KR20220102128A - Novel Ghrelin Analogues and Pharmaceutical Composition for Prophylaxix or Treatment of Disease Related Amyloid β Accumulation - Google Patents

Abstract

The present invention relates to a novel ghrelin analogue having an effect of inhibiting amyloid β aggregation and a pharmaceutical composition for preventing or treating amyloid β aggregation-related diseases using the same, and more particularly, to a ghrelin analog in which the N-terminus and C-terminus of a peptide comprising the active region of ghrelin are cyclized via a sulfide bond derived from a thiol group of cysteine and a pharmaceutical composition for preventing or treating amyloid β aggregation-related diseases using the same.

Description

Novel Ghrelin Analogs and Pharmaceutical Compositions for Preventing or Treating Amyloid Beta Aggregation-Related Diseases Containing the Same

The present invention relates to a novel ghrelin analog having an effect of inhibiting amyloid beta aggregation and a pharmaceutical composition for preventing or treating amyloid beta aggregation-related diseases using the same.

Alzheimer's disease is a representative senile dementia, a progressive chronic neurodegenerative disease with a high prevalence rate that is high enough to occur in one-third of the population over 85 years of age. Recently, not only the elderly but also the younger generation of dementia patients in their 30s and 50s are on the rise.

Alzheimer's disease causes a variety of symptoms, such as memory impairment, speech impairment, loss of cognitive function, neurodegeneration and neuroinflammation. As Alzheimer's disease progresses, more functional impairments appear, and neuropsychiatric symptoms also increase. Currently, as the level of amyloid beta (Aβ) in the brain increases, it causes deposition to form an aggregated plaque, and the accumulated amyloid beta plaque blocks the signal transduction pathway between brain cells, leading to brain cell death and eventually dementia. The 'amyloid beta hypothesis' is accepted as the orthodoxy of the mechanism of dementia. Recently, the American Dementia Treatment Research Institute reported the results of a study that showed that when amyloid beta levels start to increase, the likelihood of transition to dementia increases even when no symptoms of dementia, such as memory loss, appear. do. For this reason, as of 2018, more than half of the drugs currently in phase III clinical trials target amyloid-related mechanisms, and clinical trials of a number of drugs, including BAN2401 and aducanumab, which directly target reducing aggregation of amyloid beta, have been conducted. It is in progress so far. Previous studies suggest that cognitive impairment in Alzheimer's is associated with neurodegeneration rather than the accumulation of amyloid beta itself. Neuronal degeneration in Alzheimer's can be characterized by loss of synapses and neurons. In particular, a recent study reported that neurodegeneration alone does not have a significant effect on cognitive decline without the deposition of amyloid beta. Therefore, a therapeutic strategy targeting the inhibition of aggregation and deposition of amyloid beta appears to be a promising strategy in itself, and treatment may be more effective if it includes inhibition of neuroinflammation and neurodegeneration along with aggregation of amyloid beta. have.

Although some drugs that target directly inhibiting the deposition of amyloid beta are in clinical trials, they are frustrated in clinical trials due to low efficacy and side effects. As a representative example, bapineuzumab received the most attention among new drug candidates targeting amyloid beta, but in phase 3 clinical trial, the effect did not meet the original expectation and development was halted due to vascular cerebral edema resulting in increased cerebral fluid. . Janssen's atabecestat is a small molecule substance that inhibits the production and aggregation of amyloid beta. It was scheduled to undergo a large-scale phase 3 trial from 2015 to 2023, but the clinical trial was suspended due to side effects confirmed by the Data Safety Review Committee in 2018. In 2012, Eli Lilly's Solanezumab, which targets amyloid beta, was also discontinued in clinical trials, but with meaningful results in some patients, clinical trials are being resumed. appeared to be Accordingly, there is still no treatment that targets amyloid beta, and currently, treatment for Alzheimer's disease is limited to those that interfere with the mechanism of neurotransmitters that control learning and memory, such as acetylcholine esterase inhibitors.

Diseases related to the deposition of amyloid beta have a common characteristic in that the fragility of nerve cells increases due to abnormal amyloid production, resulting in neurodegeneration, and neuroinflammation by activation of an inflammatory mechanism. Diseases characterized by the deposition of amyloid beta include, in addition to Alzheimer's, prion diseases such as Down syndrome, Creutzfeldt-Jacob disease, Gerstmann-Straussler syndrome, amyloid angiopathy, cerebral amyloid angiopathy, Lewy body dementia, Parkinson's in Guam Dementia complex, systemic amyloid disease, Dutch amyloidosis, inclusion body myositis, reactive amyloidosis, idiopathic amyloidosis, familial amyloidosis, amyloidosis-associated hereditary cerebral hemorrhage, mild cognitive impairment, frontotemporal dementia, age-related macular degeneration, Pick's disease and brain amyloid Vasculopathy is included. Therefore, drugs that inhibit the aggregation and deposition of amyloid beta can be usefully used in the treatment of diseases related to the amyloid beta deposition in addition to Alzheimer's.

Ghrelin, a new peptide hormone discovered in 1999, is composed of 28 amino acids and is mainly secreted by gastrointestinal endocrine cells to induce hunger. Ghrelin is known to promote the secretion of growth hormone from the pituitary gland through the receptor GHS-R1a (growth hormone secretagogue receptor 1a), and to increase food intake and fat accumulation. Recently, secretion from various organs including the brain as well as the gastrointestinal tract and various physiological functions by this have been revealed. In particular, it is known that Alzheimer's disease can directly affect brain functions that are changed in relation to lesions appearing in the brain. That is, ghrelin-induced learning and cognitive ability increase, neuronal protection, anti-inflammatory effect, synapse number increase, circadian rhythm regulation, appetite promotion, depression suppression, metabolism promotion, adult neurogenesis in the hippocampus The improvement effect has been proven. For example, Kunath et al. (Scientific reports, 5: 11452, 2015) reported that LY444711, a ghrelin agonist, improved cognitive function in an animal model of Alzheimer's disease when administered for 4 months. In addition, as the expression of ghrelin is known to decrease in Alzheimer's disease patients, the potential as a therapeutic agent for Alzheimer's disease is continuously being raised. However, ghrelin has a half-life of only 30 minutes, making it unsuitable for use in actual treatment.

Patent Publication No. 10-2019-0077915

Scientific reports, 5: 11452, 2015. Nature medicine, 2002, 8(12), pp. 1390-1397.

An object of the present invention is to provide a novel ghrelin analog that binds to a ghrelin receptor and is stable and has an increased half-life.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases caused by amyloid beta aggregation comprising the novel ghrelin analog as an active ingredient.

The present invention for achieving the above object relates to a ghrelin analogue, characterized in that the N-terminus and C-terminus of a peptide containing an active region of ghrelin are cyclized through a sulfide bond derived from a thiol group of cysteine. .

Ghrelin is a peptide consisting of 28 amino acids, and the compound of the present invention may include the entire sequence of ghrelin, but if it contains the amino acid sequence of "GSS(-octyl)FLSPEHQ", which is known to exhibit ghrelin activity, some region of ghrelin regardless of including In the compound of the present invention, the N-terminus and C-terminus of the peptide are cyclized via a sulfide bond derived from the thiol group of cysteine. Directly bound by a sulfide bond, of course, means that other amino acids or other amino acid analogs are included between the N-terminus and C-terminus of ghrelin. The ghrelin analogue of the present invention is characterized in that cysteine is introduced into the C-terminal side of ghrelin, and the -SH group derived from cysteine participates in a cyclization reaction to form a cyclic structure. The compound of the present invention having a cyclic structure has greatly increased stability, as can be seen in Examples, and can solve the problem of short half-life, one of the biggest problems of ghrelin when used as a drug.

In the compound of the present invention, 1 to 3 6-aminohexanoic acids may be additionally introduced into the N-terminal, C-terminal or ghrelin sequence of ghrelin. 6-aminohexanoic acid was introduced, the compound exhibited a better neuroprotective effect against cytotoxicity due to amyloid beta aggregation compared to the compound not introduced 6-aminohexanoic acid. This can be inferred because it serves to alleviate the structural rigidity due to the cyclic compound structure by the introduction of 6-aminohexanoic acid. 6-aminohexanoic acid is introduced, the period showing the amyloid beta aggregation efficacy when stored in solution is increased compared to the compound in which 6-aminohexanoic acid is not introduced, and the 6-aminohexanoic acid residue is a ghrelin analogue. It was confirmed that it also affects the stability.

Specifically, the present invention may have a structure represented by the following Chemical Formulas 1 to 3, but is not limited thereto.

[Formula 1]

[Formula 2]

[Formula 3]

The ghrelin analogue of the present invention comprises the steps of (A) preparing a linear peptide having a C-terminal amino acid of cysteine and comprising the sequence of the ghrelin active region; (B) reacting the N-terminus of the linear peptide with a carboxylic acid having a halogen substituent; and (C) coupling the sulfide group of the cysteine at the C-terminus with the halogen group at the N-terminus to cyclize in the presence of a base catalyst. 6-aminohexanoic acid in the N-terminal, C-terminal or ghrelin sequence of the ghrelin during the preparation of the linear peptide of step (A) for the preparation of the ghrelin analog into which the structure derived from 6-aminohexanoic acid is introduced. By introducing three, a linear peptide can be prepared. For example, in GSS2, two 6-aminohexanoic acids are introduced at the C-terminus, and in GSS3, one 6-aminohexanoic acid is introduced at each of the C-terminus and the N-terminus.

In the present invention, the peptides of steps (A) and (B) can be prepared by a conventional peptide preparation method. In the following examples, a solid-phase synthesis method using a resin was used, but the present invention is not limited thereto.

It was confirmed that the ghrelin analogues inhibit cell death by protecting cells from the cytotoxicity of aggregated amyloid beta. Accordingly, it can be usefully used for the prevention and treatment of diseases related to aggregation and deposition of amyloid beta. Diseases related to aggregation of amyloid beta include prion diseases such as Alzheimer's disease, Creutzfeldt-Jacob disease, Gerstmann-Straussler syndrome, amyloid angiopathy, cerebral amyloid angiopathy, Lewy body dementia, Parkinson's dementia complex in Guam, Systemic amyloid disease, Dutch amyloidosis, inclusion body myositis, reactive amyloidosis, idiopathic amyloidosis, familial amyloidosis, amyloidosis-related hereditary cerebral hemorrhage, mild cognitive impairment, frontotemporal dementia, age-related macular degeneration, Pick's disease, cerebral amyloid angiopathy may be mentioned, but is not limited thereto.

The composition of the present invention may also be usefully used for the prevention of diseases related to amyloid beta aggregation-related diseases in the risk group. The above risk group is a group genetically vulnerable to amyloid beta aggregation-related diseases with diseases such as Alzheimer's disease patients or familial amyloidosis in the family, an age group with a high incidence of amyloid beta aggregation-related diseases, or amyloid beta aggregation and deposition are not clearly observed. However, there may be a group showing suspicious symptoms of a related disease.

In order to use the composition of the present invention as a medicament for the prevention or treatment of diseases caused by amyloid beta aggregation, it is mixed with a pharmaceutically acceptable carrier (carrier), excipient (forming agent), diluent and the like to powder, granule, tablet , capsules or injections may be prepared and used. The pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount. In the present invention, the 'pharmaceutically effective amount' means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment. Effective dose levels depend on the patient's disease type, severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. It may depend on factors. The composition of the present invention may be administered alone or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art. In general, the compound may be administered once to several times in an amount of 0.1 to 100 mg per 1 kg of body weight to an adult.

As described above, the novel ghrelin analogue of the present invention is a cyclic ghrelin derivative and has a longer half-life than ghrelin while binding to the ghrelin receptor. It can be used more effectively by replacing ghrelin as a pharmaceutical composition for the prevention and treatment of diseases known to exhibit.

In particular, the composition containing the novel ghrelin analogue of the present invention has excellent neuronal protective effect against cytotoxicity caused by aggregation and deposition of amyloid beta, so it can be used for the prevention and treatment of diseases caused by amyloid beta aggregation, including Alzheimer's disease. have.

1 is a graph showing the neuroprotective effect of ghrelin analogs on the cytotoxicity of amyloid beta aggregates.
Figure 2 is a graph showing the inhibitory efficacy of ghrelin analogs on aggregation of amyloid beta.
3 is a graph showing the stability of ghrelin analogs.

Hereinafter, the present invention will be described in more detail with reference to the accompanying examples. However, these embodiments are merely examples for easily explaining the content and scope of the technical idea of the present invention, and thereby the technical scope of the present invention is not limited or changed. It will be natural for those skilled in the art that various modifications and changes can be made within the scope of the technical spirit of the present invention based on these examples.

[Example]

Example 1: Preparation of ghrelin standard and ghrelin analogue

Cysteine and aminohexanoic acid (Ahx) were introduced into the ghrelin standard (GSS) consisting of 10 amino acids known as the active site of ghrelin and cyclized.

1) Preparation Example 1: Preparation of ghrelin (GSS) standard

150 mg of Rink amide resin (capacity: 0.50mmol/g , amount: 150mg, 0.075mmol) and 2 ml of DMF were put in a reaction vessel and allowed to swell for 30 minutes.

Then, Fmoc-protected amino acid (5 eq.) dissolved in 1 ml of DMF and coupling reagents dissolved in 1 ml of DMF, namely, HOBt (Hydroxybenzotriazole, 5 eq.), HBTU (hexafluorophosphate benzotriazole tetramethyl uronium, 5 eq.), DIEA (diisopropylethylamine) , 5 eq.) was put into the reaction vessel containing the resin. After vortexing for 1 hour to allow the Fmoc-protected amino acid to bind to the resin, it was washed 3 times with DMF.

Fmoc-protected amino acid-bound resin was treated with 2 ml of DMF containing 20% (v/v) piperidine for 10 minutes, 3 minutes, and 3 minutes to remove the protecting group Fmoc. When the deprotection reaction was completed, Q-H-E was sequentially coupled to the resin by repeating the same method by adding the following Fmoc-protected amino acids.

Since synthesis is difficult when the sequence is long, from proline (P), Fmoc-protected amino acids (5 eq.) dissolved in 1 ml of DMF, HOBt (5 eq.), HBTU (5 eq.), DIEA ( 5 eq.) was repeated twice for 30 minutes each using the same amino acid, and coupling was carried out by a double coupling method of deprotection reaction, thereby preparing a Resin-Q-H-E-P-S-L-F sequence.

For coupling of octyl serin, Fmoc-protected octylserine (10 eq.) dissolved in 1 ml of DMF was dissolved in 1 ml of DMF, HOBt (10 eq.), HBTU (10 eq.), DIEA (10 eq.) .) and reacted for 3 hours. When the reaction was completed, Fmoc was removed by the deprotection reaction, S and G were sequentially coupled by applying a double coupling method, and the deprotection reaction was performed.

When the deprotection reaction of G was completed, it was washed sequentially with DMF, DCM (dichloromethane), and ether and dried under reduced pressure. 100 μl of 5% triisopropylsilane (TIS) and 2 ml of TFA (trifluoroacetic acid) containing 5% (v/v) water were added to the dried resin and reacted for 2 hours to separate the synthesized peptide from the resin.

The reaction solution excluding the resin was added to 20 ml of cold ether, and then centrifuged to obtain the synthesized peptide by precipitation. The precipitate was dissolved in 0.05% TFA and purified to a purity of 95% or more using RP-HPLC on an analytical Vydac C ₁₈ column. The purified sample was analyzed by MALDI-TOF MS to determine the molecular weight.

2) Preparation Example 2: Preparation of GSS1

After attaching cysteine to the resin using Fmoc-protected cysteine, Resin-C-Q-H-E-P-S-L-F-S-octyl-S-G was synthesized in the same manner as in Preparation Example 1. When the deprotection reaction of glycine (G) is completed, bromoacetic acid (10 eq.) dissolved in 1 ml of DMF, HOBt (10 eq.), HBTU (10 eq.), and DIEA (10 eq.) dissolved in 1 ml of DMF. ) was added and reacted for 3 hours, followed by deprotection reaction.

When the deprotection reaction was completed, the peptide (Linear GSS1) synthesized from the resin was isolated from the resin by the same method as in Preparation Example 1, purified, and analyzed by MALDI-TOF MS to confirm that the synthesis was successful in terms of molecular weight.

5 mg of Linear GSS1 having a purity of 95% or more, 1 ml of DMSO, and 50 μl of DIEA were placed in an Eppendor tube and stirred at 80° C. at a speed of 500 rpm for 30 minutes to conduct a cyclization reaction. When the reaction was completed, it was purified by RP-HPLC on an analytical Vydac C ₁₈ column, and molecular weight (Mw. 1356.5) was confirmed by MALDI-TOF. The purified sample was freeze-dried to obtain powdery GSS1.

3) Preparation Example 3: Preparation of GSS2

GSS2 was prepared in the same manner as in Preparation Example 2, except that Ahx-Ahx was additionally introduced using F-moc protected 6-aminohexanoic acid (Ahx) between cysteine and glutamine during the preparation of the peptide. (Mw. 1582.82).

4) Preparation Example 4: Preparation of GSS3

GSS3 was prepared in the same manner as in Preparation Example 3 except that Ahx was introduced between C and Q and after G (Mw. 1582.82).

Example 2: Evaluation of the effect on neuronal cell death by amyloid beta

Human neuroblastoma SH-SY5Y cells (ATCC CRL-2266) purchased from the Korea Cell Line Bank were prepared in DMEM medium supplemented with 10% (v/v) FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin. (Gibco-BRL Technologies, CA, USA) was used and cultured at 37° C., 5% CO ₂ conditions.

Aβ25-35 (Sigma-Aldrich) was dissolved in water at a concentration of 1 mM, aliquoted, and stored at -20°C before use. The stock solution was diluted to 100 μM with PBS (pH 7.4) buffer, and then treated at 37° C. for 7 to 10 days to form aggregate Aβ25-35.

The effect of the ghrelin analogues on the cell viability by agglutination Aβ25-35 was evaluated by MTT assay. Specifically, SH-SY5Y cells were first seeded in a 96-well plate at a concentration of 1×10 ⁴ cells/well and cultured for 24 hours. The cultured cells were treated with 10 μM of aggregated Aβ25-35 and/or GSS1 10, GSS2 100, or GSS3 50 μg of one of the ghrelin analogues prepared in Example 1 and further cultured for 48 hours. Cell viability after culture was evaluated by MTT assay, and the results are shown in FIG. 1 . In addition, for comparison with ghrelin, the ghrelin standard (G) of Preparation Example 1 was treated at the same concentration instead of the ghrelin analog to evaluate the cytotoxicity, and it is shown together in FIG. 1(A).

From the result of FIG. 1, it was confirmed that the ghrelin analogue according to the present invention significantly inhibited cytotoxicity due to aggregation of amyloid beta with superior efficacy than the ghrelin standard of Preparation Example 1. In particular, GSS2 showed cytotoxicity of aggregation Aβ25-35 completely neutralized.

Example 3: Evaluation of the effect on amyloid beta aggregation

When Aβ1-42 Peptide, a major biomarker and causative agent of Alzheimer's disease, is incubated at 37° C. for 3 days, Aβ fibrils and oligomers are generated by aggregation. The inhibitory effect of the ghrelin analog compounds on the formation of Aβ aggregation was confirmed by thioflavin T assay using the Thioflavin T Aβ42 aggregation kit (ANASPEC, Fremont, CA, USA). Thioflavin T test quantitatively shows the amount of aggregated protein because the higher the number of aggregates formed as a fluorescent substance that responds to the β-Sheet generated by the aggregation of Peptide, the higher the number.

The assay buffer was stored at 4 °C to prevent premature aggregation of Aβ, and other solutions were stored at room temperature. A test solution containing 2 mM ThT (thioflavin T) was prepared using cold assay buffer. Similarly, an Aβ42 peptide solution containing 0.25 mg/ml Aβ42 was prepared using a cold assay buffer. After standing at room temperature for 5 minutes to hydrate the protein, it was sonicated for 5 minutes to completely dissolve the peptide. Thereafter, the solution was centrifuged at 10,000 rpm at 4° C. for 5 minutes to remove the residue.

10 μl of assay buffer and 85 μl of Aβ42 peptide solution were dispensed into each well of a 96-well-plate, and GSS1, GSS2, or GSS3 compounds were added to a concentration of 1, 10, 50 or 100 μg/ml. As a positive control, morin or phenol red (PheR) was added to a concentration of 100 μM, or ghrelin prepared in Preparation Example 1 was added to a concentration of 100 μg/ml. As a negative control, 5 μl of assay buffer was added. After incubation at 37° C. for 3 days, the fluorescence intensity was measured at Ex/Em=440 nm/484 nm using an Infinite M200 Fluorescence Microplate Reader (Tecan, Mannedorf, Switzerland).

All of the compounds of the present invention showed that they inhibit aggregation of amyloid beta in a concentration-dependent manner, and all of them showed significant inhibitory efficacy compared to ghrelin. In particular, GSS2 and GSS3 into which 6-aminohexanoic acid was introduced had better inhibitory efficacy than GSS1 which did not contain 6-aminohexanoic acid.

Example 4: Stability evaluation

The stability of the ghrelin analogues prepared in Preparation Example was evaluated in vitro. To this end, thioflavin in the same manner as in Example 2, except that the ghrelin or ghrelin analog prepared in Preparation Example was previously dissolved in the assay buffer and then left at room temperature for 2, 4, 8 or 16 hours. A T test was performed. 3 is a graph showing the results, suggesting that the ghrelin of Preparation Example 1 was decomposed due to the loss of all amyloid beta aggregation inhibitory activity within 8 hours. On the other hand, GSS1, GSS2, and GSS3 were all maintained even at 16 hours, showing that stability was significantly improved compared to ghrelin. In particular, it was confirmed that GSS3 maintained a very stable state in solution because there was no significant difference in activity between 2 and 16 hours.

Claims (8)

A ghrelin analogue, characterized in that the N-terminus and C-terminus of the peptide containing the active region of ghrelin are cyclized through a sulfide bond derived from the thiol group of cysteine.
The method according to claim 1,
A ghrelin analogue, characterized in that 1 to 3 6-aminohexanoic acids are additionally introduced into the N-terminal, C-terminal or ghrelin sequence of ghrelin.
The method according to claim 1 or 2,
The ghrelin analogue is a ghrelin analogue, characterized in that it is represented by any one of Chemical Formulas 1 to 3.
[Formula 1]

[Formula 2]

[Formula 3]

(A) preparing a linear peptide in which cysteine is introduced at the C-terminus of ghrelin;
(B) reacting the N-terminus of the linear peptide with a carboxylic acid having a halogen substituent; and
(C) cyclization by coupling the sulfide group of the cysteine at the C-terminus with the halogen group at the N-terminus in the presence of a base catalyst;
A method for producing the ghrelin analog of claim 1, comprising a.
5. The method according to claim 4,
When preparing the linear peptide of step (A),
A method for producing the ghrelin analogue of claim 1, characterized in that 1 to 3 6-aminohexanoic acids are introduced into the N-terminal, C-terminal or ghrelin sequence of ghrelin.
6. The method according to claim 4 or 5,
The reaction of steps (A) and (B) is a method for producing a ghrelin analog, characterized in that it is prepared in a solid phase.
Comprising the compound of claim 1 as an active ingredient,
A pharmaceutical composition for preventing or treating diseases caused by amyloid beta aggregation.
8. The method of claim 7,
The disease caused by amyloid beta aggregation is,
Prion diseases such as Alzheimer's disease, Creutzfeldt-Jacob disease, Gerstmann-Strausler syndrome, amyloid angiopathy, cerebral amyloid angiopathy, Lewy body dementia, Parkinson's dementia complex in Guam, systemic amyloid disease, Dutch type A pharmaceutical composition, characterized in that it is amyloidosis, inclusion body myositis, reactive amyloidosis, idiopathic amyloidosis, familial amyloidosis, amyloidosis-related hereditary cerebral hemorrhage, mild cognitive impairment, frontotemporal dementia, age-related macular degeneration, Pick's disease or cerebral amyloid angiopathy.

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