(en)The present invention is aimed for providing a novel peptide with a high drug efficacy and strong effect, a medicament or external preparation comprising it, specifically a prophylactic or therapeutic for dermatitis, rhinitis or alopecia, or a hair growth stimulant, a hair growing agent, an antipruritic or a skin-care product. The present invention achieved said aim by providing a cyclic peptide having an amino acid sequence expressed by the Formula I or a derivative thereof or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence does not have a peptide bond that is not between the amino acids constituting the amino acid sequence.

1.ApplicationNumber: US-201615122753-A
1.PublishNumber: US-2018066020-A1
2.Date Publish: 20180308
3.Inventor: ENDO KYOKO
ENDO YORI

4.Inventor Harmonized: ENDO KYOKO(JP)
ENDO YORI(JP)

5.Country: US
6.Claims:
(en)The present invention is aimed for providing a novel peptide with a high drug efficacy and strong effect, a medicament or external preparation comprising it, specifically a prophylactic or therapeutic for dermatitis, rhinitis or alopecia, or a hair growth stimulant, a hair growing agent, an antipruritic or a skin-care product. The present invention achieved said aim by providing a cyclic peptide having an amino acid sequence expressed by the Formula I or a derivative thereof or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence does not have a peptide bond that is not between the amino acids constituting the amino acid sequence.

7.Description:
(en)TECHNICAL FIELD
The present invention relates to a cyclic peptide and a medicament, external preparation and cosmetic comprising said cyclic peptide.
BACKGROUND ARTS
BNP (brain natriuretic peptide) is a hormone which is synthesized and secreted in heart (predominantly in ventricles). BNP was isolated from and identified in pig brain in 1988, and it has been known since that BNP is secreted from ventricular myocardium of human, etc. An increase in cardiac stress or development of myocardial hypertrophy induces BNP secretion and increased in blood concentration. In general, BNP has activities such as diuretic activity, vasodilation, renin-aldosterone secretion suppressing action, sympatholytic activity, hypertrophy suppressing action. BNP is considered to a hormone which acts to protect the myocardium against damages caused by cardiac stress.
Amino acids constituting BNP differ slightly from species to species who produce BNP. Nevertheless it been revealed that its structure possesses a cyclic part and a tail part as a common structure (Non-Patent Literatures 1-2). For instance, wild-type human BNP has been known to consist 32 amino acid residues, and its fragments and derivatives are further proposed (Patent Literatures 1-6).
In Japan, BNP is currently not used as a therapeutic, but widely used in clinical practice as a biochemical marker for cardiac failure. In United States, however, it is used as a drug for alleviating symptoms of cardiac failure (trade name: Natrecor®). Recently, it has been proposed to utilize an external preparation comprising BNP for treating dermatitis, rhinitis, alopecia, etc., and also as a skin-improving agent (Patent Literature7-9).
PRIOR ART REFERENCES
Patent Literature


[Patent Literature1] JP A 2007-525213
[Patent Literature2] JP A 2008-509746
[Patent Literature3] WO 2008/032450
[Patent Literature4] U.S. Pat. No. 6,028,055
[Patent Literature5] U.S. Pat. No. 5,114,923
[Patent Literature6] U.S. Pat. No. 6,818,619
[Patent Literature7] WO 2011/010732
[Patent Literature8] WO 2011/024973
[Patent Literature9] WO 2012/099258


Non-Patent Literature


[Non-Patent Literature1] N. Akizukia, K. Kangawaa, N. Minaminob, H. Matsou, FEBS Letters 1991 280 (2):357-362.
[Non-Patent Literature2] Takei Y, Fukuzawa A, Itahara Y, Watanabe T X, Yoshizawa Kumagaye K, Nakajima K, Yasuda A, Smith M P, Duff D W, Olson K R. FEBS Lett. 1997 Sep. 8; 414 (2):377-80.


SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
As described above, BNP is considered to be a useful substance for the therapy of various diseases. However, in order to obtain a better therapeutic effect there is a need for developing a substance with a higher drug efficacy and stronger effect and a faster- and longer-acting substance.
An improvement in the duration and fast-acting properties of the drug efficacy and effect is quite meaningful in light of reducing required number/frequency of treatment, relieving the sufferings by the patient/user, and improving the quality of life (QOL) and the mental, physical, social or intellectual satisfaction in dairy living of the user. For example, dermatitis is often accompanied by symptoms such as an itch, infiltration or hot flush in the affected site; the sufferings by the patient will be reduced if these symptoms can quickly be alleviated. When an alopecia therapeutic, hair growing agent or hair growth stimulant is employed, the QOL of the user can be improved if the therapeutic effect, hair growth-stimulating or hair growing effect can be achieved immediately.
The inventors focused on these points and tried to search for a novel substance by reference to the structure of BNP.
Accordingly, the object of the present invention is to provide a novel peptide having a strong drug efficacy and effect, and a medicament or an external preparation comprising such peptide, specifically a prophylactic or therapeutic for dermatitis, rhinitis and alopecia, and a hair growth stimulant, a hair growing agent, an antipruritic, a cosmetic and a skin-care product, etc.
Means to Solve the Problems
It was previously considered that the tail part of the wild-type BNP peptide structure (which is composed of a cyclic part and tail part) plays at least certain important role in the binding and selectivity of BNP to its receptor. However, the inventors focused on the cyclic part of said structure, deleted the tail part, and made an intensive research on the resulting cyclic peptide. Accordingly we have obtained totally new findings including the effects of such cyclic peptide, its high drug efficacy, faster-acting and longer-lasting effect, and further continued the study and finally completed the invention.
Accordingly, the present invention relates to the followings:
[1] A cyclic peptide having an amino acid sequence expressed by Formula I:









wherein,
X 1 denotes Gly, Val, Ala, Ser or Thr,
X 2 denotes Arg, Gln or His,
X 3 denotes Lys or Arg,
X 4 denotes Met, Leu or Ile,
X 5 denotes Ile or Val,
X 6 denotes Ser or Gly,
X 7 denotes Ser or Ala,
X 8 denotes Ser, Gln, Val, Ala, Thr, Leu, Ile or Met,
X 9 denotes Ser, Val, Ala or Thr,
X 10 denotes Gly or Arg,
X 11 denotes Leu, Met, Ile, Val or Ala,
X 12 denotes Gly, Ser or Ala, and
the line connecting two Cys denotes a disulfide bond,
and wherein the amino acid sequence does not have a peptide bond that is not between the amino acids constituting the amino acid sequence,
or a derivative thereof or a pharmaceutically acceptable salt thereof.
[2] The cyclic peptide according to [1], wherein
X 2 denotes Arg, X 4 denotes Met, X 6 denotes Ser, X 7 denotes Ser, and X 10 denotes Gly,

or a derivative thereof or a pharmaceutically acceptable salt thereof.
[3] The cyclic peptide according to [1], wherein the amino acid sequence is selected from the amino acid sequences expressed by Formula (I-a)-Formula (I-e):









wherein, the line connecting two Cys denotes a disulfide bond,
or a derivative thereof or a pharmaceutically acceptable salt thereof.
[4] The cyclic peptide according to any one of [1]-[3] or a derivative thereof or a pharmaceutically acceptable salt thereof, wherein the derivative is substituted by a substituent which is capable of replacing a hydrogen atom, hydroxyl group, carboxy group, amino group or imino group in the cyclic peptide.
[5] The cyclic peptide or a derivative thereof or a pharmaceutically acceptable salt thereof, which is formed by deleting 1 to 4 amino acids in the cyclic peptide expressed by any one of the Formulae (I-a)-(I-e) according to [3], or by replacing them with or adding them other amino acids, and which has an equal function with the cyclic peptide expressed by each of said formulae.
[6] An external preparation comprising one or more cyclic peptides according to any one of [1]-[5] and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof.
[7] The external preparation according to [6], wherein the external preparation is an ingredient for a dermatitis therapeutic, dermatitis prophylactic, antipruritic, antiphlogistic, epidermis regeneration accelerating agent, wound epithelialization-accelerating agent or skin-care product.
[8] The external preparation according to [7], wherein the skin-care product is for moisturizing, and/or for preventing or improving rough skin, and/or for sebum/acne care, and/or for irritation alleviation/anti-inflammation, and/or for skin-lightening, and/or for anti-aging, and/or for preventing/alleviating ultraviolet lesion, and/or for slimming, and/or for skin-cleansing.
[9] The external preparation according to [6], wherein the external preparation is a bath agent, a body-cleansing agent or a hair-cleansing agent.
[10] The external preparation according to [6], wherein the external preparation is an alopecia therapeutic, an alopecia prophylactic, a hair growing agent and/or a hair growth stimulant.
[11] The external preparation according to [6], wherein the external preparation is a rhinitis therapeutic and/or a rhinitis prophylactic.
[12] The external preparation according to [6], wherein the external preparation is a cosmetic.
[13] The external preparation according to any one of [6]-[12], wherein the formulation is a solid, semi-solid, powder, liquid, spray, ointment, cream, emulsion, gel or patch formulation.
[14] The external preparation according to any one of [6]-[13], wherein the external preparation is used as a pharmaceutical product, a quasi-drug or a cosmetic product.
[15] A use of the cyclic peptide according to any one of [1]-[4] and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof for preparing the external preparation.
[16] A method of using the external preparation comprising applying the external preparation according to any one of [6]-[14] to the skin and/or mucosa of a subject.
[17] The method of using the external preparation according to [16], wherein the mucosa is labial, oral, nasal, ocular or vaginal mucosa.
[18] The method of using the external preparation according to [16], wherein the method is a method of treating and/or preventing dermatitis, a method of alleviating or resolving itch, a method of treating eczematous or other erosion or ulcer, or a method for skin-care.
[19] The method of using the external preparation according to [16], wherein the method is a method of treating and/or preventing alopecia, and/or a method of stimulating hair growth, and/or a method of growing hair.
[20] The method of using the external preparation according to [16], wherein the method is a method of treating and/or preventing rhinitis.
[21] A medicament comprising one or more cyclic peptides according to any one of [1]-[5] or a derivative thereof or a pharmaceutically acceptable salt thereof.
[22] The medicament according to [21], wherein the medicament is a therapeutic for hypertension, unstable angina, acute myocardial infarction, edematous diseases, renal failure, cardiac failure, immune diseases, obesity or metabolic syndrome.
The cyclic peptide described herein (hereinafter also referred to as “BNP cyclic peptide”, “B ring” or “B ring-compound”) is, as described above, derived from wild-type BNP. The wild-type BNP encompasses BNP from human, as well as BNPs from monkeys, pigs, birds and rats. Therefore, the B ring-compound also encompasses the B ring-compound from human, as well as those from monkeys, pigs, birds and rats.
Effect by the Invention
According to the present invention, novel cyclic peptides and compositions comprising the same can be provided. Such compositions are applied to an external preparation for preventing or treating dermatitis, rhinitis or alopecia, and further applied to a hair growth stimulant, a hair growing agent or an antipruritic, each of which are provided as a medicament, a quasi-drug, a skin-care product, or a cosmetic product. When an “external preparation” is referred alone in the present invention, it means an agent that is applied to skin or mucosa, whose utility is not limited to a medicament, quasi-drug, skin-care product and cosmetic product.
In general, the external preparation of the present invention has a significantly higher efficacy against dermatitis as compared to a conventional steroid external preparation, as well as an excellent immediate effect such that the symptoms start to be improved within three minutes in general. Its effect is great and long-lasting, and the amelioration period is long.
Furthermore, the external preparation of the present invention can, upon being applied onto the subject's skin or mucosa suffering a dermatitis, quickly relieve or eliminate perceptible symptoms that are or can be caused by dermatitis such as pruritus, soreness (pain), hot sensation, tautness, infiltration and erythema, improving the symptoms of the dermatitis.
Furthermore, the external preparation of the present invention exerts a moisturizing effect on the applied site, while exerting an effect to improve skin texture where the corneum layer is present at the applied site. It can thus exerts effects to improve skin texture, improves dry or rough skin, softens and moisturizes skin, reduce and diminish wrinkles, and improve roughened lip.
The external preparation of the present invention also has an effect of stimulating hair growth or promoting hair growth and at the same time preventing hair loss at the applied site when being applied to a decalvant site or a site where hair growth is stimulated. In this case, stimulated hairs tend to become terminal hairs and not white hairs. These effects are exhibited relatively quick as compared to other active agents that have previously been used in the therapeutics for alopecia, e.g., BNP, and the obtained effects are more significant.
Yet the external preparation of the present invention has an immediate, large and long-lasting effect effect on rhinitis with a long amelioration period.
Besides, since the cyclic peptide of the invention is a peptide sharing a part of its structure with BNP which is a hormone inherent in body, it causes little concern about side effects. It is also considered to have a small influence on homodynamics as long as used in an appropriate amount or used externally onto skin, etc. It therefore can be administered for prolonged period to a patient in need thereof, e.g., a patient with chronic dermatitis. Moreover, the external preparation of the invention causes no irritation when being taken externally and can be applied safely to a patient with sensitive skin, as well as to a child or woman, on face or neck, etc.
The reason why the cyclic peptide of the invention has a superior drug efficacy and effect as compared with wild-type BNP is not yet clear. However, according to our in silico analysis, it is suggested that the cyclic peptide of the invention is more easily and strongly bound to NPR-A receptor (GC-A) as compared with wild-type BNP. It is thus predicted that the cyclic peptide would have an excellent drug efficacy and effect, an excellent immediate effect in particular. As described above, B ring-compound is derived not only from human, but there also are B rings from animals of other species such as monkeys, pigs, birds and rat, and it has been confirmed that they have similar structures. From the results of the structural analysis described hereinbelow, it is strongly speculated that all these B rings exert similar effects as those of the B ring-compound from human.



BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

A diagram showing the result of applying BNP cyclic peptide (A) or a gel formulation (B) on either left or right side of the face of a subject having large wrinkles on the face.

FIG. 2

A diagram showing the effect before and after the application of either B ring gel formulation or BNP gel formulation to a patient with female pattern alopecia and alopecia pityroides.



MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention is explained in detail based on its suitable embodiments.
1. The cyclic peptide, a derivative thereof and a pharmaceutically acceptable salt thereof Firstly, the cyclic peptide of the invention, a derivative thereof and a pharmaceutically acceptable salt thereof are explained.
The cyclic peptide of the present invention has an amino acid sequence expressed by Formula I:









wherein,
X 1 denotes Gly, Val, Ala, Ser or Thr,
X 2 denotes Arg, Gln or His,
X 3 denotes Lys or Arg,
X 4 denotes Met, Leu or Ile,
X 5 denotes Ile or Val,
X 6 denotes Ser or Gly,
X 7 denotes Ser or Ala,
X 8 denotes Ser, Gln, Val, Ala, Thr, Leu, Ile or Met,
X 9 denotes Ser, Val, Ala or Thr,
X 10 denotes Gly or Arg,
X 11 denotes Leu, Met, Ile, Val or Ala,
X 12 denotes Gly, Ser or Ala,
the line connecting two Cys denotes a disulfide bond,
and wherein the amino acid sequence does not have a peptide bond that is not between the amino acids constituting the amino acid sequence.
Such cyclic peptide is, similar to previously known BNPs, considered to bind to a receptor NPR-A (also known as GC-A) having a guanylate cyclase domain and promote the production of cyclic guanosine monophosphate (cGMP), and has activities such as, for example, diuretic action, vasodilation, renin-aldosterone secretion suppressing action, sympatholytic activity and hypertrophy suppressing action. It has a superior drug efficacy and effect, particularly an excellent immediate effect, as compared to BNP.
In addition, as described hereinbelow, the above cyclic peptide can be used as an ingredient of an external preparation for a dermatitis therapeutic/prophylactic, a rhinitis therapeutic/prophylactic, an alopecia therapeutic/prophylactic, a hair growing agent, a hair growth stimulant, an antipruritic, etc., or as an ingredient of a skin-care product, a quasi-drug or a cosmetic product. The above cyclic peptide can also be used as an alternative for BNP in an medicament utilizing the activity of BNP as above, e.g., in an medicament for hypertension, unstable angina, acute myocardial infarction, edematous diseases, renal failure, cardiac failure, immune diseases, obesity or metabolic syndrome.
In another embodiment of the present invention, in the cyclic peptide of the invention, X 1 -X 12 in the Formula (I) may be defined as one or more selected from the group consisting of following (1)-(12):
(1) X 1 denotes Gly, Val, Ala, Ser or Thr,
(2) X 2 denotes Arg, Gln or His,
(3) X 3 denotes Lys or Arg,
(4) X 4 denotes Met, Leu or Ile,
(5) X 5 denotes Ile or Val,
(6) X 6 denotes Ser or Gly,
(7) X 7 denotes Ser or Ala,
(8) X 8 denotes Ser, Gln, Val, Ala, Thr, Leu, Ile or Met,
(9) X 9 denotes Ser, Val, Ala or Thr,
(10) X 10 denotes Gly or Arg,
(11) X 11 denotes Leu, Met, Ile, Val or Ala, and
(12) X 12 denotes Gly, Ser or Ala.
A preferred cyclic peptide of the present invention is a cyclic peptide of Formula (I), wherein X 2 denotes Arg, X 4 denotes Met, X 6 denotes Ser, X 7 denotes Ser, and/or X 10 denotes Gly (SEQ ID NO: 2).
Furthermore, in another embodiment of the invention, in the cyclic peptide of the invention, the amino acid sequence expressed by Formula (I) may be selected from SEQ ID NOs: 3-8 and SEQ ID NOs: 16-75.
Furthermore, in the cyclic peptide of the invention, the amino acid sequence expressed by Formula (I) is preferably selected from the amino acid sequences expressed by the Formula (I-a)-Formula (I-e):









wherein, the line connecting two Cys denotes a disulfide bond.
Each of the amino acid sequences expressed by these Formulae (I-a)-(I-e) is the cyclic part of human BNP (Formula (I-a)), swine BNP (Formula (I-b)), rat BNP (Formula (I-c)), rabbit BNP (Formula (I-d)) and murine BNP (Formula (I-e)), respectively. Therefore, the cyclic peptide having such an amino acid sequence will exhibit the aforementioned effects with more certainty. Because swine BNP cyclic peptide is consistent with those of avian (SEQ ID NO: 8,
Cys-Phe-Gly-Arg-Arg-Ile-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Met-Gly-Cys, wherein, 1st Cys and 17th Cys form a disulfide bond), bovine (SEQ ID NO: 9,
Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, wherein, 1st Cys and 17th Cys form a disulfide bond), feline (SEQ ID NO: 10,
Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, wherein, 1st Cys and 17th Cys form a disulfide bond), canine (SEQ ID NO: 11,
Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, wherein, 1st Cys and 17th Cys form a disulfide bond), and ovine (SEQ ID NO: 12,
Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, wherein, 1st Cys and 17th Cys form a disulfide bond), BNP cyclic peptides derived from these organisms also have similar effects as the cyclic peptide of the present invention. Among those mentioned above, it is particularly preferred that the amino acid sequence expressed by Formula (I) is the amino acid sequence expressed by Formula (I-a). In another embodiment of the invention, the cyclic peptide may be expressed by SEQ ID NOs: 16-75.
The cyclic peptide of the present invention encompasses a derivatized form of said cyclic peptide. Such a derivative can be used as such, as an active substance, or also used as a prodrug.
A derivative of the present invention can be obtained by adding (modifying) a known substituent to certain group of an amino acid in the cyclic peptide such as, for example, a hydrogen atom, hydroxyl group, carboxy group, amino group or imino group, or replacing it with a known replaceable substituent. Modification include, but not limited to, chemical modifications such as, for example, glycosylation, acetylation, phosphorylation and lipidation, to an amino acid within the cyclic peptide.
The addition (modification) or replacement of an amino acid within the cyclic peptide can occur in one group, or can occur in more than one group at the same time. Any known substituents can be used as long as being capable of replacing above groups and it goes without saying that such substituents naturally include, for example, protecting groups such as BOC.
The cyclic peptide of the invention further includes a mutated form of said cyclic peptide. Namely, a mutant of the present invention can be obtained by deleting an amino acid in the cyclic peptide, replacing it with or adding it another amino acid. The number of amino acid to be deleted, replaced with other amino acids or added is 4 or less, more preferably 3 or less, even more preferably 2 or less, and particularly preferable 1 or less. It also goes without saying that the deletion, replacement or addition of amino acids in the cyclic peptide may occur concurrently and independently to each other.
An amino acid that is capable of being replaced with another amino acid can be, in the case of human BNP (Formula (I-a)), exemplified as follows, without being limited thereto. The third amino acid from left, Gly, may be replaced with either Val, Ala, Ser or Thr. The forth amino acid from left, Arg, may be replaced with either Gln or His. The fifth amino acid from left, Lys, may be replaced with Arg. The sixth amino acid from left, amino acid, Met, may be replaced with either Leu or Ile. The ninth amino acid from left, Ile, may be replaced with Val. The twelfth amino acid from left, Ser, may be replaced with either Gln, Val, Ala, Thr, Leu, Ile or Met. The thirteenth amino acid from left, Ser, may be replaced with either Val, Ala or Thr. The fourteenth amino acid from left, Gly, may be replaced with Arg. The fifteenth amino acid from left, Leu, may be replaced with either Met, Ile, Val or Ala. The sixteenth amino acid from left, Gly, may be replaced with either Ser or Ala. Table 32 summarizes examples of replaceable amino acids in the cyclic peptide of the invention, though the invention will not be limited thereto.
Cyclic peptides in which one amino acid has been replaced include such as, for example, SEQ ID NOs: 16-44, though the invention will not be limited thereto. Cyclic peptides in which two amino acids have been replaced include such as, for example, SEQ ID NO: 45-58, though the invention will not be limited thereto. Yet cyclic peptides in which three amino acids have been replaced include such as, for example, SEQ ID NOs: 59-70, though the invention will not be limited thereto. Furthermore, cyclic peptides in which four amino acids have been replaced include such as, for example, SEQ ID NOs: 71-75, though the invention will not be limited thereto. It also goes without saying that five or more amino acids can be replaced by appropriately combining the aforementioned amino acid.
Deletion of one to several amino acids can take place in similar manner to the replacement of the aforementioned amino acids with other amino acids.
The number of amino acid which are to be deleted or replaced with other amino acids or added may be determined such that the cyclic peptide will have 80% homology, preferably 90% homology to the cyclic peptide of the invention.
In addition, the present invention may be a mutant as described above, and a derivative thereof. As long as it retains the effect of the invention, any mutant or a derivative thereof is encompassed in the cyclic peptide according to the invention. In another embodiment, it has at least a BNP activity. For purpose of improving the activity of the cyclic peptide of the invention, prolonging the effect of the invention, and/or increasing storage stability of the cyclic peptide of the invention, the cyclic peptide of the invention or the amino acids constituting said peptide may be altered in an appropriate manner. For example, an amino acid in the cyclic peptide of the invention may be chemically modified, some amino acids constituting the cyclic peptide may be deleted or replaced with other amino acids, and/or new amino acids may be added.
For example, the C-terminal group —COOH of one Cys of the cyclic peptide may be replaced with —COOR 1 , —CONHR 1 or —CONR 12 , and/or the N-terminal group NH 2 of the other Cys of the cyclic peptide may be replaced with —NHC(O))R 1 or —N(C(O)R 1 ) 2 . Here, each appearance of R 1 is independently a branched or straight hydrocarbon group or an alkylene glycol chain or sugar chain having 1 to 20 carbon atoms. The number of carbon atoms in R 1 is preferably 1 to 10, more preferably 1 to 5, yet more preferably 1 to 2.
Pharmaceutically acceptable salts include, without being particularly limited, as the cyclic peptide of the invention or a derivative, such as for example, a salt with an inorganic base, a salt formed with an organic base, a salt formed with an inorganic acid, a salt formed with an organic acid, a salt formed with a basic or acidic amino acid. Examples of suitable salt formed with inorganic base include such as, for example, an alkaline metal salt such as a sodium salt and potassium salt; an alkaline earth meal salt such as a calcium salt and magnesium salt; and an aluminum salt and ammonium salt. Examples of suitable salt formed with organic base include such as, for example, a salt with an alkyl amine such as trimethyl amine or triethyl amine; a salt formed with a heterocyclic amine such as pyridine and picoline; a salt formed with an alkanol amine such as ethanol amine, diethanol amine and triethanol amine; a salt formed with a cycloalkyl amine such as cyclohexyl amine and dicyclohexyl amine; a salt formed with an alkylene diamine derivative such as N,N′-dibenzylethylenediamine. Examples of suitable salt formed with inorganic acid include such as, for example, a salt formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid or phosphoric acid. Examples of suitable salt formed with organic acid include such as, for example, a salt formed with a monocarboxylic acid such as formic acid, acetic acid, trifluoroacetic acid and propionic acid; a salt formed with a polyvalent carboxylic acid such as fumaric acid, oxalic acid and maleic acid; a salt formed with a oxycarboxylic acid such as tartaric acid, citric acid, succinic acid and malic acid; a salt formed with a sulfonic acid such as methane sulfonic acid, benzezne sulfonic acid and p-toluene sulfonic acid; and a salt formed with benzoic acid. Examples of suitable salt formed with a neutral amino acid include such as, for example, a salt formed with glycine, valine or leucine; examples of suitable salt formed with a basic amino acid include such as, for example, a salt formed with arginine, lysine or ornithine; and examples of suitable salt formed with an acidic amino acid include such as, for example, a salt formed with aspartic acid acid or glutamic acid.
Among those mentioned above, a cyclic peptide composed of the amino acid sequence expressed by Formula (I) or a pharmaceutically acceptable salt is preferred. Namely, it is preferred that the amino acid sequence expressed by Formula (I) is not replaced.
A method for producing the cyclic peptide of the present invention, a derivative and pharmaceutically acceptable salt thereof may employ, without being particularly limited, for example, any known chemical synthetic or genetic engineering methods.
When the amino acid sequence as above is chemically synthesized, it may be synthesized by any chemical synthetic method, or any known method for peptide synthesis, for example, or solid-phase or liquid-phase synthetic method. Moreover, any commercial synthesizer (e.g., SHIMADZU Corporation: PSSM-8) may be used for synthesis.
A disulfide bond can be formed in the amino acid sequence, for example, by DMSO oxidation method or iodine oxidation method without being particularly limited. In this case, an intramolecular disulfide bond can be formed by treating a free sulfhydryl group or a sulfhydryl group protected by a protecting group with either DMSO or iodine (I2) to result in said cyclic peptide.
A protecting group includes such as, for example, 4-methylbenzyl group (Bzl (4Me)), trityl group (Trt), tert-butyl group, N-(acetyl) aminomethyl group (Acm). Deprotection can be carried out through appropriate treatment corresponding to these protecting group, for example, for 4-methylbenzyl group by treating with a strong acid, and for N-(acetyl) aminomethyl group by treating with iodine.
Next, if necessary, the cyclic peptide is derivatized to result in a derivative. Derivatization can be carried out by known method. Alternatively, a derivative of the cyclic peptide can be produced at the time of peptide synthesis by introducing in advance a substituent into the amino acid constituting the cyclic peptide.
Then, if necessary, a pharmaceutically acceptable salt is produced by ion exchange of the cyclic peptide or derivative thereof. Ion exchange can be carried out, for example, by bringing into the contact the cyclic peptide or derivative thereof with a desired acid or base.
On the other hand, when the peptide is synthesized by genetic engineering, it can be synthesized by any known method such as, for example, methods described in Sambrook J. et al., Molecular Cloning, A Laboratory Manual (4th edition) (Cold Spring Harbor Laboratory Press (2012)). For instance, a DNA fragment coding for the amino acid sequence expressed by Formula (I) is prepared at first. Preparation of the DNA fragment can be performed, for example, if it is a case in which the DNA fragment codes for the amino acid sequence expressed by Formula (I-a), by amplifying the DNA fragment by PCR using a vector comprising a full-length human BNP gene as template and primers designed to synthesize a defined DNA region. Alternatively, a DNA fragment can be chemically synthesized.
Then, the amplified DNA fragment is ligated into an appropriate vector to obtain a recombinant vector for protein expression. Next, the vector for protein expression is allowed to be taken up by target cells and the transformed cells are selected. Finally, the protein produced by the cells (i.e., the protein composed of the amino acid sequence expressed by Formula (I)) is collected.
Formation of disulfide bond, derivatization and salt formation in the collected protein can be carried out as described above.
The identification of the compound of interest such as a cyclic peptide can be confirmed by known procedures such as, for example, reverse-phase HPLC or mass spectroscopy.
The presence or absence of BNP activity in the obtained compound can readily be confirmed by known means. For instance, it can be confirmed by examining cGMP producing activity in NPR-A receptor-expressing cells.
2. External Preparation
Secondly, the external preparation of the present invention is explained.
The external preparation of the present invention comprises one or more cyclic peptide and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof as described above. If two or more cyclic peptides are used, the number of different cyclic peptide to be mixed is not limited, though 2 to 3 are preferred in terms of preparation cost and convenience. It includes SEQ ID NO: 19, 29, 33, 36, 39 and 43, for example, but the invention is not limited to these and these cyclic peptide can appropriately be combined as long as it does not interfere the effect of the invention.
2.1 Application
The external preparation of the present invention can be used for following uses, for example, without being particularly limited, and in each case exerts remarkable effect which had not previously been achieved. The external preparation of the present invention is not limited to any particular use, and can be used as a pharmaceutical product, a quasi-drug and/or a cosmetic product depending on its drug efficacy and effect.
Hereinbelow, the invention will be described in detail for each of its use.
(1) Therapeutic/Prophylactic for Dermatitis
The external preparation of the present invention can be a a therapeutic and/or prophylactic for dermatitis.
The external preparation of the present invention can, upon being applied onto the subject's skin or mucosa suffering dermatitis, quickly relieve or eliminate various perceptible symptoms and conditions that are or can be caused by dermatitis such as pruritus, soreness (pain), hot sensation, tautness, erythema, infiltration, papule, lichenification, crust, exudation or skin desiccation, improving the symptoms of dermatitis. In addition, the external preparation of the present invention does not cause any irritation at the site of application.
Furthermore, such action of the external preparation comprising the cyclic peptide of the invention is effected more quickly and for prolonged time period as compared to that of an external preparation comprising BNP having the tail part. Although reasons for these advantageous effects of the cyclic peptide over such BNP are not clear, the cyclic peptide of the invention is considered to have an advantageous structure for binding to its receptor (NPR-A receptor) over BNP, thereby enabling faster binding to the NPR-A receptor in the vicinity of the affected site and at the same time prolonging its binding time. Moreover, it is considered that its relatively quick binding to the NPR-A receptor near the affected site can prevents the cyclic peptide from diffusing out of the affected site via bloodstream, etc., enabling it to stay around the affected site for relatively long time.
The external preparation of the present invention doese not only suppress or prevent inflammation in dermatitis but also acts to restore and/or retain the barrier function of skin. Here, skin's barrier function works for protecting skin against entry of stimuli and saprophytes from external environment or for retaining moisture. The external preparation restores the barrier function, and thus able to prevent the progress or exacerbation of inflammation. The barrier function of skin is greatly affected by the alignment state of corneocytes in the corneum, i.e., state of skin texture and moisturization. The external preparation of the present invention acts to improve skin texture and to moisturize skin. Therefore, its effects of restoring and maintaining skin barrier function are also obvious.
The external preparation of the present invention is also effective on skin symptoms commonly called acne, i.e., such as comedones, red papule, pustula, cysts/tuberosity.
Moreover, a quick relief or elimination of perceptible symptoms or conditions will reduce the burden of the subject (patient) and improve the QOL of the patient, while preventing the patient from being bothered by itch or soreness and from acting to damage the affected site, for example, touching or scratching. This effect of the external preparation of the present invention to relieve or eliminate perceptible symptoms or conditions is, in general, exhibited within 10 minutes, preferably within 5 minutes, more preferably within 3 minutes after application.
In addition, the external preparation of the present invention can treat dermatitis in which steroid dermatosis has been developed, or dermatitis that is intractable by other common drugs for dermatitis therapy such as steroid and tacrolimus, for example dermatitis for which a sufficient therapeutic effect cannot be achieved by these formulations, or dermatitis which is resistant to these formulations, or dermatitis for which the use of these formulations is not suitable or desirable. Conventional widely-used steroid external preparations have significant problems upon terminating application that the severity returns to the pre-application level and that a rebound phenomenon may occur and exacerbate the condition. The external preparation of the present invention has no such problems as a rebound phenomenon.
Dermatitis is in general a disease which causes inflammation in skin or mucosa. Dermatitis is normally accompanied with one or more symptoms selected from acute eczema symptoms such as erythema, infiltrative erythema, papule, vesicle, pustula, infiltration, incrustation and desquamation; chronic eczema symptoms such as lichenification and pigmentation; and scales, crust (scab) attachment, scratching, scratch scar, prurigo nodularis, erosion, edema, oozing and squamatization.
Dermatitis herein is not particularly limited as long as it is a disease which is accompanied with inflammation in skin or mucosa and includes such as, for example, eczema and atopic dermatitis such as chronic eczema, dyshidrotic eczema, infantile xerotic eczema; contact dermatitis such as allergic contact dermatitis and primary irritant contact dermatitis; seborrheic dermatitis; asteatotic dermatitis; autosensitization dermatitis; stasis dermatitis; urticaria such as allergic urticaria (e.g., alimentary urticaria and drug-induced urticaria) and nonallergic urticaria (e.g., physical urticaria, solar urticaria and cholinergic urticaria); insect bite; drug eruption; psoriasis such as plaque psoriasis, guttate psoriasis, erythrodermic psoriasis, pustular psoriasis and psoriasis arthropathica; prurigo such as chronic prurigo, acute prurigo, gestational prurigo and nodular prurigo; rosacea; rosacea-like dermatitis; cutaneous vasculitis such as cutaneous allergic vasculitis; cutaneous pruritus such as systemic cutaneous pruritus, localized cutaneous pruritus, senile cutaneous pruritus and gestational pruritus; solar dermatitis; erythrosis; nummular dermatitis; localized scratching dermatitis; perioral dermatitis; pompholyx; keratosis pilaris; lichen planus, dyshidrotic eczema, dyshidrosis, miliaria and acne vulgarisacne vulgaris. The external preparation of the present invention can be applied to the therapy and prophylaxis of any of these diseases.
The external preparation of the present invention exerts an excellent effect especially on eczema, atopic dermatitis, contact dermatitis, seborrheic dermatitis, insect bite, allergic or nonallergic urticaria and psoriasis, preferably on eczema, atopic dermatitis, contact dermatitis and insect bite, more preferably on eczema and atopic dermatitis. Therefore, the external preparation of the present invention can be used for the purpose of the therapy and prophylaxis of at least one or more dermatitis selected from above-mentioned group.
(2) Ingredients of Cosmetic or Skin-Care Product
The external preparation of the present invention may be an ingredient for a cosmetic or skin-care product.
The external preparation of the present invention will, upon being applied to skin and/or mucosa, provide moisture to the applied site and exert moisturizing effect, preventing skin from dryness and roughness. In cases where corneum is present at the site of application, it exerts an effect of improving skin texture there. It also provides and retains moderate elasticity and flexibility of skin and mucosa, softens skin, gives skin and mucosa resilience and firmness. Moreover, it improves wrinkles (including fine and large wrinkles) and flabbiness at the applied site, and further diminishes the appearance of dullness and spots. By diminishing dullness and spots away, it consequently provides a skin-lightening effect.
As a result, the external preparation of the present invention can be used for the purpose of maintaining or improving skin and/or mucosal condition, without being particularly limited, for example, for obtaining at least one effects selected from a group consisting of preventing or improving dry skin, skin roughness, sensitive skin, roughened lips and appearance of large and fine wrinkles, maintaining skin or mucosal condition, anti-aging, and skin-lightening, or obtaining for at least one effects selected from the above effects.
Skin roughness herein includes miliaria, chilblain, cracked skin, chapped skin, acne, diaper rashes, festering, chafed inner thighs and razor burns.
Specific utilities of the external preparation of the present invention as an ingredient for the cosmetic or skin-care product includes, without being particularly limited, for example, cosmetics for make-up such as face lotion, emulsion, serum, cream, cold cream, gel, mask, pack, powder, hand soap, perfume, deodorant, as well as foundation, face powder, eye shadow, eyeliner, mascara, eyebrow, blush, makeup base, lip stick, lip cream and nail polish, and furthermore cosmetics for hair such as shampoo, rinse, conditioner, hair color, hair tonic, styling agent and perm chemical, body cleansers such as face wash, cleansing and body soap, skin-care products such as body powder, after-shave lotion and pre-shave lotion, and bath agents.
The above effects of the external preparation of the present invention are quickly exhibited, within 10 minutes in general, preferably within 5 minutes, more preferably within 3 minutes after application depending on the type of the effect.
The above effects of the external preparation of the present invention last for relatively long time. In general, the effect lasts for 4 hours or more, preferably, 8 hours or more, more preferably 24 hours or more after it started to effect depending on the type of the effect.
(3) Antipruritic
The external preparation of the present invention can be an antipruritic.
As mentioned above, the external preparation of the present invention is suitable for using as antipruritic because it quickly exerts an excellent antipruritic effect at the applied site when being applied to skin or mucosa.
The antipruritic effect of the external preparation of the present invention as mentioned above is exhibited within 10 minutes in general, preferably within 5 minutes, more preferably within 3 minutes after application.
(4) Alopecia Therapeutic, Alopecia Prophylactic, Hair Growing Agent and Hair Growth Stimulant
The external preparation of the present invention can also be an alopecia therapeutic, alopecia prophylactic, hair growing agent and/or hair growth stimulant.
When the external preparation of the present invention is applied to a site of hair loss or a site where hair growth is to be stimulated, it acts to prevent hair loss and promote hair growth stimulation and hair growing at the applied site. It has effects such as nourishing hair, promoting hair growth, and improving or preventing hair thinning at the applied site. In this case, hairs to be stimulated to grow tend to become terminal hairs or hairs which are not white hairs. These effects are exhibited in relatively early stage as compared to other active ingredients previously used in alopecia therapeutics e.g., BNP, and the effects obtained are significant.
The external preparation of the present invention also has an improving or preventing effect on dermatitis as mentioned above. Therefore, it can improve or prevent skin inflammation associated with alopecia. Such effects are particularly advantageous when alopecia has been exacerbated due to skin condition of the site of application (such as scalp).
Moreover, the external preparation of the present invention exerts moisturizing and skin texture-improving effects at the applied site when being applied to skin as mentioned above. It can remove and suppress dandruff and itching, while giving moisture to hair and scalp, improving and preventing dryness and keeping hair and scalp healthy. It also can improve seborrhea.
The external preparation of the present invention also acts to alleviate or eliminate the perceptible symptoms or conditions as mentioned above, thereby reducing the burden of the subject (patient) and improving QOL of the patient. Moreover, it can prevent the patient from being bothered by itch or soreness and acting to damage the affected site, for example, touching or scratching, thereby preventing exacerbation of skin condition which may induce alopecia.
When the external preparation of the present invention is used as alopecia therapeutic or prophylactic, applicable alopecia includes such as, for example, alopecia as listed below, without being particularly limited. The external preparation can be used for the purpose of treating or preventing one ore more of these alopecia.
(Acquired Alopecia)
(i) Alopecia without accompanying scarring or skin lesion (alopecia areata, male pattern alopecia, seborrheic alopecia, alopecia pityroides, female pattern alopecia, gestational alopecia, malignant alopecia, senile alopecia, alopecia totalis, alopecia areata multilocularis, ophiasis, drug-induced alopecia, cancer chemotherapy-induced alopecia and radiation exposure-induced alopecia, traumatic/mechanical alopecia, malnutrition/metabolic disorder associated alopecia, endocrine dysfunction associated alopecia and telogen effluvium (post partum alopecia, alopecia after high fever)).
(ii) Alopecia observed on skin lesion or pathologic skin (infection-induced alopecia, tumor-induced alopecia, inflammation-induced alopecia)
(iii) Scarring alopecia (skin infection-induced alopecia, alopecia induced by infiltration of inflammatory cells)
(Congenital Alopecia)
Diffuse alopecia, congenital atrichia, alopecia in hereditary syndromes, localized alopecia, phakomatosis, aplasia cutis, congenital alopecia triangularis.
Among those mentioned above, the external preparation of the present invention exerts an excellent effect particularly on acquired alopecia, preferably on alopecia without accompanying scarring or skin lesion, more preferably on alopecia areata, male pattern alopecia, seborrheic alopecia, alopecia pityroides, female pattern alopecia, gestational alopecia, malignant alopecia, senile alopecia, alopecia totalis, alopecia areata multilocularis, ophiasis, drug-induced alopecia, cancer chemotherapy-induced alopecia and radiation exposure-induced alopecia, traumatic/mechanical alopecia, malnutrition/metabolic disorder associated alopecia, endocrine dysfunction associated alopecia and telogen effluvium. Therefore, the external preparation of the present invention can be used for the purpose of treating or preventing at least one or more alopecia selected from the above-mentioned group.
(5) Rhinitis Therapeutic and/or Prophylactic
The external preparation of the present invention can also be a rhinitis therapeutic and/or prophylactic.
Rhinitis is a disease caused by mucosal inflammation in nasal cavity and/or paranasal cavity and induces main symptoms such as nasal obstruction, rhinorrhea, sudden recurrent sneezing, as well as symptoms such as pruritus. In the present invention, “rhinitis” does not only includes rhinitis in a narrow definition which are accompanied with mucosal inflammation in nasal cavity, but also includes sinusitis accompanied with mucosal inflammation in paranasal cavity.
The external preparation of the present invention can improve or prevent various symptoms associated with rhinitis such as nasal obstruction, rhinorrhea, sneezing and pruritus, when being applied to nasal cavity mucosa and/or paranasal cavity mucosa. Especially these effects are exhibited quickly and last for a prolonged time as compared to a rhinitis therapeutic comprising BNP having the tail part.
Such effects of the external preparation of the present invention on rhinitis are exhibited within 8 minutes in general, preferably within 5 minutes, more preferably within 3 minutes after its application.
Also the effect of the external preparation of the present invention on rhinitis as described above lasts for 4 hours or more in general, preferably for 8 hours or more, more preferably for 24 hours or more after it started to effect.
When the external preparation of the present invention is used as a rhinitis therapeutic and/or prophylactic, applicable rhinitis includes such as, without being particularly limited, for example, infectious rhinitis including acute rhinitis and chronic rhinitis; hypersensitive non-infectious rhinitis including combined (flower hypersensitivity) rhinitis, rhinitis with rhinorrhea, congestive rhinitis, edematous rhinitis and dry-nose type rhinitis; irritant-induced rhinitis including physical rhinitis, chemical rhinitis and radiation rhinitis; and atrophic rhinitis and idiopathic granulomatous rhinitis; and sinusitis such as acute sinusitis, chronic sinusitis (maxillary empyema), eosinophilic sinusitis and paranasal cavity mycosis, The external preparation can be used for the purpose of treating or preventing one ore more of the above.
Combined rhinitis includes, for example, allergic rhinitis including perennial allergic rhinitis and seasonal allergic rhinitis, and nonallergic rhinitis including vasomotor (essential) rhinitis and eosinophilic rhinitis.
Rhinitis with rhinorrhea includes, for example, gustatory rhinitis, cold air-induced rhinitis and senile rhinitis.
Congestive rhinitis includes, for example, drug-induced rhinitis, psychogenic rhinitis, gestational rhinitis, endocrine rhinitis and cold rhinitis.
Edematous rhinitis includes, for example, aspirin-hypersensitive rhinitis.
Among those mentioned above, the external preparation of the present invention exerts an excellent effect particularly on hypersensitive non-infectious rhinitis, irritant-induced rhinitis and sinusitis, preferably on hypersensitive non-infectious rhinitis and chronic sinusitis, more preferably on combined (flower hypersensitivity) rhinitis, rhinitis with rhinorrhea, congestive rhinitis, edematous rhinitis and dry-nose type rhinitis, chronic sinusitis. Therefore, the external preparation of the present invention can be used for the purpose of treating or preventing at least one or more rhinitis selected from the above-mentioned group.
In terms of symptoms, since the external preparation of the present invention acts as described above, it can be used for any of rhinitis with sneezing/rhinorrhea, rhinitis with nasal obstruction and rhinitis with both symptoms.
In addition, the external preparation of the present invention exhibits an effect of improving rhinitis which cannot be cured by a rhinitis therapeutic that has conventionally been used such as steroid drug.
(6) Other External Preparations
The external preparation of the present invention can also be used for an application other than those described above for the effect of the cyclic peptide as described above. In this case, the external preparation of the present invention can set an objective that is not the effects of the cyclic peptide as described above. In this case, the cyclic peptide of the invention, a derivative and/or a pharmaceutically acceptable salt thereof is used for the purpose of assisting the main effect of the external preparation or adding another effect to the main effect.
Such application include such as, without being particularly limited, for example, body powder, deodorant, depilation agent, soap, body shampoo, bath agent, hand soap, perfume, sunscreen, and antiinflammatory agent and antifungal agent.
2.2 Formulation
The external preparation of the present invention can exert the effect of its active ingredient, i.e., the cyclic peptide or a derivative and/or pharmaceutically acceptable salt thereof, certainly and quickly in the vicinity of the applied site by being locally applied to the site of the interest (e.g., affected site) on skin or mucosa.
Such external preparation can be, without being particularly limited, for example, an agent for integument, eye drop, ear drop, nasal drop, buccal agent or suppository. Among these, when the external preparation of the present invention is a dermatitis therapeutic, a dermatitis prophylactic, an antipruritic, a skin-care product, an alopecia therapeutic, an alopecia prophylactic, a hair growing agent or a hair growth stimulant, it is preferably an agent for integument. On the other hand, when the external preparation of the present invention is a rhinitis therapeutic and/or prophylactic, it is preferably a nasal drop. Furthermore, when it is a therapeutic/prophylactic of a corneal disease, it is preferably an eye drop.
When the external preparation of the present invention is an agent for integument, it can be, without being particularly limited, for example, an external solid formulation, an external liquid formulation, a spray formulation, an ointment, an emulsion, a cream, a gel formulation or a patch.
An external solid formulation is a solid formulation for applying or spraying onto such as skin Such an external solid formulation includes, for example, a powder form external formulation.
An external liquid formulation is a liquid formulation for applying onto such as skin Such an external liquid formulation includes, for example, a lotion and liniment.
A spray formulation is a formulation for spraying an active ingredient in a mist, powder, foam or paste form onto skin. Such spray formulation includes, for example, an external aerosol and a pump spray formulation.
An ointment is a semi-solid formulation which is applied to skin and comprises an active ingredient dissolved or dispersed in a base. The ointment can also be a lip cream for locally applying to lips, etc.
A cream is a semi-solid formulation which is applied to skin and emulsified as either oil-in-water or water-in-oil type.
A gel formulation is a gelled formulation which is applied to skin. The gel formulation includes, for example, aqueous gel formulation and oil-based gel formulation.
A patch is a formulation which is attached to skin. The patch includes, for example, a tape or plaster.
A nasal drop is a formulation which is administered to nasal cavity or nasal mucosa. The nasal drop includes, for example, nasal powder formulation and a nasal drop. Among these, nasal drop is preferred.
In any formulation described above, the external preparation of the present invention comprises the cyclic peptide of the present invention and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof as described above.
When the formulation is an external liquid formulation, an ointment, a cream or a gel formulation, the external preparation of the present invention comprises the cyclic peptide and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof at a concentration of, for example, 0.0001 to 1000000 μg/g, preferably 0.001 to 10000 μg/g, more preferably 0.01 to 1000 μg/g, yet more preferably 0.1 to 100 μg/g. In another embodiment, it may comprises the cyclic peptide at a concentration of 1 to 800 μg/g or 3 to 500 μg/g.
When the formulation is a spray formulation, the external preparation of the present invention comprises in the stock solution of the spray formulation the cyclic peptide and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof at a concentration of, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, 0.01 to 1000 μg/mL, more preferably 0.1 to 100 μg/mL, yet more preferably 1 to 100 μg/mL. In another embodiment, it may comprises the cyclic peptide at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.
When the formulation is a patch, the external preparation of the present invention comprises the cyclic peptide and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof at a concentration of, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, it may comprises the cyclic peptide at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.
When the formulation is a nasal drop, especially a liquid nasal drop, the external preparation of the present invention comprises in the nasal drop solution (nasal drop) the cyclic peptide and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof at a concentration of, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, it may comprises the cyclic peptide at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.
The external preparation of the present invention can be formulated, for any formulation as described above, by using methods and constituent materials known to those skilled in the art.
Available constituent materials include such as, without being particularly limited, for example, a gelator, oily ingredient, higher alcohol, fatty acid, ultraviolet absorbing agent, ultraviolet scattering agent, powder, pigment, surfactant, polyhydric alcohol/sugar, polymer, bioactive ingredient, solvent, antioxidant, flavor and antiseptic agent.
Various organic or inorganic gelator compounds can be used.
An inorganic gelator compound includes such as, for example, hydrous or water-absorbable silicate, for example, aluminum silicate (e.g., bentonite), magnesium-aluminum silicate and colloidal silica.
As an organic gelator compound, a natural, semisynthetic or synthetic polymer can be used. natural and semisynthetic polymers include such as, for example, polysaccharides such as cellulose, starch, tragacanth, gum arabic, xanthane gum, agar, gelatin, alginic acid and a salt thereof (e.g., sodium alginate and a derivative thereof), lower alkylcellulose (e.g., methylcellulose or ethylcellulose), carboxy- or hydroxy-lower alkylcellulose (e.g., carboxymethylcellulose or hydroxypropylcellulose). A synthetic polymer includes such as, for example, carboxylvinyl polymer, sodium polyacrylate, (vinylmethyl ether/ethyl maleate) copolymer, polymethacrylate, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylate or polymethacrylate. Alternatively, as a gel formulation, commercially available gelators such as, for example, Lubrajel® NP, Lubrajel® CG, Lubrajel® DV, Lubrajel® MS, Lubrajel® OIL, Lubrajel® TW, Lubrajel® DS (Ashland Inc.) can also be used.
As an oily ingredient, for example, various ester, ether, hydrocarbon, silicone and fluorine oil phase ingredient, as well as animal and plant oils and a hardened oil thereof, and waxes of natural origin.
The ester oil phase ingredients include such as, for example, glyceryl tri(2-ethyl hexanoate), cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, butyl stearate, butyl myristate, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldocecyl myristate, isocetyl isostearate, diethyl sebacate, diisopropyl adipate, isoalkyl neopentanoate, glyceryl tri(capryl-caprinate), trimethylol propane tri(2-ethylhexanoate), trimethylol propane triisostearate, pentaerythritol tetra(2-ethylhexaonate), cetyl caprylate, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinoleate, isocetyl myristate, isostearyl myristate, isocetyl palmitate, isostearyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, cetostearyl 2-ethylhexanoate, stearyl 2-ethylhexanoate, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicaprinate, propylene glycol di(capryl-caprinate), propylene glycol dicaprylate, neopentyl glycol dicaprinate, neopentyl glycol dioctanoate, glyceryl tricaprylate, glyceryl triundecanoate, glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate, isostearyl octanoate, octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isostearyl isostearate, octyldecyl isostearate, polyglycerol oleic acid ester, polyglycerol isostearic acid ester, dipropyl carbonate, dialkyl (C12-18) carbonate, triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate, acetyltriethyl citrate, acetyltributyl citrate, trioctyl citrate, diisostearyl malate, 2-ethylhexyl hydroxystearate, di(2-ethylhexyl) succinate, diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, cholesteryl stearate, cholesteryl isostearate, cholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate, phytosteryl isostearate, phytosteryl oleate, isocetyl 12-stearoylhydroxystearate, stearyl 12-stearoylhydroxystearate and isostearyl 12-stearoylhydroxystearate.
Hydrocarbon oil phase ingredients include such as, for example, squalane, liquid paraffin, α-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, solid paraffin, polybutene, microcrystalline wax and vaseline.
Silicone oil phase ingredients include such as, for example, dimethylpolysiloxane, methylphenylpolysiloxane, methylcyclopolysiloxane, octamethylpolysiloxane, dacamethylpolysiloxane, dodecamethylcyclosiloxane, methyl hydrogen polysiloxane, polyether-denatured organo(polysiloxane), dimethylsiloxane-methylcetyloxysiloxane copolymer, dimethylsiloxane/methylstearoxysiloxane copolymer, alkyl-denatured organo(polysiloxane), terminal-denatured organo(polysiloxane), amino-denatured silicone oil, amino-denatured organo(polysiloxane), dimethiconol, silicone gel, acryl silicone, trimethyl siloxysilicate, silicone RTV gum.
Fluorine oil phase ingredients include such as, for example, perfluoropolyether, fluorine-denatured organo(polysiloxane), pitch fluoride, fluorocarbon, fluoroalcohol and fluoroalkyl-polyoxyalkylene co-denatured organo(polysiloxane).
Animal and plant oils and a hardened oil thereof, and waxes of natural origin include such as, for example, animal and plant oils and the hardened oil thereof such as beef tallow, hardened beef tallow, lard, hardened lard, horse oil, mink oil, orange roughy oil, fish oil, hardened fish oil, egg yolk, jojoba oil; plant oils and a hardened oil thereof such as avocado oil, almond oil, olive oil, cacao butter, apricot kernel oil, kukui nut oil, sesame oil, wheat germ oil, rice germ oil, rice bran oil, safflower oil, shea butter, soybean oil, evening primrose oil, camellia oil, corn oil, rapeseed oil, hardened rapeseed oil, palm kernel oil, hardened palm kernel oil, palm oil, hardened palm oil, peanut oil, hardened peanut oil, castor oil, hardened castor oil, sunflower oil, grape seed oil, jojoba oil, hardened jojoba oil, macadamia nut oil, meadowfoam oil, cottonseed oil, hardened cottonseed oil, palm oil, hardened palm oil, rose hip oil; waxes such as beeswax, beeswax having high acid value, lanolin, reduced lanolin, hardened lanolin, liquid lanolin, carnauba wax and montan wax.
Higher alcohols include such as, for example, lauryl alcohol, myristyl alcohol, cetyl alcohol, stearyl alcohol, isostearyl alcohol, oleyl alcohol, behenyl alcohol, 2-ethyl hexanol, hexadecyl alcohol, octyl dodecanol.
Fatty acids include such as, for example, caprylic acid, capric acid, undecylenic acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, linolenic acid, arachic acid, arachidonic acid, behenic acid, erucic acid, 2-ethyl hexanoic acid.
Ultraviolet absorbing agents include such as, for example, paraminobenzoate, amyl paraminobenzoate, ethyl dihydroxypropyl paraminobenzoate, glyceryl paraminobenzoate, ethyl paraminobenzoate, octyl paraminobenzoate, octyl dimethyl paraminobenzoate, ethylene glycol salicylate, octyl salicylate, triethanol aminesalicylate, phenyl salicylate, butyl phenyl salicylate, benzyl salicylate, menthyl salicylate, benzyl cinnamate, octyl paramethoxy cinnamate, 2-ethylhexyl paramethoxy cinnamate, glyceryl diparamethoxy cinnamate mono(2-ethylhexanoate), isopropyl paramethoxy cinnamate, paramethoxyhydrocinnamate diethanol amine salt, diisopropyl-disopropyl cinnamate ester mixture, urocanate, urocanate ethyl, hydroxymethoxy benzophenone, hydroxymethoxy benzophenone sulfonate and a salt thereof, dihydroxymethoxybenzophenone, sodium dihydroxymethoxybenzophenone sulfonate, dihydroxybenzophenone, dihydroxy dimethoxybenzophenone, hydroxyoctoxybenzophenone, tetrahydroxybenzophenone, butyl methoxy-dibenzoyl methan, 2,4,6-trianilino-p-(carbo-2-ethylhexyl-1-oxy)-1,3,5-triazine, 2-(2-hydroxy-5-methylphenyl) benzotriazole, methyl-O-aminobenzoate, 2-ethylhexyl-2-cyano-3,3-diphenyl acrylate, phenylbenzimidazole sulfate, 3-(4-methyl benzylidene) camphor, isopropyl dibenzoylmethan, 4-(3,4-dimethoxyphenylmethylene)-2,5-dioxo-1-imidazolidine propionate 2-ethylhexyl, and polymer derivatives or silane derivative thereof, titanium oxide and zinc oxide and dispersion thereof. The zinc oxide and titanium oxide may be surface-treated.
Dermal absorption auxiliary agents include such as, for example, acetic acid, sodium acetate, limonene, menthol, salicylic acid, hyaluronic acid, oleic acid, N,N-diethyl-m-toluamide (N,N-diethyl-3-methyl benzamide), n-butyl stearate, benzyl alcohol, isopropyl myristate, isopropyl palmitate, polypropylene glycol, crotamiton, diethyl sebacate, N-methylpyrrolidone, N-ethylpyrrolidone and lauryl alcohol.
Powders and pigments include such as, for example, pigments such as Pigment Red 104, Pigment Red 201, Pigment Yellow 4, Pigment Blue 1, Pigment Black 401, basic dyes, HC colors, disperse dyes, direct colors, lake pigments such as Pigment Yellow 4 AL lake, Pigment Yellow 203 BA lake; polymers such as nylon powder, silk powder, urethane powder, silicone powder, methyl polymethacrylate powder, cellulose powder, starch, silicone elastomer spherical powder and polyethylene powder; colored pigments such as yellow iron oxide, red iron oxide, black iron oxide, chromic oxide, carbon black, ultramarine, iron blue; white pigments such zinc oxide, titanium oxide, cerium oxide; extender pigment such as tare, mica, sericite, kaolin and tabular barium sulfate; pearl pigment such as titanium mica; metal salt such as barium sulfate, calcium carbonate, magnesium carbonate, aluminum silicate and magnesium silicate; inorganic powder such as silica, alumina; metal soap such as aluminum stearate, magnesium stearate, zinc palmitate, zinc myristate, magnesium myristate, zinc laurate, zinc undecylenate; bentonite, smectite, boron nitride, etc. The shape (spherical, rod-like, needle-like, tabular, amorphous, scaly, spindle-shaped) and particle diameter of these powders are not particularly limited.
These powders and pigments may be pre-treated by known conventional surface processing such as, for example, fluorine compound processing, silicone processing, silicone resin processing, pendant processing, processing with silane coupling agent, processing with titanium coupling agent, oil solution processing, N-acylated lysine processing, polyacrylic acid processing, metal soap processing, amino acid processing, lecithin processing, inorganic compound processing, plasma processing and mechanochemical processing.
As the surfactant, any of an anionic surfactant, a cationic surfactant, ampholytic surfactant and non-ionic surfactant can be used as appropriate.
Anionic surfactants include such as, for example, fatty acid soap, α-acylsulfonate, alkylsulfonate, alkylallylsulfonate, alkylnaphthalenesulfonate, alkylsulfate, POE alkylethersulfate, alkylamide sulfate, alkylphosphate, POE alkylphosphate, alkylamidephosphate, alkyloylalkyl taurine salt, N-acylamino acid salt, POE alkylether carboxylate, alkylsulfosuccinate, sodium alkylsulfoacetate, acylisethionate, acylated hydrolyzed collagen peptide salt and perfluoroalkyl phosphate ester.
Cationic surfactants include such as, for example, alkyltrimethylammonium chloride, stearyltrimethylammonium chloride, stearyltrimethylammonium bromide, cetostearyltrimethylammonium chloride, distearyldimethylammonium chloride, stearyldimethylbenzylammonium chloride, behenyltrimethylammonium bromide, benzalkonium chloride, propyldimethylhydroxypropylammonium behenic amide chloride, diethylaminoethyl-stearamide, dimethylaminopropyl-stearamide and lanolin derivative quaternary ammonium salt. Cationic surfactants also include tertiary amines such as fatty acid amide dialkyl amine and salts thereof.
Ampholytic surfactants include various ampholytic surfactants, for example, those of carboxybetain type, amidebetain type, sulfobetain type, hydroxysulfobetain type, amidesulfobetain type, phoshobetain type, aminocarboxylate type, imidazoline derivative type and amideamine type.
Non-ionic surfactants include such as, for example, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, POE sorbitan fatty acid ester, POE sorbitol fatty acid ester, POE glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hardened castor oil, POE castor oil, POE •POP copolymer, POE •POP alkyl ether, polyether-denatured silicone alkanolamide laurate, alkylamine oxide, hydrogenated soybean phospholipid, hydrogenized soybean phospholipid, polymer surfactant and biosurfactant.
Natural surfactants may also be used, including such as, for example, lecithin, saponin and saccharide surfactant.
Polyhydric alcohols and sugars include such as, for example, ethylene glycol, diethylene glycol, polyethylene glycol, propylene glycol, dipropylene glycol, polypropylene glycol, glycerin, diglycerin, polyglycerin, 3-methyl-1,3-butanediol, 1,3-butylene glycol, sorbitol, mannitol, raffinose, erythritol, glucose, sucrose, fructose, xylitol, lactose, maltose, maltitol, trehalose, alkylated trehalose, mixed isomerized sugar, sulfated trehalose and pullulan. Chemically modificated forms of these can also be used.
Polymers include such as, for example, anionic polymer compounds such as acrylate ester/methacrylate ester copolymer, vinyl acetate/crotonate copolymer, vinyl acetate/crotonate/vinyl neodecanoate copolymer, methylvinyl ether maleate half ester, t-butyl acrylate/ethyl acrylate/methacrylate copolymer, vinylpyrrolidone/vinyl acetate/vinyl propionate copolymer, vinyl acetate/crotonatecopolymer (RUBISET CA: BASF), vinyl acetate/crotonate/vinylpyrrolidone copolymer, vinylpyrrolidone/acrylate copolymer, acrylate/acrylamide copolymer, vinyl acetate/butyl maleate/isoisobornyl acrylate copolymer, carboxyvinyl polymer, acrylate/methacrylate alkyl copolymer, and ampholytic acetate of dialkyl aminoethyl methacrylate polymer, ampholytic polymer compound such as octyl acrylamide acrylate/hydroxypropyl acrylate/butyl methacrylateaminoethyl copolymer, quaternized compound of vinylpyrrolidone/dimethylaminoethyl methacrylate, cationic polymer compounds such as methylvinyl imidazolium chloride/vinylpyrrolidone copolymer, non-ionic polymer compounds suchs as polyvinylpyrrolidone, vinylpyrrolidone/vinyl acetate copolymer, vinylpyrrolidone/dimethylaminoethyl methacrylate copolymer, vinylcaprolactam/vinylpyrrolidone/dimethylaminoethyl methacrylate copolymer. Polymer compounds of natural origin such as cellulose or derivatives thereof, keratin and collagen or derivatives thereof, calcium alginate, pullulan, agar, gelatin, tamarind seed polysaccharides, xanthan gum, carrageenan, high-methoxyl pectin, low methoxyl pectin, guar gum, gum arabic, crystalline cellulose, arabino galactan, can be karaya, gum tragacanth, alginates, albumin, casein, curdlan, gellan gum and dextran, and glucooligosaccharide, fucose containing polysaccharide, rhamnose containing polysaccharide can also suitably be combined.
Bioactive ingredients include substances which provide skin with some bioactivity when being applied to skin Bioactive ingredients include those with such as, for example, skin-lightening, anti-inflammatory, anti-aging, ultraviolet protection, slimming, firming, antioxidation, hair growth stimulating/hair growing, suppressing hair growth, moisturizing, promoting circulation, antimicrobial/sterilization, cool/warm feeling, promoting wound cure, alleviating irritation, analgesic and cell-activating effects. Bioactive ingredients include such as plant extracts, seaweed extracts, vitamins and derivatives thereof, amino acids, various peptides other than the cyclic peptide, biopolymers such as sodium hyaluronate and mucopolysaccharide, intercellular lipid constituents such as ceramide, phytosphingosine, cholesterol and phytosterol, and analogues thereof, and enzymatic ingredients.
Examples of suitable combining ingredient include such as, for example, Angelica keiskei extract, avocado extract, sweet Hydrangea leaf extract, althea extract, arnica extract, aloe extract, apricot extract, apricot kernel extract, isoflavones, ginkgo extract, fennel extract, turmeric extract, oolong tea extract, Rose Fruit extract, echinacea extract, Baikal skullcup extract, Amur Corktree extract, Coptis japonica extract, barley extract, Hypericum erectum extract, Lamium album extract, cress extract, orange extract, cacao extract, desiccated sea water, seaweed extract, hydrolyzed elastin, hydrolyzed wheat powder, hydrolyzed silk, pumpkin seed extract, chamomile extract, carrot extract, Artemisia capillaris extract, licorice extract, hibiscus extract, Pyracantha fortuneana extract, kiwi extract, cinchona extract, cucumber extract, guanosine, gardenia extract, Sasa veitchii extract, Sophora flavescens extract, cranberry extract, walnut extract, grapefruit extract, clematis extract, chlorella extract, mulberry extract, gentian extract, tea extract, yeast extract, burdock extract, rice bran fermentation extract, rice germ oil, comfrey extract, collagen, cowberry extract, Asarum sieboldii Miq. extract, bupleurum extract, umbilical cord extract, salvia extract, soapwort extract, bamboo grass extract, crataegus extract, zanthoxylum extract, shiitake mushroom extract, Rehmannia root extract, Lithospermum root extract, Perilla frutescens extract, Tilia japonica (Miq.) extract, Filipendula m ultijuga extract, Paeonia lactiflora extract, calamus extract, white birch extract, Equisetum arvense extract, Hedera helix extract, Crataegus oxyacantha extract, European elder extract, yarrow extract, peppermint extract, sage extract, tree mallow extract, cnidium extract, extract of Swertia japonica (Schult.) Makino, soybean extract, jujube extract, soybean fermentation extract, thyme extract, tea extract, clove extract, cogon extract, orange peel extract, Oenothera tetraptera extract, Centella asiatica extract, Terminalia sericea extract, dong quai extract, common marigold extract, peach kernel extract, bitter orange peel extract, extract of Houttuynia cordata Thunb., tomato extract, natto extract, ginseng extract, garlic extract, wild rose extract, hibiscus extract, Ophiopogon japonicus extract, parsley extract, honey, banana flower extract, hamamelis extract, parietaria extract, Isodon japonicus (Burm.) Hara extract, bisabolol, loquat extract, coltsfoot extract, butterbur flower extract, Poria Sclerotium extract, butcher's-broom extract, grape extract, propolis, luffa extract, safflower extract, peppermint extract, linden extract, tree peonyextract, hop extract, pine extract, horse chestnut extract, Lysichiton camtschatcense (L.) Schott extract, Sapindus mukurossi Gaertn. extract, Melissa extract, peach extract, bluebottle extract, eucalyptus extract, Saxifraga stolonifera extract, Citrus junos extract, coix seed extract, mugwort extract, lavender extract, apple extract, lytchee (Litchi) extract, lettuce extract, lemon extract, Chinese milk vetch extract, rose extract, rosemary extract, Roman chamomile extract and royal jelly extract.
Examples also include such as biopolymers such as deoxyribonucleic acid, mucopolysaccharides, sodium hyaluronate, sodium chondroitin sulfate, collagen, elastin, chitin, chitosan and hydrolyzed eggshell membrane; moisturizing ingredients such as amino acids, hydrolyzed peptides, sodium lactate, urea, sodium carbonate pyrrolidone, betain, whey, trimethyl glycine, polypeptides such as lysine/arginine condensate etc.; intercellular lipid constituents such as sphingolipid, ceramide, phytosphingosine, cholesterol, cholesterol derivative, phytosterol derivative, phospholipid and analogues thereof; anti-inflammatory agents such as ε-aminoaminocaproic acid, glycyrrhizic acid, β-glycyrrhetinic acid, lysozyme chloride, guaiazulen, hydrocortisone, tea tree oil; vitamins such as vitamin A and derivatives thereof, vitamin B2 and derivatives thereof, vitamin B6 and derivatives thereof, vitamin C and derivatives thereof, vitamin D and derivatives thereof, vitamin E and derivatives thereof, calcium pantothenate, biotin and nicotinic acid; active agents such as allantoin, diisopropylamine dichloroacetate, 4-aminomethylcyclohexane carbonate; antioxidants such as tocopherol, carotenoids, flavonoids, tannin, lignan and saponin; cell activating agents such as α-hydroxy acid and 6-hydroxy acid; circulation accelerating agents such as γ-oryzanol and vitamin E derivative; wound curing agents such as retinol and retinol derivative; skin-lightening agents such as arbutin, kojic acid, placenta extract, sulfur, ellagic acid, linolenic acid, tranexamic acid, glutathione; hair growing agents such as cepharanthine, glycyrrhiza extract, capsicum tincture, hinokitiol, garlic extract iodide, pyridoxine hydrochloride, DL-α-tocopherol, DL-α-tocopherol acetate, nicotinic ac id, nicotinic acid derivative, calcium pantothenate, D-pantothenyl alcohol, acetyl pantothenyl ethyl ether, biotin, allantoin, isopropylmethylphenol, estradiol, ethinylestradiol, carpronium chloride, Benzalkonium chloride, diphenhydramine hydrochloride, Takanal, camphor, salicylic acid, vanillyl nonylate amide, vanillyl nonanoate amide, piroctone olamine, glyceryl pentadecanoate, L-menthol, mononitroguaiacol, resorcin, γ-aminobutyric acid, benzethonium chloride, mexiletine hydrochloride, auxin, female hormones, cantharides tincture, Cyclosporine, zinc pyrithione, hydrocortisone, minoxidil, polyoxyethylene sorbitan monostearate, peppermint oil, Sasanishiki extract.
Antioxidants include such as, for example, sodium bisulfite, sodium sulfite, erythorbic acid, sodium erythorbate, dilauryl thiodipropionate, tocopherol, tolyl biguanide, nordihydroguaiaretinoic acid, parahydroxyanisole, butyl hydroxyanisole, dibutyl hydroxytoluene, ascorbyl stearate, ascorbyl palmitate, octyl gallate, propyl gallate, carotenoids, flavonoids, tannin, lignans, saponins and plant extracts in which the antioxidative effect is recognized such as apple extract or clove extract.
Solvents include such as physiological saline, purified water, ethanol, lower alcohols, ethers, LPG, fluorocarbons, N-methylpyrrolidone, fluoroalcohols, volatile straight chain silicones and next-generation chlorofluorocarbons.
The cyclic peptide of the invention and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof can be used for preparation of the external preparation as described above, and therefore the present invention also relates to a use of the cyclic peptide of the invention and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof for preparing the external preparation.
3. Method of Using the External Preparation
Next, methods of using the external preparation of the invention is explained.
A method of using the external preparation of the invention comprises applying the external preparation of the present invention as described above to the skin and/or mucosa of a subject.
Subjects include human and vertebrates such as, without being particularly limited, for example, birds and mammals. Animals specifically include, for example, experimental animals including rodents such as mice, rats, gerbils, hamsters and guinea pigs, domestic animals such as pigs, goats, horses, sheep and minks, pet animals such as dogs and cats, primates such as human, monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees. On the other hand, human may be excluded from the subjects.
Skin and/or mucosa of a subject to which the external preparation is to be applied may be skin or mucosa at any site of the subject, and the external preparation is applicable, for example, to skin or mucosa of head (scalp), face, neck, arms, torso, arms, hands or feet, etc.
Specific method for using the external preparation of the present invention is as follows.
(1) Method of Treating and Preventing Dermatitis
When the external preparation of the present invention is used as a therapeutic/prophylactic of dermatitis, the external preparation can be applied directly to the aimed site of skin and/or mucosa (e.g., the affected site where dermatitis has been developed).
The application frequency is, without being particularly limited, for example, 1 to 10 times a day, preferably 1 to 5 times a day, yet more preferably 1 to 3 times a day. Because the external preparation of the present invention has a long lasting time, it exerts a sufficient effect even if it is applied at relatively low frequency, for example, once a day.
In terms of dosage, the total amount of the cyclic peptide of the invention and a derivative thereof and a pharmaceutically acceptable salt thereof for one application can be, without being particularly limited, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL.
In another embodiment, each dose may comprise the cyclic peptide of the invention at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.
(2) Method of Alleviating or Resolving Itch
When the external preparation of the present invention is used as an antipruritic, the external preparation can be applied directly to the aimed site of skin and/or mucosa.
The application frequency is, without being particularly limited, for example, 1 to 10 times a day, preferably 1 to 5 times a day, yet more preferably 1 to 3 times a day.
In terms of dosage, the total amount of the cyclic peptide of the invention and a derivative thereof and a pharmaceutically acceptable salt thereof for one application can be, without being particularly limited, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, each dose may comprise the cyclic peptide of the invention at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.
(3) Method of Using as a Cosmetic
When the external preparation of the present invention is used as a cosmetic, the external preparation can be applied directly to the aimed site of skin and/or mucosa.
The application frequency is, without being particularly limited, for example, 1 to 10 times a day, preferably 1 to 5 times a day, yet more preferably 1 to 3 times a day.
In terms of dosage, the total amount of the cyclic peptide of the invention and a derivative thereof and a pharmaceutically acceptable salt thereof for one application can be, without being particularly limited, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, each dose may comprise the cyclic peptide of the invention at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.
(4) Method of Treating and Preventing Alopecia, and Method of Stimulating Hair Growth and Method of Growing Hair
When the external preparation of the present invention is used as a therapeutic/prophylactic of alopecia, a hair growth stimulant or a hair growing agent, the external preparation can be applied directly to the aimed site (e.g., decalvant site of scalp or skin)
The application frequency is, without being particularly limited, for example, 1 to 10 times a day, preferably 1 to 5 times a day, yet more preferably 1 to 3 times a day.
In terms of dosage, the total amount of the cyclic peptide of the invention and a derivative thereof and a pharmaceutically acceptable salt thereof for one application can be, without being particularly limited, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, each dose may comprise the cyclic peptide of the invention at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.
(5) Method of Treating and Preventing Rhinitis
When the external preparation of the present invention is used as a therapeutic/prophylactic of rhinitis, the external preparation can be applied directly to the aimed site (e.g., nasal cavity mucosa).
The application frequency is, without being particularly limited, for example, 1 to 10 times a day, preferably 1 to 5 times a day, yet more preferably 1 to 3 times a day.
In terms of dosage, without being particularly limited, for example, for one application to each nasal cavity, the total amount of the cyclic peptide of the invention and a derivative thereof and a pharmaceutically acceptable salt thereof can be 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, each dose may comprise the cyclic peptide of the invention at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.
4. Medicament
Next, the medicament of the present invention is explained.
The medicament of the present invention comprises one or more cyclic peptides of the invention or derivatives thereof or pharmaceutically acceptable salts thereof.
As mentioned above, similarly to BNP, the cyclic peptide of the present invention or a derivative thereof or a pharmaceutically acceptable salt thereof binds to the receptor NPR-A (also known as GC-A), which has a guanylate cyclase domain, and promotes the production of cyclic guanosine monophosphate (cGMP). It is considered to have effects such as, for example, diuretic action, vasodilation, renin-aldosterone secretion suppressing action, sympatholytic activity and hypertrophy suppressing action. Also, the compounds of the invention is considered to have a superior efficacy and effect, particularly a superior immediate effect, as compared with BNP.
Therefore, the medicament of the present invention can be used for a similar object as a conventional medicament comprising BNP as an active agent.
Diseases for which the medicament of the present invention is applicable include such as, without being particularly limited, for example, the aforementioned various diseases as well as hypertension, unstable angina, acute myocardial infarction, edematous diseases, renal failure, cardiac failure, immune diseases, obesity and metabolic syndrome.
A formulation of the medicament of the present invention can be, without being particularly limited, addingly to the external preparations as mentioned above, for example, formulations for oral administration such as tablets, capsules, granules, powders, oral solution, syrup and oral jelly, formulations for oral application such as buccal tablets, buccal spray formulation, buccal semi-solid formulation and mouthwash, formulations for administration via injection such as an injection and infusion, dialysis formulation such as dialysis solution, as well as an inhalant, eye drop, ocular ointment, ear drop, vaginal tablet and vaginal suppository.
Working Examples
Hereinbelow, the present invention is further specifically illustrated by working examples. It should be noted that the present invention is not limited by these working examples.
1. Preparation of the Cyclic Peptide
Firstly, a cyclic peptide composed of the amino acid sequence expressed by Formula I-a is synthesized.
Specifically, a linear peptide consisting of 17 amino acids was formed by sequentially binding amino acids by solid-phase peptide synthesis using a peptide synthesizer. Subsequently, protecting groups at Cys1 and Cys17 were detached before treating with iodine GO to form a cysteine binding between oxidatively same amino acid residues, thereby forming a cyclic peptide.
A composition comprising the obtained cyclic peptide was purified by reverse-phase high performance liquid chromatography (reverse-phase HPLC) and then lyophilized to yield a purified cyclic peptide as white powder.
Mass spectroscopy was performed on the obtained cyclic peptide.
Conditions for HPLC are shown below:
Apparatus: Agilent 1100
Flow rate: 1.0 ml/min
Eluent A: 0.1% trifluoroacetic acid/water
Eluent B: 0.1% trifluoroacetic acid/acetonitrile
Gradient: 80% Eluent B, isocratic
Conditions for Mass spectroscopy (MS) are shown below:
Apparatus: Thermo Finnigan LCQ Advantage
Ionization method: electrospray ionization
Analytical method: ion trapping
The observed results, m/z=901.83 ([M+2H] 2+ ), m/z=1801.84 ([M+H] + ), confirmed that above peptide is in agreement with the theoretical values of the molecular weight (1802.07) and the mass number (1800.8069) calculated from the composition formula of the cyclic peptide of interest (C 72 H 120 N 24 O 24 S 3 ).
Furthermore, the purity of the above peptide was measured by HPLC using following conditions:
Column: Discovery C18, 4.6 mm×250 mm, particle diameter 5 micron
Column temperature: room temperature
Eluent A: 0.1% trifluoroacetic acid/water
Eluent B: 0.1% trifluoroacetic acid/acetonitrile
Gradient: 10 to 30% Eluent B/20 minutes
Flow rate: 1.2 ml/min
Temperature: room temperature
Injected volume: 20 μl
Detector: UV detector (detection wavelength: 215 nm)
The result of measurement confirmed that the purity of the obtained protein was 99.2%.
2. Preparation of Formulation
2.1 Preparation of Gel Formulation
Preparation of a gel-based formulation comprising a cyclic peptide composed of the amino acid sequence expressed by Formula I-a (in the working examples below, “B ring” is referred to mean a cyclic peptide expressed by such Formula I-a) (B ring gel formulation, Working Examples) and a gel-based formulation comprising human BNP (BNP gel formulation, Comparative Examples) were carried out as follows.
0.1 g of methyl-p-hydroxybenzoate (trade name: Mekkins-M, Ueno Fine Chemicals Industry, Ltd.), 0.2 g of phenoxyethanol and 3.0 g of 1,2-pentanediol were weighed into one same vessel, heated to 60 to 70° C. to make a homogenous solution, and this solution was poured into a mixer.
Next, added into the mixer 6.0 g of concentrated glycerin, and then the mixture of 0.44 g of carboxyvinyl polymer (trade name: Carbopol® 940, Lubrizol Advanced Materials Corporation) and 0.08 g of xanthane gum (trade name: KELTROL® T, CP Kelco, Inc.), and the mixuter was stirred with a paddle until they dispersed sufficiently.
Then 83.95 g of purified water was gradually added with stirring with a paddle. The mixer was heated to 70 to 80° C. while stirring with a paddle or disper until the dispersed contents were dissolved to give a solution. Subsequently, the disper was stopped and the solution was cooled immediately after confirming that the contents in the solution were dissolved. When the temperature of the mixer reached approximately 40° C., 6.0 g of Lubrajel® NP from Ashland Inc. (glycerin 2.7 g, carboxyvinyl polymer 0.06 g, sodium polyacrylate 0.018 g, water 3.222 g) was added to the solution, mixed uniformly with a paddle. Subsequently, 0.230 g of potassium hydroxide was further added to neutralize the solution, then the rotation of the paddle was stopped when the temperature of the mixer reached 25° C. to prepare a gel base.
Next, 20.1 mg of the the cyclic peptide (B ring) composed of the amino acid sequence expressed by Formula I-a was dissolved in 144 mL of physiological saline to obtain B ring solution. 0.131 mL of this solution was admixed with 10 g of the gel base obtained as described above, and the mixture was stirred uniformly to prepare a gel based formulation (B ring gel formulation) containing B ring at a concentration of about 1 μM (about 1.8 μg/g). Similarly, gel based formulations containing B ring at concentrations of about 0.3 μM (about 0.54 μg/g), about 0.5 μM (about 0.9 μg/g) and about 2.0 μM (about 3.6 μg/g) were prepared. In the working examples below, as long as it is specifically described otherwise, “B ring gel formulation” used contained B ring at a concentration of about 1 μM.
Next, 20.5 mg of human BNP-32 (American Peptide Company) was dissolved in 118 mL of physiological saline to obtain BNP solution. 0.2 mL of this solution was admixed with 10 g of the gel base obtained as described above, and the mixture was stirred uniformly to prepare a gel based formulation (BNP gel formulation) containing BNP-32 at a concentration of about 1 μM. Similarly, gel based formulations containing BNP-32 at concentrations of about 0.5 μM and 2.0 μM were prepared. In the working examples below, as long as being specifically described otherwise, “BNP gel formulation” used contained BNP at a concentration of about 1 μM.
2.2 Preparation of Nasal Drop
A nasal drop comprising the cyclic peptide (B ring) composed of the amino acid sequence expressed by Formula I-a (B ring nasal drop, Working Examples) and a nasal drop comprising human BNP (BNP nasal drop, Comparative Examples) were prepared as follows.
Firstly, the cyclic peptide (B ring) composed of the amino acid sequence expressed by Formula I-a was dissolved in physiological saline, the concentration was adjusted to prepare B ring nasal drop containing B ring at a concentration of about 1 μmol/l (about 1.8 μg/g).
Next, B ring nasal drop was filled in a quantitative nasal spray container (AS ONE Corporation) and the amount ro be sprayed for each administration was adjusted to 100 μl (0.1 ml), resulting B ring nasal drop.
Similarly, a BNP nasal drop comprising human BNP (American Peptide Company) at a concentration of 1 μmol/1 was obtained, filled in a quantitative nasal spray container (AS ONE Corporation), and the amount ro be sprayed for each administration was adjusted to 100 μl (0.1 ml), resulting BNP nasal drop.
3. Confirming Therapeutic Effect on Dermatitis
3.1 Confirming the Effect of B Ring Gel Formulation
For subjects suffering various dermatitis, B ring gel formulation as described was applied onto the affected site, and changes in symptoms before and after the application were observed. When possible, as a comparative example, BNP gel formulation as described was applied onto another affected site on the same subject where B ring gel formulation had not been applied, and changes in symptoms before and after the application were observed. For pruritus, VAS (Visual Analogue Scale) was used for evaluating each affected site on 10 stages.
The examination results are shown in Tables 1 to 7 along with age, sexuality, symptoms of the subject, and formulation given to the subject.











TABLE 1








Pruritus before





Pre-application symptoms
Post-application symptoms
and after


Case
Treatment
Disease
(severity)
(severity)
application (VAS)
Notes



D1
2 μm
atopic dermititis
Face: presented with
Face: itches removed 2 min after 1
face


Age: 30's
B ring gel

erythema, scales and skin
application. Desiccation, scales and
8 → 0


Sex: male
formulation

desiccation (moderate
erythema remitted (mild). After 3 days of
right arm



1 application

disease).
application, erythema, scales, infiltration






Limbs: presented with
and desiccation remarkably remitted
7 → 0





erythema, lichenification, skin
(insignificant).






desiccation and scratch scar
Right forearm: itches stopped to some






(severe).
extent after 1.5 min on B ring-applied







site. No itching or tingling after 2 min.







Lichenification/stiffness remitted after 4







min. Desiccation improved. Skin







moisturized and soft, with natural







appearance. No subjective discomfort







(moderate).




2 μm


Face: itches realized after 4 min,
face



BNP gel


erythema and scales more distinguished
8 → 4



formulation


than B ring side.
left arm



1 application


Left forearm: after 4 min, skin was still
7 → 4






dry. No improvement in lichenification.







Rough and stiff skin (severe).



D2
1 μm
atopic dermititis
Flushing, infiltratring
Right back: itches stopped after 3 min.
8 → 0


Age: 20's
B ring gel
erythroderma
erythema, scales, many scratch
Flushing erythema, infiltration and scales



Sex: male
formulation

scars (severe) accompanied by
remitted. Skin became soft (moderate).




1 application

strong itches causing sleep





1 μm

disruption and desire to
Left back: still itching after 3 min. no
8 → 4



BNP gel

scratch.
remission of flushing erythema,




formulation


infiltration and scales (severe).




1 application






D3
0.5 μm
atopic dermititis
Presented with infiltrating
Right back: no tingling itches 30 sec after
6 → 0


Age: 10's
B ring gel

erythema, scales, papules,
application. Skin felt normal. Erythema,



Sex: male
formulation

(moderate) accompanied by
infiltration and scales remitted after 4




1 application

tingling itches.
min (mild).




0.5 μm


Left back: still itching after 4 min, with no
6 → 6



BNP gel


remission of erythema, infiltration and




formulation


scales (moderate).




1 application

































































TABLE 2








Pruritus before





Pre-application symptoms
Post-application symptoms
and after


Case
Treatment
Disease
(severity)
(severity)
application (VAS)
Notes



D4
2 μm
atopic dermititis
Presented with infiltrating
Right back: itches relieved just after
10 → 0


Age: 20's
B ring gel

erthema, scales, many
applicaation. No itches after 2 min with



Sex: male
formutation

scratch scars with exudation
remission of erythema and infiltration




1 application

(severe) accompanied by
(moderate).






strong itches that cause sleep
Forehead: itches reduced after 1.5 min.






disruption on the back,
After 5 min skin was soft with no






lichenification, erythema,
lichenification. Erythema and scales






scales and scratch scars on
remitted. Wound quickly healed. Scratch






forehead (severe).
scars epithelized.




2 μm

Back: presented with many
Left back: still itching after 4 min with no
10 → 3



BNP gel

scratch scars with exudation,
remission of erythema and infiltration




formulation

infiltration, erythema, crusts
(severe).




1 application

(severe).
Forehead: no improvement in







lichenification, desiccation and scales,







with remaining itches (severe).



D5
1 μm
atopic dermititis
Presented with infiltrating
Right face: itches, edema, and infiltration
9 → 0


Age: 20's
B ring gel

erythema, edema and crusts
remitted/relieved just after application.



Sex: male
formulation

(severe) accompanied by
Skin felt no itch and moisturized after 1




1 application

strong tingling itches.
min (moderate).






Back: presented with many
No itches at all after 2 min. Edema and






scratch scars with exudation,
erythema remitted. Right eye became






infiltration, erythema, crusts
easy to open




1 μm

and papules (severe).
Left face: still itching after 3 min with no
9 → 4



BNP gel


remission of erythema, infiltration and




formulation


edema. Being ifficult to open left eye




1 application


(severe).



D6
1 μm
atopic dermititis
Presented with lichenification,
Right back: itches improved/relieved
8 → 0


Age: 10's
B ring gel
prurigo nodularis
prurigo nodularis, erythema,
after 3 min. Lichenification, prurigo



Sex: male
formulation

scales, papules and many
nodularis, erythema, scales, scratch




1 application

scratch scars (severe)
scars all improved as compared to left.






accompanied by strong itches.
Skin was soft and moisturized






Back: presented with many
(moderate).




1 μm

scratch scars with exudation,
Left back: still itching after 4 min with no
8 → 3



BNP gel

infiltration, erythema, crusts
remission of erythema and infiltration




formulation

and papules (severe).
(severe).




1 application


Forehead: lichenification, desiccation







and scales not improved, with itches left







(severe).



































































TABLE 3








Pruritus before







and after





Pre-application symptoms
Post-application symptoms
application


Case
Treatment
Disease
(severity)
(severity)
(VAS)
Notes



D7
0.3 μm
atopic
Presented with infiltrating
No itches after 1 min. Erythema remitted
10 → 0
intractable

Age: 20's
B ring gel
dermititis
erythema, papules, exudation
and exudation stopped after 10 min. 1

steroid-

Sex: male
formulation

and many scratch scars with
application/day for 3 days cleared off

ressitance


1 application

crust (severe) accompanied
infiltrating erythema, papules, exudation






by strong itches that cause
and scratch scars. Remarkably improved






sleep disruption.
crusts, partly left mild erythema (mild).



D8
0.5 μm
atopic
Presented with erythema,
5 days after application, erythema,
8 → 1
intractable

Sex: male
B ring
dermititis
infiltration, scales, crusts,
infiltration, scales, crusts and wound

steroid-


formulation

scratch scars and skin
quickly cured, with little scratch scars left

resistance


1 application,

desiccation (severe).
(mild). Desiccation remarkably remitted.




5 days


Skin moisturized and soft.



D9
B ring gel
atopic
left forearm presented with
Itches removed in 2 min (VAS0).
10 → 0


Age: 30's
formulation
dermititis
many scratch scars with
Erythema and exudation improved.



Sex: male
2 applications/

exudation, infiltration,
Severity of rash improved to moderate




day

erythema, papules,
disease level after 5 mmin. After 1 more




1 day

accompanied with tingling
application given on the same night,






strong itches (VAS10)
there was no itches for 3 days thereafter.






(severe).
Scratch scars epithelized.



D10
1 μm
atopic
Presented with infiltrating
Right forearm: itches stopped after 3
8 → 0


Age: 50's
B ring gel
dermititis
erythema, scales, papules and
min. Erythema and infiltration improved



Sex: female
formulation

many scratch scars (severe)
(moderate).




1 application

accompanied by inremediable





1 μm

strong itches
Left forearm: still itching after 4 min with
8 → 4



BNP gel


no remission of erythema and infiltration




formulation


(severe).




1 application






D11
1 μm
atopic
Presented with infiltrating
Right face: immediately after application
10 → 0


Age: 40's
B ring gel
dermititis
erythema, scales and many
itches and skin tautness relieved, with



Sex: female
formulation

scratch scars (severe)
erythema getting better. No itching after




1 application

accompanied by drastic itches
1 min. Erythema, infiltration and






and tautness.
desiccation remitted (moderate).




1 μm

Back: presented with many
Left face: still itching after 3 min, having
10 → 4



BNP gel

scratch scars with exudation,
erythema, infiltration and edema with




formulation

infiltration, erythema, crusts
swollen feeling (severe).




1 application

and papules (severe).

































































TABLE 4








Pruritus before







and after





Pre-application symptoms
Post-application symptoms
application


Case
Treatment
Disease
(severity)
(severity)
(VAS)
Notes



D12
1 μm
atopic
Strong tingling itches,
Right face: itches remitted after 3 min.
10 → 1


Age: 40's
B ring gel
dermititis
disrupted sleep, infiltrating
Edema, erythema and infiltration started



Sex:
formulation

erythema, scales, exudation,
to be remitted. After 30 min, anti-



female
1 application

cracks, crusts, scratch scars;
inflammatory effect observed, exudation






eyes cannot be opened due to
stopped, wound epithelization promoted,






exudate sealing eyelids
cracks almost epithelized. Edema,






(severe).
erythema and infiltration remitted to







some extent. Eyelids became openable







steadily.




1 μm


Left face: still having tingling itches after
10 → 6



BNP gel


3 min with no remission of erythema and




formulation


infiltration. Itches till left after 30 min.




1 application






D13
1 μm
atopic
Obvious infiltration, erythema
Right face: erythema started to be
8 → 0


Age: 20's
B ring gel
dermititis
and papules all over the face,
remitted and itches removed just after
(immediate


Sex:
formulation

accompanied with strong
application. Erythema and infiltration
after


female
1 application

itches and hot flash (severe).
remitted after 4 min (moderate).
application)



1 μm


Left face: itching with hot flush and
8 → 0



BNP gel


redness just after application. Itches
(after 4



formulation


removed after 4 min.
minutes)



1 application






D14
1 μm
atopic
Hand eczema of which main
Right hand: itches removed after 3 min
9 → 0
Steroid-

Age: 20's
B ring gel
dermititis
symptom is contact dermititis
(VAS0). Lichenification improved, skin

resistance;

Sex:
formulation

due to detergent and
got soft, erythema and infiltration

intractable

female
1 application

disinfectant. Presented with
remitted (moderate).

lichenification


1 μm

lichenification, erythema,
Left hand: no improvement in
9 → 4
which cannot


BNP gel

scales and scratch scars
lichenification and erythema after 10 min

be remitted


formulation

(severe). Sleep disruption by
(severe).

within this


1 application

strong itches (VAS9).


short time (3







min) by







conventional







therapy.






























































TABLE 5








Pruritus before







and after





Pre-application symptoms
Post-application symptoms
apllication


Case
Treatment
Disease
(severity)
(severity)
(VAS)
Notes



D15
1 μm
atopic
Presented with erythema,
Erythema, infiltration, scales and crusts
9 → 1


Age: 20's
B ring gel
dermititis
infiltration, scales, crusts,
wound epithelization improved 3 days



Sex:
formulation

scratch scars (severe), with
after application. Scratch scars



female
1 application/

strong itches.
significantly improved. Only mild




day


erythema and scales observed (mild).




3 days


Desiccation relieved, improved and







moisturzed skin texture. Itches and







scratch scars significantly improved.



D16
1 μm
contact
Contact dermititis due to hair
Right side: tingling itches removed after
8 → 0


Age: 60's
B ring gel
dermititis
dye with erythena, edema
1 min. Itches eliminated after 3 min



Sex:
formulation

and infiltration along hairline
(VAS0), felt better than left. Erythema



female
1 application

(moderate disease)
and infiltration remitted.






accompanied by strong
Anti-inflammatoryeffect observed.






tingling itches (VAS8)
Edema improved (mild).




1 μm


Left side: symptoms less improved than
8 → 4



BNP gel


right (moderate).




formulation







1 application






D17
1 μm
chronic
Presented with infiltrating
Right face: itches stopped just after
10 → 0


Age: 40's
B ring gel
eczema
erythema, scales,
application. Erythema remitted. No



Sex:
formulation

lichenification, crusts, papules
scales and desiccation after 2 min. Skin



female
1 application

and skin desiccation (severe)
was moisturized (moderate).






accompanied by drastic itches
Forehead: itches controlled after 1.5 min.






and tautness.
No lichenification after 5 min. Skin got







soft. Erythema and scales remitted,







scratch scars epithelized and wound quickly healed.




1 μm


Left face: still itching and taut after 4 min
10 → 5



BNP gel


with no remission of desiccation, scales,




formulation


erythema and infiltration (severe).




1 application






D18
1 μm
eczema
Presented with erythema,
After 15 min, erythema, edema,
8 → 0


Age:
B ring gel

edema, infiltration, scales and
infiltration and scales significantly



Sex:
formulation

papules (severe).
remitted and skin texture improved



female
1application


(mild).




1 μm


After 15 min, still many scales,
8 → 4



BNP gel


erythema, edema and infiltration left.




formulation


Skin texture is rough and still itching.




1 application




































































TABLE 6








Pruritus before





Pre-application symptoms
Post-applicationsymptoms
and after


Case
Treatment
Disease
(severity)
(severity)
application (VAS)
Notes



D20
1 μm
wheal,
Presented with multiple
Right lower limb: itches removes just
10 → 0


Age: 20's
B ring gel
insect bites
wheals, erythema and very
after application. Erythema started to be



Sex:
formulation

strong itches on both lower
remitted after 1.5 min. Wheals



female
1 application

limbs.
disappeared in about 1 hour.



D31
1 μm
wheal,
Presented with multiple
Left lower limb: itches removed 1 min
10 → 0


Age: 30's
B ring gel
insect bites
wheals and very strong itches
after application, wheals remitted after 2



Sex:
formulation

on right lower limb.
min.



female
1 application






D21
1 μm
childhood
Presented with desiccation,
Right back: itches stopped after 30 sec.
8 → 0


Age: 10's
B ring gel
xerotic
many milary large papules,
Desiccation, scales and erythema



Sex:
formulation
eczema
desquamation, strong pruritus,
improved in a few minutes, Skin got



female
2 applications/

and many scratch scars on
moisturized. After 3 days, wounds




day

trunk; incrustation, erythema
quickly epithelized leaving one scratch




3 days

and lichenification due to
scar. Skin is smooth and moisterized






scratching (severe).
with no desiccation (insignificant).




1 μm


Left back: desiccation, scales and
8 → 3



BNP gel


erythema left after few minutes.




formulation







1 application






D32
1 μm
parapsoriasis
Presented with multiple
Scales, erythema and infiltration remitted
N/A


Age: 40's
B ring gel
guttata
erythrokeratoderma with
after 4 min.



Sex:
formulation

sticking small scales on back.




female
1 application






D33
0.5 μm
psoriasis
Both lower limbs presented
Scales and erythema started to be
N/A


Age: 60's
B ring gel
vulgaris
with many red plaques with
remitted 10 min after 1st application.



Sex: male
formulation

attached small scales having
After 1 week, no scales left.




2 applications/

clear margin.





day







1 week






D34
2 μm
psoriasis
Many red plaques with
Back psoriasis: thick scales started to be
N/A


Age: 30's
B ring gel
vulgaris
attached thick scales having
remitted in 3 min after application,



Sex: male
formulation

clear margin presented on
almost disappeared after 30 min.




1 application

back and neck.
Neck psoriasis: erythema and scales







remitted in 3 min after application.































































TABLE 7








Pruritus before







and after





Pre-application symptoms
Post-application symptoms
application


Case
Treatment
Disease
(severity)
(severity)
(VAS)
Notes



D23
2 μm
dyshidrotic
Fingers presented with many
Erythema and bullae remitted in 2 min
N/A
Intractable

Age: 30's
B ring gel
eczema
intractable bullae, being
after application. After 1 day, erythema

with

Sex: male
formulation
dyshidrosis
eczematous with erythema
and scales remitted and bullae

steroid


2 applications/

and cracks.
disappeared.

ointment.


day







1 day






D24
2 μm
miliaria
Red small papules and flare
Pruritus, erythema and red small
N/A


Age: 50's
B ring gel
hyper-
with itches.
papules remitted after 3 min.



Sex: male
formulation
hidrosis






1 application






D25
1 μm
chronic
Many wheals with drastic
Pruritus removed just after application.
10 → 0


Age: 50's
B ring gel
urticaria
itches.
Wheals started to be gradually remitted



Sex: male
formulation


just after applicationand disappeared




1 application


after 7 min.



D26
1 μm
rosacea
Suffering from rosacea for 2
Tautness and tingling itches removed
N/A
Intractable

Age: 30's
B ring gel

years. 2 months of facial
just after application. Diffuse flare,

after 2 yrs of

Sex:
formulation

application of a steroid
follicular papule and pustule remitted

conventional

female
1 application

ointment exacerbated the
after 1 min. After 1 week of 2

therapy




symptoms, developing
application/day, anti-inflammatory effect

without




telangiectasia, diffuse flare,
was observed. Diffuse flare, follicular

satisfactory




follicular papule and pustule
papule and pustule remarkably

therapeutic




on cheeks. Subjective
improved. Itches and hot flush removed.

effect.




symptoms include tingling







itches and tautness.




D35
4 μm
perioral
2 years of facial application of
Diffuse flare and follicular papule
N/A


Age: 60's
B ring gel
dermititis
a steroid ointment resulted in
remarkably remitted 2 min after



Sex:
formulation

circumoral telangiectasia,
application. After 2 application/day for 2



female
1 application

diffuse flare and follicular
days flare improved and scales






papule. Subjective symptoms
removed.






include heat sensation.




























































TABLE 8








Pruritus before







and after





Pre-application symptoms
Post-application symptoms
application


Case
Treatment
Disease
(severity)
(severity)
(VAS)
Notes



D36
2 μm
rosacea
Presented with circumoral
Diffuse flare and follicular papule



Age: 50's
B ring gel

telangiectasia, diffuse flare
remarkably remitted 2 min after



Sex: male
formulation

and follicular papule.
application




1 application






D37
1 μm
acne
Oily skin accompanied by
Seborrhea remitted 2 min after
N/A
Faster and

Age: 20's
B ring gel
vulgaris
suppurative inflammation with
application. Anti-inflammatory effect

stronger

Sex: female
formulation

many pustules and comedos
remitted suppurative inflammation and

anti-


1 application/

on lower jaw.
pustules on lower jaw. Pustules reduced

inflammatory


day


and dried 3 days after application, and

effect than


3 days


domedos disappeared.

applying/







administrating







conventional







antibiotics.

D38
Right cheek:
acne
Multiple follicular papules and
Anti-inflammatory effect observed 3 min
N/A


Age: 40's
1 μm
vulgaris
pustules on both cheak. Oily
after application, pustules and papules



Sex: female
B ring gel

skin.
started to be reduced and flattened, and




formulation


flare started to be removed. After 1




1 application/


week pustules and papules substantially




day


remitted and flare further remitted. After




2 weeks


2 weeks pustules, papules and flare







almost disappeared.




Left cheek:


No change in symptoms 3 min after




BNP gel


application. After switching to B ring




single


formulation, similar effects as right




application,


cheek were obtained.




then 1 μm







B ring gel







formulation







1 application/







day







2 weeks






D39
Right cheek:
acne
Oily skin accompanied by
Anti-inflammatory effect by B ring



Age: 10's
2 μm
vulgaris
sppurativeinflammation with
application was observed on right cheek



Sex: male
B ring gel

multiple red papules and
in 3 min. Suppurative inflammation




formulation

pustules on both cheeks.
remitted.




1 application







Left cheek:


No anti-inflammatory effect on




gel formulation


gel-applied left side.




1 application






D40
4 μm
acne
Seborrheic skin with multiple
Comedos faded in 5 min after



Age: 10's
B ring gel
vulgaris
comedos and follicular
application. Oily skin improved to better



Sex: female
formulation

papules/pustules on forehead.
texture. Red papules and pustules




1 application


reduced.






























































As shown in Tables 1 to 8, in all subjects, or in all dermatitis of any symptoms, emission or elimination of various symptoms of dermatitis was observed at the site where B ring gel formulation was applied.
Specifically, when B ring gel formulation was applied, remission or elimination of pruritus was observed immediately after the application. Generally in dermatitis, patients tend to scratch where pruritus was felt, potentially causing severer dermatitis. Such a remarkable remission or elimination of pruritus in this manner can consequently prevent aggravation of dermatitis.
In all cases, the effects of B ring gel formulation were superior to those with BNP gel formulation. Specifically, B ring gel formulation had faster-acting and longer-lasting effects as well as more potent antipruritic effect as compared with BNP gel formulation. It was confirmed that these effect cannot be obtained by the gel base.
3.2 Comparing B Ring Gel Formulation to Gel Base by Two-Sided Application
For subjects suffering various dermatitis, either B ring gel formulation or the gel base was applied to the affected site, and changes in symptoms before and after the application were observed (Table below).










TABLE 9



Case
Applied site
Treatment
Disease
Pre-application symptoms
Post-applications symptoms



E1
Right
B ring
atopic dermatitis
Face presented with infiltrating
Flare and pigmentation removed 2.5 min after

Age: 20's



erythema, scales and pigmentation,
application. Skin elasticity and desiccation

Sex: female



with intractable itches
improved. Itches eliminated.


Left
gel base


No remission of pigmentation, infiltrating erythema,






scales and desiccation, with itches left.

E2
Right
B ring
chronic eczema
Stiffness and hard lichenification
Flare remitted soon after application. After 3 min

Age: 30's



on both forearms, with infilitrating
skin softened and moisturized. Itches eliminated.

Sex: male
Left
gel base

erythema and desiccation.
No improvement in lichenification, itching and flare






after 3 min.

E3
Right
B ring
atopic dermatitis
Presented with hot flush and
Flare started to fade soon after application, and

Age: 10's



puffiness
was remarkably remitted after 3 min.

Sex: female
Left
gel base


No improvement in flushing after 3 min.

E4
Right
B ring
chronic eczema
Stiffness and hard lichenification
Flare remitted soon after application, remarkably

Age: 40's



on both forearms, with infiltrating
remitted after 3 min with improved infiltration and

Sex: male



erythema, scales and hot sensation.
hot sensation, and softened skin.


Left
gel base


Infiltration felt 3 min after appication. Skin was hard






with unimproved flare and hot sensation.

































4. Confirming Cosmetic Effect
Effects of a cosmetic comprising B ring were evaluated from the results obtained using a gel formulations, a bath agent and a hair cosmetic product (shampoo or rinse) or body soap comprising the B ring to confirm their effects.
4.1.1 Confirming Skin-Improving Effect (Comparing B Ring Gel Formulation to BNP Gel Formulation)
The B ring gel formulation or BNP gel formulation was applied to subjects. The B ring gel formulation was applied on the right cheek or eye of each subject, the BNP gel formulation on the left cheek or eye, and changes in skin conditions were observed and compared (Table below). The changes in skin conditions were determined based on physical sensory evaluation by the subject and our objective observation as physicians.








TABLE 10



Case
Applied site: method
Pre-application symptoms
Post-application symptoms



F1
Right: B ring gel formulation
Presented with wrinkles, cheek
2 min after application, wrinkles on eye corner

Age: 69
0.5 μmol, 1 application
flabbiness, pigmentation on
and forehead were shallowed, skin was full.

Sex: female

eye corner.
After 3 min, cheek flabiness and skin texture




were improved, and pigmentation on eye




corner was less prominent. After application




for 3 days, large wrinkle on cheek was shallowed,




flabbiness was improved, skin was firm,




resilient, full and moisturized.


Left: applied gel base

No improvement in wrinkles after 3 min.

F2
Right: B ring gel formulation
Presented with wrinkles, cheek
2 min after application, wrinkles at eye corner due

Age: 45
0.5 μmol, 1 application
flabbiness and desiccation.
to desiccation was less prominent, skin texture

Sex: female


was improved, moisturized and softened.


Left: applied gel base

No improvement in wrinkles and desiccation after 2 min.

F3
Right: B ring gel formulation
Presented with desiccation, light
30 sec after application, skin desiccation was improved,

Age: 21
2 μmol, 1 application
erythema, tautness and itch.
skin was moisturized and flexible with no tautness.

Sex: female


After 1 min, flare was remitted and itches were




removed. After 4 min, further softness was given




and desiccation was remarkably improved.


Left: applied gel base

No improvement observed in desiccation, itching and




flare after 4 min.

F4
Right: B ring gel formulation
Presented with desiccation and
3 min after application, skin desiccation was improved,

Age: 24
2 μmol, 1 application
light erythema.
skin was moisturized and firm, with improved texture.

Sex: female


Skin was flexible, soft and moisturized.


Left: applied gel base

No improvement observed in desiccation and flare




after 3 min.

F5
Right: B ring gel formulation
Presented with wrinkles on both
2 min after application, wrinkles on both corners of

Age: 28
2 μmol, 1 application
corners of eye, and desiccation.
eyes were less prominent, cheek dryness was improved,

Sex: female


skin was moisturized and flexible. Perioral desiccation was




remitted and skin became full and flexible.


Left: applied gel base

No improvement observed in desiccation and wrinkles




after 2 min at gel-applied site on the left.

F6
Right: B ring gel formulation
Presented with skin desiccation.
1 min after application, itches were remarkably remitted.

Age: 30
2 μmol, 1 application
oily skin and red papules around
After 3 min, skin desiccation and flare were improved,

Sex: female

nose, and light erythema and
and skin was moisturized and flexible with improved



rough skin texture.
texture. Red papules around nose almost disappeared,




combination state of desiccation and oily skin was




improved, and skin is kept healthy.


Left: applied gel base

3 min after application, desiccation, flare and itches are left,




and seborrhea and papules around nose were not remitted.























































Table below summarizes examples in which B ring gel formulation was applied without applying two-sided. The changes in skin conditions were determined by similar method as described above.








TABLE 11



Case
Method of application
Pre-application symptoms
Post-application symptoms



G1
B ring gel formulation 1 μmol
Presented with cheek flare, open
After B ring application, flare was repressed, skin

Age: 50
1 application
pores, rough and hard skin texture.
texture was improved, skin was moisturized and

Sex: female


softened.

G2
B ring gel formulation 2 μmol
Presented with wrinkles and
3 min after application at B ring-applied site, wrinkles

Age: 49
1 application
desiccation on forehead and eye
on forehead and eye corner were shallowed and

Sex: female

corner.
less prominent, skin desiccation was improved,




and skin was moisturized and flexible.

G3
B ring gel formulation 1 μmol
Presented with male skin
1 day after application at ring-applied site, skin

Age: 40's
1 application
desiccation and roughness.
desiccation and roughness were improved, and razor

Sex: male


burn was prevented.

G4
B ring gel formulation 2 μmol
Presented with miliaria and
3 min after application at B ring-applied site, miliaria

Age: 50's
1 application
pruritus accompanied with flare.
was prevented, flare and itches were remitted.

Sex: male





























Accordingly, it was shown that B ring of the present invention has a skin moisturizing effect and is capable of improving skin texture, and that it is also effective in improving skin, for instance in lightening (spots, dullness) and anti-aging (flabbiness, resilience, large wrinkles) as well as in improving mucosal condition such as roughened lips.
In all subjects, a remarkable improving effect in skin and its persistence was observed at the site where B ring gel formulation had been applied as compared to where BNP gel formulation had been applied. Specifically, effects as follows were observed at the site where B ring gel formulation had been applied as compared to where BNP gel formulation had been applied with significance: effects of improving skin, moisturizing skin, improving skin texture, providing skin with resilience and firmness, softening skin, suppressing skin desiccation, fading fine wrinkles, supplying and keeping skin with water and oil, shallowing and diminishing crow's feet, eliminating skin rawness, eliminating and suppressing itches, relieving skin roughness and maintaining skin condition healthy.
Particularly, in subjects in their 20's or 30's, remarkable effects such as improving or eliminating wrinkles, improving cheek flabbiness, and supplying and keeping skin with moisture, water and oil, and the effects such as giving skin firmness and tightness, lifting up cheeks, eliminating skin rawness and relieving skin roughness were observed at the site where B ring gel formulation had been applied as compared to the site where BNP gel formulation had been applied, and these effects were greater as compared to those at BNP gel-applied site.
Moreover, in subjects in their 40's and 50's, remarkable effects such as improving flabbiness, large wrinkles and desiccation, giving skin firmness, fading dullness and spots were exhibited immediately after the application of B ring gel formulation, and these effects were greater as compared to those upon applying BNP gel formulation.
The effects exhibited significantly faster in B ring gel formulation as compared to BNP gel formulation. Specifically, effects were confirmed in most cases such that wrinkles were vanished or started to vanish and faded and skin was made full and resilient. These effects lasted for at least several hours thereafter. No irritating symptoms were observed with application of B ring gel formulation.
FIG. 1 shows a photograph of the right-side face of a subject in her 60's after the application of B ring gel formulation. In this subject, effects were observed at 20 minutes after the application of B ring gel formulation such that the skin were moist, provided with moisture, complexion and softness, given firmness, improved texture, provided with resilience, and large wrinkles faded. This result was compared to BNP gel formulation as a comparative example, indicating that the cyclic peptide of the invention is not only effective on fine wrinkles but also is effective on wrinkles and flabbiness, and that it is also effective in fading spots and dullness.
4.1.2 Confirming Skin-Improving Effect (Comparing B Ring Gel Formulation to Gel Base)
In order to examine the presence or absence of the contribution by the gel base to the effects of B ring gel formulation and BNP gel formulation, three subjects were administered B ring gel formulation on their right face and the gel base on the left, and changes in skin conditions on the subject's faces were observed (Table below).








TABLE 12



Case
Applied site: method
Pre-application symptoms
Post-application symptoms



H1
Right: B ring gel formulation
Rough, stiff and dry skin texture.
After 2 min skin was felt soft.

Age: 48
Left: applied gel base

Skin was tingling. Open pores and rough skin texture were

Sex: female


not improved.

H2
Right: B ring
Sensitive dry skin with roughness
1 min after application, skin tingling sensation stopped. After

Age: 26

and tingling.
2 min, B ring-applied side was felt more moisturized.

Sex: female
Left: applied gel base

1 min after application, skin was still tingling. After 2 min, the




subject felt that moisturizing effect is weak.

H3
Right:
Sensitive skin with tingling.

Age: 30
Left: applied gel base

Skin was tingling and temporally red. Skin desiccation was

Sex: female


not improved. White scales were observed.


























As a result, all subjects felt tingling sensation at the site where the gel base was applied, and dry feeling of skin was not improved. Also, in all subjects, no effect (e.g., improved skin texture or closed pores) was observed at the site where the gel base was applied. On the other hand, in all subjects, the skin area on which B ring gel formulation was applied has been moisturized and softened.
4.2 Observation on Mucosal Condition Upon Application of B Ring Gel Formulation.
The B ring gel formulation was applied to normal subjects who had symptoms of roughened lips, and changes in mucosal condition were observed (Table below). The changes in mucosal conditions were determined based on physical sensory evaluation by the subject and our objective observation as physicians.








TABLE 13



Case
Applied site: method
Pre-application symptoms
Post-application symptoms



I1
B ring 1 μmol
Cracks on mouth corner with pain.
Skin became moisturized soon after application. After 2 min,

Age: 20's
1 application
Desiccation and wrinkles of lips.
cracks on mouth corner were remitted. After 5 min, lips were

Sex: female


soft and no longer felt dry or tingling.

I2
B ring 0.5 μmol
Annoying lip desiccation.
3 min after application, lips became moisturized.

Age: 30's
1 application

Sex: female

I4
B ring 2 μmol
Annoying lip desiccation.
After 2 min, lips were full and soft, and wrinkles were less

Age: 30’s
1 application

prominent. Moisturized.

Sex: female

I3
B ring 2 μmol
Lips were dry and tingling, with
After 2 min, lips were full, soft and moisturized, and wrinkles

Age: 30's
1 application
wrinkles and scales.
were less prominent. Tingling sensation stopped.

Sex: female

I4
Right: B ring 1 μmol
Cracks on mouth corner with pain.
2 min after application, cracks on mouth corner were remitted

Age: 20's
1 application
Dry and roughened lips.
with no pain, and moisturized.

Sex: male
Left: gel base

2 min after application, cracks on mouth corner and pain were


1 application

not improved.
































Accordingly, it was confirmed that the cyclic peptide of the invention has an activity of improving mucosal condition and it also has an immediate effect in such activity.
4.3 Observation on Scalp/Hair Condition Upon Application of B Ring Gel Formulation.
The B ring gel formulation was applied to normal subjects who had symptoms of scalp/hair, and changes in conditions of scalp and hair were observed (Table below). The changes in scalp/hair were determined based on physical sensory evaluation by the subject and our objective observation as physicians.








TABLE 14



Case
Applied site: method
Pre-application symptoms
Post-application symptoms



J1
B ring 0.5 μmol
Presented with desiccation and
After 2 min, itches were removed, scalp was relieved from roughness

Age: 40's
1 application
itches of head.
and desiccation and moisturized. After that scalp was prevented from

Sex: male


desiccation and kept moisturized.

J2
B ring 1 μmol
Presented with erythema and itch
Hair growth was stimulated, and an increase in hair volume was

Age: 50's
1 application/day, 3 weeks
of head, and thinning and falling of
observed, and thinning of hair was improved. Hair was provided with

Sex: female

hair.
moisture, flexibility and radiance.

J3
B ring 1 μmol
Presented with thinning and falling
A decrease in falling hair was observed, showing an effect of

Age: 40's
1 application/day, 5 days
of hair.
preventing alopecia. Hair growth was stimulated, and an increase

Sex: female


in hair volume was observed, and thinning of hair started to be




improved. Hair was provided with moisture, flexibility




and radiance.

J4
B ring 2 μmol
Presented with seborrheic dandruff
After 2 min, itches were removed, scalp seborrhea and dandruff

Age: 40's
1 application
and itches.
started to be improved. After that scalp became and was

Sex: female


kept clean.

J5
B ring 2 μmol
Presented with desiccation, mild
After 1 min, itches were removed. After 2 min, scalp seborrhea and

Age: 20's
1 application
erythema and itches.
dandruff started to be improved. After 4 min, erythema and skin

Sex: male


roughness of scalp was improved. After that skin roughness




was prevented.

J6
B ring 2 μmol
Presented with scalp seborrhea,
After 2 min, itches were removed, scalp seborrhea was improved.

Age: 60's
1 application
dandruff and itches.
After that dandruff was removed, scalp became healthy and

Sex: male


clean.

J7
B ring 1 μmol
From the beginning of 5th month of
Upon being applied to alopecia 5 month post partum, an effect was

Age: 30's
To decalvant site on head
3rd pregnancy, significant hair loss
exhibited on hair loss in eyebrows and head where hair growth

Sex: female
and brows of subject:
in eyebrows, lashes and scalp
was stimulated, improving thinning of hair.


1 application/day post
occurred, loosing almost all hair.


partum, continued










































Accordingly, BNP cyclic peptide of the present invention can provide and keep scalp and hair with moisture, give them moderate water and oil, improve and prevent desiccation, and clean scalp and hair. It is further effective for suppressing itch and dandruff of scalp and also effective for preventing thinning or loosing hair, for growing hair and promoting or stimulating hair growth, and forcing hair growth, and furthermore effective for an improvement in alopecia after illness or postnatal alopecia.
4.4 Observation on Skin Condition Upon Using Bath Agent Comprising B Ring
Subjects were given bath in 37 to 41° C. hot water comprising B ring dissolved at 0.01 μM (100 ml of 20 μM B ring solution dissolved in 200 L hot water) once a day for 14 days, and skin condition of the subjects was compared to the case of hot water alone (Table 14). The changes in skin conditions were determined based on physical sensory evaluation by the subject and our objective observation as physicians.









TABLE 15





Pre-application





symptoms (miliaria,

Case
Treatment
cracks, chaps and eczema)
Post-application symptoms
Changes in subjective symptoms



K1
B ring addition
Eczema
Gradual remisssion of erythema
Itches after bath was remitted. Skin was

Age: 30's


and skin desiccation.
moisturized.

Sex: female

K2
Hot water
Eczema
Flare and desiccation exacerbated.
Enhanced itches and desiccation after bath.

Age: 30's

Sex: female

K3
BNP addition
Eczema
No remission of skin desiccation or
No subjective remission of skin desiccation at this

Age: 30's


erythema.
concentration.

Sex: female

K4
B ring addition
Chaps rough skin of hands,
Remission of desiccation.
Remission of itch and pain. Skin became smooth.

Age: 60's

itches with tingling.

Sex: female

K5
Hot water
Chaps and rough skin of hands
Exacerbation of desiccation.
Exacerbation of itches and lingling after some

Age: 60's



time after bath.

Sex: female

K6
B ring addition
Miliaria and itches
Remission of flare and miliaria.
Remission of itches and improvement in skin

Age: 20's



texture.

Sex: male

K7
Hot water
Miliaria and itches
No improvement in miliaria.
No remission of itches.

Age: 20's

Sex: male

K8
B ring addition
Cracks and pain on sole
Cracks gradually became
Relief of pain. Skin was moisturized.

Age: 40's


shallower and improved upon

Sex: female


application.

K9
Hot water
Cracks and pain on sole
No improvement in cracks.
No remission of pain.

Age: 40's

Sex: female













































Accordingly, it was confirmed that the bath agent comprising B ring of the present invention has an ameliorating effect on miliaria, cracked or chapped skin, and also an improving effect on eczema. Furthermore, it was shown to improve desiccation, pain and itch of the skin, indicating that it is effective for improvement of skin condition.
4.5 Observation on Conditions of Scalp and Hair or Skin Upon Using Hair Cosmetics (Shampoo, Rinse) or Body Soap Comprising B Ring
4.5.1 a Shampoo Comprising B Ring was Mixed and Prepared in Composition as Follows.








TABLE 16






Content


Name of ingredient (tradename)
(%)










A
Sodium cocoylmethyltaurine solution NIKKOL CMT-30)
10.0


Sodium laureth lactate (NIKKOL SBL-2N-27)
20.0


Lauryl betaine solution (NIKKOL AM-301)
10.0


Cocamide DEA
4.0


Antiseptic
q.l.

B
Citric acid
0.1


Propylene glycol
2.0


Guar hydroxypropyl trimonium chloride
0.5


Water to fill up
100.0

C
Water, B ring (B ring concentration 1 μM)
1 mL































A and B were dissolved with heat at 70° C. To A, B was added, stirred and mixed. C was added at 40 to 35° C. while being further stirred, and allowed to cool to room temperature as kept being stirred.
4.5.2 a Conditioner Comprising B Ring was Mixed and Prepared in Composition as Follows.








TABLE 17





Name of ingredient (tradename)
Content (%)










A
Pentylene glycol
1.50


BG
3.50


Glycerin
1.00


Methyl paraben
0.20


Carbomer
0.20


EDTA-2Na
0.10


Water to fill up
100.0

B
Composite emulsifier (NIKKOL Nikkomulese LC)
4.00


Methyl heptyl laurate
3.50


Squalane
0.50


Cetearyl alcohol
1.50


Macadamia nut oil
0.50


Avocado oil
0.50


Shea oil
0.50


Propyl paraben
0.10

C
Water, B ring (B ring concentration 1 μM)
1 mL




































A and B were dissolved with heat at 80° C. Then A was stirred by a homomixer while B was gradually added thereto, and the mixture was emulsified. C was further added to the mixture, and the mixture was allowed to cool to 35° C. as kept being stirred.
4.5.3 a Body Soap Comprising B Ring was Mixed and Prepared in Composition as Follows.








TABLE 18






Content


Name of ingredient (tradename)
(%)










A
Cocoyl glutamic acid TEA solution
30.0


Sodium trideceth-4 carboxylate (KIKKOL ECTD-3 NEX)
5.0


Sodium cocoamphoacetate solution (KIKKOL AM-101)
10.0


PEG-50 hydrogenated castor oil (KIKKOL HCO-50)
0.5


1,3-butylene glycol
5.0


Antiseptic
q.l.

B
EDTA-2Na
q.l.


Water to fill up
100.0

C
Water, B ring (B ring concentration 1 μM)
mL






























A and B were dissolved with heat at 80° C. Then B was added to A while being stirred. C was further added to the mixture at 40 to 35° C. while kept being stirred, and the mixture was allowed to cool to room temperature as kept being stirred.
Subjects used shampoo, rinse or body soap prepared as above once a day for 14 days, and resulted conditions of scalp and hair or skin of the subjects were assessed (Table below). Changes in skin condition was assessed by dandruff of scalp. Changes in erythema was assessed by visual observation by the subject. Itches, moisturized feeling, combing, glow, resilience and elasticity of hair were assessed by subject's physical sensory evaluation.









TABLE 19






Post-application


Case
Treatment
Pre-application symptoms
symptoms
Subjective changes



L1
shampoo/treatment
Presented with erythema and
Remission of scalp
Improvement in itches. Hair was

Age: 60's

itches on scalp, and thinning
erythema and a decrease
moisturized and provided with

Sex: male

and falling hair.
in hair falling.
resilience, radiance and elasticity.

L2
shampoo
Presented with desiccation
Improvement in dandruff
Improvement in itches. Hair was

Age: 20's

and dandruff on scalp
and desiccation of scalp.
moisturized and provided with

Sex: female

accompanied with itches

resilience, radiance and elasticity.





Improved. With better combing.

L3
body soap
Dry and sensitive skin.
Improvement in dry skin,
Improvement in itches after bath.

Age: 60's


relief of itching after bath.

Sex: female

L4
body soap
Dry and sensitive skin.
Improvement in dry skin,
Improvement in itches after bath.

Age: 60's


relief of itching after bath.

Sex: female

L5
body soap
Dry skin.
Improvement in dry skin.
Skin was moisturized after bath.

Age: 20's

Sex: male

































Accordingly, the use of the hair agent comprising B ring of the present invention had an fast-acting improving effect on itch, erythema and desiccation of scalp. Moreover, it was shown to be effective in improving symptoms of thinning and falling hair when it was used continuously for at least 2 weeks. Furthermore, it was shown that the use of the body soap comprising B ring of the present invention has an improving effect on dry or sensitive skin.
5. Confirming Therapeutic Effect on Alopecia
Either B ring gel formulation or BNP gel formulation was applied, and their effects on symptoms associated to various alopecia were observed (Table below). The changes in symptoms were determined based on physical sensory evaluation by the subject and our objective observation as physicians.








TABLE 20



Case
Treatment
Disease
Post-application symptoms



A21
Right: B ring 1 μmol
Male alopecia
7 weeks after application, hair became elastic and thick, a drastic

Sex: male
1 application/day, continued.

decrease in hair-falling. Consequently, hair become dense and

Age: 50's
Left: BNP gel formulation

thinning less prominent. B ring-containing gel formulation exhibited


1 application/day, continued.

more remarkable hair-growing effect.

A5
B ring gel formulation
Male alopecia and alopecia
2 weeks after starting applying B gel formulation, the existing soft

Sex: male
1 application/day onto head
pityroides accompanied with
hair in parietal area became thicker and longer and turned black..

Age: 50's
decalvant site, continued.
dendruff. Previous use of
Hair got resilient and elastic. Newly grown terminal hairs remarkably



minoxidil-containing drug caused
decreased thinning area. Dandruff was suppressed overall,



strong irritation, itches and
particularly in pariental area. Pruritus was eliminated.



erythema to the subject and its use



was terminated.

A22
B ring gel formulation
Seborrheic alopecia accompenied
2 weeks after application, thinning of hair was improved, exhibiting

Sex: female
2 application/day onto
with dandruff.
no more showing-through of scalp confirming hair-growing

Age: 40's
parietal decalvant site.

stimulation. Dandruff stopped, scalp seborrhea was improved and




pruitus was removed. Furthermore, scalp and skin became clean.







































TABLE 21



A10
B ring gel formulation
Alopecia
2 weeks after starting application, on both sides of head, regeneration of pores and

Sex: male
0.5 μmol
universalis.
growth of terminal hair were confirmed, which were more remarkable and hair growth

Age: 20's
BNP gel formulation
Alopecia on
area was wider on the B ring gel formulation-applied right side. Hair growth area kept


0.5 μmol
whole head.
increasing to the whole head. The density, elongation rate and thickness of hair were



Previous
greater on the B ring gel formulation-applied right side. Upon increasing the



therapy with
concentration of application to 1 μmol, rate of growth and elongation were increased.



carpronium



chloride and



steriod pulse



first exerted



hair growth-



stimulating



effect, but



caused



complete



hair loss



after 1 month.

A11
Right side of head decalvant
Ophiasis and
Right forehead ophiasis site: 7 days after starting applying B ring gel formulation, a hair

Sex: female
site: B ring gel formulation
multiple
growth stimulation effect was observed and hair became thicker terminal hair.

Age: 40's
1 application/day for 1 week.
alopecia.
hair-growing area kept increasing.


1 or 2 applications/week for 1

Left BNP gel formulation-applied site: few soft hair observed, whose growth was slow


year thereafter.

and in obviously smaller area.


Left side of head decalvant

After continuing 1 or 2 applications/week for 1 year:


site: BNP gel formulation

Right forehead B ring gel formulation-applied site: hair-growing area kept increasing and


1 application/day for 1 week.

hair elongation rate was fast (FIG. 2).


1 or 2 applications/week for 1

Left BNP formulation-applied site: hair growth was observed though its area was


year thereafter.

smaller as compared to the right side, and majority of hairs was thin and soft (FIG. 2).

















































TABLE 22



A12
Head decalvant site:
Alopecia
1 week after application, remarkable stimulation and growth of terminal hair was

Sex: female
B ring gel formulation
areata
observed.

Age: 20's
1 application/day, continued.

A12
Subject's head decalvant
Alopecia
1 week after application, remarkable stimulation, growth and elongation of terminal hair

Sex: female
site: B ring gel formulation
areata
was observed.

Age: 30's
1 application/day, continued.

A24
Right half of head decalvant
Drug-induced
Pre-application: 1 month after termination of anticancer agent therapy. Bilateral severe

Sex: female
site: B ring gel formulation
alopecia
diffuse alopecia on whole head. Thinning of hair on whole head. Hair was short and thin,

Age: 50's
(1 μmol). 2 applications/day

mostly white.


for 2 weeks

1 day after application at B ring-applied site, subjective hair elongation and growth


Left half of head decalvant

stimulation were observed, and growth hairs were predominantly black (non-white).


site: BNP gel formulation

After 2 weeks, hair growth was observed at both sides. Hair were elongated with less


(1 μmol). 2 applications/day

falling hairs. Hair elongation rate is faster on B ring-applied site. Hairs are also thicker


for 2 weeks

and denser, and contain more non-white hairs as compared to BNP-applied site. 3




weeks after application, remarkable growth of terminal hairs was observed.





























5.2 Case Summary
In the cases of the subjects having female pattern alopecia, male pattern alopecia, alopecia universalis, ophiasis or alopecia areata multilocularis as described above, a significant decrease in falling hair was observed, and the area of terminal hair growth was expanded, and the growth of the terminal hair was also faster, when B ring gel formulation of the working example was applied compared with BNP gel formulation as a comparative control (See, FIG. 2 ). Addingly, the existing hairs obtained resilience and their elasticity was increased. In all cases, symptoms were significantly improved as compared to the cases when BNP gel formulation of the comparative example was applied. Accordingly, B ring gel formulation had superior effects on the symptoms described above as compared to BNP gel formulation.
In addition, the B ring gel formulation could improve seborrhea, clean scalp and suppress dandruff. It also prevents hairs from turning white, grows black hairs, and improves thinning of hair. Moreover, it exhibits an antipruritic effect within 10 minutes, demonstrating an excellent immediate effect. It could suppress ithes within 3 minutes after application in some cases.
6. Confirming Therapeutic Effect on Rhinitis
6.1 Cases
Either ring nasal drop, BNP gel nasal drop was sprayed, and their effects on rhinitis in the subject was observed (Table 17). The changes in symptoms were determined based on physical sensory evaluation by the subject and our objective observation as physicians.








TABLE 23



Case
Treatment
Disease
Post-application symptoms (severity)



R1
Right nasal
Perennial chronic rhinitis
Right nasal cavity (B ring nasal drop-treated side): Subject fell breath goes through right

Sex: female
cavity: B ring
and rhinorrhea.
nasal cavity soon after application. After 30 sec, air goes through nasal cavity. After 2 min,

Age: 20's
nasal
20 or more blow/day
nose was clear and the subject could breath comfortably. After 3 min fell fresh


formulation

and retained nasal dischared was removed. No discharge by blowing. A day after


0.1 ml, 1 spray

application, no discharge from right nasal cavity.


Left nasal

Left nasal cavity (BNP nasal drop-treated side): 3 min after application, the discharge


cavity: BNP

still retained in nasal cavity and drained by blowing. A day after application, mild nasal


nasal

obstruction and rhinorrhea symptoms in left nasal cavity with small amount of retained


formulation

discharge.


0.1 ml, 1 spray

R2
Right nasal
Rhinitis with severe
Right nasal cavity (B ring nasal drop-treated side): Subject felt breath goes through right

Sex: female
cavity: B ring
nasal obstruction and
nasal cavity soon after application. After 30 sec, obstruction was improved, subject's

Age: 30's
nasal
feeling heavy. Right
suffering in breathing was reduced in right nasal cavity. After 1.5 min, suffering in


formulation
nasal cavity has
breathing was removed in right nasal cavity and breath went though. A day after


0.1 ml, 1 spray
severer obstruction
application, still no obstruction or rhinorrhea. The subject felt no irritation upon treatment


Left nasal
symptom. The subject
in right nasal cavity.


cavaity: BNP
feels dry and tingling by
Left nasal cavity (BNP nasal drop-treated side): 30 sec after application, obstruction of


nasal
steroid nasal drops.
left nasal cavity was slightly improved, through the subject did not feel breath goes


formulation

through as in the right cavity. A day after application, obstruction or rhinorrhea in left


0.1 ml, 1 spray

nasal cavity were removed. The subject felt mild dryness in left nasal cavity.

R3
Right nasal
Perennial allergic
Right nasal cavity: Subject felt breath goes through nasal cavity 1 min after application.

Sex: female
cavity: B ring
rhinitis with rhinorrhea
After 3 min nasal obstruction was improved, the subject could breathe through nose.

Age: 20's
nasal formulation
and nasal obstruction.
After 5 min the subject could breathe comfortably through nose, with no nasal discharge


0.1 ml, 1 spray
Right nasal cavity has
by blowing.



severer obstruction with



retention of nasal



discharge.



















































TABLE 24



R4
Right nasal
Chronic rhinitis with severe
Right nasal cavity (B ring nasal drop-treated side): 1 min after treatment, nasal

Sex: female
cavity: B ring
rhinorrhea and nasal
obstruction was improved to some extent. After 2 min, obstruction was improved, the

Age: 30's
nasal
obstruction, Application of
subject could breathe through nose as usual, and rhinorrhea stopped. After 3 min, no


formulation
steroid nasal drops could not
nasal discharge from the right nasal cavity by blowing.


0.1 ml, 1 spray
improve symptoms when they
Left nasal cavity (BNP nasal drop-treated side): 1 min after treatment, nasal discharge


Left nasal
were severe.
was retainded in the left nasal cavity with no improvement in obstruction. After 2 min,


cavity: BNP

obstruction was improved to some extent, though discharge was retained, showing


nasal

rhinorrhea symptom. After 3 min, nasal discharge was not drained by blowing from left


formulation

nasal cavity due to obstruction. After 8 min, nasal discharge was drained by blowing.


0.1 ml, 1 spray

R5
Right nasal
Allergic rhinitis with
Right nasal cavity (B ring nasal drop-treated side): 30 sec after treatment, obstruction

Sex: female
cavity: B ring
rhinorrhea as main symptom
was improved and the subject could breathe through nose. After 2 min, the subject

Age: 20's
nasal
and mild obstruction.
could easily breathe through nose. After 8 min, no nasal discharge was drained by


formulation

blowing from the right nasal cavity. A day after treatment (after 26 hours), nasal


0.1 ml, 1 spray

discharge was controlled, and breathing was easier through the right nasal cavity as


Left nasal

compared to the left.


cavity: BNP

Left nasal cavity (BNP nasal drop-treated side): 2 min after treatment, obstruction was


nasal

still observed. After 8 min, small amount of discharge was drained by blowing. A day


formulation

after treatment (after 26 hours), nasal discharge was controlled.


0.1 ml, 1 spray

R6
Right nasal
Allergic rhinitis with both
Right nasal cavity (B ring nasal drop-treated side): nasal obstruction was slightly

Sex: female
cavity: B ring
rhinorrhea and nasal
improved soon after treatment. After 1 min, the subject could easily breathe though

Age: 20's
nasal
obstruction having constant
nose. After 4 min, no nasal discharge was drained by blowing from the right nasal cavity.


formulation
obstruction and severe
Nasal obstruction was resolved. After 10 min, pruritus was removed. A day after


0.1 ml, 1 spray
rhinorrhea. 10-20 blow/day.
treatment (after 28 hours), no obstruction or rhinorrhea was observed.


Left nasal
Regular dose of oral anti-
Left nasal cavity (BNP nasal drop-treated side): 1 min after treatment, nasal obstruction


cavity: BNP
allergic drug did not
was not improved. After 3 min, obstruction was still not improved and rhinorrhea was


nasal
sufficiently improve nasal
observed. After 3 min, small amount of discharge was drained by blowing. After 10 min,


formulation
obstruction and rhinorrhea.
obstruction was slightly improved but not resolved. After 2 hours, obstruction was


0.1 ml, 1 spray
Steroid nasal spray causes
resolved and the subject could breathe though nose. A day after treatment (after 28



mucosa pain.
hours), no obstruction or rhinorrhea was observed.




















































TABLE 25



R21
Right nasal cavity: B ring nasal
Rhinitis with continuous rhinorrhea

Sex: female
formulation 0.1 ml, 1 spray
and sneeze. Nasal discharge was drained

Age: 20's

by facing down.

R8
Right nasal cavity: B ring nasal
Chronic allergic rhinitis (with both

Sex: female
formulation 0.1 ml, 1 spray +1 spray
rhinorrhea and obstruction). The

Age: 20's
20 min later
subject regularly feels nasal


Left nasal cavity: BNP nasal
obstruction. Application of steroid


formulation 0.1 ml, 1 spray +1
nasal formulation causes the


spray 20 min later
subject strong irritation.



Accompanied with rhinorrhea and eye



pruritus.

R9
Right nasal
Allergic rhinitis (with rhinorrhea).

Sex: female
cavity: B ring nasal formulation (1 μmol)
The subject has rhinorrhea all day

Age: 30's
0.1 ml, 1 spray
and usually blows 20 or more times


Left nasal cavity: BNP nasal
a day. Application of steroid nasal


formulation (1 μmol) 0.1 ml, 1
formulation causes the subject


spray
strong dry feeling on mucosae of



nasal cavity and pharynx. The



effect of the steroid nasal



formulation lasts only 3-4 hours,



and after the effect ended,



sneezing and nasal discharge last



for several hours and need frequent



blowing.






R21
Right nasal cavity (B ring nasal drop-treated side): 30 sec after treatment, the subject

Sex: female
could easily breathe through nose. After 1 min rhinorrhea stopped. No nasal discharge

Age: 20's
by facing down.

R8
Right nasal cavity (B ring nasal drop-treated side): 1 min after treatment, obstruction

Sex: female
was improved and the subject could easily breathe through nose. The subject felt no

Age: 20's
irritation by treatment. The effect lasts for 1 week thereafter, and rhinorrhea and


obstruction were controlled. No nasal discharge by blowing.


Commerical steroid nasal formulation caused a sharp irritation and recurrence of


symptoms within 2 hours.


Left nasal cavity (BNP nasal drop-treated side):

R9
Right nasal cavity (B ring nasal drop-treated side): 2 min after treatment, the subject

Sex: female
could breathe through nose, with no irritation in the right nasal cavity. After 4 min the

Age: 30's
subject could breathe through nose, with no nasal discharge drained.


Left nasal cavity (BNP nasal drop-treated side): 2 min after treatment, obstruction was


improved and the subject could breathe through nose. After 4 min, in the left nasal


cavity, the subject drained small amount of nasal discharge by blowing.


In the right nasal cavity, the effect of the nasal treatment lasted for 4 hours. Blowing


caused obstruction in the left nasal cavity but not in the right.


The subject felt that B ring nasal formulation is much better to the steroid nasal


formulation regarding the effect on the symptoms, long-lasting and fast-acting effect,


lower frequency of use required, and absence of irritating symptom upon use.







































































TABLE 26



R10
Right nasal
Allergic rhinitis with rhinorrhea
Right nasal cavity (B ring nasal drop-treated side): 2 min after nasal treatment,

Sex: male
cavity:
and obstruction complicated
obstruction was improved and the subject could breathe through nose. The subject

Age: 30's
B ring
with rhinosnusitis. Obstruction
could breathe more comfortably in the right nasal cavity as compared to the left. The


nasal
in the right nasal cavity due to
subject felt no irritation in the right nasal cavity. After 4 min, the subject could very easily


formulation
rhinosinusitis is severer than the
breathe, with no nasal discharge drained by blowing.


(1 μmol)
left and could not be improved
Left nasal cavity (BNP nasal drop-treated side): 4 min after nasal treatment, nasal


0.1 ml,
by steroid nasal formulation,
obstruction was not resolved. Nasal discharge was decreased as compared to


1 spray
making the subject incapable of
pre-treatment, though draining was still observed.


Left nasal
breathing through nose. If left


cavity: BNP
untreated, nasal discharge


nasal
is drained from nostrilis


formulation
spontaneously.


(1 μmol)


0.1 ml,


1 spray

R11
Right nasal
Perennial rhinitis with rhinorrhea
Right nasal cavity (B ring nasal drop-treated side): 2 min after nasal treatment, the

Sex: male
cavity:
and obstruction complicated
subject could breathe through nose. After 5 min, nasal discharge stopped, enabling the

Age: 20's
B ring
with rhinosinusitis, having
subject to breathe though nose. The subject felt no obstruction and was able to breathe


nasal
severe obstruction. Very high
through nose throughout 1 week after nasal treatment.


formulation
frequency of blowing. These
Left nasal cavity (BNP nasal drop-treated side): 5 min after nasal treatment, obstruction


0.1 ml,
symptoms were not improved
was not resolved, with nasal discharge present in the left nasal cavity. After 10 min,


1 spray
by any previously used nasal
obstruction was not resolved, making the subject incapable of breathing through nose.


Left nasal
formulation.


cavity: BNP


nasal


formualtion


0.1 ml,


1 spray










































6.2 Case Summary
In all cases described above, both rhinorrhea and nasal obstruction were quickly improved or eliminated when B ring nasal drop of the working example had been applied as compared to the case when BNP nasal drop of comparative example had been applied. Accordingly, B ring nasal drop had faster effect than BNP nasal drop. Addingly, the effect of B ring nasal drop was more than equal to that of BNP nasal drop, and because the effect lasted for longer time period, it can suppressing recurrence of symptoms.
The following table summarizes information from the cases described above, within the range recognizable at the time when symptoms of nasal obstruction and rhinorrhea were improved.










TABLE 27








No


within
within
within
resolution


3 min
5 min
10 min
within 10 min










Resolution of rhinorrhea







B ring nasal formulation
3
4
1
0

BNP nasal formulation
0
0
0
7





Resolution of nasal obstruction







B ring nasal formulation
6
5
0
0

BNP nasal formulation
0
0
0
8





































7. Analysis of Binding State of Human BNP and Type A Receptor
The superior pharmacological effects of the B ring-compound of the invention on various diseases relative to conventional BNP has been explained so far with reference to data based on respective clinical cases. Such pharmacological effects is also supported by the result of the conformation analysis of the compound performed by the inventors, which is explained by following experimental report for reference.
In order to investigate binding state of human BNP (BNP-32) and its receptor Type A receptor (NPR-A), an in silico analysis was performed by homology modeling using conformation. Swiss-Pdb viewer and SWISS-MODEL were used for modeling.
7.1 Template Structure
Firstly, prior to the above analysis, a template structure for investigation on binding state of human BNP and Type A receptor was selected. As this template structure, the conformation of the complex of rat NPR-A and rat ANP peptide (PDB ID: 1T34) were used. Rat NPR-A is a homodimer composed of A and B strands. The conformation of such rat NPR-A has been determined by X-ray crystalline structural analysis such that 21 residues from Cys7 to Arg 27 of rat ANP were bound. The conformation of rat NPR-A was obtained from the database of protein conformation, Protein Data Bank. Amino acid homology between human NPR-A and rat NPR-A is 85%.
7.2 BNP Peptide Model
In the present study, as BNP peptide model, a resion from Cys10 to Arg30 in human BNP (BNP-32, SEQ ID NO: 13, Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Le u-Gly-Cys-Lys-Val-Leu-Arg-Arg-His), i.e., an amino acid sequence (SEQ ID NO: 14, Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg) was used. In the peptide model above, the amino acid sequence of B ring of the invention corresponds to a resion from Cys10 to Cys26 of human BNP above, i.e., Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys (SEQ ID NO: 15).
7.3 Homology Modeling
In order to speculate the amino acid residues that are involved in binding of human NPR-A and human BNP, a complex model in which BNP peptide is bound to human NPR-A was constructed by homology modeling. Specifically, human BNP peptide model was constructed based on the template structure of rat ANP peptide, and human NPR-A model structure based on the template structure of rat NPR-A.
Next, residues that are involved in the interaction was speculated from the amino acid residues detected between human BNP peptide model and human NPR-A.
The results showed that BNP peptide model is bound to human NPR-A dimer being sandwiched between the A strand and B strand. Amino acids in the amino acid sequence, e.g., the amino acid sequence used as the model (SEQ ID NO: 14, Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg) are expressed as “amino acid (number)”. Namely, the number in parentheses denotes the relative position counted from the N-terminal of the amino acid sequence, for example, Phe2 indicates an amino acid that is the second Phe counted from the N-terminal of the peptide model.
In the constructed complex model, the presence of a hydrophobic bond by Phe2 side chain, a hydrogen bond by Phe2 main chain, and hydrogen bonds by side chains of Arg4, Met6, Arg8, Ser10 and Ser11 were speculated between BNP peptide model and the A strand of NPR-A. Phe2, Arg4, Met6, Arg8, Ser10 and Ser11 in BNP peptide model correspond to Phell, Arg13, Met15, Arg17, Ser19 and Ser20 in human BNP respectively. Namely, this result suggests that these amino acid residues in human BNP is likely to be the residues that contribute to NPR-A activation.
7.4 Discussion
Thus, by in silico conformation analysis, it was speculated that, in human BNP, the amino acid residues that are present in its cyclic part are likely to contribute to NPR-A activation, whereas the amino acid residues present in the tail part are not. It was also suggested that BNP cyclic structure is a smaller molecule than BNP-32 and therefore capable of binding easily and quickly. In fact, a clinical application of a peptide having BNP cyclic structure confirmed that BNP cyclic structure provides faster therapeutic effect than BNP-32. This clinical result is consistent with that of the in silico analysis that BNP cyclic structure contributes to NPR-A activation and can bind to it more easily and quickly than BNP-32. Thus, BNP cyclic structure has a superior therapeutic effect than a general BNP peptide, and therefore is a different substance.
On the other hand, an NMR analysis revealed that ANP does not take any particular conformation in a solution where its conformation greatly wobbles; this is considered to be similar for BNP. Namely, in human BNP, assuming that the amino acid residues in the cyclic part contribute to its binding to human NPR-A, it is speculated that the amino acid residues in the tail part rather prevent human BNP from entering into the narrow BNP binding site sandwiched between A and B strands of human NPR-A due to its large wobbling. Therefore, it is considered that the cyclic part of human BNP is a relatively smaller molecule than conventionally known human BNP, enabling itself to enter into BNP binding site of NPR-A more easily and quickly. Moreover, it is speculated that the the cyclic part of human BNP has a higher affinity to human NPR-A than BNP-32. This supports the clinical outcomes described herein that the peptide having only the cyclic part of BNP exhibits therapeutic effect faster than BNP-32.
In order to speculate the effect of BNP cyclic structure (B ring) from non-human species on human Type A receptor, complex models of human NPR-A and BNP rings from pig, bird or rat were generated to surmise interaction. The results suggested that a sufficient effect of the invention can be expected by using BNP ring from non-human animal species (e.g., pigs, birds or rats), as long as it shows an affinity to human NPR-A.
8. Speculating Replaceable Amino Acid Residues in BNP Cyclic Moiety
Using the constructed model structure of the complex, replaceablity of amino acid residues other than those considered to be involved in the interaction was investigated.
Specifically, in order to investigate whether the peptide in which amino acid residue that is not assumed to be involved in the interaction has been replaced with another amino acid is capable of binding to NPR-A, a mutant model of BNP was generated to analyze the interaction. Swiss-Pdb viewer was used for modeling.
Specifically, amino acid residues Gly12, Lys14, Asp16, Ile18, Ser21, Ser22, Gly23, Leu24 and Gly25 in human BNP were targeted for the investigation on their replaceability with other amino acids in terms of following points:
No steric hindrance caused by binding to NPR-A. No interatomic collision observed in the model structure of NPR-A and BNP. No influence on electrostatic potential on surface. No large increase in intramolecular energy value (no unnatural angle or twist caused in intermolecular binding). No non-naturally occurring hydrogen bond formed between the strands of NPR-A and BNP and within BNP strand. No cavity (cavity, niche) formation.

Among those described above, intramolecular energy was calculated by ComputeEnergy command of Swiss-Pdb viewer. Intramolecular energy was calculated from the sum of the length of binding, bond angle, twist and binding energy, etc. in the unit kilojoule/mol (Kj/mol).
The results of analysis indicated the presence of replaceable amino acid residues in Gly12, Lys14, Ile18, Ser21, Ser22, Leu24 and Gly25 among the amino acid residues of human BNP (Table below).








TABLE 28





human BNP
Replaceable amino acid





Gly12
Ala, Val, Ser, Thr


Lys14
Arg


Asp16
none


Ile18
Val


Ser21
Thr, Ala, Val, Gln, Leu, Ile, Met


Ser22
Thr, Ala, Val


Gly23
none


Leu24
Ala, Val, Ile, Met


Gly25
Ala, Ser


























Accordingly, even when the replacement of the amino acid corresponding to those described in the table above took place in the cyclic part of human BNP, i.e., the peptide expressed by the Formula I-a, it was suggested that the peptide replaced in such a way exerts a similar effect as the peptide composed of the amino acid sequence expressed by Formula I-a.
9. Confirming Therapeutic Effect of the Cyclic Peptide with Amino Acid Replacement
Next, cyclic peptides with some replaced amino acid were subjected to following examination in order to confirm their effect. The methods for preparing the above cyclic peptide and confirming amino acid sequence of the prepared peptide were same as the method for preparing the cyclic peptide as described above and Mass spectroscopy method.
9.1 Confirming Effects on Scalp or Hair
The obtained cyclic peptide was prepared in purified water at 3, 15, 30, 100 or 500 μg/ml and applied to the affected site. Each of formulations A to E in the table indicates the concentration of cyclic peptide at 3, 15, 30, 100 or 500 μg/ml, respectively. The absence of the effect upon the application of purified water confirmed the effect was not a placebo effect.











TABLE 29-1



SEQ ID NO







(amino







acid sequence)




Form-


of the cyclic




ula-


peptide used
ID
Sex
Age
Cases
tion
Treatement



SEQ ID NO: 16:
S1
male
50's
multiple alopecia
C
once a day

(CFVRKMDRISSSS



areata



GLGC)
S2
female
40's
seborrheic
C
once a day





dermatitis







male AGA




S3
female
50's
female AGA
C
once a day


S4
female
50's
hair thinning after
C
once a day





anticancer agent







therapy




S5
female
20's
multiple alopecia
C
once a day





areata




S6
female
60's
healthy skin (scalp)
C
once a day

SEQ ID NO 17:
S7
male
40's
multiple alopecia
B
once a day

(CFGQKMDRISSSS



areata



GLGC)
S8
female
20's
multiple alopecia
B
once a day





areata




S9
male
30's
male AGA
B
once a day


S10
female
30's
female alopecia
C
once a day

SEQ ID NO 18:
S11
male
50's
male alopecia
B
once a day

(CFGHKMDRISSSS
S12
male
70's
healthy skin (scalp)
C
once a day

GLGC)
S13
female
40's
alopecia areata
B
once a day


S14
female
50's
alopecia areata
B
once a day

SEQ ID NO 19:
S15
female
20's
multiple alopecia
B
once a day

(CFGRRMDRISSSS



areatea



GLGC)
S16
female
50's
hair thinning after
C
once a day





anticancer agent







therapy




S17
male
50's
male AGA
C
once a day


S18
female
40's
female alopecia
D
once a day


S19
female
40's
scalp seborrheic
C
once a day





dermatitis



SEQ ID NO 20:
S20
male
50's
male AGA
C
once a day

(CFGRKLDRISSSS
S21
male
40's
multiple alopecia
C
once a day

GLGC)



areata



SEQ ID NO 21:
S22
male
40's
multiple alopecia
C
once a day

(CFGRKIDRISSSS



areata



GLGC)
S23
male
40's
male alopecia
C
1 application/







day on







parietal







male thinning







area


S24
female
40's
female AGA
C
once a day


S25
female
30's
multiple alopecia
C
once a day





areata




S26
female
10's
multiple ophiasis
C
once a day

SEQ ID NO 22:
S27
male
40's
male AGA
B
1 application/

(CFGRKMDRVSSSS





day on fore-

GLGC)





head thinning







area


S28
male
20's
multiple alopecia
C
once a day


S29
female
50's
female AGA
C
once a day


S30
female
70's
female alopecia
C
once a day


S31
female
20's
ophiasis
B
once a day


S32
female
30's
multiple alopecia
C
once a day





areata



SEQ ID NO 23:
S33
male
30's
alopecia areata
C
once a day

(CFGRKMDRIGSSS



universalis



GLGC)
S34
female
50's
hair thinning after
C
once a day





anticancer agent







therapy




S35
female
50's
female AGA
C
once a day


S36
female
40's
female alopecia
B
1 application/







day on







parietal







and forehead







thinning area


S37
male
50's
multiple alopecia
C
once a day





areata




S38
female
40's
female alopecia
B
once a day





seborrheic







dermatitis




S39
female
10's
ophiasis
B
once a day

SEQ ID NO 24:
S40
female
10's
alopecia areata
A
once a day

(CFGRKMDRISASS
S41
female
50's
female AGA
C
once a day

GLGC)
S42
male
30's
multiple alopecia
B
once a day





areata




S43
female
30's
multiple alopecia
C
once a day





areata



SEQ ID NO 25:
S44
male
30's
multiple alopecia
B
once a day

(CFGRKMDRISSQS



areata



GLGC)
S45
male
50's
male AGA
C
once a day


S46
female
60's
multiple alopecia
B
once a day





areata




S47
female
40's
female alopecia
C
once a day

SEQ ID NO 26:
S48
female
30's
female alopecia
C
once a day

(CFGRKMDRISSVS
S49
male
30's
male alopecia
B
once a day

GLGC)
S50
female
20's
female AGA
C
once a day


S51
female
60's
female AGA
C
once a day


S52
male
20's
multiple alopecia
C
once a day


S53
female
30's
multiple alopecia
C
once a day





areata



SEQ ID NO 27:
S54
female
50's
hair thinning after
B
once a day

(CFGRKMDRISSLS



anticancer agent



GLGC)



therapy




S55
male
50's
male AGA
C
once a day


S56
male
20's
multiple alopecia
E
once a day





areata



SEQ ID NO 28:
S57
female
40's
female AGA
C
once a day

(CFGRKMDRISSIS
S58
male
20's
multiple alopecia
C
once a day

GLGC)



areata




S59
male
20's
multiple alopecia
C
once a day





areata




S60
female
50's
health skin (scalp)
C
once a day


S61
female
40's
multiple alopecia
C
once a day





areata




S62
female
40's
alopecia areate
B
once a day

SEQ ID NO 29:
S63
female
40's
female alopecia
C
once a day

(CFGRKMDRISSMS
S64
male
40's
healthy skin (scalp)
C
once a day

GLGC)
S65
male
40's
male AGA
C
once a day


S66
male
30's
male alopecia
C
2 application/







day for 3 days


S67
female
70's
female alopecia
C
once a day

SEQ ID NO: 30:
S68
female
20's
alopecia areata
B
once a day

(CFGRKMDRISSSV



ophiasis



GLGC)
S69
male
40's
male AGA
B
once a day


S70
male
30's
alopecia universalis
B
once a day


S71
female
30's
multiple alopecia
C
once a day





areata



SEQ ID NO 31:
S72
female
20's
alopecia areata
C
once a day

(CFGRKMDRISSSS



ophiasis



RLGC)
S73
male
40's
healthy skin (scalp)
C
once a day


S74
male
30's
alopecia areata
C
2 application/





universalis

day for 3







days


S75
female
60's
multiple alopecia
B
once a day

SEQ ID NO 32:
S76
male
30's
male alopecia
B
once a day

(CFGRKMDRISSSS
S77
female
50's
female AGA
C
once a day

GMGC)
S78
male
40's
alopecia universalis
C
once a day


S79
female
50's
hair thinning after
C
once a day





anticancer agent







therapy



SEQ ID NO 33:
S80
female
50's
multiple alopecia
B
once a day

(CFGRKMDRISSSS



areate



GIGC)
S81
male
50's
healthy skin (scalp)
C
once a day


S82
female
40's
multiple alopecia
B
once a day





areata




S83
male
50's
alopecia areata
C
once a day

SEQ ID NO 34:
S84
female
50's
hair thinning after
B
once a day

(CFGRKMDRISSSS



anticancer agent



GVGC)



therapy




S85
male
50's
male AGA
C
once a day


S86
male
40's
male AGA
C
once a day


S87
male
30's
male alopecia
C
once a day


S88
male
20's
multiple alopecia
C
once a day





areata



SEQ ID NO 35:
S89
female
40's
hair thinning after
B
once a day

(CFGRKMDRISSSS



anticancer agent



GAGC)



therapy




S90
male
20's
multiple alopecia
C
once a day





areata




S91
female
50's
female AGA
C
once a day


S92
female
60's
multiple alopecia
B
once a day





areata




S93
female
30's
multiple alopecia
C
once a day





areata



SEQ ID NO 36:
S94
male
30's
alopecia universalis
C
once a day

(CFGRKMDRISSSS
S95
male
40's
healthy skin (scalp)
C
once a day

GLSC)
S96
male
50's
seborrheic
C
once a day





dermatitis




S97
male
50's
male alopecia
B
once a day

SEQ ID NO 37:
S98
female
40's
hair thinning after
B
once a day

(CFGRKMDRISSSS



anticancer agent



GLAC)



therapy




S99
male
40's
male AGA
B
1 application/







day on







forehead







thinning area


S100
male
20's
multiple alopecia
C
once a day





areata




S101
female
20's
alopecia areata
A
once a day

SEQ ID NO 38:
S102
female
50's
female AGA
C
once a day

(CFARKMDRISSSS
S103
female
50's
dandruff, itches
C
once a day

GLGC)
S104
female
10's
octopic multiple
C
once a day





alopecia



SEQ ID NO 39:
S105
male
10's
alopecia universalis
C
once a day

(CFSRKMDRISSSS
S106
female
50's
dandruff, itches
C
once a day

GLGC)
S107
female
60's
healthy skin (scalp)
C
once a day


S108
female
60's
multiple alopecia
C
once a day

SEQ ID NO 40:
S109
male
50's
male AGA
C
once a day

(CFTRKMDRISSSS
S110
male
20's
multiple alopecia
C
once a day

GLGC)



areata



SEQ ID NO 41:
S111
female
50's
female AGA
C
once a day

(CFGRKMDRISSTS
S112
male
20's
multiple alopecia
C
once a day

GLGC)



areata



SEQ ID NO 42:
S113
male
30's
multiple alopecia
C
once a day

(CFGRKMDRISSAS



areata



GLGC)
S114
male
40's
healthy skin (scalp)
C
once a day


S115
female
60's
multiple alopecia
C
once a day





areata



SEQ ID NO 43:
S116
male
40's
male AGA
B
1 application/

(CFGRKMDRISSST





day on

GLGC)





forehead







thinning area


S117
male
20's
multiple alopecia
C
once a day





areata



SEQ ID NO 44:
S118
female
60's
female AGA
C
once a day

(CFGRKMDRISSSA
S119
female
20's
multiple alopecia
C
once a day

GLGC)



areata



SEQ ID NO 45:
S120
male
40's
male AGA
C
once a day

(CFSRRMDRISSSS
S121
female
40's
healthy skin (scalp)
C
once a day

GLGC)







SEQ ID NO 46:
S122
female
60's
female AGA
C
once a day

(CFSRKMDRISSTS
S123
female
60's
healthy skin (scalp)
C
once a day

GLGC)







SEQ ID NO 47:
S124
male
40's
healthy skin (scalp)
C
once a day

(CFSRKMDRISSST
S125
female
50's
female AGA
C
once a day

GLGC)







SEQ ID NO 48:
S126
female
40's
multiple alopecia
B
once a day

(CFSRKMDRISSSS



areata



GIGC)
S127
female
50's
healthy skin (scalp)
C
once a day


S128
female
50's
female AGA
C
once a day

SEQ ID NO 49:
S129
female
50's
female AGA
C
once a day

(CFSRKMDRISSSS
S130
female
50's
alopecia areata
B
once a day

GLAC)
S131
female
40's
healthy skin (scalp)
C
once a day

SEQ ID NO 50:
S132
male
50's
male AGA
C
once a day

(CFGRRMDRISSTS
S133
male
40's
healthy skin (scalp)
C
once a day

GLGC)







SEQ ID NO 51:
S134
male
40's
male AGA
B
1 application/

(CFGRRMDRISSST





day on

GLGC)





forehead







thinning area


S135
male
50's
healthy skin (scalp)
C
once a day

SEQ ID NO 52:
S136
male
60's
male AGA
C
once a day

(CFGRRMDRISSSS
S137
female
40's
multiple alopecia
B
once a day

GIGC)



areata




S138
female
60's
healthy skin (scalp)
C
once a day

SEQ ID NO 53:
S139
female
40's
female AGA
C
once a day

(CFGRRMDRISSSS
S140
male
40's
healthy skin (scalp)
C
once a day

GLAC)







SEQ ID NO 54:
S141
male
40's
male AGA
B
1 application/

(CFGRKMDRISSTS





day on

GIGC)





forehead







thinning area


S142
female
50's
healthy skin (scalp)
C
once a day

SEQ ID NO 55:
S143
male
50's
male AGA
C
once a day

(CFGRKMDRISSTS
S144
female
60's
healthy skin (scalp)
C
once a day

GLAC)







SEQ ID NO 56:
S145
male
40's
male AGA
C
once a day

(CFGRKMDRISSST
S146
male
40's
healthy skin (scalp)
C
once a day

GIGC)







SEQ ID NO 57:
S147
male
50's
male AGA
C
once a day

(CFGRKMDRISSST
S148
male
40's
healthy skin (scalp)
C
once a day

GLAC)







SEQ ID NO 58:
S149
female
40's
female AGA
C
once a day

(CFGRKMDRISSSS
S150
female
40's
healthy skin (scalp)
C
once a day

GIAC)







SEQ ID NO 59:
S151
male
50's
male AGA
C
once a day

(CFSRRMDRISSTS
S152
male
40's
healthy skin (scalp)
C
once a day

GLGC)







SEQ ID NO 60:
S153
male
60's
male AGA
C
once a day

(CFSRRMDRISSST
S154
male
40's
healthy skin (scalp)
C
once a day

GLGC)







SEQ ID NO 61:
S155
female
60's
female AGA
C
once a day

(CFSRRMDRISSSS
S156
male
60's
healthy skin (scalp)
C
once a day

GIGC)







SEQ ID NO 62:
S157
male
50's
male AGA
C
once a day

(CFSRRMDRISSSS
S158
male
40's
healthy skin (scalp)
C
once a day

GLAC)







SEQ ID NO 63:
S159
male
60's
male AGA
B
1 application/

(CFSRKMDRISSTS





day on

GIGC)





forehead







thinning area


S160
male
40's
healthy skin (scalp)
C
once a day

SEQ ID NO 64:
S161
male
60's
healthy skin (scalp)
C
once a day

(CFSRKMDRISSST
S162
female
50's
female AGA
C
once a day

GIGC)







SEQ ID NO 65:
S163
male
40's
male AGA
B
1 application/

(CFSRKMDRISSST





day on

GLAC)





forehead







thinning area


S164
male
40's
healthy skin (scalp)
C
once a day

SEQ ID NO 66:
S165
male
60's
male AGA
C
once a day

(CFSRKMDRISSSS
S166
male
40's
healthy skin (scalp)
C
once a day

GIAC)







SEQ ID NO 67:
S167
male
40's
male AGA
B
1 application/

(CFGRRMDRISSTS





day on

GIGC)





forehead







thinning area


S168
female
60's
healthy skin (scalp)
C
once a day

SEQ ID NO 68:
S169
female
40's
female AGA
C
once a day

(CFGRRMDRISSTS
S170
male
40's
healthy skin (scalp)
C
once a day

GLAC)







SEQ ID NO 69:
S171
male
60's
male AGA
B
1 application/

(CFGRRMDRISSST





day on

GIGC)





forehead







thinning area


S172
male
30's
multiple alopecia
C
once a day


S173
male
60's
healthy skin (scalp)
C
once a day

SEQ ID NO 70:
S174
female
50's
female AGA
C
once a day

(CFGRRMDRISSST
S175
male
40's
healthy skin (scalp)
C
once a day

GLAC)







SEQ ID NO 71:
S176
male
50's
male AGA
C
once a day

(CFSRRMDRISSTS
S177
male
20's
multiple alopecia
C
once a day

GIGC)



areata




S178
male
50's
healthy skin (scalp)
C
once a day

SEQ ID NO 72:
S179
male
40's
male AGA
B
1 application/

(CFSRRMDRISSTS





day on

GLAC)





forehead







thinning area


S180
male
40's
healthy skin (scalp)
C
once a day

SEQ ID NO 73:
S181
female
40's
female AGA
C
once a day

(CFSRRMDRISSST
S182
female
40's
alopecia areata
B
once a day

GIGC)
S183
female
60's
healthy skin (scalp)
C


SEQ ID NO 74:
S184
male
60's
male AGA
C
once a day

(CFSRRMDRISSST
S185
male
40's
healthy skin (scalp)
C
once a day

GLAC)







SEQ ID NO 75:
S186
male
40's
male AGA
B
1 application/

(CFSRRMDRISSSS





day on

GIAC)





forehead







thinning area


S187
female
40's
multiple alopecia
B
once a day





areata




S188
female
50's
healthy skin (scalp)
C
once a day





SEQ ID NO



(amino



acid sequence)



of the cyclic



peptide used
ID
Diagnostic Impression of hair and scalp after application



SEQ ID NO: 16:
S1
After 1 day, fluffy hairs started to grow.

(CFVRKMDRISSSS

After 3 days, obviously Hair started to grow, restoration and elongation were

GLGC)

observed. Hair gained elasticity and resilence, thickened. Hair root regeneration



confirmed.


S2
Itches were remitted immediately after application



After 3 minutes, seborrhea and erythema remitted.



After 1 day, dandruff, redness and seborrhea of scalp improved, falling hairs



reduced.



After 7 days, hairs stimulated to grow, restored hair and hair growth confirmed.


S3
After 1 day, hair gained elasticity, resilience and increased volume.


S4
After 5 days, hair stimulation and growth confirmed. Hair became dense and



thick.


S5
Itches were improved immediately after application.



After 1 day, fluffy hairs started to grow.



After 3 days, obviously Hair growth stimulated and restored elongation of hair



observed, hair gained elasticity and resilience, thickened. Hair root regeneration



observed.


S6
After 1 day, hair became fuller with increased volume. Hair gained elasticity and



resilience. After 7 days, grown hairs were predominantly black over white hairs.



White hairs turned black.

SEQ ID NO 17:
S7
After 3 days, remarkable terminal hair growth was observed.

(CFGQKMDRISSSS
S8
After 5 days, newly born hair roots and grown hairs were observed.

GLGC)
S9
After 2 or 3 days, hair gained elasticity and resilience.



After 7 days, hair gained volume, restoration was confirmed.


S10
Hair gained elasticity and resilience, hair volume was increased on the night of



first application.



After 5 days, appearance of scalp through hair were improved.



The effect lasted for 1 week after stopping application

SEQ ID NO 18:
S11
After 1 week, hair gained thickness and elasticity, hair density was increased.

(CFGHKMDRISSSS
S12
After 1 day, hair became fuller with increased volume. Hair gained elasticity and

GLGC)

resilience. After 7 days, grown hairs were predominantly black over white hairs.



White hairs turned black.


S13
After 5 days, hair started to grow, elongation and growth of hair were observed.


S14
After 5 days, hair started to grow, hair density was increased.

SEQ ID NO 19:
S15
After 5 days, hair started to grow, elongation and restoration of hair were

(CFGRRMDRISSSS

observed.

GLGC)
S16
After 2 days, hair gained elasticity and resilience. Volume of hair increased.


S17
After 1 day, hair gained elasticity, resilience and increased volume. Hair started



to grow, restoration and elongation were observed.


S18
After 3 days, scalp shown through hair due to thinning got obscure. Reduced



falling hairs.


S19
After 3 days, seborrhea and erythema were improved, so was dandruff.

SEQ ID NO 20:
S20
After 1 day, hair gained elasticity, resilience and increased volume.

(CFGRKLDRISSSS
S21
After 3 days, hair density was increased. Hair started to grow and grew.

GLGC)



SEQ ID NO 21:
S22
After 3 days, hair started to grow at the site of alopecia areata and grew.

(CFGRKIDRISSSS
S23
After 3 days, hair started to gain resilience, felt thick.

GLGC)

After 7 days, parietal thinning were improved and hair volume started to increase


S24
After 1 day, hair gained elasticity, resilience and increased volume. Hair started



to grow, restoration and elongation were observed.


S25
After 1 day, hair started to grow.



After 2 weeks, obvious lots of hairs started to grow, restoration, elongation and



growth were observed.


S26
After 2 days, stimulated hair growth was confirmed.



After 7 days, obvious stimulated hair growth was observed, elongated and grown



hair was also observed.

SEQ ID NO 22:
S27
After 2 or 3 days, hair gained elasticity and resilience. After 7 days, hair

(CFGRKMDRVSSSS

gained volume, restoration was confirmed.

GLGC)
S28
After 3 days, hair started grow and grew.


S29
After 1 day, hair gained elasticity, resilience and increased volume.


S30
After 7 days, increased hair volume. Appearance of scalp shown through hair got



obscure.


S31
After 5 days, stimulated hair confirmed along intractable hairline, and elongation



of hair observed.


S32
After 1 day, hair started to grow.



After 6 weeks, obvious stimulated hair growth, elongation and growth of hair were



observed.

SEQ ID NO 23:
S33
After 3 days, stimulated and elongated hairs were observed.

(CFGRKMDRIGSSS
S34
After 1 day, hair gained elasticity and resilience.

GLGC)
S35
After 1 day, hair gained elasticity, resilience and increased volume. Hair started



to grow, restoration and elongation were observed.


S36
After 5 days, hair gained elasticity, resilience and increased volume. Appearance



of scalp shown through hair got obscure.


S37
After 1 day, stimulated hair was observed. After 3 days, application was stopped,



but stimulation and elongation of hair contitued. White hairs turned black again,



black hairs started to grow.


S38
After 7 days, hair gained volume, appearance of scalp shown through hair got



obscure. Seborrhea of scalp was improved.


S39
After 4 days, regardless of highly intractable ophiasis, stimulated soft hairs



were observed.

SEQ ID NO 24:
S40
After 7 days, stimulated and grown soft hairs were observed.

(CFGRKMDRISASS
S41
After 1 day, hair gained elasticity, resilience and increased volume.

GLGC)
S42
After 4 days, stimulation and elongation of hair observed.


S43
After 1 day, hair started to grow. A numbe of stimulated, elongated and grown



hairs were observed in 3 weeks.

SEQ ID NO 25:
S44
After 5 days, hairs started to grow and grew.

(CFGRKMDRISSQS
S45
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

GLGC)

to grow, restoration and elongation were observed.


S46
After 5 days, hair root regeneration and hair growth stimulation were observed.



Application continued and a remarkable restoration, elongation and growth of hair



were observed. Hair elongation rate was fast.


S47
After 1 day, hair gained elasticity and resilience. Application continued for 3



days and stopped, but hair kept elasticity and resilience. The effect lasted for



1 week.

SEQ ID NO 26:
S48
After 3 days, appearance of scalp shown through hair got obscure, hair gained

(CFGRKMDRISSVS

elasticity, resilience and increased volume. Hairs were restored.

GLGC)
S49
After 5 days, volume of hair increased. Hair gained elasticity and resilience.


S50
Volume of hair increased and scalp does not show through after 3 days. The



effect went on.


S51
After 1 day, hair gained elasticity, resilience and increased volume. Hair started



to grow, restoration and elongation were observed.


S52
After 3 days, stimulated hair was observed.



After 7 days, further stimulation and growth of hair were confirmed.


S53
After 1 day, hair started to grow. Terminal hairs started to grow, and



restoration, elongation and growth of hair were observed in 2 weeks.

SEQ ID NO 27:
S54
After 5 days, hair got thick and dense.

(CFGRKMDRISSLS
S55
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

GLGC)

to grow, restoration and elongation were observed.


S56
After 3 days, hair started to grow. After 7 days, stimulation and growth of hair



were confirmed.

SEQ ID NO 28:
S57
After 1 day, hair gained elasticity, resilience and increased volume.

(CFGRKMDRISSIS
S58
After 1 day, hair falling was reduced and hair started to grow.

GLGC)

After 7 days, remarkable stimulation, elongation and growth of hair were



confirmed.


S59
After 1 day, hair started to grow and falling hairs reduced. After 4 days,



application was stopped, but stimulation, elongation and restoration of hair



persisted.


S60
After 1 day, hair became fuller with increased volume. Hair gained elasticity and



resilience.



After 7 days, grown hairs were predominantly black over white hairs. White hairs



turned black.


S61
After 3 days, hairs started to grow and grew.


S62
After 5 days, stimulated hair growth was confirmed.



After 2 weeks, further growth of terminal hair, elongation and growth of hair were



confirmed all over alopecia site.

SEQ ID NO 29:
S63
After 4 days, hair gained elasticity, resilience and increased volume.

(CFGRKMDRISSMS
S64
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

GLGC)
S65
Hair gained resilience and volume after 3 days. Application continued for 1 week,



and the effect persisted for 1.5 monthhs thereafter.


S66
After 3 days, hair gained elasticity, resilience and increased volume at thinning



area on forehead.



After a few days, hair growth stimulation and hair growth were confirmed.


S67
After 2 days, hair gained elasticity and resilience.



After 5 days, volume of hiar increased. It was confirmed that the stimulated hairs



are predominantly black over white hair.

SEQ ID NO: 30:
S68
After 7 days, stimulated hair growth was confirmed. Even 2 weeks after stopping

(CFGRKMDRISSSV

application, hair growth stimulation and restoration persisted, and there were

GLGC)

little falling hairs.


S69
After 2-3 days, hair gained elasticity and resilience.



After 7 days, hair gained volume, restoration was confirmed.


S70
After 3 days, hairs started to grow and grew.


S71
After 1 day, stimulated hair growth was confirmed.

SEQ ID NO 31:
S72
After 7 days, stimulated hair growth was confirmed. Even 2 weeks after stopping

(CFGRKMDRISSSS

application, hair growth stimulation and restoration persisted, and falling hairs

RLGC)

significantly decreased.


S73
After 1 day, hair volume was increased. Hair gained elasticity and resilience.


S74
After 3 days, stimulation and elongation of hair were confirmed.


S75
After 5 days, hairs stimulated to grow, restored hair and hair growth were



confirmed. It was confirmed that tair elongation rate is fast.

SEQ ID NO 32:
S76
After 7 days, hair gained elasticity. Hair started to grow, and volume of hair on

(CFGRKMDRISSSS

parietal forehead was increased.

GMGC)
S77
After 1 day, hair gained elasticity, resilience and increased volume. Hair started



to grow, restoration and elongation were observed.


S78
After 3 days, hair stimulation and growth were confirmed.


S79
After 2 days, hair gained elasticity, resilience and increased volume. Appearance



of scalp shown through hair got obscure. Hair increase was confirmed.

SEQ ID NO 33:
S80
After 7 days, stimulated hair growth was confirmed.

(CFGRKMDRISSSS
S81
After 1 day, hair became fuller with increased volume. Hair gained elasticity and

GIGC)

resilience.



After 7 days, grown hairs were predominantly black over white hairs. White hairs



turned black.


S82
After 5 days, hair root regeneration and hair growth stimulation were confirmed.


S83
After 1 day, apparent hair growth stimulation and restoration were confirmed.



After 4 days, hair elongation were confirmed.

SEQ ID NO 34:
S84
After 5 days, thin and fuzzy hairs along hairline got thicker and denser.

(CFGRKMDRISSSS
S85
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

GVGC)

to grow, restoration and elongation were observed.


S86
Hair gained resilience and volume after 3 days. Application continued for 1 week,



and the effect persisted for 1.5 months thereafter.


S87
After 3 days, hair gained resilience and volume of hair increased in parietal



thinnig area


S88
After 1 day, hairs started to grow and grow.

SEQ ID NO 35:
S89
After 5 days, hair gained elasticity, resilience, thickness, and increased volume.

(CFGRKMDRISSSS
S90
After 3 days, stimulated hair growth was confirmed.

GAGC)
S91
After 1 day, hair gained elasticity, resilience and increased volume. Hair started



to grow, restoration and elongation were observed.


S92
After 5 days, hair root regeneration and hair growth stimulation were observed.



Then, remarkable restoration, elongation and growth of hair were observed.


S93
After 1 day, stimulated hair was observed. After 2 weeks, there is remarkable



elongation of hair area around large decalvant spot, and new hair



started to grow in center part.

SEQ ID NO 36:
S94
After 4 days, hair stimulation and growth were confirmed.

(CFGRKMDRISSSS
S95
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

GLSC)
S96
After 3 days, seborrhea and erythema were improved.


S97
After 5 days, hair gained elasticity, resilience and increased volume.

SEQ ID NO 37:
S98
After 5 days, hair gained elasticity, resilience, thickness and increased volume.

(CFGRKMDRISSSS
S99
After 2 or 3 days, hair gained elasticity and resilience. After 7 days, hair

GLAC)

gained volume, restoration was confirmed.


S100
After 5 days, stimulated hair growth was confirmed.


S101
After 10 days, hair growth stimulation elongation were confirmed.

SEQ ID NO 38:
S102
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFARKMDRISSSS

to grow, restoration and elongation were observed.

GLGC)
S103
Itches stopped in 3 minutes, dandruff was reduced, redness in scalp was



significantly improved.


S104
After 3 days, stimulated hair growth was confirmed in eyebrows.

SEQ ID NO 39:
S105
After 3 days, stimulated hair growth was confirmed.

(CFSRKMDRISSSS
S106
Itches stopped in 1 minute, dandruff was reduced, redness in scalp was

GLGC)

significantly improved.


S107
After 1 day, hair became fuller with increased volume. Hair gained elasticity and



resilience.



After 7 days, grown hairs were predominantly black over white hairs. White hairs



turned black.


S108
After 3 days, stimulated hair growth was confirmed. More black hairs started to



grow than white hairs, and hair elongation rate was fast.

SEQ ID NO 40:
S109
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFTRKMDRISSSS

to grow, restoration and elongation were observed.

GLGC)
S110
After 4 days, stimulated hair growth was confirmed.

SEQ ID NO 41:
S111
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFGRKMDRISSTS

to grow, restoration and elongation were observed.

GLGC)
S112
After 4 days, stimulated hair growth was confirmed.

SEQ ID NO 42:
S113
After 3 days, stimulated hair growth was confirmed.

(CFGRKMDRISSAS
S114
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

GLGC)
S115
After 3 days, stimulated hair growth was confirmed. Black hairs started to grow



and hair elongation was fast.

SEQ ID NO 43:
S116
After 2-3 days, hair gained elasticity and resilience.

(CFGRKMDRISSST

After 7 days, hair gained volume, restoration was confirmed.

GLGC)
S117
After 3 days, stimulated hair growth was confirmed.

SEQ ID NO 44:
S118
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFGRKMDRISSSA

to grow, restoration and elongation were observed.

GLGC)
S119
After 3 days, stimulated hair growth was confirmed.

SEQ ID NO 45:
S120
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFSRRMDRISSSS

to grow, restoration and elongation were observed.

GLGC)
S121
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

SEQ ID NO 46:
S122
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFSRKMDRISSTS

to grow, restoration and elongation were observed.

GLGC)
S123
After 1 day, hair became fuller with increased volume. Hair gained elasticity and



resilience.



After 7 days, grown hairs were predominantly black over white hairs. White hairs



turned black

SEQ ID NO 47:
S124
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

(CFSRKMDRISSST
S125
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

GLGC)

to grow, restoration and elongation were observed.

SEQ ID NO 48:
S126
After 5 days, hair root regeneration and hair growth stimulation were confirmed.

(CFSRKMDRISSSS
S127
After 1 day, hair became fuller with increased volume. Hair gained elasticity and

GIGC)

resilience.



After 7 days, grown hairs were predominantly black over white hairs. White hairs



turned black.


S128
After 1 day, hair gained elasticity, resilience and increased volume. Hair started



to grow, restoration and elongation were observed.

SEQ ID NO 49:
S129
After 1 day, hair gained elasticity, resilience and increased volume.

(CFSRKMDRISSSS
S130
After 5 days, hair started to grow and hair density was increased.

GLAC)
S131
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

SEQ ID NO 50:
S132
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFGRRMDRISSTS

to grow, restoration and elongation were observed.

GLGC)
S133
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

SEQ ID NO 51:
S134
After 2 or 3 days, hair gained elasticity and resilience.

(CFGRRMDRISSST

After 7 days, hair gained volume, restoration was confirmed.

GLGC)
S135
After 1 day, hair became fuller with increased volume. Hair gained elasticity and



resilience.

SEQ ID NO 52:
S136
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFGRRMDRISSSS

to grow, restoration and elongation were observed.

GIGC)
S137
After 5 days, hair root regeneration and hair growth stimulation were confirmed.


S138
After 1 day, hair became fuller with increased volume. Hair gained elasticity and



resilience.



After 7 days, grown hairs were predominantly black over white hairs. White hairs



turned black.

SEQ ID NO 53:
S139
After 1 day, hair gained elasticity, resilience and increased volume.

(CFGRRMDRISSSS
S140
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

GLAC)

GLAC)

SEQ ID NO 54:
S141
After 2 or 3 days, hair gained elasticity and resilience. After 7 days, hair

(CFGRKMDRISSTS

gained volume, restoratoin was confirmed.

GIGC)
S142
After 1 day, hair becaome fuller with increased volume. Hair gained elasticity and



resilience. After 7 days, grown hairs were predominantly black over white hairs.



White hairs turned black.

SEQ ID NO 55:
S143
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFGRKMDRISSTS

to grow, restoration and elongation were observed.

GLAC)
S144
After 1 day, hair became fuller with increased volume. Hair gained elasticity and



resilience.



After 7 days, grown hairs were predominantly black over white hairs. White hairs



turned black.

SEQ ID NO 56:
S145
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFGRKMDRISSST

to grow, restoration and elongation were observed.

GIGC)
S146
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

SEQ ID NO 57:
S147
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFGRKMDRISSST

to grow, restoration and elongation were observed.

GLAC)
S148
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

SEQ ID NO 58:
S149
After 1 day, hair gained elasticity, resilience and increased volume.

(CFGRKMDRISSSS
S150
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

GIAC)



SEQ ID NO 59:
S151
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFSRRMDRISSTS

to grow, restoration and elongation were observed.

GLGC)
S152
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

SEQ ID NO 60:
S153
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFSRRMDRISSST

to grow, restoration and elongation were observed.

GLGC)
S154
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

SEQ ID NO 61:
S155
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFSRRMDRISSSS

to grow, restoration and elongation were observed.

GIGC)
S156
After 1 day, hair became fuller with increased volume. Hair gained elasticity and



resilience.



After 7 days, grown hairs were predominantly black over white hairs. White hairs



turned black.

SEQ ID NO 62:
S157
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFSRRMDRISSSS

to grow, restoration and elongation were observed.

GLAC)
S158
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

SEQ ID NO 63:
S159
After 2 or 3 days, hair gained elasticity an dresilience. After 7 days, hair

(CFSRKMDRISSTS

gained volume, restoration was confirmed.

GIGC)
S160
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

SEQ ID NO 64:
S161
After 1 day, hair became fuller with increased volume. Hair gained elasticity and

(CFSRKMDRISSST

resilience.

GIGC)

After 7 days, grown hairs were predominantly black over white hairs. White hairs



turned black.


S162
After 1 day, hair gained elasticity, resilience and increased volume. Hair started



to grow, restoration and elongation were observed.

SEQ ID NO 65:
S163
After 2 or 3 days, hair gained elasticity and resilience.

(CFSRKMDRISSST

After 7 days, hair gained volume, restoration was confirmed.

GLAC)
S164
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

SEQ ID NO 66:
S165
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFSRKMDRISSSS

to grow, restoration and elongation were observed.

GIAC)
S166
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

SEQ ID NO 67:
S167
After 2-3 days, hair gained elasticity and resilience.

(CFGRRMDRISSTS

After 7 days, hair gained volume, restoration was confirmed.

GIGC)
S168
After 1 day, hair became fuller with increaserd volume. Hair gained elasticity and



resilience.



After 7 days, grown hairs were predominantly black over white hairs. White hairs



turned black.

SEQ ID NO 68:
S169
After 1 day, hair gained elasticity, resilience and increased volume.

(CFGRRMDRISSTS
S170
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

GLAC)



SEQ ID NO 69:
S171
After 2 or 3 days, hair gained elasticity and resilience.

(CFGRRMDRISSST

After 7 days, hair gained volume, restoration was confirmed.

GIGC)
S172
After 1 day, hairs started to grow and grew.


S173
After 1 day, hair became fuller with increased volume. Hair gained elasticity and



resilience. After 7 days, grown hairs were predominantly black over white hairs.



White hairs turned black.

SEQ ID NO 70:
S174
After 1 day, hair gained elasticity, resilience and increased volume.

(CFGRRMDRISSST
S175
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

GLAC)



SEQ ID NO 71:
S176
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFSRRMDRISSTS

to grow, restoration and elongation were observed.

GIGC)
S177
After 1 day, hairs started to grow and grew.


S178
After 1 day, hair became fuller with increaserd volume. Hair gained elasticity and



resilience.



After 7 days, grown hairs were predominantly black over white hairs. White hairs



turned black.

SEQ ID NO 72:
S179
After 2 or 3 days, hair gained elasticity and resilience. After 7 days, hair

(CFSRRMDRISSTS

gained volume, restoration was confirmed.

GLAC)
S180
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

SEQ ID NO 73:
S181
After 1 day, hair gained elasticity, resilience and increased volume.

(CFSRRMDRISSST
S182
After 5 days, hair started to grow and hair density was increased.

GIGC)
S183
After 1 day, hair became fuller with increased volume. Hair gained elasticity and



resilience.



After 7 days, grown hairs werer predominantly black over white hairs. White hairs



turned black.

SEQ ID NO 74:
S184
After 1 day, hair gained elasticity, resilience and increased volume. Hair started

(CFSRRMDRISSST

to grow, restoration and elongation were observed.

GLAC)
S185
After 1 day, hair volume was increased. Hair gained elasticity and resilience.

SEQ ID NO 75:
S186
After 2 or 3 days, hair gained elasticity and resilience.

(CFSRRMDRISSSS

After 7 days, hair gained volume, restoration was confirmed.

GIAC)
S187
After 5 days, hair root regeneration and hair growth stimulation were confirmed.


S188
After 1 day, hair became fuller with increaserd volume. Hair gained elasticity and



resilience.



After 7 days, grown hairs were predominantly black over white hairs. White hairs



turned black.



In SEQ ID NOS 16-75, 1st Cys and 17th Cys from left form a disulfide bond






















































































































































































































































































































































































































































































































































































































































































































The result described above demonstrated that the cyclic peptide of the invention exhibits a similar effect as the cyclic peptide expressed by Formula I-a even if some amino acids of the cyclic peptide have been replaced.
Specifically, it has effects of preventing hair loss at the applied site, stimulating hair growth or promoting hair growth, giving hair resilience and elasticity, increasing volume of hair and dramatically decreasing falling hairs when it was applied to the site of alopecia or hair growth stimulation, etc. It also has effects of nourishing hair, promoting hair growth stimulation, improving/preventing thinning of hair at the applied site. In this case, the stimulated hair tends to become a terminal (non-white) hair. Moreover, these effect exhibit significantly faster as compared to other active agents conventionally used in alopecia therapeutics such as BNP such that stimulated hair growth can be confirmed one day after only one application even in the case of an intractable alopecia areata multilocularis or ophiasis. In addition, resulting effects persists remarkably even after terminating application and the stimulated hair keep growing. In the case of AGA or healthy subject it exert immediate effect of providing hair with resilience and elasticity on the following day, increasing the volume, and dramatically decreasing falling hairs, which effects last even after terminating application. It also has an improving and preventing effect on dermatitis. Therefore, it can improve or prevent skin inflammation associated with alopecia. Such effect is advantageous in cases where alopecia has been exacerbated due to skin condition of the applied site (e.g., scalp). Moreover, the external preparation of the present invention exerts a moisturizing and skin texture-improving effects at the applied site when being applied to skin as described above. It can remove and suppress dandruff and itching, while giving moisture to hair and scalp, improving and preventing dryness and keeping hair and scalp healthy. It also can improve seborrhea. On the other hand, when it is used as an alopecia therapeutic/prophylactic, it can be used for the purpose of treating or preventing one or more of applicable alopecia including, for example, those described below, without being particularly limited thereto.
(Acquired Alopecia)
(i) Alopecia without accompanying scarring or skin lesion (alopecia areata, male pattern alopecia, seborrheic alopecia, alopecia pityroides, female pattern alopecia, gestational alopecia, malignant alopecia, senile alopecia, alopecia totalis, alopecia areata multilocularis, ophiasis, drug-induced alopecia, cancer chemotherapy-induced alopecia and radiation exposure-induced alopecia, traumatic/mechanical alopecia, malnutrition/metabolic disorder associated alopecia, endocrine dysfunction associated alopecia and telogen effluvium (post partum alopecia, alopecia after high fever)).
(ii) Alopecia observed in skin lesion or pathologic skin (infection-induced alopecia, tumor-induced alopecia, inflammation-induced alopecia).
(iii) Scarring alopecia (skin infection-induced alopecia, alopecia induced by infiltration of inflammatory cells).
(Congenital Alopecia)
Diffuse alopecia, congenital atrichia, alopecia in hereditary syndromes, localized alopecia, phakomatosis, aplasia cutis, congenital alopecia triangularis.
It exerts an excellent effect especially on acquired alopecia, preferably on alopecia without accompanying scarring or skin lesion, more preferably on alopecia areata, male pattern alopecia, seborrheic alopecia, alopecia pityroides, female pattern alopecia, gestational alopecia, malignant alopecia, senile alopecia, alopecia totalis, alopecia areata multilocularis, ophiasis, drug-induced alopecia, cancer chemotherapy-induced alopecia and radiation exposure-induced alopecia, traumatic/mechanical alopecia, malnutrition/metabolic disorder associated alopecia, endocrine dysfunction associated alopecia and telogen effluvium. Therefore, the external preparation of the present invention can be used for the purpose of treating or preventing at least one or more alopecia selected from the above-mentioned group.
One simple application onto aforementioned affected site at 3 μg/ml to 500 μg/ml improved or eliminated itch, dandruff, scalp redness and inflammation immediately thereafter (immediate effect). A single application stimulated hair growth, grew hair, provided hair with resilience, elasticity and increased volume, and dramatically decreased falling hairs, and these effects started on the day after the application and persisted for one week or more (long-lasting effect). If application was carried on for about one week, these effects are further improved, and even after terminating application, the effects lasted from 1 to 2 weeks such that stimulation of hair growth and restoration of hair continued and there were little or no falling hairs.
9.2 Confirming Effects on Skin
The obtained cyclic peptide was prepared in purified water at 3, 15, 30, 100 or 500 μg/ml and applied to affected site. Each of formulations A to E in the table indicates the concentration of the cyclic peptide at 3, 15, 30, 100 or 500 μg/ml, respectively. The absence of the effect upon the application of purified water without the peptide confirmed the effect was not a placebo effect.













TABLE 30



Amino acid









sequence of









cyclic peptide



Diagnostic


Diagnostic impression on
Itches after

mutant used
ID
Sex
Age
Impression
Formulation
Treatment
skin after application
application



SEQ ID NO 16:
Q1
female
30's
eczema
B
once
After 3 minutes, red
Iches were remitted

(CFVRKMDRISSSSGLGC)





a day
papule was reduced and
immediately after








remitted.
application.


Q2
male
20's
atopic
B
once
After 2 minutes,
Iches were eliminated





dermatitis

a day
erythema, edema and
immediately after








infiltration was
application.








remitted.



Q3
male
60's
plaque
C
once
After 3 minutes, crusts,






psoriasis

a day,
scales, erythema and








2 days
infiltration were









remitted.



Q4
male
40's
plaque
C
once
After 10 minutes,






psoriasis

a day
scales, erythema and









infiltration were









remitted.



Q5
female
80's
eczema
C
once
After 3 minutes,








a day
erythema was remitted.



Q6
male
20's
atopic
B
once
Erythema, infiltration
Iches were remitted





dermatitis

a day,
and papule were
immediately after







7 days
remitted.
application.


Q7
female
60's
eczema
C
once
After 2 minutes,








a day
erythema, infiltration









and papule were









remitted. After 20









minutes, they were









ameliorated.



Q8
female
20's
atopic
C
once
After 2 minutes,
Iches were





dermatitis

a day
erythema and
eliminated








infiltration were
in 1 minute.








remitted.



Q9
male
10's
atopic
C
once
Redness vanished
Itches were





dermatitis

a day
immediately after
eliminated








application.
in 1 minute.



SEQ ID NO 17:
Q10
male
40's
atopic
C
once
After 3 minutes,
After 3 minutes,

(CFGQKMDRISSSSGLGC)



dermatitis

a day
erythema and
iches were remitted.








infiltration were









remitted.



Q11
male
10's
nummular
C
once
After 3 minutes,






eczema

a day
infiltration, erythema









exudate and scales were









remitted.



Q12
male
20's
eczema
B
once
After 2 minutes, scales
Iches were







a day
and erythema were
eliminatted








remitted. With only 1
immediately after








application, no
application.








recurrence for 7 days









thereafter.




SEQ ID NO 18:
Q13
male
60's
milaria
C
once
After 2 minutes,


(CFGHKMDRISSSSGLGC)





a day
redness was remitted.



Q14
female
40's
roughened
A
once
After 2 minutes, zits,






skin

a day
dryness and rawness on









skin were reduced, skin









was moisturized and it









texture improved. After









7 days, large wrinkles









and nasolabial fold









became less deep.




SEQ ID NO 19:
Q15
male
20's
eczema,
C
once
After 2 minutes,


(CFGRRMDRISSSSGLGC)



atopic

a day
erythema and scales were






dermatitis


remitted. After 20









minutes, skin got









moisturized. After only









1 application, erythema









vanishes and kept in a









good condition for 1









week.



Q16
male
20's
atopic
C
once
After 5 minutes, wound






dermatitis

a day
was epithelized,









erythema and papule were









improved.



Q17
female
40's
roughened
B
once
After 2 minutes, redness






skin

a day
was remitted. Roughness









and skin texture were









also improved.



Q18
female
30's
roughened
B
once
After 2 minutes, redness






skin

a day
was remitted. Roughness









and skin texture were









also improved. Rawness









got better. Iches









vanished immediately









after application, skin









were moisturized and









smooth.



Q19
male
40's
plaque
C
once
After 3 minutes,






psoriasis

a day
erythema, crusts, scales









and infiltration were









remitted.



Q20
male
40's
eczema
C
once
Erythema was remitted
Itches were extincted







a day
immediately after
immediately after








application.
application.



SEQ ID NO 20:
Q21
female
60's
eczema
C
once
After 1 minute, red
After 3 minutes,

(CFGRKLDRISSSSGLGC)





a day
papule was reduced and
iches were remitted.








extincted.



Q22
male
20's
atopic
B
once
After 2 minutes,
Itches vanished





dermatitis

a day
erythema and scales were
completely








remitted.
immediately after









application.


Q23
female
40's
eczema
C
once
After 2 minutes,
Rawness vanished







a day
erythema, scales and
immediately after








papule were remitted.
application.


Q24
female
30's
roughened
B
once
After 3 minutes, zits,
Iches were eliminated





skin, acne

a day
ruggedness, dry skin and
after application.








redness were remitted,









skin got full and its









texture improved. Acnes









were reduced and redness









was remitted.



Q25
female
60's
wrinkles,
B
once
After 2 minutes,






dry skin

a day
wrinkles became less









deep, dry skin was









remitted.



Q26
female
60's
eczema
C
once
After 3 minutes,








a day
erythema and scales were









remitted.




SEQ ID NO 21:
Q27
male
20's
eczema,
C
once
After 3 days, erythema,
Iches were eliminated

(CFGRKIDRISSSSGLGC)



atopic

a day
scales and papule were
immediately after





dermatitis


improved. Cracks were
application.








epithelized, skin got









moisturized and









improved.



Q28
female
40's
eczema
B
once
After 2 minutes,
After 1 minute, iches







a day
infiltration, erythema,
were eliminated.








papule and scratch wound









were remitted.



Q29
male
40's
atopic
B
once
Immediately after






dermatitis

a day
application, erythema









and scales were









remitted.




SEQ ID NO 22:
Q30
female
30's
atopic
C
once
After 3 minutes, 1
After 30 seconds,

(CFGRKMDRVSSSSGLGC)



dermatitis

a day
application, erythema
iches were








and infiltration were
eliminated.








remarkably remitted, dry









skin, scales and cracks









were also improved.



Q31
female
70's
eczema
C
once
immediately after
Facial rawness







a day
application, erythema
vanished immediately








and papules remitted.
after application.


Q32
female
30's
atopic
B
once
A remarkable improvement






dermatitis

a day,
after 2 days, and








7 days
erythema was eliminated.



Q33
female
40's
atopic
C
once
After 1 minute, erythema
Iches were eliminated





dermatitis

a day,
was remitted. After 2
immediately after







2 days
minutes, scratch wound
application.








was epithelized. After









3 minutes, skin texture









was improved. With only









1-day application,









erythema was eliminated,









skin texture was









improved with no









dryness.



Q34
male
20's
atopic
C
once
After 2 minutes,
Iches were remitted





dermatitis

a day
erythema and scales were
immediately after








remitted.
application.


Q35
female
50's
chronic
C
once
After 2 applications
Iches were eliminated





eczema

a day
lichenificated erythema
immediately after








was remitted. No
application.








recurrence for 3 weeks









thereafter.



Q36
female
50's
roughened
C
once
Skin roughness was cured






skin

a day
immediately after









application. After 2









minutes, skin got









moisturized, its resness









faded. Skin texture was









finely improved. After 7









days, skin tone became









brighter, spots faded









and flabiness was









improved.



Q37
female
30's
roughened
B
once
After 2 minutes, zits,
Rawness vanished





skin

a day
redness was remitted.
immediately after








After 1 day, skin became
application.








normal tone and its









texture was improved.




SEQ ID NO 23:
Q38
female
40's
atopic
C
once
After 2 minutes, scratch
After 1 minute, iches

(CFGRKMDRIGSSSGLGC)



dermatitis

a day
wound was epithelized
were eliminated.








and cured. Erythema was









improved.



Q39
male
20's
eczema
C
once
After 2 minutes,
Itches were absied







a day
erythema, dry skin and
immediately after








scales were improved,
application.








and skin got









moisturized.



Q40
male
20's
atopic
C
once
After 2 minutes,
Itches were extincted





dermatitis

a day
erythema, scales and
immediately after








infiltration were
application.








remitted. With 1









application, no









recurrence for 7 days.



Q41
male
40's
plaque
C
once
After 3 minutes, crusts,






psoriasis

a day
scales, erythema and









infiltration were









remitted.



Q42
female
30's
roughened
B
once
After 2 minutes,
After 2 minutes,





skin

a day
roughness, zits and
itches were








redness were reduced,
extincted.








skin got moisturized and









its texture was









improved.



Q43
female
50's
rosacea,
C
once
After 1 minute,






acne

a day
erythema, prolong









eclasia and acne was









remitted. After 2









minutes, papule,









infiltration, acne, dry









skin and redness was









remitted.




SEQ ID NO 24:
Q44
male
10's
atopic
C
once
After 3 minutes,


(CFGRKMDRISASSGLGC)



dermatitis

a day
erythema, scale and









infiltration were









remitted. Further









improvement after 40









minutes. Scratch wound









was epithelized.



Q45
male
20's
atopic
C
once
After 3 minutes,
After 3 minutes,





dermatitis

a day
erythema and
iches were








infiltration were
eliminated.








remitted.



Q46
male
20's
eczema
C
once
After 3 days, cracks








a day
were epithelized.









Erythema, scales and









infiltration were









remitted.



Q47
male
20's
atopic
C
once
After 2 minutes, wound
Iches were eliminated





dermatitis

a day
was epithelized.
immediately after








Erythema, scales and
application.








lichenification was









remitted. After 5









minutes, skin got









softened, erythema and









infiltration were









improved.




SEQ ID NO 25:
Q48
male
20's
eczema,
C
once
After 2 minutes,
Iches were remitted

(CFGRKMDRISSQSGLGC)



atopic

a day
erythema, scales and
immediately after





dermatitis


infiltration were
application.








remitted. Skin got









moisturized.



Q49
female
40's
roughened
B
once
After 3 minutes, deep
Rawness was





skin

a day
wrinkles were less deep,
eliminated








dry skin and redness
immediately after








were improved, skin got
application.








moisturized and full. By









using for t week, large









wrinkles and nasolabial









fold became less deep.









skin became brighter.









Spots faded. Flabiness









was improved.




SEQ ID NO 26:
Q50
male
60's
plaque
C
once
After 2 minutes, scales


(CFGRKMDRISSVSGLGC)



psoriasis

a day
and infiltration









remitted.



Q51
male
30's
miliaria
C
once
After 2 minutes,








a day
remitted.



Q52
male
20's
acne
C
once
After 6 minutes, acne






vulgaris

a day
became dry and was









reduced in size.



Q53
female
60's
eczema
C
once
After 3 minutes,








a day
erythema and









infiltration were









remitted. After 4









minutes, ameliorated.



Q54
female
10's
atopic
C
once
After 2 minutes, strong






dermatitis

a day
itches almost vanished.









Erythema and









lichenification were









improved. Skin got









softened.



Q55
female
40's
roughened
C
once
After 2 minutes, zits,






skin

a day
redness and roughness









were improved. Skin got









moisturized and its









texture was improved.









skin was improved.




SEQ ID NO 27:
Q56
female
50's
roughened
C
once
After 3 minutes, dry
Rawness vanished

(CFGRKMDRISSLSGLGC)



skin, acne

a day
skin was improved, skin
immediately after








got moisturized and its
application.








texture was finely









improved. Redness









vanished. Inframmation









in acne was remitted,









redness faded and dried.









By using for 1 week,









skin became brighter.



Q57
male
20's
atopic
C
once
After 3 minutes,
Iches were eliminated





dermatitis

a day
erythema, infiltration
immediately after








and lichenification were
application.








improved.




SEQ ID NO 28:
Q58
female
30's
eczema
C
once
After 1 minute,
After 1 minute, iches

(CFGRKMDRISSISGLGC)





a day
erythema and
were eliminated.








infiltration were









remitted.



Q59
female
20's
roughened
A
once
After 7-day continuous






skin

a day
application, skin









roughness vanished,









wrinkles were less deep,









and skin became









brighter.



Q60
female
20's
roughened
C
once
After 2 minutes, scales






skin

a day
were improved. With 1









application/day,









roughness was improved









and skin could be kept









moisturized.




SEQ ID NO 29:
Q61
female
30's
eczema
C
once
After 1 minute,
Iches were eliminated

(CFGRKMDRISSMSGLGC)





a day
erythema and
immediately after








infiltration were
application.








remitted.




SEQ ID NO 30:
Q62
female
20's
atopic
B
once
After 3 days,


(CFGRKMDRISSSVGLGC)



dermatitis

a day,
erythema and








7 days
infiltration were









remitted.



Q63
male
60's
eczema
C
once
After 1 minute,
Iches were eliminated







a day
erythema and papule were
immediately after








remitted. After 3 days,
application.








they were almost cured.



Q64
female
50's
dry skin
C
once
Immediately after








a day
application, skin got









promptly moisturized and









its texture was









improved. Zits, erythema









and roughened skin were









improved.



Q65
male
20's
atopic
C
once
After 10 minutes,
Iches were eliminated





dermatitis

a day
lichenification was
immediately after








improved. Skin got
application.








softened. erythema and









scales were improved.



Q66
female
40's
roughened
A
once
After 1 day with 1






skin

a day
application, skin









conditions of roughness,









zits and itches were









well improved, and skin









texture was finely









improved. The effect









lasted for 7 days









thereafter.




SEQ ID NO 31:
Q67
male
20's
atopic
C
once
After 2 minutes,


(CFGRKMDRISSSSRLGC)



dermatitis

a day
erythema and papule was









remitted.




SEQ ID NO 32:
Q68
male
30's
miliaria
C
once
After 2 minutes, red


(CFGRKMDRISSSSGMGC)





a day
papule was reduced in









size, skin dryness was









remitted.



Q69
male
10's
atopic
C
once
After 3 minutes, exudate






dermatitis,

a day
stopped, erythema and






nummular


infiltration were









improved.




SEQ ID NO 33:
Q70
female
30's
contact
C
2
After 3 minutes,
After 1 minute, iches

(CFGRKMDRISSSSGIGC)



dermatitis

appli-
erythema, infiltration
were eliminated.







cation
and eczema were









remitted.



Q71
female
30's
atopic
E
once
Immediately after
Iches were eliminated





dermatitis

a day
application, erythema
immediately after








and scales were
application.








improved.



Q72
female
70's
plaque
C
2
After 5 minutes,






psoriasis

appli-
erythema, scales and








cation
infiltration were









remitted.



Q73
female
40's
roughened
B
once
In 2 minutes after






skin

a day
application, skin was









improved. Skin texture









was finely improved.









Skin got softened.




SEQ ID NO 34:
Q74
female
30's
eczema
C
2
After 3 minutes,
After 1 minute, iches

(CFGRKMDRISSSSGVGC)





appli-
erythema, infiltration
were eliminated.







cation
and edema was remitted.



Q75
male
20's
eczema,
C
2
After 5 minutes, acne






acne

appli-
was dried and reduced in






vulgaris

cation
size. Erythema and









scales were remitted.



Q76
female
70's
plaque
C
2
After 5 minutes,






psoriasis

appli-
erythema, scales and








cation
infiltration were









remitted.



Q77
female
40's
roughened
C
once
After 2 minutes, skin






skin

a day
texture was improved,









skin got full, and









redness and dryness were









improved.



Q78
female
50's
roughened
B
once
After 3 minutes, skin






skin

a day
got moisturized and its









texture was improved.



Q79
male
20's
atopic
C
once
After 3 minutes,
Itches were improved





dermatitis

a day
erythema and scales
immediately after








were improved.
application.


Q80
female
50's
roughened
B
once
After 3 minutes, skin
Iches were eliminated





skin

a day
roughness was improved,
immediately after








skin got moisturized and
application.








its texture was finely









improved. Skin gained









resilience. After 7









days, large wrinkles and









nasolabial fold became









less deep. By continuing









for 4 weeks, spots faded









and skin tone became









brighter. Skin









resilience was improved.









Large wrinkles became









less deep.



Q81
male
30's
roughened
B
once
After 3 minutes, skin






skin

a day
roughness and redness









were removed. Skin









texture was finely









improved. By continuing









for 7 days, skin









resilience was improved.




SEQ ID NO 35:
Q82
female
40's
roughened
B
once
After 3 minutes,


(CFGRKMDRISSSSGAGC)



skin

a day
roughened skin and dry









skin were promptly









improved, skin texture









was improved.



Q83
male
60's
plaque
C
twice
After 30 minutes,






psoriasis

a day
erythema, scales and









infiltration were









improved.



Q84
male
40's
plaque
C
twice
After 20 minutes,






psoriasis

a day
erythema and scales were









improved.



Q85
female
70's
plaque
C
twice
After 5 minutes,






psoriasis

a day
erythema and scales were









remitted.




SEQ ID NO 36:
Q86
female
10's
atopic
C
once
After 2 minutes,
After 2 minutes,

(CFGRKMDRISSSSGLSC)



dermatitis

a day
lichenification was
strong iches were








remarkably remitted.
eliminated.








erythema, scales,









infiltration, cracks,









scratch wound, etc. all









remitted. After 5









minutes, improved with









only mild infiltration









left.



Q87
female
20's
atopic
B
once
After 1 minute,
After 2 minutes,





dermatitis

a day
erythema, infiltration
iches were








and papule remitted.
eliminated.








After 4 minutes,









infiltration was









remitted, with only mild









erythema left.



Q88
male
10's
atopic
C
once
After 3 minutes,
After 3 minutes,





dermatitis

a day
erythema, scales,
iches were








infiltration and scratch
eliminated.








wound were remitted.



Q89
female
40's
roughened
B
once
Immediately after






skin

a day
application, itches and









redness were removed.









After 3 minutes, skin









got full and resilient,









and skin was improved.



Q90
male
50's
seborrheic
C
once
Immediately after
Itches were remitted





dermatitis

a day,
application, seborrhes
immediately after







3 days
were remitted. After 3
application.








days, erythema and









seborrhes were remitted.




SEQ ID NO 37:
Q91
male
60's
plaque
C
once
After 2 minutes,


(CFGRKMDRISSSSGLAC)



psoriasis

a day
erythema and scales were









remitted.



Q92
male
20's
atopic
C
once
After 2 minutes,
Iches were eliminated





dermatitis,

a day
erythema and
immediately after





prurigo


infiltration were
application.





nodularis


remitted. Applied twice.









After 15 minutes,









prurigo nodularis on hip









was remitted.



Q93
female
40's
roughened
B
once
After 3 minutes,






skin

a day
roughened skin and dry









skin were improved









promptly, and skin









texture was improved.



Q94
male
40's
plaque
C
once
After 20 minutes,






psoriasis

a day
erythema and scales were









remitted.



Q95
male
30's
atopic
C
once
After 2 minutes, scratch






dermatitis

a day
wound epithelized,









erythema was remitted.




SEQ ID NO 38:
Q96
male
20's
atopic
C
once
After 3 minutes, cracks
immediately after

(CFARKMDRISSSSGLGC)



dermatitis

a day
was reduced in size.
application, itches








Erythema, scales and
and rawness were








lichenification were
removed.








remitted.



Q97
female
40's
roughened
B
once
After 1 minute, dry skin
immediately after





skin

a day
was improved, skin got
application, itches








moisturized and full,
were removed.








and skin texture was









improved.



Q98
female
20's
chronic
C
once
Immediately after






eczema

a day
application, erythema









and scales were









remitted.



Q99
male
20's
atopic
C
once
After 2 minutes,
After 1 minute, iches





dermatitis

a day
redness was improved.
were eliminated.








After 3 minutes,
After 1 application,








erythema, infiltration
mo itches for 8 days








and papule were
thereafter.








remitted. After 1









application, the effect









lasted for 8 days with









no recurrence.



Q100
female
50's
prurigo
C
once
After 3 minutes, nodes
Iches were eliminated





nodularis,

a day
were flattened. scratch
immediately after





eczema


wound became dry and
application.








epithelized with no more









exudate.




SEQ ID NO 39:
Q101
female
50'S
prurigo
C
once
After 3 minutes, nodes


(CFSRKMDRISSSSGLGC)



nodularis,

a day
were fattened.






eczema






Q102
female
10's
chronic
C
once
After 2 minutes, an






eczema

a day
intractable eczema that









cannot be remitted by









applying strong steroid









ointments was remitted,









and erythema, scales and









papule were reduced.



Q103
female
50's
eczema
C
once
After 2 minutes,








a day
erythema, scales and









pigmentation were









remitted. Skin got full









and soft, with fresh









color.



Q104
female
20's
atopic
D
once
Scratch wound was
Iches were eliminated





dermatitis

a day
epithelized. Exudate was
immediately after








dried and
application.








lichenification was









remitted. erythema and









infiltration were









remitted.



Q105
male
20's
atopic
B
once
After 3 minutes,
After 2 minutes,





dermatitis

a day
erythema and
itches and tingling








infiltration were
pain were removed.








remitted. With 1
The effect persisted








application, the effect
for 7 days with no








persisted for 1 week
itches.








with no recurrence.




SEQ ID NO 40:
Q106
male
40's
atopic
C
once
After 2 minutes,
Iches were eliminated

(CFTRKMDRISSSSGLGC)



dermatitis

a day
erythema, infiltration
immediately after








and lichenification
application.








was remitted.



Q107
female
20's
roughened
C
1
After 1 minute, dry skin






lips

appli-
was improved, cracks








cation
were less deep and









epithelized.



Q108
female
40's
roughened
B
once
After 3 minutes, redness






skin

a day
and roughness were









improved.



Q109
female
30's
healthy
C
once
After 3 minutes, dark-






skin

a day
reddish skin tone was









improved to turn fresh









color. Dryness,









tautness, rawness and









hot flash of skin were









removed. With 1









application, the effect









persisted for 1 week.




SEQ ID NO 41:
Q110
male
10's
atopic
C
once
immediately after
Iches were eliminated

(CFGRKMDRISSTSGLGC)



dermatitis/

a day
application, erythema
immediately after





acne


and infiltration of
application.








eczema were remitted.









The effects were









exhibited remarkably









fast. After 3 minutes,









erythema was further









remitted. Acne was dried









and reduced in size.



Q111
male
20's
atopic
C
once
immediately after
Iches were eliminated





dermatitis

a day
application, erythema
immediately after








and infiltration were
application.








remitted. After 20









minutes, deep scratch









wound was epithelized.



Q112
female
40's
roughened
B
1
After 1 minute, skin






lips

appli-
tautness and rawness








cation
were improved and skin









got moisturized.



Q113
female
50's
atopic
B
once
Iches stopped in 30






dermatitis

a day
seconds. Redness and









edema were remitted in









1 minute.



Q114
female
10's
roughened
B
once
After 1 minute, redness
After 3 minutes, skin





skin/acne

a day
was remitted. After 3
tautness was removed.








minutes, skin roughness,









zits, rough and stiff









skin texture were









improved, skin got full









and its texture was









finely improved. Acne









became dry and reduced









in size.



Q115
female
40's
roughened
B
once
Rawness vanished in 1






skin

a day
minute. Skin was









moisturized. After 2









minutes, redness,









roughness and stiffness









were remarkably









improved. By using for 1









week, skin became









brighter, flabbiness was









improved.




SEQ ID NO 42:
Q116
female
40's
acne
C
once
After 1 minute, redness


(CRGRKMDRISSASGLGC)





a day
was remitted.





Q117
male
10's
atopic
B
once
After 3 minutes,
Iches were eliminated





dermatitis

a day
erythema and
immediately after








infiltration were
application.








remitted.



Q118
female
60's
eczema
C
once
After 3 minutes,








a day
erythema and dry skin









remitted.




SEQ ID NO 43:
Q119
male
40's
atopic
C
once
After 2 minutes, no
After 3 minutes,

(CFGRKMDRISSSTGLGC)



dermatitis

a day
discomfort, felt fresh.
roughness and








Erythema, infiltration
stiffness were








and lichenification
removed, and itches








were remitted.
vanished.


Q120
female
30's
roughened
C
once
1 minute after






lips

a day
application, dry skin









was improved, cracks









became less deep and









epithelized.



Q121
female
40's
roughened
B
once
After 3 minutes, redness






skin

a day
and dry skin were









improved. Skin got









moisturized promptly,









and skin texture was









improved. Skin rawness,









stiffness and irritation









were eliminated. Pores









became less









distinguished.



Q122
female
40's
rosecea/
C
once
After 7 minutes, acne






acne

a day
was reduced and became









dry.



Q123
female
30's
eczema
C
once
After 1 minute, redness
Iches were eliminated







a day
and infiltration was
immediately after








remitted.
application.



SEQ ID NO 44:
Q124
female
50's
healthy
B
once
Skin got moisturized


(CFGRKMDRISSSAGLGC)



skin

a day
promptly, wrinkles









became less deep, skin









texture was improved. By









using for 4 weeks, large









wrinkles and nasolabial









fold became less deep.









Skin resilience and









flabbiness were









improved. Spots faded.









Skin tone became









brighter.



Q125
female
60's
eczema
C
once
After 1 minute,
Iches were remitted







a day
erythema and
immediately after








infiltration were
application.








remitted.




SEQ ID NO 45:
Q126
female
40's
roughened
B
once
After 2 minutes,


(CFSRRMDRISSSSGLGC)



skin

a day
dryness, tautness and









rawness of skin were









eliminated, skin got









full and wrinkles became









less deep. Skin gained









elasticity. By using for









5 days, flabbiness was









improved. Skin became









brighter, nasolabial









fold became less deep.









By using for 4 weeks,









nasolabial fold became









less deep. Skin









resilience and









flabbiness were









improved. Spots faded









and skin tone became









brighter.




SEQ ID NO 46:
Q127
female
40's
eczema
C
once
After 2 minutes,


(CFSRKMDRISSTSGLGC)





a day
erythema, scales and









infiltration were









remitted. Skin got









softened.




SEQ ID NO 47:
Q128
female
30's
chronic
C
once
immediately after


(CFSRKMDRISSSTGLGC)



eczema

a day
application, erythema









and scales were









remitted.




SEQ ID NO 48:
Q129
male
50's
eczema
C
once
After 2 minutes,


(CFSRKMDRISSSSGIGC)





a day
erythema, scales and









infiltration were









remitted. Skin got









moisturized.




SEQ ID NO 49:
Q130
male
30's
chronic
C
once
Immediately after


(CFSRKMDRISSSSGLAC)



eczema

a day
application, erythema









and scales were









remitted.




SEQ ID NO 50:
Q131
male
50's
eczema
C
once
After 2 minutes,


(CFGRRMDRISSTSGLGC)





a day
erythema, scales and









infiltration were









remitted.




SEQ ID NO 51:
Q132
female
60's
eczema
B
once
After 3 minutes,


(CFGRRMDRISSSTGLGC)





a day
erythema and









infiltration were









remitted.




SEQ ID NO 52:
Q133
female
50's
eczema
C
once
After 2 minutes,


(CFGRRMDRISSSSGIGC)





a day
erythema, scales and









infiltration were









remitted. Skin got









moisturized.




SEQ ID NO 53:
Q134
female
20's
eczema
C
once
After 1 minute,


(CFGRRMDRISSSSGLAC)





a day
erythema and









infiltration were









remitted.




SEQ ID NO 54:
Q135
female
30's
roughened
B
once
After 2 minutes,


(CFGRKMDRISSTSGIGC)



skin

a day
dryness, tautness and









rawness of skin were









eliminated, skin got









full and wrinkles









become less deep. Skin









gained elasticity.




SEQ ID NO 55:
Q136
female
50's
eczema
C
once
After 2 minutes,


(CFGRKMDRISSTSGLAC)





a day
erythema, scale and









infiltration were









remitted.




SEQ ID NO 56:
Q137
male
20's
roughened
B
once
After 2 minutes,


(CFGRKMDRISSSTGIGC)



skin

a day
dryness, tautness and









rawness of skin were









eliminated, skin got









full, and wrinkles









became less deep. Skin









gained elasticity.




SEQ ID NO 57:
Q138
female
40's
eczema
C
once
After 2 minutes,


(CFGRKMDRISSSTGLAC)





a day
erythema, scales and









infiltration were









remitted, scratch wound









was epithelized and









dried.




SEQ ID NO 58:
Q139
female
30's
eczema
C
once
After 2 minutes,


(CFGRKMDRISSSSGIAC)





a day
erythema, scales, papule









and infiltration were









remitted.




SEQ ID NO 59:
Q140
male
60's
roughened
B
once
After 2 minutes,


(CFSRRMDRISSTSGLGC)



skin

a day
dryness, tautness and









rawness of skin were









eliminated. Skin gained









elasticity, wrinkles









became less deep




SEQ ID NO 60:
Q141
female
50's
chronic
C
once
Immediately after


(CFSRRMDRISSSTGLGC)



eczema

a day
application, erythema









and scales were









remitted.




SEQ ID NO 61:
Q142
female
30's
chronic
C
once
Immediately after


(CFSRRMDRISSSSGIGC)



eczema

a day
application, erythema









and scales were









remitted.




SEQ ID NO 62:
Q143
female
40's
roughened
B
once
After 2 minutes,


(CFSRRMDRISSSSGLAC)



skin

a day
dryness, tautness and









rawness of skin were









eliminated, skin got









full, and wrinkles









became less deep. Skin









gained elasticity.




SEQ ID NO 63:
Q144
male
50's
eczema
B
once
After 3 minutes,


(CFSRKMDRISSTSGIGC)





a day
erythema and scales/









infiltration were









remitted, and skin got









softened.




SEQ ID NO 64:
Q145
male
40's
chronic
C
once
Immediately after


(CFSRKMDRISSSTGIGC)



eczema

a day
application, erythema









and scales were









remitted.




SEQ ID NO 65:
Q146
female
40's
roughened
C
once
After 1 minute, skin got


(CFSRKMDRISSSTGLAC)



skin

a day
moisturized and









resilient, skin texture









was improved.




SEQ ID NO 66:
Q147
male
50's
eczema
C
once
After 2 minutes,


(CFSRKMDRISSSSGIAC)





a day
erythema, scales and









infiltration were









remitted.




SEQ ID NO 67:
Q148
female
70's
roughened
B
once
After 2 minutes,


(CFGRRMDRISSTSGIGC)



skin

a day
dryness, tautness and









rawness of skin were









eliminated, skin got









full, and wrinkles









became less deep. Skin









gained elasticity.




SEQ ID NO 68:
Q149
female
30's
chronic
C
once
Immediately after


(CFGRRMDRISSTSGLAC)



eczema

a day
application, erythema









and scales were









remitted.




SEQ ID NO 69:
Q150
female
20's
eczema
C
once
After 2 minutes,


(CFGRRMDRISSSTGIGC)





a day
erythema, scales,









infiltration were









improved.




SEQ ID NO 70:
Q151
female
60's
chronic
C
once
Immediately after


(CFGRRMDRISSSTGLAC)



eczema

a day
application, erythema









and scales were









remitted.



Q152
female
30's
roughened
C
once
Redness vanished in 30






skin

a day
seconds. After 1 minute,









skin texture improved









finely. Pores became









less distinguished.




SEQ ID NO 71:
Q153
male
50's
roughened
A
once
After 2 minutes,


(CFSRRMDRISSTSGIGC)



skin

a day
dryness, tautness and









rawness of skin were









eliminated, skin got









full, and wrinkles









became less deep. Skin









gained elasticity.




SEQ ID NO 72:
Q154
male
50's
chronic
C
once
Immediately after


(CFSRRMDRISSTSGLAC)



eczema

a day
application, erythema,









scales and papule were









remitted.




SEQ ID NO 73:
Q155
male
20's
chronic
C
once
Immediately after


(CFSRRMDRISSSTGIGC)



eczema

a day
application, erythema









and scales were









remitted.




SEQ ID NO 74:
Q156
female
30's
roughened
C
once
After 1 minute, rawness


(CFSRRMDRISSSTGLAC)



skin

a day
and hot flash were









eliminated, skin got









moisturized and its









texture improved,









wrinkles faded and skin









gained elasticity.



Q157
male
60's
chronic
D
once
Iches were eliminated






eczema

a day
immediately after









application. Redness was









remitted.




SEQ ID NO 75:
Q158
female
60's
eczema
C
once
After 2 minutes,


(CFSRRMDRISSSSGIAC)





a day
erythema, scales,









papule, infiltration









were remitted.




SEQ ID NO 43:
Q258
male
40's
atopic
C
once
After 2 minutes, cracks


(CFGRKMDRISSSTGLGC)



dermatitis

a day
were epithelized with no


and






more pains and itches.


SEQ ID NO 19:






Erythema and scales were


(CFGRRMDRISSSSGLGC)






also remitted.




SEQ ID NO 43:
Q358
male
40's
eczema
C
once
After 2 minutes, wound


(CFGRKMDRISSSTGLGC)





a day
was epithelized quickly,


and






and erythema, scales and


SEQ ID NO 39:






infiltration were









remarkably remitted.



In SEQ ID NOS 16-75, 1st Cys and 17th Cys from left form a disulfide bond.


















































































































































































































































































































































































































































































































































































































































































































































































































































































































































The result described above demonstrated that the cyclic peptide of the invention exhibits a similar effect as the cyclic peptide expressed by Formula I-a even if some amino acids of the cyclic peptide have been replaced. Moreover, a similar effect could be obtained by applying the replaced cyclic peptide in a mixture as long as an effective amount of the cyclic peptide is applied.
Specifically, in subjects having atopic dermatitis or eczema, effects were observed immediately after applying 3 μg/ml to 500 μg/ml to affected site, including elimination of itches, epithelialization at infiltrated site, papule or scratch, termination of exudation, epithelialization of a crack. Moreover, a single application induced a persistent effect for a week or more.
When it was applied to healthy or roughened skin, it exerted a moisturizing effect immediately or within a few minutes after application, improving dryness, providing and keeping skin and mucosa with moderate elasticity and flexibility, softening skin, giving skin or mucosa resilience and firmness. Also, fine wrinkles and flabbiness are improved at the applied site, and dullness and spots are further faded. A single application induced a persistent effect for a week or more.
9.3 Confirming Nasal Effect
The obtained cyclic peptide was prepared in physiological saline at 3, 15, 30, 100 or 500 μg/ml, and 0.1 ml each was applied to nasal cavity. Each of formulations A to E in the table indicates the concentration of the cyclic peptide at 3, 15, 30, 100 or 500 μg/ml, respectively. The absence of the effect upon the application of physiological saline without the peptide confirmed the effect was not a placebo effect.















SEQ ID NO









(amino acid sequence)



Diagnostic


Diagnostic impression on
Itches after

of cyclic peptides
ID
Sex
Age
impression
Formulation
Treatment
skin after application
application



SEQ ID NO 16:
P1
female
10's
allergic
C
once a day,
From immediately after
Itches were remitted

(CFVRKMDRISSSSGLGC)



rhinitis

0.1 ml applied
nasal application, and
immediately after







to each nasal
nose got cleared,
nasal application,







cavity
rhinorrhea stopped. There
and eliminated after








was no irritating symptoms,
1 minute.








and the effect persisted









for all day by 1 nasal









application.




SEQ ID NO 17:
P2
male
30's
allergic
D
once a day,
From immediately after
Itches were remitted

(CFGQKMDRISSSSGLGC)



rhinitis

0.1 ml applied
nasal application, and
immediately after







to each nasal
nose got cleared,
nasal application,







cavity
rhinorrhea stopped. There
and eliminated after








was no irritating symptoms,
1 minute.








and the effect persisted









for all day by 1 nasal









application.




SEQ ID NO 18:
P3
male
50's
allergic
C
once a day,
After 3 minutes, and nose


(CFGHKMDRISSSSGLGC)



rhinitis

0.1 ml applied
got cleared, rhinorrhea








to each nasal
stopped. There was no








cavity
irritating symptoms, and









the effect persisted for









all day by 1 nasal









application.




SEQ ID NO 19:
P4
female
40's
chronic
B
once a day,
After 1 minute, and nose


(CFGRRMDRISSSSGLGC)



rhinitis,

0.1 ml applied
got cleared, nasal






perennial

to each nasal
discharge stopped. There






rhinitis

cavity
was no irritating symptoms.



P5
female
70's
allergic
B
once a day,
After 2 minutes, and nose
Itches were remitted





rhinitis

0.1 ml applied
got cleared, and no nasal
immediately after







to each nasal
discharge came out by
nasal application.







cavity
blowing nose.




SEQ ID NO 20:
P6
female
40's
allergic
B
once a day,
After 1 minute, nasal


(CFGRKLDRISSSSGLGC)



rhinitis

0.1 ml applied
obstruction was improved,








to each nasal
and nose got cleared. The








cavity
effect persisted all day.




SEQ ID NO 21:
P7
female
20's
allergic
C
once a day,
After 1 minute, and nose


(CFGRKIDRISSSSGLGC)



rhinitis

0.1 ml applied
got cleared. Nasal








to each nasal
discharge stopped.








cavity





SEQ ID NO 22:
P8
female
50's
allergic
E
once a day,
Immediately after nasal
Immediately after

(CFGRKMDRVSSSSGLGC)



rhinitis

0.1 ml applied
application, and nose got
nasal application,







to each nasal
cleared.
restless itches and







cavity

sneezing stopped.


P9
female
40's
chronic
C
once a day,
After 20 seconds, and nose






rhinitis,

0.1 ml applied
got cleared. No nasal






perennial

to each nasal
discharge after 1 minute.






rhinitis

cavity
There was no irritating









symptoms.




SEQ ID NO 23:
P10
female
30's
allergic
C
once a day,
After 1 minute, and nose


(CFGRKMDRIGSSSGLGC)



rhinitis

0.1 ml applied
got cleared. Nasal








to each nasal
discharge stopped.








cavity





SEQ ID NO 24:
P11
female
20's
allergic
C
once a day,
After 1 minute, and nose


(CFGRKMDRISASSGLGC)



rhinitis

0.1 ml applied
got cleared. Nasal








to each nasal
discharge stopped.








cavity





SEQ ID NO 25:
P12
female
20's
allergic
A
once a day,
After 2.5 minutes, and nose


(CFGRKMDRISSQSGLGC)



rhinitis

0.1 ml applied
got cleared.








to each nasal









cavity





SEQ ID NO 26:
P13
female
50's
allergic
C
once a day,
After 1 minute, nose got


(CFGRKMDRISSVSGLGC)



rhinitis

0.1 ml applied
cleared and refreshed.








to each nasal
After 2 minutes, no nasal








cavity
discharge by blowing.




SEQ ID NO 27:
P14
male
20's
allergic
C
once a day,
After 1 minute, and nose


(CFGRKMDRISSLSGLGC)



rhinitis

0.1 ml applied
got cleared.








to each nasal







SEQ ID NO 28:
P15
female
40's
allergic
C
once a day,
After 1 minute, and nose


(CFGRKMDRISSISGLGC)



rhinitis

0.1 ml applied
got cleared. Nasal








to each nasal
discharge stopped.








cavity





SEQ ID NO 29:
P16
male
50's
allergic
C
once a day,
After 1 minute, and nose


(CFGRKMDRISSMSGLGC)



rhinitis

0.1 ml applied
got cleared. Nasal








to each nasal
discharge stopped.








cavity





SEQ ID NO 30:
P17
female
30's
allergic
C
once a day,
After 1 minute, rhinorrhea


(CFGRKMDRISSSVGLGC)



rhinitis

0.1 ml applied
stopped.








to each nasal









cavity





SEQ ID NO 31:
P18
female
10's
allergic
D
once a day,
After 30 seconds,


(CFGRKMDRISSSSRLGC)



rhinitis

0.1 ml applied
rhinorrhea stopped. After 1








to each nasal
minute, no nasal discharge








cavity
by blowing.




SEQ ID NO 32:
P19
male
40's
allergic
C
once a day,
After 1 minute, nasal


(CFGRKMDRISSSSGMGC)



rhinitis

0.1 ml applied
obstruction was improved,








to each nasal
and nose got cleared. The








cavity
effect persisted for all









day.



P20
female
40's
chronic
C
once a day,
Immediately after nasal






rhinitis,

0.1 ml applied
application nose got






perennial

to each nasal
cleared. The effect was






rhinitis

cavity
remarkable and persisted









for 7 days after 1 nasal









application.




SEQ ID NO 33:
P21
female
40's
allergic
C
once a day,
After 30 seconds,
Sneezing and itches

(CFGRKMDRISSSSGIGC)



rhinitis

0.1 ml applied
rhinorrhea stopped. After 1
in deep inside nose







to each nasal
minute, no nasal discharge
stopped.







cavity
by blowing.




SEQ ID NO 34:
P22
female
40's
allergic
C
once a day,
After 2.5 minutes, and nose


(CFGRKMDRISSSSGVGC)



rhinitis

0.1 ml applied
got cleared.








to each nasal





SEQ ID NO 35:
P23
female
30's
allergic
C
once a day,
After 1 minute, nasal


(CFGRKMDRISSSSGAGC)



rhinitis

0.1 ml applied
obstruction was improved,








to each nasal
and nose got cleared. The








cavity
effect persisted for all









day.



P24
female
40's
chronic
D
once a day,
Immediately after nasal






rhinitis,

0.1 ml applied
application, nose got






perennial

to each nasal
refreshed and cleared.






rhinitis

cavity





SEQ ID NO 36:
P25
female
40's
allergic
C
once a day,
Immediately after nasal
Immediately after

(CFGRKMDRISSSSGLSC)



rhinitis

0.1 ml applied
application, nose got
nasal application,







to each nasal
cleared.
itches were removed.







cavity

No Irritating









symptoms.



SEQ ID NO 37:
P26
female
40's
allergic
C
once a day,
After 30 seconds,
Immediate effect to

(CFGRKMDRISSSSGLAC)



rhinitis

0.1 ml applied
rhinorrhea stopped. Nose
remove itches.







to each nasal
got cleared and felt good.








cavity
Other nose drops causes









this subject to sneeze due









to some irritant, but the









inventive nose drop had no









irritation. The effect









persisted The effect









persisted for 1 week by 1









nasal application.



P27
female
40's
chronic
C
once a day,
Nose got cleared and






rhinitis,

0.1 ml applied
rhinorrhea stopped in 30






perennial

to each nasal
seconds.






rhinitis

cavity





SEQ ID NO 38:
P28
male
30's
chronic
C
once a day,
After 2 minutes, rhinorrhea


(CFARKMDRISSSSGLGC)



rhinitis

0.1 ml applied
stopped. Nasal obstruction








to each nasal
was improved to allow,








cavity
nasal breathing.




SEQ ID NO 39:
P29
male
40's
allergic
C
once a day,
Immediately after nasal


(CFSRKMDRISSSSGLGC)



rhinitis

0.1 ml applied
application, nose got








to each nasal
refreshed and cleared.








cavity
No nasal discharge after 30









seconds.




SEQ ID NO 40:
P30
female
30's
allergic
C
once a day,
After 2.5 minutes, and nose


(CFTRKMDRISSSSGLGC)



rhinitis

0.1 ml applied
got cleared.








to each nasal





SEQ ID NO 41:
P31
female
20's
allergic
C
once a day,
After 1 minute, rhinorrhea


(CFGRKMDRISSTSGLGC)



rhinitis

0.1 ml applied
stopped. There was no








to each nasal
irritating symptoms.








cavity





SEQ ID NO 42:
P32
female
10's
allergic
C
once a day,
Immediately after nasal


(CFGRKMDRISSASGLGC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared.








cavity





SEQ ID NO 43:
P33
male
20's
allergic
C
once a day,
Immediately after nasal


(CFGRKMDRISSSTGLGC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared. After 1 minute, no








cavity
nasal discharge even by









twisting nose.




SEQ ID NO 44:
P34
male
40's
allergic
C
once a day,
After 1 minute, rhinorrhea


(CFGRKMDRISSSAGLGC)



rhinitis

0.1 ml applied
stopped. There was no








to each nasal
irritating symptoms.








cavity





SEQ ID NO 45:
P35
male
60's
allergic
C
once a day,
After 1 minute, and nose


(CFSRRMDRISSSSGLGC)



rhinitis

0.1 ml applied
got cleared.








to each nasal





SEQ ID NO 47:
P36
male
50's
allergic
C
once a day,
After 1 minute, rhinorrhea


(CFSRKMDRISSSTGLGC)



rhinitis

0.1 ml applied
stopped. There was no








to each nasal
irritating symptoms.








cavity





SEQ ID NO 48:
P37
female
50's
allergic
C
once a day,
After 1 minute, and nose


(CFSRKMDRISSSSGIGC)



rhinitis

0.1 ml applied
got cleared. Nasal








to each nasal
discharge stopped.








cavity





SEQ ID NO 49:
P38
female
40's
allergic
C
once a day,
Immediately after nasal


(CFSRKMDRISSSSGLAC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared.








cavity





SEQ ID NO 50:
P39
female
10's
allergic
C
once a day,
Immediately after nasal


(CFGRRMDRISSTSGLGC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared.








cavity





SEQ ID NO 51:
P40
male
20's
chronic
C
once a day,
After 1.5 minutes, nasal


(CFGRRMDRISSSTGLGC)



rhinitis

0.1 ml applied
obstruction was improved,








to each nasal
and nose got cleared.








cavity





SEQ ID NO 52:
P41
male
30's
allergic
C
once a day,
After 1 minute, rhinorrhea


(CFGRRMDRISSSSGIGC)



rhinitis

0.1 ml applied
stopped. There was no








to each nasal
irritating symptoms.








cavity





SEQ ID NO 53:
P42
male
40's
allergic
C
once a day,
Immediately after nasal


(CFGRRMDRISSSSGLAC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared.








cavity





SEQ ID NO 54:
P43
female
30's
allergic
C
once a day,
Immediately after nasal


(CFGRKMDRISSTSGIGC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared.








cavity





SEQ ID NO 55:
P44
male
50's
allergic
C
once a day,
After 1 minute, and nose


(CFGRKMDRISSTSGLAC)



rhinitis

0.1 ml applied
got cleared. Nasal








to each nasal
discharge stopped.








cavity





SEQ ID NO 56:
P45
male
30's
allergic
C
once a day,
Immediately after nasal


(CFGRKMDRISSSTGIGC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared.








cavity





SEQ ID NO 57:
P46
female
40's
allergic
C
once a day,
After 1 minute, and nose


(CFGRKMDRISSSTGLAC)



rhinitis

0.1 ml applied
got cleared. Nasal








to each nasal
discharge stopped.








cavity





SEQ ID NO 58:
P47
female
40's
allergic
C
once a day,
After 30 seconds,
Itches were removed

(CFGRKMDRISSSSGIAC)



rhinitis

0.1 ml applied
rhinorrhea stopped, with no
in 30 seconds.







to each nasal
more sneezing. there was no








cavity
irritating symptoms.




SEQ ID NO 59:
P48
female
20's
allergic
C
once a day,
Immediately after nasal


(CFSRRMDRISSTSGLGC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared.








cavity





SEQ ID NO 60:
P49
female
40's
allergic
C
once a day,
After 1 minute, and nose


(CFSRRMDRISSSTGLGC)



rhinitis

0.1 ml applied
got cleared. Nasal








to each nasal
discharge stopped.








cavity





SEQ ID NO 61:
P50
male
30's
allergic
C
once a day,
Immediately after nasal


(CFSRRMDRISSSSGIGC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared.








cavity





SEQ ID NO 62:
P51
female
40's
allergic
C
once a day,
After 1 minute, rhinorrhea


(CFSRRMDRISSSSGLAC)



rhinitis

0.1 ml applied
stopped, with no more








to each nasal
sneezing. There was no








cavity
irritating symptoms.




SEQ ID NO 63:
P52
female
30's
allergic
C
once a day,
Immediately after nasal


(CFSRKMDRISSTSGIGC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared.








cavity





SEQ ID NO 64:
P53
male
60's
allergic
C
once a day,
After 1 minute, rhinorrhea


(CFSRKMDRISSSTGIGC)



rhinitis

0.1 ml applied
stopped, with no more








to each nasal
sneezing. There was no








cavity
irritating symptoms.




SEQ ID NO 65:
P54
female
30's
allergic
C
once a day,
After 1 minute, and nose


(CFSRKMDRISSSTGLAC)



rhinitis

0.1 ml applied
got cleared. Nasal








to each nasal
discharge stopped.








cavity





SEQ ID NO 66:
P55
female
30's
allergic
C
once a day,
Immediately after nasal


(CFSRKMDRISSSSGIAC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared.








cavity





SEQ ID NO 67:
P56
female
40's
allergic
C
once a day,
After 1 minute, and nose


(CFGRRMDRISSTSGIGC)



rhinitis

0.1 ml applied
got cleared. Nasal








to each nasal
discharge stopped.








cavity





SEQ ID NO 68:
P57
male
60's
allergic
C
once a day,
After 1 minute, rhinorrhea


(CFGRRMDRISSTSGLAC)



rhinitis

0.1 ml applied
stopped, with no more








to each nasal
sneezing. There was no








cavity
irritating symptoms.




SEQ ID NO 69:
P58
male
20's
allergic
C
once a day,
Immediately after nasal


(CFGRRMDRISSSTGIGC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared.








cavity





SEQ ID NO 70:
P59
female
40's
allergic
C
once a day,
Immediately after nasal


(CFGRRMDRISSSTGLAC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared.








cavity





SEQ ID NO 71:
P60
female
30's
allergic
C
once a day,
After 1 minute, rhinorrhea


(CFSRRMDRISSTSGIGC)



rhinitis

0.1 ml applied
stopped, with no more








to each nasal
sneezing. There was no








cavity
irritating symptoms.




SEQ ID NO 72:
P61
female
40's
allergic
C
once a day,
After 1 minute, rhinorrhea


(CFSRRMDRISSTSGLAC)



rhinitis

0.1 ml applied
stopped, with no more








to each nasal
sneezing. There was no








cavity
irritating symptoms.




SEQ ID NO 73:
P62
female
20's
allergic
C
once a day,
Immediately after nasal


(CFSRRMDRISSSTGIGC)



rhinitis

0.1 ml applied
application, and nose got








to each nasal
cleared.








cavity





SEQ ID NO 74:
P63
female
40's
allergic
C
once a day,
After 1 minute, rhinorrhea


(CFSRRMDRISSSTGLAC)



rhinitis

0.1 ml applied
stopped, with no more








to each nasal
sneezing. There was no








cavity
irritating symptoms.




SEQ ID NO 75:
P64
male
20's
allergic
C
once a day,
After 1 minute, and nose


(CFSRRMDRISSSSGIAC)



rhinitis

0.1 ml applied
got cleared. Nasal








to each nasal
discharge stopped.








cavity



In SEQ ID NOS 16-75, 1st Cys and 17th Cys from left form a disulfide bond.

































































































































































































































































































































































The result described above demonstrated that the cyclic peptide of the invention exhibits a similar effect as the cyclic peptide expressed by Formula I-a even if some amino acids of the cyclic peptide have been replaced.
Specifically, when it was applied to nasal cavity mucosa and/or paranasal cavity mucosa, it can improve or prevent various symptoms associated with rhinitis such as nasal obstruction, rhinorrhea, sneezing and pruritus. Rhinitis also includes, without being particularly limited, for example, infectious rhinitis including acute rhinitis and chronic rhinitis; hypersensitive non-infectious rhinitis including combined (flower hypersensitivity) rhinitis, rhinitis with rhinorrhea, congestive rhinitis, edematous rhinitis and dry-nose type rhinitis; irritant-induced rhinitis including physical rhinitis, chemical rhinitis and radiation rhinitis; and atrophic rhinitis and idiopathic granulomatous rhinitis; sinusitis such as acute sinusitis, chronic sinusitis (maxillary empyema), eosinophilic sinusitis and paranasal cavity mycosis. The external preparation can be used for the purpose of treating or preventing one ore more of the above.
Combined rhinitis includes, for example, allergic rhinitis including perennial allergic rhinitis and seasonal allergic rhinitis, and nonallergic rhinitis including vasomotor (essential) rhinitis and eosinophilic rhinitis. Rhinitis with rhinorrhea includes, for example, gustatory rhinitis, cold air-induced rhinitis and senile rhinitis.
Congestive rhinitis includes, for example, drug-induced rhinitis, psychogenic rhinitis, gestational rhinitis, endocrine rhinitis and cold rhinitis.
Edematous rhinitis includes, for example, aspirin-hypersensitive rhinitis.
Among those mentioned above, it exerts an excellent effect specifically on hypersensitive non-infectious rhinitis, irritant-induced rhinitis and sinusitis, preferably on hypersensitive non-infectious rhinitis and chronic sinusitis, more preferably on combined (flower hypersensitivity) rhinitis, rhinitis with rhinorrhea, congestive rhinitis, edematous rhinitis and dry-nose type rhinitis, and chronic sinusitis. Therefore, the external preparation of the present invention can be used for the purpose of treating or preventing at least one or more rhinitis selected from the above-mentioned group.
In terms of its symptoms, for its effects as described above, it can be used for any of rhinitis with sneezing/rhinorrhea, rhinitis with nasal obstruction and rhinitis with both symptoms.
Immediately after a single application of 0.1 ml of 3 μg/ml to 500 μg/ml preparation was applied to the affected site in each nasal cavity, nasal discharge was stopped, nasal obstruction was improved, itches in nasal cavity mucosa or sneezing were stopped (immediate effect). A single application of a nasal drop induced persistent effects lasting for one day, or for a week or more in some cases (long-lasting effect). In addition, because it causes no local irritation at all, it can be used safely even in a subject who is not desired to use a nasal drop comprising rhinitis therapeutic that has conventionally been used such as steroid drug due to its strong local irritation.
Examples of multiple replaceable amino acids in the cyclic peptide of the invention are summarized below, based on working examples as described above, without not being limited thereto.













TABLE 30-3



SEQ ID
Q30
female
30's
atopic
C
once
After 3 minutes, 1 application,
After 30 seconds,

NO 22:



dermatitis

a day
erythema and infiltration were
iches were eliminated.

(CFGRKMDRV






remarkably remitted. dry skin,


SSSSGLGC)






scaled and cracks were also









improved.



Q31
female
70's
eczema
C
once
Immediately after application,
Facial rawness vanished







a day
erythema and papule remitted.
immediately after









application.


Q32
female
30's
atopic
B
once
A remarkable improvement after






dermatitis

a day,
2 days, and erythema was








7 days
oliminated.



Q33
female
40's
atopic
C
once
After 1 minute, erythema was
Iches were eliminated





dermatitis

a day,
remitted. After 2 minutes,
immediately after







2 days
scratch wound was epithelized.
application.








After 3 minutes, skin texture









was improved. With only 1-day









application, erythema was









eliminated, skin texture was









improved with no dryness.



Q34
male
20's
atopic
C
once
After 2 minutes, erythema and
Iches were remitted





dermatitis

a day
scales were remitted. No
immediately after








reoccurance for 7 days
application.








thereafter.



Q35
female
50's
chronic
C
once
After 2 applications,






eczema

a day
lichenificated erythema was









remitted. No recurrence for 3









weeks thereafter.



Q36
female
50's
roughened
C
once
Skin roughness was cured






skin

a day
immediately after application.









After 2 minutes, skin got









moisturized, its redness faded.









Skin texture was finely









improved. After 7 days, skin









tone became brighter, spots









faded and flabbiness was









improved.



Q37
female
30's
roughened
B
once
After 2 minutes, zits, redness
Rawness vanished





skin

a day
was remitted. After 1 day, skin
immediately after








became normal tone and its
application.








texture was improved.




SEQ ID
Q38
female
40's
atopic
C
once
After 2 minutes, scratch wound
After 1 minute, iches

NO 23:



dermatitis

a day
was epithelized and cured.
were eliminated.

(CFGRKMDRI






Erythema was improved.


GSSSGLGC)
Q39
male
20's
eczema
C
once
After 2 minutes, erythema, dry
Itches were abated







a day
skin and scales were improved,
immediately after








and skin got moisturized.
application.


Q40
male
20's
atopic
C
once
After 2 minutes, erythema,
Itches were extincted





dermatitis

a day
scales and infiltration were
immediately after








remitted. With 1 application,
application.








no recurrance for 7 days.



Q41
male
40's
plaque
C
once
After 3 minutes, crusty scales/






psoriosis

a day
erythema and infiltration were









remitted.



Q42
female
30's
roughened
B
once
After 2 minutes, roughness,
After 2 minutes,





skin

a day
zits and redness were reduced,
itches were extincted.








skin got moisturized and its









texture was improved.



Q43
female
50's
rosecea,
C
once
After 1 minute, erythema,






acne

a day
tolangiaclasia and acne were









remitted. After 2 minutes,









papule, infiltration, acne,









dry skin and redness was









remitted.