include Alzhemed ™ (Neurochem), scylo-inositol, also known as AZD-103 (Ellipsis), SP-235 (Samaritan), benzofurans (GE), 9-acridinones (Warner-Lambert), cyclodextrins (State University) Illinois, Chicago), RS-406 (Sankyo), clioquinol (Rgapa) and curcumin (UCLA / UCI). The standard conditions for analysis of RPEF are used, as described in Example 1. The inability of the Alzhemed ™ compound to inhibit the assembly of Aβ _1-42 monomer in Aβ oligomers is shown in FIG. 2. Such results are characteristic of all compounds listed above, which were originally developed as inhibitors of amyloid fibrils.

Example 3. A model based on a rat, carrying out successive pressing of levers in a cyclic sequence

Introduction

The preparations of the Aβ _1-42 oligomer (ADDL) and the potential therapeutic compound are tested in a rat model with AD, alternately pressing the levers in a cyclic sequence (ALCR), in order to evaluate in vivo efficacy. Using this susceptible model, cognitive impairment can be detected by direct injection of Aβ oligomers of cell origin into the rat brain. In this study, a direct injection of ADDL consisting of synthetic Aβ _1-42 and the intended therapeutic compound tested by the ALCR technique is tested.

The ALCR test has been proven to be much more sensitive than previously published methods for measuring the effects of a drug on cognitive function. When performing this task, rats should be accustomed to a complex sequence of requests for pressing a lever, in which they receive food reinforcement in an experimental chamber with two levers. Subjects must press two levers in turn, and switching to the second lever should be carried out only after sufficient pressure on the first lever, for which they receive food rewards. Food is replaced after a certain number of taps, which initially rises from 2 to 56 responses per nutritional tablet, and then decreases back to 2 responses per nutritional tablet. Intermediate values are calculated by a quadratic function × 2. One cycle is a general sequence of increasing and decreasing the number of requests for pressing a lever (for example, 2, 6, 12, 20, 30, 42, 56, 56, 42, 30, 20, 12, 6 and 2 clicks for one food reward ) In one day session, six complete cycles of this type are carried out. Errors are counted, which include those cases when the subject stubbornly continues to press the lever after the initial pressing, sufficient to receive a food reward, i.e. he does not switch to another lever (perseverative error), or when the subject moves from one lever to another until the request is completed by clicking on the first lever (transition error).

Materials and methods

Synthetic powder Aβ _{1-42 is} dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), obtaining a solution of Aβ _1-42 in HFIP with a concentration of about 1 mm, which is kept at ambient temperature for 1 h. The resulting solution was cooled in an ice bath for 5-10 minutes, and then aliquots were taken into Eppendorf tubes in an amount of 50 μl of solution per tube. The tubes are then placed in a chemical fume hood and allowed to stand overnight to allow the HFIP to evaporate under a gentle stream of nitrogen. To remove the remaining traces of HFIP, the tubes are evacuated at room temperature and a pressure of 15-25 mm Hg. in two fast cycles lasting 15 minutes The resulting films of monomeric Aβ _1-42 peptide are stored over a desiccant at -80 ° C until use.

The tube with the monomeric Aβ _1-42 peptide is warmed to room temperature, and the Aβ _1-42 peptide is dissolved in anhydrous DMSO to obtain a mother solution of the Aβ _1-42 peptide in DMSO with a concentration of 10 to 100 μM. To 998 μl of a neural basal medium (without phenol red dye, Gibco 12348-017), 2 μl of a 20 mM stock solution of the test compound in anhydrous DMSO was added, thereby obtaining a 40 mM solution of the test compound in a neural basal medium.

When treating with only Aβ _1-42 peptide, the mother solution of the peptide in DMSO is added at 37 ° C to the neural basal medium, bringing the concentration of Aβ _1-42 peptide monomer to the desired value, provided that the maximum concentration in DMSO is 1% or less , then the contents of the tube are mixed on a vortex for 30-60 seconds, after which they rotate in a microcentrifuge for a short time and the solution is kept at 37 ° C for 15 minutes before the start of injection.

When treating Aβ _{1-42 with a} peptide and a test compound, the mother solution of the peptide in DMSO is added at 37 ° C to a solution of the compound in a neural basal medium, bringing the concentration of Aβ _1-42 monomeric peptide to the required value, provided that the maximum concentration in DMSO is 1% or less, then the contents of the tube are mixed on a vortex for 30-36 seconds, after which they rotate in a microcentrifuge for a short time and the solution is kept at 37 ° C for 15 minutes before the start of injection.

Upon control injection, the solution of the compound in a neural basal medium is maintained at 37 ° C for 15 minutes before the start of injection.

Rats: Rats are trained in the ALCR procedure until the error rate is constant. After the rats transition to the final ALCR procedure, daily training sessions are carried out until the end of the study.

Surgery: All rats are given a single size 28 cannula, fixing it on the skull and directing it to the lateral ventricle. Half of the rats have a cannula inserted into the region of the right ventricle, and the other half into the region of the left ventricle. Rats are given 5 days to recover from surgery, and then resume training. Test Injection and ALCR Test: The test is performed every fifth day. The animals are injected with 20 μl injection of a control, peptide or peptide solution together with a solution of the compound through an implanted cannula for 3-4 minutes. Animals are examined approximately 3 hours after the injection.

Analysis of the error rate: All error rates are compared with the baseline for the error rate, consisting of at least a 3-day period of absence of treatment, which follows immediately after the injection. When analyzing effects, Student's test is used for statistical inference. results

A significant increase in the frequency of occurrence of perseverative errors is observed between the base frequencies and the frequencies obtained with the introduction of the necessary concentration of Aβ _1-42 ADDL (Table 2). Tested compound C taken separately does not increase the error rate. When mixing test compound C with the required concentration of Aβ _1-42 ADDL, the increase in the frequency of perseverative errors ceases. Thus, the test compound C eliminates errors due to Aβ _1-42 ADDL taken in the required concentration. Test compound A is compound 24 of Table 1.

table 2 Error rate in the ALCR procedure Injectable Zoom error Perserative Error Average SSM p value Average SSM p value Aβ _1-42 ADDL 1,02 0,07 NS 1.33 0.16 0,045 Test compound C 0.96 0,07 NS 0.95 0.15 NS Aβ _1-42 ADDL + test compound C 1.00 0.08 NS 0.92 0.10 NS

SMS is the standard average error.

The base error is assumed to be 1.00. NS means that there is no statistically significant difference.

When reading the foregoing description, those skilled in the art will recognize various modifications and changes that can be made to the compositions or methods of the invention. It is understood that all such modifications falling within the scope of the attached claims are included in this invention.

Claims (20)

1. A method for improving cognitive function in a patient with reduced cognitive function resulting from neurotoxicity of ADDL proteins, comprising administering to the patient a therapeutically effective amount of a compound of formula I

where R is phenyl or sulfur-containing heteroaryl, each of which is substituted at position 2 with a substituent selected from the group consisting of hydroxyl, methoxy and C ₁ -C ₂ carboxyl ether;
R ¹ selected from the group consisting of hydrogen and trifluoromethyl;
R ^{2 is} selected from the group consisting of nitrogen, oxygen or sulfur containing heteroaryl and C ₆ -C ₁₀ aryl, wherein said heteroaryl and aryl are optionally substituted with one to three groups selected from hydroxyl, methyl, methoxy and halo;
L is selected from the group consisting of covalent bonds and —NHC (O) -; and
L 'is selected from the group consisting of covalent bonds and —CH ₂ C (O) -. 2. The method according to claim 1, in which a violation of cognitive function in a patient is associated with a disease or a risk of developing a disease mediated by the formation and / or activity of ADDL proteins. 3. The method according to claim 2, in which the disease is selected from the group comprising Alzheimer's disease, Down syndrome, stroke or mild cognitive impairment. 4. The method according to claim 1, in which the compound is administered in an amount of from 0.05 to 1000 mg at least once a day. 5. The method according to claim 4, in which the compound is administered in the form of a pharmaceutical composition, which further comprises a pharmaceutically acceptable excipient. 6. The method according to claim 1, in which the deterioration in cognitive function in a patient is a consequence of a disease associated with insoluble amyloid fibrils, senile plaques and / or neurofibrillary tangles. 7. The method according to claim 1, in which the deterioration in cognitive function in a patient is a consequence of a disease associated with overexpression of Aβ _1-42 protein. 8. The method according to claim 7, in which the disease is selected from the group including focal ischemia associated with dementia, and neuronal dystrophy. 9. The method according to claim 1, wherein said compound inhibits the formation of ADDL proteins neurotoxic to nerve cells with an IC _{50 of} at least about 5 μM or less. 10. The method according to claim 9, in which these nerve cells are isolated from the brain tissue of an animal and grown in tissue culture. 11. A method for improving cognitive function in a patient with reduced cognitive function resulting from neurotoxicity of ADDL proteins, comprising administering to the patient a therapeutically effective amount of a compound of formula I

where R is phenyl or 4,5,6,7-tetrahydrobenzo [b] thiophenyl, also substituted at position 2 with a substituent selected from the group consisting of hydroxyl, methoxy and C ₁ -C ₂ carboxyl ether, and where indicated 4, 5,6,7-tetrahydrobenzo [b] thiophenyl is substituted at position 7 by a methyl group, R ^{1 is} selected from the group consisting of hydrogen and trifluoromethyl;
R ^{2 is} selected from the group consisting of thiazolyl and C ₆ -C ₁₀ aryl, wherein said heteroaryl and aryl are optionally substituted with one to three groups selected from hydroxyl, methyl, methoxy and halo;
L is selected from the group consisting of covalent bonds, and —NHC (O) —L ′ is selected from the group consisting of covalent bonds and —CH ₂ C (O) -. 12. The method according to claim 11, in which the violation of cognitive function in a patient is associated with a disease or a risk of developing a disease mediated by the formation and / or activity of ADDL proteins. 13. The method of claim 12, wherein the disease is selected from the group comprising Alzheimer's disease, Down syndrome, stroke, or mild cognitive impairment. 14. The method according to item 13, in which the compound is administered in an amount of from 0.05 to 1000 mg at least once a day. 15. The method according to 14, in which the compound is administered in the form of a pharmaceutical composition, which further comprises a pharmaceutically acceptable excipient. 16. The method according to claim 11, in which the deterioration in cognitive function in a patient is a consequence of a disease associated with insoluble amyloid fibrils, senile plaques and / or neurofibrillary tangles. 17. The method according to claim 11, in which the deterioration in cognitive function in a patient is a consequence of a disease associated with overexpression of Aβ _1-42 protein. 18. The method according to 17, in which the disease is selected from the group including focal ischemia associated with dementia, and neuronal dystrophy. 19. The method of claim 11, wherein said compound inhibits the formation of neurotoxic to nerve cell ADDL proteins with an IC _{50 of} at least about 5 μM or less. 20. The method according to claim 19, in which these nerve cells are isolated from the brain tissue of an animal and grown in tissue culture.

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