JP5010913B2 - Tissue regeneration preparation containing atrial diuretic hormone family molecule as active substance, tissue regeneration method using the preparation, hair growth, hair growth, hair restorer and skin tissue repair improving agent containing atrial diuretic hormone family molecule as active substance Hair growth, hair thickening, hair growth promoting method and skin tissue repair improving method using the preparation - Google Patents

Description

The present invention relates to a tissue regeneration technique that promotes the restoration of biological tissues and cell proliferation, and further, a preparation for improving hair growth and restoration of skin damage and a method for improving the same.

In recent years, medical techniques have been attempted to regenerate parts of human tissues and organs or specific cells to complement and substitute for organs that have suffered severe illness or have suffered devastating damage, making functional recovery difficult. .

Most of the regenerative medicine is at the stage of animal experiments, and the tissue regeneration in the human body that has been put to practical use is still only about cultured skin.

The tissue and organ regeneration methods currently being attempted include a technique for treating and culturing an object to be regenerated, such as ES cells, germinal stem cells, or similar tissues, under the administration of various stimulating factors and with feeder cells. Adopted.

However, the problem with the prior art is that differentiated cells are less likely to be regenerated. In other words, in the case of cells / tissues with advanced differentiation, such as the central nervous system, heart, and kidney, in the living body, once suffering some kind of injury, it is almost impossible to regenerate by conventional treatment.
On the other hand, among various devices, a growth factor that acts on the S phase in the cell cycle and promotes DNA synthesis has been isolated and purified from living tissue. Representatively, it is known that epidermal growth factor (EGF) and hepatocyte growth factor (HGF) exert a DNA synthesis promoting action. Other known growth factors include, for example, IGF-1 (insulin-like growth factor-1), IGF-2 (insulin-like growth factor-2), TGF-α (transforming growth factor-α), etc. Also plays an important biochemical role in the process of promoting cell proliferation.

A mitogenic agent such as EGF or HGF binds to a specific receptor and initiates a cascade of protein kinases, that is, MEK (MAP kinase kinase = MAPK by phosphorylation and activation of MAP (mitogen-activated protein) kinase). -It acts on -ERK kinase, MEK phosphorylates and activates other MAP kinases: p44 (ERK-1: extracellular signal-regulated kinase 1) and p42. And p42 (ERK-2) sends the signal of proliferation and differentiation to cytoplasm and a nucleus. For example, this MAP kinase cascade reaction is a key signaling pathway in the control of the cell cycle of hepatocytes. Activation of ERKs by growth factors is known to lead to DNA synthesis in primary rat hepatocyte cultures. Phosphatidylinositol 3-kinase (P13K) and protein kinase B (PKB, Akt), a downstream signal kinase, are major regulators of cell survival in response to growth factors. Recently, activation of PKB serine threonine kinase is said to phosphorylate and inactivate pro-apoptotic proteins such as BAD and caspase-9. Thus, growth factors play an important role in the control of cell proliferation and survival. As described above, the growth factor binds to a specific receptor of the cell, and the cascade reaction of protein kinase proceeds, starting with MAP kinase. As a result, cell DNA synthesis, cell cycle control and cell regeneration, compensation It is known to induce cell proliferation ability such as hypertrophy.

On the other hand, ANP is a peptide hormone that is secreted mainly from the atria and exhibits strong natriuretic action, vasodilatation and blood pressure lowering action, and is classified into three types, α-type, β-type, and _Y- type, depending on the difference in molecular weight. . The α-form is a single-chain polypeptide chain composed of 28 amino acids and has one disulfide bond [Cys (7) -Cys (23)] in the molecule (Biochem. Biophys. Res. Commun., 118 , 131-139, 1984). β-type is anti-parallel dimer of α-type, and _Y- type is a high molecular protein composed of 126 amino acids containing α-type sequence in C-terminal region [99-126]. It has been suggested to be a precursor (Nature, 313 , 397-400, 1985).

Reports of drugs that modify or suppress the bioactivity of various ANPs against multiple organs by competing with ANP for receptor binding reactions have lacked the N-terminal, C-terminal, and part of the cyclic structure of rat ANP Synthetic ligand C-ANF (Science, 238 , 675-678, 1987) and an analog (Analog) in which one pair of disulfide bonds of anti-parallel dimer of human ANP [7-28] is replaced with L-α-aminosuberic acid ) III (FEBS Lett., 248 , 28-34, 1989). On the other hand, the inventors have found that ANP has a chicken embryonic cardiomyocyte proliferation promoting action or the like as its biological activity. However, the ANP family molecule cell and tissue organ regeneration or skin hair and bone And its effect on cartilage formation is not yet known.

A problem with conventional cell tissue repair techniques is that differentiated cells are less likely to be regenerated. That is, in the case of cells / tissues with particularly advanced differentiation, such as the central nervous system and myocardium, once they are injured in vivo, it is almost impossible to regenerate by conventional treatment. In addition, baldness represents a reduced state of hair and is characterized by various changes such as hair loss, hair loss, hair thickness and color, elasticity and stiffness, while skin and subcutaneous tissue injury is trauma, burns, Skin or subcutaneous tissue loss or roughening caused by chemical irritation, allergy, inflammation, infection, circulatory failure, or physiological aging, and damage to the epidermis, dermis, or supporting tissue. Minoxidil, adenosine, and FGF7 are known as treatment methods for improving baldness, but in reality, there are not many cases that still show remarkable effects. For wounds and skin tissue injuries, various countermeasures such as various ointments, blood flow improvers, FGF, wound treatment surgery, cosmetics, etc. are taken. The cleanliness and repairing ability of this product are basically affected, and the effect of the conventional method is not sufficient. That is, there is a limit to the improvement of hair growth and skin tissue by conventional methods, and the development of new effective formulations and treatment methods is awaited. Next, in healthy bones, a strong external force exceeding the limit or repeated external force is applied, and the bone is destroyed and a fracture occurs. On the other hand, pathological bones are fragile and cause fractures even with a slight external force. Fractures include complete fractures, incomplete fractures, simple fractures, and complex fractures depending on the form and degree, but the basic treatment is to fix and stay at rest for a certain period after reduction. The shortening of the treatment period for fractures has great benefits for patients because they can return to daily life and social activities at an early stage. However, there are no known drugs that have been used to shorten the fracture treatment period. Osteoarthritis and rheumatism are known to involve cartilage damage, and many drugs have been developed. However, drugs for growing cartilage tissue are not yet commercially available. Implants are known as a treatment method for making up for teeth lost due to cavities, gingival diseases, or injuries. For implants, artificial bone graft materials such as hydroxyapatite are used, but in order to increase the success rate of surgery, it is desired to use a protein preparation having bone growth ability. BMP-2 is a growth factor clinically used in the United States for bone tissue regeneration, but it has not yet been approved in Japan. There is a need for formulations that have been clinically confirmed to be safe and effective.

The inventor uses peptide hormones ANP, BNP, CNP, urodilatin, and combinations thereof, as well as preparations that function in a common manner with them as preparations for repair and regeneration of biological tissues including the myocardium , and hair growth. It was conceived to be used for the preparation of hair growth and / or for the improvement and repair of cutaneous mucosa and subcutaneous / bone / cartilage tissue injury such as wounds, rough skin, burns, necrosis, psoriasis. In addition, in the case of the preparation for repair and regeneration, the inventors also invented a preparation containing a cell growth factor or tissue repair factor other than the NP family molecule group and / or a cell tissue growth promoter. In addition, the inventors have invented a preparation for regenerating and regenerating a living tissue, further comprising a neutral endopeptidase inhibitor, an anti-atrial diuretic hormone family molecular antibody, or an atrial diuretic hormone action modifier. In addition, the present inventor has also identified any of atrial diuretic hormone (ANP), brain natriuretic peptide (BNP), C-type diuretic hormone (CNP), phosphorylated urodilatin (P-Uro), and precursors and derivatives thereof. A composition containing a gene and a vector containing a nucleotide sequence that expresses or a combination thereof as an active ingredient, and may contain a diluent, an excipient, a filler or an auxiliary agent usually used in pharmacology We have invented a preparation for repair and regeneration of tissue organs, a preparation for hair growth and / or hair growth, and / or a preparation for improving repair of injuries to skin mucosa and subcutaneous / bone / cartilage tissues such as wounds, rough skin, and burns. In addition, in the case of the above-mentioned preparations for improving hair growth and hair growth, preparations for improving and repairing skin mucosa and subcutaneous tissue injury such as wounds, rough skin, and burns, in addition to the above active ingredients, other growth factors and / or hair growth promotion An agent may further be included. Further, it may further contain a neutral endopeptidase inhibitor, an anti-atrial diuretic hormone family molecular antibody, or an atrial diuretic hormone action modifier.

In cardiomyocytes, for the first time, ANP's cell proliferation action, cell type-specific growth induction action (Koide et al. Circulation 88, 4, I-129, 1993, Koide et al. Differentiation, 61, 1-11, In recent years, it has been known that other lineage cells (hepatocytes and nerve cells) have an ANP apoptosis inhibitory effect and protective effect. However, the use of ANP family molecules in living organisms and organs is still only performed based on the mechanism of diuretic action and vasodilatory action, and the role of ANP family molecules in the repair and regeneration of living tissues and organs is unknown. In addition, ANP family molecules have never been used for the repair and regeneration of living tissues and organs.

In the present invention, the action of ANP family molecules on tissue organ regeneration and repair was first confirmed using living organisms and various cultured cells. Next, we confirmed the regenerative activation effect on the thinned myocardium by continuous repeated administration of ANP in patients with dilated cardiomyopathy. In addition, we examined the effects of ANP family molecules on hair growth, skin regeneration, and skin recovery in the process of ectodermal cell and tissue damage, especially bald hair loss and / or skin injury, and postoperative tissue recovery and regeneration. The new formulation and the treatment method with the new formulation were tested.

The active action on the thin myocardium of the preparation of the present invention starts to show an effect in several weeks, increases in the thickness of the myocardium in about 3 months, and increases the contractility. Catecholamine and renin-angiotensin It is clearly more effective than known preparations such as drugs acting on the system and digitalis, and the mode of action is different.

Also, the effects of the formulations of the present invention on post-operative wounds are represented by earlier wound bulge and closure healing and less scarring compared to the control natural healing site. In particular, early closure of the ulcer site is useful for the treatment of gangrene due to obstructive arteriosclerosis or long-term bedrock. This effect is different from the blood flow dependency as shown by the results of cultured cells, and the direct repair to target cells and the proliferation effect are useful for the regeneration and repair of various tissue organs.

The physiological activity on the hair of the preparation of the present invention is clearly higher than that of known preparations such as minoxidil and adenosine since the effect starts to appear in several weeks and the hair increases in one month. In addition, it enhances the suppleness, elasticity, thickness, etc. of hair, so it is more useful than existing hair restorers. The effect on the skin is excellent in that it is difficult to make red and rough skin even after water work, or it is cured in a short period of time, and the usefulness of the active ingredient as a skin external preparation or cosmetics is shown. It was.

In tests using cultured cells, the cytotoxicity of the cell group with cytotoxicity was clearly higher when ANP was administered compared to the control, and formation of cell colonies and cell sequence networks was possible only with ANP. It has demonstrated the ability of NP family molecules to induce tissue repair and regeneration. The addition of ANP to the cultured cells increased BMP-2 production over time. Since BMP-2 is a growth factor that plays an important role in the bone / cartilage formation cascade, it was found that ANP has an important role that triggers the formation / regeneration of bone / cartilage. Furthermore, the fact that ANP modified the expression of growth factors such as BMP2 indicated the role of the ANP family as a regulator of known growth factor group actions. This means that even when regeneration and repair involve growth factor groups that cannot function by exogenous administration, ANP family molecules can regulate the endogenous production and functional expression of these growth factors to induce regeneration and repair. Squeezed. The ANP family also increased the expression levels of cell cycle-related factor (cyclin D1), cell type-specific protein (KRT15), and epimorphin, making tissue repair and regeneration ideal for cells and tissues. It was shown that it can be accelerated and guided. In fact, when the ANP was administered topically, the cracking of the skin and the repair of the surgical wound were quicker than when they were not used, and the fact that the skin was smooth, firm and moist was better It was consistent with the test results.

This NP family composition is administered systemically, such as by intravenous infusion or intravenous bolus administration. In the case of chronic heart failure including dilated cardiomyopathy, it is desirable to continuously administer 100 μg to 1000 μg of ANP intravenously from 1 to 7 times a week for 2 months or longer. In relatively mild cases, administration once every two weeks may be continued for 3 months or more. In addition, the preparation of the present invention is a preparation for improving and repairing skin mucosa and subcutaneous tissue injury such as hair growth, hair growth, debris, rough skin, wound, gangrene, surgical wound, fracture, cartilage damage, burn, etc. , Alone or in combination with a protective agent, can be administered transdermally, subcutaneously, intramuscularly, inhalation or applied locally to the affected area by injection, spraying, or the like. For topical administration , it can be administered as a lotion or mist, or as a cream or gel patch around the area. In the case of wrinkles and rough skin, it is preferable to transdermally administer by lotion, mist, cream or ODT patch. The dose is preferably 10 ng to 100 μg per ANP, particularly preferably 10 ng to 1 μg.

In the case of cell culture such as regenerative medicine, it is preferable to add to the culture solution or add the culture solution after exposure to the NP family when removing the culture solution.

In administration, the active ingredient can be mixed with a liquid non-toxic pharmaceutical carrier and administered in the form of a conventional pharmaceutical preparation. Examples of such preparations include solid preparations such as tablets, granules, powders and capsules, solutions such as solutions, suspensions and emulsions, freeze-dried preparations, and the like. It can be prepared at the time of use by conventional means. Non-toxic pharmaceutical carriers include, for example, glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, Water, physiological saline, etc. are mentioned. Further, if necessary, conventional additives such as a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent can be appropriately added.

NP family ligand molecules are recognized by three different receptors, Atype, Btype, and Ctype, and affect cell function. It is preferable to set the dosage in consideration of the fact that if such a ligand / receptor reacts, other combinations will exhibit more or less activity.

The NP group of the present invention can be used in place of a known preparation, or can be used in combination with a known preparation. For example, in dilated cardiomyopathy, it can be administered in combination with drugs such as diuretics, β-blockers, and ACE inhibitors. For hair growth, it can be blended with various hair growth ingredients such as minoxidil, finasteride, carpronium chloride, adenosine, natural product extract, amino acids, horse mackerel oil and the like. It can also be used with other bone growth factors such as BMP-2 in bone and cartilage repair and regeneration.

ANP, BNP, CNP, urodilatin and their precursors or derivatives or combinations thereof according to the present invention are formulated as active ingredients and contain diluents, excipients, fillers or auxiliaries commonly used in pharmacology In addition, a drug capable of controlling these activities (neutral endogenous proteolytic enzyme inhibitor, various anti-NP family molecule antibodies and NP receptor binding substances such as HS232) and an existing hair growth composition are formulated. May be. The NP family compounding amount of the present invention can be appropriately determined depending on the age, weight, symptom, lesion site, size of the subject, administration route, administration schedule, formulation form, etc. of the subject.

Moreover, the cosmetic material which mix | blended the NP family molecule | numerator of this invention, and the form of cosmetics can be manufactured. For cosmetics, fats and oils such as vegetable oils, higher fatty acids, higher alcohols, silicones, anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, preservatives, sugars, sequestering agents Further, a polymer such as a water-soluble polymer, a thickener, a powder component, an ultraviolet absorber, an ultraviolet blocker, a moisturizing agent such as hyaluronic acid, a fragrance, a pH adjuster and the like can be contained. Other medicinal and physiologically active ingredients such as vitamins, cell growth factors such as EGF, skin activators, blood circulation promoters, resident bacteria control agents, active oxygen scavengers, anti-inflammatory agents, whitening agents, bactericides It can also be contained.

As cosmetics, skin cosmetics such as lotion, milky lotion, cream and pack, body cosmetics such as hair scalp cream, hair and body lotion, bathing agents, oral cosmetics and hair cosmetics can be used. . As the cosmetics containing the composition having an active ingredient obtained by the method of the present invention, for example, milky lotion, cosmetic liquid, face cream, hand cream, lotion, essence and the like are preferable from the functional aspect.

It is also preferable to use an emulsifying means with liposomes in order to maintain the substance activity in the living body or enhance tissue migration in the external application. Liposomes that can be used in the present invention are, for example, liposomes prepared from lipid molecules such as phospholipids, glycolipids, or cholesterol, and both single-layer and multilamellar liposomes are effective. Phospholipids from which liposomes can be prepared generally include, for example, glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, or cardiopine), or sphingophospholipids (sphingomyelin). , Ceramide phosphorylethanolamine, or ceramide phosphorylglycerol). Examples of glycolipids that can be used to prepare liposomes include glyceroglycolipid (digalactosyl diglyceride or seminolipid), or sphingoglycolipid (galactosylceramide or lactosylceramide).

Examples of the liposome include neutral liposomes, cationic liposomes, anionic liposomes, and pH-sensitive liposomes depending on the charge state of the polar part. For example, cationic liposomes are artificial mixtures of cationic head lipids and helper lipids, for example: (1) (a) monocationic heads such as DOTMA {i.e. N- [1- (2,3-dioleyloxy) propyl] -N, N, N-triethylammonium}, DMRIE {ie, N- [1- (2,3-dimyristyloxy) propyl] -N, N- Dimethyl-N- (2-hydroxyethyl) ammonium bromide}, DOTAP {ie, 1,2-dioleoyloxy-3- (trimethylammonio) propane}, DDAB {ie, dimethyldioctadecylammonium bromide}, PC-cholesterol {Ie, 3β- [N- (N ′, N′-dimethylaminoethane) -carbamoyl] co Sterol} or DOTIM {ie, 1- [2-9 (Z) -octadecenoyloxy) -ethyl-2- (8 (Z) -heptadecenyl) -3- (2-hydroxyethyl) -imidazolinium Chloride}, or (b) a polycationic head such as DOGS {ie dioctadecylamidoglycylspermine} or DOSPA {ie 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propaneammonium trifluoroacetate} and a cation consisting of (2) a helper lipid such as DOPE {ie dioleoyl-phosphatidylethanolamine} or DOPC {ie dioleoylphosphatidylchlorine}. To use sex liposomes it can.

In the tissue repair or hair growth agent according to the present invention, the existence form thereof is not particularly limited as long as the active ingredient and the liposome are contained in the form of a complex at the same time. Examples of the complex include a mixture of the active ingredient and liposome, an NP family molecule embedded body or an encapsulated body of the liposome, and the embedded body is preferable.

The embedded body can be prepared by, for example, a method in which an NP family molecule is encapsulated in a liposome. That is, using a lipid such as phosphatidylserine, a multilayer liposome is prepared with a vortex mixer or the like, and then sonicated to prepare a single membrane liposome. The active ingredient can be added to the obtained single membrane liposome, lightly applied to a vortex mixer or the like, then freeze-dried and rehydrated. The encapsulated product can also be prepared by a known method.

The tissue repair or hair growth agent of the present invention contains a complex of the active ingredient and the liposome as an active ingredient, and optionally contains a normal carrier that is pharmaceutically or veterinarily acceptable. be able to. The dosage form of the liposome is not particularly limited, and for example, oral agents such as powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, or pills, Or, parenteral preparations such as injections, liquids for external use, ointments, suppositories, creams for topical administration, and eye drops may be mentioned, and in particular, drugs (for example, injections or sustained-release pellets) administered locally. Etc.) is preferable.

Moreover, the tissue repair or hair growth-promoting agent of the present invention can be administered using a sustained-release preparation technique using a sustained-release polymer, cyclodextrin and the like. For example, an ANP family molecule of the present invention can be incorporated into a pellet of ethylene vinyl acetate polymer and the pellet can be surgically implanted into the tissue to be treated.

An ANP family that may contain cyclodextrin, or a preparation comprising an ANP family cyclodextrin inclusion compound, which is freeze-dried, further freeze-dried containing maltose or trehalose, and 1 part by weight of active ingredient A lyophilized product containing about 10 to about 10,000 parts by weight of maltose or trehalose is preferable.

The oil / fat component used in the fat emulsion is not particularly limited as long as it is an oil / fat base such as vegetable oil / fat, animal oil / fat, or mineral oil / fat, but vegetable oil / fat is preferable. Examples of vegetable oils include olive oil, soybean oil, sesame oil, castor oil, corn oil, safflower oil, rapeseed oil, eucalyptus oil, and examples of animal oils include liver oil, seal oil, sardine oil, docosahexaene. Examples of mineral oils include acid, eicosapentaenoic acid, docosapentaenoic acid, and liquid paraffin. In particular, olive oil, soybean oil, and sesame oil are preferable.

Furthermore, as the phospholipid, a naturally derived phospholipid can be used as it is or after further purification. Specifically, egg yolk lecithin, soybean lecithin, or the like can be used. Naturally derived phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingoemilin, and the like.

In addition to the above components and water, fatty acids can be added to the fat emulsion. This fatty acid functions as an emulsification aid and gives excellent emulsion stability to the NP family-containing emulsion of the present invention. The fatty acid may be natural or synthetic, and may be either a saturated acid or an unsaturated acid, but is preferably an unsaturated medium chain fatty acid, such as oleic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, etc. It is done. When oleic acid is used as the fatty acid, the purity is preferably 99% or more.

The fat emulsion is 5 to 50% by weight, preferably 10 to 20% by weight of fats and oils, 0.05 to 10% by weight, preferably 0.5 to 5% by weight of phospholipid, 0 to 1% by weight, preferably It can manufacture by emulsifying by a conventional method using 0.2-0.5 mass% fatty acid and the balance water.

Since the NP family-containing emulsion of the present invention may be directly injected or administered locally to a living body, emulsification is preferably performed so that the maximum particle size of the resulting fat granules does not exceed 1 μm, and the maximum particle size is 0. It is more preferable to carry out so as not to exceed 7 μm. When the maximum particle size is 1 μm, for example, there is a possibility that problems such as peripheral blood vessel closure occur. For example, it is preferable to blend ANP into an O / W type fat emulsion using polyethylene glycol-linked phospholipid and lecithin as an emulsifier.

The polyethylene glycol-linked phospholipid used as an emulsifier in the NP family-containing emulsion of the present invention is a phospholipid having a polyethylene glycol (PEG) chain bound thereto, and having a PEG molecular weight of 1000 to 10,000. 1000 to 5000 are particularly preferred. This is because when the molecular weight of PEG is less than 1000, it is difficult to produce an emulsion, and when it exceeds 10,000, the viscosity of the fat emulsion increases, making it difficult to administer as an injection. Any terminal such as a hydroxyl group, an alkoxy group, or a carboxyl group can be used for the terminal of the PEG chain of the polyethylene glycol-linked phospholipid that is not bonded to the phospholipid.

Examples of the phospholipid to which the PEG chain binds in the polyethylene glycol-linked phospholipid include lecithin, phosphatidylcholine, hydrogenated phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and derivatives thereof. Distearoylphosphatidylethanolamine is particularly preferable.

Egg yolk lecithin and soybean lecithin can be used as the lecithin used as another type of emulsifier in the NP family-containing emulsion of the present invention, with egg yolk lecithin being particularly preferred. Egg yolk lecithin is preferably purified until it has a content of about 70% or more of phosphatidylcholine because it is an injection. Here, from the viewpoint of the stability of the fat emulsion, it is considered possible to replace lecithin with other glycerophospholipids, but there is currently no substitute for the injection.

The emulsifier used in the fat emulsion contains polyethylene glycol-linked phospholipid and lecithin as essential components, but other glycerophospholipids that can be used for injections can also be mixed and used. The amount of these emulsifiers is preferably 1-30 W / V% of the whole preparation.

As the fatty oil used in the fat emulsion, those generally used for injections can be used. Specific examples include soybean oil, sesame oil, safflower oil, olive oil and the like. The amount of the fatty oil is preferably 1 to 30 W / V% of the whole preparation.

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention, and functionally equivalent methods and ingredients are included within the scope of the invention.
Example 1

A 44-year-old male with chronic heart failure who was diagnosed with dilated cardiomyopathy was administered a small amount of ANP once a week for 1 mg / 4 hours via intravenous vein. The left ventricular contraction rate, which was 19% before the start of ANP administration, improved to 40% after 6 months, and the body movement ability, which was NYHAIV before the treatment, improved to NYHAII. The myocardium in which thin fibers were observed on the ultrasound increased in thickness and was judged to be a granular scar inside the normal myocardium. With continued administration once a week, NYHAI became 2 years later, and the prognosis estimated to be half a year in the case of existing therapies was that they could spend a normal family life for more than 10 years. During this process, both myocardial wall thickness and contractility are good.
Example 2

In a 49-year-old woman who had ablated a large number of moles of the entire face by laser, ANP 0.1 to 5 μg (isotonic aqueous solution 0.01 ml to 0.5 ml) was applied to the left facial treatment wound and the surrounding area after washing twice a day The right facial treatment wound was used as a control. As a result, the subcutaneous tissue was remodeled clearly earlier in the left wound than in the right wound, and one week after administration, the left wound was mostly blocked by ANP administration, whereas the right wound was still red with craters remaining. The subcutaneous tissue was directly viewed (FIG. 1). In addition, administration of ANP healed traces that were less prominent and less noticeable. In addition, the skin on the left face became more moist and smoother than the right.
Example 3

Using 54-year-old and 89-year-old men with thin hair, ANP was applied to the scalp by applying 0.5 to 5 μg (0.5 ml of isotonic aqueous solution) after washing the hair twice a day. Before and after administration, the scalp, hair follicle, hair, and whole hair were observed. Moreover, hair evaluation was performed before administration and 40 days and 60 days later. In both cases, hair growth-like hair growth was observed in the frontal region at 1 week after hANP administration, and a site where a black hair papilla was visible after hair loss began to appear. After 2-3 weeks, the elasticity of hair and the rigidity were enhanced as the whole hair. After one month, the scalp that had been seen through before use was clearly difficult to see. The front row of the hair that was receding moved forward, and the new hair that began to emerge reached 5-7 mm. In addition, a second short hair was found from the side of the long single hair. After 40 days, as seen from the whole picture, the whole hair became firm and thick. In addition, the spirally dark hair can be seen through under the hair follicle. When this hair protrudes from the body, it was initially swirled, so when it was upright, it already showed a length of 3-4 mm. After 60 days, it became difficult to see the entire scalp, hair follicles were seen in most hair follicles, and hair growth increased around and around the thinnest toothpicks before administration.
Example 4

Every day 0.5 to 5 μg of ANP was applied to both palms and palms of potters engaged in water mud work. As a result, cracks and rough skin were always noticeable every winter since before, but both cracks and rough skin improved after application of ANP.
Example 5

Falcon flat bottom 96 well dish (# 3072) so that human normal keratinocyte cell line HaCaT becomes 5 × 10 ² cells / well (A−) and 10 ⁴ cells / well (B−), 2 × 10 ⁴ cells / well (C−). After 3 days and 1 day, HANP (carperitide / Daiichi Pharmaceutical Co., Ltd.) is added every day to a concentration of 1 μM (diluted with PBS). After 4 days (A) and 3 days (B, C), cell proliferation measurement reagent WST- 1 (Roche) was added, and after 2 hours at 37 ° C., the OD450 was measured with a microplate reader MTP-120 (Corona Denki), and the number of living cells was compared. HANP inhibited cell proliferation in the rat smooth muscle cell line A10 as a control, whereas HaCaT showed 3-10% cell proliferation action (FIG. 2).
Example 6

A human normal keratinocyte cell line HaCaT was seeded in a 3.5 cm falcon dish (3001) so as to be 2 × 10 ⁴ cells / dish, and 3 days later, HANP was added to 1 μM, and 0, 30 minutes, 4 hours later RNA was recovered from the cells using RNeasy plus (Qiagen). CDNA was synthesized from 0.2 μg of RNA using Superscript III (Invitrogen) and used as a template for PCR. Using GAPDH as an internal control, after PCR, agarose gel electrophoresis was performed, stained with Cyber Safe (Invitrogen), and then electrophoresed on a UV transilluminator with a digital camera (Olympus C5080WZ). Measurement was performed using Image J (densitometer analysis). Analysis of changes in the expression levels of keratinocyte differentiation markers KRT15, Invorculin, apoptosis inhibitor Bcl-2, apoptosis-related molecule HSPCO16, cell cycle-related molecule CyclinD1, and growth differentiation regulator BMP-2 revealed that the basal layer and hair matrix KRT15, a stem cell marker, showed a transient increase in expression of about 20% after 30 minutes of HANP treatment. BMP-2 was found to increase expression about 1.5 times after 4 hours of HANP treatment compared to no treatment. CyclinD1 showed a transient decrease in expression in the control at 30 minutes but recovered thereafter, whereas the expression level decreased to 1/3 in the HANP treatment (FIG. 3). Since BMP-2, which has keratinocyte growth suppression and differentiation promoting effects, increases and Cyclin D1 that acts to promote cell division decreases, it was considered that keratinocyte differentiation was promoted.
Example 7

The human normal keratinocyte cell line HaCaT was seeded in a 6-well falcon dish (3046) so as to be 2 × 10 ⁵ cells / dish. After 3 days, confluent cells were linearly scratched with the tip of a pipetteman tip and twice with PBS. After washing, HANP was added to 1 μM (in Opti-MEM serum free), and cell behavior after 24 hours was observed (FIG. 4). In the HANP treatment, many rounding-like cells were observed in the wound cell groups.
Example 8

The human normal keratinocyte cell line HaCaT was spotted on a falcon dish so as to be 5 × 10 ⁴ cells / 20 μl drop and 2 × 10 ⁴ cells / 10 μl drop, and the cell colony forming ability in the presence of HANP 1 μM was compared. In the case of 10 μl drop, no colony formation was observed, but in the HANP treatment, it was observed that the cells extended their legs to form a colony (FIG. 5).
Example 9

In order to clarify the NPP-NPR signal molecule in HaCaT, the expression of the receptor NPR-1,2,3 and ligand NPPA, B, C genes was analyzed by RT-PCR, and only NPR2 was detected (FIG. 6). .

The present invention disclosed that the atrial diuretic peptide group, which was conventionally thought to act on blood vessels, is useful for improving the recovery of various tissues including regenerative medicine, baldness, and skin injury. This use makes it possible to develop procedures and preparations that have not been realized in the field of medical biology.

In the area where ANP is applied, the recovery of the surgical wound after laser surgery is much accelerated. HANP inhibited cell proliferation in the rat smooth muscle cell line A10 as a control, whereas HaCaT showed 3-10% cell proliferation action. BMP-2 was found to increase expression about 1.5 times after 4 hours of HANP treatment compared to no treatment. CyclinD1 showed a transient decrease in expression in the control at 30 minutes but recovered thereafter, whereas the expression level decreased to 1/3 in the HANP treatment. In the HANP treatment, many rounding-like cells were observed in the wound cell groups. That is, the cell motility of HaCaT is increased, which works preferentially for wound repair. In the HANP treatment, it was observed that cells extended their legs and formed colonies. From this, it can be said that a protective action, a cell back attachment, and a sequence-forming action worked against stress. It is thought that NPR2 is mainly expressed and works to promote differentiation under the dominance of? NPR2 paracrine signal from NP family molecules.

Other

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Atrial diuretic hormone family molecules are useful in tissue organ repair and regeneration formulations and tissue organ repair and regeneration methods, and are not available with conventional substances / techniques for hair loss, hair loss, skin and subcutaneous tissue injury Demonstrate the improvement effect.

Claims (1)

A preparation for hair growth, hair growth, and hair growth that contains atrial diuretic hormone (ANP) as an active ingredient and may contain a diluent, excipient, filler, or auxiliary agent commonly used in pharmacology.