JPH09157294A - Parathyroid hormone derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new parathyroid hormone derivative, comprising a peptide having a specific amino acid sequence, having strong cyclic adenosine 3',5'- monophosphate (cAMP) production activities and osteogenetic activities and useful for treatment, etc., of bone diseases such as fracture, osteopathy, osteomalacia or osteoarthritis. SOLUTION: This new parathroid hormone derivative comprises a peptide (salt) represented by the formula (R1 is an acidic amino acid; R2 is a hydrophobic α-amino acid or a basic amino acid; R3 is Gly, D or L-isomer Ala, Ser, Lys, Orn or Trp; R4 and R5 are each a basic amino acid; R6 is an aliphatic neutral amino acid or a basic amino acid; R7 is a nonchargeable hydrophilic amino acid, a basic amino acid or a dipeptide thereof; R8 is an acidic amino acid or a basic amino acid; R9 is an aliphatic neutral amino acid or a basic amino acid; R10 is a basic amino acid; R11 is a nonchargeable hydrophilic amino acid or a basic amino acid; R12 is an acidic amino acid or an aliphatic neutral amino acid; R13 is an aromatic amino acid, a peptide, etc., corresponding to a part of the parathroid hormone). The derivative is useful for treatment, etc., of bone diseases.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel derivative of parathyroid hormone and its use.

[0002]

BACKGROUND OF THE INVENTION Parathyroid hormone (PTH) is synthesized in the parathyroid gland and then acts on its target organs, bone and kidney, and plays an important role in mainly controlling the concentration of calcium and phosphate ions in blood. Are doing PTH is a peptide hormone consisting of 84 amino acids, but its biological action is N
It is known that it can be reproduced with a peptide fragment at the end (positions 1-34) [GW Tregear et al., Endocrinology, 93, 1349-135.
P. 3 (1973)]. N-terminal (1-
Peptide fragment at position 34 (hereinafter, human PTH
The amino acid sequence of (abbreviated as (1-34)) is as follows. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn -Ser- 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His- Asn-Phe-OH (SEQ ID NO: 1) To understand the structure-activity relationship of the hormone, PTH (1-
34) Various derivatives of fragments have been synthesized. Conventionally, researches on bovine PTH (1-34) have been mainly conducted, but recently researches on human PTH (1-34) have been increasing. For example, human PTH (1
Increase in activity when converting the C-terminal Phe in Phe-NH ₂ are known that seen is -34) (JP 58-96
052). However, this is probably because the degradation by carboxypeptidase was suppressed, and as a result, an apparent increase in activity was observed. In addition, human PTH (1-34)
Contains 2 residues of Met, and it is known that a molecule obtained by substituting these with Nle prevents the disappearance of hormone activity due to oxidation (Japanese Patent Laid-Open No. 61-24598). See also F.
E. Cohen et al. (The Journal of Biological Chemistr
y), 266 , 1997-2004 (1991); WO
92/00753] is human bovine PTH (1-34)
In Ser., Ser at position 3 was substituted with various L-amino acids, but the Ala-substituted form showed almost the same activity as the natural type, and the activity was remarkably reduced by other amino acid substitutions. Further, even a derivative in which the amino acids at the 6th and 9th positions have been substituted, a derivative having an activity suitable for use as a medicine has not been obtained. Even if the continuous sequence of basic amino acids at the 25th to 27th positions of PTH (1-34) is replaced with the sequence of another amino acid, its biological activity is retained, but its action on blood pressure and smooth muscle is reduced. (WO93 / 06845).
It is also said that analogs in which the 23rd position is substituted with another amino acid have the same effect (WO93 / 06846). In addition, JP-A-6-184198 (WO94 / 02510) discloses various amino acid-substituted analogs in addition to analogs having a modified side chain amino group.

From the biological action of PTH, it is expected that if it is used as a medicine, it can be a useful medicine for various bone diseases, but the following properties of the peptide make it difficult. I have to. 1. It is susceptible to degradation by various enzymes in the body. 2. The absorption efficiency into the body by various routes is very low. 3. It is unstable to various physicochemical conditions such as oxidation. In order to solve such problems and to elucidate the structure-activity relationship of the hormone, various derivatives of PTH (1-34) active fragment have been synthesized. In the measurement of the biological activity of these compounds, in the compound in which any one of the above problems 1 to 3 was avoided, the Phe-N at position 34 was added in the previous section.
Derivatives with increased activity may be found, as described for H ₂ derivatives. Further, for example, the derivative whose original activity is increased by increasing the affinity with the receptor can compensate the above-mentioned problems 1 to 3 due to its high activity.

Previously, the present inventors have found that human PTH (1-3
The amino acid substitution in 4) is performed chemically synthetically, and the 1st position, 8th position, 11th position, 12th position of human PTH (1-34)
13th, 18th, 19th, 21st, 23rd, 25th
(1) by performing amino acid substitution in consideration of resistance to various proteolytic enzymes to any of the amino acids at positions 26, 27, 34 and (2), the expected two-dimensional structure, By enhancing the activity of the hormone by amino acid substitution considering hydrophilic / hydrophobic or ionic environment, (3) further reduce the activity of amino acids that are unstable to acid, alkaline conditions, oxidation conditions, etc. However, it was discovered that this object can be achieved by substituting an amino acid that is stable against these conditions, and submitted an excellent human PTH (1-34) derivative (JP-A-05-032696). In addition, human PTH (1
-34) 3rd, 14th, 15th, 16th in the sequence
It has been found that the peptide derivative consisting of any of the amino acids at positions 17, 17, 25, 26, 27 and 34, or a combination of these substitutions has excellent activity (JP-A-05-320193). . Furthermore, human PTH
The peptide derivative in which any of the amino acids at the (34-47) position in (1-84) is substituted with Cys is capable of forming a dimer, and is introduced with another functional group to be a more excellent compound. It was found that conversion is possible (Japanese Patent Laid-Open No. 271/1993).
279).

[0005]

[Problems to be Solved by the Invention] Human PTH (1-3
It was an object of the present invention to find out, as the substitution product of 4), one having more excellent properties.

[0006]

As a result of further studies, it was found that the amino acid Asn at position 10 in human PTH (1-34) was replaced with an acidic amino acid to give a derivative excellent in its properties. . Further, by combining this knowledge with the results of the above-mentioned inventions of the present inventors, they succeeded in finding a more excellent compound, and completed the present invention. That is, the present invention has an amino acid sequence of Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-R ₁ -R ₂ -R ₃ -R ₄ -R ₅
-R ₆ -R ₇ -Met-R ₈ -Arg-R ₉ -Glu-Trp-Leu-Arg-R ₁₀ -R ₁₁ -Leu-G
ln-R ₁₂ -Val-His-Asn-R ₁₃ (SEQ ID NO: 2) [wherein R ₁ is an acidic amino acid, R ₂ is a hydrophobic α-amino acid or basic amino acid, and R ₃ is Gly or D]. Alternatively, L-form Ala, Ser, Lys, Orn or Trp, R ₄ is a basic amino acid, R ₅ is a basic amino acid, R ₆ is an aliphatic neutral amino acid or basic amino acid, and R ₇ is uncharged. A dipeptide consisting of a hydrophilic amino acid, a basic amino acid or a combination thereof, R ₈ is an acidic amino acid or a basic amino acid, R ₉ is an aliphatic neutral amino acid or a basic amino acid, R ₁₀ is a basic amino acid, ₁₁ is an uncharged hydrophilic amino acid or basic amino acid, R ₁₂ is an acidic amino acid or an aliphatic neutral amino acid, R ₁₃ is an aromatic amino acid, human PTH
(34-35), (34-36), (34-37),
(34-38), (34-39) or (34-4)
Peptide corresponding to 0) position, or peptide sequence corresponding to (34-84) position in which at least one amino acid between positions 35 and 45 may be substituted with Cys in D or L form, The carboxyl groups of the aromatic amino acid and the C-terminal amino acid of the peptide may be amidated. ] A peptide or a salt thereof, a pharmaceutical composition characterized by containing the peptide or a salt thereof, and a prophylactic / therapeutic agent for a bone disease characterized by containing the peptide or a salt thereof. .

In the above definition, R _{1 to} R ₁₃ will be described more specifically. R ₁ may be any acidic amino acid, natural or non-natural. Among such acidic amino acids are especially the formula

[0008]

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(Where R _a is H, OH or COOH,
n _a represents an integer of 0 to 4). The hydrophobic α-amino acid in R ₂ means Ala, Val, Leu, Ile, Pro, Met which constitutes a protein.
Such as amino acids having an alkyl group which may be substituted in the side chain, aromatic amino acids such as Phe, Trp, and Tyr, and amino acids that do not constitute proteins such as Nle (norleucine), naphthylalanine, and 4-chlorophenylalanine. included. The basic amino acid for R ₂ may be any basic amino acid, natural or non-natural, and may be of any of the formulas

[0010]

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[Wherein Z _a is NH ₂ , NHC (NH) NH ₂
Or, an imidazolyl group and m _a represents an integer of 1 to 5. ] The basic amino acid represented by these is mentioned. R ₃ is Gly or D or L form Ala, Ser, L
ys, Orn or Trp. The basic amino acid in R ₄ may be any basic amino acid, natural or non-natural,

[0012]

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[In the formula, Z _b is NH ₂ , NHC (NH) NH
₂ or an imidazolyl group, and m _b is an integer of 1 to 5. ] The basic amino acid represented by these is mentioned. The basic amino acid represented by R ₅ may be any basic amino acid, natural or non-natural,

[0014]

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[Wherein Z _c is NH ₂ , NHC (NH) NH
₂ or an imidazolyl group, m _c represents an integer of 1 to 5. ] The basic amino acid represented by these is mentioned. The aliphatic neutral amino acid for R ₆ may be natural or non-natural, and any aliphatic neutral amino acid may be used.

[0016]

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[In the formula, each of J _a and U _a represents H or an alkyl group having 1 to 4 carbon atoms. ] The aliphatic neutral amino acid represented by these is mentioned. R ₆ can also be a basic amino acid, in which case it may be any basic amino acid, natural or non-natural, in particular of the formula

[0018]

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[Wherein Z _d is NH ₂ , NHC (NH) NH
₂ or an imidazolyl group, and m _d is an integer of 1 to 5. ] The basic amino acid represented by these is mentioned. The uncharged hydrophilic amino acids constituting the R ₇ dipeptide include (1) Gly, (2) L or D-form Ser, Th
r, Cys, Asn or Gln is specifically mentioned,
(3) Whether the basic amino acid is natural or unnatural,
Any basic amino acid may be used, but in particular the formula

[0020]

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[Wherein Z _e is NH ₂ , NHC (NH) NH
₂ or an imidazolyl group, and m _e is an integer of 1 to 5. ] The basic amino acid represented by these is mentioned. Examples of R ₇ include the above-mentioned (1), (2) or (3), and (4) a dipeptide comprising a combination thereof. R ₈
The acidic amino acid may be any acidic amino acid, natural or non-natural,

[0022]

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[Wherein R _b is H, OH or COOH,
n _b represents an integer of 0 to 4. ] An acidic amino acid represented by the following is mentioned, and the basic amino acid may be any basic amino acid, natural or non-natural,

[0024]

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[Wherein Z _f is NH ₂ , NHC (NH) NH
₂ or an imidazolyl group, and m _f is an integer of 1 to 5. ] The basic amino acid represented by these is mentioned. The aliphatic neutral amino acid for R ₉ may be natural or non-natural and may be any aliphatic neutral amino acid.

[0026]

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[In the formula, each of J _b and U _b represents H or an alkyl group having 1 to 4 carbon atoms. ] The aliphatic neutral amino acid represented by these is mentioned. The basic amino acid may be any natural or non-natural basic amino acid.

[0028]

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[Wherein Z _g is NH ₂ , NHC (NH) NH
₂ or an imidazolyl group, and _mg represents an integer of 1 to 5. ] The basic amino acid represented by these is mentioned. The basic amino acid for R ₁₀ may be any basic amino acid, natural or non-natural, and may have any formula,

[0030]

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[In the formula, Z _h is NH ₂ , NHC (NH) NH
₂ or an imidazolyl group, m _h represents an integer of 1 to 5. ] The basic amino acid represented by these is mentioned. The uncharged hydrophilic amino acids of R ₁₁ include (1) Gly and (2) L
Or D-type Ser, Thr, Cys, Asn or G
ln is specifically mentioned, and as the basic amino acid (3), any of basic amino acids may be used, whether natural or unnatural,

[0032]

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[Wherein Z _i is NH ₂ , NHC (NH) NH
₂ or an imidazolyl group, and m _i is an integer of 1 to 5. ] The basic amino acid represented by these is mentioned. The acidic amino acid of R ₁₂ may be any acidic amino acid, natural or non-natural,

[0034]

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[Wherein R _c is H, OH or COOH,
n _c represents an integer of 0 to 4. ] R ₁₂ may be an aliphatic neutral amino acid, and the aliphatic neutral amino acid may be natural or unnatural, provided that it is an aliphatic neutral amino acid. Either, but especially the formula

[0036]

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[In the formula, each of J _c and U _c represents H or an alkyl group having 1 to 4 carbon atoms. ] The aliphatic neutral amino acid represented by these is mentioned. R ₁₃ is Phe, Tyr, Phe-Val, in which the carboxyl group of the C-terminal amino acid may be substituted with an amide group or an N—C _1-4 alkylamide group.
Phe-Val-Ala, Phe-Val-Ala-Leu (SEQ ID NO: 3), Phe-Val-Al
a-Leu-Gly (SEQ ID NO: 4), Phe-Val-Ala-Leu-Gly-Ala (SEQ ID NO: 5), Phe-Val-Ala-Leu-Gly-Ala-Pro (SEQ ID NO: 6), Alternatively, Phe-Val-Ala-Leu-Gly-Ala-Pro-Leu-Ala-Pro-, in which at least one amino acid between the 2nd to 12th amino acids in the sequence may be substituted with D- or L-form Cys Arg-As
p-Ala-Gly-Ser-Gln-Arg-Pro-Arg-Lys-Lys-Glu-Asp-Asn-
Val-Leu-Val-Glu-Ser-His-Glu-Lys-Ser-Leu-Gly-Glu-Al
a-Asp-Lys-Ala-Asp-Val-Asn-Val-Leu-Thr-Lys-Ala-Lys-
One that is Ser-Gln (SEQ ID NO: 7).

The R _{1 to} R ₁₃ will be described more specifically. Specific examples of R ₁ include Asp, Glu, aminoadipic acid, aminosuberic acid, and 4-carboxyglutamic acid, and Asp and Glu are particularly preferable. A specific example of R ₂ is L
eu, Phe, Lys or naphthylalanine are mentioned, and among them, Leu, Phe or Ly is preferable.
s. Specific examples of R ₃ include Gly, DT
rp, D-Ala or D-Ser is mentioned, and among them, Gly, D-Ala or D-Ser is preferable. Specific examples of R ₄ include Lys or Orn. Specific examples of R ₅ include His and Lys, and His is particularly preferable. Specific examples of R ₆ include Leu and Lys, and among them, Leu is preferable. R
_As specific examples of ₇ , Asn-Ser, Lys-Lys,
Asn-Lys, Lys-Ser or Ser-Ser can be mentioned, and among them, Asn-Ser,
Lys-Lys, Lys-Ser or Ser-Ser are mentioned. Specific examples and preferred examples of R ₈ include Gl
u or Arg. Specific examples and preferable examples of R ₉ include Val and Arg. Specific examples and preferred examples of R ₁₀ include Lys or Arg. Specific examples and preferred examples of R ₁₁ are Lys or G
In may be mentioned. Specific examples and preferred examples of R ₁₂ include Asp or 2-aminoisobutyric acid.

Specific examples and preferred examples of R ₁₃ include Ph
e.

As a specific example of the peptide of the present invention or a salt thereof, in the SEQ ID NO: 2, R ₁ is Asp, Glu, aminoadipic acid, aminosuberic acid or 4-carboxyglutamic acid, and R ₂ is Leu, Phe, Lys or naphthylalanine, R ₃ is Gly, D-Trp, D
-Ala or D-Ser and R ₄ is Lys or Or
n, R ₅ is His or Lys, and R ₆ is Leu
Or Lys and R ₇ is Asn-Ser, Lys-L
ys, Asn-Lys, Lys-Ser or Ser-S
er, R ₈ is Glu or Arg, and R ₉ is Va
1 or Arg, R ₁₀ is Lys or Arg,
R ₁₁ is Lys or Gln, and R ₁₂ is Asp or 2-
Examples include aminoisobutyric acid in which R ₁₃ is Phe.

As a specific example of the peptide of the present invention or a salt thereof, in the SEQ ID NO: 2, R ₁ is an acidic amino acid and R _{2 is}
Is a hydrophobic α-amino acid or a basic amino acid, and R ₃
Is Gly or D or L Ala, Ser, L
ys or Orn, R ₄ is Lys, R ₅ is Hi
s, R ₆ is Leu, R ₇ is a dipeptide consisting of an uncharged hydrophilic amino acid, a basic amino acid or a combination thereof, R ₈ is Glu, R ₉ is Val and R ₁₀ is Lys, R ₁₁ is an uncharged hydrophilic amino acid or basic amino acid, R ₁₂ is Asp and R ₁₃
Is an aromatic amino acid, human PTH (34-35), (34
-36), (34-37), (34-38), (34-
39) or the peptide corresponding to the (34-40) position,
Or at least one amino acid between positions 35 and 45 is D
Alternatively, it may be substituted with L-form Cys (34-
The peptide sequence corresponding to position 84) is shown, and the aromatic amino acid and the carboxyl group of the C-terminal amino acid of the peptide may each be amidated. ] What is.

As a specific example of the peptide of the present invention or a salt thereof, in the SEQ ID NO: 2, R ₁ is represented by the formula:

[0043]

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[Wherein R _a is H, OH or COOH
And n _a represents an integer of 0 to 4. ] The acidic amino acid represented by the following, R ₂ is Ala, Val, Leu, Ile,
Pro, Met, Phe, Trp, Tyr, Nle, naphthylalanine, 4-chlorophenylalanine or formula

[0045]

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[In the formula, Z _a is NH ₂ , NHC (NH) NH
₂ or an imidazolyl group, and m _a represents an integer of 1 to 5. ] And R ₃ is Gly.
Alternatively, D or L form Ala, Ser, Lys or Orn, R ₄ is Lys, R ₅ is His, R ₆ is Leu, R ₇ is (1) Gly, (2) L
Or D-type Ser, Thr, Cys, Asn or G
ln, formula (3)

[0047]

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[Wherein Z _e is NH ₂ , NHC (NH) NH
₂ or an imidazolyl group, and m _e is an integer of 1 to 5. ] A basic amino acid represented by the following, or (4) a dipeptide consisting of a combination thereof, wherein R ₈ is Gl
u, R ₉ is Val, R ₁₀ is Lys,
R ₁₁ is (1) Gly, (2) L or D body Ser,
Thr, Cys, Asn or Gln, formula (3)

[0049]

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[Wherein Z _i is NH ₂ , NHC (NH) NH
₂ or an imidazolyl group, and m _i is an integer of 1 to 5. ] It is a basic amino acid represented by these, and R ₁₂ is Asp
And R ₁₃ is Phe, Tyr, Phe-Val, Phe-Val-Ala, Ph in which the carboxyl group of the C-terminal amino acid may be substituted with an amide group or an N-C1-4 alkylamido group.
e-Val-Ala-Leu, Phe-Val-Ala-Leu-Gly, Phe-Val-Ala-Leu-
Gly-Ala, Phe-Val-Ala-Leu-Gly-Ala-Pro, or 2 in the sequence
At least one of the amino acids between the 12th and 12th is D
Or Phe-Val-, which may be substituted with L-form Cys
Ala-Leu-Gly-Ala-Pro-Leu-Ala-Pro-Arg-Asp-Ala-Gly-Se
r-Gln-Arg-Pro-Arg-Lys-Lys-Glu-Asp-Asn-Val-Leu-Val-
Glu-Ser-His-Glu-Lys-Ser-Leu-Gly-Glu-Ala-Asp-Lys-Al
a-Asp-Val-Asn-Val-Leu-Thr-Lys-Ala-Lys-Ser-Gln, in which R ₁ is Asp, Glu, aminoadipic acid, aminosuberic acid or 4-carboxy Peptides that are glutamic acid or salts thereof are preferred. Examples of the amidated carboxyl group include an amide group and an N—C _1-4 alkylamide group, and examples of the N—C _1-4 alkylamide group include a methylamide, an ethylamide, a propylamide and a butylamide. . Ja, Jb, Jc, Ua,
Examples of the alkyl group having 1 to 4 carbon atoms represented by Ub or Uc include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and sec-butyl.

These substitutions in the compound of the present invention are not limited to one substitution, but a combination of substitutions at several substitutions is possible, and a combination of substitutions up to 4 substitutions is particularly preferable. Specifically, for example, [Asp ¹⁰ , Lys ¹¹ ] hPTH (1-3
4) [Asp ^10] hPTH (1-34) [Glu ^10] hPTH (1-34) [Asp ^10, Phe ^11] hPTH (1-34) ^{[Asp 10, Ala (2-Naph} ) 11 ] hPTH (1
-34) [Glu ^10] hPTH (1-34) methylamide [Glu ^10, Lys ^{16, 17]} hPTH (1-34) [Glu ^10, Ser ^16] hPTH (1-34) [Glu ^10, Tyr ^34] hPTH (1-34) [Glu ¹⁰ , Cys ³⁵ ] hPTH (1-84) [Glu ¹⁰ , Orn ¹³ ] hPTH (1-34), [Gl
u ¹⁰ , D-Ala ¹² ] hPTH (1-34) [Glu ¹⁰ , Lys ^16,17 , Gln ²⁷ ] hPTH (1-
34) [Glu ¹⁰ , Phe ¹¹ , D-Ala ¹² ] hPTH (1-
34), [Glu ¹⁰ , Phe ¹¹ , Lys ¹⁶ , Gln ²⁷ ].
hPTH (1-34), [Glu ¹⁰ ] hPTH (1-8
4) and the like, among which [Asp ¹⁰ , Lys ¹¹ ] hP
TH (1-34), [Glu ¹⁰ ] hPTH (1-3
4), [Glu ¹⁰ , Phe ¹¹ , Lys ¹⁶ , Gln ²⁷ ] h
PTH (1-34), [Glu ¹⁰ , Ser ¹⁶ ] hPTH
(1-34), [Glu ¹⁰ , Orn ¹³ ] hPTH (1-
34), [Glu ¹⁰ , Phe ¹¹ , D-Ala ¹² ] hPT
H (1-34), [Asp ¹⁰ , Phe ¹¹ ] hPTH (1
-34) or [Asp ¹⁰ ] hPTH (1-34) or a salt thereof is mentioned as a preferred embodiment of the present invention.

The peptide compound of the present invention can be synthesized by a gene recombination method or a chemical synthesis method. In particular, the latter method can be mainly carried out using an automatic peptide synthesizer. Regarding the production of peptides by the gene recombination method, see JP-A-5-32019.
3, No. 5,271,279, No. 5-304976 and the like. A brief description of them is as follows. In order to produce the parathyroid hormone derivative of the present invention by the gene recombination method, a gene encoding the amino acid sequence of human PTH (1-84) [eg European Patent Application Publication No. 483509] or its C-terminal deletion is used. The gene encoding the corresponding amino acid sequence is converted to the gene encoding the derivative of interest using conventional DNA techniques, such as site-directed mutagenesis techniques. Site-directed mutagenesis techniques are well known and are
Racer (Lather, RF) and Jay P. Lecock
(Lecoq, JP), Genetic Engineering (G
Enetic Engineering), Academic Press (198
3 years) on pages 31-50. Oligonucleotide-directed mutagenesis is by Smith, M.
And Gillam, S., Genetic Engineering: Principles and Methods, Plenum Phums (198).
1 year) 3 vol. 1-32. In order to produce a structural gene encoding a parathyroid hormone derivative having various chain lengths with amino acid substitution according to the present invention, for example, (a) human PTH or its C-terminal deleted structural gene Hybridizing the resulting DNA with a mutated oligonucleotide primer, (b) extending the primer with a DNA polymerase to form a mutagenic heteroduplex, followed by (c) the mutagenic heterodimer. There is a method of replicating the body. Following this replication, the mutant gene is isolated from the progeny of the mutant chain, inserted into a suitable vector, and the vector used to transform a suitable host organism or cell. The phage DNA carrying the mutated gene is then isolated and integrated into a plasmid. The gene thus cloned can be linked to the downstream of the promoter in a vehicle (vector) suitable for expression to obtain an expression type vector. Vectors include E. coli-derived plasmids (eg, pBR322, pBR32
5, pUC12, pUC13), a Bacillus subtilis-derived plasmid (eg, pUB110, pTP5, pC194), a yeast-derived plasmid (eg, pSH19, pSH15), bacteriophages such as λ phage, and retroviruses, animal viruses such as vaccinia virus, etc. Can be given. The gene may have ATG as a translation initiation codon at its 5'end and TAA, TGA or TAG as a translation stop codon at its 3'end. In order to further express the gene, a promoter is connected upstream thereof. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.

The vector containing the recombinant DNA having the nucleotide sequence encoding the parathyroid hormone derivative of the present invention thus constructed is used to produce a transformant carrying the vector. Examples of the host include Escherichia, Bacillus, yeast, animal cells and the like. A parathyroid hormone derivative is produced by culturing a transformant carrying a vector containing a recombinant DNA having a nucleotide sequence encoding the obtained parathyroid hormone derivative in a medium. Separation and purification of the parathyroid hormone derivative from the above culture can be performed, for example, by the following method. First, the cultured cells or cells are destroyed and the contents are extracted. This is a French press,
Ultrasonic waves, lysozyme, freeze-thaw, glass beads and the like can be used. When destroying the cells or cells, 1 M to 8 M urea or 1 M to 6 M guanidine hydrochloride may be added to the buffer solution, or if a reducing agent such as dithiothreitol is added, the target secondary Recovery of thyroid hormone derivatives may increase. The reducing agent is added after allowing lysozyme to act. Next, the cell extract is separated into a supernatant and a precipitate by centrifugation. When the parathyroid hormone derivative is recovered in the supernatant, for example,
M. Iwane et al. [Biochem. Biophys.
Res. Commun.) 146 , 470-477 (1987)]. When the parathyroid hormone derivative is recovered as a precipitate, the precipitate can be dissolved in a solution containing a protein denaturant such as guanidine hydrochloride or urea, and then dialyzed or diluted to reduce the concentration of the denaturant. A thyroid hormone derivative can be obtained. The parathyroid hormone derivative recovered from the precipitate becomes a highly pure and highly active product as well as the parathyroid hormone derivative recovered from the supernatant by performing a purification operation as necessary. Examples of further separation and purification means include column filtration such as gel filtration, ion exchange chromatography using a cation exchange resin or anion exchange resin, hydrophobic chromatography, partition adsorption chromatography and high performance liquid chromatography.

Basic synthesis using an automatic peptide synthesizer is described in, for example, RB Merrifield [Advances in Enzymology 32 , 22.
1-296 (1969)]. In this method, the amino acid at the carboxyl terminus is covalently bonded to the resin carrier, and the protective group for the amino group is removed.
The principle is that condensation of protected amino acids is sequentially repeated to extend the peptide chain toward the amino terminus to obtain a protected peptide resin having a target amino acid sequence. Condensation of each amino acid and removal of the amino-protecting group are carried out under substantially the same conditions, and no intermediate purification is carried out. Therefore, generally no high skill is required in the synthesis. Moreover, this method is rapid and is a very convenient method for synthesizing various peptides. The protected peptide resin thus obtained is reacted with, for example, anhydrous hydrogen fluoride, trifluoromethanesulfonic acid or trifluoroacetic acid in the presence of various additives to remove the peptide from the resin and remove all protecting groups. Can be done in one step.
As an automatic peptide synthesizer, the conditions can usually be set according to the protocol of each apparatus. The obtained crude peptide product can be purified by a known means for purifying a peptide or protein. For example, gel filtration, cation exchange, or ion exchange chromatography using anion exchange resin, and further hydrophobic chromatography,
Column chromatography and high performance liquid chromatography based on various principles such as partition adsorption chromatography can be mentioned.

The peptides of the present invention can be obtained in the form of various salts. As the salt, a physiologically acceptable salt or a salt that can be used as a raw material is used, and examples thereof include a salt with an inorganic acid or an organic acid such as formic acid, acetic acid, tartaric acid or citric acid, and an inorganic salt such as sodium or ammonia. Examples thereof include bases and salts with organic bases such as triethylamine, ethylamine or methylamine. When the desired product is obtained in a free state, it may be converted into a salt according to a conventional method, and when the desired product is obtained as a salt, it may be converted into a free form or another salt according to a conventional method. You can also

Human PTH represented by the general formula of the present invention
Since the (1-34) derivative peptide has low toxicity and is safe, it can be used as a medicine alone or in combination with an excipient or the like. Among other medicines, bone disease prevention and
Therapeutic agent, therapeutic agent for hypoparathyroidism, therapeutic agent for hypertension,
It can be used as a therapeutic agent for menopausal disorders (including menopausal disorders caused by the use of other drugs). Prevention of bone disease
Treatment includes improvement of bone formation, that is, fixation of calcium in bone and osteoporosis due to various causes (e.g., juvenile, menopausal, postmenopausal, posttraumatic, old age, estrogen deficiency, growth hormone deficiency, Bone disorders, including acute and chronic conditions associated with hypoparathyroidism, hyperthyroidism, nutritional or metabolic disorders, or due to corticosteroid therapy or inactivity), bone fracture, skeletal demineralization, and All prophylaxis or treatment of bone disorders such as osteomalacia, periodontal bone loss or bone loss due to arthritis or osteoarthritis, or treatment of hypoparathyroidism is included. Examples of the dosage form include injections, nasal absorbents, rectal absorbents, vaginal absorbents, and transdermal absorbents, but they may be orally administered depending on the case. When the peptide is used as such a therapeutic agent, an effective amount thereof is used for mammals (for example, humans, mice, rats, dogs, cats, cows, pigs, monkeys, etc.). Generally 1ng-100
Range of μg / kg of body weight, preferably 5-100 μg / kg of body weight
However, the exact amount is appropriately determined by those skilled in the art. When this peptide is used as a prophylactic / therapeutic agent, it is sufficiently purified so that bacteria and pyrogens do not exist, but the method itself may be carried out according to known means. When this peptide is used as a prophylactic or therapeutic drug for osteoporosis or the like, it may be used as it is or after being mixed with a pharmacologically acceptable additive, and then the above injection, nasal absorbent, rectal absorbent, vaginal absorbent or transdermal absorption. It can be administered parenterally in the form of a drug. The injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like. Such an injection is prepared by a method known per se, that is, by dissolving, suspending or emulsifying the compound of the present invention in a sterile aqueous liquid or oily liquid. A saline solution as an aqueous solution for injection,
Isotonic solutions containing glucose and other adjuvants (eg D-
Sorbitol, D-mannitol, sodium chloride, etc., and suitable solubilizers such as alcohols (eg ethanol), polyalcohols (eg propylene glycol, polyethylene glycol), nonionic surfactants (eg polysorbate 80, You may use together with HCO-50). Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg,
Human serum albumin, polyethylene glycol, etc.),
You may mix | blend with preservatives (for example, benzyl alcohol, phenol, etc.). The prepared injection is usually
Fill into a suitable ampoule. In some cases, it may be administered orally. When producing oral preparations such as powders, tablets, granules and capsules, pharmaceutically acceptable carriers can be blended. Examples of the carrier include excipients (eg lactose, starch etc.), lubricants (eg magnesium stearate, talc etc.),
Binders (hydroxypropylcellulose, hydroxypropylmethylcellulose, macrogol, etc.), disintegrating agents (eg starch, carboxymethylcellulose calcium, etc.) and the like are used. Also, if necessary,
Additives such as preservatives (for example, benzyl alcohol, chlorobutanol, methyl paraoxybenzoate, propyl paraoxybenzoate, etc.), antioxidants, colorants, sweeteners and the like can be used. The dose of the peptide of the present invention is 50 ng to 5 mg, preferably 20 to 300 μg, once a day for adults and once a day for injections. The concentration of the peptide of the present invention is 10 in the injection.
100 μg / ml is suitable. When used as a percutaneous absorption agent, it can be absorbed from the skin by iontophoresis and is 50 ng to 5 mg per dose, preferably 20
It is suitable that the dose is from 1 μg to 1 mg, more preferably from 20 μg to 400 μg, three times a day to once every three days.

In the present specification, when amino acids and the like are represented by abbreviations, IUPAC-IUB Commision on
It is based on the abbreviations according to Biochemical Nomenclature or abbreviations commonly used in this field, and examples are given below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified. Gly or G: Glycine Ala or A: Alanine Val or V: Valine Leu or L: Leucine Ile or I: Isoleucine Ser or S: Serine Thr or T: Threonine Cys or C: Cysteine Met or M: Methionine Glu or E: Glutamic acid Asp or D: aspartic acid Lys or K: lysine Arg or R: arginine His or H: histidine Phe or F: phenylalanine Tyr or Y: tyrosine Trp or W: tryptophan Pro or P: proline Asn or N: asparagine Gln or Q : Glutamine Nle: Norleucine Orn: Ornithine Gla: 4-Carboxyglutamic acid Ala (2-Naph): 2-Naphtylalanine Aad: 2-Aminoadipic acid Asu: 2-Aminosuberic acid Aib 2-aminoisobutyric acid hPTH: human PTH

[0058]

By substituting hPTH (1-34) as in the present invention, a derivative showing a strong PTH action can be obtained. First, the activity was increased by changing the 10th amino acid to an acidic amino acid, and the 11th, 13th, 14th,
15th, 16th, 17th, 19th, 21st, 26th, 2
This effect is retained or enhanced by combining with substitution at the 7-position and 30-position. Substitution of the D-amino acid at position 12 increases resistance to various proteolytic enzymes,
A sustained activity in the blood is obtained.

[0059]

EXAMPLES The present invention will be specifically described below with reference to examples, but the examples of typical amino acid substitutions listed here should not be construed as limiting the present invention. Example 1 Synthesis and Purification of PTH Peptide (1-34) Derivative The synthesis of this peptide was carried out by the solid phase synthesis method for peptides (RB Merrifield. Advances in Enzymology) 32 developed by Merrifield et al. roll,
221-296, 1969), using an automated peptide synthesizer 430A (Applied Biosystems). The protected peptide-resin was synthesized using the protocol specified by Applied Biosystems. A protected amino acid-p-oxymethylphenylacetamidomethyl resin (polystyrene-1% divinylbenzene) is used to obtain a derivative of a free carboxylic acid at the carboxyl terminal, and a 4-methylbenzhydryl resin is used to obtain a derivative of a carboxylamide. Was used as a starting material, and a protected amino acid was condensed to this as a starting material. A tertiary butoxycarbonyl (BOC) group was used to protect the α-amino group of each amino acid during the condensation. Side functional group protection was performed as follows. The hydroxyl group of serine and threonine is O-benzyl ether, and the hydroxyl group of tyrosine is p-
As bromobenzyloxycarbonyl ester, the carboxyl groups of glutamic acid and aspartic acid are as benzyl esters, the imidazole nitrogen of histidine is benzyloxymethyl, the side chain amino group of lysine is 2-chlorobenzyloxycarbonyl, and the side chain amino group of ornithine is The group was protected with benzyloxycarbonyl, the guanidine functional group of arginine was p-toluenesulfonyl group, and the indoleimine of tryptophan was protected with formyl group. All amino acids were obtained from Applied Biosystems Japan, Nova Biochem or Bachem Chemicals. After condensing all amino acids on the resin, remove the protected peptide resin from the synthesizer,
Dried. Peptide resin (1 g) was reacted with anhydrous hydrogen fluoride (8 ml) containing p-cresol (1 ml), 1,2-ethanedithiol (1 ml) and 2-mercaptopyridine (100 mg) at 0 ° C. for 2 hours. Let After the reaction,
Hydrogen fluoride was distilled off and the residue was washed with diethyl ether to remove most of the mixed reagents. The peptide was extracted with 3% acetic acid (10 ml) and the resin was removed by filtration. The filtrate was purified by gel filtration using Sephadex G-25. The conditions for gel filtration are column size 2.6 x 66 cm,
The detection wavelength was 280 nm, the solvent was 3% acetic acid, and the flow rate was 30 ml / hour. Fractions containing the peptide were collected and lyophilized, and the resulting powder preparation was further purified by reverse phase high performance liquid chromatography (HPLC). Column YMC-Pack R & D D-ODS-5 S-5 120A OD
S (20 × 250 mm) elution solvent A, 0.1% trifluoroacetic acid-99.9% water; elution solvent B, 0.1% trifluoroacetic acid-99.9% acetonitrile; elution gradient program, 0 min ( 80% A + 20% B), 30 minutes (50%
(A + 50% B) (However, other elution programs may be used if necessary.) Elution rate 5.0 ml / min, detection wavelength 230 or 280 nm. Biorad AG 1x8 (acetic acid type, 2.5x2c
After passing through the column of m), the washings were collected, the acetonitrile was distilled off, and the residue was freeze-dried. For automated peptide synthesis, 9-fluorenylmethoxycarbonyl (Fm
The oc) group was also used. In this method, an automatic peptide synthesizer 431A (Applied Biosystems) was used. The protected peptide-resin was synthesized using the protocol specified by Applied Biosystems.
The protected amino acid-p-alkoxybenzyl alcohol resin was used as a starting material to obtain a derivative of a free carboxylic acid at the carboxyl terminal, and a protected amino acid was subsequently condensed with this. A 9-fluorenylmethoxycarbonyl (Fmoc) group was used to protect the α-amino group of each amino acid during condensation. Side chain functional group protection was performed as follows. Serine,
The hydroxyl group of threonine and tyrosine is an O-tertiary butyl ether, the side chain carboxyl group is a tertiary butyl ester, the imidazole nitrogen of histidine is a trityl group, and the side chain amino group such as lysine is a tertiary butoxycarbonyl group, and arginine. The guanidine functional group of is 2,2,
Protected with a 5,7,8-pentamethylchroman-6-sulfonyl group. The protected amino acid resin was obtained from Watanabe Chemical Industry, and the amino acid was obtained from Watanabe Chemical Industry, Peptide Research Institute, Applied Biosystems Japan, Nova Biochem, and Vaticem Chemicals. After condensation of all amino acids on the resin and removal of the N-terminal Fmoc group, the protected peptide resin was removed from the synthesizer and dried. Peptide resin (0.5g) on crystalline phenol (0.375g),
1,2-Ethanedithiol (0.125 ml), thioanisole (0.25 ml), distilled water (0.25 ml) and trifluoroacetic acid (5 ml) were sequentially added under ice-cooling, and then the mixture was returned to room temperature and reacted for 2 hours. Let After completion of the reaction, trifluoroacetic acid was distilled off, the residue was washed with diethyl ether, and most of the mixed reagents were removed. Peptide 30% acetic acid (7 ml)
The resin was removed by filtration. The filtrate was purified by gel filtration using Sephadex G-25. Gel filtration followed by purification by reverse phase HPLC was performed in the same manner as described above.

The peptides (1) to (2 obtained in this way
5) is as follows. (1) [Asp ¹⁰ , Lys ¹¹ ] hPTH (1-34)
(SEQ ID NO: 8) (2) [Asp ¹⁰ , Lys ¹¹ , D-Trp ¹² ] hPTH
(1-34) (3) [Asp ¹⁰ ] hPTH (1-34) (SEQ ID NO:
9) (4) [Glu ¹⁰ ] hPTH (1-34) (SEQ ID NO:
10) (5) [Asp ¹⁰ , Phe ¹¹ ] hPTH (1-34)
(SEQ ID NO: 11) (6) [Asp ¹⁰ , Ala (2-Naph) ¹¹ ] hPT
H (1-34) (SEQ ID NO: 12) (7) [Gla ¹⁰ ] hPTH (1-34) (SEQ ID NO:
13) (8) [Asu ¹⁰ ] hPTH (1-34) (SEQ ID NO:
14) (9) [Aad ¹⁰ ] hPTH (1-34) (SEQ ID NO:
15) (10) [Glu ¹⁰ , Phe ¹¹ , D-Ala ¹² ] hPT
H (1-34) (11) [Glu ¹⁰ , D-Ser ¹² ] hPTH (1-3
4) (12) [Glu ¹⁰ , Lys ^16,17 ] hPTH (1-3
4) (SEQ ID NO: 16) (13) [Glu ¹⁰ , Lys ¹⁷ ] hPTH (1-34)
(SEQ ID NO: 17) (14) [Glu ¹⁰ , Lys ¹⁶ ] hPTH (1-34)
(SEQ ID NO: 18) (15) [Glu ¹⁰ , Ser ¹⁶ ] hPTH (1-34)
(SEQ ID NO: 19) (16) [Glu ¹⁰ , Lys ¹⁶ , Gln ²⁷ ] hPTH
(1-34) (SEQ ID NO: 20) (17) [Glu ¹⁰ , Phe ¹¹ , Lys ¹⁶ , Gln ²⁷ ]
hPTH (1-34) (SEQ ID NO: 21) (18) [Asp ¹⁰ , Phe ¹¹ , Lys ¹⁶ , Gln ²⁷ ,
Aib ³⁰ ] hPTH (1-34) (SEQ ID NO: 22) (19) [Asp ¹⁰ , Phe ¹¹ , Lys ^16,17 , Gln
²⁷ , Aib ³⁰ ] hPTH (1-34) (SEQ ID NO: 2
3) (20) [Asp ¹⁰ , Phe ¹¹ , Lys ^15,16 , Gln
²⁷ , Aib ³⁰ ] hPTH (1-34) (SEQ ID NO: 2
4) (21) [Glu ¹⁰ , Lys ¹⁴ ] hPTH (1-34)
(SEQ ID NO: 25) (22) [Glu ¹⁰ , Orn ¹³ ] hPTH (1-34)
(SEQ ID NO: 26) (23) [Asp ¹⁰ , Arg ¹⁹ ] hPTH (1-34)
(SEQ ID NO: 27) (24) [Asp ¹⁰ , Arg ²¹ ] hPTH (1-34)
(SEQ ID NO: 28) (25) [Glu ¹⁰ , Arg ²⁶ ] hPTH (1-34)
(SEQ ID NO: 29) A list of this peptide is shown in [Table 1], and the amino acid analysis results and retention times in reverse phase high performance liquid chromatography are shown in [Table 2-1] to [Table 2-5]. In addition,
A, b, and c in [Table 2] are as follows. a: In the presence of 4% thioglycolic acid, hydrolysis was performed with 6 N hydrochloric acid in a vacuum sealed tube at 110 ° C. for 24 hours and then subjected to amino acid analysis. Theoretical values are in parentheses. b: Test compound (carboxylic acid type has nothing at the end): c: Retention time of derivative by high performance liquid chromatography. Analytical conditions: Waters 'M600E high performance liquid chromatogram was used by connecting a Waters' 717 plus autosampler. Column TMC-pack
R & D R-ODS-5 S-5 120A (4.6x
250 mm); elution solvent A, 0.1% trifluoroacetic acid-99.9% water; elution solvent B, 0.1% trifluoroacetic acid-99.9% acetonitrile; elution concentration program,
0 minutes (80% A + 20% B), 30 minutes (50% A + 50)
% B); flow rate 1.0 ml / min; detection wavelength 230 nm.

[0061]

[Table 1]

[0062]

[Table 2-1]

[0063]

[Table 2-2]

[0064]

[Table 2-3]

[0065]

[Table 2-4]

[0066]

[Table 2-5]

Example 2 PTH peptide (1-34
In vitro biological activity of PTH partial peptide (1-34 position) analogs, Shigeno et al., The Journal of Biological Chemistry, Vol. 263, pp. 18369-18377, 1980 〔Shi
geno et al., The Journal of Biological Chemistry, 263: 1.
8369-18377 (1988)] and modified and evaluated. 96-well multi plate (Nunclo
n), Nunc] mouse skull-derived osteoblast-like cell line, MC3T3-EI cells, 0.01, 0.1, 1, 10 or 10
100 μl of culture medium containing 0 nM peptide derivative [20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 0.1% bovine serum albumin (BSA) and 0.5 mM
Of Hank's solution containing isobutylmethylxanthine was added and reacted at room temperature for 30 minutes. 0.2 normality hydrochloric acid 100 μl
Then, the cells were immersed in boiling water for two and a half minutes, and cyclic adenosine monophosphate (cAMP) produced by the PTH receptor was extracted from the cells. The total cAMP measurement in the culture solution and the cells was carried out using a commercially available radioimmunoassay kit [Cyclic AMP [ ¹²⁵ I] kit “DuPont-Daiichi”, Daiichi Pure Chemicals). Regarding the biological activity of the PTH peptide (1-34 position) derivative, Table 3 shows the increased amount of cAMP produced by the analog 1 nM.

[0068]

[Table 3]

[Example 3] PTH peptide (1-34)
The biological activity of the derivative was measured by subcutaneously administering the compound synthesized in Example 1 (4.9 nmol / kg) to male Sprangue Dawley rats aged 4 weeks, for 2 weeks, to increase the bone mass in the femur.
(0.15M salt, 0.001N hydrochloric acid, heat inactivated 2%
(Rat serum) administration group. After administration, the right femur is taken out, the surrounding tissues are removed, and dried at 100 ° C. for 3 hours,
Weighed. Table 4 shows the increase in bone mass in the rats to which the compound of 4.9 nmol / kg was administered daily.

[0070]

[Table 4]

[0071]

INDUSTRIAL APPLICABILITY The novel PTH (1-34) derivative of the present invention has a strong cAMP producing activity and osteogenic activity and can be a useful drug for bone diseases and the like.

[0072]

[Sequence list]

[0073]

[SEQ ID NO: 1] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0074]

[SEQ ID NO: 2] Sequence length: 34 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics: Location: 10 Other information: Xaa = acidic amino acid, Location: 11 other Information: Xaa = hydrophobic α-amino acid, basic amino acid Location: 12 Other information: Xaa = Gly, Ala, Ser, Lys, Orn Location: 13 Other information: Xaa = basic amino acid Location: 14 other Information: Xaa = basic amino acid location: 15 Other information: Xaa = aliphatic neutral amino acid, basic amino acid Location: 16,17 Other information: Xaa = uncharged hydrophilic amino acid-basic amino acid location : 19 Other information: Xaa = acidic amino acid, basic amino acid Location: 21 Other information: Xaa = aliphatic neutral amino acid, basic amino acid Location: 26 Other information: Xaa = basic amino acid Location: 27 Other information: Xaa = uncharged hydrophilic amino acid, basic amino acid Location: 30 Other information: Xaa = acidic amino acid Aliphatic neutral amino acids present position: 34 Other Information: Xaa = aromatic amino acids, Phe-Val, Phe-Val-Ala, Ph
e-Val-Ala-Leu, Phe-Val-Ala-Leu-Gly, Phe-Val-Ala-Leu-
Gly-Ala, Phe-Val-Ala-Leu-Gly-Ala-Pro, Val-Ala-Leu-Gl
y-Ala-Pro-Leu-Ala-Pro-Arg-Asp-Ala-Gly-Ser-Gln-Arg-
Pro-Arg-Lys-Lys-Glu-Asp-Asn-Val-Leu-Val-Glu-Ser-Hi
s-Glu-Lys-Ser-Leu-Gly-Glu-Ala-Asp-Lys-Ala-Asp-Val-
Asn-Val-Leu-Thr-Lys-Ala-Lys-Ser-Gln Sequence: Ser Val Ser Glu Ile Gln Leu Met His Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Met Xaa Arg Xaa Glu Trp Leu Arg Xaa Xaa Leu Gln Xaa Val His 20 25 30 Asn Xaa 34

[0075]

[SEQ ID NO: 3] Sequence length: 4 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Phe Val Ala Leu 1

[0076]

[SEQ ID NO: 4] Sequence length: 5 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Phe Val Ala Leu Gly 1 5

[0077]

[SEQ ID NO: 5] Sequence length: 6 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Phe Val Ala Leu Gly Ala 1 5

[0078]

[SEQ ID NO: 6] Sequence length: 7 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Phe Val Ala Leu Gly Ala Pro 1 5

[0079]

[SEQ ID NO: 7] Sequence length: 51 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser Gln 1 5 10 15 Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu Lys 20 25 30 Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys Ala 35 40 45 Lys Ser Gln 50

[0080]

[SEQ ID NO: 8] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Asp Lys Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0081]

[SEQ ID NO: 9] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Asp Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0082]

[SEQ ID NO: 10] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Glu Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe.

[0083]

[SEQ ID NO: 11] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Asp Phe Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0084]

[SEQ ID NO: 12] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Asp Xaa Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe Sequence features: Location: 11 Other information: Xaa = Ala (2-Naph)

[0085]

[SEQ ID NO: 13] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence features: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Gla Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0086]

[SEQ ID NO: 14] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Asu Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0087]

[SEQ ID NO: 15] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Aad Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0088]

[SEQ ID NO: 16] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Glu Leu Gly Lys His Leu Lys 1 5 10 15 Lys Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0089]

[SEQ ID NO: 17] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Glu Leu Gly Lys His Leu Asn 1 5 10 15 Lys Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0090]

[SEQ ID NO: 18] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Glu Leu Gly Lys His Leu Lys 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0091]

[SEQ ID NO: 19] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Glu Leu Gly Lys His Leu Ser 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0092]

[SEQ ID NO: 20] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Glu Leu Gly Lys His Leu Lys 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Gln Leu Gln Asp Val His 20 25 30 Asn Phe

[0093]

[SEQ ID NO: 21] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Glu Phe Gly Lys His Leu Lys 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Gln Leu Gln Asp Val His 20 25 30 Asn Phe

[0094]

[SEQ ID NO: 22] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Asp Phe Gly Lys His Leu Lys 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Gln Leu Gln Aib Val His 20 25 30 Asn Phe

[0095]

[SEQ ID NO: 23] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Asp Phe Gly Lys His Leu Lys 1 5 10 15 Lys Met Glu Arg Val Glu Trp Leu Arg Lys Gln Leu Gln Aib Val His 20 25 30 Asn Phe

[0096]

[SEQ ID NO: 24] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence features: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Asp Phe Gly Lys His Lys Lys 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Gln Leu Gln Aib Val His 20 25 30 Asn Phe

[0097]

[SEQ ID NO: 25] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Glu Leu Gly Lys Lys Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0098]

[SEQ ID NO: 26] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Glu Leu Gly Orn His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0099]

[SEQ ID NO: 27] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Asp Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Arg Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0100]

[SEQ ID NO: 28] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Asp Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Arg Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe

[0101]

[SEQ ID NO: 29] Sequence length: 34 Sequence type: Peptide Topology: Linear Sequence type: Amino acid Sequence characteristics: Partial peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Glu Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Arg Lys Leu Gln Asp Val His 20 25 30 Asn Phe

Claims (36)

[Claims] 1. Amino acid sequence Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-R ₁ -R ₂ -R ₃ -R ₄ -R ₅
-R ₆ -R ₇ -Met-R ₈ -Arg-R ₉ -Glu-Trp-Leu-Arg-R ₁₀ -R ₁₁ -Leu-G
ln-R ₁₂ -Val-His-Asn-R ₁₃ [wherein R ₁ is an acidic amino acid, R ₂ is a hydrophobic α-amino acid or a basic amino acid, and R ₃ is Gly or a D or L form of Ala, Ser, Lys,
Orn or Trp, R ₄ is a basic amino acid, R ₅ is a basic amino acid, R ₆ is an aliphatic neutral amino acid or a basic amino acid, R ₇ is an uncharged hydrophilic amino acid, a basic amino acid or their A dipeptide consisting of a combination, R ₈ is an acidic amino acid or basic amino acid, R ₉ is an aliphatic neutral amino acid or basic amino acid, R ₁₀ is a basic amino acid, R ₁₁ is an uncharged hydrophilic amino acid or basic amino acid. R _{12 for} amino acids
Is an acidic amino acid or an aliphatic neutral amino acid, R ₁₃ is an aromatic amino acid, human PTH (34-35), (34-3
6), (34-37), (34-38), (34-3)
9) or the peptide corresponding to the (34-40) position, or at least one of the amino acids between the 35th position and the 45th position may be substituted with Cys in the D or L form (34-8).
The peptide sequence corresponding to position 4) is shown, and the aromatic amino acid and the carboxyl group of the C-terminal amino acid of the peptide may each be amidated. ] The peptide or its salt represented by these. 2. R ₁ is of the formula: [In the formula, R _a represents H, OH or COOH, and n _a represents an integer of 0 to 4. ] The peptide or its salt of Claim 1 which is an acidic amino acid represented by these. 3. R ₁ is of the formula: [In the formula, R _a represents H, OH or COOH, and n _a represents an integer of 0 to 4. ] The acidic amino acid represented by these, R < ₂ > is Ala, Val, Leu, Ile, Pro, Me.
t, Phe, Trp, Tyr, Nle, naphthylalanine, 4-chlorophenylalanine or the formula: [In the formula, Z _a represents an NH ₂ , NHC (NH) NH ₂ or imidazolyl group, and m _a represents an integer of 1 to 5. ] A basic amino acid represented by the following, wherein R ₃ is Gly or a D or L form of Ala or Se.
r, Lys, Orn or Trp, and R ₄ is of the formula: [In the formula, Z _b represents NH ₂ , NHC (NH) NH ₂ or an imidazolyl group, and m _b represents an integer of 1 to 5. ] A basic amino acid represented by the formula, wherein R ₅ is of the formula: [In the formula, Z _c represents NH ₂ , NHC (NH) NH ₂ or an imidazolyl group, and m _c represents an integer of 1 to 5. ] Is a basic amino acid represented by the formula, wherein R ₆ is of the formula: [In the formula, each of J _a and U _a represents H or an alkyl group having 1 to 4 carbon atoms. ] An aliphatic neutral amino acid represented by the following formula or [In the formula, Z _d represents NH ₂ , NHC (NH) NH ₂ or an imidazolyl group, and m _d represents an integer of 1 to 5. ] And R ₇ is (1) Gly, (2) L or D-form Ser,
Thr, Cys, Asn or Gln, Formula (3): [In the formula, Z _e represents NH ₂ , NHC (NH) NH ₂ or an imidazolyl group, and m _e represents an integer of 1 to 5. ] A basic amino acid represented by the following, or (4) a dipeptide consisting of a combination thereof, wherein R ₈ is of the formula: [In the formula, R _b represents H, OH or COOH, and n _b represents an integer of 0 to 4. ] The acidic amino acid represented by [In the formula, Z _f represents NH ₂ , NHC (NH) NH ₂ or an imidazolyl group, and m _f represents an integer of 1 to 5. ] A basic amino acid represented by the formula, wherein R ₉ is represented by the formula: [In the formula, each of J _b and U _b represents H or an alkyl group having 1 to 4 carbon atoms. ] An aliphatic neutral amino acid represented by the following formula or [In the formula, Z _g represents NH ₂ , NHC (NH) NH ₂ or an imidazolyl group, and _mg represents an integer of 1 to 5. ] A basic amino acid represented by the formula, wherein R ₁₀ is of the formula: [In the formula, Z _h represents NH ₂ , NHC (NH) NH ₂ or an imidazolyl group, and m _h represents an integer of 1 to 5. ] And R ₁₁ is (1) Gly, (2) L or D-form Ser,
Thr, Cys, Asn or Gln, Formula (3): [In the formula, Z _i represents NH ₂ , NHC (NH) NH ₂ or an imidazolyl group, and m _i represents an integer of 1 to 5. ] A basic amino acid represented by the following formula, wherein R ₁₂ has the formula: [In the formula, R _c represents H, OH or COOH, and n _c represents an integer of 0 to 4. ] The acidic amino acid represented by the formula or [In the formula, each of J _c and U _c represents H or an alkyl group having 1 to 4 carbon atoms. ] R ₁₃ is an aliphatic neutral amino acid, wherein R ₁₃ may have a carboxyl group of the C-terminal amino acid substituted with an amide group or an N—C _1-4 alkylamide group, Phe, Tyr, Phe-Val, Phe-Val-Ala, Phe-Val-Ala-
Leu, Phe-Val-Ala-Leu-Gly, Phe-Val-Ala-Leu-Gly-Ala, Ph
e-Val-Ala-Leu-Gly-Ala-Pro, or the 2nd to 1st in the sequence
At least one of the amino acids between the second is D or L
Phe-Val-Ala-Leu-Gl optionally substituted by Cys in the body
y-Ala-Pro-Leu-Ala-Pro-Arg-Asp-Ala-Gly-Ser-Gln-Arg-
Pro-Arg-Lys-Lys-Glu-Asp-Asn-Val-Leu-Val-Glu-Ser-Hi
s-Glu-Lys-Ser-Leu-Gly-Glu-Ala-Asp-Lys-Ala-Asp-Val-
Asn-Val-Leu-Thr-Lys-Ala-Lys-Ser-Gln.
The described peptide or a salt thereof. 4. The peptide or salt thereof according to claim 1, wherein R ₁ is Asp, Glu, aminoadipic acid, aminosuberic acid or 4-carboxyglutamic acid. 5. The peptide or a salt thereof according to claim 1, wherein R ₁ is Asp or Glu. 6. The peptide or salt thereof according to claim 1, wherein R ₂ is Leu, Phe, Lys or naphthylalanine. 7. The peptide according to claim 1, wherein R ₂ is Leu, Phe or Lys, or a salt thereof. 8. The peptide or salt thereof according to claim 1, wherein R ₃ is Gly, D-Trp, D-Ala or D-Ser. 9. R ₃ is Gly, D-Ala or D-Ser
The peptide or salt thereof according to claim 1, which is 10. The method of claim 1 R ₄ is Lys or Orn
The described peptide or a salt thereof. 11. R ₁ is His or Lys.
The described peptide or a salt thereof. 12. The peptide according to claim 1, wherein R ₅ is His, or a salt thereof. 13. The peptide according to claim 1, wherein R ₆ is Leu or Lys, or a salt thereof. 14. The peptide according to claim 1, wherein R ₆ is Leu, or a salt thereof. 15. R ₇ is Asn-Ser, Lys-Ly
s, Asn-Lys, Lys-Ser or Ser-Se
The peptide or salt thereof according to claim 1, which is r. 16. R ₇ is Asn-Ser, Lys-Ly
s, Lys-Ser or Ser-Ser.
The described peptide or a salt thereof. 17. The method according to claim 1, wherein R ₈ is Glu or Arg.
The described peptide or a salt thereof. 18. The method according to claim 1, wherein R ₉ is Val or Arg.
The described peptide or a salt thereof. 19. The method according to claim 1, wherein R ₁₀ is Lys or Arg.
The described peptide or a salt thereof. 20. The method of claim 1 R ₁₁ is Lys or Gln
The described peptide or a salt thereof. 21. The peptide according to claim 1, wherein R ₁₂ is Asp or 2-aminoisobutyric acid, or a salt thereof. 22. The peptide according to claim 1, wherein R ₁₃ is Phe, or a salt thereof. 23. R ₁ is Asp, Glu, aminoadipic acid, aminosuberic acid or 4-carboxyglutamic acid, R ₂ is Leu, Phe, Lys or naphthylalanine, and R ₃ is Gly, D-Trp, D-Ala or D-Ser
R ₄ is Lys or Orn, R ₅ is His or Lys, R ₆ is Leu or Lys, and R ₇ is Asn-Ser, Lys-Lys, Asn-Ly.
s, Lys-Ser or Ser-Ser, R ₈ is Glu or Arg, R ₉ is Val or Arg, R ₁₀ is Lys or Arg, R ₁₁ is Lys or Gln, R ₁₂ Is Asp or 2-aminoisobutyric acid and R ₁₃ is Ph
The peptide or salt thereof according to claim 1, which is e. 24. R ₁ is an acidic amino acid, R ₂ is a hydrophobic α-amino acid or a basic amino acid, and R ₃ is Gly or a D or L form of Ala or Se.
r, Lys or Orn, R ₄ is Lys, R ₅ is His, R ₆ is Leu, and R ₇ is a dipeptide consisting of an uncharged hydrophilic amino acid, a basic amino acid or a combination thereof. R ₈ is Glu, R ₉ is Val, R ₁₀ is Lys, R ₁₁ is an uncharged hydrophilic amino acid or basic amino acid, R ₁₂ is Asp and R ₁₃ is an aromatic amino acid, Human PTH
(34-35), (34-36), (34-37),
(34-38), (34-39) or (34-4)
The peptide sequence corresponding to the (0) position or the peptide sequence corresponding to the (34-84) position in which at least one of the amino acids between the 35th position and the 45th position may be substituted with Cys in the D or L form is shown. The carboxyl groups of the aromatic amino acid and the C-terminal amino acid of the peptide may be amidated. ] The peptide or its salt according to claim 1, which is 25. R ₁ is of the formula: [In the formula, R _a represents H, OH or COOH, and n _a represents an integer of 0 to 4. Acidic amino acids represented by], R ₂ is Ala, Val, Leu, Ile, Pro, Me
t, Phe, Trp, Tyr, Nle, naphthylalanine, 4-chlorophenylalanine or the formula: [In the formula, Z _a represents an NH ₂ , NHC (NH) NH ₂ or imidazolyl group, and m _a represents an integer of 1 to 5. ] A basic amino acid represented by the following, wherein R ₃ is Gly or a D or L form of Ala or Se.
r, Lys or Orn, R ₄ is Lys, R ₅ is His, R ₆ is Leu, R ₇ is (1) Gly, (2) L or D-form Ser,
Thr, Cys, Asn or Gln, Formula (3): [In the formula, Z _e represents NH ₂ , NHC (NH) NH ₂ or an imidazolyl group, and m _e represents an integer of 1 to 5. ] A basic amino acid represented by the following, or (4) a dipeptide consisting of a combination thereof, R ₈ is Glu, R ₉ is Val, R ₁₀ is Lys, and R ₁₁ is (1) Gly. , (2) L or D body Ser,
Thr, Cys, Asn or Gln, Formula (3): [In the formula, Z _i represents NH ₂ , NHC (NH) NH ₂ or an imidazolyl group, and m _i represents an integer of 1 to 5. ] A basic amino acid represented by the formula, R ₁₂ is Asp, and R ₁₃ is Phe or Tyr in which the carboxyl group of the C-terminal amino acid may be substituted with an amide group or an N—C _1-4 alkylamide group. ， Phe-Val, Phe-Val-Ala, Phe-Val-Ala-
Leu, Phe-Val-Ala-Leu-Gly, Phe-Val-Ala-Leu-Gly-Ala, Ph
e-Val-Ala-Leu-Gly-Ala-Pro, or the 2nd to 1st in the sequence
At least one of the amino acids between the second is D or L
Phe-Val-Ala-Leu-Gl optionally substituted by Cys in the body
y-Ala-Pro-Leu-Ala-Pro-Arg-Asp-Ala-Gly-Ser-Gln-Arg-
Pro-Arg-Lys-Lys-Glu-Asp-Asn-Val-Leu-Val-Glu-Ser-Hi
s-Glu-Lys-Ser-Leu-Gly-Glu-Ala-Asp-Lys-Ala-Asp-Val-
Asn-Val-Leu-Thr-Lys-Ala-Lys-Ser-Gln.
The described peptide or a salt thereof. 26. The peptide according to claim 25 or a salt thereof, wherein R ₁ is Asp, Glu, aminoadipic acid, aminosuberic acid or 4-carboxyglutamic acid. 27. [Asp ¹⁰ , Lys ¹¹ ] hPTH (1-
34) The peptide according to claim 1 or a salt thereof. 28. The peptide according to claim 1, which is [Glu ¹⁰ ] hPTH (1-34), or a salt thereof. 29. [Glu ¹⁰ , Phe ¹¹ , Lys ¹⁶ , Gl
The peptide or a salt thereof according to claim 1, which is n ²⁷ ] hPTH (1-34). 30. [Glu ¹⁰ , Ser ¹⁶ ] hPTH (1-
34) The peptide according to claim 1 or a salt thereof. 31. [Glu ¹⁰ , Orn ¹³ ] hPTH (1-
34) The peptide according to claim 1 or a salt thereof. 32. [Glu ¹⁰ , Phe ¹¹ , D-Ala ¹² ]
The peptide or salt thereof according to claim 1, which is hPTH (1-34). 33. [Asp ¹⁰ , Phe ¹¹ ] hPTH (1-
34) The peptide according to claim 1 or a salt thereof. 34. The peptide according to claim 1, which is [Asp ¹⁰ ] hPTH (1-34), or a salt thereof. 35. A pharmaceutical composition comprising the peptide according to claim 1 or a salt thereof. 36. A preventive / therapeutic agent for bone diseases, which comprises the peptide according to claim 1 or a salt thereof.