HK1165195A - Method of inducing cleavage of amyloid precursor protein to form a novel fragment - Google Patents

Description

Method for inducing amyloid precursor protein cleavage to form novel fragments

Technical Field

The present invention relates to modulating the processing of Amyloid Precursor Protein (APP), and in particular to inducing cleavage of APP to form novel protein fragments.

Background

Alzheimer's Disease (AD) is a degenerative disease of nervous tissue for which there is only symptomatic treatment and has limited efficacy. Certain amyloid beta (A β) fragments of APP, particularly A β_1-40And Abeta_1-42Pathology involving AD. A reduction of A β has been performed as a method of modifying the AD process (Barten, D. and C. Albright, mol. Neurobiol. 37: 171-. However, to date, this approach has not resulted in an approved therapy.

Attempts have been made to treat AD using active passive immunization against a β. One such immunization method has failed in human trials (Holmes, c. et al, Lancet 372: 216-23 (2008)). A limitation of a β immunotherapy may be that it only targets a β that has already formed. It does not slow down or stop the production of new a β and, indeed, may even stimulate increased production of new a β.

Other attempts to treat AD involve inhibiting enzymes known in APP processing before they are able to produce deleterious Α β fragments. These enzymes target gamma-secretase and beta-secretase. Since many such inhibitors affect the cleavage of other γ -secretase substrates and are therefore toxic, γ -secretase inhibitors have not proven useful (Czirr, E. and S.Weggen, neuro-generative Dis. (Neurodegenerative diseases) 3: 298-; milano, j, et al, Toxicological Sciences 82: 341-358(2004)).

Gamma-secretase modulators have not proven useful. Examples of gamma-secretase modulators include non-steroidal anti-inflammatory drugs (NSAIDs), which are allosteric modulators of gamma-secretase. Such compounds are not toxic, but compounds that have entered clinical trials have only high micromolar in vitro potency and are therefore too weak to have sufficient clinical effect (Czirr, E. and S.Weggen, neuroregenerative Dis.3: 298-. Recently, the prototype gamma-secretase modulator flurbiprofen failed phase III clinical trials.

Furthermore, previous attempts to treat AD using beta-secretase inhibitors have not proven useful because the large binding pocket of beta-secretase that binds to its membrane site creates a challenge to design inhibitors that pass through the blood-brain barrier (Barten, D. and C. Albright, mol. neurobiol 37: 171-.

Thus, there remains a need in the art for effective treatment of AD.

Summary of The Invention

The present invention provides a heterocyclic compound of the general formula (I) or a pharmaceutically acceptable salt, hydrate or prodrug thereof for use in inducing cleavage of APP to produce an approximately 17 kilodalton (kDa) carboxy-terminal fragment of APP:

wherein the about 17kDA fragment comprises the carboxy-terminal amino acid sequence of APP and the amyloid-beta amino acid sequence, and wherein each R_x、R₁、R₂、R₃、R₄As defined herein.

The invention also provides an about 17kDa APP fragment comprising the carboxy-terminal amino acid sequence of APP and the beta-amyloid amino acid sequence.

The present invention also provides a method for screening compounds that cleave APP to generate an APP fragment of about 17kDa, the method comprising: (a) exposing cells that produce APP or a fragment thereof to a test compound, and (b) determining the amount of the about 17kDa fragment, wherein the about 17kDa fragment comprises the carboxy-terminal amino acid sequence of APP and an amyloid-beta amino acid sequence, and wherein an increase in the amount of the about 17kDa fragment in cells exposed to the compound, relative to the amount of the about 17kDa fragment in cells not exposed to the compound, indicates that the compound cleaves APP to produce the about 17kDa fragment.

The present invention also provides a compound other than a heterocyclic compound of formula (I), or a pharmaceutically acceptable salt, hydrate or prodrug thereof, for use in inducing cleavage of APP to produce an approximately 17 kilodalton (kDa) carboxy-terminal fragment of APP:

wherein the about 17kDA fragment comprises the carboxy-terminal amino acid sequence of APP and the amyloid-beta amino acid sequence, and wherein each R_x、R₁、R₂、R₃、R₄As defined herein.

Brief description of the drawings

Fig. 1A and 1B are bar graphs depicting the effect of compound ST101 on a β production by N2a cells. Figure 1A is a bar graph depicting a β concentration in cell culture medium as a function of ST101 concentration after 24 hours of treatment compared to control. FIG. 1B is a graph depicting A.beta.versus control_1-42And Abeta_1-40The ratio of (d) was plotted as a bar graph of ST101 concentration function.

FIGS. 2A, 2B and 2C are graphs depicting the effect of ST101 on 3 × Tg-AD mice in the Morris water maze. Fig. 2A is a line graph depicting latency (in seconds) during seven days of training compared to control mice. Fig. 2B and 2C are bar graphs depicting latency (in seconds) and number of passes through the platform position at 24 and 72 hours after training in ST101 treated animals and control mice.

FIGS. 3A and 3B are bar graphs depicting the effect of ST101 on A β in brain tissue from 3 × Tg-AD mice. FIG. 3A is a graph depicting the relative position of control miceSoluble A beta in brain tissue of mice treated with ST101_1-40And Abeta_1-42Bar graph of the amount of (c). FIG. 3B is a graph depicting insoluble A β in mice treated with ST101 relative to control mice_1-40And Abeta_1-42Bar graph of the amount of (formic acid extract).

FIG. 4 is a Western immunoblot (Western blot) depicting the carboxyl-terminal fragment of APP as determined by antibody CT20 in the brain of ST 101-treated (S)3 × Tg-AD mice relative to untreated (C)3 × Tg-AD mice.

FIG. 5 is a Western immunoblot depicting APP and degradation fragments determined by antibody CT20 in the brain of ST 101-treated (S)3 × Tg-AD mice relative to untreated (C)3 × Tg-AD mice.^*Represents the full-length of the APP,^**the major degradation products are indicated, and "actin" represents an anti- β -actin antibody as a protein loading control.

FIG. 6 is a graph depicting the proposed amyloid processing pathway leading to a novel carboxy-terminal fragment of APP.

FIGS. 7A-C are Western immunoblots depicting the C-terminal fragment of APP determined by antibody CT20 in the brain of ST 101-treated (S)3 × Tg-AD mice, relative to untreated (C)3 × Tg-AD mice.

FIG. 8 is a chart depicting Western immunoblotting of C-terminal fragments determined by antibody 1565-1 (Epitomics) in the brain of ST 101-treated (S)3 × Tg-AD mice versus untreated (C)3 × Tg mouse-AD.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The present invention provides a method of inducing cleavage of APP to prepare an approximately 17kDa carboxy-terminal fragment of APP in a subject, the method comprising administering to a subject in need thereof a heterocyclic compound of formula (I):

wherein the about 17kDa fragment comprises the carboxy-terminal amino acid sequence of APP and the amyloid-beta amino acid sequence, and wherein each R_x、R₁、R₂、R₃、R₄As defined herein.

The present invention also provides a heterocyclic compound of the general formula (I) or a pharmaceutically acceptable salt, hydrate or prodrug thereof for use in inducing cleavage of APP to produce an approximately 17 kilodalton (kDa) carboxy-terminal fragment of APP:

wherein the about 17kDA fragment comprises the carboxy-terminal amino acid sequence of APP and the amyloid-beta amino acid sequence, and wherein each R_x、R₁、R₂、R₃、R₄As defined herein.

In one embodiment, upon administration of the compound to an individual in need thereof, a fragment of about 17kDA is formed in the individual.

The present invention also provides the use of a heterocyclic compound of general formula (I) or a pharmaceutically acceptable salt, hydrate or prodrug thereof for the manufacture of a medicament for inducing cleavage of APP to yield an approximately 17 kilodalton (kDa) carboxy-terminal fragment of APP:

wherein the about 17kDA fragment comprises the carboxy-terminal amino acid sequence of APP and the beta-amyloid amino acid sequence, and wherein each R_x、R₁、R₂、R₃、R₄As defined herein.

In one embodiment, administration of a heterocyclic compound of formula (I) results in A β of APP_1-42、Aβ_1-40One or more of C99 fragment and/or C83 fragment of APP.

In another embodiment, the individual to whom the heterocyclic compound of formula (I) is administered suffers from AD. In another embodiment, the individual is diagnosed with or has been diagnosed with AD. In another embodiment, the individual suffers from mild cognitive impairment. In another embodiment, the individual is diagnosed or has been diagnosed with mild cognitive impairment.

In another embodiment, AD is treated. In another embodiment, mild cognitive impairment is treated. As used herein, "treatment" refers to any method of ameliorating or otherwise beneficially altering a condition, disorder, or symptom of a disease.

In one embodiment, AD is prevented. In another embodiment, mild cognitive impairment is prevented. As used herein, "preventing" AD or cognitive disorders refers to preventing the occurrence of one or more symptoms of AD in an individual.

As used herein, ameliorating a particular disease symptom by administering a particular pharmaceutical composition refers to any alleviation, whether permanent or temporary, sustained or transient, attributed to or associated with the administration of the pharmaceutical composition.

In another embodiment, the individual is or has been screened to determine whether the individual has AD. Screening can be performed by examining the individual. In addition, screening can be performed by testing for one or more AD biomarkers.

In another embodiment, the individual has been diagnosed as predisposed to AD. In another embodiment, an individual is or has been screened to determine whether the individual is predisposed to having AD. Screening can be performed by examining the individual. In addition, screening can be performed by testing for one or more biomarkers that are predisposed to AD.

In another embodiment, the subject is a human subject.

The invention also provides an isolated about 17kDa APP fragment comprising the carboxy-terminal amino acid sequence of APP and β -amyloid amino acid sequence.

The invention also provides compositions comprising the about 17kDA fragments of the invention. In another embodiment, the composition further comprises a cell culture lysate and/or culture medium.

The invention also provides a container comprising the about 17kDA fragment of the invention. In another embodiment, the vessel is a microtube. In another embodiment, the container is a test tube. In another embodiment, the container is a pipette or micropipette. In another embodiment, the container is a microarray device. In another embodiment, the container is a microtiter plate. In another embodiment, the container is a component of a screening assay device.

The invention also provides a method for screening a compound that cleaves APP to produce an about 17kDA fragment of APP, the method comprising (a) exposing a cell that produces APP or a fragment thereof to a test compound, and (b) determining the amount of the about 17kDA fragment, wherein the about 17kDA fragment comprises the carboxy-terminal amino acid sequence of APP and the β -amyloid amino acid sequence, and wherein an increase in the amount of the about 17kDA fragment in a cell exposed to the compound relative to the amount of the about 17kDA fragment in a cell not exposed to the compound indicates that the compound induces cleavage of APP to produce the about 17kDA fragment.

Furthermore, one can determine the presence of the free amino terminus of the approximately 17kDa fragment, or one can determine the free carboxyl terminus of the APP generated by cleavage resulting in the 17kDa fragment.

The present invention also provides a method for screening for compounds that cleave APP to produce an about 17kDa fragment of APP, the method comprising: (a) exposing cells that produce amyloid precursor protein or a fragment thereof to a test compound, and (b) determining the approximately 17kDa fragment, wherein the approximately 17kDa fragment comprises the carboxy-terminal amino acid sequence of the APP and an amyloid-beta amino acid sequence, and wherein the presence of the approximately 17kDa fragment in cells exposed to the compound relative to the approximately 17kDa fragment absent from cells not exposed to the compound indicates that the compound induces cleavage of APP to produce the approximately 17kDa fragment.

In one embodiment, the screening method of the present invention further comprises (c) a β relative to APP in cells not exposed to said compound_1-42、Aβ_1-40C99 fragment or C83 fragment of said APP, determining A β of APP in cells exposed to said compound_1-42、Aβ_1-40Whether one or more of the C99 fragments or the C83 fragment of APP is reduced.

In one embodiment, the screening method of the invention is performed in vivo.

In another embodiment, the screening method of the invention is performed in vitro. In this embodiment, the presence or amount of the about 17kDa fragment in cell culture can be detected for cells exposed to the compound and control cells not exposed to the compound. For example, an increase in the amount of the about 17kDa fragment in the cell culture of cells exposed to the compound relative to the amount of the about 17kDa fragment in the cell culture of cells not exposed to the compound indicates that the compound cleaves APP to generate the about 17kDa fragment. Furthermore, the presence of the about 17kDa fragment in the cell culture of cells exposed to the compound, relative to the absence of the about 17kDa fragment in the cell culture of cells not exposed to the compound, indicates that the compound cleaves APP to generate the about 17kDa fragment.

In another embodiment, the screening method of the invention is performed in cells in culture.

In another embodiment, the screening method of the invention further comprises culturing the cells in a cell culture medium relative to cells not exposed to the compoundAbeta of APP_1-42、Aβ_1-40C99 fragment or the C83 fragment of APP, determining a β of APP in cell culture lysates of cells exposed to said compound_1-42、Aβ_1-40Whether one or more of the C99 fragments or the C83 fragment of APP is reduced.

In another embodiment, the screening method of the invention is performed in a high throughput manner. In another embodiment, the screening method of the invention is automated. In another embodiment, the screening method of the invention is computer controlled.

In another embodiment, a plurality of cultured cells are separately exposed to a plurality of test compounds, e.g., in different wells of a microtiter plate. In this embodiment, a large number of test compounds can be screened simultaneously.

The test compound may be provided to a cell or cell line dissolved in a solvent. Examples of solvents include DMSO, water, and/or buffers. DMSO can be used in amounts below 2%. In addition, DMSO can be used in an amount of 1% or less than 1%. At this concentration, DMSO acts as a solubilizer for the test compound rather than a permeation reagent. The amount of solvent that the cells can tolerate must first be checked by separately measuring the activity of the cells at different solvent amounts to ensure that the amount of solvent has no effect on the cell properties being measured.

Suitable buffers include cell growth media, such as Iscove's medium (Invitrogen) with or without 10% fetal bovine serum. Other known cell incubation buffers include phosphate, PIPES or HEPES buffers. Other suitable buffers can be determined by one of ordinary skill in the art using no more than routine experimentation.

Cells from which APP or fragments thereof are prepared include, but are not limited to, IMR-32, BV-2, T98G, NT2N and N2A cells. In another embodiment, the cell is an N2A cell.

In another embodiment, the cell from which the APP or fragment thereof is prepared comprises a cell into which a nucleic acid encoding APP has been introduced or into which APP has been mutated, such as by transfection.

Can also determine about 17kDa APP fragment and APP Abeta_1-42、Aβ_1-40C99 fragment and/or C83 fragment of APP, for example using gel electrophoresis. The 17kDa APP fragment and the APP Abeta can also be determined by using a double-antibody sandwich ELISA test method_1-42、Aβ_1-40C99 fragment and/or C83 fragment of APP using a first monoclonal antibody directed against the N-terminus, such as the 17kDA fragment, and a second monoclonal antibody directed against another region, such as the 17kDA fragment, e.g. the carboxy terminus of the 17kDA fragment.

Can also detect APP fragments of about 17kDa and A beta of APP through an antibody_1-42、Aβ_1-40C99 fragment, and/or C83 fragment of APP, e.g., using mass spectrometry with or without prior immunoprecipitation.

In another embodiment, the about 17kDA fragment is isolated. The term "isolated" as used herein means isolated from the brain of an individual. In another embodiment, the about 17kDA fragment is present in an electrophoresis gel. In another embodiment, the about 17kDA fragment is present in a cell culture lysate or medium.

An "about 17kDa fragment" of APP is a fragment of APP that comprises the C-terminal sequence of APP and the β -amyloid sequence of APP. The about 17kDA fragment is not a C99 fragment of APP or a C83 fragment of APP.

The present invention also provides a method of inducing cleavage of APP in a subject to produce an about 17kDa carboxy-terminal fragment of APP, the method comprising administering a compound other than of formula (I):

wherein each R_x、R₁、R₂、R₃、R₄As used hereinAnd (4) defining. In one embodiment, the compound is not disclosed in U.S. application No. 11/872,408 (published as US 2008/0103157 a 1); US application No. 11/872,418 (published as US2008/0103158 a 1); U.S. patent application No. 6,635,652; U.S. patent application No. 7,141,579; and PCT/JP2007/070962 (published as WO2008/047951), each of which is incorporated by reference in its entirety. In another embodiment, the compound is not spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2' -indan).

The present invention also provides a compound other than a heterocyclic compound of formula (I), or a pharmaceutically acceptable salt, hydrate or prodrug thereof, for use in inducing cleavage of APP to produce a carboxy-terminal fragment of about 17 kilodaltons (kDa) APP:

wherein the about 17kDA fragment comprises the carboxy-terminal amino acid sequence of APP and the amyloid-beta amino acid sequence, and wherein each R_x、R₁、R₂、R₃、R₄As defined herein.

In one embodiment, upon administration of a compound other than of formula (I) to a subject in need thereof, a fragment of about 17kDA is formed in the subject.

The present invention also provides the use of a compound other than a heterocyclic compound of formula (I), or a pharmaceutically acceptable salt, hydrate or prodrug thereof, in the manufacture of a medicament for inducing cleavage of APP to produce a carboxy-terminal fragment of APP of about 17 kilodaltons (kDa):

wherein the about 17kDA fragment comprises the carboxy-terminal amino acid sequence of APPAnd beta-amyloid amino acid sequence, and wherein each R_x、R₁、R₂、R₃、R₄As defined herein.

In another embodiment, administration of a compound other than formula (I) results in A β of APP_1-42、Aβ_1-40One or more of C99 fragment and/or C83 fragment of APP.

In another embodiment, the individual to whom the heterocyclic compound of formula (I) is administered suffers from AD. In another embodiment, the individual is diagnosed with or has been diagnosed with AD. In another embodiment, the individual suffers from mild cognitive impairment. In another embodiment, the individual is diagnosed or has been diagnosed with mild cognitive impairment.

In another embodiment, AD is treated. In another embodiment, mild cognitive impairment is treated. As used herein, "treating" refers to any method of alleviating or otherwise beneficially altering a condition, disorder, or symptom of a disease.

In one embodiment, AD is prevented. In another embodiment, mild cognitive impairment is prevented. As used herein, "preventing" AD or cognitive disorders refers to preventing the development of one or more symptoms of AD in an individual.

As used herein, amelioration of a particular disease symptom by administration of a particular pharmaceutical composition refers to any alleviation, whether permanent or temporary, sustained or transient, attributed to or associated with administration of the composition.

In another embodiment, the individual is or has been screened to determine whether the individual has AD. Screening can be performed by examining the individual. In addition, screening can be performed by testing for one or more biomarkers for AD.

In another embodiment, the individual has been diagnosed as predisposed to AD. In another embodiment, an individual is or has been screened to determine whether the individual is predisposed to having AD. Screening can be performed by examining the individual. In addition, screening can be performed by testing for one or more biomarkers that are predisposed to AD.

In another embodiment, the subject is a human subject.

The heterocyclic compounds of the present invention can be administered in effective oral doses of 0.0005mg or more per kilogram body weight. In one embodiment, the compound is administered as the active ingredient as part of a unit dosage form comprising 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100mg of the compound.

All compositions encompassed by the compositions used in the present invention comprise the active ingredient in an amount effective to achieve the intended use of the active ingredient. It is within the skill of the art to determine the optimal range of effective amounts of each component as individual needs vary. Generally, the active ingredient can be administered orally to a mammal, such as a human, at a dosage of 0.001mg/kg to 3mg/kg per day of the body weight of the mammal to treat AD, or an equivalent amount of a pharmaceutically acceptable salt thereof. The active ingredient can be administered intravenously or intramuscularly to a mammal, such as a human, at a dosage of 0.001mg/kg to 3mg/kg daily of the body weight of the mammal to treat AD, or an equivalent amount of a pharmaceutically acceptable salt thereof. About 0.001mg/kg to about 3mg/kg can be administered orally to treat or prevent such diseases. If another agent is also administered, the active ingredient can be administered in an amount effective to achieve its intended use.

A unit oral dose may comprise from about 0.001mg to about 200mg, or from about 0.5mg to about 180mg, of a composition of the invention. The unit dose may be administered once or more times daily in the form of one or more tablets each comprising from about 0.1mg to about 90mg, suitably from about 10mg to 180mg, of the composition or solvate thereof. In one embodiment, the unit oral dose is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180mg of the compound.

In topical formulations, the active ingredient can be present at a concentration of about 0.01mg to 100mg per gram of carrier.

In addition to administering the active ingredient as a chemical raw material, the active ingredient can be administered as part of a pharmaceutical formulation containing a suitable pharmaceutically acceptable carrier, including excipients and auxiliaries that facilitate processing of the active ingredient into preparations that can be used pharmaceutically. Formulations, particularly those capable of oral administration such as tablets, dragees and capsules, and also capable of rectal administration such as suppositories, and solutions suitable for injection or oral administration can contain from about 0.01% to 99%, or from about 0.25% to 75% of the active ingredient together with excipients.

The heterocyclic compounds of the general formula (I) can be used as pharmaceutically acceptable salts in the form of hydrates or acid addition salts. Possible acid addition salts include inorganic acid salts such as hydrochloride, sulfate, hydrobromide, nitrate and phosphate and organic acid salts such as acetate, oxalate, propionate, glycolate, lactate, pyruvate, malonate, succinate, maleate, fumarate, malate, tartrate, citrate, benzoate, cinnamate, methanesulfonate, benzenesulfonate, p-toluenesulfonate and salicylate.

Acid addition salts are formed by mixing a solution of a particular compound of the present invention with a solution of a pharmaceutically acceptable non-toxic acid such as hydrochloric acid, hydrobromic acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, lactic acid, tartaric acid, carbonic acid, phosphoric acid, oxalic acid and the like. Basic salts are formed by mixing a solution of a particular compound of the invention with a solution of a pharmaceutically acceptable non-toxic base such as sodium hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate, tris, N-methyl-glucosamine and the like.

The pharmaceutical compositions of the present invention can be administered to any animal that can experience the beneficial effects of the active ingredient. Although the present invention is not intended to be so limited, mammals such as humans, as well as veterinary animals (veteriary animals) are preferred among such animals.

The pharmaceutical compositions of the present invention may be administered by any method that achieves their intended use. For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal, or topical routes. Alternatively, or simultaneously, administration can be by the oral route. The dose administered will depend on the age, health and weight of the recipient, the type of concurrent treatment present, the frequency of treatment, and the nature of the effect desired.

The pharmaceutical preparations of the invention are manufactured in a manner known per se, for example by means of conventional mixing, granulating, dragee-making, dissolving or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active ingredient with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as sugars, for example lactose or sucrose, mannitol or sorbitol; cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate; and binders such as starch paste, using for example corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone. If desired, disintegrating agents may be added, such as the starches mentioned above, as well as carboxymethyl starch, cross-linked polyvinyl pyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, firstly, flow regulators and lubricants, for example silicon dioxide, talc, stearic acid or its salts, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. If desired, dragee cores are provided with suitable coatings which are resistant to gastric juices. For this purpose, concentrated solutions of sugars may be used, which may optionally comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. For the preparation of coatings resistant to gastric juices, solutions of suitable cellulose preparations, for example cellulose acetate phthalate or hydroxypropylmethylcellulose phthalate, are used. For example, dyes or pigments may be added to the tablets or dragee coatings for identification or to characterize combinations of active ingredient doses.

Other pharmaceutical formulations that can be used orally include compression-bonded capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The compression-molded capsules can contain the active ingredient in the form of granules, which can be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredient can be dissolved or suspended in suitable liquids, such as fatty oils or liquid paraffin. In addition, stabilizers may be added.

Possible pharmaceutical preparations for rectal administration include, for example, suppositories that consist of a combination of one or more active ingredients with a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides or paraffin hydrocarbons. In addition, gelatin rectal capsules consisting of a combination of the active ingredient with a base can also be used. Possible basic materials include, for example, liquid triglycerides, polyethylene glycols or paraffins.

Suitable formulations for parenteral administration include aqueous solutions of the active ingredients in water-soluble form, e.g. water-soluble salts and alkaline solutions. In addition, suspensions of the active ingredients can be administered as suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides or polyethylene glycol-400. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may further comprise a stabilizer.

As used herein, a prodrug is a compound that is metabolized or otherwise converted to the biological, pharmaceutical, or therapeutically active form of the compound upon in vivo administration. To prepare prodrugs, pharmaceutically active compounds are modified so that the active compounds are regenerated by metabolic processes. Prodrugs can be designed to alter the metabolic stability or transport properties of a drug, thereby masking side effects or toxicity, improving the taste of a drug, or altering other properties or attributes of a drug. With knowledge of the pharmacodynamic processes and drug metabolism in vivo, the skilled worker is able to design prodrugs of the compounds once the pharmaceutically active compounds are known (see, for example, Nogrady, Medicinal Chemistry: ABiochemical Approach, Oxford university Press, New York, page 388-.

Also included within the scope of the present invention are dosage forms of the active ingredient wherein the oral pharmaceutical formulation comprises an enteric coating. The term "enteric coating" as used herein refers to any coating on an oral pharmaceutical dosage form that inhibits dissolution of the active ingredient in acidic media, but which dissolves rapidly in neutral to alkaline media and has good stability for long term storage. In addition, dosage forms having an enteric coating may also comprise a water-soluble separating layer between the enteric coating and the core.

The core of the enteric dosage form contains the active ingredient. Optionally, the core further comprises pharmaceutical additives and/or excipients. The separating layer may be a water-soluble inert active ingredient or polymer for film coating applications. The separation layer is coated on the core by any conventional coating technique known to those of ordinary skill in the art. Examples of separating layers include, but are not limited to, sugars, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropyl cellulose, polyvinyl acetal diethylaminoethyl ester, and hydroxypropyl methylcellulose. The enteric coating is applied to the separating layer by any conventional coating technique. Examples of enteric coatings include, but are not limited to, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate, carboxymethyl ethylcellulose, and copolymers of methacrylic acid and methyl methacrylate, such as EudragitL12, 5 or EudragitL 100(Rohm Pharma), such as Aquateric(FMC Corporation)、EudragitAqueous dispersions of L100-55 (Rohm Pharma) and Coating CE 5142(BASF), and dispersions such as Citroflex(Pfizer) of (co) polymers containing water-soluble plasticizers. The final dosage form is enteric coated tablet, capsule or pill.

Examples of prodrugs of compounds of the invention include simple esters of carboxylic acids comprising the compounds (e.g., by reaction with C according to methods known in the art)_1-4Those obtained by alcohol condensation); containing hydroxy esters of compounds (e.g. by reaction with C according to methods known in the art)_1-4Carboxylic acid, C_3-6Those obtained by condensation of diacids or anhydrides thereof (e.g., succinic anhydride and fumaric anhydride); aminoimines comprising compounds (e.g. by reaction with C according to methods known in the art)_1-4Those obtained by condensation of aldehydes or ketones); and acetals or ketals of alcohols containing the compounds (such as those obtained by condensation with chloromethyl methyl ether or chloromethyl ethyl ether according to methods known in the art).

AD symptoms include confusion, disorganization of short-term memory, problems with attention, problems with spatial localization, personality changes, language disorders and mood swings. It is understood that as pharmaceutical science continues to advance, the list of AD symptoms may increase in the future. Thus, the term "AD symptoms" is not limited to the list of symptoms provided herein.

An effective amount of a compound for treating a particular disease as used herein is an amount sufficient to ameliorate or to some extent reduce the symptoms associated with the disease. Such an amount can be administered in the form of a single dose or according to a course of treatment, whereby it is effective. The amount may cure the disease, but is generally administered to ameliorate the disease. Often, repeated administrations are required to achieve the desired improvement in symptoms.

In the general formula (I), the structural unit having the general formula (II) may be one or more structural units selected from a plurality of types of structural units having the general formula (III).

In the general formula (I), R_xIs methyl or absent. In the general formula (I) and the general formula (II), R₁And R₂Each independently selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxyl group, an amino group, an acetamido group, a benzylamino group, a trifluoromethyl group, and C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₂-C₆Alkenyl radical, C₃-C₈Cycloalkyl, benzyloxy, CH₂-R₅(wherein R is₅Is phenyl (which may be represented by C)₁-C₆Alkyl, halogen atom or cyano substituted) or thienyl) and-O- (CH)₂)_n-R₆Wherein R is a hydrogen atom, wherein R is a hydrogen atom₆Is vinyl, C₃-C₈Cycloalkyl or phenyl, and n is 0 or 1.

In the general formula (I) and the general formula (II), R₃And R₄Each independently selected from hydrogen atom, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₃-C₈Cycloalkyl radical, CH₂-R₅(wherein R is₅Is phenyl (which may be represented by C)₁-C₆Alkyl, halogen atom or cyano substituted); naphthyl or thienyl) and-CH (R)₈)-R₇One or more functional groups of (a). Furthermore, R₃And R₄Together form a spiro ring having the general formula (IV):

R₇is selected from vinyl; an ethynyl group; optionally from C₁-C₆Alkyl radical, C₁-C₆Alkoxy, hydroxy, 1 or 2 halogen atoms, di-C₁-C₆Phenyl substituted with alkylamino, cyano, nitro, carboxy or phenyl; a phenethyl group; a pyridyl group; a thienyl group; and one or more functional groups of a furyl group. R is as defined above₈Is a hydrogen atom or C₁-C₆An alkyl group.

Further, in the general formula (IV), the structural unit B may be one or more structural units selected from a plurality of types of structural units having the general formula (V). In formula (V) the structural units B are bound at the positions marked by x to form a spiro ring.

Wherein R is₉Is selected from hydrogen atom, halogen atom, hydroxyl, C₁-C₆One or more functional groups of alkoxy, cyano and trifluoromethyl.

When the heterocyclic compound of the general formula (I) has an asymmetric carbon atom in the structure, isomers from the asymmetric carbon atom thereof and mixtures thereof (racemic modification) exist. In such cases, they are all contained in the heterocyclic compounds used in the embodiments described herein.

Unless otherwise defined, the term "C₁-C₆"means 1 to 6 carbon atoms. Unless otherwise defined, the term "C₃-C₈"means 3 to 8 carbon atoms. The term "C₁-C₆Alkyl "includes straight or branched chain alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, sec-butyl, n-pentyl and n-hexyl. The term "C₁-C₆Alkoxy "includes straight or branched chain alkoxy groups such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxyN-pentyloxy and n-hexyloxy. The term "C₃-C₈Cycloalkyl "includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. The term "halogen atom" includes fluorine, chlorine, bromine and iodine.

In another embodiment of any of the methods of the present invention, the heterocyclic compound actually used in the present invention is selected from the group consisting of:

3, 3-dimethylimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dipropylimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibutylimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-diallylimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-diallyl-8-benzyloxyimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (2-propynyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzylimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzyl-8-methylimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzyl-5, 7-dimethylimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzyl-8-hydroxyimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzyl-8-methoxyimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzyl-8-ethoxyimidazo (1, 2-a) pyridin-2 (3H) -one,

8-allyloxy-3, 3-dibenzylimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzyl-8-isopropoxyimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzyl-8-cyclopropylmethyloxoimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzyl-8-cycloheptyloxyimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzyl-6-chloroimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzyl-6, 8-dichloroimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzyl-8-chloro-6-trifluoromethylimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzyl-8-benzyloxyimidazo (1, 2-a) pyridin-2 (3H) -one,

8-amino-3, 3-dibenzylimidazo (1, 2-a) pyridin-2 (3H) -one,

8-acetylamino-3, 3-dibenzylimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibenzyl-8-benzylamino-imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (3-chlorobenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (3-fluorobenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (4-fluorobenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (2, 4-dichlorobenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (4-dimethylaminobenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (4-methoxybenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (4-biphenylmethyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (4-cyanobenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (4-hydroxy-benzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (3-phenyl-1-propyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (2, 4-difluorobenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (4-nitrobenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (4-carboxybenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

8-benzyloxy-3, 3-bis (1-phenylethyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

8-benzyloxy-3, 3-bis (3-methylbenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

8-benzyloxy-3, 3-bis (4-methylbenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3-benzyl-3- (4-fluorobenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3-ethyl-3- (4-fluorobenzyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

8-methyl-3, 3-bis (3-pyridylmethyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

8-methyl-3, 3-bis (4-pyridylmethyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (2-thienylmethyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (2-furylmethyl) imidazo (1, 2-a) pyridin-2 (3H) -one,

spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2' -indane),

spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2' - (2, 3) dihydrophenalene)),

spiro (imidazo (2, 1-b) thiazol-6 (5H) -one-5, 2' -benzo (f) indan),

spiro (imidazo (1, 2-b) thiazol-6 (5H) -one-5, 2' -indan),

spiro (2-methylimidazo (1, 2-b) thiazol-6 (5H) -one-5, 2' -benzo (f) indane),

5, 5-bis (4-fluorobenzyl) imidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-dibenzylimidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-bis (4-methylbenzyl) imidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-bis (4-cyanobenzyl) imidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-dibenzyl-2-methylimidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-bis (4-fluorobenzyl) -2-methylimidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-dicyclohexyl-2-methylimidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-bis (4-cyanobenzyl) -2-methylimidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-bis (2-butenyl) imidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-dibutylimidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-dicyclohexylimidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-bis (2-thienylmethyl) imidazo (2, 1-b) thiazol-6 (5H) -one,

spiro (2, 3-dihydroimidazo (2, 1-b) thiazol-6 (5H) -one-5, 2' -benzo (f) indane),

5, 5-dibutyl-2, 3-dihydroimidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-bis (2-butenyl) -2, 3-dihydroimidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-bis (4-methylbenzyl) -2, 3-dihydroimidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-bis (2-thienylmethyl) -2, 3-dihydroimidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-bis (4-fluorobenzyl) -2, 3-dihydroimidazo (2, 1-b) thiazol-6 (5H) -one,

5, 5-dibenzyl-2, 3-dihydroimidazo (2, 1-b) thiazol-6 (5H) -one,

spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2' -benzo (f) indane),

2-hydroxy-3- (2-naphthylmethyl) -imidazo (1, 2-a) pyridine,

3-benzylimidazo (1, 2-a) pyridin-2 (3H) -one,

spiro (5, 6, 7, 8-tetrahydroimidazo (1, 2-a) pyridin-2 (3H) -one-3, 2' -benzo (f) indane),

3, 3-dicyclohexyl-5, 6, 7, 8-tetrahydroimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-bis (2-thienylmethyl) -5, 6, 7, 8-tetrahydroimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dibutyl-5, 6, 7, 8-tetrahydroimidazo (1, 2-a) pyridin-2 (3H) -one,

3, 3-dipropyl-5, 6, 7, 8-tetrahydroimidazo (1, 2-a) pyridin-2 (3H) -one,

spiro (imidazo (1, 2-a) pyrimidin-2 (3H) -one-3, 2' -benzo (f) indane),

3, 3-bis (2-butenyl) imidazo (1, 2-a) pyrimidin-2 (3H) -one,

3, 3-bis (2-thienylmethyl) imidazo (1, 2-a) pyrimidin-2 (3H) -one,

3, 3-bis (4-fluorobenzyl) imidazo (1, 2-a) pyrimidin-2 (3H) -one,

3, 3-dicyclohexylimidazo (1, 2-a) pyrimidin-2 (3H) -one,

3, 3-bis (4-cyanobenzyl) imidazo (1, 2-a) pyrimidin-2 (3H) -one,

3, 3-bis (4-methylbenzyl) imidazo (1, 2-a) pyrimidin-2 (3H) -one,

4, 4-dibenzyl-1-methyl-5-oxo-4, 5-dihydroimidazole,

spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2 '- (4' -fluoroindane)),

spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2 '- (5' -methoxyindane)),

spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2 '- (5' -iodoindane)),

spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2 '- (4' -cyanoindane)),

spiro (imidazo (2, 1-a) isoquinolin-2 (3H) -one-3, 2' -indan),

spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2' - ((1, 2, 5-thiadiazole) (4, 5-c) indane)),

spiro (imidazo (2, 1-a) isoquinolin-2 (3H) -one-3, 2' - ((1, 2, 5-thiadiazole) (4, 5-c) indane)),

spiro (imidazo (1, 2-a) pyrimidin-2 (3H) -one-3, 4' - (1-cyclopentene)),

spiro (imidazo (1, 2-a) pyrimidin-2 (3H) -one-3, 2' -indane),

spiro (imidazo (1, 2-a) pyrimidin-2 (3H) -one-3, 2' - ((1, 2, 5-thiadiazole) (4, 5-c) indane)),

spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2 '- (5' -trifluoromethylindane)),

spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2' -benzo (e) indane),

spiro (imidazo (2, 1-a) isoquinolin-2 (3H) -one-3, 1 '- (3' -cyclopentene)),

spiro (8-benzyloxyimidazo (1, 2-a) pyridin-2 (3H) -one-3, 1 '- (3' -cyclopentene)),

spiro (7, 8, 9, 10-tetrahydroimidazo (2, 1-a) isoquinolin-2 (3H) -one-3, 1' -cyclopentane),

spiro (imidazo (2, 1-a) isoquinolin-2 (3H) -one-3, 1' -cyclopentane), and

spiro (5, 6, 7, 8-tetrahydroimidazo (1, 2-a) pyridin-2 (3H) -one-3, 2' -indane).

In another embodiment, the compound is spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2' -indan).

In another embodiment, the compounds disclosed in U.S. application No. 11/872,408 (published as US 2008/0103157 a 1); U.S. application No. 11/872,418 (published as US2008/0103158 a 1); U.S. patent application No. 6,635,652; U.S. patent application No. 7,141,579; and international application No. PCT/JP2007/070962 (published as WO2008/047951), each of which is incorporated by reference in its entirety.

Compound ST101, also known as ZSET 1446, has shown pharmacological activity in rodent models of learning and memory associated with AD, both after short-term (single dose) and long-term administration. The chemical name of ST101 is spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2' -indan).

For example, ST101 significantly improves age-related memory impairment and attenuates memory loss induced by the loss of memory of chemical agents such as methamphetamine, glutamate receptor antagonists, MK-801 and muscarinic antagonists, hyoscyamine. (Yamaguchi Y., et al, J.Pharmacol. exp. Ther.317: 1079-87 (2006); Ito Y., et al, J.Pharmacol. exp. Ther.320: 819-27 (2007)).

Experiments have shown that ST101 enhances the stimulated release of nicotine from acetylcholine (ACh), increases extracellular ACh concentrations in the cerebral cortex, and increases the intracellular concentrations of ACh and dopamine in the hippocampus. The width of the model through which ST101 exerts its effect indicates the likelihood of participating in an upstream target in the signaling pathway associated with these processes.

ST101 also has a demonstrated effect in aging-accelerated mice 8(SAMP8), a mouse strain that develops age-related deficits in learning and memory with the accumulation of a β -like deposits in brain tissue. In Morley, j.e., Biogerontology 3: SAMP8 mice are discussed in 57-60 (2002). ST101 reduced the accumulation of a β -like deposits and also resulted in improved learning and memory functions, suggesting that the behavioral effects of ST101 may be associated with a reduction in a β production and/or deposits. See US 2008/103158 a 1.

All patents, patent applications, and publications discussed herein are incorporated by reference in their entirety.

Example 1

In vitro Effect of ST101 on beta-amyloid in N2a cultured cells

N2a is known to prepare amyloid peptide A β in quantities measurable by ELISA assays_1-40And Abeta_1-42Murine neuroblastoma cell of (1). These forms of a β are associated with pathology in the AD brain, and it is specifically assumed that a β_1-42Has the ability to block the alpha 7 nicotinic receptor and produce direct neurotoxic effects. N2a cells were treated for 24 hours using ST101 added to tissue culture medium. For the presence of a β, tissue culture medium was collected and analyzed by ELISA.

Fig. 1A and 1B are bar graphs depicting the effect of compound ST101 on a β production by N2a cells. Figure 1A is a bar graph depicting a β concentration in cell culture medium as a function of ST101 concentration compared to control. FIG. 1B is a graph depicting A.beta.versus control_1-42To Abeta_1-40As the concentration of ST101Bar graph of the function. As shown in FIGS. 1A and 1B, ST101 significantly reduced A β_1-42To A beta_1-40Without major impact (figure 1).

Example 2

Effect of ST101 on 3 × Tg-AD mice in the Morris Water maze

FrankLaFerla laboratories, University of California, at the University of California, has developed transgenic mice (β APPSwwe, PS1M146V and tauP301L) containing 3 mutants associated with Alzheimer's disease pathology (Oddo et al, Triple-transgenic model of AD with sequences and variants: intracellular A β and synthetic dysfunction, Neuron 39 (3): 409-21 (2003)). These mutants transfer APP cleavage from alpha-secretase to beta-secretase, increasing A beta_1-42And promoting aggregation of tau into paired helix-like fibers. The basic features of developing AD in an age-dependent manner in 3 × Tg-AD animals are defects in memory-related behavioral functions, plaque and tangle lesions, and synaptic dysfunction, including defects of long-term potentiation, activities believed to be critical for memory (odd et al, 2003). In addition, plaque formation precedes tangle formation, thus mimicking human AD development. To date, 3 × Tg-AD mice represent one of the closest mature animal models of AD.

ST101 dosing and testing methods

3 × Tg-AD mice aged about one year were treated with ST101 for 2 months. The average dose of 5 mg/kg/day was given as drinking water (calculated dose based on average water consumption). Behavioral effects were tested by evaluating the behavior of the Morris water maze. Biochemical effects were examined by measuring brain content of a β and APP by ELISA and Western immunoblotting.

Behavioral effects: behaviour of 3 × Tg-AD mice in the Morris Water Maze (MWM), adapted from Roozendaal et al, Proc.Natl.Acad.Sci U.S.A.100: 1328-1333(2003).

MWM tests spatial memory (i.e., hippocampal dependency) and clue learning (i.e., non-hippocampal) in rodents. The labyrinth is a circulating water trough filled with opaque water. The mice were placed in water and had to swim to find and escape onto a platform submerged 1.5cm below the water surface. The time (in seconds) required to find the platform was recorded. Animals rely on visual cues in the room containing the sink to find the platform on successive trials. Training was performed daily for seven consecutive days.

Memory of the training was evaluated twice at 24 and 72 hours after the final training experiment. Animals were allowed to swim freely in the sink with the platform removed for 60 seconds. Parameters tested included (1) latency: time required to reach previous stage position and (2) number of passes: number of times the animal roamed past the previous platform position. A decrease in latency and an increase in the number of passes indicate an increase in spatial memory and lead learning.

FIGS. 2A, 2B and 2C are graphs depicting the effect of ST101 on 3 × Tg-AD mice in MWM. Fig. 2A is a line graph depicting latency (in seconds) during training compared to control mice. Fig. 2B and 2C are bar graphs depicting latency (in seconds) at 24 and 72 hours after training in ST101 treated animals and control mice.

As shown in fig. 2A, ST101 and control animals had similar latency on the first day of training. However, ST101 treated mice trained for several consecutive days showed lower latency compared to controls. Figures 2B and 2C also demonstrate a decrease in latency and an increase in number of passes during the 24 and 72 hour memory tests. These data demonstrate that ST101 improves learning and memory behavior of 3 × Tg-AD mouse strains that closely resemble human AD.

Example 3

Effect of ST101 on A.beta.in brain tissue from 3 × Tg mouse-AD

Biochemical effects: ST101 and amyloid processing pathways

At the end of the 2 month treatment period, 3 × Tg mice were sacrificed and brain tissue was processed. In the first set of assays, soluble A.beta.was quantified by ELISA_1-40And Abeta_1-42And insoluble a β (after formic acid extraction). Soluble a β represents a protein that has been processed and released from full-length APP. Insoluble a β represents the fibrous aggregates that eventually precipitate in amyloid plaques.

FIGS. 3A and 3B are bar graphs depicting the effect of ST101 on A β in brain tissue from 3 × Tg-AD mice. FIG. 3A is a graph depicting soluble A β in brain tissue of mice treated with ST101 relative to control mice_1-40And Abeta_1-42Bar graph of the amount of (c). FIG. 3B is a graph depicting insoluble A β in mice treated with ST101 relative to control mice_1-40And Abeta_1-42Bar graph of the amount of (formic acid extract). One animal in the group treated with ST101 in plate a was excluded due to analysis of artifacts.

As shown in FIGS. 3A and 3B, ST101 treated mice induced soluble A β_1-42Significantly reduced levels and soluble a β_1-40And is suitably reduced. Insoluble a β is not affected. These results indicate that ST101 may affect the production or release of a β, but it has no measurable effect on a β that has formed insoluble aggregates.

Example 4

C-terminal fragment of APP determined by antibody CT20

In an attempt to determine which portion of the a β processing/release pathway ST101 was effective, a series of Western immunoblot analyses from the same mouse brain extracts were performed. These Western immunoblots examine intact APP and its products of post-translational processing and subsequent degradation.

FIG. 4 is a Western immunoblot depicting the C-terminal fragment of APP determined by antibody CT20 in the brain of ST 101-treated (S)3 × Tg-AD mice relative to untreated (C)3 × Tg-AD mice.

As shown in figure 4, antibody CT20 (directed to the C-terminus of APP) showed a substantial reduction in C99 and C83C-terminal APP fragments. These fragments are by-products of beta-secretase and alpha-secretase cleavage, respectively. It also shows the appearance of a new, longer C-terminal fragment with a molecular weight of about 17kDa(by^*Representation).

Example 5

APP and degraded fragments determined by antibody CT20

FIG. 5 is a Western immunoblot depicting APP and degraded fragments determined by antibody CT20 in the brain of ST 101-treated (S)3 × Tg-AD mice relative to untreated (C)3 × Tg-AD mice. "CT 20" represents full-length APP and "actin" represents an anti- β -actin antibody as a protein loading control.

Western immunoblot analysis full-length untreated APP was determined in all extracts (figure 5,^*). Subtle band shifts indicate additional ST 101-induced APP modifications, such as a slight decrease in molecular weight of certain full-length molecules (possible changes in glycosylation, phosphorylation, or other post-translational modifications) and the disappearance or significant reduction of the major APP degradation intermediate (-50 kDa) (figure 5,^**)。

example 6

Alternative starchy body processing routes

FIG. 6 is a graph depicting proposed amyloid processing pathways leading to novel C-terminal fragments of APP. The proposed pathway illustrates the appearance of a new approximately 17kD fragment shown in the Western immunoblot of FIG. 4. The fragment is generated by N-terminal cleavage of the non-characteristic position at about 60 amino acids to the beta-secretase cleavage site.

Since the general products of these cleavages are significantly reduced, the new pathway appears to preferentially replace alpha-secretase cleavage and beta-secretase cleavage (C83 and C99 are used for alpha-secretase and beta-secretase, respectively), so the cleavage sites targeting these enzymes remain unchanged.

This change in APP metabolism induced by ST101 is accompanied by a significant improvement in learning and memory tasks in animal models arguably considered to be the closest representative of clinical AD. When considered in conjunction with earlier non-clinical data, it was shown that ST101 can be manipulated upstream of the physiological processing of commercially available agents and those in scientific research currently having known mechanisms of action, and thus represent a new approach to AD treatment.

Example 7

C-terminal fragment of APP determined by antibody CT20

3 × Tg-AD mice aged about 3 months were treated with ST101 for 10 months. The average dose administered as drinking water was 5 mg/kg/day or 1 mg/kg/day or 0.1 mg/kg/day (calculated doses based on average water consumption).

FIGS. 7A-C are Western immunoblots depicting APP C-terminal fragments determined by antibody CT20 in the brain of ST 101-treated (S)3 × Tg-AD mice relative to untreated (C)3 × Tg-AD mice.

As shown in FIGS. 7A-7C, antibody CT20 (directed to the C-terminus of APP) is shown in FIG. 5,^**the effects shown are similar in some samples with disappearance or significant reduction of the major APP degradation intermediate (-50 kDa). The appearance of a new, longer C-terminal fragment of approximately 17kDa molecular weight in some samples similar to the effect shown in FIG. 4 is also shown.

Example 8

APP C-terminal fragments determined by antibody 1565-1

3 × Tg-AD mice aged about 12 months were treated with ST101 for 2.5 months. The average dose administered as drinking water was 5 mg/kg/day (calculated dose based on average water consumption).

FIG. 8 is a Western immunoblot depicting C-terminal fragments determined by antibody 1565-1 (Epitomics) in the brain of ST 101-treated (S)3 × Tg-AD mice relative to untreated (C)3 × Tg mouse-AD.

As shown in fig. 8, antibody 1565-1 (directed against a peptide near the C-terminus of APP) showed disappearance or significant reduction of the APPC99 fragment. No longer C-terminal fragment with a molecular weight of about 17kDa was observed.

The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims (21)

1. A heterocyclic compound of the general formula (I) or a pharmaceutically acceptable salt, hydrate or prodrug thereof for use in inducing cleavage of APP to produce a carboxy-terminal fragment of APP of about 17 kilodaltons (kDa):

wherein the about 17kDA fragment comprises the carboxy-terminal amino acid sequence of APP and the beta-amyloid amino acid sequence, and

wherein each R_x、R₁、R₂、R₃、R₄As defined herein.

2. The use according to claim 1, wherein the heterocyclic compound is spiro (imidazo (1, 2-a) pyridin-2 (3H) -one-3, 2' -indan).

3. The use of claim 1, wherein said use of said heterocyclic compound results in a β of amyloid precursor protein_1-42、Aβ_1-40One or more of fragment C99 or fragment C83 of amyloid precursor protein.

4. The use of claim 1, wherein the subject to whom the compound is administered has alzheimer's disease.

5. The use of claim 4, wherein the subject has been diagnosed with Alzheimer's disease.

6. An isolated approximately 17 kilodalton amyloid precursor protein fragment comprising the carboxy-terminal amino acid sequence of amyloid precursor protein and an amyloid-beta amino acid sequence.

7. A composition comprising the fragment of claim 6.

8. A method for screening compounds that cleave amyloid precursor protein to produce an approximately 17 kilodalton fragment of amyloid precursor protein, the method comprising:

(a) exposing cells producing amyloid precursor protein or fragments thereof to a test compound, and

(b) the amount of about 17 kilodalton fragments was determined,

wherein the about 17 kilodalton fragment comprises the carboxy-terminal amino acid sequence of amyloid precursor protein and the amyloid-beta amino acid sequence, and

wherein an increase in the amount of the about 17 kilodalton fragment in a cell exposed to the compound relative to the amount of the about 17 kilodalton fragment in a cell not exposed to the compound indicates that the compound induces amyloid precursor protein cleavage to produce the about 17 kilodalton fragment.

9. The method of claim 8, further comprising (c) A β relative to amyloid precursor protein in cells not exposed to the compound_1-42、Aβ_1-40C99 fragment or the C83 fragment of amyloid precursor protein, determining the A beta of amyloid precursor protein in cells exposed to said compound_1-42、Aβ_1-40Or a fragment of C99 or a fragment of C83 of the amyloid precursor protein.

10. The method of claim 1, wherein the screening method is performed in vitro.

11. The method of claim 8, further comprising (c) A β relative to amyloid precursor protein in cell culture medium of cells not exposed to the compound_1-42、Aβ_1-40C99 fragment or the C83 fragment of amyloid precursor protein, and determining the A β of amyloid precursor protein in a cell culture lysate of cells exposed to the compound_1-42、Aβ_1-40Or a fragment of C99 or a fragment of C83 of the amyloid precursor protein.

12. The method of claim 8, wherein the screening method is performed in a high throughput manner.

13. A method for screening compounds that cleave amyloid precursor protein to generate an APP fragment of about 17 kilodaltons, the method comprising:

(a) exposing cells producing amyloid precursor protein or fragments thereof to a test compound, and

(b) determining said about 17 kilodaltons fragment,

wherein said about 17 kilodalton fragment comprises the carboxy-terminal amino acid sequence of said amyloid precursor protein and the amyloid-beta amino acid sequence, and

wherein the presence of the about 17 kilodalton fragment in a cell exposed to the compound relative to the absence of the about 17 kilodalton fragment in a cell not exposed to the compound indicates that the compound induces cleavage of amyloid precursor protein to produce the about 17 kilodalton fragment.

14. The method of claim 13, further comprising (c) a β relative to amyloid precursor protein in cells not exposed to the compound_1-42、Aβ_1-40C99 fragment or the C83 fragment of amyloid precursor protein, and determining the A β of amyloid precursor protein in cells exposed to the compound_1-42、Aβ_1-40Or a fragment of C99 or a fragment of C83 of the amyloid precursor protein.

15. The method of claim 13, wherein the screening method is performed in vitro.

16. The method of claim 13, further comprising (c) a β relative to amyloid precursor protein in cell culture medium of cells not exposed to the compound_1-42、Aβ_1-40C99 fragment or the C83 fragment of amyloid precursor protein, and determining the A β of amyloid precursor protein in a cell culture lysate of cells exposed to the compound_1-42、Aβ_1-40Or a fragment of C99 or a fragment of C83 of the amyloid precursor protein.

17. The method of claim 13, wherein the screening method is performed in a high throughput manner.

18. A compound other than a heterocyclic compound of formula (I), or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, for use in inducing cleavage of APP to produce a carboxy-terminal fragment of APP of about 17 kilodaltons (kDa):

wherein the about 17kDA fragment comprises the carboxy-terminal amino acid sequence of APP and the beta-amyloid amino acid sequence, and

wherein each R_x、R₁、R₂、R₃、R₄As defined herein.

19. The use of claim 18, wherein the compound causes a β of amyloid precursor protein_1-42、Aβ_1-40One or more of fragment C99 or the fragment C83 of the amyloid precursor protein.

20. The use of claim 1, wherein the subject to whom the compound is administered has alzheimer's disease.

21. The use of claim 20, wherein the subject has been diagnosed with alzheimer's disease.