JP2003221400A - New peptide and therapeutic agent for bone disease containing the peptide as active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new substance which is useful as a therapeutic agent for bone diseases. <P>SOLUTION: A peptide having a specific amino acid sequence and its derivatives are provided. The peptide has a growth promoting effect and an activity promoting effect on osteoblasts. This new peptide provided in this invention has the growth promoting effect and the activity promoting effect on osteoblasts and has an activity as a therapeutic agent for bone diseases. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel peptide and a therapeutic agent for bone diseases containing the peptide as an active ingredient.

[0002]

2. Description of the Related Art It is a well-known fact that many physiologically active substances are present in the living body and are closely related to the maintenance of normal living activities of the living body. The in-vivo physiologically active substance and the synthetic physiologically active substance themselves have the potential as a new drug and also provide the knowledge for developing a new drug. Therefore, the search for these physiologically active substances is extremely important.

On the other hand, calcitonin, female hormone, activated vitamin D3 and the like are currently used for the treatment of bone diseases such as osteoporosis, but the therapeutic effects are not always satisfactory.

[0004]

An object of the present invention is to provide a novel substance useful as a therapeutic agent for bone diseases.

[0005]

Means for Solving the Problems The present inventor found a novel peptide having this action when searching for a substance that promotes the activity of osteoblasts involved in bone formation. Completed the invention.

That is, according to the present invention, SEQ ID NO: 1 in the sequence listing
There is provided a peptide having an amino acid sequence represented by and a derivative thereof having a growth promoting action and an activity promoting action on osteoblasts. The present invention also provides a therapeutic agent for bone diseases, which contains the peptide of the present invention as an active ingredient.

The present invention will be described in detail below.

The peptide of the present invention basically has the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.

Further, in the scope of the present invention, in addition to the above-mentioned peptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, it is a derivative of the peptide and has a proliferation promoting activity and an activity promoting activity on osteoblasts. Peptides are also included. For example, a peptide obtained by chemically modifying the above-mentioned peptide without impairing the above-mentioned action is included in the "derivative" referred to herein. For example, it is known that stability in vivo can be enhanced by changing the C-terminal carboxyl group of a peptide to an amino group, and such chemically modified peptides are also included in the scope of the present invention. .

Further, in general, in a peptide having physiological activity, the physiological activity is maintained even when the small number of amino acids are substituted with other amino acids, the small number of amino acids are added, or the small number of amino acids are deleted. It is a fact well known to those skilled in the art that there are cases. Therefore, within the scope of the present invention, among the amino acids constituting the peptide having the amino acid sequence represented by SEQ ID NO: 1, a small number of amino acids are substituted with other amino acids, a small number of amino acids are added, or a small number of amino acids are added. Are also included in the “derivative” referred to in the present invention, and of these, peptides having a growth promoting action and an activity promoting action on osteoblasts are included in the scope of the present invention. For example, a peptide having 5 or less amino acids added, substituted or deleted, and a peptide having an amino acid sequence homology of 70% or more and having a growth promoting action and an activity promoting action on osteoblasts are included in the scope of the present invention. Be done. Whether or not the peptide derivative has a growth promoting action and an activity promoting action on osteoblasts can be examined by the methods described in Examples 2 and 4 below.

Since the peptide of the present invention represented by SEQ ID NO: 1 has a relatively small number of constituent amino acids of 16, it can be easily produced by chemical synthesis based on a conventional method.
For example, it can be easily produced using a commercially available peptide synthesizer. Alternatively, a conventional genetic engineering method of synthesizing a DNA encoding the amino acid sequence by a DNA synthesizer, inserting the DNA into a cloning site of a commercially available cloning vector, transforming a host microorganism with this, and culturing Can also be easily manufactured.

As confirmed experimentally in the following Examples, the peptide of the present invention has a growth promoting action and an activity promoting action on osteoblasts. Therefore, the peptide of the present invention is useful as a therapeutic agent for bone diseases such as osteoporosis. Examples of bone diseases treatable by the peptide of the present invention include osteoporosis and osteomalacia.

Since the peptide of the present invention has a relatively small molecular weight, it can be administered intravenously, subcutaneously and intramuscularly, as well as orally and transdermally by a formulation technique.

The dose may be appropriately determined depending on the condition of the patient, but usually, the daily dose of the peptide of the present invention of 0.
It is about 1 to 10 mg.

In the case of intravenous administration, subcutaneous administration and intramuscular administration, the peptide of the present invention is added to a citrate buffer solution (pH).
4-6) or a weakly acidic buffer such as an acetate buffer (pH 4-6) is preferably administered. In this case, the concentration of peptide in the buffer is usually 0.1 mg / ml.
It is about 10 mg / ml. In addition, in the case of oral and transdermal administration, a fat-soluble substance (eg petrolatum)
It is desirable to dissolve it in water and improve its absorbability. In this case, the concentration of the peptide of the present invention is usually 0.1 mg / ml-
It is about 100 mg / ml.

[0016]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Example 1 Production of Peptide A peptide having the amino acid sequence shown in SEQ ID NO: 1 was synthesized using a commercially available peptide synthesizer.

Example 2 Osteoblast Growth Promoting Action Rat-derived ROS cells, which are osteoblasts (obtained from ATC)
C) was cultured in F10 medium containing 10% fetal bovine serum (obtained from Dainippon Pharmaceutical Co., Ltd.) and grown in a 37 ° C. incubator humidified with 5% carbon dioxide. A 24-well culture plate was seeded with 1 × 10 5 cells / well by trypsin treatment, and when it became confluent, the medium was replaced with 1% fetal bovine serum F10 medium and cultured for 24 hours. After that, the peptide of the present invention produced in Example 1 was dissolved in 1% fetal bovine serum F10 medium, added to the well while changing the volume, and further cultured for 24 hours. After culturing, the cell growth-promoting effect of this peptide was measured by the MTT assay, and the growth-promoting effect for the untreated group was determined. The MTT assay and the calculation of the growth promotion rate were specifically performed as follows. MTF-Cel commercially available from Funakoshi Co., Ltd.
According to the procedure of the 1-Growth Assay kit, this substance was added to the well while changing the dose, and left for one day, and then M by the enzyme existing in mitochondria of living cells.
TT (3-4,5Dimethylthiazol-2YL) -2,5Diphenyl Tetraz
Viable cells were counted by the colorimetric method utilizing the phenomenon that olium bromide is cleaved into dark blue formazan. The control group to which this substance was not added was set to 100%, and the colorimetric value of the group to which the dose was changed was calculated as follows. The results are shown in Table 1 below.

[0019]

[Table 1] Table 1

As is clear from Table 1, it was confirmed that the peptide of the present invention acts on osteoblasts in a growth promoting manner. Therefore, the peptide of the present invention is considered to be associated with an increase in bone mass, and is useful for treating bone diseases such as osteoporosis.

Example 3 Presence of Receptor for this Peptide on Osteoblast Since it was revealed that this peptide has a growth-promoting effect on osteoblast, the osteoblast has a receptor for this peptide. It is inferred that If the receptor is present, this peptide is considered to be a vital substance for life, and then it was investigated whether or not the receptor was present in osteoblasts.

The peptide obtained in Example 1 was labeled with biotin, and the ROS cells cultured in the same manner as in Example 2 were added with a certain amount of the labeled peptide, and then the unlabeled peptide was added for further competitive reaction. Was dissolved in 10% fetal bovine serum F10 medium and added while changing the volume to see a competitive reaction. Specifically, this experimental operation was performed as follows. SU
This peptide was biotinylated according to the procedure of the protein biotinylated labeling kit manufactured by MILON, and a fixed amount of biotinylated peptide was added to a fixed number of cells seeded in the well, and unlabeled peptide 0 to 0.512 μg / well was added to each. The competitive reaction is carried out for 6 hours, and then the cells are washed with PBS and reacted with the biotinylated peptide bound to the receptor on the cell surface with streptavidin-labeled peroxidase to observe the color reaction. If the receptor of this peptide is present on the cell surface, a competitive reaction occurs with the unlabeled peptide and the intensity of color development decreases. The results are shown in Table 2 below.

[0023]

[Table 2] Table 2

As shown in Table 2, since the ratio of the labeled substance to the added labeled substance was changed depending on the amount of the unlabeled substance added, it is clear that the osteoblast has a receptor for this peptide. Therefore, it is inferred that this substance plays a fundamental role.

Acute toxicity test The peptide prepared in Example 1 was tested for its acute toxicity using male ddy mice (body weight 40 to 45 g). This peptide is dissolved in physiological saline (pH 6.0),
This was administered from the tail vein of mice and observed for 14 days.
The dose was 1, 10, 100 μg / kg. The results are shown in Table 3 below.

[0026]

[Table 3] Table 3

As described above, no deaths were observed in the range up to 100 μg / kg.

[0028]

INDUSTRIAL APPLICABILITY The present invention provides a novel peptide. As experimentally confirmed by the above-mentioned examples, the peptide of the present invention has a proliferation promoting action and an activity promoting action on osteoblasts, and it is clear that it has an action as a therapeutic agent for bone diseases. became.

[0029]

[Sequence list]                             SEQUENCE LISTING <110> Kenji SAKAMOTO and Hideo NAKOSHI <120> Novel peptides and drugs for treating bone diseases containing th e same as effective ingredients <130> 95374DA <160> 1

[0030] <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> peptide having an activity to accelerate the growth of osteoblast s <400> 1 Lys Leu Thr Thr Ile Phe Pro Leu Asn Trp Lys Tyr Arg Lys Ala Leu   1 5 10 15

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[Procedure amendment]

[Submission date] January 8, 2003 (2003.1.8)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction content]

[Document name] Statement

Title: Novel peptide and therapeutic agent for bone disease containing the same as an active ingredient

[Claims]

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for bone diseases containing a novel peptide as an active ingredient.

[0002]

2. Description of the Related Art It is a well-known fact that many physiologically active substances are present in the living body and are closely related to the maintenance of normal living activities of the living body. The in-vivo physiologically active substance and the synthetic physiologically active substance themselves have the potential as a new drug and also provide the knowledge for developing a new drug. Therefore, the search for these physiologically active substances is extremely important.

On the other hand, calcitonin, female hormone, activated vitamin D3 and the like are currently used for the treatment of bone diseases such as osteoporosis, but the therapeutic effects are not always satisfactory.

[0004]

The object of the present invention is to provide a novel
To provide a therapeutic agent for various bone diseases.

[0005]

Means for Solving the Problems The present inventor found a novel peptide having this action when searching for a substance that promotes the activity of osteoblasts involved in bone formation. Completed the invention.

That is, according to the present invention, SEQ ID NO: 1 in the sequence listing
Peptide or 該A having the amino acid sequence shown in
Addition of 1 to 5 amino acids in the mino acid sequence,
A peptide with a substituted or deleted amino acid sequence
Proliferation-promoting and activity-promoting effects on osteoblasts
To the peptides having
As an active ingredient, a derivative with chemical modification
Contains (however, the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing)
Except when a peptide having a no acid sequence is used as the active ingredient
Providing therapeutic agents for bone diseases .

The present invention will be described in detail below.

The peptide, which is an active ingredient of the therapeutic agent for bone diseases of the present invention, basically has the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.

[0009] The scope of the present invention also includes a peptide which is a derivative of the above-mentioned peptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing and which has a growth promoting action and an activity promoting action on osteoblasts. For example, a peptide obtained by chemically modifying the above-mentioned peptide without impairing the above-mentioned action is included in the "derivative" referred to herein. For example, it is known that stability in vivo can be enhanced by changing the C-terminal carboxyl group of a peptide to an amino group, and such chemically modified peptides are also included in the scope of the present invention. .

Further, in general, in a peptide having physiological activity, the physiological activity is maintained even when the small number of amino acids are substituted with other amino acids, the small number of amino acids are added, or the small number of amino acids are deleted. It is a fact well known to those skilled in the art that there are cases. Therefore, within the scope of the present invention, one of the amino acid sequences shown in SEQ ID NO: 1 is
No more than 5 amino acids added, substituted or deleted
A peptide having an amino acid sequence
Peptide having growth-promoting action and activity-promoting action
(Hereinafter sometimes referred to as "modified peptide") is the active ingredient
The therapeutic agent for bone diseases contained as is included in the scope of the present invention. Whether or not the peptide derivative or modified peptide has a growth promoting action and an activity promoting action on osteoblasts can be examined by the methods described in Examples 2 and 4 below.

The peptide represented by SEQ ID NO: 1 or its modification
Since the decorative peptide has a relatively small number of constituent amino acids of 16, it can be easily produced by chemical synthesis based on a conventional method. For example, it can be easily produced using a commercially available peptide synthesizer. Alternatively, DNA encoding the amino acid sequence is synthesized by a DNA synthesizer, inserted into a cloning site of a commercially available cloning vector, and a host microorganism is transformed with this and cultured.
It can also be easily produced by conventional genetic engineering techniques.

As experimentally confirmed in the following Examples, the peptide having the amino acid sequence represented by SEQ ID NO: 1 has a growth promoting action and an activity promoting action on osteoblasts. Therefore, the therapeutic agent for bone diseases of the present invention is useful as a therapeutic agent for bone diseases such as osteoporosis. Bone of the present invention
Examples of bone diseases that can be treated with the disease therapeutic agent include osteoporosis and osteomalacia.

Used as an active ingredient of the therapeutic agent for bone diseases of the present invention
Since the peptide that can be used has a relatively small molecular weight, it can be administered intravenously, subcutaneously and intramuscularly, as well as orally and transdermally by a formulation technique.

The dose may be appropriately determined depending on the condition of the patient, but usually, the daily dose of the peptide of the present invention of 0.
It is about 1 to 10 mg.

In the case of intravenous administration, subcutaneous administration and intramuscular administration, the peptide of the present invention is added to a citrate buffer solution (pH).
4-6) or a weakly acidic buffer such as an acetate buffer (pH 4-6) is preferably administered. In this case, the concentration of peptide in the buffer is usually 0.1 mg / ml.
It is about 10 mg / ml. In addition, in the case of oral and transdermal administration, a fat-soluble substance (eg petrolatum)
It is desirable to dissolve it in water and improve its absorbability. In this case, the concentration of the peptide of the present invention is usually 0.1 mg / ml-
It is about 100 mg / ml.

[0016]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Example 1 Production of Peptide A peptide having the amino acid sequence shown in SEQ ID NO: 1 was synthesized using a commercially available peptide synthesizer.

Example 2 Osteoblast Growth Promoting Action Rat-derived ROS cells, which are osteoblasts (obtained from ATC)
C) was cultured in F10 medium containing 10% fetal bovine serum (obtained from Dainippon Pharmaceutical Co., Ltd.) and grown in a 37 ° C. incubator humidified with 5% carbon dioxide. A 24-well culture plate was seeded with 1 × 10 5 cells / well by trypsin treatment, and when it became confluent, the medium was replaced with 1% fetal bovine serum F10 medium and cultured for 24 hours. After that, the peptide of the present invention produced in Example 1 was dissolved in 1% fetal bovine serum F10 medium, added to the well while changing the volume, and further cultured for 24 hours. After culturing, the cell growth-promoting effect of this peptide was measured by the MTT assay, and the growth-promoting effect for the untreated group was determined. The MTT assay and the calculation of the growth promotion rate were specifically performed as follows. MTF-Cel commercially available from Funakoshi Co., Ltd.
According to the procedure of the 1-Growth Assay kit, this substance was added to the well while changing the dose, and left for one day, and then M by the enzyme existing in mitochondria of living cells.
TT (3-4,5Dimethylthiazol-2YL) -2,5Diphenyl Tetraz
Viable cells were counted by the colorimetric method utilizing the phenomenon that olium bromide is cleaved into dark blue formazan. The control group to which this substance was not added was set to 100%, and the colorimetric value of the group to which the dose was changed was calculated as follows. The results are shown in Table 1 below.

[0019]

[Table 1] Table 1

As is clear from Table 1, it was confirmed that the peptide of the present invention acts on osteoblasts in a growth promoting manner. Therefore, the peptide of the present invention is considered to be associated with an increase in bone mass, and is useful for treating bone diseases such as osteoporosis.

Example 3 Presence of Receptor for this Peptide on Osteoblast Since it was revealed that this peptide has a growth-promoting effect on osteoblast, the osteoblast has a receptor for this peptide. It is inferred that If the receptor is present, this peptide is considered to be a vital substance for life, and then it was investigated whether or not the receptor was present in osteoblasts.

The peptide obtained in Example 1 was labeled with biotin, and the ROS cells cultured in the same manner as in Example 2 were added with a certain amount of the labeled peptide, and then the unlabeled peptide was added for further competitive reaction. Was dissolved in 10% fetal bovine serum F10 medium and added while changing the volume to see a competitive reaction. Specifically, this experimental operation was performed as follows. SU
This peptide was biotinylated according to the procedure of the protein biotinylated labeling kit manufactured by MILON, and a fixed amount of biotinylated peptide was added to a fixed number of cells seeded in the well, and unlabeled peptide 0 to 0.512 μg / well was added to each. The competitive reaction is carried out for 6 hours, and then the cells are washed with PBS and reacted with the biotinylated peptide bound to the receptor on the cell surface with streptavidin-labeled peroxidase to observe the color reaction. If the receptor of this peptide is present on the cell surface, a competitive reaction occurs with the unlabeled peptide and the intensity of color development decreases. The results are shown in Table 2 below.

[0023]

[Table 2] Table 2

As shown in Table 2, since the ratio of the labeled substance to the added labeled substance was changed depending on the amount of the unlabeled substance added, it is clear that the osteoblast has a receptor for this peptide. Therefore, it is inferred that this substance plays a fundamental role.

Acute toxicity test The peptide prepared in Example 1 was tested for its acute toxicity using male ddy mice (body weight 40 to 45 g). This peptide is dissolved in physiological saline (pH 6.0),
This was administered from the tail vein of mice and observed for 14 days.
The dose was 1, 10, 100 μg / kg. The results are shown in Table 3 below.

[0026]

[Table 3] Table 3

As described above, no deaths were observed in the range up to 100 μg / kg.

[0028]

INDUSTRIAL APPLICABILITY The present invention provides a novel peptide. As experimentally confirmed by the above-mentioned examples, the peptide of the present invention has a proliferation promoting action and an activity promoting action on osteoblasts, and it is clear that it has an action as a therapeutic agent for bone diseases. became.

[0029]

[Sequence list]                             SEQUENCE LISTING <110> Kenji SAKAMOTO and Hideo NAKOSHI <120> Novel peptides and drugs for treating bone diseases containing th e same as effective ingredients <130> 95374DA <160> 1

[0030] <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> peptide having an activity to accelerate the growth of osteoblast s <400> 1 Lys Leu Thr Thr Ile Phe Pro Leu Asn Trp Lys Tyr Arg Lys Ala Leu   1 5 10 15

Claims (3)

[Claims] 1. A peptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing and a derivative thereof, which has a growth promoting action and an activity promoting action on osteoblasts. 2. The peptide according to claim 1, which has the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 3. A therapeutic agent for bone diseases, which comprises the peptide according to claim 1 or 2 as an active ingredient.