KR101721608B1 - Pharmaceutical composition for treating or preventing degenerative brain disease comprising multi-targeting compound - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment or prevention of degenerative brain diseases comprising as an active ingredient a compound targeting multiple targets for further improving the therapeutic efficiency of degenerative brain diseases such as Alzheimer's disease. The compound represented by formula (2) not only induces the assembly process of the amyloid beta peptide to form an aggregate that does not exhibit toxicity under all or no conditions of metal such as copper or zinc, but also forms amyloid beta peptide, metal-amyloid beta peptide, Can be used as a very useful therapeutic or health food for degenerative brain diseases including Alzheimer's disease because it can react with multiple targets of Alzheimer ' s disease at the same time and suppress toxicity.

Description

[0001] The present invention relates to a pharmaceutical composition for treating or preventing a degenerative brain disease comprising a compound targeting a multiple target as an active ingredient,

The present invention relates to a pharmaceutical composition for the treatment or prevention of degenerative brain diseases comprising as an active ingredient a compound targeting multiple targets for further improving the therapeutic efficiency of degenerative brain diseases such as Alzheimer's disease.

Degenerative brain disease is an aging-related disease caused by the inability of neuronal cells to function, and with the rapid increase of the aging population, social interest in degenerative brain diseases is growing.

Degenerative brain diseases are classified according to the clinical symptoms and the area of the affected brain. Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis and amyotrophic sclerosis lateral sclerosis).

Alzheimer's disease destroys the nerves of the central nervous system, especially the fore brain, amygdala, hippocampus and cerebral cortex, and the limbic system, which are responsible for learning, memory, thinking, Emotional regulation, and so on. In particular, the lack of neurotransmitters such as acetylcholine in Alzheimer's disease is the most important indicator, and restoring this is one of the goals of Alzheimer's disease treatment.

As Alzheimer's disease, parasympathetic drugs can be divided into muscarinic agonists or nicotine agonists, cholinesterase inhibitors (ChEIs), and drugs that indirectly control acetylcholine release. Many of these drugs have been developed and used as ChEIs, including tacrine, physostigmine, donepezil, rivastigmin, and memantin.

Second-generation drugs, such as donepezil and rivastigmin, have longer duration and stability and higher blood-brain barrier (BBB) permeability than first-generation drugs such as tacrine, There is a high tendency. While the first-generation drugs selectively inhibit AChE, butyrylcholinesterase, and other peripheral cholinesterases, some newer drugs are known to have a high selectivity for AChE and thus to reduce the incidence of peripheral adverse events. The mechanism of action of these drugs is known to be due to the increase of ACh concentration in synapses by inhibiting the decomposition of acetylcholine (ACh).

Such conventional treatments for Alzheimer's disease have serious side effects due to long-term use. Therefore, it is necessary to develop new drugs having similar or better pharmacological efficacy and fewer side effects.

Korean Patent Laid-Open Publication No. 20100-009415 (published on Jan. 21, 2010)

It is an object of the present invention to provide a pharmaceutical composition for the treatment or prevention of degenerative brain diseases comprising as an active ingredient a compound targeting multiple targets for further improving the therapeutic efficiency of degenerative brain diseases such as Alzheimer's disease.

Another object of the present invention is to provide a health food for preventing or ameliorating degenerative brain diseases, which contains a compound targeting a multiple target as an active ingredient.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for treating or preventing degenerative brain diseases, which comprises a compound represented by the following general formula (1) or (2) as an active ingredient:

[Chemical Formula 1]

Wherein R ¹ to R ³ are the same or different from each other and are any one selected from the group consisting of hydrogen, halogen, di (C 1 to C 4 alkyl) amino and carboxyl, R ⁴ and R ⁵ are the same as or different from each other, and any one selected from the group consisting of hydrogen or C1 to C4 alkyl, n ₁ and n ₂ are each the same or different from one another, may be an integer of 0 or 1.

(2)

The present invention also provides a health food for preventing or ameliorating degenerative brain diseases comprising the compound represented by the above formula (1) or (2) as an active ingredient.

The present invention relates to a therapeutic agent for degenerative brain diseases comprising, as an active ingredient, a compound targeting multiple targets for further improving the therapeutic efficiency of degenerative brain diseases such as Alzheimer's disease. The compound according to the present invention is characterized in that the aggregation process of amyloid beta peptide Or aggregation that does not exhibit toxicity in all conditions with or without metals such as zinc, as well as toxicity by reacting simultaneously with multiple targets of Alzheimer's disease, such as amyloid beta peptide, metal-amyloid beta peptide, metal and activated oxidizing species It can be used as a very useful therapeutic or health food in degenerative brain diseases including Alzheimer's disease.

Figure 1 is N, N- dimethyl -p- phenylenediamine for the metal or the metal in a variety of conditions induced Aβ aggregation ₄₀ [N, N-dimethyl-p -phenylenediamine; DMPD] and the effect of DMPD on metal-free or metal-treated Aβ ₄₀ triggered cytotoxicity.
Figure 2 shows the interaction between DMPD and monomer A [beta] ₄₀ .
Figure 3 shows the bond between DMPD and Zn (II).
Figure 4 shows the interaction between Cu (I) - / Cu (II) -A fibril and DMPD via copper K-boundary X-ray absorption spectroscopy.
Figure 5 shows the variation of DMPD with or without Cu (II) or A? ₄₀ by UV-vis.
FIG. 6 shows the result of mass spectrometry and mass spectrometry analysis of the interaction between DMPD and A? ₄₀ .
FIG. 7 is a histopathological evaluation of the amount of A? ₄₀ / A? ₄₂ and the load of amyloid deposition in the brain after administration of DMPD in mice.
FIG. 8 shows the effect of improving the cognitive disorder after administering DMPD to the mouse.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for treating or preventing a degenerative brain disease comprising a compound represented by the following formula (1) or (2) as an active ingredient:

[Chemical Formula 1]

Wherein R ¹ to R ³ are the same or different from each other and are any one selected from the group consisting of hydrogen, halogen, di (C 1 to C 4 alkyl) amino and carboxyl, R ⁴ and R ⁵ are the same as or different from each other, and any one selected from the group consisting of hydrogen or C1 to C4 alkyl, n ₁ and n ₂ are each the same or different from one another, may be an integer of 0 or 1.

(2)

Wherein R ¹ to R ² are any one selected from hydrogen, halogen or di (C1 to C4 alkyl) amino, and R ³ is any one selected from hydrogen, di (C1 to C4 alkyl) amino, .

In Formula 1, one of R ¹ to R ³ is selected from dimethylamino or carboxyl, and the remaining substituents are the same or different, and may be selected from hydrogen or halogen.

The compound represented by the above formula (1) or (2) may be prepared by reacting N, N-dimethyl-p-phenylenediamine, N ¹ , N ¹ -dimethylbenzene- ^{[N 1, N 1 -dimethylbenzene-} 1,2-diamine], N 1, N 1 - dimethyl-benzene-1,3-diamine ^{[N 1, N 1 -dimethylbenzene-} 1,3-diamine], 4- ( amino methyl) -N, N- dimethylaniline [4- (aminomethyl) -N, N -dimethylaniline], 4 - (( dimethylamino) methyl) aniline [4 - ((dimethylamino) methyl ) aniline], N 1, N 1 -dimethyl-1,4-cyclohexane-diamine ^{[N 1, N 1 -dimethyl-} 1,4-cyclohexanediamine], 4- ( dimethylamino) benzoic acid [4- (dimethylamino) benzoic acid], 4-aminobenzoic acid [4- aminobenzoic acid], 2- (4-aminophenyl) acetic acid [2- (4- (dimethylamino) phenyl) acetic acid] ], 2-fluoro--N ^1, N ¹ - dimethyl-benzene-1,4-diamine [2-fluoro-N1, -N 1, with N1-dimethylbenzene-1,4-diamine], and 3-fluoro-N ¹ - Dimethylbenzene-1,4-diamine [3-fluoro-N1, N1-dimethylbenze ne-1,4-diamine].

The compound represented by Formula 1 or Formula 2 induces the aggregation process of the amyloid beta peptide to induce aggregation that does not exhibit toxicity under all conditions with or without metal such as copper or zinc, as well as amyloid beta peptide, metal-amyloid beta Peptide, metal, and activated oxidative species can be used as a therapeutic or healthful food for degenerative brain diseases including Alzheimer's disease because they can react with multiple targets of Alzheimer's disease all at once to inhibit toxicity.

The degenerative brain disease may be any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, dementia, Huntington's disease, multiple sclerosis, proximal lateral sclerosis, stroke, stroke and hardness cognitive disorders, Lt; / RTI >

The pharmaceutical compositions may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions.

Examples of the carrier, excipient or diluent which can be used in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose sucrose), lactose, gelatin, and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The amount of the compound which is an active ingredient of the pharmaceutical composition according to the present invention may vary depending on the age, sex, body weight and disease of the patient, but it is preferably 0.001 to 100 mg / kg, preferably 0.01 to 10 mg / Or several times.

Further, the dose of the compound according to the present invention may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intratracheal, intrauterine or intracerebroventricular injections.

The present invention also provides a health food for preventing or ameliorating degenerative brain diseases comprising a compound represented by the following general formula (1) or (2) as an active ingredient:

[Chemical Formula 1]

Wherein R ¹ to R ³ are the same or different from each other and are any one selected from the group consisting of hydrogen, halogen, di (C 1 to C 4 alkyl) amino and carboxyl, R ⁴ and R ⁵ are the same as or different from each other, and any one selected from the group consisting of hydrogen or C1 to C4 alkyl, n ₁ and n ₂ are each the same or different from one another, may be an integer of 0 or 1.

(2)

The degenerative brain disease may be any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, dementia, Huntington's disease, multiple sclerosis, proximal lateral sclerosis, stroke, stroke and mild cognitive impairment.

The health food may be provided in the form of powder, granules, tablets, capsules, syrups or beverages. The health food may be used together with food or food additives other than the compound according to the present invention as an active ingredient, And the like. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.

The effective dose of the compound contained in the health food may be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range for the purpose of health and hygiene or long-term intake for the purpose of health control, Since the active ingredient has no problem in terms of safety, it can be used in an amount exceeding the above range.

There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

<Reference example> Preparation of reagents and instruments

All reagents were purchased from commercial vendors and used as is, unless otherwise specified. N, N- dimethyl - p - phenylene diamine [N, N-dimethyl-p -phenylenediamine; DMPD] were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aβ ₄₀ and Aβ ₄₂ were purchased from AnaSpec (Fremont, CA, USA) (Aβ ₄₂ = DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA).

An Agilent 8453 UV-vis spectrophotometer (Santa Clara, Calif., USA) was used to measure the optical spectra and the anaerobic reaction was monitored using a glove box filled with N ₂ (Korea Kiyon, Bucheon-si , Gyeonggi-do, Republic of Korea). Thermodynamic parameters were measured with a VP isothermal titration calorimeter (MicroCal, Northampton, MA, USA).

Transmission electron microscopy (TEM) images were acquired using a Philips CM-100 transmission electron microscope (Philips CM-100 transmission electron microscope, Microscopy and Image Analysis Laboratory, University of Michigan, Ann Arbor, Was obtained using a transmission electron microscope (UNIST Central Research Facilities, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea).

Absorbance values for cell viability analysis were determined using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, Calif., USA).

The Bruker HCT basic system mass spectrometer was equipped with an electrospray ionization (ESI) ion source and was used to obtain mass spectra over time of DMPD incubated with Aβ.

All ion mobility-mass spectrometry (IM-MS) experiments were performed on Synapt G2 (Waters, Milford, Mass., USA).

NMR analysis of DMPD was performed using a 400 MHz Agilent NMR spectrometer with or without Zn (II). NMR analysis of DMPD and Aβ was performed using a TCI triple-resonance inverse detection CryoProbe (Michigan State University, Lansing, MI, USA) Was carried out using a 900 MHz Bruker spectrophotometer.

Example 1: Analysis of the effect of DMPD on Aβ aggregation

1) Aβ aggregation experiment

Experiments on Aβ existing known methods (Proc Natl Acad Sci USA 107, 21990-21995, 2010;....... Proc Natl Acad Sci USA 110, 3743-3748, 2013;.. J. Am Chem. Soc . 136, 299-310, 2014; J. Am. Chem . Soc . 131, 16663-16665, 2009; Inorg. , 51, 12959-12967, 2012).

Aβ ₄₀ or Aβ ₄₂ were dissolved in ammonium hydroxide (NH ₄ OH, 1% v / v aq), lyophilized overnight and stored at -80 ° C. For this experiment, a Stock solution of Aβ was prepared by dissolving lyophilized peptides in 1% NH ₄ OH (10 μL) and diluting with ddH ₂ O (deionized distilled water).

The concentration of Aβ peptide in the solution was determined by measuring the absorbance of the solution at ^{280 nm (ε = 1450 M -1} cm -1 for Aβ 40; ε = 1490 M -1 cm -1 for Aβ 42).

The peptide stock solution contained HEPES [4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid; 20 [mu] M; pH 6.6 for Cu (II) samples; pH 7.4 for metal-free and Zn (II) samples] and NaCl (150 μM) to a final concentration of 25 μM in a buffered solution.

To review DMPD influence of the inhibition Aβ aggregate formation, metal chloride salts (CuCl ₂ or ZnCL _2, 25 μM) samples of Aβ in the case two types of the non-existent or non-existent, as in the Figure 1b (i) ( 25 μM) was added DMPD (50 μM; 1% v / v dimethyl sulfoxide (DMSO)) and reacted at 37 ° C. for 24 hours with constant stirring.

To investigate the effect of DMPD on the degradation of A [beta] aggregation, A [beta] was prepared in two cases in which metal ions were present or not before treatment of DMPD (50 [mu] M) And reacted at 37 캜 for 24 hours with stirring.

2) Gel electrophoresis and western blot

Samples were analyzed by gel electrophoresis and Western blotting using an anti-Aβ antibody (6E10).

Each sample (10 μL) was separated on a 10-20% tris-tricine gel (Invitrogen, Grand island, NY, USA) and the separated protein sample was loaded onto a Tris buffer (Bovine serum albumin, BSA, 3% w / v, Sigma-Aldrich, St. Louis, Mo., USA) in physiological saline (TBS-T, 1.00 mM Tris base, pH 8.0, 1.50 mM NaCl) Transferred to a nitrocellulose membrane at room temperature for 2 hours.

The membranes were then reacted overnight at 4 ° C with primary antibody (6E10, Covance, Princeton, NJ, USA; 1: 2000) in a solution of 2% w / v BSA (in TBS-T). After washing with TBS-T (10 min, 3 times), the cells were treated with a goat anti-mous (HBS) conjugated with horseradish peroxidase in 2% w / v BSA ) Secondary antibody (1: 5000; Cayman Chemical Company, Ann Arbor, MI, USA) for 1 hour at room temperature.

Protein bands were then visualized using a SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, Ill., USA).

3) Transmission Electron Microscopy (TEM)

Aβ samples in inhibition and degradation experiments were treated with glow-discharged grids (Formar / Carbon 300-mesh, Electron Microscopy Sciences, Hatfield, PA, USA) for 2 min at room temperature.

Excess buffer was carefully removed using filter paper and blotting, washed twice with ddH ₂ O and each grid was washed with uranyl acetate staining solution (1% w / v ddH ₂ O, 5 mL) for 1 min Lt; / RTI &gt; After excess dyeing solution was drawn, the grid was air dried at room temperature for at least 20 minutes.

Images of each sample were taken at 25,000x magnification using Philips CM-100 (80 kV) or JEOL JEM-2100 TEM (200 kV).

4) Measurement of cell viability

The SK-N-BE (2) -M17 (M17) cell line, a human neuroblastoma, was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Cells were cultured in a medium containing 1: 1 of a minimum essential medium (MEM; GIBCO, Life Technologies, Grand Island, NY, USA) and Ham's F12K Kaighn's Modification Media (F12K; GIBCO), 10% ml penicillin (GIBCO), and 100 mg / mL streptomycin (GIBCO) in the presence of 5% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, % CO ₂ is growing in a humidity of 37 ℃ supplied and maintained environment was maintained.

M17 cells were cultured in 96 well-plates according to the known method ( Proc . Natl . Acad . Sci . USA 107, 21990-21995, 2010; J. Am. Chem . Soc . 131, 16663-16665, 2009) 15,000 cells / 100 μL.

At this time, CuCl ₂ or ZnCl ₂ to the cells: the presence of a (1: 1 metal / ligand ratio) or in the absence, Aβ ₄₀ in the presence or absence of various concentrations under (Aβ: metal: ligand = 10:: 10 20 μM) DMPD ( 0-10 [mu] M, 1% v / v DMSO).

After incubation at 37 ° C for 24 hours, 25 μL of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide; Phosphate buffered saline (PBS, pH 7.4, GIBCO) supplemented with 5 mg / mL was added to each well and reacted at 37 ° C for 4 hours.

Formazan formed by the cells was dissolved in N, N-dimethylformamide (DMF, 50% v / v aq, pH 4.5) and sodium dodecyl sulfate (SDS, 20% w / v) at room temperature overnight, and the absorbance was measured at 600 nm with a microplate reader.

5) Experimental results

The inhibition of aggregation formation showed different molecular weight distributions in DMPD-treated Aβ ₄₀ / Aβ ₄₂ with or without metal (left side in FIG. 1 c), compared with the group without DMPD treatment, and was either amorphous Aβ aggregates or shorter, (Fig. 1D, left). And, in the aggregate degradation experiments DMPD is previously produced metal-free Aβ ₄₀ / Aβ ₄₂ aggregates and metal -Aβ ₄₀ / Aβ ₄₂ shows the effect of interest to focus on agglomerate strain (Fig. 1c on the right), whether metallic or not for the Aβ aggregates ₄₂ Showed a lower reactivity and a mixture of amorphous A [beta] aggregates, shorter fibrils or two A [beta] sequences was observed in the sample treated with DMPD (Fig. 1d, right).

In addition, even when A [beta] ₄₀ was treated with DMPD in cell culture medium, a distinct change in the molecular weight distribution of A [beta] species was observed (Fig. Thus, it has been found that the treatment of DMPD not only inhibits A [beta] aggregation formation in metal-free or metal-induced A [beta] aggregates, but also transforms already formed aggregates into relatively smaller, less structurally toxic A [beta] aggregates .

Moreover, the Aβ DMPD ₄₀ - or metal -Aβ ₄₀ - was increased to 10-20% cell viability compared to the cells without any treatment, even when the introduction to the treated M17 cells (Fig. 1f).

Example 2: Analysis of interaction between A? And DMPD

Interaction of DMPD with metal free Aβ was analyzed by isothermal titration calorimetry (ITC) analysis, 2D NMR spectroscopy and MD simulation.

1) Isothermal titration calorimetry (ITC) analysis

A solution of DMPD (200 μM, 10% v / v DMSO) and Aβ ₄₀ (20 μM) dissolved in 20 mM HEPES (pH 7.4, 150 mM NaCl) was prepared as a ligand solution and degassed by ITC Analysis was performed.

The ligand solution (10 μL) was titrated into Aβ ₄₀ solution (1.4 mL) at 25 ° C. for 25 seconds at 1 second, with a constant interval of 200 s using a 250 μL syringe rotation at 310 rpm. In a control experiment, the same titrant solution was injected into the same buffer together with A? ₄₀ to measure the heat of the diluted solution.

The rational row of combined values was calculated by subtracting the column of diluted solution values from the total column variation. Titration data were analyzed using MicroCal Origin (v.7.0). Ka values (binding constants) and ΔH (binding heat exchange) of each ligand upon binding to Aβ ₄₀ were measured in a suitable fitting model. Bonding curves were most suitable for the three bond sites and sequential bond models. The values of -TΔS and ΔG were calculated from Gibb's free energy relationship [( ΔG = ΔH - TΔS ; ΔG = - RT ln ( Ka )].

Thermodynamic parameters for A [beta] _40- DMPD interactions are shown in Table 1 below and the binding between A [beta] _40- DMPD was thermodynamically favorable due to the significant contribution of hydrophobic binding.

[Table 1]

2) 2D Band-Selective Optimized Flip-Angle Short Transient (SOFAST) - Heteronuclear Multiple Quantum Correlation (HMQC) NMR Spectroscopy

NMR titration experiments were carried out according to known methods ( Proc . Natl . Acad . Sci . USA 110, 3743-3748, 2013; J. Am. Chem . Soc . 136, 299-310, 2014; Chem . Commun . 50, 5301-5303 , 2014).

NMR samples with 1 mM NaOH (pH 10) and frozen at 1% NH ₄ OH and again suspended in 100 μL dry peptide ¹⁵ N- labeled ^{_{Aβ 40 (15 N-labeled Aβ}} 40, rPeptide, Bogart, GA, USA) of Prepared.

At this time, the peptide was diluted with 200 mM of phosphate buffer (pH 7.4) and 1 M NaCl, D ₂ O, and water to a final concentration of 80 μM. Each spectrum was obtained using 256 complex t1 points (256 complex t1 points) and 1 second recycle delay at 4 ° C.

2D data were processed using TopSpin 2.1 (TopSpin 2.1, Bruker, Billerica, MA, USA). The resonant frequency is specified guidelines (Biochem Biophys Res Commun 411, 312-316 , 2011;........ Angew Chem Int Ed 50, 5110-5115, 2011) a machine with the specified issued for Aβ according to the And a computer-aided resonance assignment (CARA).

The compiled chemical shift perturbation (CSP) was calculated using the following equation:

Titration of DMPD with ¹⁵ N-labeled A [beta] ₄₀ resulted in considerable chemical shift values (CSPs) in the six amino acid residues, particularly L17, F20, G33, G37, V39 and V40 as shown in FIGS. 2A and 2B. These residues correspond to self-recognition (residues 17-21) and the C-terminal hydrophobic region and are known to be important for Aβ aggregation and cross-b-sheet formation through hydrophobic interactions. CSP for V40 is thought to be due to the endogenous C-terminal problem rather than by interaction with DMPD. The distribution of observed CSP suggests that DMPD interacts with the self-recognition residues of A [beta] ₄₀ and the peptide framework near the hydrophobic residues, which is also supported by thermodynamic data on A [beta] _40- DMPD interaction.

3) Molecular dynamics (MD) simulation

A multistage computational strategy was used to explore the interaction of Aβ ₄₀ with DMPD.

In step 1, a 100 ns MD simulation in aqueous solution was performed to obtain the equilibrated structure of the A [beta] ₄₀ monomer. The simulation was performed using the GROMACS program (version 4.0.5) and the GROMOS 96 53A6 force field. The starting structure of the Aβ ₄₀ monomer was extracted from the NMR structure (model 2, PDB 1BA4) determined by aqueous SDS micelle at pH 5.1. The root-mean-square deviations (rmsd) showed that the reaction system reached equilibrium during the simulation time.

In the next step, through the simulation to include the flexibility of the Aβ ₄₀ monomers in the docking procedure was measured 100 times a snapshot of 1 nanosecond (ns) intervals. These snapshots were used for rigorous docking of DMPD molecules using AutoDock Vina 1.1.2 software. In this procedure, the receptor was held stationary, but the ligand was changed in its shape. The DMPD molecules were constructed using the GaussView program (B3LYP / Lanl3DZ) and optimized at the theoretical level using the Gaussian03 program. In the docking process, the size of the grid was chosen to occupy the entire receptor-ligand complex. Each docking trial produced 20 poses with 20 exhaustiveness values. The docking process provided 2000 poses. Based on the binding energy and the composition of the interaction sites, 20 different poses were selected for short-term (5 ns) MD simulations in aqueous solution.

From these 20 different simulations, five structures were derived and a 20 ns simulation was performed using the same program and force field. The simulation is provided a binding site comprising the L17, F19 and G38 residue of Aβ ₄₀ monomers. To analyze the trajectory and simulation structure, useful tools were used in the GROMACS program package and YASAFA software (v. 13.2.2).

The starting structure of all simulations is in a cubic box cut to a size of 7.0x7.0x7.0 nm. This excludes the undesirable effects that may arise from the applied periodic boundary conditions (PBC). The box was filled with a single point charge (SPC) water molecule. The reaction system was neutralized by replacing water molecules with little sodium and chlorine ions. The starting structure then minimized energy by a steep descent method for 3000 steps. As a result of this minimization, we have created a starting structure for MD simulation. The MD simulation was then performed with a constant number of particles (N), pressure (P) and temperature (T) (i.e., NPT ensemble).

The LINCS algorithm was used to limit the bond length of the peptide while the SETTLE algorithm was used to limit the bond length and angle of the water molecule. The Particle-Mesh Ewald (PME) method is implemented to handle long-range electrostatic interactions. A constant pressure of one bar was applied to the coupling constant of 1.0 ps. The peptides, water molecules and ions were bound to the water bath at 300 K, respectively, with a coupling constant of 0.1 ps. The kinetic equations were integrated at each 2 fs time step using the leap-frog algorithm.

The simulation results show various interactions as shown in FIG. 2C. (Ii) the aromatic ring of DMPD forms an NH-pi interaction (3.16 &lt; RTI ID = 0.0 &gt; A &lt; / RTI &gt; ), And (iii) the methyl group of the dimethylamino group of DMPD stabilized the A [beta] -DMPD interaction through CH-pi (having an aromatic ring of F19) interaction (4.10 A).

Therefore, ITC, 2D NMR and docking / MD simulation analysis confirmed the direct interaction between DMPD and metal-free Aβ.

Example 3: Analysis of interaction between metal-A? Monomer and fibril and DMPD

1) Copper K-boundary X-ray absorption spectroscopy (XAS) analysis

Previously known (Biochemistry 44, 5478-5487, 2005; ... J. Am Chem Soc 126, 13534-13538, 2004) the created a Aβ ₄₂ monomers as described, fibrils of Aβ ₄₂ are known (Biochemistry, 47 , 5006-5016, 2008). After fibrillation or monomers, all samples were run in an anaerobic atmosphere (N ₂ ) in a COY anaerobic chamber (COY Laboratory, Grass Lake, MI, USA).

Aβ ₄₂ was dissolved in a mixture of 10 mM N-ethylmorpholine buffer (pH 7.4) and glycerol (used as an ice-breaking agent) in a ratio of 4: 1, followed by the addition of the same equivalent amount of CuCl ₂ . The A [beta] ₂ monomer was kept at 5 [deg.] C and all procedures were carried out rapidly to prevent flocculation. After addition of CuCl ₂ , two equivalents of ascorbate were added to form Cu (II) -loaded Aβ ₄₂ The peptide was reduced with Cu (I). DMPD (2 equivalents, dissolved in DMSO) was then added to each solution. The final concentration of A [beta] ₄₂ was 250 [mu] M. DMPD was incubated with copper-loaded fibrils for 24 hours. In order to prevent aggregation, copper-loaded A [beta] ₄₂ monomers were reacted for 15 minutes, which was confirmed by gel permeation chromatography (GPC).

After reacting with DMPD, the solution was poured into clear plastic sample holders with a capton tape window and quickly frozen in liquid nitrogen. All data were recorded with Beamline X-3b (beamline X-3b) from National Synchrotron Light Source, Brookhaven National Laboratories, Upton, NY, USA.

Samples were maintained at ~ 18 K through data collection with He Displex cryostat. The energy monochromatization was performed with a Si (111) double crystal monochromator and a low angle Ni mirror was used to remove harmonics.

The data were detected with a fluorescence spectra using a Canberra 31 element Ge solid-state detector with three micron Ni filters placed between the sample and the detector and the simultaneous collection of copper-foil (first inflection point 8980.3 eV) Lt; / RTI &gt; spectra.

The count ratio was between 15 and 30 kHz, and deadtime corrections did not further improve the quality of the spectrum.

Data were collected at 200-20 eV below the boundary (over 1 sec on average), 20 eV below the boundary (over 3 sec on average) and 30 eV above the boundary eV (greater than or equal to 5 seconds on average), and 5 eV steps (an average of more than 5 seconds) of 13 k at 300 eV above the boundary. Each data set represents an average of 16 individual spectra. Known defects were removed from the mean spectrum. The X-ray beam was repositioned every 4 scans and there was no noticeable photodamage / photoreduction. Data were analyzed using known software packages EXAFS123 and FEFF 7.02, and the error was reported as the value of ε ² .

2) Spectroscopic analysis

All samples contained HEPES [20 mM; was prepared in a Chelex-treated buffer containing either pH 6.6 (Cu (II) sample) or pH 7.4 (metal-free sample and Zn (II) sample) and NaCl (150 mM). For samples without Aβ, DMPD (50 μM) and CuCl ₂ or ZnCl ₂ (25 μM) were treated for 2 min. For Aβ-containing samples, Aβ ₄₀ (25 μM) and CuCl ₂ or ZnCl ₂ (25 μM) were treated for 2 min and then DMPD was added. The absorption spectra of the solutions thus obtained were obtained at intervals of 2 hours at room temperature for 24 hours without stirring. Metal-free samples with and without A [beta] ₄₀ were monitored using UV-vis in anaerobic environments. All solvents required for the anaerobic sample preparation is freeze-pump-thaw degassed three times using the (freeze-pump-thaw) cycles and was stored in a glove box (glove box) is filled with N _2, the anaerobic samples were prepared in a glove box Respectively. The UV-vis spectra were recorded at 0, 4 and 24 hours of reaction time at room temperature without agitation.

3) Mass spectrometry

For this experiment, samples containing DMPD (50 μM) and Aβ ₄₀ (25 μM) were prepared in 100 μL of 1 mM NH ₄ OAc (pH 7.4). The resulting solution was reacted at 37 ° C for 0, 2, 4, 8 and 24 hours under constant stirring. The sample was injected directly into the mass spectrometer at a flow rate of 240 mL / h. The ESI interface was operated in a cationic mode, with a spray voltage of 4.5 kV, a capillary temperature of 300 ° C, and a capillary exit voltage of 101V. The mass spectrum was obtained in the range of m / z 50-500.

Ion mobility-mass spectrometry (IM-MS) experiments were performed on Synapt G2 (Waters, Milford, Mass., USA). Samples were ionized using a nano-electrospray source fabricated in cationic mode. The MS instrument was operated at a back pressure of 2.7 mbar and a sample cone voltage of 40 V. Data were analyzed using MassLynx 4.1 and DriftScope 2.0 (Waters, Milford, Mass., USA). Collision cross-section (CCS) measurements were calibrated using a database of known proteins and the CCS value of the protein complex of helium. Lyophilized Aβ ₄₀ peptide (AnaSpec, Fremont, CA, USA) was prepared in 1 mM ammonium acetate (pH 7.0) at a staining concentration of 25 μM. A portion of A [beta] ₄₀ was reacted at 25 [deg.] C for 24 hours without or with 50 [mu] M DMPD (1% v / v DMSO) without constant agitation. After the reaction, all samples were freeze-dried overnight hexafluoro-2-propanol (HFIP, Sigma-Aldrich, St Louis, MO, USA) ([Aβ 40] = 50 μM) samples prior to reproduction-suspended in 5 Lt; / RTI &gt; min. Samples were diluted to 50% with 1 mM ammonium acetate (final concentration 0.5 mM) to give a final A [beta] ₄₀ concentration of 25 [mu] M followed by mass spectrometry.

4) Experimental results

i) Analysis of binding of DMPD to metal

As a result of confirming the binding between DMPD and Zn (II) using UV-vis and ¹ H NMR, it was confirmed that Zn (II) interacted via the N atom of the amino group of DMPD as shown in FIGS. 3A and 3B.

ii) Binding analysis of DMPD with metal-A? monomer and fibril

The XAS values of the Cu (I) -loaded Aβ ₄₂ fibrils after DMPD treatment were consistent with the linear two-coordination Cu (I) (N / O) ₂ environment as shown in FIG. The XANES region near the X-ray absorption edge of the XAS spectrum showed an overwhelming free-edge characteristic at 8985.2 (2) eV, which is the same as the Cu (1s → 4p _z ) transformation, and this characteristic is characteristic of the linear Cu (I) . As shown in Figure 4 (blue), Cu (II) - indicate the fully reduced in the case of handling a load DMPD Aβ ₄₂ fibrils linear two-coordinate Cu (I) (N / O ) Cu (II) on a _second center . From the XAS analysis, it was confirmed that the interaction between the copper-Aβ complex and DMPD shows a possible reduction reaction between the DMPD and the copper center surrounded by Aβ.

iii) Control mechanism of DMPD Aβ aggregation

In order to clarify the control mechanism for DMPD Aβ aggregation, the chemical modification of DMPD by Aβ under various conditions was examined. As a result, DMPD was observed under the condition that CuCl ₂ was present or absent and Aβ ₄₀ was present or absent in the buffer as shown in FIGS. a time-dependent result of monitoring the changes in the optical DMPD treated with Aβ _40, as opposed to the absence of Aβ ₄₀ was a spectral movement (Fig. 5a, Fig. 5b). The formation index of cation radicals formed through the oxidative degradation pathway of DMPD, ca. The optical band at 513 and 550 nm did not react with DMPD and Aβ and was not observed after 24 hours. In a solution containing Aβ ₄₀ in accordance with the addition of DMPD and an optical band of DMPD immediately moved from 295 nm to 305 nm, when the 4 hours ca. The strong optical band at 250 nm is ca. 280 and 330 nm (FIG. 5A, upper). After more than 4 hours of reaction, ca. Ca. New optical bands at 340 or 350 nm began to occur (FIGS. 5A and 5B, below). ca. The optical band at 250 and 340 or 350 nm is predicted as an indicator of the adduct production of benzoquinoneimine (BQI) or benzoquinone (BQ) and their proteins (FIG. 6). Thus, DMPD is modified through different pathways in the presence of A [beta] to produce a modified DMPD conjugate coupled to A [beta].

Also, in order to verify the relationship of oxygen in the conversion of DMPD, UV-vis analysis of DMPD in the presence or absence of Aβ resulted in the spectral change of DMPD under anaerobic conditions It was not. In addition, the regulation of Aβ aggregation by DMPD was not observed under anaerobic conditions, unlike aerobic conditions (FIG. 1c). Thus, oxygen is an essential element in the formation of A [beta] aggregates that do not exhibit oxidative deformation and toxicity of DMPD.

Analysis of the reaction between DMPD and Aβ for 0, 2, 4, 8, and 24 hours revealed that the signal for DMPD at m / z 137 and the loss of amine group at m / z 122 The signal decreased with the reaction time. A time-dependent decrease in the MS signal means that the interaction between DMPD and A [beta] occurs through the formation of the conversion product.

MS analysis of Aβ ₄₀ samples treated with DMPD was also performed to confirm the formation of Aβ ₄₀ -ligand complexes. As a result, a new peak occurs (Fig. 6 (a)) due to the addition of Aβ at 103.93 ± 0.04 Da, which is presumed to be BQ, the covalent modification product of DMPD.

To demonstrate the mode of A [beta] -DMPD interaction through modification of DMPD, the interaction of structurally consistent BQ with A [beta] ₄₀ was investigated under the same experimental conditions. As a result, BQ was bound to A? ₄₀ (Fig. 6b), and mass change (104.1 ± 0.1 Da) also occurred in accordance with DMPD treatment. Was performed for the tandem MS (MS / MS) coupled to collision caused separation (CID) for the 5-level charge binding charge state, Aβ ₄₀ through which-the properties of the _modified DMPD (Aβ ₄₀ -DMPD _transformed) was determined ( 6c). MS / MS results showed that the covalently bound to Aβ ₄₀ through the DMPD and BQ all K16 or K28 strain similarly to the above result. While this ligated mass difference is too small to support a single covalent bond formation between A? And _modified DMPD / BQ (106.1 Da expected), formation of a second covalent bond between A? And _modified DMPD / BQ (104.1 Da) is suitable to support. Thus, it can be shown that the _modified DMPD / BQ can be crosslinked to A [beta], and this result is consistent with the previously known (FEBS Journal 272, 3661-3672 (2005)) fora-synuclein Match.

In addition, ion mobility-mass spectrometry (IM-MS) was performed in the fourth-charge state to evaluate the conformers of the Aβ-bound state employed. When compared in the apo (apo) conditions, _{the modified} Aβ- DMPD conjugate showed a substantially reduced ion migration also (ion mobility, IM) arrival time and, more compact structure of Aβ ₄₀ (Figure 6a, ii). As with the above results, A [beta] ₄₀ -BQ binding also led to decreased IM arrival time and support for the generation of a more compact species than the form adopted through apopeptides (Fig. 6b, ii).

By incorporating the MS / MS analysis results and the results, the _modified DMPD / BQ cross-linked trap Aβ is relatively concomitantly conformational, which is related to the off-pathway to amyloid fibril formation .

In addition, on the basis of the optical results and MS result, Aβ- _modified DMPD complex is determined to be caused by two possible mechanisms of, as shown in FIG. 6d.

Under aerobic conditions, in the presence of Aβ, DMPD can first form a cationic imine (CI) -Aβ complex through two electron-oxidative modifications ( i ). CI is hydrolyzed to BQI and ( ii ) its imine is hydrolyzed to produce BQ ( iii ). BQ can form a covalent bond Aβ-BQ adduct through interaction with an initial amine containing residue (Aβ + 106.1 Da, IV ) such as K16 and a similar functional group (Aβ + 104.1 Da , V ). &Lt; / RTI &gt; The covalent complexes of A? And BQ that can direct structural compaction were identified in the IM-MS analysis (Fig. 6a, b), through which redirection of DMPD in the peptide aggregation pathway to amorphous A? Aggregation ) Can be explained. The shared hybridization of Aβ and BQ that can direct structural compaction was confirmed in the IM-MS analysis (FIG. 6a, b), and it was confirmed that the peptides from the gel / western blot and the amorphous Aβ aggregation identified in the TEM study The redirection of DMPD in the coagulation pathway can be explained.

< Example  4> In vivo effects of DMPD on amyloid pathology and cognitive impairment

To demonstrate the effect of DMPD on AD pathology in vivo, DMPD was administered via the intraperitoneal route of 3-month old 5xFAD mice at a dose of 1 mg / kg / day for 30 days. After 30 days of DMPD administration, a biochemical analysis was performed to assess the amount of Aβ ₄₀ / Aβ _{42 in the} brain and the load of amyloid deposits histopathologically. For this purpose, mice showing severe phenotypic and behavioral dysfunction of AD after the initial progression of 5xFAD mice were screened. At the end of DMPD treatment, there was no significant difference in body weight or appearance of vehicle- and DMPD treated 5xFAD mice.

The A [beta] peptide of mouse brain was quantitated by ELISA (enzyme-linked immunosorbent assay).

Aβ ₄₀ / Aβ ₄₂ contained in PBS- and sodium dodecyl sulfate (SDS) of 5x FAD mice treated with DMPD And formic acid (FA) were significantly lower than those in vehicle treated 5xFAD mice with ca. 65% and 47%, respectively (Fig. 7A).

The level of Aβ ₄₀ / Aβ ₄₂ was dissolved in SDS was (ca. 68% and 67%) FA of Aβ drastically reduced by ₄₀ / Aβ ₄₂ than (ca. 50% and 32%) dissolved in DMPD. In addition, significant reductions in amyloid accumulation have been identified in 5xFAD mice treated with DMPD, and the loads of amyloid precursor protein (APP) / immunoreactive 4G8- / Aβ-immunoreactive and Congo red- Analysis of amyloid plaques (Congo red-stained amyloid plaques) revealed that ca. 23% and 20%, respectively (Figs. 7 (b) and 7 (c)

Thus, it has been confirmed that DMPD can delay or reverse amyloid development in the brain of AD model mice.

In addition, in order to confirm whether DMPD can improve cognitive impairment in AD model mice, 4-month-old 5xFAD mice were treated with DMPD and subjected to the last four consecutive days with the Morris water maze test).

5xFAD mice showed impaired spatial learning when compared to wild-type mice born on the same abdomen, and it was more difficult to locate the escape platform hidden in the water pool, whereas DMPD Showed that the 5xFAD mice repeatedly administered had improved learning and memory ability when compared to vehicle-treated normal mice (Fig. 8A).

Three hours after the experiment of the underwater maze, probe trials were carried out to observe the performance of long - term memory, and the escape platform was removed.

The DPMD-treated mice had less distinguished time to reach the platform area when compared to the vehicle-treated 5xFAD mouse and had more time in the target quadrant (North West, NW) where the platform was hidden (Fig. 8B, C).

Thus, these results confirm that DMPD improves cognitive impairment in 5xFAD mice.

Hereinafter, formulation examples of DMPD-containing compositions will be described, but the present invention is not intended to be limited thereto but is specifically described.

&Lt; Prescription Example 1 > Prescription Example of Pharmaceutical Composition

&Lt; Prescription Example 1-1 > Preparation of powder

20 mg of DMPD, 100 mg of lactose, and 10 mg of talc were mixed and filled in airtight bags to prepare powders.

&Lt; Prescription Example 1-2 > Preparation of tablets

20 mg of DMPD, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed and tableted according to a conventional preparation method.

&Lt; Prescription Example 1-3 > Preparation of capsules

10 mg of DMPD, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed, and the above components were mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

&Lt; Prescription Example 1-4 > Preparation of injection

DMPD 10 mg, sterilized distilled water suitable amount for injection, and pH adjuster were mixed, and the contents were adjusted to the above contents in the amount of 2 ml per ampoule according to the usual injection preparation method.

&Lt; Prescription Example 1-5 > Preparation of ointment preparation

DMPD 10 mg, PEG-4000 250 mg, PEG-400 650 mg, white petrolatum 10 mg, manganese p-hydroxybenzoate 1.44 mg, p-hydroxybenzoyl propyl 0.18 mg and the remaining amount of purified water were mixed, An ointment agent was prepared.

&Lt; Prescription Example 2 >

&Lt; Prescription Example 2-1 > Preparation of health food

DMPD 1 mg, Vitamin A acetate 70,, Vitamin E 1.0 mg, Vitamin B 1 0.13 mg, Vitamin B 2 0.15 mg, Vitamin B 6 0.5 mg, Vitamin B 12 0.2,, Vitamin C 10 mg, Biotin 10 (Ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium phosphate monobasic 15 mg, dibasic calcium phosphate 55 mg, nicotinate amide 1.7 mg, folic acid 50 mg, calcium pantothenate 0.5 mg) Mg of calcium citrate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride) were mixed to prepare a granule, and a health food was prepared according to a conventional method.

&Lt; Prescription Example 2-2 > Preparation of health drink

1 mg of DMPD, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of a plum concentrate, 1 g of taurine and purified water were added to a total of 900 ml, and the above components were mixed according to a conventional health drink manufacturing method, The solution was filtered and sterilized in a sterilized 2 L container, and then refrigerated.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is to be understood that various modifications and changes may be made without departing from the scope of the appended claims.

Claims (9)

N, N-dimethyl-p-phenylenediamine; DMPD], wherein the DMPD inhibits the toxicity of a metal-amyloid beta peptide complex in vitro. The composition for inhibiting metal-amyloid beta peptide complex in vitro. delete delete delete The method according to claim 1,
A composition for inhibiting metal-amyloid beta peptide complexes in vitro, wherein the DMPD induces aggregation that does not exhibit toxicity during the aggregation process of the metal-amyloid beta peptide complex. The method according to claim 1,
A composition for inhibiting metal-amyloid beta peptide complexes in vitro, wherein the metal is zinc or copper. delete In vitro, N, N-dimethyl-p-phenylenediamine (N, N-dimethyl-p-phenylenediamine; DMPD] to a neuronal cell, comprising the step of administering to the neural cell a therapeutically effective amount of a compound of the invention.
delete

Images