KR102515986B1 - Pharmaceutical compositions comprising melanin-like particles for inhibiting amyloid-beta aggregation - Google Patents

Abstract

The present invention relates to a composition that inhibits the aggregation of amyloid-beta containing melanin analogues. Specifically, the composition has an effect of inhibiting the aggregation of amyloid-beta or removing the already aggregated fibrous structure of amyloid-beta.

Description

Pharmaceutical compositions comprising melanin-like particles for inhibiting amyloid-beta aggregation}

The present disclosure relates to a pharmaceutical composition for inhibiting amyloid-beta aggregation comprising a melanin analogue. According to another embodiment, the composition relates to a pharmaceutical composition for removing already aggregated fibers of amyloid-beta.

Melanin is a biological polymer present in various parts of various organisms such as plants, animals, and protists, and is classified into dark brown eumelanin and yellowish red pheomelanin. Eumelanin is 3,4-dihydroxy-L-phenylalanine (L-DOPA) or 2-(3,4-dihydroxyphenyl)ethylamine [2-(3, 4-dihydroxyphenyl)ethylamine, dopamine], and pheomelanin is derived from L-DOPA or dopamine in the presence of thiol group (-SH)-containing substances such as cysteine and glutathione.

Biological functions of melanin known so far include photoprotection that absorbs a wide range of electromagnetic radiation, photosensitization, metal ion chelation, antibiotic action ( Its functions are diverse, such as antibiotic, thermoregulation, and free radical quenching. It is used in various application fields that can utilize the function of such melanin.

The formation of amyloid due to abnormal unwinding of proteins and subsequent aggregation has received much attention as it is considered to be associated with the development of various neurodegenerative diseases. Amyloid-beta is composed of 36-43 amino acids, and the accumulation of amyloid-beta peptide is considered an early etiology that precedes brain damage and the development of neurofibrillary tangles, and active research related to this is ongoing. As a result of intensive research for about 30 years, various substances for inhibiting protein aggregation have been developed, but a fundamental solution to improve the disease has not yet been presented.

On the other hand, molecules containing catechol such as dopamine, L-dopa, EGCG, a type of green tea extract, and pyrogalol are naturally derived natural products that have the potential to decompose amyloid or inhibit its production. It has been continuously studied by researchers. However, studies on how they chemically interact with amyloid structures have been lacking, and substances that can be used as protein aggregation inhibitors have not been presented.

According to one embodiment of the present disclosure, it is to provide a pharmaceutical composition for inhibiting amyloid-beta aggregation of melanin analogs containing polydopamine.

According to another embodiment, it is to provide a method for producing a melanin analogue containing the polydopamine.

According to one specific example of the present disclosure,

A pharmaceutical composition for inhibiting amyloid-beta aggregation comprising a melanin analogue is provided.

Specifically, the pharmaceutical composition containing at least one of eumelanin analogues (EMPs) and pheomelanin analogues (PMPs) is provided as the melanin analogue.

According to another embodiment of the present disclosure,

(a) providing dopamine or L-dopa to a solvent containing at least one of an alcohol having 1 to 4 carbon atoms and distilled water;

(b) providing hydrochloric acid to the solution;

(c) a method for preparing a melanin analogue comprising the step of isolating the prepared melanin analogue is provided.

According to one embodiment of the present disclosure, the pharmaceutical composition containing the melanin analogue can significantly inhibit the aggregation of amyloid-beta. Specifically, the composition may inhibit fiber formation by acting on amyloid-beta monomers.

According to another embodiment, the pharmaceutical composition containing the melanin analogue can remove already formed amyloid-beta aggregated fibers. Specifically, the composition can remove already formed amyloid-beta aggregated fibers and transform them into a mesh form. More specifically, these effects can be seen immediately within 2 hours.

According to another embodiment, the melanin analogue of the present disclosure may be more stable than DHI and thus likely to be a pharmaceutical composition for inhibiting amyloid-beta aggregation. In addition, it is expected that the possibility of pharmaceutical utilization is higher than that of DHI.

1 is a photograph of the melanin analogue of the present invention with a scanning electron microscope.
Figure 2 will confirm the effect of inhibiting amyloid-beta aggregation of EMPs.
Figure 3 confirms the amyloid-beta aggregation inhibitory effect of catechol and PMPs.
Figure 4 is a transmission electron microscope image of the pattern of amyloid-beta aggregation inhibition of catechol and melanin analogues.
5 confirms the effect of removing amyloid-beta in which melanin analogues and catechol are aggregated.
6 is a transmission electron microscope image of the removal of amyloid-beta in which melanin analogues and catechol are aggregated.

The present invention can all be achieved by the following description. The following description should be understood as describing embodiments of the present invention, and the present invention is not necessarily limited thereto. In addition, the accompanying drawings are for illustrative purposes only, and it should be understood that the present invention is not limited thereto.

Embodiments of the present disclosure may be subject to various changes and implemented in various forms. Therefore, it should be understood to include all modifications, equivalents, or substitutes to the extent that the sameness of spirit and technical features of the present disclosure is recognized.

Unless otherwise specified, all numbers used in this disclosure are to be understood as modifying the term "about" in all instances. The modifier “about” is intended to have a commonly recognized approximate meaning, which can be more accurately interpreted as a meaning within a certain percentage of the modified value, and more specifically ±20%, ±10%, ± It can mean 5%, ±2% or ±1% or less.

In the present disclosure, inhibition of amyloid-beta aggregation can be confirmed by fluorescence expression of Thioflavin T (ThT) related to the beta-sheet structure of amyloid-beta. Specifically, when a beta-sheet structure is generated due to aggregation of amyloid-beta, the intensity of fluorescence by ThT increases, and therefore, the ability to inhibit amyloid-beta aggregation can be confirmed by reducing fluorescence emission.

The term 'prevention' used in the present disclosure refers to any activity that inhibits or delays diseases related thereto by inhibiting aggregation of amyloid-beta.

The term 'treatment' used in the present disclosure refers to any activity that improves or beneficially changes the symptoms of a disease by inhibiting aggregation of amyloid-beta.

As used in the present disclosure, the term 'melanin analogue' refers to black or brown pigments present in tissues such as the skin or eyes of animals, and includes pheomelanin and eumelanin. Specifically, in the present disclosure, polydopamine is contained. It means any composition that is black to brown in color. More specifically, the melanin analogue may include at least one of eumelanin-like particles (EMPs) and pheomelanin-like particles (PMPs).

According to one specific example of the present disclosure,

A pharmaceutical composition comprising a melanin analogue can inhibit the aggregation of amyloid-beta. For example, the melanin analogue can prevent amyloid-beta from aggregating to form a fibrous structure, and for example, the melanin analogue can aggregate to release a fibrous structure that has already been formed.

The melanin analogue may include polydopamine. Specifically, polydopamine may be produced by polymerization of 5,6-dihydroxyindole (DHI). More specifically, the polydopamine may be prepared by oxidation and polymerization of at least one of dopamine and levodopa (L-dopa). On the other hand, in the case of DHI, it is a small molecule substance, and it is very unstable in the presence of oxygen and can easily change into other molecules, so it may be difficult to use it alone. On the other hand, the melanin analogue of the present disclosure is more stable than DHI, and may be a pharmaceutical composition for inhibiting amyloid-beta aggregation that is more stable than DHI.

The diameter of the eumelanin analogues (EMPs) may be 50 to 1,000 nm, specifically 100 to 500 nm, and more specifically 135 to 450 nm. Therapeutic concentrations of eumelanin analogs can be, for example, 0.0001 to 10 equivalents of 6.25 μM amyloid-beta, such as 0.001 to 5 equivalents, such as 0.01 to 3 equivalents, e.g. For example, it may be 0.1 to 1 equivalent.

The diameter of the pheomelanin analogues (PMPs) may be 10 to 1,000 nm, specifically 50 to 500 nm, and more specifically 100 to 200 nm. The therapeutic concentration of the pheomelanin analog can be, for example, 0.0001 to 10 equivalents, such as 0.001 to 5 equivalents, such as 0.01 to 3 equivalents of 6.25 μM amyloid-beta, e.g. For example, it may be 0.1 to 1 equivalent.

The melanin analogue can maintain the ability to inhibit the aggregation of amyloid-beta for 5 hours or more. Specifically, the holding time may be, for example, 5 hours to 100 hours, for example, 5 hours to 50 hours, for example, 5 hours to 20 hours, for example, 5 hours to 15 hours For example, it may be 5 hours to 10 hours, for example, it may be 10 hours to 50 hours, for example, it may be 10 hours to 20 hours, for example it may be 15 hours to 50 hours. And, for example, it may be 15 hours to 20 hours.

The melanin analogue may prevent the formation of amyloid-beta fibrous structure. Specifically, the melanin analogue may act on the amyloid-beta monomer to prevent aggregation. In addition, melanin analogues can remove the fibrous structure of amyloid-beta. Specifically, it may be to reduce the fibrous structure of amyloid-beta or to form a mesh form with no fibrous structure. More specifically, beta-sheet bonds can be released. The melanin analogue may be an eumelanin analogue or a pheomelanin analogue.

According to another embodiment of the present disclosure,

A composition comprising a melanin analogue can inhibit the expression of ThT fluorescence by already aggregated amyloid-beta. Specifically, the composition can remove the already aggregated fibrous structure of amyloid-beta. For example, the composition can inhibit ThT fluorescence expression simultaneously with treatment. Specifically, 50% of fluorescence expression may be inhibited within 4 hours of treatment. More specifically, 50% of fluorescence expression may be inhibited within 2 hours of treatment. According to another embodiment, 50% of the aggregated amyloid-beta fibrous structure may be removed within 4 hours of treatment, and 50% of the aggregated amyloid-beta fibrous structure may be removed within 2 hours of treatment.

The pharmaceutical composition may include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may use at least one selected from the group consisting of a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant, and a flavoring agent for oral administration, but is limited thereto. It is not. In the case of injection, a buffer, preservative, analgesic agent, solubilizer, isotonic agent or stabilizer may be mixed and used, and in the case of topical administration, a base, excipient, lubricant or preservative may be mixed and used.

Formulations of the pharmaceutical compositions of the present disclosure may be variously prepared by mixing with pharmaceutically acceptable carriers as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. It can also be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, and the like.

On the other hand, examples of carriers, excipients or diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like may be further included.

Routes of administration of the pharmaceutical composition according to the present disclosure include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, These include topical, sublingual or rectal. Oral or parenteral administration is preferred. The administration may be administered systemically or locally.

In this disclosure, “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intracapsular, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. A pharmaceutical composition of the present disclosure may also be administered in the form of a suppository for rectal administration. The administration is 0.1 mg to 1,000 mg, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg may be administered.

The dosage of the composition may be, for example, about 0.001 mg/kg to about 100 mg/kg, for example, about 0.01 mg/kg to about 10 mg/kg, or, for example, about 0.1 mg/kg to about 1 mg/kg. The frequency of administration may be once a day, multiple times a day, 2 to 3 times a week, 1 to 4 times a month, or 1 to 12 times a year.

According to the present disclosure, at least one separate composition may be further included in the pharmaceutical composition. Specifically, a separate composition may be a composition capable of alleviating or treating symptoms that may occur due to amyloid-beta aggregation. More specifically, it may be a composition for inhibiting nerve damage, it may be a composition for promoting nerve regeneration, it may be a composition for improving memory, it may be a composition for preventing, alleviating or treating ataxia, and it may be a composition for preventing, alleviating or treating Parkinson's disease. It may be a composition for Alzheimer's disease, but may be a composition for preventing, alleviating or treating Alzheimer's disease, but is not limited thereto. The composition may be a pharmaceutical composition, a herbal extract or a fraction thereof, or a drug.

Specifically, the content ratio of the pharmaceutical composition of the present disclosure and the separate composition may be, for example, 1: about 0.001 to 1000, for example 1: about 0.01 to 100, or, for example, 1: about It can be 0.1 to 10.

The composition may be included in health functional foods for preventing, improving or treating amyloid-beta aggregation. The health functional food may be used alone or in combination with a composition for inhibiting amyloid-beta aggregation containing a melanin analogue or other food or food ingredient, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw material. There is no particular limitation on the type of health food. Beverage compositions among types of health foods may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The health food composition may also contain nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonated beverages. carbonation agent used, or a combination thereof. The health food composition may also contain natural fruit juice, fruit juice beverages, fruit flesh for preparing vegetable beverages, or a combination thereof.

Example 1

Example 1-1: Preparation of EMPs

A mixture of water and ethanol in a volume ratio of 1:2 was added to 130 ml of an ammonia solvent having a concentration of about 28 to 30%, and mixed for 30 minutes. In addition, 0.5 g of dopamine-hydrochloride was completely dissolved in 10 ml of distilled water and added to the solvent. After culturing for about 24 hours at room temperature, the resulting EMPs were prepared by washing with distilled water and centrifuging. The diameter size of the EMPs was adjusted according to the amount of ammonium hydroxy groups added. This produced EMPs with diameters of 135 nm, 210 nm, 315 nm and 450 nm.

Example 1-2: Preparation of PMPs

85.6 mg of L-cysteine and 360 ml of 1N KMnO ₄ were added to a solution in which 70 mg of L-dopa was completely dissolved by heating in 50 ml of distilled water. After reacting for 24 hours, the product was centrifuged and dissolved again in 50 ml of distilled water. After removing Mn ²⁺ from the solution by adding 1N hydrochloric acid, the product was washed and centrifuged three times to separate PMPs.

Example 1-3: Procedure for measuring amyloid-beta aggregation inhibitory effect

After dissolving amyloid-beta(Aβ42) in ammonium hydroxy group, it was subdivided and fat-solubilized. The lipid-solubilized peptide was dissolved in 10 ml of 1% NH ₄ OH, and then diluted with water and phosphate buffer solution (pH 7.4). To visualize amyloid species, thioflavin T (ThT) was diluted to 6.25 μM. When aggregation was complete, centrifugation was performed at 12,000 rpm to separate amyloid-beta having a fibrous structure and non-aggregated amyloid-beta, and their respective concentrations were measured using a spectrophotometer and Bicinchoninic acid (BCA) analysis. Melanin analogues and catechol monomers were treated with unaggregated amyloid-beta, and incubated at 378° C. for 24 hours, and ThT fluorescence excitation at 450 nm and ThT fluorescence emission at 490 nm were measured. In addition, melanin analogues and catechol monomers were treated with aggregated amyloid-beta, and the same measurement was performed.

Example 2: Amyloid-beta aggregation inhibitory effect of melanin analogs

EMPs and PMPs were prepared according to Example 1, and were prepared with the properties shown in Table 1.

Diameter-SEM (nm) Diameter-DLS (nm) PDI Zeta Potential(mV) EMP ₁₃₅ 134.67±7.94 194.3 0.147 -25.3 EMP ₂₁₀ 210.27±9.85 290.4 0.120 -23.3 EMP ₃₁₅ 314.43±14.36 513.4 0.234 -33.7 EMP ₄₅₀ 450.45±12.98 572.1 0.269 -34.5 PMP 118.45±9.79 165.4 0.062 -21.7

According to Figure 1, EMPs and PMPs with different diameters were confirmed by scanning electron microscopy (SEM) that they were prepared in a round shape, and according to Figure 2, EMPs significantly inhibited amyloid-beta aggregation due to the difference in diameter. There was no effect, and it was confirmed that aggregation was inhibited in a dose-dependent manner. According to FIG. 3, PMPs inhibited aggregation of amyloid-beta in a dose-dependent manner, and it was confirmed that the time required for the fluorescence expression of ThT to appear at 2,000 au was longer than that of catechol monomers such as dopamine, levodopa, and tyrosine. That is, it was confirmed that the duration of the effect of inhibiting the aggregation of amyloid-beta was longer than that of the catechol monomer. According to FIG. 4, the pattern of amyloid-beta was confirmed by transmission electron microscopy when catechol monomers and melanin analogues were treated. Specifically, dopamine and PMPs formed a network without a fibrous structure, but EMPs and tyrosine clearly showed a fibrous structure.

Example 3: Effect of removing the fibrous structure of aggregated amyloid-beta

EMPs and PMPs prepared according to Example 1 were treated with already aggregated amyloid-beta to confirm whether fibers could be removed by removing already formed beta-sheets (β-sheet). According to FIG. 5, unlike tyrosine, levodopa, dopamine, and epinephrine, which had no effect within 2 hours of treatment, EMPs and PMPs immediately suppressed ThT fluorescence expression within 2 hours of treatment, and all melanin analogues were 50% of the control within 4 hours. It was confirmed that fluorescence expression of % or less appeared. According to Figure 6, the pattern of the amyloid-beta fiber structure according to the melanin analog and catechol treatment is shown. Specifically, when treated with dopamine, levodopa, 5,6-dihydroxyindole (DHI) or PMPs, it was confirmed that the fiber structure was significantly reduced and a network form appeared, whereas when treated with tyrosine or EMPs, the fiber structure remained intact. confirmed to appear. Therefore, EMPs only have the effect of masking ThT fluorescence expressed in beta-sheets, but PMPs, DHI, dopamine, and levodopa have been confirmed to have the effect of unraveling the fiber structure composed of beta-sheets.

However, DHI is a small molecule substance and is very unstable in the presence of oxygen, so it is very difficult to use it alone as it easily changes into other molecules. However, the melanin analogue of the present invention is not a small molecule and is therefore more stable than DHI.

Claims (12)

In the pharmaceutical composition for preventing, treating or improving Alzheimer's containing eumelanin analogues (EMPs) or pheomelanin analogues (PMPs),
The eumelanin analogues (EMPs) or pheomelanin analogues (PMPs)
(a) providing dopamine or 3,4-dihydroxy-L-phenylalanine (L-dopa) in a solvent containing at least one of an alcohol having 1 to 4 carbon atoms and distilled water;
(b) providing hydrochloric acid to the solution; and
(c) A pharmaceutical composition characterized in that it is prepared, including the step of isolating the prepared eumelanin analogues (EMPs) or pheomelanin analogues (PMPs). delete delete According to claim 1,
The pharmaceutical composition wherein the eumelanin analogues (EMPs) or pheomelanin analogues (PMPs) contain polydopamine. According to claim 4,
The polydopamine is a pharmaceutical composition comprising 5,6-dihydroxyindole (5,6-dihydroxyindole, DHI). The method of any one of claims 1 and 4 to 5,
The composition is a pharmaceutical composition that inhibits the formation of amyloid-beta aggregated fibrils. The method of any one of claims 1 and 4 to 5,
The composition is a pharmaceutical composition for removing amyloid-beta aggregated fibers. According to claim 7,
The composition is a pharmaceutical composition that begins to remove amyloid-beta aggregated fibers within 2 hours. According to claim 7,
The composition is a pharmaceutical composition that removes 50% or more of amyloid-beta aggregated fibers within 4 hours. (a) providing dopamine or 3,4-dihydroxy-L-phenylalanine (L-dopa) in a solvent containing at least one of an alcohol having 1 to 4 carbon atoms and distilled water;
(b) providing hydrochloric acid to the solution; and
(c) A method for preparing eumelanin analogues (EMPs) or pheomelanin analogues (PMPs) comprising the step of isolating the prepared eumelanin analogues (EMPs) or pheomelanin analogues (PMPs). According to claim 10,
Method for producing eumelanin analogues (EMPs) or pheomelanin analogues (PMPs) further providing cysteine (L-cysteine) and KMnO ₄ in step (a). In the health functional food for preventing or improving Alzheimer's containing eumelanin analogues (EMPs) or pheomelanin analogues (PMPs),
The eumelanin analogues (EMPs) or pheomelanin analogues (PMPs)
(a) providing dopamine or 3,4-dihydroxy-L-phenylalanine (L-dopa) in a solvent containing at least one of an alcohol having 1 to 4 carbon atoms and distilled water;
(b) providing hydrochloric acid to the solution; and
(c) A health functional food characterized in that it is prepared by including the step of isolating the manufactured eumelanin analogues (EMPs) or pheomelanin analogues (PMPs).

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