CN1823086A

CN1823086A - Oligopeptide

Info

Publication number: CN1823086A
Application number: CN 200480020469
Authority: CN
Inventors: 平井洋平; 中岛喜一郎
Original assignee: Sumitomo Electric Industries Ltd
Current assignee: Sumitomo Electric Industries Ltd
Priority date: 2003-05-16
Filing date: 2004-05-14
Publication date: 2006-08-23

Abstract

An oligopeptide having the activity of promoting hair growth. In particular, the oligopeptide is represented by, for example, the formula (I): X1-X2-X3-X4-X5-X6-X7 (wherein X1 is a residue of amino acid such as Ser; X2 is a residue of amino acid such as Ile; X3 is a residue of amino acid such as Glu; X4 is a residue of amino acid such as Gln; X5 is a residue of amino acid such as Ser; X6 is an amino acid residue of Cys having a carbonylated modification group bonded thereto or an amino acid residue of Lys having a carbonylated modification group bonded thereto; and X7 is a residue of amino acid such as Glu or Ala).

Description

Oligopeptides

Technical field

The present invention relates to have the oligopeptides of hair growth promoter action, and contain the pharmaceuticals of this oligopeptides as effective constituent.

Background technology

Existing prompting shows, the normal morphology of epithelium forms the regulation and control of the factor that is subjected to being present in the mesenchymal cell source around the epithelium, in addition, be that in view of much resulting from the cause of disease that the epithelium form forms unusual disease mesenchymal cell is unusual, thereby the illustrating of morphogenetic mechanism of mesenchymal cell regulation and control epithelium is subjected to extensive concern.Yet, because what participate in the morphogenetic material group of regulation and control epithelium is expressed in the time that is subjected in the complex system and spatiality regulation and control, with these separating substances and to its function resolve be very the difficulty, in addition, the form of epithelium is formed simplification, make up the experimental model system and also be difficult to, thereby the research in this field is up till now for to yet there are no remarkable progress.To result from the pathogenesis of the morphogenetic disease of epithelium in order illustrating, to set up these treatment of diseases methods etc., the morphogenetic regulatory mechanism of epithelium resolve to the eager expectation of people institute.

In this case, participate in the separated and purifying (European patent discloses No. 0562123 (United States Patent (USP) the 5th, 726, No. 298)) of the control morphogenetic epimorphin of epithelium.Show that this material is with by the physiologically active substance of 277 to 289 protein that amino acid constitutes as core protein, mainly by the mesenchymal cell biosynthesizing.Show also that in addition epimorphin acts on epithelial cell, have the morphogenetic effect of the epithelium of promotion, and under the situation that epimorphin does not play a role, tissue forms and can not normally carry out.

For the structure of epimorphin, found that the epimorphin molecule structurally roughly is divided into 4 fragments (European patent discloses No. 0698666).That is, constitute the polypeptide of epimorphin total length, can be divided into coiled coil (coiled-coil) zone (1), functional area (2), coiled coil zone (3) and the terminal water repellent region of C-from the N-end side.In these fragments, for functional area (in people's epimorphin, from terminal the 104th to 187 the amino acid whose specific region of N-), existing prompting shows, this zone participates in the physiologically active of cell adhesion and epimorphin and expresses closely related (above-mentioned European patent is open).

Because epimorphin has the effect that promotes that normal morphology forms, this material results from form as prevention and treatment and forms the pharmaceuticals of unusual disease etc., or the effective constituent of pharmaceuticals such as hair growth promoter is to be worth expectation.Yet the natural type epimorphin that is obtained by Mammals is insoluble in water-soluble solvents such as physiological saline, and its practical application has difficulties as pharmaceuticals.Thereby people are just attempting development and exploitation solvability excellence, have kept the epimorphin derivative of the form formation promoter action of natural type epimorphin simultaneously basically.For example known, remove the varient (fragment 123) (European patent discloses No. 0562123 (United States Patent (USP) the 5th, 726, No. 298)) that C-terminal part water repellent region obtains.

Show that the polypeptide with epimorphin part-structure acts on epithelial cell, promote the epithelium form to form (European patent discloses No. 1008603).This polypeptide is the polypeptide that dissolves in aqueous solvents such as physiological saline, have in the above-mentioned publication this polypeptide have the hair growth promoter action instruction and enlightenment.In addition, European patent discloses in No. 1288221 and discloses the oligopeptides with hair growth promoter action, and it is the part in the structure of epimorphin.

Summary of the invention

Problem of the present invention is to provide the oligopeptides with hair growth promoter action.The above-mentioned oligopeptides that provides purification efficiency high is provided another problem of the present invention.Another problem of the present invention is to provide and contains the pharmaceuticals of above-mentioned oligopeptides as effective constituent.

The inventor is for solving above-mentioned problem found that of effort with keen determination, by with the modification group that contains carbonyl to modifying as Cys residue or Lys residue the epimorphin part-structure, in the oligopeptides, the oligopeptides that obtains has good promotion hair growth effect.And then, the inventor finds, by import linking group in the aminoacid sequence as the oligopeptides of the part-structure of epimorphin, forms the resulting oligopeptides of intramolecularly ring texture, when showing good purification efficiency, has good hair growth promoter action.The present invention is based on these discoveries and finishes.

That is, according to the present invention, provide oligopeptides or its modification oligopeptides of hair growth promoter action, it is the oligopeptides that is made of 5 to 100 amino-acid residues that comprise the arbitrary amino acid sequence in the following formula (I) to (IV).

X1-X2-X3-X4-X5-X6-X7 (I)

X1-X2-X3-X4-X6-X5-X7 (II)

X1-X2-X3-X6-X4-X5-X7 (III)

X1-X2-X6-X3-X4-X5-X7 (IV)

(in the formula, X1 represents amino-acid residue Ser, Ala, Tyr, Thr, Pro, Phe, Val, Gly, Leu, Ile or Met, or disappearance, and X2 represents amino-acid residue Ile, Gly, Asn, Thr, Val, Ser, Phe, Leu, Ala, Pro, Cys or Met, or disappearance

X3 represents amino-acid residue Glu, Lys, Gln, Arg, Ala, Val, Trp, Cys or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser, His, Cys or Lys,

X5 represents amino-acid residue Ser, Trp, Phe, Thr, Cys, Tyr, Pro, Ala, Gly, Val, Leu, Ile or Met,

X6 represents the Cys amino-acid residue that is connected with the modification group that contains carbonyl, or the Lys amino-acid residue that is connected with the modification group that contains carbonyl, and

X7 represents amino-acid residue Asp, Glu, His, Ser, Ala, Gly, Asn, Tyr, Arg or Leu, or disappearance.)

Preferred X1 represents the Ser amino-acid residue, X2 represents the Ile amino-acid residue, X3 represents the Glu amino-acid residue, X4 represents the Gln amino-acid residue, X5 represents the Ser amino-acid residue, X6 represents the Cys amino-acid residue that is connected with the modification group that contains carbonyl, and X7 represents Glu or Ala amino-acid residue.

The oligopeptides that comprises 5 to 7 amino-acid residues that is made of above-mentioned formula (I) to (IV) arbitrary amino acid sequence preferably is provided.

According to other aspects of the invention, provide oligopeptides or its modification oligopeptides with hair growth promoter action, it is the oligopeptides by 6 to 100 amino-acid residues formations of the arbitrary amino acid sequence that comprises following formula (XI) to (XIV).

X1-X2-X3-X4-X5-X6-X7-X8-X9 (XI)

XI-X2-X3-X4-X6-X5-X7-X8-X9 (XII)

X1-X2-X3-X6-X4-X5-X7-X8-X9 (XIII)

X1-X2-X6-X3-X4-X5-X7-X8-X9 (XIV)

(in the formula, X1 represents amino-acid residue Ser, Ala, Tyr, Thr, Pro, Phe, Val, Gly, Leu, Ile or Met, or disappearance,

X2 represents amino-acid residue Ile, Gly, Asn, Thr, Val, Ser, Phe, Leu, Ala, Pro, Cys or Met, or disappearance,

X3 represents amino-acid residue Glu, Lys, Gln, Arg, Ala, Val, Trp, Cys or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser, His, Cys or Lys,

X6 represents the Cys amino-acid residue that is connected with the modification group that contains carbonyl, or the Lys amino-acid residue that is connected with the modification group that contains carbonyl,

X7 represents amino-acid residue Asp, Glu, His, Ser, Ala, Gly, Asn, Tyr, Arg or Leu, or disappearance,

X8 represents amino-acid residue Gln, and

X9 represents amino-acid residue Asp, Glu, His, Ser, Ala, Gly, Asn, Tyr, Arg or Leu.)

Among preferred X7 and the X9 at least one is the amino-acid residue beyond the amino-acid residue Asp.

And then preferred X1 represents the Ser amino-acid residue, X2 represents the Ile amino-acid residue, X3 represents the Glu amino-acid residue, X4 represents the Gln amino-acid residue, X5 represents the Ser amino-acid residue, and X6 represents the Cys amino-acid residue that is connected with the modification group that contains carbonyl, and X7 represents Glu or Ala amino-acid residue, X8 represents the Gln amino-acid residue, and X9 represents the Glu amino-acid residue.

The oligopeptides that comprises 7 to 9 amino-acid residues that is made of the arbitrary amino acid sequence in the above-mentioned formula (XI) to (XIV) preferably is provided.

Preferred X6 represents that end is connected with the Cys amino-acid residue of the modification group with carbonyl, or the terminal Lys amino-acid residue that is connected with the modification group with carbonyl.

Preferably being connected in carbonatoms at the inner carbonyl of modification group is 2 to 10 hydrocarbon chain.

Preferably being connected in carbonatoms at the inner carbonyl of modification group is 3 to 7 hydrocarbon chain.

Preferred modification group is the modification group in N-β-maleimide propionic acid source, the modification group in N-ε-maleimide caproic acid source, or the modification group in N-κ-maleimide undeeanoic acid source.

Preferably provide following any oligopeptides or its to modify oligopeptides.

Ser-Ile-Glu-Gln-Ser-Xaa-Ala-Gln-Glu (sequence 1)

Ser-Ile-Glu-Gln-Ser-Xaa-Glu-Gln-Glu (sequence 2)

Ser-Ile-Glu-Gln-Ser-Xaa-Glu-Gln-Glu-Ala (sequence 3)

(in the formula, Xaa represents to be connected with the Cys amino-acid residue of the modification group in N-ε-maleimide caproic acid source.)

According to other aspects of the invention, provide any oligopeptides of comprising above-mentioned record or its to modify on oligopeptides or its physiology acceptable salt as the pharmaceuticals and the hair growth promoter of effective constituent.

According to another aspect of the invention, provide above-mentioned oligopeptides or its to modify acceptable salt on oligopeptides or its physiology, the purposes in above-mentioned pharmaceuticals of preparation or hair growth promoter; And the growth method of hair, this method comprises modifies on oligopeptides or its physiology acceptable salt with the above-mentioned oligopeptides of significant quantity or its and is applied to and comprises the people in interior mammiferous step.

According to another aspect of the invention, provide the cyclic oligopeptides that constitutes by the aminoacid sequence of following formula (1) or its to modify oligopeptides with hair growth promoter action.

X2 represents amino-acid residue Ile, Gly, Asn, Thr, Val, Ser, Phe, Leu, Ala, Pro or Met, or disappearance,

X3 represents amino-acid residue Glu, Lys, Gln, Arg, Ala, Val, Trp or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser, His or Lys,

X5 represents amino-acid residue Ser, Trp, Phe, Thr, Tyr, Pro, Ala, Gly, Val, Leu, Ile or Met,

X6 represents amino-acid residue Cys,

Y disappearance or 1-90 aminoacid sequence of expression,

L represents linking group.)

Preferred X1 represents the Ser amino-acid residue, and X2 represents the Ile amino-acid residue, and X3 represents the Glu amino-acid residue, and X4 represents the Gln amino-acid residue, and X5 represents the Ser amino-acid residue, and X6 represents the Cys amino-acid residue, and X7 represents Glu or Ala amino-acid residue.

Preferred Y disappearance or 1-12 aminoacid sequence of expression.

Preferably provide to have the aminoacid sequence that constitutes by following formula (2), have the cyclic oligopeptides of hair growth promoter action or it modifies oligopeptides.

X3 represents amino-acid residue Glu, Lys, Gln, Arg, Ala, Val, Trp or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser, His or Lys,

X6 represents amino-acid residue Cys,

X8 represents amino-acid residue Gln,

X9 represents amino-acid residue Asp, Glu, His, Ser, Ala, Gly, Asn, Tyr, Arg or Leu,

L represents linking group.)

At least one side among preferred X7 and the X9 is the amino-acid residue beyond the amino-acid residue Asp.

Preferred X1 represents the Ser amino-acid residue, X2 represents the Ile amino-acid residue, X3 represents the Glu amino-acid residue, X4 represents the Gln amino-acid residue, X5 represents the Ser amino-acid residue, and X6 represents the Cys amino-acid residue, and X7 represents Glu or Ala amino-acid residue, X8 represents the Gln amino-acid residue, and X9 represents the Glu amino-acid residue.

According to other aspects of the invention, provide by the aminoacid sequence of following formula (3) to constitute, have the cyclic oligopeptides of hair growth promoter action or it modifies oligopeptides.

X3 represents amino-acid residue Glu, Gln, Arg, Ala, Val, Trp or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser or his,

X6 represents amino-acid residue Lys,

Y disappearance or 1-90 aminoacid sequence of expression,

L represents linking group,

Z represents the group that can be connected with Lys reaction.)

Preferred X1 represents the Ser amino-acid residue, and X2 represents the Ile amino-acid residue, and X3 represents the Glu amino-acid residue, and X4 represents the Gln amino-acid residue, and X5 represents the Ser amino-acid residue, and X6 represents the Cys amino-acid residue, X7 table Glu or Ala amino-acid residue.

Preferred Y disappearance or 1-12 aminoacid sequence of expression.

Preferably provide to have the aminoacid sequence that constitutes by following formula (4), have cyclic oligopeptides or its modification oligopeptides of hair growth promoter action.

X3 represents amino-acid residue Glu, Gln, Arg, Ala, Val, Trp or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser or his,

X6 represents amino-acid residue Lys,

X8 represents amino-acid residue Gln,

L represents linking group,

Z represents the group that can be connected with Lys reaction.)

Preferred X1 represents the Ser amino-acid residue, X2 represents the Ile amino-acid residue, X3 represents the Glu amino-acid residue, X4 represents the Gln amino-acid residue, X5 represents the Ser amino-acid residue, and X6 represents the Cys amino-acid residue that is connected with the modification group that contains carbonyl, and X7 represents Glu or Ala amino-acid residue, X8 represents the Gln amino-acid residue, and X9 represents the Glu amino-acid residue.

The carbonatoms of preferred linking group L is 6 to 8.

Preferably in linking group L carbonyl to be connected in carbonatoms be 5 to 7 hydrocarbon chain.

Preferred linking group L is-CO-(CH ₂) _n-NH-(in the formula, n represents the integer of 5-7).

Among the preferred linking group L-CO is connected with X1, and the NH-among the linking group L and the Cys residue that is connected in X6 or the group that Z represents are connected.

Especially preferably provide following any cyclic oligopeptides or its to modify oligopeptides.

According to another aspect of the invention, provide and comprise above-mentioned any cyclic oligopeptides or its and modify on oligopeptides or its physiology acceptable salt as the pharmaceuticals and the hair growth promoter of effective constituent.

According to another aspect of the invention, provide to comprise acceptable salt on above-mentioned any cyclic oligopeptides or its modification oligopeptides or its physiology, and minoxidil is as the hair growth promoter of effective constituent.

Description of drawings

Fig. 1 represents with liquid phase chromatography the synthetic oligopeptides, and and modifies the result that the reaction product of sample is resolved.

Fig. 2 represents with liquid phase chromatography the synthetic oligopeptides, and and modifies the result that the reaction product of sample is resolved.

Fig. 3 represents to adopt liquid phase chromatography to the synthetic oligopeptides, and and modifies the result that the reaction product of sample is resolved.

Fig. 4 represents the evaluation result of the hair growth promoter action of various oligopeptides.

Fig. 5 represents the evaluation result of the hair growth promoter action of various oligopeptides.

Fig. 6 represents the HPLC analytical results of compound (11).

Fig. 7 represents the HPLC analytical results of compound (12).

Fig. 8 represents the HPLC analytical results of compound (13).

Fig. 9 represents the HPLC analytical results of compound (14).

Figure 10 represents the hair growth promoter action of cyclic oligopeptides of the present invention.

Figure 11 represents the hair growth promoter action of cyclic oligopeptides of the present invention.

Figure 12 represents the hair growth promoter action of cyclic oligopeptides of the present invention and minoxidil and time spent.

Embodiment

Below embodiments of the present invention are elaborated.

The oligopeptides of first kind of embodiment of the present invention be comprise following formula (I) to (IV) the arbitrary amino acid sequence by 5 to 100 amino-acid residue oligopeptides that constitute, that have the hair growth promoter action.

X1-X2-X3-X4-X5-X6-X7 (I)

X1-X2-X3-X4-X6-X5-X7 (II)

X1-X2-X3-X6-X4-X5-X7 (III)

X1-X2-X6-X3-X4-X5-X7 (IV)

X3 represents amino-acid residue Glu, Lys, Gln, Arg, Ala, Val, Trp, Cys or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser, His, Cys or Lys,

The oligopeptides of second kind of embodiment of the present invention be comprise following formula (XI) to (XIV) the arbitrary amino acid sequence by 6 to 100 oligopeptides that amino-acid residue constitutes, be oligopeptides with hair growth promoter action.

X1-X2-X3-X4-X5-X6-X7-X8-X9 (XI)

X1-X2-X3-X4-X6-X5-X7-X8-X9 (XII)

X1-X2-X3-X6-X4-X5-X7-X8-X9 (XIII)

X1-X2-X6-X3-X4-X5-X7-X8-X9 (XIV)

(in the formula, X1 represents and above-mentioned the same amino-acid residue that to X7 X8 represents the Gln amino-acid residue, and X9 represents amino-acid residue Asp, Glu, His, Ser, Ala, Gly, Asn, Tyr, Arg or Leu.)

In addition, among the present invention, at least one side among preferred X7 and the X9 is the amino-acid residue beyond the amino-acid residue Asp.Reason is, when at least one side among X7 and the X9 is amino-acid residue beyond the amino-acid residue Asp, the β conversion (succinimide) that results from aspartic acid that can not take place to be occurred when X7 and X9 are the Asp amino-acid residue makes synthesis yield improve.Especially preferred X7 is the amino-acid residue beyond the amino-acid residue Asp.This is because the β conversion often takes place the Asp of X7.

Above-mentioned aminoacid sequence (I) is to (IV) and (XI) to (XIV), be that European patent is disclosed transforming from 11 terminal amino-acid residues of C-of No. 1008603 disclosed polypeptide (II), be about to it from terminal 1 amino-acid residue (Lys) of N-and aminoacid sequence from obtaining after 2 amino-acid residues (Asp-Glu) of C-end are removed, or in this aminoacid sequence, further the position of cysteine residues (Cys) is changed and the aminoacid sequence that obtains, be that this Cys residue is replaced with the Cys amino-acid residue that is connected with the modification group that contains carbonyl, or be connected with the Lys aminoacid sequence of the modification group that contains carbonyl and the aminoacid sequence that obtains as the basis, disappearance again, replace, with and/or add the 1-several amino acid and the aminoacid sequence that obtains.Comprise above-mentioned aminoacid sequence (I)-(IV) and, as the concrete demonstration of following examples institute, have good hair growth promoter action (XI) to the oligopeptides of (XIV).

Arrive in (XIV) to (IV) and (XI) at aminoacid sequence (I), X6 represents to be connected with the Cys amino-acid residue of the modification group that contains carbonyl, or is connected with the Lys amino-acid residue of the modification group that contains carbonyl.

According to preferred implementation of the present invention, modification group is the modification group that end has carbonyl.In addition, according to the present invention further optimization embodiment, being connected in carbonatoms at the inner carbonyl of modification group is 2 to 10 hydrocarbon chain, more preferably the hydrocarbon chain of carbonatoms 3 to 7.

Among the present invention, when the Cys amino-acid residue that X6 represents to be connected with the modification group that contains carbonyl, object lesson as modification group, can enumerate the modification group in preferred N-β-maleimide propionic acid source, the modification group in N-ε-maleimide caproic acid source, or the modification group in N-κ-maleimide undeeanoic acid source.This modification group is by making reagent and oligopeptides reactions such as N-β-maleimide propionic acid, N-ε-maleimide caproic acid or N-κ-maleimide undeeanoic acid, make the sulfydryl and the mentioned reagent reaction of the cysteine residues in the oligopeptides, thereby can import to cysteine residues in the oligopeptides.

In addition, among the present invention, when the Lys amino-acid residue that X6 represents to be connected with the modification group that contains carbonyl,, represent that with above-mentioned X6 the situation of the Cys amino-acid residue that is connected with the modification group that contains carbonyl is the same as the object lesson of modification group.

Oligopeptides of the present invention, be comprise formula (I) to (IV) or (XI) to (XIV) arbitrary amino acid sequence by 5 to 100 oligopeptides that amino-acid residue constitutes.For example, the number lower value of amino-acid residue can be 5,6,7,8,9,10,11,12,13,14 or 15, and higher limit can be 15,20,25,30,35,40,45,50,60,70,80,90 or 100.The number of amino-acid residue preferably 5 to 40, more preferably 6 to 30, especially preferred 7-20, and then preferred 7-15, and then preferred 7-12, preferred especially 7-10.Select at the number of amino-acid residue, expectation makes oligopeptides dissolve in aqueous solvents such as physiological saline or water, has biologic activity.

Polypeptide in the third embodiment of the present invention is aminoacid sequence cyclic oligopeptides that constitute, that have the hair growth promoter action or its modification oligopeptides by following formula (I).

X3 represents amino-acid residue Glu, Lys, Gln, Arg, Ala, Val, Trp or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser, His or Lys,

X6 represents amino-acid residue Cys,

Y disappearance or 1-90 aminoacid sequence of expression,

L represents linking group.)

As the preferred object lesson of above-mentioned oligopeptides, can enumerate and have the aminoacid sequence that constitutes by following formula (2), have the cyclic oligopeptides of hair growth promoter action or it modifies oligopeptides.

(in the formula, X1-X7 and L and formula (1) synonym,

X8 represents amino-acid residue Gln,

The oligopeptides of the 4th kind of embodiment of the present invention is the cyclic oligopeptides with hair growth promoter action or its modification oligopeptides by the aminoacid sequence formation of following formula (3).

X3 represents amino-acid residue Glu, Gln, Arg, Ala, Val, Trp or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser or his,

X6 represents amino-acid residue Lys,

Y disappearance or 1-90 aminoacid sequence of expression,

L represents linking group,

Z represents the group that can be connected with Lys reaction.)

As the preferred object lesson of above-mentioned oligopeptides, can enumerate the cyclic oligopeptides or its modification oligopeptides that have the aminoacid sequence that constitutes by following formula (4) and have the hair growth promoter action.

(in the formula, X1-X7, L and Z and formula (3) synonym,

X8 represents amino-acid residue Gln,

In addition, among the present invention, at least one side is the amino-acid residue beyond the amino-acid residue Asp among preferred X7 and the X9.Reason is, when at least one side was a amino-acid residue beyond the amino-acid residue Asp in X7 and X9, the β conversion (succinimide) that results from aspartic acid that can not take place to be occurred when X7 and X9 are the Asp amino-acid residue made the synthesis yield raising.Especially preferred X7 is the amino-acid residue beyond the amino-acid residue Asp.This is because the β conversion often takes place the Asp of X7.

Above-mentioned aminoacid sequence is that European patent is disclosed transforming from 11 terminal amino-acid residues of C-of No. 1008603 disclosed polypeptide (II), be about to it and remove, based on the aminoacid sequence of this aminoacid sequence that obtains from 1 amino-acid residue (Lys) of N-end with from terminal 2 amino-acid residues (Asp-Glu) of C-.

The cyclic oligopeptides of the invention described above handling with 30% acetonitrile, 0.1% trifluoroacetic acid, after the drying, also can be brought into play good hair growth promoter action then as the concrete demonstration of following embodiment institute.Handle with 30% acetonitrile, 0.1% trifluoroacetic acid, owing to also be the supply conditions of oligopeptides during with the anti-phase chromatography purification, thereby cyclic oligopeptides of the present invention can use the anti-phase chromatogram purification, thereby can further improve purification efficiency.

In (4), X6 represents Cys or Lys amino-acid residue at aminoacid sequence (1), and especially preferred X6 represents the Cys amino-acid residue.

Cyclic oligopeptides of the present invention is characterised in that, X1 and X6 are via-L-Cys-(in the formula, L represents the linking group that is connected with X1, Cys represents the Cys residue that is connected with X6) or-L-Z is (in the formula, L represents that the linking group that is connected with X1, Z represent the group that can be connected with the Lys reaction) and be connected to form the intramolecularly ring texture.As can with the object lesson of the represented group that can be connected of Z with Lys reaction, can enumerate the N-hydroxy-succinamide ester group, imide ester group, hydroxyl methylphosphine etc.

As the object lesson of the represented linking group of L, can enumerate carbonatoms and be 6 to 8 linking group, for example, can enumerating carbonyl, to be connected in carbonatoms be linking group of 5 to 7 hydrocarbon chain etc.As the object lesson of linking group L, can enumerate-CO-(CH ₂) _n-NH-(in the formula, n represents the integer of 5-7).In above-mentioned linking group L ,-Co can be connected with X1, and NH-can be connected with the Cys residue or the Z that are connected in X6.

Cyclic oligopeptides of the present invention, be have formula (1) or (3) aminoacid sequence by 5 to 98 oligopeptides that amino-acid residue constitutes.The number preferable range of amino-acid residue is 5 to 20, is more preferably 7 to 15, and then preferably 7 to 12, especially preferably 7 to 10.On the total number of atnino acid purpose was selected, expectation made oligopeptides dissolve in aqueous solvents such as physiological saline or water, and has biologic activity.

Below, will be from oligopeptides (that is 5 to 100 amino-acid residues oligopeptides that constitute, that have the hair growth promoter action that, comprises the arbitrary amino acid sequence in the formula (I) to (IV) of four kinds of embodiments of above-mentioned first kind of embodiment to the; Comprise formula (XI) to (XIV) the arbitrary amino acid sequence by 5 to 100 amino-acid residue oligopeptides that constitute, that have the hair growth promoter action; Aminoacid sequence cyclic oligopeptides that constitute, that have the hair growth promoter action by formula (1); And the cyclic oligopeptides that constitutes by formula (3) aminoacid sequence) is called oligopeptides of the present invention with hair growth promoter action.

The kind of the amino-acid residue in the oligopeptides of the present invention is not particularly limited, and can be natural type amino-acid residue, non-natural type amino-acid residue or their derivative.That is, amino acid can be L-amino acid, also can be D-amino acid, can also be their mixture.In addition, as amino acid whose kind, can be a-amino acid, beta-amino acids, gamma-amino acid, δ-amino acid, natural type amino acid a-amino acid preferably.

Said non-natural type amino acid is meant in this specification sheets, except constituting 20 kinds of natural type amino acid (glycine of natural protein, the L-L-Ala, the L-Xie Ansuan, the L-leucine, the L-Isoleucine, the L-Serine, the L-Threonine, the L-aspartic acid, L-L-glutamic acid, altheine, L-glutaminate, L-Methionin, the L-arginine, the L-halfcystine, the L-methionine(Met), the L-phenylalanine, L-tyrosine, the L-tryptophane, the L-Histidine, the L-proline(Pro)) whole amino acid in addition, specifically can enumerate, (1) atom in the natural type amino acid is replaced with other materials and the non-natural type amino acid that obtains, (2) optical isomer of natural type amino acid side chain, (3) import substituting group in the natural type amino acid side chain and the non-natural type amino acid that obtains, and (4) replaced the amino acid whose side chain of natural type, thereby change hydrophobicity, reactive, state of charge, molecular size, hydrogen bound energy etc. and non-natural type amino acid of obtaining etc.

Other embodiments of oligopeptides of the present invention, be comprise above-mentioned aminoacid sequence (I) to (IV), (XI) to (XIV) by 5 to 100 oligopeptides that amino-acid residue constitutes, being the modification oligopeptides with oligopeptides of hair growth promoter action, is the modification oligopeptides that has identical hair growth promoter action with the oligopeptides of the invention described above.

Other embodiments of oligopeptides of the present invention, be comprise above-mentioned aminoacid sequence (1) to (4) arbitrary sequence by 7 to 98 oligopeptides that amino acid constitutes, being the modification oligopeptides with oligopeptides of hair growth promoter action, is the modification oligopeptides that has identical hair growth promoter action with the oligopeptides of the invention described above.

By among this specification sheets embodiment in detail and the experimental technique of concrete record, or this is with the suitable change of above-mentioned experimental technique or adjusts, those skilled in the art can be easy to confirm, oligopeptides of the present invention or modify oligopeptides and have the hair growth promoter action.As the object lesson of this kind method, can enumerate following method, but be not limited to these methods.

Know that (1) C3H and C57BL/6 mouse have 50 days resting stage approximately from the 45th day to the 95 days front and back, back of being born.The color of skin is a pink in this resting stage, and is grey or black in the growth stage, thereby is easy to judge hair cycle.Material to be checked is applied to this mouse, whether promotes to advance, can estimate and educate knit to the growth stage by judging it.

(2) antigen that uses specific recognition to be present in the new saccus setigerus of epithelium (for example can be enumerated, the antigen of about 220Kda) monoclonal antibody (for example, preserving number is the monoclonal antibody of the hybridoma generation of FERM BP-8121) or its fragment evaluation are educated knit.More specifically, in the presence of material to be checked, cultivate the skin histology in organism source, reclaim this skin histology sheet then, itself and said monoclonal antibody or its fragment are reacted, this monoclonal antibody or its fragment by detection or mensuration and the reaction of skin histology sheet, can measure the antigenic expression amount that is present in the new saccus setigerus of epithelium, estimate and educate knit.Or, downcut skin, it is cultivated in the presence of material to be checked, again with protein in the culture and aforementioned monoclonal antibody reactive, can estimate by this reaction result and to educate knit.

In the modification oligopeptides of the present invention, " modification " speech is to comprise chemically modified and biology to modify, and should be interpreted as generalized and modify.As the example of modifying, for example can enumerate, the importing of functional groups such as alkylation, ester groupization, halogenation or amination, oxidation, reduction, addition or functional group such as dissociate change, the importing of sugar compounds (monose, disaccharides, oligosaccharides or polysaccharide) or lipid compounds etc., phosphorylation, or biotinylation etc., but be not limited to these.

As preferred modification oligopeptides, can enumerate biotinylated oligopeptides, as preferred modification oligopeptides, can enumerate via introns (spacer) or without introns vitamin H is connected in the terminal and oligopeptides that obtains of N-.In above-mentioned modification oligopeptides, also can be in the limit of not damaging desirable physiologically active to the vitamin H chemically modified that suits.In order vitamin H to be imported to the N-end of oligopeptides via the introns of suitable length, for example can use NHS-Biotin or NHS-LC-Biotin (all can obtain) from Pierce company.

The various oligopeptides of the invention described above (also comprise modify oligopeptides) can be unbound states, also can acid salt or the form of subsalt provide.As acid salt, for example can enumerate inorganic acid salts such as hydrochloride, vitriol, nitrate, phosphoric acid salt, or organic acid salt such as tosilate, metilsulfate, Citrate trianion, oxalate, maleate, tartrate.As subsalt, for example can enumerate metal-salts such as sodium salt, sylvite, calcium salt, magnesium salts, organic ammonium salts such as ammonium salt, ammonium carbamate, leptodactyline.Sometimes form salt with amino acid such as glycine, form companion ion at intramolecularly sometimes.

In addition, these oligopeptides or its salt exist as hydrate or solvate sometimes.Above-mentioned oligopeptides has a plurality of chiral carbon.The three-dimensional arrangement of each chiral carbon is not particularly limited, but the preferred amino acid residue is a L-amino acid.Based on any mixture of steric isomers such as the optically active body of these chiral carbon, non-mapping (diastero) isomer, steric isomer, racemic modification etc. also are included in the scope of the invention.

Oligopeptides of the present invention, chemical processes such as normal solid phase method that uses and liquid phase method are synthetic in can synthesizing by peptide.About the condensing agent of protecting group such as the amino in the peptide building-up process and condensation reaction, there are a large amount of ready-made books can reference.Solid phase method can use commercially available various peptide synthesizer.As required, can be by the protection and the deprotection of functional group, more effective building-up reactions of carrying out.About the importing and the removal method of protecting group, for example can be with reference to Protective Groupsin Organic Synthesis, T.W.Greene, John Wiley ﹠amp; Sons, Inc. (1981).In addition, comprise the manufacture method of the modification oligopeptides of chemically modified or bio-modification etc., known by those skilled in the art, can use any method.

In addition, according to common biological methods such as genetic expression operation, after preparing the recombinant vectors of the dna sequence dna that comprises the above-mentioned oligopeptides of encoding, through microorganism transformed (transformant), can from the substratum of cultivating this transformant, separation and purification obtain desired oligopeptides by this preparing carriers.But the manufacture method of above-mentioned oligopeptides is not limited to these chemistry and biology methods.In addition, comprise the manufacture method of the modification oligopeptides of chemically modified or biology modification etc., known by those skilled in the art, can use any method.

Manufacture method when then, oligopeptides of the present invention being cyclic oligopeptides more specifically illustrates.Cyclic oligopeptides of the present invention can be synthetic according to the order of following (1)-(3).

(1) at first, chemosynthesis is equivalent to linking group L (CO-(CH ₂) _n-NH-) HOOC-(CH ₂) _n-NH ₂(in the formula, n represents the integer of 5-7).

(2) then, according to the synthetic ordinary method of peptide as described above, synthetic represented oligopeptides or the Z-L-X1-X2-X3-X4-X5-X6-X7 (when X6 is Lys) of Cys-L-X1-X2-X3-X4-X5-X6-X7 (when X6 is Cys).For example, use commercially available automatic peptide synthesizer (for example, the automatic peptide synthesizer 430A type of applied Biosystem company), amenable to process prolongs peptide chain by the beginning of C end successively with the BOC method, can synthesize desired protection peptide resin.

(3) last, when X6 was Cys, the Cys residue of N-terminal was connected by covalent linkage with the represented Cys of X6.The formation of this covalent linkage can be carried out according to ordinary method.Particularly, by making the oligopeptides that has free sulfhydryl groups the iodine oxidizing reaction taking place in acetum, thereby carry out cyclization, can form above-mentioned covalent linkage.

In addition, when X6 was Lys, the Z residue of N-terminal was connected by covalent linkage with the represented Lys of X6.The formation of this covalent linkage can be carried out according to ordinary method.

Oligopeptides of the present invention can be used as the effective constituent of pharmaceuticals.Pharmaceuticals one speech in this specification sheets except being used to comprise the people the prevention or treatment of interior mammal disease, incorporates the hair promotor of outside pharmaceutical product or makeup etc. into when also including, meaning is very extensive.

The object lesson of the disease treatment object of the subject of pharmaceuticals of the present invention, drug effect and pharmaceuticals of the present invention is listed below:

(1) oligopeptides of the present invention shows role of Vasculogenesis, regeneration promotion effect, cardiovascular regeneration, vascular endothelial cell inducing action etc., can effectively prevent and treat (ExpCell Res 1996 Jan 10,222 (1): 189-98) such as chronic occlusion arteriosclerosis, thromboangiitis obliterans, prinzmetal angina, arteriosclerosis.

(2) the oligopeptides of the present invention form that participates in the kidney epithelium forms, can effectively prevent and treat diabetes etc. (J Cell Bio 2001, Mar 5; 152 (5): 911-22).

(3) oligopeptides of the present invention participates in the formation (regeneration) of liver, can effectively prevent and treat hepatic metabolism obstacle (Biochem Biophys Res Commun 1998 Sep18; 250 (2): 486-90).

(4) oligopeptides of the present invention participates in bone and tooth formation, can effectively prevent and treat periodontopathy, fracture, osteocarcinoma, damaged, osteoporosis (the Arch Oral Biol 1995Feb of bone; 40 (2): 161-4).

(5) oligopeptides of the present invention participates in formation of lung branching morphogenesis and lung fiber disease, can effectively prevent and treat tuberculosis (Biochem Biophys Res Commun 1997 May19; 234 (2): 522-5; And Am J Respir Cell Mol Biol 2000Aug; 23 (2): 168-74).

(6) oligopeptides of the present invention participates in the formation of intestines fold, can effectively prevent and treat intestinal tract disease (AM J Physiol 1998 Jul; 275 (1pt1): G 114-24).

(7) oligopeptides of the present invention participates in keeping of muscle structure, for example, can effectively prevent and treat muscle distrophy (Histochem J 1998 Dec; 30 (12): 903-8).

(8) oligopeptides of the present invention participates in the formation of gall-bladder epithelium, can effectively prevent and treat gallbladder disease (Cell Tissue Res 2000 May; 300 (2): 331-44).

(9) oligopeptides of the present invention participates in the formation formation of breast epithelium, can effectively prevent and treat mastopathy (J Cell Biol 2001 May 14; 153 (4): 785-94).

Among these, most preferably use as hair growth promoter.

" hair growth promoter " speech that uses in this specification sheets is construed as and comprises that new piliation promotes, hair growth promotes and very long wide implication such as trichomadesis prevention, and can not be interpreted as and be confined to any implication.

In addition, cyclic oligopeptides of the present invention or its are modified when acceptable salt uses as hair growth promoter on oligopeptides or its physiology, also can be used in combination with minoxidil.

As pharmaceuticals of the present invention, the material more than a kind or a kind that can select the acceptable salt on above-mentioned oligopeptides or its physiology directly uses, but usually preferred with the above-mentioned substance more than a kind or a kind as effective constituent, and the preparation additive more than a kind or a kind that allows on the interpolation technology of pharmaceutics, make medical composition and use.Contain above-mentioned oligopeptides more than a kind or a kind as the hair growth promoter of effective constituent, can be used as ointment, sprays, smear with external application agent such as solution or patches and use, also can be used as injection and be directly used in the affected part, can be made into the preparation that is applicable to the suitable form of using as hair growth promoter.

For example, can cooperate the agent of shampoo, nutrition hair care to use above-mentioned oligopeptides, also above-mentioned oligopeptides can be enclosed in the liposome etc. and make preparation as effective constituent.The composition of this form is also contained in the pharmaceuticals of the present invention.In order to make as the effective Transdermal absorption of the percutaneous stratum corneum of the oligopeptides of effective constituent, it is very suitable being used suitable tensio-active agent or lipid-soluble substance etc. in ointment etc.

For to the Mammals topical application, composition of the present invention can be provided as the formulation of the variform that comprises (but being not limited to these) such as washing lotion, emulsifiable paste, gelifying agent, swab (sticks), sprays, ointment, pasty state agent and use.These formulations comprise the multiple mediation form that comprises solution, emulsion, gelifying agent, solid and liposome etc.

Composition of the present invention is mixed with when solution is local to be used, and its useful composition generally can be enumerated pharmaceutically acceptable water-based or organic solvent." pharmaceutically acceptable organic solvent " is meant and can disperses or dissolves oligopeptides of the present invention and security (for example, pungency and susceptibility etc.) satisfactory solvent.As the object lesson of suitable organic solvent, can enumerate propylene glycol, polyoxyethylene glycol (200-600), polypropylene glycol (425-2025), glycerine, 1,2,4-trihydroxybutane, sorbitol ester, 1,2,6-hexanetriol, ethanol, Virahol, butyleneglycol and their mixture etc.

Topical compositions is being mixed with sol, when being used for skin, in liquid composite, is adding propelling agent as sprays.As the object lesson of propelling agent, can enumerate chlorination, fluoridize or fluorine chlorating low molecular weight hydrocarbon, but be not limited thereto.

Topical compositions can be mixed with the solution that comprises tenderizer.Tenderizer is meant and is used to prevent or alleviate drying, and the material of skin care.Known multiple suitable tenderizer all can be used for the present invention.

As preparing other formulations, ointment and lotion are arranged by the composition that comprises oligopeptides of the present invention.

As can ointment being arranged by other formulations of the composition preparation that comprises oligopeptides of the present invention.Ointment can comprise animal or plant oil or semi-solid hydrocarbon (oiliness) base.Ointment also can comprise the absorption ointment base that absorbs water and form the milkiness thing.Ointment is also water miscible.

Other formulations are opacifying agent.Emulsifying agent can be nonionic, cationic or anionic property, and as the example of emulsifying agent, for example United States Patent (USP) the 3rd, 755, No. 560 and 4,421, and record in No. 769.

Emulsion and ointment can be mixed with opacifying agent and solution.The single opacifying agent that is used for local composites such as oil-in-water-type and water-in-oil emulsion and ointment is known for everyone.Heterogeneous milkiness compositions such as water-in-oil-in-water type also are known, and for example United States Patent (USP) the 4th, 254, in No. 105 put down in writing.3 heavy opacifying agent also can be used for local use the of the present invention, can enumerate silicon bag oil-in-water liq milkiness thing etc., and for example United States Patent (USP) the 4th, 960, No. 764 put down in writing.

Other opacifying agent that can be used for topical composition are microemulsion systems.For example, the squalane that comprises about 9-about 15%, the system of the polyoxyethylene glycol sorbyl alcohol mono fatty acid of the silicone oil of about 25%-about 40%, the Fatty Alcohol(C12-C14 and C12-C18) of about 8-about 20%, about 15%-about 30% (commercial goods name TWEENS) or other nonionics and about 20% water of about 7-.

Liposomal agents also can be used for comprising the composition of oligopeptides of the present invention.Liposome composition, phosphatide, cholesterol and water such as the composition by will comprising oligopeptides of the present invention and dipalmitoyl phosphatidylcholine are mixed with according to prescriptive procedure.The also available epidermis lipid of the composition that liposome forms that is applicable to replaces phosphatide.The liposome composite is immersed in above-mentioned a kind of local using in the blender (for example, gelifying agent or oil-in-water-type opacifying agent), can obtains Liposomal formulation.About local other compositions and pharmaceutical use with liposome, Mezei (1985) Topics inPharmaceutical Sciences for example, Bremer et al.eds., Elsevier Science, New York, N.Y., pp345-358 puts down in writing.

The usage quantity of above-mentioned hair growth promoter can for example, can be selected with reference to the concrete usage quantity that shows of this specification sheets embodiment according to the suitably selections such as kind of application target, formulation, effective constituent.For example, the usage quantity of the effective constituent of being grown up one day is at 1 μ g/kg/ days-10mg/kg/ days, in the scope about preferred 10 μ g/kg/ days-1mg/kg/ days.

In addition, aspect enforcement the present invention, can use common technologies such as molecular biology (comprising gene recombination technology), cytobiology, biological chemistry and immunology, these all are the knowledge that those skilled in the art grasped.This technology, Molecular Cloning:ALaboratory Manual for example, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J.Gait, ed., 1984); Animal CellCulture (R.I.Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M.Wei ﹠amp; C.C.Blackwell, eds.); Gene Transfer Vectors forMammalial Cells (J.M.Miller ﹠amp; M.P.Calos, eds., 1987); CurrentProtocols in Molecular Biology (F.M.Ausubel et al., eds., 1987and annual updates); PCR:The Polymerase Chain Reaction (Mulliset al., eds., 1994); Current Protocols in Immunology (J.E.Coligan et al., eds., 1991 and annual updates).

Embodiment

Below, further specify the present invention by embodiment, but scope of the present invention is not limited to following examples.

Embodiment 1: the preparation of modifying oligopeptides

Synthetic with the oligopeptides that following aminoacid sequence is represented by the solid phase method that has used Fmoc.

Ser-Ile-Glu-Gln-Ser-Cys-Ala-Gln-Glu (sequence 4)

Sef-Ile-Glu-Gln-Ser-Cys-Glu-Gln-Glu (sequence 5)

Ser-Ile-Glu-Gln-Ser-Cys-Glu-Gln-Glu-Ala (sequence 6)

Then, synthetic oligopeptides and following modification reagent are reacted according to the working instructions of this reagent.The ratio of oligopeptides and modification reagent is reacted with 1: 1 mol ratio just.

EMCA (N-ε-maleimide caproic acid): Pierce company produces

KMUA (N-κ-maleimide undeeanoic acid): Pierce company produces

BMPA (N-β-maleimide propionic acid): Pierce company produces

Phenyl maleimide: Sigma company produces

With the reaction product of synthetic oligopeptides and modification reagent, resolve with liquid chromatography, the results are shown in Fig. 1 to Fig. 3.The condition of liquid chromatography is as follows.

Inject sample: 20 μ l

Elution requirement TFA, acetonitrile 20-75%

Absorb the wash-out position of observing peptide at 210nm.

What Fig. 1 represented to Fig. 3 is to make oligopeptides and modify reagent (EMCA) just with the reaction of 1: 1 ratio of mol ratio, the liquid chromatography before and after the reaction.

Left figure among Fig. 1 represents is result with the synthetic oligopeptide of sequence 1 expression, and right figure represents is the result of the oligopeptides of its cysteine residues after with the EMCA modification.

Left figure among Fig. 2 represents is result with the synthetic oligopeptide of sequence 2 expressions, and right figure represents is the result of the oligopeptides of its cysteine residues after with the EMCA modification.

Left figure among Fig. 3 represents is result with the synthetic oligopeptide of sequence 3 expressions, and right figure represents is the result of the oligopeptides of its cysteine residues after with the EMCA modification.

Embodiment 2: the preparation of new saccus setigerus monoclonal antibody specific

Extract the mouse hair from the skin of the B57BL mouse that is in the growth stage (the 48th day to 50 days), be placed among the PBS that contains 8M urea, 2%SDS, 100mM DTT 37 ℃ hatch a night after, extract protein.In addition, under stereomicroscope, gather hair follicle (the hair follicle bulb has the pigment person of B56BL mouse beard; Growth stage), homogenate in PBS.With above-mentioned 2 kinds of test portions (in proteinic amount, 0.5mg) after and the auxiliary adjuvant of equivalent mix the preparation colloid.

The above-mentioned colloid that obtains (0.2mg) is used in the subcutaneous branch of rat (Wister) 3 places, carried out immunity.After the first immunisation 1 month, with the above-mentioned the same enhancing immunity of carrying out.Then in two all backs and the above-mentioned the same enhancing immunity second time of carrying out.In the second time of enhancing immunity after 3 days, take out the immune rat spleen, reclaim the hemocyte composition with screen cloth.In this hemocyte composition, contain antibody producing cells.After the hemocyte composition (full dose) of above-mentioned recovery and mouse multiple myeloma P3U1 mixed with polyethylene glycol 1500, be suspended in the Dulbecco/Ham F12 mixed culture medium, join in 96 well culture plates 100 μ l/ holes.Second day, equivalent (100 μ l) HAT substratum (Sigma company) is added into each hole.After 2 days, draw 100 μ l respectively from each hole and discard, in each hole, add the new substratum of 150 μ l again.96 well culture plates are statically placed in 37 ℃ of CO ₂In the incubator.

The hair follicle of the beard of growth stage B57BL mouse is pulverized dissolving with the ultrasonic grinding machine in 8M urea.Nitrocellulose filter soaked 5 minutes in this solution after, fully clean with PBS, install on the Biorad dot blot, once screen reclaiming the hybridoma supernatant that obtains from each hole of above-mentioned 96 well culture plates.At first, with the nitrocellulose filter of above-mentioned preparation with Tris damping fluid (TBST) sealing that is dissolved with 5% skim-milk after, in each hole that with this nitrocellulose filter is the end, add hybridoma supernatant 100 μ l.After hatching 1 hour, clean, add the mouse IgG of the Chinese People's Anti-Japanese Military and Political College (1 μ g/ml TBST) of two anti-horseradish peroxidase-labeled with the Tris damping fluid.Add chromogenic substrate ECL reagent (productions of Amersham pharmacia company), filter out antibody (50 kinds altogether) (once screening) by whether developing the color with the beard hair follicle reaction in vegetative period.

Antibody (50 kinds) at above-mentioned once screening obtains with the freezing microtome section (10 μ m) of growth stage beard hair follicle, filters out the antibody (postsearch screening) of specific reaction.Particularly, the freezing microtome section of growth stage beard hair follicle is placed on the slide glass, add the hybridoma supernatant that once screening obtains, with two anti-colour developings.Particularly, will use the growth stage beard hair follicle freezing microtome section of Cryostat freezing-microtome (Bright company) preparation to handle with-20 ℃ of methyl alcohol after, with TBST sealing clearance response 1 hour after 1 hour and on the hybridoma.After cleaning with the Tris damping fluid and the mouse IgG of the Chinese People's Anti-Japanese Military and Political College of FITC mark (among the TBST with 100 μ g/ml dissolving) reaction.After cleaning with the Tris damping fluid, covered is with fluorescence microscope.

The result of postsearch screening is that screening obtains 8 kinds of antibody altogether.These antibody and epidermis do not react, and and the hair follicle specific reaction.These 8 kinds of antibody are cloned with limiting dilution assay.

At these 8 kinds of antibody, the Western blotting observe its to growth stage beard hair follicle or resting stage the beard hair follicle reactivity, and then use the section test portion of the 14 age in days mouse fetal skins that hair follicle forming to observe its reactivity.Consequently, obtain with growth stage beard hair follicle and forming process in hair follicle (new saccus setigerus) specific reaction, and and the nonreactive monoclonal antibody mAb27 of beard hair follicle resting stage.Produce the hybridoma of monoclonal antibody mAb27, be preserved in Independent Administrative Leged Industrial Technology Complex Inst November 2 calendar year 2001 and specially permit biological sustenance center (a kind of ground of one fourth order, ripple city east, 1 central authorities the 6th are built in the Ibaraki, Japan) in putting down into, preserving number is FERM P-18578, and transferring international preservation on July 22nd, 2002 in putting down into, preserving number is FERM BP-8121.

Embodiment 3: the evaluation of the hair growth promoter action of various oligopeptides

Use the various oligopeptides of embodiment preparation, the assessing hair growth-promoting effect.The oligopeptides that uses is as follows.

Ser-Ile-Glu-Gln-Ser-Xaa-Asp (sequence 7)

In the above-mentioned formula, Xaa represents to be connected with halfcystine (Cys) residue of the modification group in EMCA (N-ε-maleimide caproic acid), KMUA (N-κ-maleimide undeeanoic acid), BMPA (N-β-maleimide propionic acid) or phenyl maleimide source.

Ser-Ile-Glu-Gln-Ser-Xaa-Asp-Gln-Asp-Glu (sequence 8)

Ser-Ile-Glu-Gln-Ser-Xaa-Ala-Gln-Glu (sequence 9)

Ser-Ile-Glu-Gln-Ser-Xaa-Glu-Gln-Glu (sequence 10)

(in the above-mentioned formula, Xaa represents to be connected with the Cys amino-acid residue of the modification group in EMCA (N-ε-maleimide caproic acid) source.)

Under stereomicroscope, take the maxilla skin histology of the 12nd day ICR mouse fetal, be divided into a left side and the right side, reclaim 5 parts.The 5 part skins that reclaim are placed on the nucleopore membranes (aperture 8 μ m, diameter 13mm) with a left side and right each mode of 5,, the outside is placed the top with entity microscope observing.Add the glubecoMEM/humF12 substratum 500 μ l that contain 1%BSA in 2 holes of 24 porose discs, add oligopeptide solution to be checked (solvent is PBS) in the hole, final concentration is 20 μ M, adds equivalent solvent (PBS) in contrast in another hole.Oligopeptides to be checked uses the unmodified oligopeptides of above-mentioned (1) preparation and modifies oligopeptides.

Make mounting have the film of skin histology to float in the solution in the above-mentioned hole, 1/hole, cultivated 6 days for 37 ℃.Reclaim 5 tissue from film, place SDS sample buffer (SDS0.02g/ml, glycerine 0.2g/ml, pH6.8) 100 μ l, dissolve with ultrasonic disintegrator.Contrast is also handled equally.The solution that above-mentioned processing obtains with SDS-PAGE (acrylamide 4-20%) electrophoresis (35mA, 1.5 hours) after, transfer to pvdf membrane, in containing the Tris damping fluid (TBST) of 5% skim-milk, hatched 1 hour again.MAb27 (10 μ g/ml among the TBST) reaction that obtains with embodiment 2 is after 1 hour, clean with TBS, with two anti-reactions, two resist the mouse IgG of the peroxidase labelling Chinese People's Anti-Japanese Military and Political College (Amersham company) for the TBST dilution being 1/1000, the reaction back is fully cleaned, and investigates the power of mAb27 reaction with ECL test kit (Amersham company).

What obtain the results are shown in Fig. 4 and Fig. 5.AHF among Fig. 4 and Fig. 5, the antigen (particularly, by 1439 protein that amino-acid residue constitutes shown in the sequence 11) of expression monoclonal antibody mAb27 identification.

Fig. 4 result shows that the band of the left side the 1st, 2 and the 3 row all band than the left side the 4th and the 5th row is dense.These results confirm, being connected with the oligopeptides that contains carbonyl modified group (EMCA, KMUA, BMPA) at cysteine residues and having the strong knit of educating in the oligopeptides especially has stronger promotion hair growth activity than the unmodified oligopeptides of correspondence, the oligopeptides that is connected with the modification group (phenyl maleimide) that does not contain carbonyl.In above-mentioned, the activity of EMCA is especially strong.

In addition, the result of Fig. 5 confirmation,

With Ser-Ile-Glu-Gln-Ser-Xaa-Asp-Gln-Asp-Glu (sequence 8)

Ser-Ile-Glu-Gln-Ser-Xaa-Ala-Gln-Glu (sequence 9)

Ser-Ile-Glu-Gln-Ser-Xaa-Glu-Gln-Glu (sequence 10)

(in the above-mentioned formula, Xaa represents to be connected with the Cys amino-acid residue of the modification group in EMCA (N-ε-maleimide caproic acid) source.) expression oligopeptides have the strong knit of educating.In above-mentioned, sequence 8 and 9 represented oligopeptides show the especially strong knit of educating.

Embodiment 4: the preparation of cyclic oligopeptides

The cyclic oligopeptides that the following aminoacid sequence of chemosynthesis is represented.In addition, also with general-CO (CH in the compound 11 ₂) ₅NH-is substituted by-CONH, carries out compound and synthesizes.

Synthetic in the following sequence.

Use the automatic peptide synthesizer (430A type Fostercity, CA, USA) of Applied Biosystem company, prolong peptide chain according to the BOC method successively from the C end, synthetic desired protection peptide resin according to program.Initial amino-acid resin carrier uses Boc-Ala-PAM (0.25mmol), and uses Cys (MeBzl), Ser (Bzl), Glu common amino acid derivatives such as (cHx).

Peptide is constructed after on the resin, the dry-run protection peptide resin.The protecting group removal of the protection peptide that obtains and peptide are to handle by anhydrous hydrogen fluoride to carry out from the isolating operation of resin carrier.The rough peptide of sulfydryl free obtains the lyophilize powder after extracting with aqueous acetic acid.Then, the rough peptide of this sulfydryl free is carried out cyclization by the iodine oxidation in acetum, synthetic above-mentioned described cyclic oligopeptides.The rough peptide that obtains adopts the system of acetonitrile-0.1% trifluoroacetic acid water with anti-phase high efficiency chromatography (Shimadzu Seisakusho Ltd., separating extraction device, LC8A type), carries out the separation and Extraction purifying, obtains the purpose cyclic oligopeptides.The purifying thing that obtains is identified with HPLC, mass analysis and amino acid analysis by analyzing.Below the determination data of above-mentioned synthetic compound was shown in, PHLC the results are shown in Fig. 6-Fig. 9.

Compound (11)

Outward appearance: white lyophilize body

The amino acid analysis value:

Hydrolysising condition: 6N HCl, 110 ℃, 22 hours

Ser(2)1.78、Glu(4)4.00、Ala(1)0.96、Ile(1)0.98、NH ₃(2)2.05、Cys(2)1.86、ε-Acp(1)1.05

Purity (HPLC): 98.4%

ESI-MS: molecular weight 1207.7 (theoretical value 1208.3)

Compound (12)

Outward appearance: white lyophilize body

The amino acid analysis value:

Hydrolysising condition: 6N HCl, 110 ℃, 22 hours

Ser (2) 1.80, Glu (4) 4.00, Ala (1) 1.00, Ile (1) 0.99, NH ₃(2) 2.08, Cys (2) 1.91,8-Aoa (1) N.D. (because under common analysis condition, can not wash-out, thereby can't be quantitatively)

Purity (HPLC): 97.8%

ESI-MS: molecular weight 1235.8 (theoretical value 1236.4)

Compound (13)

Outward appearance: white lyophilize body

The amino acid analysis value:

Hydrolysising condition: 6N HCl, 110 ℃, 22 hours

Ser(2)1.79、Glu(2)2.01、Ala(1)1.00、Ile(1)0.97、NH ₃(1)1.20、Cys(2)1.94、ε-Acp(1)1.01

Purity (HPLC): 99.0%

ESI-MS: molecular weight 950.5 (theoretical value 951.1)

Compound (14)

Outward appearance: white lyophilize body

The amino acid analysis value:

Hydrolysising condition: 6N HCl, 110 ℃, 22 hours

Ser(2)1.79、Glu(2)2.00、Ile(1)0.97、NH ₃(1)1.18、Cys(2)1.92、ε-Acp(1)1.01

Purity (HPLC): 97.9%

ESI-MS: molecular weight 879.6 (theoretical value 880.0)

Embodiment 5: the evaluation of the hair growth promoter action of various oligopeptides

After embodiment 4 synthetic cyclic oligopeptides 30% acetonitrile, 0.1% trifluoroacetic acid are handled, drying.This cyclic oligopeptides is dissolved in the phosphate buffer normal saline (PBS), and final concentration is 0.00002 weight %, adds isopyknic 100% ethanol in this solution, is mixed with 50% ethanol-PBS solution of 0.00001 weight %.PBS is solution in contrast.

Known C3H and C57BL/6 mouse have 50 days resting stage approximately before and after the 45th day to the 95th day after birth.In addition and since resting stage skin color be pink, then become grey or black in the growth stage, thereby mao cycle is judged easily.Use this mouse,, estimate whether promoted dividing a word with a hyphen at the end of a line to the growth stage by using cyclic oligopeptides of the present invention.Buy 7 the week ages (48-50 age in days, female) C 56BL/6 mouse, with animal with electric shear carefully with back (about 3 * 2.5cm ²) hair cut off, confirm hair resting stage in cycle by skin color.The cyclic oligopeptides solution (0.00001 weight %) of above-mentioned preparation is coated and respectively organized mouse, and 0.2ml/ only 10/group, every day 1 time, 5 days weekly, finished until experiment begins the back on the 38th day.Be coated with syringe not with syringe needle.

Observe mouse weekly 2 times, the ratio of the area that cuts off with respect to fleece according to hair regeneration area is set at 6 grades with mark, is judged by a plurality of people (2 people) naked eyes, estimates.In addition, also take pictures.Scared mark calculates by following main points.Among the part that cuts off at fleece, skin color becomes the ratio of grey or black part, uses following mark.0-20％：1、20-40％：2、40-60％：3、60-80％：4、80-100％：5。Each organizes above-mentioned fractional summation as scared mark.The scared fractional maximum value that each evaluation person gets is 50, owing to be two evaluation persons, thereby scared mark maximum value is 100.

The group of coating cyclic oligopeptides is compared with control group, and its hair regeneration obtains promoting.The results are shown in Figure 10 and Figure 11.These results confirm that cyclic oligopeptides of the present invention has the hair growth promoter action.

Embodiment 6: cyclic oligopeptides of the present invention and minoxidil together use

Formula (13) cyclic oligopeptides (0.00001%) that preparation 1: embodiment 4 makes and the mixed solution of minoxidil (1%)

Preparation 2: minoxidil (1%) liquid (directly using commercially available " RiUP " (Taisho Pharmaceutical Co., Ltd))

Whether preparation 1 is the same with embodiment 5 respectively with preparation 2, is used in hair follicle and is in the back of the body of C57BL/6 mouse of resting stage, estimate to promote hair follicle to divide a word with a hyphen at the end of a line to the growth stage.The results are shown in Figure 12.The situation of using minoxidil separately compares, and when cyclic oligopeptides of the present invention and minoxidil are together used, has significantly promoted hair follicle dividing a word with a hyphen at the end of a line to the growth stage.Evaluate both difference with the Wilson method, there is significant difference P＜0.05.

Embodiment 7: Cytotoxic evaluation

Formula (13) cyclic oligopeptides that embodiment 4 makes is carried out cytotoxicity experiment respectively with the concentration of 0.1 weight % solution, 0.01 weight % solution, 0.001 weight % solution, 0.0001 weight % solution, 0.00001 weight % solution and 0.000001 weight % solution.Cell to be checked is that the HSC-5 in skin source is (available from the affiliated institutions of Human Scineces Development Organization, be human sciences's resources for research storehouse (http://www.jhsf.or.jp/index_b.html)), with 1 * 100,000 cells/well is inoculated in 24 well culture plates.Substratum is the ISCOV improvement DMEM substratum that contains 10%FCS, cultivates 2 days.After cultivating end, calculate the ratio of viable cell.Consequently, cell is all survived in all cases, does not see cytotoxicity.

Utilize possibility on the industry

Oligopeptides of the present invention has the hair growth facilitation, can be used as the doctors such as hair growth promoting agents Pharmaceutically active ingredient. The present invention is specific by modification group is carried out, so that its hair growth promotes Effect strengthens than the peptide that international open WO01/094382 puts down in writing. And then, by with X7 and X9 is substituted by other amino acid beyond the Asp, can improve yield. In addition, ring-type of the present invention Oligopeptides has ring structure in the molecule, also is stable in the organic solvent even be exposed to, thereby, Also stable under the stringent conditions such as anti-phase chromatogram, especially have the high advantage of purification efficiency.

Sequence table

<110>Sumitomo?Electric?Industries，Ltd.

＜120〉oligopeptides

<130>A41291A

<160>14

<210>1

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>1

Ser?Ile?Glu?Gln?Ser?Xaa?Ala?Gln?Glu

1 5

<210>2

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>2

Ser?Ile?Glu?Gln?Ser?Xaa?Glu?Gln?Glu

1 5

<210>3

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>3

Ser?Ile?Glu?Gln?Ser?Xaa?Glu?Gln?Glu?Ala

1 5 10

<210>4

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>4

Ser?Ile?Glu?Gln?Ser?Cys?Ala?Gln?Glu

1 5

<210>5

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>5

Ser?Ile?Glu?Gln?Ser?Cys?Glu?Gln?Glu

1 5

<210>6

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>6

Ser?Ile?Glu?Gln?Ser?Cys?Glu?Gln?Glu?Ala

1 5 10

<210>7

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>7

Ser?Ile?Glu?Gln?Ser?Xaa?Asp

1 5

<210>8

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>8

Ser?Ile?Glu?Gln?Ser?Xaa?Asp?Gln?Asp?Glu

1 5 10

<210>9

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>9

Ser?Ile?Glu?Gln?Ser?Xaa?Ala?Gln?Glu

1 5

<210>10

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>10

Ser?Ile?Glu?Gln?Ser?Xaa?Glu?Gln?Glu

1 5

<210>11

<211>1439

<212>PRT

＜213〉house mouse (home mouse)

<400>11

Met?Ser?Pro?Leu?Ile?Arg?Ser?Ile?Val?Asp?Ile?Thr?Glu?Val?Phe?Asn

1 5 10 15

Gln?Tyr?Ala?Ser?Gln?Ser?Cys?Asp?Gly?Ala?Ser?Leu?Ser?Lys?Lys?Asp

20 25 30

Leu?Lys?Asn?Leu?Leu?Glu?Arg?Glu?Leu?Gly?Asp?Val?Leu?Gln?Arg?Pro

35 40 45

His?Asp?Pro?Glu?Thr?Ile?Asp?Leu?Thr?Leu?Glu?Leu?Leu?Asp?Arg?Asp

50 55 60

Cys?Asn?Gly?Arg?Val?Asp?Phe?Asn?Glu?Phe?Leu?Leu?Phe?Leu?Phe?Lys

65 70 75 80

Ile?Ala?Gln?Ala?Cys?Tyr?Tyr?Ala?Leu?Asp?Glr?Ala?Ala?Glu?Leu?Gly

85 90 95

Glu?Lys?Arg?Ala?Leu?Pro?Asn?Glu?Lys?Arg?Asn?Leu?Ser?Gln?Asp?Arg

100 105 110

Arg?Gln?Glu?Asp?Gln?Arg?Arg?Phe?Glu?Pro?Arg?Ser?Arg?Gln?Leu?Asp

115 120 125

Glu?Glu?Pro?Gly?Arg?Arg?Ser?Trp?Gln?Lys?Arg?Arg?Glu?Gln?Glu?Glu

130 135 140

Arg?Ala?Glu?Glu?Gln?Arg?Leu?Glu?Gln?Arg?Tyr?Arg?Gln?His?Arg?Asp

145 150 155 160

Glu?Glu?Gln?Arg?Leu?Gln?Arg?Arg?Glu?Leu?Gln?Glu?Leu?Glu?Glu?Arg

165 170 175

Leu?Ala?Glu?Lys?Glu?Pro?Leu?Gly?Trp?Ser?Lys?Gly?Arg?Asp?Ala?Glu

180 185 190

Glu?Phe?Ser?Glu?Val?Glu?Glu?Gln?Gln?Arg?Gln?Glu?Arg?Gln?Glu?Leu

195 200 205

Lys?Gly?Lys?Gly?Gln?Thr?Glu?Glu?Arg?Arg?Leu?Gln?Lys?Arg?Arg?Gln

210 215 220

Glu?Glu?Leu?Arg?Glu?Pro?Leu?Leu?Arg?Arg?Asp?Leu?Glu?Leu?Arg?Arg

225 230 235 240

Glu?Gln?Glu?Leu?Arg?Arg?Glu?Gln?Glu?Leu?Arg?Gln?Glu?Gln?Arg?Arg

245 250 255

Glu?Gln?Glu?Leu?Arg?Arg?Glu?Gln?Glu?Leu?Arg?Gln?Glu?Leu?Arg?Arg

260 265 270

Glu?Gln?Glu?Leu?Asn?Arg?Arg?Gln?Glu?Leu?Arg?Arg?Glu?Gln?Glu?Leu

275 280 285

Arg?Arg?Glu?Gln?Glu?Leu?Arg?Gln?Glu?Leu?Arg?Arg?Glu?Gln?Glu?Leu

290 295 300

Arg?Arg?Glu?Gln?Glu?Leu?Arg?Gln?Glu?Leu?Arg?Arg?Glu?Gln?Glu?Leu

305 310 315 320

Arg?Arg?Glu?Gln?Glu?Leu?Arg?Gln?Glu?Leu?Arg?Arg?Glu?Gln?Glu?Leu

325 330 335

Arg?Arg?Glu?Gln?Glu?Leu?Arg?Gln?Glu?Leu?Arg?Arg?Glu?Gln?Glu?Leu

340 345 350

Arg?Arg?Glu?Gln?Glu?Leu?Arg?Gln?Glu?Leu?Ala?Glu?Glu?Asp?Glu?Leu

355 360 365

Thr?Arg?Ile?Arg?Glu?Pro?Asp?Glu?Ser?Ile?Thr?Gln?Arg?Trp?Gln?Trp

370 375 380

Gln?Leu?Glu?Asn?Glu?Ala?Asp?Ala?Arg?Gln?Asn?Lys?Val?Tyr?Ser?Arg

385 390 395 400

Pro?Ser?Arg?Gln?Glu?Gln?Arg?Leu?Arg?Gln?Glu?Leu?Gly?Glu?Arg?Gln

405 410 415

Leu?Arg?Glu?Gln?Glu?Glu?Gln?Arg?Arg?Asp?Leu?Gln?Gln?Glu?Arg?Pro

420 425 430

Ala?Glu?Glu?Ala?Arg?Gln?Arg?Asn?Gln?Trp?Glu?Arg?Pro?Gln?Arg?Ala

435 440 445

Glu?Glu?Arg?Leu?Glu?Gln?Glu?Gln?Arg?Phe?Arg?Asp?Arg?Glu?Glu?Gln

450 455 460

Arg?Phe?Arg?Glu?Glu?Lys?Leu?Gln?Arg?Ala?Glu?Leu?Gln?Asp?Ser?Leu

465 470 475 480

Leu?Asp?Glu?Glu?Gln?Arg?Arg?Leu?Gln?Glu?Glu?Arg?Arg?Glu?Pro?Asn

485 490 495

Arg?Ser?Arg?Gln?Leu?Arg?Glu?Glu?Ser?Gln?Arg?Arg?Arg?Thr?Leu?Tyr

500 505 510

Ala?Lys?Pro?Ser?Gln?Arg?Gln?Gln?Arg?Arg?Arg?Leu?Gln?Gln?Glu?Arg

515 520 525

Gln?Tyr?Gln?Glu?Glu?Asp?Leu?Gln?Arg?Leu?Arg?Asp?Glu?Asp?Gln?Arg

530 535 540

Arg?Asp?Leu?Lys?Trp?Gln?Trp?Gln?Pro?Arg?Lys?Glu?Asn?Glu?Val?Arg

545 550 555 560

Ser?Asn?Arg?Leu?Phe?Thr?Lys?Arg?Arg?Gly?Asp?Glu?Glu?Pro?Ile?Gln

565 570 575

Gln?Leu?Glu?Asp?Ser?Gln?Glu?Arg?Glu?Arg?Arg?Gln?Asp?Arg?Arg?Pro

580 585 590

Leu?Gln?Asp?Glu?Glu?Glu?Glu?Lys?Arg?Glu?Leu?Glu?Gln?Glu?Arg?Arg

595 600 605

Arg?Arg?Gln?Gln?Arg?Asp?Arg?Gln?Ile?Leu?Glu?Glu?Glu?Gln?Phe?Gln

610 615 620

Arg?Glu?His?Gln?Arg?Glu?Ala?Arg?Arg?Arg?Asp?Glu?Thr?Phe?Gln?Glu

625 630 635 640

Glu?Glu?Gln?Leu?Gln?Gly?Glu?Ser?Arg?Arg?Arg?Gln?Gln?Glu?Arg?Glu

645 650 655

Gly?Lys?Phe?Leu?Glu?Glu?Glu?Arg?Gln?Leu?Arg?Thr?Glu?Arg?Glu?Glu

660 665 670

Gln?Arg?Arg?Arg?Gln?Glu?Gln?Glu?Arg?Glu?Phe?Gln?Glu?Glu?Glu?Glu

675 680 685

His?Leu?Gln?Glu?Arg?Glu?Lys?Glu?Leu?Arg?Gln?Glu?Cys?Asp?Arg?Lys

690 695 700

Ser?Arg?Glu?Gln?Glu?Arg?Arg?Gln?Gln?Arg?Glu?Glu?Glu?Gln?Leu?Arg

705 710 715 720

Arg?Gln?Glu?Arg?Asp?Gln?Arg?Phe?Arg?Arg?Glu?Gln?Glu?Arg?His?Leu

725 730 735

Glu?Arg?Glu?Glu?Glu?Gln?Leu?Arg?Asp?Arg?Pro?Ser?Arg?Arg?Glu?Gln

740 745 750

Glu?Arg?His?Gln?Glu?Arg?Glu?Glu?Glu?Gln?Leu?Arg?Asp?Arg?Pro?Ser

755 760 765

Arg?Arg?Glu?Gln?Glu?Arg?His?Gln?Glu?Arg?Glu?Glu?Glu?Gln?Leu?Arg

770 775 780

Asp?Arg?Pro?Ser?Arg?Arg?Glu?Gln?Glu?Arg?His?Gln?Glu?Arg?Glu?Glu

785 790 795 800

Glu?Gln?Leu?Arg?Asp?Arg?Pro?Phe?Arg?Arg?Glu?Gln?Glu?Arg?Arg?Leu

805 810 815

Glu?Arg?Glu?Glu?Glu?Gln?Leu?Arg?Asp?Arg?Pro?Ser?Arg?Arg?Glu?Gln

820 825 830

Glu?Arg?His?Gln?Glu?Arg?Glu?Glu?Glu?Gln?Leu?Arg?Asp?Arg?Pro?Ser

835 840 845

Arg?Arg?Glu?Gln?Glu?Arg?Arg?Leu?Glu?Arg?Glu?Glu?Glu?Gln?Leu?Arg

850 855 860

Asp?Arg?Ser?Phe?Arg?Arg?Glu?Gln?Glu?Leu?Arg?Arg?Asp?Arg?Lys?Phe

865 870 875 880

His?Glu?Glu?Glu?Glu?Arg?Arg?Glu?Glu?Leu?Glu?Glu?Glu?Gln?Arg?Gly

885 890 895

Gln?Glu?Arg?Asp?Arg?Leu?Arg?Val?Glu?Glu?Gln?Leu?Arg?Gly?Gln?Arg

900 905 910

Glu?Glu?Glu?Gln?Arg?Arg?Arg?Gln?Glu?Cys?Asp?Arg?Lys?Leu?His?Arg

915 920 925

Glu?Leu?Glu?Val?Arg?Gln?Glu?Leu?Glu?Glu?Glu?Arg?Leu?Arg?Asp?Arg

930 935 940

Lys?Leu?Arg?Arg?Glu?Gln?Glu?Leu?Arg?Arg?Asp?Arg?Lys?Phe?His?Glu

945 950 955 960

Glu?Glu?Glu?Arg?Arg?His?Glu?Glu?Phe?Glu?Glu?Lys?Gln?Leu?Arg?Leu

965 970 975

Gln?Glu?Pro?Asp?Arg?Arg?Phe?Arg?Arg?Glu?Gln?Glu?Leu?Arg?Gln?Glu

980 985 990

Cys?Val?Glu?Glu?Glu?Arg?Leu?Arg?Asp?Ser?Lys?Ile?Arg?Arg?Glu?Gln

995 1000 1005

Glu?Leu?Arg?Arg?Glu?Arg?Glu?Glu?Glu?Arg?Leu?Arg?Asp?Arg?Lys?Ile

1010 1015 1020

Arg?Arg?Asp?Gln?Glu?Leu?Arg?Gln?Gly?Leu?Glu?Glu?Glu?Gln?Leu?Arg

1025 1030 1035 1040

Arg?Gln?Glu?Leu?Asp?Arg?Lys?Phe?Arg?Glu?Glu?Gln?Glu?Leu?Asp?Gln

1045 1050 1055

Glu?Leu?Glu?Glu?Glu?Arg?Leu?Arg?Asp?Arg?Lys?Ile?Arg?Arg?Glu?Gln

1060 1065 1070

Glu?Leu?Arg?Arg?Glu?Gln?Glu?Leu?Arg?Arg?Glu?Gln?Glu?Phe?Arg?Arg

1075 1080 1085

Glu?Gln?Glu?Leu?Arg?Arg?Glu?Gln?Glu?Phe?Arg?Arg?Glu?Gln?Glu?Leu

1090 1095 1100

Arg?Gln?Glu?Arg?Glu?Glu?Glu?Arg?Leu?Arg?Asp?Arg?Lys?Ile?Arg?Arg

1105 1110 1115 1120

Asp?Gln?Glu?Leu?Arg?Gln?Gly?Leu?Glu?Glu?Glu?Gln?Leu?Arg?Arg?Gln

1125 1130 1135

Glu?Arg?Asp?Arg?Lys?Phe?Arg?Glu?Glu?Gln?Glu?Leu?Gly?Gln?Glu?Leu

1140 1145 1150

Glu?Glu?Glu?Arg?Leu?Arg?Asp?Arg?Lys?Ile?Arg?Arg?Glu?Gln?Glu?Leu

1155 1160 1165

Arg?Arg?Glu?Arg?Glu?Gln?Glu?Gln?Arg?Arg?Arg?Leu?Glu?Arg?Glu?Glu

1170 1175 1180

Glu?Gln?Gln?Arg?Leu?His?Glu?Arg?Glu?Glu?Glu?Gln?Arg?Arg?Arg?Gln

1185 1190 1195 1200

Glu?Arg?Glu?Gln?Glu?Gln?Gln?Arg?Cys?Leu?Glu?Arg?Glu?Glu?Glu?Gln

1205 1210 1215

Phe?Arg?Phe?Glu?Glu?Gln?Gln?Arg?Arg?Arg?Gln?Glu?Arg?Glu?Gln?Gln

1220 1225 1230

Leu?Arg?Gln?Glu?Arg?Asp?Arg?Arg?Val?Leu?Glu?Glu?Glu?Glu?Leu?Arg

1235 1240 1245

Gln?Glu?Arg?Glu?Glu?Leu?Leu?His?Arg?Gln?Val?Gly?Gly?Arg?Lys?Phe

1250 1255 1260

Arg?Glu?Glu?Glu?Arg?Leu?Arg?Leu?Glu?Arg?Glu?Glu?Gln?Gln?Arg?Arg

1265 1270 1275 1280

Leu?Gln?Glu?Arg?Asp?Asn?Arg?Arg?Phe?Arg?Glu?Glu?Val?Glu?Leu?Arg

1285 1290 1295

Gln?Glu?Arg?Glu?Gly?Gln?Gln?Leu?Arg?Gln?Glu?Arg?Asp?Arg?Lys?Phe

1300 1305 1310

Arg?Glu?Val?Glu?Glu?Leu?Arg?Gln?Glu?Glu?Gln?Arg?Arg?Arg?Gln?Glu

1315 1320 1325

Arg?Asp?Arg?Lys?Phe?Arg?Glu?Glu?Lys?His?Pro?Arg?Glu?Glu?Arg?Glu

1330 1335 1340

Glu?Gln?Gln?Leu?Arg?Arg?Glu?Lys?Arg?Asp?Gly?Gln?Tyr?Leu?Ala?Glu

1345 1350 1355 1360

Glu?Gln?Phe?Ala?Arg?Asp?Thr?Ile?Arg?Arg?Gln?Glu?Gln?Glu?Leu?Arg

1365 1370 1375

Gln?Glu?Glu?Glu?Gln?Arg?Arg?Arg?Gln?Glu?Arg?Glu?Arg?Lys?Phe?Gln

1380 1385 1390

Glu?Glu?Gln?Ile?Arg?Arg?Arg?Gln?Glu?Glu?Gln?Arg?Arg?Arg?Gln?Ile

1395 1400 1405

Leu?Glu?Pro?Gly?Thr?Arg?Gln?Phe?Ala?Asn?Val?Pro?Val?Arg?Ser?Ser

1410 1415 1420

Pro?Leu?Tyr?Glu?Tyr?Ile?Gln?Glu?Gln?Arg?Ser?Gln?Tyr?Arg?Pro

1425 1430 1435

<210>12

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>12

Ser?Ile?Glu?Gln?Ser?Cys?Ala?Gln?Glu

1 5

<210>13

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>13

Ser?Ile?Glu?Gln?Ser?Cys?Ala

1 5

<210>14

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>14

Ser?Ile?Glu?Gln?Ser?Cys

1 5

Claims

1. the oligopeptides with hair growth promoter action or its are modified oligopeptides, its be comprise following formula (I) to (IV) arbitrary aminoacid sequence by 5 to 100 oligopeptides that amino-acid residue constitutes,

X1-X2-X3-X4-X5-X6-X7 (I)

X1-X2-X3-X4-X6-X5-X7 (II)

X1-X2-X3-X6-X4-X5-X7 (III)

In X1-X2-X6-X3-X4-X5-X7 (IV) formula, X1 represents amino-acid residue Ser, Ala, Tyr, Thr, Pro, Phe, Val, Gly, Leu, Ile or Met, or disappearance,

X3 represents amino-acid residue Glu, Lys, Gln, Arg, Ala, Val, Trp, Cys or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser, His, Cys or Lys,

X7 represents amino-acid residue Asp, Glu, His, Ser, Ala, Gly, Asn, Tyr, Arg or Leu, or disappearance.

2. modify oligopeptides according to the described oligopeptides of claim 1 or its, wherein, X1 represents the Ser amino-acid residue, X2 represents the Ile amino-acid residue, X3 represents the Glu amino-acid residue, and X4 represents the Gln amino-acid residue, and X5 represents the Ser amino-acid residue, X6 represents the Cys amino-acid residue that is connected with the modification group that contains carbonyl, and X7 represents Glu or Ala amino-acid residue.

3. according to claim 1 or 2 described oligopeptides or its modification oligopeptides, wherein, it is the oligopeptides that comprises 5 to 7 amino-acid residues that is made of formula (I) to (IV) arbitrary amino acid sequence.

4. the oligopeptides with hair growth promoter action or its are modified oligopeptides, its be comprise following formula (XI) to (XIV) arbitrary aminoacid sequence by 6 to 100 oligopeptides that amino-acid residue constitutes;

X1-X2-X3-X4-X5-X6-X7-X8-X9 (XI)

X1-X2-X3-X4-X6-X5-X7-X8-X9 (XII)

X1-X2-X3-X6-X4-X5-X7-X8-X9 (XIII)

In X1-X2-X6-X3-X4-X5-X7-X8-X9 (XIV) formula, X1 represents amino-acid residue Ser, Ala, Tyr, Thr, Pro, Phe, Val, Gly, Leu, Ile or Met, or disappearance,

X3 represents amino-acid residue Glu, Lys, Gln, Arg, Ala, Val, Trp, Cys or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser, His, Cys or Lys,

X8 represents amino-acid residue Gln, and

X9 represents amino-acid residue Asp, Glu, His, Ser, Ala, Gly, Asn, Tyr, Arg or Leu.

5. modify oligopeptides according to the described oligopeptides of claim 4 or its, wherein, at least one among X7 and the X9 is the amino-acid residue beyond the amino-acid residue Asp.

6. according to claim 4 or 5 described oligopeptides or its modification oligopeptides, wherein, X1 represents the Ser amino-acid residue, and X2 represents the Ile amino-acid residue, and X3 represents the Glu amino-acid residue, X4 represents the Gln amino-acid residue, X5 represents the Ser amino-acid residue, and X6 represents the Cys amino-acid residue that is connected with the modification group that contains carbonyl, and X7 represents Glu or Ala amino-acid residue, X8 represents the Gln amino-acid residue, and X9 represents the Glu amino-acid residue.

7. modify oligopeptides according to each described oligopeptides of claim 4-6 or its, wherein, it is the oligopeptides that comprises 7 to 9 amino-acid residues that is made of the arbitrary aminoacid sequence in the formula (XI) to (XIV).

8. modify oligopeptides according to each described oligopeptides of claim 1-7 or its, wherein, X6 represents that end is connected with the Cys amino-acid residue of the modification group with carbonyl, or the terminal Lys amino-acid residue that is connected with the modification group with carbonyl.

9. modify oligopeptides according to each described oligopeptides of claim 1-8 or its, wherein, being connected in carbonatoms at the inner carbonyl of modification group is 2 to 10 hydrocarbon chain.

10. modify oligopeptides according to each described oligopeptides of claim 1-9 or its, wherein, being connected in carbonatoms at the inner carbonyl of modification group is 3 to 7 hydrocarbon chain.

11. modify oligopeptides according to each described oligopeptides of claim 1-10 or its, wherein, modification group is the modification group in N-β-maleimide propionic acid source, the modification group in N-ε-maleimide caproic acid source, or the modification group in N-κ-maleimide undeeanoic acid source.

12. following any oligopeptides or its are modified oligopeptides,

Ser-Ile-Glu-Gln-Ser-Xaa-Ala-Gln-Glu

Ser-Ile-Glu-Gln-Ser-Xaa-Glu one Gln-Glu

In the Ser-Ile-Glu-Gln-Ser-Xaa-Glu-Gln-Glu-Ala formula, Xaa represents to be connected with the Cys amino-acid residue of the modification group in N-ε-maleimide caproic acid source.

13. pharmaceuticals, it contains each described oligopeptides of claim 1-12 or its modifies on oligopeptides or its physiology acceptable salt as effective constituent.

14. hair growth promoter, it contains each described oligopeptides of claim 1-12 or its modifies on oligopeptides or its physiology acceptable salt as effective constituent.

15. the oligopeptides with hair growth promoter action or its modification oligopeptides that constitutes by the aminoacid sequence of following formula (1),

In the formula, X1 represents amino-acid residue Ser, Ala, Tyr, Thr, Pro, Phe, Val, Gly, Leu, Ile or Met, or disappearance,

X3 represents amino-acid residue Glu, Lys, Gln, Arg, Ala, Val, Trp or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser, His or Lys,

X6 represents amino-acid residue Cys,

Y disappearance or 1-90 aminoacid sequence of expression,

L represents linking group.

16. modify oligopeptides according to the described cyclic oligopeptides of claim 15 or its, wherein, X1 represents the Ser amino-acid residue, X2 represents the Ile amino-acid residue, X3 represents the Glu amino-acid residue, and X4 represents the Gln amino-acid residue, and X5 represents the Ser amino-acid residue, X6 represents the Cys amino-acid residue, and X7 represents Glu or Ala amino-acid residue.

17. according to claim 15 or 16 described cyclic oligopeptides or its modification oligopeptides, wherein Y disappearance.

18. according to claim 15 or 16 described cyclic oligopeptides or its modification oligopeptides, wherein 1-12 aminoacid sequence of Y table.

19. the oligopeptides that constitute, that have the hair growth promoter action of the aminoacid sequence by following formula (2) or its modification oligopeptides,

X3 represents amino-acid residue Glu, Lys, Gln, Arg, Ala, Val, Trp or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser, His or Lys,

X6 represents amino-acid residue Cys,

X8 represents amino-acid residue Gln,

L represents linking group.

20. modify oligopeptides according to the described cyclic oligopeptides of claim 19 or its, wherein, at least one side among X7 and the X9 is the amino-acid residue beyond the amino-acid residue Asp.

21. according to claim 19 or 20 described oligopeptides or its modification oligopeptides, wherein X1 represents the Ser amino-acid residue, X2 represents the Ile amino-acid residue, X3 table Glu amino-acid residue, X4 represents the Gln amino-acid residue, X5 represents the Ser amino-acid residue, X6 represents the Cys amino-acid residue, X7 represents Glu or Ala amino-acid residue, and X8 represents the Gln amino-acid residue, and X9 represents the Glu amino-acid residue.

22. the cyclic oligopeptides that constitute, that have the hair growth promoter action of the aminoacid sequence by following formula (3) or its modification oligopeptides,

X3 represents amino-acid residue Glu, Gln, Arg, Ala, Val, Trp or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser or His,

X6 represents amino-acid residue Lys,

Y disappearance or 1-90 aminoacid sequence of expression,

L represents linking group,

Z represents the group that can be connected with Lys reaction.

23. modify oligopeptides according to the described oligopeptides of claim 22 or its, wherein, X1 represents the Ser amino-acid residue, X2 represents the Ile amino-acid residue, X3 represents the Glu amino-acid residue, and X4 represents the Gln amino-acid residue, and X5 represents the Ser amino-acid residue, X6 represents the Cys amino-acid residue, and X7 represents Glu or Ala amino-acid residue.

24. according to claim 22 or 23 described oligopeptides or its modification oligopeptides, wherein Y disappearance.

25. according to claim 22 or 23 described oligopeptides or its modification oligopeptides, wherein Y represents 1-12 aminoacid sequence.

26. the cyclic oligopeptides that constitute, that have the hair growth promoter action of the aminoacid sequence by following formula (4) or its modification oligopeptides,

X3 represents amino-acid residue Glu, Gln, Arg, Ala, Val, Trp or Asp,

X4 represents amino-acid residue Gln, Pro, Glu, Thr, Arg, Ser or his,

X6 represents amino-acid residue Lys,

X8 represents amino-acid residue Gln,

L represents linking group,

Z represents the group that can be connected with Lys reaction.

27. modify oligopeptides according to the described oligopeptides of claim 26 or its, wherein, at least one side among X7 and the X9 is the amino-acid residue beyond the amino-acid residue Asp.

28. according to claim 26 or 27 described oligopeptides or its modification oligopeptides, wherein, X1 represents the Ser amino-acid residue, and X2 represents the Ile amino-acid residue, and X3 represents the Glu amino-acid residue, X4 represents the Gln amino-acid residue, X5 represents the Ser amino-acid residue, and X6 represents the Cys amino-acid residue, and X7 represents Glu or Ala amino-acid residue, X8 represents the Gln amino-acid residue, and X9 represents the Glu amino-acid residue.

29. modify oligopeptides according to each described oligopeptides of claim 15 to 28 or its, wherein, the carbonatoms of linking group L is 6 to 8.

30. modify oligopeptides according to each described oligopeptides of claim 15 to 29 or its, wherein, to be connected in carbonatoms be 5 to 7 hydrocarbon chain to carbonyl in linking group L.

31. modify oligopeptides according to each described oligopeptides of claim 15 to 30 or its, wherein, linking group L is-CO-(CH ₂) _n-NH-(in the formula, n represents the integer of 5-7).

32. modify oligopeptides according to the described oligopeptides of claim 31 or its, wherein, among the linking group L-CO is connected with X1, and the NH-among the linking group L and the Cys residue that is connected in X6 or the group that Z represents are connected.

33. following any cyclic oligopeptides or its are modified oligopeptides:

34. pharmaceuticals, it contains each described cyclic oligopeptides of claim 15-33 or its modifies on oligopeptides or its physiology acceptable salt as effective constituent.

35. a hair growth promoter, it contains each described cyclic oligopeptides of claim 15-33 or its modifies on oligopeptides or its physiology acceptable salt as effective constituent.

36. a hair growth promoter, it contains each described cyclic oligopeptides of claim 15-33 or its modifies on oligopeptides or its physiology acceptable salt and minoxidil as effective constituent.