JP7323162B2 - nucleic acid aptamer - Google Patents

Description

The present invention relates to a nucleic acid aptamer, a hair restorer and a hair restorer composition.

Many people suffer from hair loss such as hair loss due to aging or genetic factors, or alopecia areata due to excessive stress. According to the 2017 edition of medical guidelines for male and female pattern hair loss (guidelines of the Japanese Dermatological Association), the incidence of male pattern hair loss, androgenetic alopecia (AGA) is about 30% on average for all ages. .
In addition, according to the 2014 government statistics patient survey (injury classification change), the total number of patients who visited hospitals due to alopecia alone exceeded 100,000, and it is believed that the number of latent alopecia patients is even greater. be done.
Against this background, various hair cosmetics, quasi-drugs, or pharmaceuticals for the purpose of suppressing hair growth, hair growth, and hair loss have been developed and are already on the market.

However, even in the case of AGA, the causes of hair loss are diverse, including the effects of male hormones, poor blood circulation in the capillary network of the dermal layer of the head, and conditions of the scalp surface (dryness, excess sebum, etc.). Most of the many hair restorers currently on the market are applied to the head with a hair restorer containing ingredients derived from natural products and chemical substances that are effective for these hair loss causes, and the scalp surface is treated. The purpose is to suppress hair loss or promote hair growth by penetrating these ingredients from. However, at present, there is no universally effective hair restorer, etc., and even if it is expected to have effects such as thickening thin hair or slowing down the hair loss cycle somewhat, it is possible. Moreover, there is a problem that there is a large individual difference in their effects.
Therefore, there is still a high need for a hair restorer that is universally effective and stronger than conventional products, and its development has been desired.

Moreover, not only human hair but also the body hair of pet animals or livestock animals, the composition for external use exhibits a hair-growth effect against alopecia symptoms caused by skin disease, aging, stress caused by breeding environment, etc. I have a demand for things.

Hair on the head and body usually grows while repeating the anagen, catagen, and telogen phases.Dermal papilla cells at the base of the hair follicle, which is the skin tissue that wraps the hair root, are activated during the anagen phase, resulting in hair matrix cells. starts dividing and hair grows, and the hair grows as the hair matrix cells extend as the hair shaft. After that, in the catagen phase, hair growth declines and irreversibly enters the telogen phase, where hair growth stops, and hair loss eventually occurs. The cycle of hair growth and hair loss from the anagen phase to the telogen phase (hair cycle: hair cycle) is a delicate biological mechanism that differs for each hair and is controlled by various genes and their products.

In recent years, research on hair growth has progressed, and attention has been focused on fibroblast growth factor (FGF) as one of the substances that affect the hair cycle. FGF, as its name suggests, is a factor involved in the proliferation of fibroblasts, and is known to support the formation of new hair follicles by fibroblasts.
There are 23 types of FGFs as family proteins, and they are known to be multifunctional secretory factors that play an important role as metabolic regulators for maintaining homeostasis of the body. FGF-1, FGF-2, FGF-5, FGF-7, FGF-10, FGF-13, FGF-18 and FGF-22 are known to be expressed in the epithelium of skin and hair follicles. When these eight FGFs bind to any of FGFR1 to FGFR4 on the target cell surface, it is believed that signals are transmitted within the cell to control the hair cycle. For example, FGF-18, which is widely known to regulate the formation and growth of bone and cartilage, is expressed in the hair follicle stem cell region and maintains the telogen phase (Non-Patent Document 1).

In addition, FGF-5, one of the FGF family proteins, is known to act suppressively on hair growth. That is, FGF-5 induces the anagen phase to the catagen phase in the hair cycle of the hair follicle. In other words, FGF-5 terminates the anagen phase of the hair follicle by its restricted expression during the late anagen phase of the hair follicle, leading to hair growth arrest. For example, the present inventors disclosed that a specific preparation of Sasa extract has inhibitory activity against the action of FGF-5, and a hair restorer composition based on the hair cycle control mechanism by FGF-5 using such Sasa extract preparation. (Patent Document 1).
Furthermore, FGF-5S, a short-chain molecule of FGF-5 that is expressed in large amounts in hair follicles during the anagen phase, exhibits antagonistic activity against FGF-5, thereby delaying the transition from the anagen phase to the catagen phase. It was also found to promote hair growth (Non-Patent Documents 2 to 5). However, the interaction mechanism between FGF-5 and its receptor has not been elucidated.

Furthermore, the studies of the present inventors revealed that FGF-5 inhibits hair growth and has the effect of shifting the hair cycle from the growth phase to the regression phase. have also invented a hair restorer composition containing phytic acid that inhibits FGF-5 (Patent Document 2).

JP 2007-238503 A JP 2017-43594 A

Kawano M et al., J Invest Dermatol.124:877-885 (2005) Ozawa K et.al., J.Biol.Chem.273:29262-29271(1998) Suzuki et. al., J. Invest. Dermatol. 111:963-972 (1998) Suzukiet.al., J.Invest.Dermatol.114:456-463(2000) Ota et.al., Biochem.Biophys.Res.Commun.290:169-176(2002)

In the course of research on the action of FGF-5, the present inventors came up with the idea that a substance having an inhibitory activity against this action could be used as a highly effective hair restorer. As a result of further intensive research in order to discover substances having an inhibitory effect on the action of FGF-5, the present inventors focused on nucleic acid aptamers as a substance that inhibits the action of FGF-5. Nucleic acid aptamers are single-stranded nucleic acid molecules that bind specifically to target molecules by adopting a unique three-dimensional structure. It can be easily chemically synthesized and chemically modified, has low immunogenicity (difficult to be eliminated by immune reactions), and has high stability. It has been put to practical use.

The present inventors have successfully developed a nucleic acid aptamer having a high inhibitory effect on FGF-5, and found that the nucleic acid aptamer has a high potential for hair growth activity.
Based on the above, an object of the present invention is to provide a nucleic acid aptamer that inhibits FGF-5, a hair growth activator, and the like.

The present invention is a nucleic acid aptamer or the like that specifically binds to FGF-5 (Fibroblast growth factor 5) protein.

That is, the present invention provides, for example, the following [1] to [23].
[1]
A nucleic acid aptamer that is a nucleic acid molecule that specifically binds to FGF-5 (Fibroblast growth factor 5), a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof.
[2]
The nucleic acid aptamer according to [1], a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof, wherein the nucleic acid aptamer inhibits the binding of the FGF-5 to the FGF-5 receptor.
[3]
The nucleic acid aptamer according to [1] or [2], which has a dissociation constant for FGF-5 of 10 pM to 100 nM, a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof.
[4]
The nucleic acid aptamer according to any one of [1] to [3], which has 10 to 100 bases, a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof.
[5]
The nucleic acid aptamer according to any one of [1] to [4], which has 50 or more bases and 60 or less bases, a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof.
[6]
said nucleic acid molecule has at least one sequence selected from the group consisting of sequences A, B, and C;
the sequence A is the sequence: 5'-ACCU-3';
The sequence B is the sequence: 5'-XGACA-3' (X represents any base),
The nucleic acid aptamer according to any one of [1] to [5], wherein the sequence C is the sequence: 5'-UCCAGA-3', and pharmaceuticals of the nucleic acid aptamer legally acceptable salts, or solvates thereof.
[7]
The nucleic acid aptamer according to [6], wherein the X is a pyrimidine base in the sequence: 5'-XGACA-3', which is the sequence B, and the pharmaceutically acceptable nucleic acid aptamer salts, or solvates thereof;
[8]
The nucleic acid aptamer according to [6] or [7], wherein X is C or U, a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof.
[9]
said nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NOS: 1-11, 13, 16, and 17; A nucleic acid selected from variants of a nucleic acid molecule having a sequence in which 1 to 5 bases are substituted, deleted, or inserted in a position other than said sequences A, B, and C that said sequence has The nucleic acid aptamer according to any one of [6] to [8], which is a molecule, a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof.
[10]
The nucleic acid aptamer according to any one of [1] to [9], wherein the nucleic acid molecule has a sequence selected from the group consisting of SEQ ID NOs: 1 to 11, 13, 16, and 17; A pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof.
[11]
Any one of [1] to [10], wherein the nucleic acid molecule has a sequence selected from the group consisting of SEQ ID NOS: 1, 4, 6, 7, 10, 13, 16, and 17 3. The nucleic acid aptamer according to 1, a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof.
[12]
The nucleic acid aptamer according to any one of claims [1] to [11], wherein the nucleic acid molecule is a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NOs: 1, 6, 16, and 17; A pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof.
[13]
The nucleic acid aptamer according to [11], wherein the nucleic acid molecule has a sequence represented by SEQ ID NO: 1 or 6, a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof.
[14]
The nucleic acid aptamer according to any one of [1] to [12], a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof, wherein the nucleic acid molecule is RNA.
[15]
The nucleic acid aptamer according to any one of [1] to [13], wherein the FGF-5 is human FGF-5, a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof.
[16]
In one or more of cytosine (C) and uracil (U) contained in the nucleotide contained in the nucleic acid aptamer, the 2' position of ribose is substituted with a fluorine atom,
The nucleic acid aptamer according to any one of [1] to [15], a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof.
[17]
The 2'-position of ribose in cytosine (C) and uracil (U) contained in the nucleotide contained in the nucleic acid aptamer is substituted with a fluorine atom,
The nucleic acid aptamer according to any one of [1] to [16], a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof.
[18]
A hair growth activator comprising at least one selected from the nucleic acid aptamer according to any one of [1] to [17], pharmaceutically acceptable salts of the nucleic acid aptamer, and solvates thereof.
[19]
The hair growth activator according to [18], which has an inhibitory activity against the action of FGF-5.
[20]
A hair restorer composition containing the hair restorer activator according to [18] or [19].
[21]
0.00001 to 10% by mass of the nucleic acid aptamer according to any one of [1] to [17], a pharmaceutically acceptable salt of the nucleic acid aptamer, and a solvate thereof, to [20] The described hair restorer composition.
[22]
Containing 0.00005 to 5% by mass of the nucleic acid aptamer according to any one of [1] to [17], a pharmaceutically acceptable salt of the nucleic acid aptamer, and a solvate thereof, [20] 3. The hair restorer composition according to .
[23]
The hair restorer composition according to any one of [20] to [22], which is a pharmaceutical.

According to the nucleic acid aptamer of the present invention, it is possible to provide a physically stable hair growth activator and a hair restorer composition that can be expected to have a more reliable hair growth effect than conventional products.

1 is a graph showing the activity of FGF-5 prepared in Preparation Example 1. FIG. Figure 2 shows the sequences of aptamers F-02, F-58, F-33, F-05, F-06, F-18, F-14, F-19, F-46, F-52 and F-39. be. The boxed bases are the consensus sequences AC. FIG. 3 shows cytosine and Sequence demonstrating that uracil is fluorinated. However, C(F) represents fluorinated cytosine, and U(F) represents fluorinated uracil. FIG. 4(A) shows F02-52, F02-42, F02-40, F02-56, which is a shortened (stabilized) aptamer F-02, and F18-, which is a shortened (stabilized) aptamer F-18. 56 arrays. FIG. 4(B) is a sequence demonstrating fluorination of cytosine and uracil in F02-52, F02-42, F02-40, F02-56 and F18-56. However, C(F) represents fluorinated cytosine, and U(F) represents fluorinated uracil. FIG. 5 shows aptamers F-02, F-05, F-14, F-18, F-19, F-39, F-52, and F-02-52 using the free tool 'mfold'. Secondary structure prediction. FIG. 6 shows secondary structure predictions by Chiba Institute of Technology for aptamer F-02 and F02-52 obtained by shortening (stabilizing) the sequence. FIG. 7 was performed to confirm the binding ability of aptamers F-02, F-05, F-14, F-18, F-19, F-39, F-51, and F-52 to FGF-5. This is the result of EMSA. FIG. 8 is a graph of the amount of intermolecular interaction measured to confirm the binding ability between aptamer F-02 and water-soluble FGF-5 protein. FIG. 9 is a graph of the amount of intermolecular interaction measured to confirm the binding ability of aptamer F-02 to FGF-1, FGF-2, FGF-4, FGF-6, and FGF-5. . FIG. 10 is a graph obtained by analyzing the intermolecular interaction between aptamer F-02 and water-soluble FGF-5 protein. FIG. 11 is a graph showing the effect of each aptamer on FGF-5-dependent cell proliferation. FIG. 12 is a graph showing the effect on FGF-2- or FGF-5-dependent cell proliferation in the presence of random primers. FIG. 13 is a graph showing the effect of each aptamer on FGF-2-dependent cell growth inhibition. FIG. 14 is a graph showing the effect of aptamer F-02 on FGF-1- or FGF-5-dependent cell proliferation. FIG. 15 is a graph showing the effect of each shortened aptamer on FGF-5-dependent cell growth inhibition. FIG. 16 is a graph showing the effect of aptamer F-02 on the transcription level of alkaline phosphatase (ALP), a hair follicle induction marker that is suppressed by FGF-5.

The "nucleic acid aptamer" of the present invention is a nucleic acid aptamer that is a nucleic acid molecule that specifically binds to a target molecule, and the aptamer may be in the form of a pharmaceutically acceptable salt or solvate. The target molecule of the nucleic acid aptamer of the present invention is FGF-5 (Fibroblast growth factor 5) protein.
The nucleic acid aptamer of the present invention directly inhibits the binding of FGF-5, which is a nucleic acid molecule that specifically binds to FGF-5, to the FGF-5 receptor, thereby inhibiting hair growth by FGF-5 signal transduction. It has an excellent effect on hair growth and hair growth by suppressing cycle progression and hair loss signals to hair papilla.

<Nucleic acid aptamer>
A nucleic acid aptamer according to the present invention is a nucleic acid molecule that specifically binds to a target molecule, FGF-5. The nucleic acid aptamer may be a pharmaceutically acceptable salt or solvate of the nucleic acid aptamer. The FGF-5 may be FGF-5 of any animal species such as mouse, rat, rabbit, guinea pig, gerbil, hamster, ferret, dog, miniature pig, monkey, cow, horse, sheep, etc. More preferred is human FGF-5. The nucleic acid molecule may be either DNA or RNA, preferably single-stranded RNA. The nucleic acid molecule may be either an artificial nucleic acid molecule or a natural nucleic acid molecule, but is preferably an artificial nucleic acid molecule.

By specifically binding to FGF-5, the nucleic acid aptamer inhibits the binding of FGF-5 to its receptor and directly suppresses FGF-5 signal transduction.
From the viewpoint of suppression of hair loss signals to dermal papilla associated with FGF-5, it is preferable that the binding between the nucleic acid aptamer and FGF-5 is strong, that is, the dissociation constant between the two is preferably small. The dissociation constant between FGF-5 and FGF-5 receptor is preferably 10 pM to 100 nM, more preferably 10 pM to 10 nM.

The number of bases contained in the nucleic acid molecule that is the nucleic acid aptamer is preferably 10 or more and 100 or less, more preferably 50 or more and 60 or less. Since the average molecular weight of one base is about 330, the molecular weight of the nucleic acid aptamer of the present invention is preferably about 3300-33000, more preferably about 16500-19800.
Adenine (abbreviation: A), guanine (abbreviation: G), cytosine (abbreviation: C), thymine (abbreviation: T), and uracil (abbreviation: U) are generally used as nucleotides contained in the nucleic acid molecule. Although the above natural nucleotides can be used as they are, it is typically preferable to use the above modified nucleotides such as fluorinated (F) or methylated from the viewpoint of stability. Natural nucleotides other than those mentioned above and non-natural nucleotides can also be used.

When modified nucleotides are used, the modification site within each nucleotide is not particularly limited, but modification is preferably performed at the 2' position of ribose in the nucleotide. In particular, when obtaining an aptamer by the SELEX method, as described later, from the viewpoint of resistance to RNases, it is a common technical means to use C and U having F-formed ribose at the 2'-position. Therefore, in one or more of cytosine (C) and uracil (U) contained in the nucleotides contained in the nucleic acid aptamer, the 2'-position of ribose is preferably substituted with a fluorine atom. It is preferred that the 2'-position of ribose in cytosine (C) and uracil (U) contained in the nucleotides contained in the nucleotide is substituted with a fluorine atom. Here, cytosine (C) and uracil (U) refer to substantially all cytosine (C) and uracil (U), specifically 95-100% cytosine (C) and uracil (U). It is preferably F-formed, more preferably 98-100% cytosine (C) and uracil (U) is F-formed, and 99-100% cytosine (C) and uracil (U) is particularly preferred to be F-modified. Nucleotides artificially endowed with desired properties by other modifications, protection, deprotection, etc. may also be used. In addition, from the viewpoint of improving stability and pharmacokinetics, the length and size of the nucleic acid molecule may be appropriately adjusted, and the ends of the nucleic acid molecule may be subjected to chemical modification such as PEG modification. In addition, PEG is generally known to be important for improving pharmacokinetics. In addition, unless there are special circumstances, the above five nucleotides are described using their respective abbreviations in this specification (including claims and drawings).

(Nucleic acid aptamer sequence-1)
A nucleic acid aptamer in which the target molecule is FGF-5 is described below.

As described above, the number of bases contained in the nucleic acid molecule is preferably 10 or more and 100 or less, more preferably 50 or more and 60 or less.
The nucleic acid molecule that is the nucleic acid aptamer has the sequence A: 5'-ACCU-3', the sequence B: 5'-XGACA-3' (X represents any base), preferably having at least one sequence of the sequence C: 5'-UCCAGA-3', particularly having all of the sequences A to C preferable. When the nucleic acid molecule has all of the sequences A to C, the number of each sequence is not particularly limited, but it is preferable to have one of each sequence. When the nucleic acid molecule has all sequences A to C one by one, the order is not particularly limited, but 5'-sequence A-sequence B-sequence C-3'("-" links each sequence linker sequence, the sequence of which is not particularly limited).

In the sequence B: 5'-XGACA-3', X is any base as described above, preferably a pyrimidine base, more preferably C or U .

The nucleic acid molecule is selected from the group consisting of SEQ ID NOS: 1-11, 13, 16, and 17, and a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NOS: 1-11, 13, 16, and 17 A nucleic acid selected from variants of a nucleic acid molecule having a sequence in which 1 to 5 bases are substituted, deleted, or inserted in a position other than said sequences A, B, and C that said sequence has Molecular is preferred. More preferably, the nucleic acid molecule is a nucleic acid molecule having a sequence selected from the group consisting of 1-11, 13, 16, and 17, SEQ ID NOs: 1, 4, 6, 7, 10, 13, 16, and more preferably a nucleic acid molecule having a sequence selected from the group consisting of 17, even more preferably a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NOS: 1, 6, 16, and 17; Nucleic acid molecules having a sequence represented by SEQ ID NO: 1 or 6 are particularly preferred. The sequences represented by SEQ ID NOs:2 and 3 are preferred because they are similar sequences to the sequence represented by SEQ ID NO:1. The sequences represented by SEQ ID NOS: 13 and 16 are preferred because they are shortened versions of the sequence represented by SEQ ID NO:1. Also, the sequence represented by SEQ ID NO: 17 is preferable because it is a shortened version of the sequence represented by SEQ ID NO: 6.
In terms of strength of binding inhibition, nucleic acid molecules having sequences represented by SEQ ID NOS: 6, 7 and 10, SEQ ID NO: 4, and its analogous sequence, SEQ ID NO: 5, are also preferred.

In the present specification, the aptamer having SEQ ID NO: 1 is F-02, the aptamer having SEQ ID NO: 2 is F-58, the aptamer having SEQ ID NO: 3 is F-33, and the aptamer having SEQ ID NO: 4 is F-05. , F-06 an aptamer having SEQ ID NO: 5, F-18 an aptamer having SEQ ID NO: 6, F-14 an aptamer having SEQ ID NO: 7, F-19 an aptamer having SEQ ID NO: 8, and SEQ ID NO: 9 Aptamer F-46, aptamer having SEQ ID NO: 10 F-52, aptamer having SEQ ID NO: 11 F-39, aptamer having SEQ ID NO: 12 F-51, aptamer having SEQ ID NO: 15 F-02- 40, the aptamer with SEQ ID NO: 14 is designated F02-42, and the aptamer with SEQ ID NO: 13 is designated F02-52. Also, the aptamer having SEQ ID NO: 16 is referred to as F-02-56, and the aptamer having SEQ ID NO: 17 is referred to as F-18-56.

The nucleic acid molecule has 1 to 5 bases, preferably 1 to 2 bases substituted, deleted, or inserted at locations other than sequences A, B, and C that may be contained in the sequence. may Examples of nucleic acid molecules in which base substitutions, deletions, or insertions have occurred include the sequences A and B of sequences selected from the group consisting of SEQ ID NOS: 1 to 11, 13, 16, and 17. , and variants of nucleic acid molecules having sequences with 1-5 base substitutions, deletions, or insertions at locations other than C, including 1-2 base substitutions, deletions, or insertions. Preferred are variants of nucleic acid molecules having the sequence defined.

(Selection of nucleic acid aptamers)
The method for obtaining the nucleic acid aptamer of the present invention is not particularly limited, but generally a method for obtaining from a nucleic acid library such as the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method is common.
The SELEX method is widely used especially when obtaining nucleic acid aptamers that specifically bind to a certain target protein. In this method, after mixing a nucleic acid library of about 10 ¹⁴ random sequences with target molecules, the nucleic acids bound to the target molecules are amplified using methods such as RT-PCR in the case of RNA. , a method of determining a sequence by sequence analysis to identify a target nucleic acid aptamer. When obtaining an aptamer by the SELEX method in this way, A, G, C, T, and U described above are used. is a common technical means.
In recent years, a method has also been used in which it is possible to select an RNA sequence that does not exist in nature and is most suitable for the desired function by performing selection while introducing mutations into the nucleic acid during amplification.

(Structure determination of nucleic acid aptamer)
Since a nucleic acid aptamer inhibits the function of a target molecule by specifically binding to the target molecule, not only the sequence but also the three-dimensional structure is important.
Regarding the secondary structure of nucleic acids, free tools such as mfold and CentroidFold, which are tools for predicting the secondary structure based on the free energy change when the bases in the nucleic acid molecule form base pairs, have been published. Predictions can be made using them as appropriate.
Techniques such as nuclear magnetic resonance spectroscopy and X-ray crystallography can be used to determine the three-dimensional or higher three-dimensional structure of the nucleic acid aptamer or the three-dimensional structure when the nucleic acid aptamer binds to the target molecule.

<Hair growth activator>
The hair restorer of the present invention contains at least one selected from the nucleic acid aptamers, pharmaceutically acceptable salts of the nucleic acid aptamers, and solvates thereof.
The hair growth activator of the present invention may be the nucleic acid aptamer itself, the salt itself, the solvate itself, or a mixture thereof. The hair growth activator of the present invention preferably consists only of one or more components selected from the nucleic acid aptamers, pharmaceutically acceptable salts of the nucleic acid aptamers, and solvates thereof.
The hair growth activator of the present invention has an activity of inhibiting the action of FGF-5, and is excellent in hair growth effects.

<Hair restorer composition>
The hair restorer composition of the present invention contains the hair restorer activator.
The amount of aptamer contained in the hair restorer composition of the present invention varies greatly depending on the form in which the hair restorer composition is provided, and is not particularly limited. In this case, the aptamer content is preferably 0.00001 to 10% by mass, more preferably 0.00005 to 5% by mass, even more preferably 0.001 to 1% by mass.

The hair restorer composition of the present invention preferably has a pH of 4.5 to 6.2 during storage and use. If the pH is too low (strongly acidic), the aptamer in the hair restorer composition will be insolubilized and easily separated. On the other hand, if the pH is too high (alkaline), the nucleic acid aptamer is likely to decompose.
Therefore, from the above point of view, it is most preferable to adjust the pH (for example, pH 5.0 to 5.6) close to the pH of the skin (about 5.3: weakly acidic).

The hair restorer composition of the present invention can take various forms such as liquid, emulsion, gel, cream, ointment, foam, mist and gel. For example, the hair restorer composition of the present invention may be prepared as an aqueous solution or an oil-in-water (O/W) emulsion obtained by dispersing a fatty phase in an aqueous phase (W/Si) in a silicone phase, It may also be produced as a water-in-oil (O/W) emulsion obtained by dispersion of an aqueous phase in a fatty phase.

Further, the emulsions may be prepared as lotions, serum-type formulations, or milky formulations with a liquid, semi-liquid consistency, or as aqueous or anhydrous suspensions with a creamy or gel-like soft consistency. good. Further, the emulsion may be contained in microcapsules, microparticles, or ionic and/or nonionic type vesicles to prepare a dispersion that is further dispersed in a liquid, or the emulsion may be formed into a foam. It may be prepared in the form of These adjustment methods can be performed by any conventionally used method. It should be noted that the amounts of the various ingredients of the composition according to the invention are those typically used in the field concerned.
Specifically, the hair restorer composition of the present invention is in the form of lotions, tonics, gels, creams, treatments, foams, mists, shampoos, conditioners, etc. for the scalp, hair, eyelashes, eyebrows or other areas where hair is to be increased. can be provided in

(Auxiliary component)
In the hair restorer composition of the present invention, various substances used in general hair restorer compositions, such as vasodilators, anti-inflammatory agents, keratolytic agents, bactericides, moisturizers, various animal and plant extracts, Antioxidants, solubilizers, metabolic activators, thickeners, adhesives, perfumes, cooling agents, and the like may be added as auxiliary ingredients within limits that do not impair the effects of the present invention. In addition, the additives described in the item of medicines described below may be added as auxiliary ingredients of the hair restorer composition. The auxiliary component may be derived from a natural product such as a plant (extract of assembly, etc.), or may be an artificially synthesized compound.

<Pharmaceuticals>
The hair restorer composition of the present invention may be a pharmaceutical. It is preferable to contain a nucleic acid aptamer (hair growth activator) as described above as an active ingredient. Depending on the mode of use or symptoms, the content may be appropriately changed, and if necessary, various pharmaceutically acceptable additives may be further included in formulations. Alternatively, it may be made into a nanocapsule by encapsulating it in a base material as appropriate.

(symptom)
The symptoms include slow hair growth, thinning hair, hair loss, etc., caused by a shortened hair cycle. "Treatment" includes not only curing the symptoms, but also alleviating (relieving) and preventing them. In addition, "prevention" includes prevention of the above-mentioned symptoms and prevention of their recurrence after healing.

The administration site of the drug is not particularly limited as long as it is desired to suppress hair growth or the above symptoms. When the administration subject is a human, it is usually administered to the scalp, but when the administration subject is an animal, it can be administered to a site where hair loss or the like occurs systemically or locally.

For example, the dermal papilla, which is important in hair growth such as scalp hair and FGF-5 is involved in hair loss, is usually present in the subcutaneous tissue containing subcutaneous fat from the dermis of the skin. It is preferably administered via Although the method is not particularly limited, ointments, liquids, and sprays can be applied directly to the epidermis, or can be administered by applying a sheet or patch coated with the drug. preferable. It can also be administered by injection such as subcutaneous injection, intradermal injection and intramuscular injection.

The dosage and frequency of administration of pharmaceuticals (or active ingredients contained therein) are determined by the patient's age, sex, symptoms, degree of therapeutic effect required, administration method, and active ingredients (nucleic acid aptamers, etc.). It varies depending on the content, etc., and can be adjusted as appropriate.

(Additive)
The drug according to the present invention may contain pharmaceutically acceptable additives as necessary.

Pharmaceutically acceptable additives are not particularly limited as long as they do not contradict the purpose of pharmaceuticals. additives such as agents (preservatives), coloring agents, etc., and depending on the symptoms and the like, moisturizing agents, antiseborrheic agents, blood circulation promoters, and the like may be included.

For example, when the dosage form of the pharmaceutical of the present invention is a coating, from the viewpoint of increasing the accessibility of the active ingredient contained in the pharmaceutical from the epidermis to the dermis or subcutaneous tissue (dermal papilla), a thickener or thickener It is preferable to prepare the desired viscosity and active ingredient concentration by blending.

When administering the drug of the present invention to an animal, the thickness and properties of the skin vary depending on the species of the animal. Therefore, it is preferable to appropriately adjust the concentration of the active ingredient and the content of the additive.

<Medical media additives>
The aptamer of the present invention can be added to a maintenance medium for hair follicles separated from the scalp for hair transplantation. In the field of regenerative medicine, it can also be used as a medium for hair follicles reconstituted using iPS cells, ES cells, cells differentiated from stem cells, etc., or normal cells such as dermal papillae isolated from living organisms. The amount to be added is not particularly limited, and can be appropriately selected depending on the medium or cells used. The medium is not particularly limited as long as it is a medium for animal cell culture, and may further contain serum, antibiotics, factors involved in growth and proliferation as appropriate.

EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited only to such Examples.
Unless otherwise specified, "%" means "% by mass".

[Production example 1]
<Preparation of water-soluble FGF-5 protein (gphm-hFGF-5(21-242)-His)>
GRP-tag and His-tag bound FGF-5 protein (GRP-3C-hFGF-5(21-242)-His) was prepared, and by further removing GRP-tag, highly water-soluble FGF-5 protein , gphm-hFGF-5(21-242)-His was prepared.
Specifically, an expression plasmid of GRP-3C-hFGF-5(21-242)-His was constructed, expressed in E. coli BL21(DE3)pLysS, and His- The preparation was done by preparing water-soluble FGF-5 protein with the tag and then removing the GRP-tag. Details are given below.

By the method of Inoue et al., Gene, 96, p23-128, 1990, Escherichia coli BL21 (DE3) pLysS (Biodynamics Laboratory Co., Ltd., product number: DS260) was transformed into plasmid pET-GRP-3C-hFGF-5 (21- E. coli colonies transformed with 242)-His (Merck, product number: 69742-3CN) were added to 2×YT medium (50 μg/mL carbenicillin disodium salt (Nacalai Tesque, product number: 07129-01) and 34 μg/mL chlorine. Ramphenicol (Nacalai Tesque, product number: 08027-14), 16 mg / ml trypsin (Nacalai Tesque, product number: 35640-95), 10 mg / 1 ml Extract Yeast Dried (Nacalai Tesque, product number: 15838-45), It was added directly to 10 mL containing 5 mg/1 mL sodium chloride (Wako Pure Chemical Industries, Ltd., product number 191-01665), and cultured with shaking at 37° C. and 170 rpm for 13 hours. After adding 150 μL of glycerol to 850 μL of this culture medium and mixing, the mixture was stored at −85° C. as a glycerol stock.

0.01 mL of the glycerol stock was added to 10 mL of 2×YT medium (containing 50 μg/mL carbenicillin disodium salt and 34 μg/mL chloramphenicol) and cultured with shaking at 37° C. and 170 rpm for 13 hours. A pre-culture solution was prepared by performing This preculture solution was centrifuged at 1,680 g at 25° C. for 10 minutes, and the obtained cells were inoculated into 1 L of 2×YT medium of the composition. This was further cultured with shaking at 37°C, 120 rpm for 2 hours, and then the culture temperature was lowered to 16°C in 1 hour.
1 mL of 0.1 M isopropyl-β-thiogalactopyranoside (IPTG, Nakarai Co., Ltd., product number 19742-94) is added (final concentration 0.1 mM), and cultured with shaking at 16° C. and 120 rpm for 24 hours. Thus, the expression of the recombinant protein (GRP-3C-hFGF-5(21-242)-His) was induced. After culturing, the culture medium was centrifuged at 8000 rpm at 4°C for 5 minutes to collect the cells, which were frozen and stored at -80°C.

Ni-NTA washing buffer (3.6 mL, 25 units/mL, Benzonase (registered trademark) nuclease (SIGMA-ALDRICH, product number: E-1014), 50 mM pH 8.0 sodium phosphate buffer, per 1 g of the frozen cells, containing 300 mM sodium chloride and 20 mM imidazole), ice-cooled for 30 minutes, and passed through a 21 G needle twice.
To this suspension, 0.4 mL of Ni-NTA washing buffer containing 10% Triton X-100 was added per 1 g of cells, ice-cooled for 30 minutes, and then passed through a 21 G needle four times. This cell lysate was centrifuged at 18000 rpm, 4°C for 30 minutes,
The resulting supernatant was passed through a Minisart (registered trademark) NML Plus membrane filter (Sartorius, Inc.).
Product number: 17829K).

The filtrate was added to a metal chelate affinity gel carrier column Ni-NTA superflow (QIAGEN, product number: 30410) (diameter 10 mm x length 100 mm, self-made column) at a flow rate of 2 mL/min, and Ni-NTA Lysis buffer (50 mM phosphate sodium buffer, pH 8.0 300 mM sodium chloride (80 mL). Thereafter, the protein (GRP-3C-hFGF-5 (21-242)-His) was eluted using a linear gradient method with 20 column volumes of Ni-NTA Lysis buffer containing 0-500 mM imidazole.

The fraction containing GRP-3C-hFGF-5(21-242)-His protein was added to a heparin carrier column HiTrap Heparin HP (bed volume 5 mL) at a flow rate of 2 mL/min, and then the column was treated with 20 mM Hepes-NaOH buffer. Washed with 50 mL of pH 7.5. Subsequently, using a linear gradient method with 20 column volumes of 20 mM Hepes-NaOH buffer pH 7.5 containing 0 to 3 M sodium chloride, GRP-3C-hFGF-5(21-242)-His adsorbed on the column was eluted. The eluted fraction was centrifuged at 3000 g and 4° C. using an ultrafiltration filter Amicon ultra 4 (molecular weight cutoff 3000) (Merck Millipore Ltd., product number: UFC800324) and concentrated to 1.2 mL. 0.6 mL of it was added to a gel filtration column Superdex 75 10/300GL (GE Healthcare, product number: 17-5174-01) equilibrated with 20 mM Hepes-NaOH buffer and pH 7.5 1 M sodium chloride for purification. .
A single peak fraction collected was taken as the fraction containing purified GRP-3C-hFGF-5(21-242)-His protein.

After adding 8 mL of 50% (v/v) curdlan beads (bead diameter: 75 to 150 μm, self-made) suspended in Ni-NTA Lysis buffer to the eluted fraction, mix by inverting at 4° C. for 1 hour. , GRP-3C-hFGF-5(21-242)-His was adsorbed to curdlan beads. The curdlan beads after adsorption were recovered by centrifugation at 1,000 g, 4° C. for 2 minutes. After washing the collected beads with 80 mL of Ni-NTA washing buffer, 20 mL of Ni-NTA washing buffer and 0.2 mL of GST-HRV3C protease were added, and mixed by inversion at 4° C. for 35 hours to cleave the GRP-tag. gphm-hFGF-5(21-242)-His was obtained. The supernatant obtained by centrifugation at 1,000 g at 4°C for 2 minutes was collected, and 20 mL of Ni-NTA washing buffer was added to the remaining beads, mixed by inversion, and centrifuged again at 1,000 g at 4°C for 2 minutes. The obtained supernatant was combined with the previous supernatant and filtered through a Minisart NML Plus membrane filter.
The filtrate was subjected to heparin affinity chromatography using a HiTrap Heparin HP column and gel filtration using a Superdex 75 10/300 GL column in the same manner as when the GRP-3C-hFGF-5(21-242)-His fraction was obtained. , the collected single peak fraction was obtained as purified gphm-hFGF-5(21-242)-His, which was used as the water-soluble FGF-5 protein in this experimental example.

[Experimental example 1]
<Activity confirmation of water-soluble FGF-5>
Using FR-Ba/F3 cells (having hFGFR1 on the cell surface), which have growth responsiveness to FGF-5, the effect of adding FGF-5 was examined from a dose-dependence standpoint.
FR-Ba/F3 cells suspended in RPMI1640 medium containing 10% fetal bovine serum and antibiotics (G418) were seeded in 96-well culture plates at 10,000 cells/well and treated with 5 μg/ml heparin (Sigma-G418). Aldrich) and RPMI 1640 (Fujifilm Wako Pure Chemical Industries, Ltd.) medium containing FGF-5 diluted to a predetermined concentration to a volume of 100 μl in each well, and cultured for 3 days at 37° C. in a 5% CO ₂ atmosphere. bottom. Then, WST-8 cell count kit (manufactured by Dojindo Laboratories) was added to each well, cultured for 2 hours at 37°C in a 5% CO ₂ atmosphere, and after color development, the absorbance was measured using a microplate reader (450 nm). It was measured and the state of cell proliferation was investigated. The results are shown in FIG. Water-soluble FGF-5 prepared by the above method (1 in the graph: gphm-hFGF-5) is a standard FGF-5 reagent used in this research field FGF-5 commercially available from R & D ( The activity was significantly higher than that of 3) in the graph, and the same level of activity as the FGF-5 reagent (4 in the graph) manufactured by Oriental Yeast Co., Ltd. under consignment.

[Production example 2]
<Screening of nucleic acid aptamers>
Using gphm-hFGF-5(21-242)-His, which is a water-soluble FGF-5 protein having a His tag prepared in Preparation Example 1, and a library of random DNA sequences, nucleic acids were prepared by the SELEX method according to the following procedure. Aptamer screening was performed.
5′-GGGTGTTAGCTGTTAGTATCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGTACGATCAGCTAGCCCTATAGTGAGTCGTATATTA-3′ (where N is an arbitrary base) was used as a template DNA (synthesis request from Genedesign Inc.), and forward primer: 5′-TAATACGA CTCACTATAGGGCTAGCTGATCGTACC-3' and reverse primer: 5'-GGGTGTTAGCTGTTAGTATC-3 ' (Hokkaido System Science), 2'-F modified UTP and CTP, natural ATP and GTP, and mutant T7 RNA polymerase was used to transcribe at 37°C for 5 hours to prepare an RNA pool.

The RNA pool and the resin beads on which gphm-hFGF-5(21-242)-His, which is a water-soluble FGF-5 protein having a His tag prepared in Preparation Example 1, was immobilized were mixed with SELEX buffer (20 mM Tris HCl ( pH 7.4), 200 mM NaCl, 5 mM MgCl ₂ , 5 mM CaCl ₂ , 0.05% TWEEN 20, 10% glycerol) at 25° C. for 30 minutes to bind the RNA pool and FGF-5. At this time, heparin and BSA were used as competitors to suppress non-specific binding. After the reaction, non-specifically bound RNA was washed away with a washing buffer (20 mM Tris HCl (pH 7.4), 500 mM NaCl, 5 _mM MgCl2, 5 mM _CaCl2 , 0.05% TWEEN20, 10% glycerol). After that, RNA bound to FGF-5 was recovered by phenol-chloroform treatment and ethanol precipitation with an elution buffer (20 mM Tris HCl (pH 7.4), 6 M guanidinium chloride), and a forward primer was used as a primer (synthesized by Hokkaido System Science Co., Ltd.). : 5'-TAATACGACTCACTATAGGGCTAGCTGATCGTACC-3', reverse primer: 5'-GGGTGTTAGCTGTTAGTATC-3', reverse transcription PCR was performed to reconstruct the DNA library. The same operation was performed 7 times (TALON Magnetic Beads (Clontech) for the second, fourth, and sixth operations as the resin beads, and Ni-NTA Agarose (Qiagen Co., Ltd.) for the first and fifth operations. ), His60 Ni Superflow Resin (Clontech) was used in the 3rd and 7th runs, and 3 M urea was added to the above composition for the washing buffer in the second half), 61 clones got

Sequence analysis was performed on each of the sequences of 61 clones. 15 clones among them were found to have the sequence of F-02 (SEQ ID NO: 1). Furthermore, it seems important from the viewpoint of having a motif similar to F-02 from the 61 clones obtained, sequence conservation and binding activity with FGF-5 based on the prediction of secondary structure. 37 clones (11 sequences including F-02) were extracted. Each of the extracted aptamers (F-02 (SEQ ID NO: 1) and similar F-58 (SEQ ID NO: 2), F-33 (SEQ ID NO: 3), F-05 (SEQ ID NO: 4) and similar F -06 (SEQ ID NO: 5), F-18 (SEQ ID NO: 6), F-14 (SEQ ID NO: 7), F-19 (SEQ ID NO: 8) and similar F-46 (SEQ ID NO: 9), F- 52 (SEQ ID NO: 10) and F-39 (SEQ ID NO: 11)) and their frequencies are shown in FIG. The boxed bases are the consensus sequences (sequences A to C described above). The double underline of F-58 and F-33 indicates a different base from F-02, the double underline of F-06 indicates a different base from F-05, and the double underline of F-46 indicates a different base from F-19. . Also shown in FIG. 3 are sequences demonstrating that cytosine and uracil are fluorinated in each sequence. In the figure, C(F) represents fluorinated cytosine and U(F) represents fluorinated uracil.

The template DNA of F-02 (SEQ ID NO: 1) was shortened at Hokkaido System Science Co., Ltd., and transcription reaction was carried out using the shortened template DNA, 2′-F modified UTP and CTP, and natural ATP and GTP. F-02-52 (SEQ ID NO: 13), F-02-42 (SEQ ID NO: 14), F-02-40 (SEQ ID NO: 15), F-02-56 ( SEQ ID NO: 16) was prepared, and F-18-56 (SEQ ID NO: 17) was prepared by shortening F-18 in the same manner. Each sequence is shown in FIG. 4(A). As shown in Experimental Example 7, F-02-40 (SEQ ID NO: 15) and F-02-42 (SEQ ID NO: 14) do not exhibit the effects of the present invention and are used as comparative examples. Also, the sequences clearly showing that cytosine and uracil are fluorinated in each sequence are shown in FIG. 4(B). In the figure, C(F) represents fluorinated cytosine and U(F) represents fluorinated uracil.

Secondary structure prediction using the free tool "mfold" is shown in FIG. (B) F-05 (SEQ ID NO: 4), (C) F-14 (SEQ ID NO: 7), (D) F-18 (SEQ ID NO: 6), (E) F-19 (SEQ ID NO: 8), (F) ) F-39 (SEQ ID NO: 11), (G) F-52 (SEQ ID NO: 10), (H) F-02-52 (SEQ ID NO: 13)).
FIG. 6 shows the secondary structure prediction of the sequence F-02 and F-02-52 (SEQ ID NO: 13) obtained by truncating the sequence, performed by Chiba Institute of Technology.
[Experimental example 2]

<Binding ability between aptamer and water-soluble FGF-5 protein>
An attempt was made to analyze the ability of nucleic acid aptamers to bind to human FGF-5 by Electrophoresis Mobility Shift Assay (EMSA). 0.3 μM of each aptamer prepared in Preparation Example 2 and the water-soluble FGF-5 prepared in Preparation Example 1 were mixed at a molar ratio of 1:1, placed in SELEX buffer at 25 ° C. for 30 minutes, and then 10% Denatured PAGE (native PAGE) was performed. After performing electrophoresis at 100 V for 40 minutes, it was stained with CYBR Gold. A staining diagram is shown in FIG.
Aptamers with the consensus sequence (sequences A to C) (F-02, F-05, F-14, F-18, F-19, F-39, F-52), when FGF-5 protein is added Although binding to FGF-5 protein was confirmed, the aptamer (F-51) having no consensus sequence did not bind to FGF-5 protein.
Moreover, among the obtained aptamers, F-02, F-05, F-14 and F-18 in particular interact with FGF-5 with high specificity.

[Experimental example 3]
<Binding ability between aptamer F-02 and water-soluble FGF-5 protein-1>
The binding between the aptamer F-02 prepared in Preparation Example 1 and FGF-5 was evaluated using BiacoreX (GE Healthcare), which is a Biacore™ system using SPR signals.
By immobilizing biotin-added polydT (16 bases) to a sensor chip on which streptavidin is immobilized, and binding an aptamer to which polyA (16 bases) added by PCR is added to polydT on the sensor chip. , immobilized the aptamer on the sensor chip.
In order to suppress non-specific binding to the chip, 4 μg/ml heparin was added to a buffer having the same composition as the SELEX buffer, and FGF-5 adjusted to 100 nM in the buffer was injected into the system. FGF-5 was reacted with the same amount of F-02 or RNA having a random sequence (RNA pool), and the amount of intermolecular interaction (Response unit value: RU) was measured. As a result, in the results of reacting F-02 with FGF-5, the Response unit value was significantly higher than in the case of reacting FGF-5 with an RNA pool. 02 was found to bind specifically. The results are shown in FIG.

[Experimental example 4]
<Binding ability between aptamer F-02 and FGF family protein-2>
Aptamer F-02 was immobilized on a sensor chip in the same manner as in Experimental Example 3, and FGF-5, FGF-1, FGF-2, FGF-4, and FGF-6 prepared to 100 nM (manufactured by the inventor or R & D Systems company) are injected into the system, respectively, FGF-5 and F-02, FGF-1 and F-02, FGF-2 and F-02, FGF-4 and F-02, and FGF-6 and The amount of intermolecular interaction (Response unit value: RU) with F-02 was measured. As a result, compared to the case of reacting FGF-1, FGF-2, FGF-4, and FGF-6, the Response unit value when FGF-5 was reacted was significantly higher. binds specifically to F-02, whereas FGF-1, FGF-2, FGF-4, and FGF-6 do not bind to F-02. The results are shown in FIG. Among these, FGF-4 and FGF-6 belong to the same subfamily as FGF-5, and compared with these FGF family proteins, which are considered to be particularly close in terms of three-dimensional structure, FGF-5 of F-02 It was found that the specificity for

[Experimental example 5]
<Binding ability between aptamer F-02 and water-soluble FGF-5 protein-3>
A dissociation constant with water-soluble FGF-5 was determined for aptamer F-02. Aptamer F-02 was immobilized on a sensor chip in the same manner as in Experimental Example 3, FGF-5 adjusted to 2.5 to 20 nM was injected into the system, and an intermolecular reaction between FGF-5 and F-02 was performed. The amount of interaction (Response unit value: RU) was measured (Fig. 10). Using the software BIAevaluation attached to BiacoreX, the dissociation constant was calculated in Langmuir (1:1) binding mode and gave a value of 0.28±0.04 nM.

[Experimental example 6]
<Effect on FGF-5-dependent cell proliferation>
FGF-5 is known to proliferate cells expressing FGFR1 by binding and interacting with FGFR1. Using NIH3T3 cells (fetal mouse fibroblasts) expressing FGFR1, the ability of each nucleic acid aptamer to inhibit FGF-5-dependent cell growth was confirmed. At least several doses of the aptamer were set by changing the dilution ratio, and the effect of addition was examined from the viewpoint of dose-dependence.

NIH3T3 cells were seeded in a 96-well culture plate at 5,000 cells/well, cultured in D-MEM medium containing 10% serum and antibiotics at 37°C in an atmosphere of 5% CO ₂ for 1 day. The supernatant was removed, washed and removed with 100 μl/well PBS.
Serum-free DMEM containing 50 ng/ml insulin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 5 μg/ml heparin (manufactured by Sigma-Aldrich, 25 mg/ml PBS solution prepared in advance) (Fujifilm Wako Pure Chemical Industries, Ltd.) Co., Ltd.) medium, FGF-5 (manufactured by R & D Systems, 100 μg / ml PBS solution was prepared in advance) was added to a final concentration of 3 nM, and each aptamer was added to each concentration. The volume of each well was adjusted to 100 μl (as a control, a medium supplemented with PBS without FGF-5 and heparin was used). After culturing for 2 days at 37°C and 5% CO ₂ atmosphere, WST-8 cell count kit (manufactured by Dojindo Laboratories) was added to each well and cultured for 2 hours at 37°C and 5% CO ₂ atmosphere. After color development, absorbance was measured using a microplate reader (450 nm) to confirm cell proliferation. The results are shown in FIG. No cell proliferation was observed in any control (PBS) in which FGF-5 was not added to the medium. On the other hand, when FGF-5 was added to the medium (FGF-5 3 nM), the aptamers of F-02, F-05, F-14, F-18, F-19, F-39, and F-52 However, the aptamer (F-51), which does not have a common sequence, could not be observed to have an effect on FGF-5-dependent cell proliferation.
Therefore, it is speculated that aptamers having these common sequences inhibit proliferation of NIH3T3 cells by binding to FGF-5.

Table 1 summarizes the performance of each aptamer in the above experiment, where IC ₅₀ (50% inhibitory concentration) indicates the aptamer concentration at which cell growth is inhibited to 50% of that in the absence of aptamer. All of the aptamers (F-02, F-05, F-14, F-18, F-19, F-39, F-52) having the above common sequences (sequences A to C) have IC ₅₀ values of 10 nM or less. It became clear that there is a cell growth inhibitory effect at such a low concentration (Table 1 middle column). The right column of Table 1 shows values obtained by dividing IC ₅₀ (molar concentration) by FGF-5 molar concentration. All aptamers except F-19 and F-39 (see Experimental Example 2 and FIG. 7), which have relatively weak binding ability to FGF-5, show small values of 2 to 3, and FGF-5 -5 suggests a good binding state with each aptamer.
From the results so far, the aptamer that exhibits the cell growth inhibitory effect binds to FGF-5, thereby inhibiting the binding of FGF-5 to its receptor FGFR1, thereby exhibiting the cell growth inhibitory effect. It is suggested.

[Comparative Example 1]
<Effect of random aptamers on FGF-5-dependent cell proliferation>
The ability of RNA aptamers having random nucleic acid sequences (random aptamers) to inhibit FGF-2- or FGF-5-dependent cell growth was confirmed. In addition to the FGF-5 test group, FGF is one of the FGF family proteins known to grow cells expressing FGFR1 by binding and interacting with FGFR1 like FGF-5 -2 (final concentration 0.1 nM: manufactured by R & D Systems, 100 μg / ml PBS solution was prepared in advance) test group and PBS not containing FGF-5 and FGF-2 were used as control test groups, Experimental Example 6 Cells were cultured in the same manner as in Experimental Example 6, except that the aptamers used in 1. were changed to random aptamers, and FGF-2- or FGF-5-dependent cell proliferation was measured. The results are shown in FIG.
As a result, regardless of the amount of random aptamer, there is no effect on FGF-2 or FGF-5 dependent cell proliferation, thus random aptamers do not cause inhibition of FGF-2 or FGF-5 dependent cell proliferation. It has been suggested.

[Comparative Example 2]
<Effect on FGF-2-dependent cell growth inhibition>
As described above, it is known that FGF-2, which is one of the FGF family proteins, also binds and interacts with FGFR1 to proliferate cells expressing FGFR1.
Cells were cultured in the same manner as in Experimental Example 6, except that FGF-2 (final concentration: 0.1 nM; manufactured by R&D Systems) was used instead of FGF-5, and FGF-2-dependent cell proliferation was measured.
As a result, the degree of proliferation of NIH3T3 cells was not affected regardless of the presence or absence of the aptamer, and therefore no inhibition of FGF-2-dependent cell proliferation by each nucleic acid aptamer was observed (Fig. 13).

[Comparative Example 3]
<Effect on FGF-1-dependent cell growth inhibition>
FGF-1, which is one of the FGF family proteins, is also known to proliferate cells expressing FGFR1 by binding and interacting with FGFR1 like FGF-5 and FGF-2. . The FGF-1-dependent cell proliferation ability of aptamer F-02 was confirmed.
Using FGF-5 (final concentration 3 nM: manufactured by R & D Systems) or FGF-1 (final concentration 0.1 nM: manufactured by R & D Systems), the cells were cultured in the same manner as in Experimental Example 6, and the degree of cell proliferation was was measured. As a result, as in Experimental Example 6, it was confirmed that F-02 inhibited FGF-5-dependent cell proliferation, but did not inhibit FGF-1-dependent cell proliferation (Fig. 14).
As described above, from the results of Experimental Example 6 and Comparative Examples 2 and 3, while F-02 inhibits FGF-5-dependent cell proliferation, FGF-2 and FGF interacting with a common receptor with FGF-5 -1 dependent cell proliferation was found not to be inhibited, suggesting that aptamer F-02 specifically binds FGF-5.

[Experimental example 7]
<Effect of shortened aptamer on FGF-5-dependent cell growth inhibition>
Furthermore, a similar experiment was performed using shortened nucleic acid aptamers to confirm whether the FGF-5-dependent cell growth inhibitory ability was maintained.
The template DNAs of F-02 (SEQ ID NO: 1) and F-18 (SEQ ID NO: 6) were shortened by Hokkaido System Science Co., Ltd., and transcription reaction was performed using the shortened template DNAs to shorten the strands. Aptamers F-02-40 (SEQ ID NO: 15), F-02-42 (SEQ ID NO: 14), F-02-52 (SEQ ID NO: 13), F-02-56 (SEQ ID NO: 16), F-18- 56 (SEQ ID NO: 17)) was prepared. It was confirmed that all the shortened aptamers have common sequences A to C. When these were used to examine the ability to bind to FGF-5 (manufactured by R & D Systems), only F-02-40 (SEQ ID NO: 15) and F-02-42 (SEQ ID NO: 14) bound to FGF-5 could not be confirmed. Furthermore, as a result of examining the ability of each aptamer to inhibit FGF-5-dependent cell growth, F-02-40 (SEQ ID NO: 15) and F-02-42 (SEQ ID NO: 14) were found to inhibit FGF-5-dependent cell growth. However, F-02-52 (SEQ ID NO: 13), F-02-56 (SEQ ID NO: 16), and F18-56 (SEQ ID NO: 17) had the ability to inhibit FGF-5-dependent cell growth ( Figure 15). Therefore, it can be said that binding to FGF-5 is necessary for FGF-5-dependent cell growth inhibitory ability. In addition, although F-02-40 (SEQ ID NO: 15) and F-02-42 (SEQ ID NO: 14) have a common sequence, binding to FGF-5 and FGF-5-dependent cell growth inhibition The reason why the ability could not be confirmed is that the sequence is shorter than other aptamers, so the structure required for binding to FGF-5 cannot be obtained, and as a result, the ability to inhibit FGF-5-dependent cell growth cannot be exhibited. It can be inferred that

[Experimental example 8]
<Alkaline phosphatase (ALP) expression analysis of dermal papilla cells>
Alkaline phosphatase (ALP) is a hair follicle induction marker that is expressed in hair follicles and is known to be an indicator of dermal papilla cell activity, including the ability to induce hair growth.
Human follicle dermal papilla cells (HFDPC cells): PromoCell) were seeded at 50,000 cells/well in a 12-well culture plate, and 10% fetal bovine serum, 20 ng/ml Wnt-3a, and antibiotics were added. The cells were cultured in D-MEM medium containing substances (100 units/ml penicillin and 100 μg/ml streptomycin) at 37° C. in a 5% CO 2 atmosphere. After 3 days, the culture supernatant in the wells was removed, and the wells were washed with 1 ml/well of PBS to remove the PBS.
D-MEM (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) medium containing 2% B-27 supplement (manufactured by Gibco) and 5 μg/ml heparin (manufactured by Sigma-Aldrich) was added to the medium to a final concentration of 3 nM. Diluted FGF-5 protein (R&D Systems) or PBS (FGF-5 protein vehicle) diluted in D-MEM medium containing 2% B-27 supplement was added.

Aptamer F-02 (Example) diluted to a final concentration of 10 nM in D-MEM medium containing 2% B-27 supplement in each well, PBS diluted in D-MEM medium containing 2% B-27 supplement (No addition group), and Random Aptamer (reference example) diluted to a final concentration of 10 nM with D-MEM medium containing 2% B-27 supplement were added to make the volume of each well 1 ml, 37 ° C., 5% Cultured for 3 days under CO2 atmosphere. After that, the culture supernatant in each well was removed, and the wells were washed with 1 mL/well of PBS to remove PBS.

RNA was extracted from human dermal papilla cells adhering to each well using Rneasy Mini Kit (manufactured by Qiagen). Further, the extracted RNA was reverse transcribed using RiverTraAce-α- (manufactured by Toyobo Co., Ltd.) to obtain cDNA. Quantitative PCR was performed for each obtained cDNA to determine the cyclic threshold (Ct) value for the ALP gene. At the same time, the Ct value of β-actin, which is a housekeeping gene, was determined, and the relative quantification value of the ALP gene transcription level was calculated by the ΔΔCt method using both Ct values. The results are shown in FIG.

In the non-addition group and the reference example, the addition of FGF-5 significantly decreased the transcription level of ALP, but no change in the transcription level of ALP was observed in the cells to which aptamer F-02 was added. This indicates that aptamer F-02 suppresses the activity of FGF-5, thereby suppressing the transcriptional repression of ALP by FGF-5, and the dermal papilla cells are activated even in the presence of FGF-5. suggesting to get

(Discussion)
As described above, the FGF-5 protein is known to inhibit hair growth and shift the hair cycle from the anagen phase to the regression phase via its receptor FGFR1. .
It was revealed that the aptamer of the present invention inhibits the binding between FGF-5 and FGFR1 by specifically binding to FGF-5. As a result, the aptamer of the present invention can suppress signal transduction between FGF-5 and FGFR1, and as a result, it can be expected to exhibit a hair loss suppressing effect by continuing the growth phase of the hair cycle. Experiments using dermal papilla cells to which aptamer F-02 was added actually suggested the effect of suppressing the suppression of the expression of ALP, a hair follicle induction marker expressed in hair follicles, by FGF-5. The hair loss inhibitory effect of is more strongly suggested. In addition, by shortening the chain of each aptamer, it is possible to obtain an aptamer that is more suitable for hair loss suppression. can be expected to be applied to

Claims (17)

A nucleic acid aptamer that is a nucleic acid molecule that specifically binds to FGF-5 (Fibroblast growth factor 5), a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof ,
said nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NOS: 1-11, 13, 16, and 17; A nucleic acid molecule selected from variants of a nucleic acid molecule having a sequence in which 1 to 5 bases are substituted, deleted, or inserted at a location other than the sequences A, B, and C of the sequence. and
the sequence A is the sequence: 5'-ACCU-3';
The sequence B is the sequence: 5'-XGACA-3' (X represents any base),
A nucleic acid aptamer, a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof, wherein said sequence C is the sequence: 5'-UCCAGA-3'. The nucleic acid aptamer, the pharmaceutically acceptable salt of the nucleic acid aptamer, or the solvate thereof according to claim 1, wherein the nucleic acid aptamer inhibits the binding of the FGF-5 to the FGF-5 receptor. . 3. The nucleic acid aptamer, a pharmaceutically acceptable salt thereof, or a solvate thereof according to claim 1 or 2, wherein the dissociation constant for said FGF-5 is 10 pM to 100 nM. The nucleic acid aptamer according to any one of claims 1 to 3 , wherein the nucleic acid molecule has a sequence selected from the group consisting of SEQ ID NOs: 1 to 11, 13, 16, and 17. A pharmaceutically acceptable salt of an aptamer, or a solvate thereof. 5. A nucleic acid molecule according to any one of claims 1 to 4 , wherein said nucleic acid molecule has a sequence selected from the group consisting of SEQ ID NOS: 1, 4, 6, 7, 10, 13, 16, and 17. , a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof. The nucleic acid aptamer according to any one of claims 1 to 5 , wherein the nucleic acid molecule is a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NOS: 1, 6, 16, and 17. Pharmaceutically acceptable salts or solvates thereof. The nucleic acid aptamer according to any one of claims 1 to 6, wherein the nucleic acid molecule has a sequence represented by SEQ ID NO: 1 or 6 , a pharmaceutically acceptable salt of the nucleic acid aptamer, or solvates of these. The nucleic acid aptamer, a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof according to any one of claims 1 to 7 , wherein the nucleic acid molecule is RNA. The nucleic acid aptamer, a pharmaceutically acceptable salt thereof, or a solvate thereof according to any one of claims 1 to 8 , wherein said FGF-5 is human FGF-5. The 2' position of ribose in one or more of cytosine (C) and uracil (U) contained in the nucleotide contained in the nucleic acid aptamer is substituted with a fluorine atom, according to any one of claims 1 to 9 The nucleic acid aptamer described, a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof. The nucleic acid aptamer according to any one of claims 1 to 10 , wherein the 2'-position of ribose in cytosine (C) and uracil (U) contained in the nucleotide contained in the nucleic acid aptamer is substituted with a fluorine atom. , a pharmaceutically acceptable salt of the nucleic acid aptamer, or a solvate thereof. A hair growth activator comprising at least one selected from the nucleic acid aptamer according to any one of claims 1 to 11 , pharmaceutically acceptable salts of the nucleic acid aptamer, and solvates thereof. 13. The hair growth activator according to claim 12 , which has an activity of inhibiting the action of FGF-5. A hair restorer composition containing the hair restorer activator according to claim 12 or 13 . The nucleic acid aptamer according to any one of claims 1 to 11 , a pharmaceutically acceptable salt of the nucleic acid aptamer, and a solvate thereof of 0.00001 to 10% by mass, according to claim 14 . Hair restorer composition. Claim 15 , containing 0.00005 to 5% by mass of the nucleic acid aptamer according to any one of claims 1 to 11 , a pharmaceutically acceptable salt of the nucleic acid aptamer, and a solvate thereof of the hair restorer composition. The hair restorer composition according to any one of claims 14 to 16 , which is a pharmaceutical.