Note: Descriptions are shown in the official language in which they were submitted.
CA 02216704 1997-09-29
NOVEL PEPTIDES AND REMEDY FOR BONE DISEASES CONTAINING
THE SAME AS ACTIVE INGREDIENT
FIELD OF THE INVENTION
The present inventi.on relates to a new peptide and a new drug containing the
same as
an effective component for treating osteopathy.
BACKGROUND OF THE INVENTION
It is well known that there are a number of physiological active substances in
a
human body, and that such substances are closely involved in the support of
non;nal
biological activities in a human body. Both of such physiological active
substances of
a human body and synthesized physiological active substances are showing
possibilities of being new drugs as well as providing new insights into the
development of new drugs. Therefore, the search for such physiological active
substances is of utmost importance.
On the other hand, for the treatment of osteopathy such as osteoporosis,
calcitonin,
female hormone, and activated vitamin D3 and the like are currently used, the
remedial effects thereof are not necessarily of satisfactory one.
SUMMARY -OF THE INVENTION
The object of the present invention is to provide a new substance which is
useful as
a drug for the remedy of osteopathy.
The present inventors found, while conducting various experiments to search
for a
substance which promotes the activities of osteoblasts, a new peptide having
foregoing effects on osteoblasts which is involved in the bone formation, and
the
present invention was made through such a finding.
In another words, the present invention provides a peptide and a derivative
thereof,
said peptide having an amino acid sequence designated by the sequence number 1
shown in the sequence table attached herewith, wherein both of said peptide
and said
derivative thereof having growth promotion effects and activity promotion
effects on
osteoblasts. In another aspect of the present invention, it provides a drug
having said
peptide of the present invention as an effective component for treating
osteopathy.
The present invention is further explained in detail hereunder.
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CA 02216704 1997-09-29
BEST MODE TO CARRY OUT THE INVENTION
The peptide of the prescnt invention has basically an amino acid sequence
designated
by sequence number 1 shown in the sequence table attached herewith.
Furthermore, within the scope of the present invention, in addition to said
peptide
having an amino acid sequence designated by the sequence number 1 shown in the
sequence table attached herewith, a derivative of said peptide is included,
wherein
said derivative having growth promotion effects and activity promotion effects
on
osteoblasts. For example, any "derivative peptide" referred to in the present
invention
may include any derivative of said peptidc obtained by any chemical
modification
thereof which may not impede any of said effects. For example, it is well know
that
by replacing the carboxyl radical at the C end of a peptide with an amino
radical, the
stability thereof in a human body is increased, and any of such chemically
modified
peptide is included in.the scope of the present invention.
Still further, it is well known to anyone skilled in the art that, in case of
a peptide
which generally has physiological activity, even when some of the amino acids
thereof are replaced by other amino acids, when some amino acids are added
thereto,
or when some of the amino acids are deleted therefrom, there are cases where
the
physiological activity is maintained. Therefore, in case of the scope of the
present
invention, the "derivative peptide" includes any peptide wherein some of the
amino
acids which form said peptide having an amino acid sequence designated by the
sequence number I are replaced by some other amino acids, wherein some amino
acids are added thereto, or wherein there are some amino acids deleted
therefrom, and
furthermore, among such derivative peptides, any peptide having growth
promotion
effects and activity promotion effects on osteoblasts are included in the
scope of the
present invention. For example, the scope of the present invention includes a
peptide
where not more than 5 amino acids are added, replaced or deleted, or a peptide
wherein the degree of homology of the amino acid sequence is not less than 70%
and
said peptide having growth promotion effects and activity promotion effects on
osteoblasts. Furthennore, whether a derivative of a peptide has growth
promotion
effects and activity promotion effects on osteoblasts may be examined by the
method
of Examples 2 and 4 described hereunder.
The peptide of the present invention designated by the sequence number 1 has a
rather small number of amino acid components, with such number being 16, it
may
be easily synthesized by a chemical synthesizing method of any known method.
For
example, it may be easily produced by using a commercially available peptide
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synthesizing machine. It may also be easily produced by any of known generic
engineering methods, wherein a DNA encoding said amino acid sequence is
synthesized by using a DNA synthesizing machine, followed by inserting said
DNA
into the cloning portion of a commercially available cloning vector thereby
transforming the host.microorganism, followed further by culturing the same.
As has been experimentally confirmcd in the examples described hereunder, the
peptide of the present invention has growth promotion effects and activity
promotion
effects on osteoblasts. Therefore, the peptide of the present invention is
useful as a
drug for the treatment of osteopathy such as osteoporosis. Osteopathy which
can be
cured by the peptide of the present invention may be found, for example, in
osteomalacia and the like other than osteoporosis.
Because the molecular weight of the peptide of the present invention is
relatively
low, it may be intravenously administered, hypodermically administered,
intramuscularly administered, as well as orally administered or percutaneously
administered after the dosage is prepared by any of the known method.
Although the amount of dosage may be appropriately decided depending on the
patient's condition, it may be generally in the order of 0.1 to 10mg of the
peptide of
the present invention per an adult per each day.
Furthermore, in case of intravenous administration, hypodermic administration
or
intramuscular administration, the peptide of the present invention is
preferably
administered after dissolving the same into a weak acid buffer solution such
as a
citric buffer solution (pH 4~- 6) or acetic buffer solution (pH 4~- 6). In
such a case,
the concentration of the pcptide in the buffer solution is generally in the
order of
0.1mg/ml - l0mg/ml. In case of oral administration or percutaneous
administration,
the peptide is preferably dissolved into a fat-soluble substance (for examplc,
petrolatum and the like) to facilitate the increase of absorption
characteristics. In such
a case, the concentration of the peptide of the present invention is generally
in the
order of from 0.1 mg/mI to 100 mg/ml.
[Examples)
The present invention is further described in detail with reference to
examples.
However, the scope of the present invention is not restricted to the examples
described hereunder.
[Example 1] Production of a peptide
A peptide having an amino acid sequence designated by the sequence number I
was
synthesized by using a commercially available peptide synthesizing machine.
[Example 21 Effects on growth promotion of osteoblasts
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ROS cells, which are osteoblasts, obtained from a rat (source: ATCC) are
cultured on
a F10 medium containing 10% of the scrum of a calf embryo, followed by a
growing
in a thermostat being set at 37 'C and under a humidification by 5% carbon
dioxide
gas. Seeding of I x 105 cells/well on a culture plate with 24 wells was
effectuated
after tripsinization, followed by replacing said medium with a F10 medium
having
1% of the serum of a calf embryo when the former became confluent, and
thereafter
it was cultured for 24 hours. Then, the peptide of the present invention
produced in
Example 1 of this invention was dissolved in a F10 medium having 1% of the
serum
of a calf embryo, and it was added to wells while the amount thereof being
varied,
followed by continuation of the culture process for another 24 hours. After
the culture
process, the cell growth promotion effects of the present peptide was measured
by
MTT assay to obtain the growth promotion effects in comparison with the
untreated
group. MTT assay and the calculation of the rate of growth promotion was, in
effect,
*
carried out as follows. According to the procedure of MTF-Cell-Growth Assay
kit
commercially available from Funakoshi Co., Ltd., after adding the present
substance
to the wells while the amount thereof being varied and followed by leaving the
same
for a whole day, the number of living cells were counted by colorimetric
analysis by
using a phenomenon wherein MTT (3-4, 5-Dimethylthiazol-2-YL)-2, 5-Diphenyl
Tetrazolium Bromide is deaved to produce formazan having dark blue color by an
enzyme present in the mitocondrion of a living cell . The colorimetric results
of the
group obtained by adding varying amount of the present substance thereto,
while the
control group to which none of the present substance was added being as 100%,
are
shown as follows. The results are shown in the following Table 1.
(Table 1)
Amount of Peptide Added Rate of Growth Promotion
( g/well) (%)
0 100.0
0.001 109.6
0.01 110.5
0.1 636.2
1.0 1317.1
As can be seen from Table l, it was confirmed that the peptide of the present
invention provides growth promotion effects ott osteoblasts. Therefore, it is
considered
that the peptide of the present invention is related to an increase of the
amount of
bone, and it is useful for lhe treatment of osteopathy such as osteoporosis
and the
*Trade-mark
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CA 02216704 1997-09-29
like.
(Example 3] Existence of a receptor for the peptide of the present invention
on an
osteoblast
Because it was found that the peptide of the present invention has growth
promo6on
effects on osteoblasts, it can be inferred that osteoblasts have receptors for
the present
peptides. If there is any receptor, the present peptide can be considered to
be the
fundamental substance for the living organisms or cells, and further analysis
was
carried out to find out whether the osteoblast has any receptor "or not.
The peptide, obtained in Example lwas marked by biotin, and then, the present
peptide after being marked to a certain degree was added to :ROS cells
cultured in the
same way as that of: Example 2, wherein the present peptide without any
marking was
dissolved, to effectuate : competing reaction, into a F10 medium having 10% of
the
serum of a calf embry ofollowed by adding the same, while varying the amount
thereof, to said cells to see the competing reaction. The actual process of
this
experiment was as follows. According to the procedures of a protein bionizing
and
labeling ldt available from LTMII.ON, the present peptide was biotinized, and
then, a
certain amount of biotinized peptide was added to a certain number of cells
seeded
in wells, then from 0 to 0.512 g/well of unlabeled peptide was added to each
cell
to effectuate the competing reaction for 6 hours, and thereafter, the cell was
washed
by PBS and then the coloration reaction, which was caused by peroxidase
labeled
with streptoabidin reacting with biotinized peptide bonded to receptors
present on the
cell surface, was observed. When any receptor for the present peptide is
present on
the cell surface, there is a reaction competing with the unlabeled peptide
whereby the
coloration degree is decreased. The results are shown in Table 2 hereunder.
(Table 2]
Amount of Unlabeled Peptide Added Ratio to the Added Labeled Peptide
( u g/well) (%)
0 100
0.032 98.4
0.064 86.5
0.128 79.6
0.256 34.1
0.512 29.5
As shown in Table 2 above, because the ratio to the added labeled peptide
varies
depending on the amount of unlabclcd pcptide, it is obvious that the
osteoblast has
receptors for the present peptide, and it is further inferred that the present
substance
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CA 02216704 1997-09-29
has a fundamental role.
Tests on Acute Toxicity
By using the peptide prepared in Example 1, its acute toxicity was tested on
ddy male
mice (with the body weight ranging from 40 to 45 g). The present peptide was
dissolved in a physiological saline (pH 6.0) and the solution thereof was
administered
intravenously into a vein in the tail of each mouse, and observation was made
for 14
days. The dosage amount was set respectively at 1, 10, and 100 u g/kg. The
results.
are shown in Table 3 hereunder.
[Table 31
Dosage Amount Number of Deceased Micc
( u g/kg)
1 0/5
0/5
100 0/5
As can be seen from the above table, there was no deceased mouse observed up
to
the dosage amount of 100 p g/kg.
INDUSTRIAL APPLICABILITY
A new peptide is provided by the present invention. The peptide of the present
invention has, as has been experimentally confirmed by the above examples,
growth
promotion effects and activity promotion effects on osteoblasts, and it has
been made
obvious that such peptide has effects as a drug for the treatment of
osteopathy.
6
CA 02216704 1997-09-29
[Sequence Table)
Sequence Number: I
Sequence Length: 16
Type of Sequencc: Amino Acid
Sequence:
Lys Leu Thr Thr Ile Phe Pro Leu Asn Trp Lys Tyr Arg Lys Ala Leu
1 5 10 15
7
CA 02216704 2003-05-01
SEQUENCE LISTING
GENERAL INFORMATION:
APPLICANT: SAKAMOTO, Kenji
TITLE OF INVENTION: Novel Peptides and Remedy for Bone Diseases
Containing the Same as Active Ingredient
NUMBER OF SEQUENCES: 1
CORRESPONDENCE ADDRESS: MacRae & Co.
P.O. Box 806 Station "B"
Ottawa, Ontario K1P-5T4
COMPUTER-READABLE FORM
COMPUTER: IBM Compatible
OPERATING SYSTEM: DOS
SOFTWARE: Generic Text
CURRENT APPLICATION DATA
APPLICATION NUMBER: CA 2,216,704
FILING DATE: April 3, 1996
CLASSIFICATION: 06CO7K-07/08
PRIOR APPLICATION DATA
APPLICATION NUMBER: Japanese Application No. 7/101681
FILING DATE: April 3, 1995
CLASSIFICATION: 06C07K-07/08
PATENT AGENT INFORMATION
NAME: MacRae & Co.
REFERENCE NUMBER: 23450
INFORMATION FOR SEQ ID NO: 1
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 Amino Acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Unknown
(ii) MOLECULE TYPE: Peptide
(iii) HYPOTHETICAL: No
1
CA 02216704 2003-05-01
(iv) ANTI-SENSE: No
(v) FRAGMENT TYPE: Entire Peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Peptide Synthesizer
(vii) IMMEDIATE SOURCE:
(B) CLONE: Peptide Synthesizer
(ix) FEATURE:
(D) OTHER INFORMATION: Promotion effects on osteoblasts
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Lys Leu Thr Thr Ile Phe Pro Leu Asn Trp Lys Tyr Arg Lys Ala Leu
10 15
2
