JP5051471B2 - Resting phase alopecia treatment - Google Patents

Description

  The present invention relates to a therapeutic agent for resting alopecia, and more specifically to a therapeutic agent for resting alopecia containing leptin or a partial peptide thereof as an active ingredient.

  Hair is distinguished from head hair, eyebrows, eyelashes, beards, eyelashes, pubic hairs, body hairs, etc., depending on the site. However, the structure is common, and a single hair grows and grows as a new hair component by cell division in the hair matrix of the root of the root, and is supported and fixed by the intrinsic epithelial hair follicle that encloses it. The

  Hair growth in the scalp does not occur continuously, but occurs by an active cycle that alternates between the period of development and rest. This cycle is mainly divided into three stages, a growth period, a regression period, and a rest period, and this is called a hair cycle. Growth is the developmental stage of the cycle, characterized by deep penetration of hair follicles into the dermis, rapid proliferation and differentiation of cells to form hair. The next stage is the regression phase, which is a transitional stage characterized by the termination of cell division, during which the hair follicles regress in the dermis and the hair growth stops. The next quiescent phase is characterized as a quiescent phase, during which the regressed hair follicle has a hair root in close proximity to the dermal papillary cell population. Thereafter, cells rapidly proliferate in the hair root, the dermal papilla expands, the basement membrane components become assimilated, and a new growth phase begins. By the way, when a sufficient growth period is not maintained, the period shifts to a rest period and the rest period is postponed, so-called rest period alopecia occurs.

Currently, as a therapeutic agent for such resting alopecia, topical minoxidil (commercially available from Pharmacia & Upjohn as ROGAINE®) for the treatment of male pattern alopecia, which is a type of resting alopecia. And oral finasteride (commercially available from Merck & Co., Inc. as PROPECIA®).
Patent Document 1 describes that administration of nicotinic acid ester increases leptin levels, leading to skin tumor degeneration, skin homoostasis and reconstruction, wound healing, and hair growth. However, this document does not describe that leptin can be administered to induce the growth phase from the resting phase of the hair cycle to treat resting alopecia.

JP-T-2004-519488

  An object of the present invention is to provide a drug useful for treating resting alopecia (particularly male pattern alopecia) by inducing a growth period from a resting period of the hair cycle.

  As a result of intensive studies to solve the above problems, the present inventors have found that leptin or a partial peptide thereof induces the growth phase from the resting phase of the hair cycle and has an excellent effect on the treatment of resting alopecia. Based on this finding, the present inventors have further studied and completed the present invention.

That is, the present invention
[1] A therapeutic agent for resting alopecia comprising leptin or a partial peptide thereof as an active ingredient,
[2] The therapeutic agent according to the above [1], wherein the active ingredient is leptin,
[3] The therapeutic agent according to the above [1], wherein the active ingredient is a partial peptide of leptin,
[4] The therapeutic agent according to the above [2], wherein the leptin is human leptin,
[5] The therapeutic agent according to the above [4], wherein the human leptin is human recombinant leptin,
[6] The therapeutic agent according to the above [3], wherein the partial peptide is represented by SEQ ID NO: 4,
[7] The therapeutic agent according to any one of the above [1] to [6], wherein the resting alopecia is androgenetic alopecia,
[8] The therapeutic agent according to any one of [1] to [7] above, wherein the formulation type is an injection for subcutaneous administration,
[9] The therapeutic agent according to any one of the above [1] to [7], wherein the formulation type is a formulation for topical application,
[10] The therapeutic agent according to the above [9], wherein the active ingredient is made into a liposome.
[11] Use of leptin or a partial peptide thereof for producing a therapeutic agent for resting alopecia, and [12] A therapeutically effective amount of leptin or a partial peptide thereof is administered to a patient with resting alopecia A method for treating resting alopecia,
About.

  The therapeutic agent for alopecia alopecia according to the present invention can be administered to humans to induce the hair cycle from the resting phase to the growing phase to treat resting alopecia.

FIG. 1 is a diagram showing a hair cycle (A) and a photograph showing a hair follicle state at 35 days after birth (growth stage) of a db / db mouse or a normal mouse. B shows a normal mouse and C shows a db / db mouse.
FIG. 2 shows photographs of the 14th day after dorsal hair removal of ob / ob mice or normal mice at 7 weeks of age (second rest period) and the follicular state of ob / ob mice or normal mice. A and B are normal mice, and C and D are ob / ob mice.
FIG. 3 is a photograph of the dorsal region on the 16th day of an ob / ob mouse administered with physiological saline or leptin. A shows a physiological saline-administered mouse, and B shows a leptin-administered mouse.
FIG. 4 is a photograph showing the hair follicle state on the 21st day after administration of physiological saline or leptin to a 7-week-old normal mouse, and a photograph of the back of the normal mouse on day 21 after administration of leptin or physiological saline. It is. A shows a physiological saline-administered mouse, and B shows a leptin-administered mouse. Above C shows leptin-administered mice, and below C shows physiological saline-administered mice.
FIG. 5 is a photograph of the dorsal region on the 21st day of normal mice in which normal saline or leptin partial peptide (SEQ ID NO: 4) was administered to 7-week-old normal mice.

  The active ingredient of the therapeutic agent for alopecia according to the present invention is leptin or a partial peptide thereof. The leptin according to the present invention may be mouse leptin or human leptin, preferably human leptin. The amino acid sequence of human leptin including the signal peptide (21 amino acids) is shown in SEQ ID NO: 1, and that of mouse is shown in SEQ ID NO: 2.

  Further, the leptin according to the present invention may be deleted, added, or substituted as long as the leptin function is maintained.

  On the other hand, the leptin partial peptide according to the present invention includes a partial peptide containing the leptin active site, that is, a low molecular weight functional peptide. As the partial peptide, a partial peptide generated by cleavage by an enzyme originally possessed by an animal (particularly human) that is the origin of leptin is preferable in that it has low antigenicity. Peptides.

  The leptin and the leptin partial peptide according to the present invention can be prepared by a conventional method, and commercially available products such as human recombinant leptin (R & D Systems) or human leptin partial peptide (BRACHEM) can also be used. When producing, it can be produced according to a conventional method by genetic engineering techniques. For example, leptin is described below as an example, but partial peptides can also be produced according to this. First, a gene encoding leptin (leptin gene) is obtained by a conventional genetic engineering method (for example, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition), Cold Acquired in accordance with Spring Harbor Laboratory publication (method described in Cold Spring Harbor Laboratory Press). The leptin gene is preferably a human leptin gene, and its DNA sequence including a sequence encoding a signal peptide is as shown in SEQ ID NO: 3. Next, using the obtained leptin gene, leptin can be produced according to an ordinary genetic engineering method.

  For example, a culture obtained by preparing a plasmid capable of expressing the leptin gene in a host cell, introducing the plasmid into the host cell, transforming it, and further culturing the transformed host cell (transformant). Leptin can be obtained from the object.

  Examples of the plasmid include a promoter that contains genetic information that can be replicated in a host cell, can be propagated autonomously, can be easily isolated and purified from the host cell, and can function in the host cell. Preferred examples include those in which a gene encoding leptin is introduced into an expression vector having a detectable marker. For example, an expression vector used for expression in E. coli is an expression vector containing a promoter such as lac, trp, tac, etc., and these are commercially available from Pharmacia, Takara Shuzo. If it is necessary to induce higher expression, a ribosome binding region may be linked upstream of the gene encoding leptin. Examples of the ribosome binding region used include Guarente L. et al. (Cell 20, p543) and Taniguchi et al. (Genetics of Industrial Microorganisms, p202, Kodansha).

  Examples of host cells include prokaryotic or eukaryotic microbial cells, insect cells, or mammalian cells. For example, from the viewpoint of facilitating mass preparation of leptin, preferred is E. coli.

The therapeutic agent of the present invention exhibits an excellent effect in the treatment of resting alopecia. Resting alopecia to which the therapeutic agent of the present invention can be applied includes androgenetic alopecia, postpartum (postpartum) alopecia, post-pill alopecia, diet / starvation alopecia, pressure alopecia, hypothyroidism Alopecia due to pituitary hypofunction, alopecia due to hypoparathyroidism, drug alopecia, seborrheic alopecia, traction alopecia, alopecia areata, alopecia associated with endocrine disease, contact Alopecia due to atopic dermatitis is mentioned, and male pattern alopecia is particularly preferable. When the therapeutic agent for resting alopecia of the present invention is locally applied externally, it is preferably applied locally or injected subcutaneously. The dose of leptin, which is an active ingredient, or its partial peptide varies depending on the subject's gender, age, weight, and condition (symptoms), but when applied locally, it should be selected appropriately according to the area of skin to be administered. However, it is usually about 10 to 1000 mg, preferably about 10 to 500 mg per day for the area of the application site of about 10 cm ² . The administration dose may be applied once or several times per day. In the case of subcutaneous injection, the dose of leptin or a partial peptide thereof can be appropriately selected depending on the skin area to be administered, but is usually about 1 to 1000 mg with respect to the area of the application site of about 10 cm ² . , Preferably about 10-100 mg. The frequency of administration is not particularly limited, but administration once a day to once every several weeks is preferable.

The therapeutic agent for alopecia of the present invention is locally administered. Examples of dosage forms include semi-solid preparations such as ointments and creams, liquid preparations such as shampoos, rinses and lotions, and tapes when applied topically, and injections when administered subcutaneously locally. Is mentioned. The formulation method for these dosage forms is in accordance with the usual method in principle. However, when manufacturing these formulations, use appropriate amounts of carriers or additives according to the formulation form. Can do.
For example, in the case of an injection, in addition to a solvent (for example, purified water), a dispersant (eg, Tween 80 (manufactured by Atlas Powder Co., USA), HCO 60 (manufactured by Nikko Chemicals), polyethylene glycol, carboxymethylcellulose , Sodium alginate, etc.), preservatives (methyl paraben, propyl paraben, benzyl alcohol, etc.), and isotonic agents (sodium chloride, mannitol, sorbitol, glucose, etc.) can be appropriately used.
On the other hand, when the preparation is suitable for topical application (eg, ointment, cream, lotion, etc.), leptin or a partial peptide thereof is made into a liposome to facilitate percutaneous absorption, and leptin or its It is preferable to make the partial peptide easy to reach the hair follicle tissue.

  Liposomeization means inclusion of a drug inside a liposome (that is, a closed vesicle composed of lipids assembled in a membrane and an internal aqueous phase), and leptin or a partial peptide thereof constitutes the liposome. It may be included in a lipid layer assembled in a film form or in an aqueous phase inside the liposome.

  In the liposome according to the present invention, various lipophilic or hydrophilic components can be encapsulated in the lipid membrane and the inner aqueous phase, respectively.

  Leptin or its partial peptide can be made into a liposome by a conventional method. For example, leptin or its partial peptide is dissolved in a lipid component of the membrane, mixed with a water-soluble non-volatile organic solvent together with the lipid component of the membrane, dissolved or suspended in an aqueous solution, etc. It can be included in a membrane or an inner aqueous phase. Liposomal leptin prepared as described above or a partial peptide thereof is dispersed in a therapeutic agent base to obtain a therapeutic agent for resting alopecia.

Examples of lipids used for liposome formation include glycerophospholipid, glyceroglycolipid, sphingophospholipid, glycosphingolipid, and sterols. Examples of the glycerophospholipid include phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine. Examples of the glyceroglycolipid include digalactosyldiglyceride and galactosyldiglyceride. Examples of the sphingophospholipid include sphingomyelin. Examples of the glycosphingolipid include cerebroside and ganglioside, and examples of the sterols include cholesterol and cholesterol hemisuccinate. Here, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, sphingomyelin, ganglioside each have two saturated or unsaturated fatty acid ester chains, but the fatty acid (alkanoyl or alkenoyl group) moiety has 10 carbon atoms. Lipids that are ˜18 can be used as liposome constituents. Examples of the alkanoyl or alkenoyl group having 10 to 18 carbon atoms include desilyl, undesilyl, lauroyl, tridesilyl, myristoyl, pentadesilyl, palmitoyl, heptadesilyl, stearoyl, oleoyl group, preferably lauroyl, Myristoyl, palmitoyl and stearoyl groups. For the aqueous phase constituting the interior of the liposome, an aqueous sodium chloride solution, buffer solution (phosphate buffer solution, acetate buffer solution, etc.), monosaccharide or disaccharide solution (glucose aqueous solution, trehalose aqueous solution, etc.) is usually used. it can.
In order to obtain a preparation suitable for topical application using the liposomal leptin or its partial peptide obtained as described above, it may be carried out according to a method employed in this field.

  Hereinafter, the present invention will be described using test examples, but the present invention is not limited thereto.

[Test Example 1]
(1) Examination of hair cycle in db / db (leptin receptor-deficient) mice 35-day-old C57BL / KsJ-db / db mice (leptin receptor-deficient mice) obtained from Nippon Claire Co., Ltd. and normal mice of the same strain The back skin of each (n = 3) was excised, and after fixing with formalin, a hematoxylin-eosin stained specimen was prepared using a known method, and the state of the hair follicle was observed. As a result, in all the individuals, the growing hair follicle was observed in the normal mouse (B in FIG. 1), but only the resting hair follicle was observed in the db / db mouse (C in FIG. 1). From this, it was verified that db / db mice cannot enter the second growth phase. FIG. 1A shows the normal hair cycle of a mouse.

(2) Growth phase induction by hair removal in ob / ob (leptin deficient) mice Seven-week-old (second resting phase C57BL / 6J-ob / ob mouse leptin-deficient mice) obtained from Nippon Claire Co., Ltd.) and the same The back body hairs of normal mice (n = 3 each) were removed, and the back skin was excised 14 days later. After fixing with formalin, hematoxylin / eosin stained specimens were prepared, and the state of the hair follicles was observed. As a result, in all the individuals, the growth follicle was observed in the normal mouse (B in FIG. 2), but only the resting follicle was observed in the ob / ob mouse (D in FIG. 2). From this, it was verified that ob / ob mice cannot induce the growth phase by hair removal stimulation. 2A and 2C are photographs of the back of the mouse immediately before the dorsal skin resection.

(3) Growth phase induction by leptin in ob / ob (leptin deficient) mice The dorsal body hair of the above-mentioned 7-week-old (second resting phase) ob / ob mice (n = 3) was removed, and mouse recombinant leptin (R & D) A single subcutaneous injection of a physiological saline solution (10 μg / 0.1 ml) manufactured by Systems, Inc. confirmed that the dorsal skin turned black on the 16th day and that the growth period was induced (FIG. 3 B). In the control group to which only physiological saline was administered, the back skin was hardly changed (A in FIG. 3). From this, it was verified that leptin induces the growth phase from the resting phase in leptin-deficient individuals.

(4) Growth period induction by leptin in C57BL / 6J mice (normal mice) The shaved back hairs of the above-mentioned 7-week-old (second rest period) C57BL / 6J mice (normal mice, n = 3) were shaved. A single subcutaneous injection of mouse recombinant leptin in physiological saline solution (10 μg / 0.1 ml) or saline alone, hair growth was observed in mice injected with leptin on the 21st day in line with the subcutaneous injection site. (C, arrow in FIG. 4) Histologically, the hair follicle was also confirmed (B, arrow in FIG. 4), but hair growth occurred in mice injected with saline subcutaneously. (C in FIG. 4, lower part) and histologically remained in a resting state (A in FIG. 4). From this, it was verified that leptin induces the growth phase from the resting phase even in normal individuals.

[Test Example 2]
Growth phase induction by partial peptides in C57BL / 6J mice (normal mice) The back body hairs of the above-mentioned 7-week-old (second resting) C57BL / 6J mice (normal mice, n = 3) were shaved, and a part of human leptin A single subcutaneous injection of a physiological saline solution (10 μg / 0.1 ml) of peptide (22-56 residues, SEQ ID NO: 4, manufactured by BRACHEM) or physiological saline alone, 21 days for mice subcutaneously injected with a partial peptide In the eyes, hair growth was observed in accordance with the site of subcutaneous injection (FIG. 5B), and no hair growth was observed in mice subcutaneously injected with physiological saline (A in FIG. 5). From this, it was verified that even in normal individuals, the partial peptide induces the growth phase from the resting phase.

(5) Discussion From the above experiment, it was found that leptin or its partial peptide has a therapeutic effect on resting alopecia.

  The therapeutic agent for resting alopecia according to the present invention can be used as a medicament for treating resting alopecia.

Claims (6)

A therapeutic agent for resting alopecia comprising a partial peptide of leptin as an active ingredient , wherein the amino acid sequence of the partial peptide is represented by SEQ ID NO: 4 .   The therapeutic agent according to claim 1, wherein the resting alopecia is male pattern alopecia. The therapeutic agent according to claim 1 or 2, wherein the formulation type is an injection for subcutaneous administration. The therapeutic agent according to claim 1 or 2, wherein the formulation type is a formulation for topical application. The therapeutic agent according to claim 4, wherein the active ingredient is made into a liposome. Use of a partial peptide of leptin represented by SEQ ID NO: 4 for producing a therapeutic agent for resting alopecia.