U.S. patent application number 14/328169 was filed with the patent office on 2016-09-22 for small molecule, indirubin-3-oxime, for prevention and treatment of bone disease.
This patent application is currently assigned to Industry-Academic Cooperation Foundation, Yonsei University. The applicant listed for this patent is Industry-Academic Cooperation Foundation, Yonsei University. Invention is credited to Kang-Yell Choi, Sehee Choi, Hyun Yi Kim.
Application Number | 20160271104 14/328169 |
Document ID | / |
Family ID | 52280228 |
Filed Date | 2016-09-22 |
United States Patent
Application |
20160271104 |
Kind Code |
A1 |
Choi; Kang-Yell ; et
al. |
September 22, 2016 |
SMALL MOLECULE, INDIRUBIN-3-OXIME, FOR PREVENTION AND TREATMENT OF
BONE DISEASE
Abstract
The present invention provides a composition comprising an
indirubin derivative for stimulating longitudinal bone growth.
Because the composition for stimulating longitudinal bone growth
according to the present invention is shown to be effective in
longitudinal bone growth, it may be used not only as a composition
for stimulating longitudinal bone growth, but also as a
pharmaceutical composition for treating or preventing short
stature, microplasia, dwarfism, or precocious puberty.
Inventors: |
Choi; Kang-Yell; (Seoul,
KR) ; Kim; Hyun Yi; (Gunpo, KR) ; Choi;
Sehee; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Industry-Academic Cooperation Foundation, Yonsei
University |
Seoul |
|
KR |
|
|
Assignee: |
Industry-Academic Cooperation
Foundation, Yonsei University
|
Family ID: |
52280228 |
Appl. No.: |
14/328169 |
Filed: |
July 10, 2014 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/404 20130101;
A23L 33/105 20160801; A23L 33/10 20160801; A23V 2002/00 20130101;
A61P 19/00 20180101; A23L 2/52 20130101 |
International
Class: |
A61K 31/404 20060101
A61K031/404; A23L 2/52 20060101 A23L002/52 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 11, 2013 |
KR |
10-2013-0081464 |
Claims
1. A composition comprising an indirubin derivative for stimulating
longitudinal bone growth.
2. The composition according to claim 1, wherein the indirubin
derivative is indirubin-3'-oxime.
3. The composition according to claim 1, wherein the longitudinal
bone growth is an increase in the length of the tibia.
4. The composition according to claim 1, wherein the longitudinal
bone growth is an increase in the activity of the growth plate.
5. A pharmaceutical composition comprising an indirubin derivative
for treating or preventing longitudinal bone growth disorders.
6. The pharmaceutical composition according to claim 5, wherein the
indirubin derivative is indirubin-3'-oxime.
7. The pharmaceutical composition according to claim 5, wherein the
longitudinal bone growth disorder is short stature, microplasia,
dwarfism, or precocious puberty.
8. A food composition comprising an indirubin derivative for
improving longitudinal bone growth.
9. The food composition according to claim 8, wherein the indirubin
derivative is indirubin-3'-oxime.
10. The food composition according to claim 8, wherein the
longitudinal bone growth is an increase in the length of the
tibia.
11. The food composition according to claim 8, wherein the
longitudinal bone growth is an increase in the activity of the
growth plate.
12. A functional beverage composition comprising an indirubin
derivative for improving longitudinal bone growth.
13. The functional beverage composition according to claim 12,
wherein the indirubin derivative is indirubin-3'-oxime.
14. The functional beverage composition according to claim 12,
wherein the longitudinal bone growth is an increase in the length
of the tibia.
15. The functional beverage composition according to claim 12,
wherein the longitudinal bone growth is an increase in the activity
of the growth plate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to Korean Application No.
10-2013-0081464 filed on Jul. 11, 2013, which is incorporated
herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to a small molecule,
indirubin-3-oxime, for prevention and treatment of bone
disease.
BACKGROUND ART
[0003] The increased awareness of the importance of normal growth,
as well as an increased aesthetic interest, in infants and
adolescents has led to an increased public interest in height
growth. In the past, height growth was thought to depend on
heredity. However, it is said that the proportion to which heredity
is responsible for actual height growth is only 23%, and also,
depending on the care during the period of growth, sufficient
height growth can be achieved. Therefore, a great deal of money is
being invested, estimated to be anywhere between 4-100 million
dollars, to achieve height growth within the time period in which
the growth plate is not closed. A public opinion survey shows that
33% of children in this growth period receive artificial care for
height growth. Thus, there is also a growing number of therapeutic
agents for growth, or hospitals that run growth clinics. However,
most of the drugs that are currently in the market simply contain
nutritional supplement components, and a question remains as to the
proportion of therapeutic agents for growth that produce a real
effect. Furthermore, virtually no drug has a target directly
related to height growth. The Fair Trade Commission reports that
even though the majority of therapeutic agents for growth that are
distributed in the market are simply general food products or
functional food products, they are labeled with terms such as
"agent for height growth" or "(functional) food product for height
growth." Developing a novel therapeutic agent for growth is
therefore critical at this point, where proper therapeutic agents
for growth are still lacking, in spite of consumption trends.
[0004] Meanwhile, height growth necessitates an increase in bone
length, and endochondral ossification is associated with such an
increase. Ossification can be largely classified as
intramembraneous ossification and endochondral ossification.
Intramembraneous ossification is a formation of bones in which
calcium and phosphorus secreted from the osteoblasts are lumped
together and gradually increase in size. It occurs in the
periosteum on the outer surface of the bone edge, and relates to
the increase in bone thickness. On the other hand, endochondral
ossification is a formation of bones in which a cartilage model is
formed and then an ossification center emerges at its center. This
occurs in the epiphyseal plate and relates to the increase in
longitudinal bone length. That is, the increase in bone thickness
and that in bone length are caused by different ossification
processes. Endochondral ossification is largely induced by
endocrine factors and paracrine factors. Growth hormone injection,
which is a drug that can stimulate the endocrine factors, is
available to date. However, growth hormone injection is not
suitable for a child with normal hormone secretion, and instead
produces side effects when used, such as hypothyroidism and
hyperpituitarism. Moreover, it is not easily accessible because of
its high cost. In addition, a recent issue concerns precocious
puberty causing early closure of the growth plate, thus shortening
the period of growth. Since paracrine factors are involved in the
actual closure of the growth plate, it can be said that targeting
them is important for enhancing the effect of related drugs. Hence,
the present inventors have investigated the Wnt/.beta.-catenin
signaling pathway, which is activated by Wnt, one of the paracrine
factors that are involved in the increase in bone length, and found
that the increase in longitudinal bone length is induced by
indirubin-3'-oxime, one of the substances that activate
.beta.-catenin, which is a core signal transducer in this signaling
pathway. The present inventors also sought to utilize the substance
to induce height growth. Indirubin-3'-oxime was synthesized as a
derivative of indirubin, a component found in the roots or leaves
of indigo plants, used for the Chinese medicine, Danggui Longhui
Wan. It is also known to be free of toxicity. The present inventors
have found that indirubin-3'-oxime, in particular, activates the
growth plate by stimulating chondrocytes, which are involved in
height growth, in the growth plate. Unlike conventional therapeutic
agents for growth, indirubin-3'-oxime regulates the signaling
pathway based on specific targets involved in height growth, and is
therefore effective in inducing height growth specifically.
Furthermore, because indirubin, which is the source substance for
indirubin-3'-oxime, is a low molecular weight compound that has
already reached the second stage of clinical trial as a therapeutic
agent for leukemia, its safety is more or less guaranteed.
Moreover, the development cost for indirubin-3'-oxime is relatively
low. Thus, the expected effect is very large for utilizing
indirubin-3'-oxime as a therapeutic agent for height growth.
SUMMARY
[0005] Thus, drugs targeting .beta.-catenin, which is one of
paracrine factors involved in the increase in bone length, are
required.
[0006] To achieve said purpose, the present invention provides in
an embodiment a composition comprising an indirubin derivative for
stimulating bone growth. Another embodiment provides a composition
wherein the indirubin derivative is indirubin-3'-oxime. Another
embodiment provides a composition wherein the bone growth is an
increase in the length of the tibia. Another embodiment provides a
composition wherein the bone growth is an increase in the activity
of the growth plate.
[0007] In addition, an embodiment of the present invention provides
a pharmaceutical composition comprising an indirubin derivative for
treating or preventing bone growth disorders. Another embodiment
provides a pharmaceutical composition wherein the indirubin
derivative is indirubin-3'-oxime. Another embodiment provides a
pharmaceutical composition wherein the bone growth disorder is
short stature, microplasia, dwarfism, or precocious puberty.
[0008] Furthermore, an embodiment of the present invention provides
a food composition comprising an indirubin derivative for improving
bone growth. Another embodiment provides a food composition wherein
the indirubin derivative is indirubin-3'-oxime. Another embodiment
provides a food composition wherein the longitudinal bone growth is
an increase in the length of the tibia. Another embodiment provides
a food composition wherein the bone growth is an increase in the
activity of the growth plate.
[0009] Moreover, an embodiment of the present invention provides a
functional beverage composition comprising an indirubin derivative
for improving longitudinal bone growth. Another embodiment provides
a functional beverage composition wherein the indirubin derivative
is indirubin-3'-oxime. Another embodiment provides a functional
beverage composition wherein the longitudinal bone growth is an
increase in the length of the tibia. Another embodiment provides a
functional beverage composition wherein the longitudinal bone
growth is an increase in the activity of the growth plate.
[0010] Indirubin is an indigoid compound which is very similar in
structure as indigo and which has a red color. It is generally
produced in small amounts as a byproduct during a production of the
blue dye indigo, using natural indigo (Polygonum tinctorium), woad
(Isatis tinctoria), etc. Danggui Longhui Wan, a prescription in
traditional Chinese medicine, is comprised of 11 types of medicines
that have been used to treat chronic leukemia. Among these,
indirubin has been found to be an effective drug. Moreover, as a
cell cycle inhibitor, it was recently reported to be a drug of
great value for medical applications, including a therapeutic agent
for chronic leukemia, neurodegenerative diseases such as
Alzheimer's, and the like (Bri. J. Haemato., 130:681-690, 2005;
Nature Cell Biol., 1:60-67, 1999). Indirubin can form many
derivatives around its parent structure, including indirubin oxime
derivatives, indirubin hydrazone derivatives, indirubin N-acetyl
derivatives, indirubin amine derivatives, etc.
[0011] The Wnt/.beta.-catenin signaling pathway is a signaling
pathway that plays a key role in the development, growth, and
maintenance of homeostasis in vertebrates. It is known that the
presence of an abnormality in the Wnt/.beta.-catenin signaling
pathway can cause an abnormality in bone formation (Liu et al.,
Cell signal., 2008). The activation of the Wnt/.beta.-catenin
signaling pathway begins with the binding of Wnt, a ligand;
Frizzled (Fz), a receptor; and lipoprotein receptor-related
proteins 5 and 6 (LRP 5/6), a co-receptor. In the absence of Wnt
signals, .beta.-catenin, which is a core signal transducer in said
signaling pathway, forms a complex with substances that interfere
with signal transduction, such as Axin, GSK3, Apc, etc., and said
complex is thus degraded by ubiquitination. Once the activation of
signal transduction is initiated by Wnt stimuli, these complexes
are disassembled, and .beta.-catenin is stabilized and accumulated
in the cytoplasm, after which it is translocated into the nucleus
to facilitate the expression of target genes through interaction
with transcription factors, such as Tcf, Lef, or the like. Here,
.beta.-catenin is the most important molecule in the
Wnt/.beta.-catenin signaling pathway, and is well known to be
involved in the increase in bone length. In the present invention,
substances that increase the amount of .beta.-catenin were tested
for their ability to stimulate the increase in bone length, and
among these, indirubin-3'-oxime was found to have such an effect.
Indirubin-3'-oxime, a GSK3 inhibitor, blocks the decomposition of
.beta.-catenin and increases .beta.-catenin translocation into the
nucleus, thus increasing the expression of genes necessary for the
proliferation and differentiation of chondrocytes. The present
inventors confirmed through ex vivo and in vivo systems that
indirubin-3'-oxime increases the length of the tibia. Because
indirubin-3'-oxime increases height by targeting a specific
mechanism, it is expected to be useful as a new therapeutic agent
for height growth.
[0012] The pharmaceutical composition according to the present
invention may further comprise suitable carriers, excipients, and
diluents that are typically used for the preparation of
pharmaceutical compositions.
[0013] The pharmaceutical composition according to the present
invention may be formulated and used in the form of oral
formulations, including powders, granules, tablets, capsules,
suspensions, emulsions, syrups, aerosols, etc., external
preparations, suppositories, or injectable solutions. Carriers,
excipients, and diluents that may be comprised in a composition
comprising an extract include lactose, dextrose, sucrose, sorbitol,
mannitol, xylitol, erythritol, maltitol, starch, acacia gum,
alginate, gelatin, calcium phosphate, calcium silicate, cellulose,
methyl cellulose, microcrystalline cellulose, polyvinyl
pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate,
talc, magnesium stearate, and mineral oil. For formulation, the
composition is prepared using common diluents or excipients, such
as fillers, extending agents, binding agents, wetting agents,
disintegrating agents, and surfactants. Solid formulations for oral
administration include tablets, pills, powders, granules, capsules,
etc. Such solid formulations are prepared by mixing at least one
excipient, e.g., starch, calcium carbonate, sucrose or lactose,
gelatin, etc., with said extract. Besides the simple excipients,
lubricants, such as magnesium stearate and talc, are also used.
Liquid formulations for oral administration include suspensions,
emulsions, syrups, etc. In addition to liquid paraffin, which is a
commonly used simple diluent, a variety of excipients may be
included, e.g., wetting agents, sweeteners, aromatics,
preservatives, etc. Formulations for parenteral administration
include sterilized solutions, non-aqueous solvents, suspensions,
emulsions, lyophilized formulations, and suppositories. For
non-aqueous solvents and suspensions, vegetable oils, such as
propylene glycol, polyethylene glycol, and olive oil, injectable
esters, such as ethyl oleate, or the like may be used. For
suppository bases, Witepsol, Macrogol, Tween 61, cacao butter,
laurinum, glycerogelatin, or the like may be used.
[0014] A suitable dosage of the pharmaceutical composition
according to the present invention may vary according to the
patient's general health and weight, the severity of disease, the
form of drug, and the route and period of administration, but may
be properly selected by a person skilled in the art. Nonetheless,
it is recommended that the composition of the present invention is
administered daily at 0.2 mg/kg to 200 mg/kg, preferably 2 mg/kg to
100 mg/kg, to obtain a desirable effect. The administration may be
once a day or spread out over multiple times a day. However, said
dosage does not, in any way, limit the scope of the present
invention.
[0015] The pharmaceutical composition according to the present
invention may be administered via a variety of routes to mammals,
such as rats, mice, livestock, and humans. Any mode of
administration may be employed; for example, the administration may
be via an oral, rectal or intravenous, intramuscular, subcutaneous,
intrauterine, or epidural or intracerebroventricular injection.
[0016] The food composition according to the present invention may
be widely used in drugs, foods, beverages, etc. for treating and
repairing wounds. Foods that may comprise the compound of the
present invention include, for example, a variety of foodstuffs,
beverages, gums, teas, vitamin complexes, dietary supplements, etc.
They may be in the form of powders, granules, tablets, capsules, or
drinks.
[0017] Concerning the amount of said compound in food or beverages
of the present invention, in general, a health food composition
according to the present invention may be added 0.01% to 15% by
weight of the total food weight; a health beverage composition
according to the present invention may be added 0.02 g to 5 g,
preferably 0.3 g to 1 g, based on 100 ml.
[0018] Besides containing said compound as an essential ingredient
at a suggested proportion, the functional beverage composition
according to the present invention is not particularly limited in
terms of the liquid component and, like common beverages, may
contain various sweeteners or natural carbohydrates as an
additional ingredient. Examples of said natural carbohydrates are
common sugars, including monosaccharides, e.g., glucose, fructose,
etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides,
e.g., dextrin, cyclodextrin, etc., and sugar alcohols, including
xylitol, sorbitol, erythritol, etc. Apart from said sweeteners,
natural sweeteners (thaumatin and stevia extracts, e.g.,
rebaudioside A, glycyrrhizin, etc.) and synthetic sweeteners
(saccharin, aspartame, etc.) may be used to an advantage. The
proportion of said natural carbohydrates is generally about 1 g to
20 g, preferably about 5 g to 12 g, per 100 ml of the composition
of the present invention.
[0019] Apart from said ingredients, the composition according to
the present invention may contain various nutritional supplements,
vitamins, minerals (electrolytes), flavorings including synthetic
flavors and natural flavors, colorings and fillers (cheese,
chocolate, etc.), pectic acids and their salts, alginic acids and
their salts, organic acids, protective colloid thickeners, pH
control agents, stabilizers, preservatives, glycerin, alcohols,
carbonating agents used for carbonated drinks, etc. In addition,
the compositions according to the present invention may contain
flesh that is used for preparing natural fruit juices, fruit juice
drinks, or vegetable drinks. Such ingredients may be used
independently or in combination. Although the proportions of these
additives are not of great importance, they are generally selected
from the range of 0 to 20 parts by weight per 100 parts by weight
of the composition of the present invention.
[0020] As described above, the composition according to the present
invention, comprising an indirubin derivative for stimulating
longitudinal bone growth, has an effect of treating or preventing
short stature, microplasia, dwarfism, or precocious puberty. In
addition, unlike conventional therapeutic agents for growth,
indirubin-3'-oxime regulates the signaling pathway based on
specific targets involved in height growth, and therefore, produces
a clear effect. Not only that, but indirubin-3'-oxime is a low
molecular weight compound that has potential as a therapeutic agent
for leukemia. Hence, it has low toxicity, as well as low
development cost.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIGS. 1A-1D illustrate that indirubin-3'-oxime
dose-dependently induces longitudinal bone growth of ex vivo tibia.
More specifically, FIG. 1A and FIG. 1B show the increase in the
total length depended on the concentration change. FIG. 1C shows
the comparison of cell conditions in the tibia, using safranin
staining procedure. And, FIG. 1D shows the dependence of cell
conditions on concentration change, which resulted in the increase
in the total length (the resting zone-RZ, the proliferating
zone-PZ, and the hypertrophic chondrocyte zone-HZ) that depended on
the concentration of indirubin-3'-oxime.
[0022] FIGS. 2A-2C illustrate that indirubin-3'-oxime enhances
chondrocyte maturation via the Wnt/.beta.-catenin pathway. More
specifically, FIG. 2A shows indirubin-3'-oxime increased the
stability of .beta.-catenin while increasing the amount of inactive
GSK.beta., according to the concentration change. FIG. 2B shows the
cell images staining with green color for .beta.-catenin, red color
for Col2a1, and blue color for nucleus. And, FIG. 2C shows that the
amount of .beta.-catenin markedly increased in the hypertrophic
chondrocyte zone of ex vivo tibiae treated with
indirubin-3'-oxime.
[0023] FIGS. 3A-3C illustrate that indirubin-3'-oxime inhibits
growth plate senescence. More specifically, FIG. 3A shows the
experimental schedules (7-week-old mice were treated with
indirubin-3'-oxime for 2 weeks, and the amount of activity of the
growth plate was compared with that in the control group at 9
weeks). FIG. 3B shows the cell conditions in the growth plate which
were observed using the hematoxylin-eosin staining procedure. And,
FIG. 3C show the IHC analysis about the indirubin-3'-oxime effect
according to FIG. 3A schedule.
[0024] FIGS. 4A-4E illustrate that indirubin-3'-oxime promotes
longitudinal bone growth. More specifically, FIG. 4A shows the
administration time of indirubin-3'-oxime to understand whether
indirubin-3'-oxime can actually produce an increase in bone length
in mice. FIG. 4B shows that the effect on the activity of the
growth plate in the middle of a period of growth was examined using
the hematoxylin-eosin staining procedure. FIG. 4C shows that
Indirubin-3'-oxime treatment increases not only the length of each
zone but also the total length of the growth plate. FIG. 4D shows
that Indirubin-3'-oxime treatment ultimately leads to an increase
in the total bone length of the tibia. And, FIG. 4E shows the above
described effect of Indirubin-3'-oxime treatment lasts even in
13-week-old mice in which height growth completely ceased.
DETAILED DESCRIPTION
[0025] Hereinafter, the present invention is described in detail
through the following examples. However, these examples are only
intended to illustrate the present invention and do not limit the
scope of the invention.
Example 1
Cell Culture and Reagents
[0026] Rat chondrosarcoma (RCS) cells were cultured in a Dulbecco's
modified Eagle's medium (DMEM; Gibco, Grand Island, N.Y.),
supplemented with 10% fetal bovine serum (FBS) and
penicillin/streptomycin.
Example 2
Western Blot Analysis
[0027] For Western blot analysis, cells were treated with
indirubin-3'-oxime for 24 hours, cultured and reached to 70%
confluency and then were harvested. After the harvested samples
underwent cytolysis, the proteins were quantified, which was
followed by electrophoresis using 10% or 12% SDA-PAGE gel.
Afterwards, immunoblot was conducted using antibodies.
Example 3
Immunocytochemistry
[0028] 2.5.times.10.sup.4 cells were adhered to a cover glass
placed in a 12-well plate. Once securely attached, cells were
treated with indirubin-3'-oxime for 24 hours, and then were rinsed
with a phosphoric acid buffer solution. After adding 4%
paraformaldehyde, cells were fixed at room temperature for 20 min.
Then, proteins expressed in the cells were observed through cell
membrane permeation, blocking, and immunofluorescent staining
processes.
Example 4
Tissue Culture and Reagents
[0029] The tibiae were separated from 15.5-embryonic-day-old
C57BL/6 mice, and were incubated in a 24-well tissue culture dish
for 7 days. The medium used was prepared by adding 10% FBS, 1 mM
beta-polyglycerol disodium phosphate, 50 ng/ml ascorbic acid, 0.3
mg/ml L-glutamine, 0.2% BSA, 100 U/ml penicillin, and 100 .mu.g/ml
streptomycin to a minimal essential medium (.alpha.-MEM, Gibco).
Then, the tibiae were separated from the mice. After being treated
in the medium for 24 hours, the tibia was treated with a drug. The
medium and the drug were changed every two days. The tibia length
was measured before and after treatment with the drug. The tibial
tissue, on which drug treatment was completed, was rinsed with PBS
and then fixed using 4% formaldehyde.
Example 5
Mouse Model
[0030] Two and six-week-old C57BL/6 mice, purchased from KOATECH
(Gyeonggi-do, South Korea), were cared for in accordance with the
guidelines established by the Institutional Animal Care and Use
Committee at Yonsei University. The mice were divided into a
control group and an experimental group, and were adapted to the
environment for 1 week. Afterwards, they were treated every day for
2 weeks with a solution containing 20% dimethyl sulfide and 30%
ethanol, or with indirubin-3'-oxime (0.05 mg/kg, 0.1 mg/kg, and 0.5
mg/kg), via intraperitoneal injection. Two weeks later, the tibiae
were separated from mice, and then the muscles were separated and
removed, after which the bones were fixed using 4% formaldehyde.
The bones were treated for 2 weeks with a 10% EDTA solution for the
decalcification process. The EDTA solution was changed every two
days.
Example 6
Staining of the Tibia
[0031] The tissue-cultured embryo tibiae and the animal-tested
tibiae were dewaxed in xylene, and were rehydrated in a
stepwise-diluted ethanol solution. The tibial tissue, which was
separated from 15.5-embryonic-day-old ICR mice and then
tissue-cultured, was stained using the safranin staining procedure.
Staining was carried out for 25 min in a 0.02% fast green solution,
and after brief dipping in acetic acid, was again carried out for 7
min in a 0.1% safranin solution. Meanwhile, the tibial tissue that
was animal-tested and decalcified in EDTA was stained via the
hematoxylin-eosin staining procedure. The tissue was stained in a
hematoxylin solution for 10 min and then rinsed in running water
for 10 min After being briefly dipped in a solution comprising 0.1%
hydrochloric acid and 70% ethanol, the tissue was stained for 30
sec in an eosin solution. The stained tibial tissues again
underwent a dehydration process in the stepwise-diluted ethanol
solution and were dewaxed in xylene.
Example 7
Immunohistochemistry
[0032] The tibiae that underwent a dewaxing process were
dehydrated. After paraffin blocks were formed, the tissues were
sectioned at 4-.mu.m thickness. The sectioned tissues were
rehydrated, and then stained using immunofluorescence.
[0033] Increase in the Length of the Tibia Due to
Indirubin-3'-Oxime
[0034] To obtain a more accurate understanding of the effect of
indirubin-3'-oxime on ex vivo tibia culture, two different
concentrations (1 .mu.M and 5 .mu.M) of indirubin-3'-oxime were
treated to tibiae for 6 days. The results show that the increase in
the total length depended on the concentration change (FIGS. 1A,
1B). Moreover, the comparison of cell conditions in the tibia,
using the safranin staining procedure (FIG. 1C), shows the
dependence of cell conditions on concentration change, which
resulted in the increase in the total length (the resting zone, the
proliferating zone, and the hypertrophic chondrocyte zone) that
depended on the concentration of indirubin-3'-oxime (FIG. 1D).
[0035] Increase in the Stability of .beta.-Catenin Due to
Indirubin-3'-Oxime
[0036] Indirubin-3'-oxime, a GSK.beta. inhibitor, is already known
to increase the stability of .beta.-catenin. To test whether such
an effect is actually produced in chondrocytes, the present
inventors conducted Western blot using RCS cells, and found that
indirubin-3'-oxime increased the stability of .beta.-catenin while
increasing the amount of inactive GSK.beta., according to the
concentration change (FIG. 2A). ICC analysis confirmed that this
increase in the stability of .beta.-catenin resulted from an
increase in the amount of .beta.-catenin that was translocated into
the nucleus. In addition, IHC analysis on ex vivo tissues showed
that the amount of .beta.-catenin markedly increased in the
hypertrophic chondrocyte zone of ex vivo tibiae treated with
indirubin-3'-oxime.
[0037] Increase in the Activity of the Growth Plate in Mice Dosed
with Indirubin-3'-Oxime
[0038] As humans reach adulthood, the growth plate is reduced in
size and eventually closes, thus preventing further height growth.
In mice, growth plates do not close. However, there is a marked
decline in the activity of cells inside the growth plate from 8
weeks of age, and height growth gradually comes to a halt. In the
present experiment, the effect of indirubin-3'-oxime on the
activity of the growth plate was investigated. 7-week-old mice were
treated with indirubin-3'-oxime for 2 weeks, and the amount of
activity of the growth plate was compared with that in the control
group at 9 weeks (FIG. 3A). Cell conditions in the growth plate
were observed using the hematoxylin-eosin staining procedure. As a
result, the activity of the growth plate was still maintained in
9-week-old mice under treatment with indirubin-3'-oxime (FIG. 3B).
This effect of indirubin-3'-oxime was identified through IHC
analysis to be an increase in the amount of Ki67, a marker related
to cell proliferation, along with an increase in the stability of
.beta.-catenin. These results suggest the possibility of
indirubin-3'-oxime as an agent for growth that can delay the time
point at which the growth plate closes in humans.
[0039] Increase in the Length of the Tibia in Mice Dosed with
Indirubin-3'-Oxime
[0040] To understand whether indirubin-3'-oxime can actually
produce an increase in bone length in mice, indirubin-3'-oxime was
administered during a period of active growth, and its effect was
examined (FIG. 4A). First of all, the effect on the activity of the
growth plate in the middle of a period of growth was examined using
the hematoxylin-eosin staining procedure (FIG. 4B).
Indirubin-3'-oxime treatment was found to result in an increase not
only in the length of each zone but also in the total length of the
growth plate (FIG. 4C). Cell proliferation markers related to these
effects were once again confirmed through PCNA (FIG. 4B). Moreover,
these results ultimately led to an increase in the total bone
length of the tibia. In addition, these effects were found to last
even in 13-week-old mice, in which height growth completely ceased
(FIGS. 4D, 4E).
[0041] In conclusion, the above results suggest that
indirubin-3'-oxime effectively increases longitudinal bone length
by activating the Wnt/.beta.-catenin signaling pathway via
inhibition of GSK-3.beta.. Furthermore, because of its ability to
delay the time point at which the growth plate closes,
indirubin-3'-oxime proves to be an effective therapeutic agent that
may overcome the current problem of early closure of the growth
plate resulting from precocious puberty.
[0042] So far, the present invention was described with reference
to the examples. However, a person skilled in the art could
understand that various changes can be made without exceeding the
scope of the present invention, and that the elements can be
replaced with their equivalents. Moreover, from the numerous
changes made without exceeding the essential scope of the present
invention, specific aspects and materials can be employed within
the teachings of the present invention. Thus, the present invention
should be construed not to be limited in terms of the specific
examples disclosed as the best modes contemplated for carrying out
the present invention, but to include all examples that fall under
the scope of the claims attached hereto.
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