Neprilysin inhibitor composition
Mycosporine-like amino acids from cyanobacteria provide a potent neprilysin inhibitor composition to address the inadequacies of existing inhibitors, effectively reducing neprilysin activity and expression for therapeutic benefits.
Patent Information
- Application Number
- JP2024096791
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2024-06-14
- Publication Date
- 2025-12-25
AI Technical Summary
Existing neprilysin inhibitors, such as ginger extract and coenzyme Q10, are insufficient in inhibiting neprilysin activity, which contributes to wrinkle formation and other pathological conditions like heart failure and Alzheimer's disease.
Development of a neprilysin inhibitor composition using mycosporine-like amino acids or their salts, particularly extracted from cyanobacteria, which effectively inhibit neprilysin activity and expression.
Mycosporine-like amino acids significantly suppress neprilysin activity and expression induced by IL-1α stimulation in fibroblasts, offering potential treatments for wrinkles, heart failure, and Alzheimer's disease.
Smart Images
Figure 2025187757000004 
Figure 2025187757000005 
Figure 2025187757000001
Abstract
Description
[Technical Field]
[0001] The present invention relates to a neprilysin inhibitor composition. [Background technology]
[0002] Neprilysin (NPL), also known as neutral endopeptidase or enkephalinase, is a transmembrane metalloprotease expressed in various tissues. It degrades and inactivates various substrates by cleaving hydrophobic amino acid residues at the amino terminus of proteins. Because neprilysin degrades and inactivates natriuretic peptides (ANP, BNP, and CNP), which are involved in vasodilation, blood pressure reduction, cardiac hypertrophy suppression, fibrosis suppression, and aldosterone secretion suppression, angiotensin receptor-neprilysin inhibitors have been used to treat heart failure and hypertension. In the skin, elastin, produced by fibroblasts in the dermis, is one of the important components for maintaining firmness and elasticity. Neprilysin degrades elastin as a substrate, and is therefore thought to be one of the enzymes responsible for wrinkle formation. Therefore, effective neprilysin inhibitors are desired. On the other hand, neprilysin also has the ability to degrade amyloid beta peptide, which accumulates in the brains of Alzheimer's disease patients and is thought to be deeply involved in the onset and progression of the disease. Therefore, there is hope that decomposition activity promoters may be useful in removing the causative substance and in treating Alzheimer's disease.
[0003] The identification of the neprilysin gene and protein structure has enabled gene and protein analysis of this enzyme, enabling the elucidation of the cell biological mechanism by which neprilysin expression is upregulated in dermal fibroblasts upon UV exposure. Analytical studies using a co-culture system of epidermal cells and dermal fibroblasts have shown that increased neprilysin expression in fibroblasts is due to the increased secretion of interleukin-1α (IL-1α) from UV-exposed epidermal cells, which penetrates the dermis and acts on fibroblasts. In other words, it is believed that wrinkles are formed through UVB stimulation, which causes interleukin-1α (IL-1α) secreted by epidermal cells to act on dermal fibroblasts, increasing neprilysin (NPL) present on the fibroblast cell membrane, cleaving surrounding elastin fibers, altering the three-dimensional structure of elastin and reducing skin elasticity.
[0004] Substances that inhibit neprilysin have been discovered, and it has been reported that ginger extract, which has a neprilysin inhibitory effect, can suppress wrinkle formation in humans. It has also been elucidated that coenzyme Q10 exerts its anti-wrinkle effect by suppressing the increased expression of neprilysin in fibroblasts stimulated by IL-1. [Prior art documents] [Patent documents]
[0005] [Non-Patent Document 1] Genji Imokawa: 23 years of research (1994-2017) into the mechanisms of UV-induced wrinkles and sagging skin. Fragrance J.6:12-44, 2017. [Non-patent document 2] Naoko Morisaki, Shigeru Moriwaki, Yoriko Sugiyama-Nakagiri, Keiichi Haketa, Yoshinori Takema, Genji Imokawa: Neprilysin is identical to skin fibroblast elastase - its role in skin aging and UV responses. J Biol Chem 2010;285(51):39819-39827. [Non-patent document 3] Hiroaki Nakajima, Shuko Terazawa, Takao Niwano, Yorihiro Yamamoto, Genji Imokawa: The Inhibitory Effects of Anti-Oxidants on Ultraviolet-Induced Up-Regulation of the Wrinkling-Inducing Enzyme Neutral Endopeptidase in Human Fibroblasts.PLoS One.DOI:10.1371 / journal.pone.0161580 September 20,2016 Summary of the Invention [Problem to be solved by the invention]
[0006] However, the neprilysin inhibitory effects of the above-mentioned ginger extract and coenzyme Q10 are not sufficient, and the development of new neprilysin inhibitors is desired. [Means for solving the problem]
[0007] The present inventors have investigated the neprilysin inhibitory activity of various naturally occurring components and have found that mycosporine-like amino acids or salts thereof, which have been known to have UV protection activity, have excellent neprilysin inhibitory activity, leading to the completion of the present invention.
[0008] That is, the present invention provides the following [1] to [4]. [1] A neprilysin inhibitor composition containing a mycosporine-like amino acid or a salt thereof as an active ingredient. [2] The neprilysin inhibitor composition according to [1], wherein the mycosporine-like amino acid or a salt thereof is a mycosporine-like amino acid or a salt thereof extracted from cyanobacteria. [3] The neprilysin inhibitor composition according to [1], wherein the mycosporine-like amino acid or a salt thereof is a mycosporine-like amino acid or a salt thereof extracted from cyanobacteria with water or a mixture of water:ethanol=1:9 to 9:1. [4] The neprilysin inhibitor composition according to any one of [1] to [3], which is an external skin composition or an oral composition. [Effects of the Invention]
[0009] Mycosporine-like amino acids or salts thereof inhibit IL-1α-stimulated neprilysin activity in fibroblasts, and are useful as active ingredients of neprilysin inhibitor compositions. [Brief explanation of the drawings]
[0010] [Figure 1] 1 shows the inhibitory effect of mycosporine-like amino acids (MAAs) on the increase in neprilysin activity (RFU: relative enzyme activity). [Figure 2] 1 shows the inhibitory effect of mycosporine-like amino acids (MAAs) on the expression of neprilysin protein (results of Western blotting). DETAILED DESCRIPTION OF THE INVENTION
[0011] Neprilysin, an enzyme encoded by the MME gene in humans, is a zinc-dependent metalloprotease that cleaves peptides such as glucagon, enkephalin, substance P, neurotensin, oxytocin, and bradykinin at the N-terminal hydrophobic residues. Neprilysin also degrades amyloid beta. Abnormal folding and aggregation of amyloid beta in neural tissues have been suggested as a cause of Alzheimer's disease. Furthermore, as mentioned above, neprilysin is also known as an enzyme that causes wrinkles.
[0012] Mycosporine-like amino acids are known to be found in fish and shellfish, seaweed, star jelly plants, freshwater organisms, and fungal cultures. The mycosporine skeleton, a metabolic product of these organisms, is a general term for amino acids that exhibit a partial structure of an amino alcohol or an amino acid bond.
[0013] One aspect of the present invention is a neprilysin inhibitor composition containing a mycosporine-like amino acid or a salt thereof as an active ingredient. The mycosporine-like amino acids used in the present invention include extracts from the aforementioned fish and shellfish, seaweed, star jelly plants, freshwater organisms, etc., and extracts from fungal cultures, etc., but it is preferable to use an extract from blue-green algae, and more preferably a mycosporine-like amino acid or a salt thereof extracted from blue-green algae with water or a mixture of water:ethanol = 1:9 to 9:1.
[0014] Mycosporine-like amino acids can be extracted from cyanobacteria by extracting them from dried or lyophilized cyanobacteria using polar solvents such as water, ethanol, acetone, methanol, isopropanol, and acetonitrile, or a mixture thereof. A water-ethanol mixture is preferred as the extraction solvent, with a water:ethanol ratio of 1:9 to 9:1 being more preferred. The resulting extract is preferably further concentrated by adsorbing the mycosporine-like amino acids using an activated carbon adsorption column, a resin column, or the like, and eluting with a water-ethanol mixture. Further purification can be performed using high-performance liquid chromatography.
[0015] Known examples of mycosporine-like amino acids include the following compounds:
[0016] [ka]
[0017] [ka]
[0018] [ka]
[0019] Examples of salts of mycosporine-like amino acids include acid addition salts such as hydrochlorides, and alkali metal salts such as sodium salts and potassium salts.
[0020] As shown in the Examples below, mycosporine-like amino acids or salts thereof inhibit IL-1α-stimulated neprilysin activity in fibroblasts, and are therefore useful as active ingredients in neprilysin inhibitor compositions. Neprilysin is known to be a causative enzyme of wrinkles. Therefore, the neprilysin inhibitor composition of the present invention can be used as a composition for preventing and / or treating heart failure and hypertension, or as a composition for inhibiting wrinkle formation, in the form of a drug, quasi-drug, food for specified health uses, food with functional claims, or cosmetic.
[0021] The neprilysin inhibitor composition of the present invention may be administered orally or transdermally. More specifically, oral compositions such as tablets, granules, fine granules, capsules, and lozenges are preferred; and topical skin compositions such as creams, ointments, emulsions, and lotions are preferred. In addition, in the case of foods for specified health uses or foods with functional claims, the composition may be in the same form as ordinary foods such as beverages and yogurt.
[0022] The content of the mycosporine-like amino acid or a salt thereof in the composition of the present invention is not particularly limited, but is preferably 0.001 to 50% by mass, more preferably 0.001 to 30% by mass.
[0023] In addition to the mycosporine-like amino acid or a salt thereof, the oral composition may contain excipients, binders, disintegrants, lubricants, colorants, flavoring agents, odorants, etc. In the case of a topical skin composition, in addition to the mycosporine-like amino acid or a salt thereof, various oils, dyes, preservatives, surfactants, thickeners, etc. may be contained. [Example]
[0024] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.
[0025] Example 1 Human dermal fibroblasts (HDFs) were cultured in serum-containing medium and the cell suspension (1.5 × 10 5 The cell suspension was dispensed into a 12-well plate at 1 mL / well and cultured for approximately 5 hours in an incubator (37°C, 5% carbon dioxide). After confirming that the cells had adhered to the bottom of the plate, the medium was replaced with serum-free medium and cultured overnight in an incubator. Mycosporine-like amino acids (MAAs)-supplemented media (9.225, 18.45, and 36.9 μg / mL, serum-free) and media supplemented with MAAs (at the same concentrations) and IL-1α (5 ng / mL) were prepared. After culturing for 1 hour in media containing only MAAs (8 wells per condition), half of the wells (4 wells) were replaced with media containing only MAAs and media containing MAAs and IL-1α. The same procedure was repeated using media without MAAs as a control. The sample used was a diluted solution of MAAs (36.9 μg / mL) extracted and purified from cyanobacteria using a 70% ethanol solution and activated carbon column, as described in JP 2007-16004. After 72 hours of culture, the medium was removed, cell lysis buffer was added, and the cells were scraped off. The cells were disrupted by ultrasound, then centrifuged, and the supernatant was collected. The resulting protein solution was measured for concentration using the BCA method, and diluted with the buffer used for extraction to make each sample a uniform concentration. Samples with uniform protein concentrations were used to measure neprilysin activity (Neprilysin Activity Assay Kit, ANA) and analyze protein expression by Western blotting.
[0026] When HDF cells were cultured in a medium containing IL-1α, neprilysin activity increased, but when they were cultured for 1 hour in a medium containing MAAs (18.5 or 36.9 μg / mL) before IL-1α treatment, the increase in NPL activity was significantly suppressed (Figure 1). Furthermore, when MAAs were added at 36.9 μg / mL, not only was activity suppressed, but protein expression was also significantly suppressed (Figure 2).
[0027] MAAs were found to have the effect of suppressing the increase in neprilysin activity and protein expression induced by IL-1α stimulation in human dermal fibroblasts.
Claims
1. A neprilysin inhibitor composition comprising a mycosporine-like amino acid or a salt thereof as an active ingredient.
2. 2. The neprilysin inhibitor composition according to claim 1, wherein the mycosporine-like amino acid or a salt thereof is a mycosporine-like amino acid or a salt thereof extracted from blue-green algae.
3. 2. The neprilysin inhibitor composition according to claim 1, wherein the mycosporine-like amino acid or a salt thereof is extracted from cyanobacteria with water or a mixture of water:ethanol=1:9 to 9:
1.
4. The neprilysin inhibitor composition according to any one of claims 1 to 3, which is a composition for external use on the skin or a composition for oral administration.