U.S. patent application number 14/362917 was filed with the patent office on 2015-01-29 for pharmaceutical composition comprising bicyclic pyridinol derivatives for preventing or treating diseases caused by angiogenesis.
The applicant listed for this patent is Research Cooperation Foundation of Yeungnam University. Invention is credited to Byeong Seon Jeong, You Ra Kang, Jung Ae Kim, Jin Mo Ku, Tae Gyu Nam, Ned A. Porter.
Application Number | 20150031890 14/362917 |
Document ID | / |
Family ID | 48861585 |
Filed Date | 2015-01-29 |
United States Patent
Application |
20150031890 |
Kind Code |
A1 |
Jeong; Byeong Seon ; et
al. |
January 29, 2015 |
PHARMACEUTICAL COMPOSITION COMPRISING BICYCLIC PYRIDINOL
DERIVATIVES FOR PREVENTING OR TREATING DISEASES CAUSED BY
ANGIOGENESIS
Abstract
There is provided a pharmaceutical composition comprising a
bicyclic pyridinol derivative as an active ingredient for
preventing or treating diseases caused by angiogenesis. The
bicyclic pyridinol derivative expressed by Chemical Formula 1 can
be effectively used as a drug for preventing and treating diseases
caused by angiogenesis since it inhibits neovascularization in a
chorioallantoic membrane model.
Inventors: |
Jeong; Byeong Seon; (Daegu,
KR) ; Kim; Jung Ae; (Daegu, KR) ; Kang; You
Ra; (Gyeongsan, KR) ; Nam; Tae Gyu; (Suwon,
KR) ; Ku; Jin Mo; (Gunpo, KR) ; Porter; Ned
A.; (Nashville, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Research Cooperation Foundation of Yeungnam University |
Gyeongsan-si, Gyeongsangbuk-do |
|
KR |
|
|
Family ID: |
48861585 |
Appl. No.: |
14/362917 |
Filed: |
December 7, 2012 |
PCT Filed: |
December 7, 2012 |
PCT NO: |
PCT/KR2012/010630 |
371 Date: |
September 5, 2014 |
Current U.S.
Class: |
546/122 ;
546/113 |
Current CPC
Class: |
A61K 9/2059 20130101;
A61P 9/00 20180101; A61K 9/2018 20130101; A61K 31/44 20130101; A61K
31/4375 20130101; C07D 471/04 20130101; A61K 31/437 20130101 |
Class at
Publication: |
546/122 ;
546/113 |
International
Class: |
C07D 471/04 20060101
C07D471/04 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 8, 2011 |
KR |
10-2011-0131290 |
Dec 7, 2012 |
KR |
10-2012-0141581 |
Claims
1. A pharmaceutical composition comprising a bicyclic pyridinol
derivative expressed by the following Chemical Formula 1 for
preventing or treating diseases caused by angiogenesis:
##STR00011## Wherein in the above formula, R.sup.1 to R.sup.3 are
identical with or different from one another and are any one of
hydrogen, C1 to C16 alkyl, acyloxymethyl, or trimethyltridecyl, and
n is an integer of 1 to 2.
2. The pharmaceutical composition for preventing or treating
diseases caused by angiogenesis of claim 1, wherein in the bicyclic
pyridinol derivative, R.sup.1 is any one of hydrogen, methyl,
ethyl, or n-C.sub.16H.sub.33, R.sup.2 is any one of hydrogen or
methyl, R.sup.3 is one of hydrogen, methyl, n-butyl, t-butyl,
acyloxymethyl, or trimethyltridecyl ##STR00012## and n is an
integer of 1 to 2.
3. The pharmaceutical composition for preventing or treating
diseases caused by angiogenesis of claim 1, wherein the bicyclic
pyridinol derivative is a compound expressed by the following
Chemical Formula 2: ##STR00013## In the above formula, n is an
integer of 1 to 2.
4. The pharmaceutical composition for preventing or treating
diseases caused by angiogenesis of claim 1, wherein the bicyclic
pyridinol derivative is a compound expressed by the following
Chemical Formula 3: ##STR00014##
5. The pharmaceutical composition for preventing and treating
diseases caused by angiogenesis of claim 1, wherein the disease
caused by angiogenesis is at least one selected from the group
consisting of rheumatic arthritis, osteoarthritis, septic
arthritis, psoriasis, corneal ulcer, senile macular degeneration,
diabetic retinopathy, proliferative vitreous body retinopathy,
premature retinopathy, ocular inflammation, conical cornea,
Sjogren's syndrome, myopia eye tumor, cornea graft rejection,
abnormal wound healing, bone disease, proteinuria, abdominal aortic
aneurysm, regressive cartilage loss due to traumatic joint injury,
demyelinating disease of nervous system, hepatic cirrhosis,
glomerular disease, premature rupture of embryonic membrane,
inflammatory bowel disease, periodontitis, atherosclerosis,
restenosis, inflammatory disease of central nervous system,
Alzheimer's disease, skin aging and infiltration metastasis of
cancer.
Description
TECHNICAL FIELD
[0001] The present invention relates to a pharmaceutical
composition comprising a bicyclic pyridinol derivative for
preventing or treating diseases caused by angiogenesis.
BACKGROUND ART
[0002] Angiogenesis is a process of forming new capillary blood
vessels from pre-existing microvessels. Angiogenesis normally
occurs during embryonic development, tissue regeneration, wound
healing, and corpus luteum development that is a cyclical change in
female reproductive system; in any case, angiogenesis is strictly
regulated to progress (Folkman J et al., Int. Rev. Exp. Pathol.,
16, pp 207-248, 1976).
[0003] The vascular endothelial cells are growing slowly and do not
divide well relatively as compared with other types of cells in
adult body. Angiogenesis is a process that generally includes
degradation of a vascular basement membrane by proteases released
by stimuli of proangiogenic factors; migration and proliferation of
vascular endothelial cells; tubular formation due to
differentiation of vascular endothelial cells; reconstruction of
blood vessels; and generation of new capillary blood vessels.
[0004] However, there are diseases induced by angiogenesis that is
not regulated autonomously but grows morbidly. Such diseases
associated with angiogenesis occurring in pathological conditions
include hemangioma, angiofibroma, vascular malformation and
cardiovascular diseases, such as arteriosclerosis, vascular
adhesion, and scleroedema. Ocular diseases associated with
angiogenesis include corneal graft angiogenesis, neovascular
glaucoma, diabetic retinopathy, corneal diseases induced by new
blood vessels, macular degeneration, pterygium, retinal
degeneration, retrolental fibroplasia, granular conjunctivitis, and
the like. Furthermore, angiogenesis-related diseases may include
chronic inflammatory diseases such as arthritis, cutaneous diseases
such as psoriasis, capillarectasia, pyogenic granuloma, seborrheic
dermatitis, acne, Alzheimer's disease, and obesity. Tumor growth
and metastases are necessarily dependent on angiogenesis (D'Amato R
J et al., Ophthalmology, 102(9), pp 1261-1262, 1995; Arbiser J L,
J. Am. Acad. Dermatol., 34(3), pp 486-497, 1996; O'Brien K D et al.
Circulation, 93(4), pp 672-682, 1996; Hanahan D et al., Cell, 86,
pp 353-364, 1996).
[0005] Especially, angiogenesis plays an important role in growth
and metastasis of cancer cells. Tumor is supplied with nutrition
and oxygen necessary for growth and proliferation through new blood
vessels, and the new blood vessels infiltrating into the tumor
allow the cancer cells being metastasized to enter the blood
circulation system and thus support metastasis of the cancer cells
(Folkman and Tyler, Cancer Invasion and metastasis, Biologic
mechanisms and Therapy (S. B. Day ed.) Raven press, New York, pp
94-103, 1977; Polverini P J, Crit. Rev. Oral. Biol. Med., 6(3), pp
230-247, 1995). The major cause of death in cancer patients is
metastasis, and the reasons why the chemotherapies or
immunotherapies being used clinically at present do not contribute
to an increase in a survival rate of cancer patients is directed to
metastasis of cancer.
[0006] Arthritis, a typical disease in inflammatory diseases, is
initiated as an autoimmune disorder. Along with progression of the
disease, chronic inflammation occurring in the synovial cavity
between joints induces angiogenesis to destroy the cartilage. That
is, proliferations of synovial cells and vascular endothelial cells
in the synovial cavity are activated by cytokines that induce
inflammations, resulting in development of angiogenesis. Finally,
the articular cartilage playing a role of a cushion is destroyed by
articular pannus as a connective tissue layer formed in a
cartilaginous part (Koch A E et al., Arthritis. Rheum., 29, pp
471-479, 1986; Stupack D G et al., Braz J. Med. Biol. Res., 32(5),
pp 578-581, 1999; Koch A E, Atrhritis. Rheum., 41(6), pp 951-962,
1998).
[0007] Many ocular diseases, from which millions of people are
losing their eyesight all over the world every year, result mainly
from angiogenesis (Jeffrey M I et al., J. Clin. Invest., 103, pp
1231-1236, 1999). Typical diseases resulting from angiogenesis
include macular degeneration, diabetic retinopathy, and the like
that occur commonly in old age, premature infant's retinopathy,
neovascular glaucoma, corneal diseases induced by new blood
vessels, and the like (Adamis A P et al., Angiogenesis, 3, pp 9-14,
1999). Among them, diabetic retinopathy that is one of the diabetic
complications and a disease that retinal capillaries invade
vitreous body to become blind.
[0008] Psoriasis characterized by red spots and scaly skin is a
chronic proliferative disease occurring in skin and is accompanied
with pain and malformation. Normally, keratinocytes proliferate
once a month, however, in a psoriasis patient, the keratinocytes
proliferate at least once a week. For such rapid proliferation, a
large quantity of blood is required, resulting in active
angiogenesis (Folkman J, J. Invest. Dermatol., 59, pp 40-48,
1972).
[0009] Since it is possible to apply angiogenesis inhibitors to
agents for treating various diseases associated with angiogenesis,
in recent years, a variety of studies aimed at treating such
diseases by inhibiting angiogenesis have been actively conducted.
Since such angiogenesis inhibitors should be administrated to
patients for a long time, the most ideal inhibitor is one that
should have low toxicity and can be orally administrated.
Accordingly, there has been a demand for development of drugs that
have low toxicity as angiogenesis inhibitors.
DISCLOSURE
Technical Problem
[0010] Hence, the inventors of the present invention confirmed that
a bicyclic pyridinol derivative having a specific structure is
highly effective in inhibiting angiogenesis, and, thus, completed
the present invention.
[0011] Therefore, an object of the present invention is to provide
a pharmaceutical composition containing a bicyclic pyridinol
derivative as an active ingredient for preventing or treating
diseases caused by angiogenesis.
Technical Solution
[0012] The present invention provides a pharmaceutical composition
containing a bicyclic pyridinol derivative expressed by the
following Chemical Formula 1 for preventing or treating diseases
caused by angiogenesis:
##STR00001##
[0013] In the above formula, R.sup.1 to R.sup.3 may be identical
with or different from one another and may be any one of hydrogen,
C1 to C16 alkyl, acyloxymethyl, or trimethyltridecyl, and n may be
an integer of 1 to 2.
[0014] In the bicyclic pyridinol derivative, R.sup.1 may be any one
of hydrogen, methyl, ethyl, or n-C.sub.16H.sub.33, R.sup.2 may be
any one of hydrogen or methyl, R.sup.3 is one of hydrogen, methyl,
n-butyl, t-butyl, acyloxymethyl, or trimethyltridecyl
##STR00002##
and n may be an integer of 1 to 2.
[0015] Further, the bicyclic pyridinol derivative may be preferably
a compound expressed by the following Chemical Formula 2 and more
preferably a compound expressed by the following Chemical Formula
3:
##STR00003##
[0016] In the above formula, n may be an integer of 1 to 2,
##STR00004##
Advantageous Effects
[0017] A bicyclic pyridinol derivative according to the present
invention can be effectively used as a drug for preventing and
treating diseases caused by angiogenesis since it inhibits
neovascularization induced by an angiogenesis inducer such as a
vascular endothelial growth factor in a chicken chorioallantoic
membrane model.
DESCRIPTION OF DRAWINGS
[0018] FIG. 1 illustrates chemical formulas of bicyclic pyridinol
derivatives according to an example of the present invention;
[0019] FIG. 2 is a value of IC.sub.50 obtained by treating a
compound 12 with a CAM by concentrations;
[0020] FIG. 3 illustrates a measurement result of a VEGF-induced
growth inhibitory effect of the compound 12 on HUVEC cells;
[0021] FIG. 4 provides photomicrographs taken at a magnification of
200 times of a bottom side of a polycarbonate filter in order to
check an infiltration inhibitory effect of the compound 12 on HUVEC
cells;
[0022] FIG. 5 provides photomicrographs taken at a magnification of
400 times of cells in order to check an ROS scavenging effect of
the compound 12;
[0023] FIG. 6 illustrates an observation result of the ROS
scavenging effect of the compound 12 by treating the adult retinal
pigment epithelium (ARPE)-19 cell line with angiotensin II, which
is a risk factor of macular degeneration;
[0024] FIG. 7 illustrates an observation result of the ROS
scavenging effect of the compound 12 by treating the adult retinal
pigment epithelium (ARPE)-19 cell line with 4-hydroxynonenal
(4-HNE), which is a risk factor of macular degeneration; and
[0025] FIG. 8 illustrates an observation result of angiogenesis
caused by tumorigenesis by inoculating A549 lung cancer cells to a
CAM and a tumor growth inhibitory effect.
BEST MODE
[0026] In order to achieve the above object, the present invention
provides a pharmaceutical composition containing a bicyclic
pyridinol derivative expressed by the following Chemical Formula 1
for preventing or treating diseases caused by angiogenesis:
##STR00005##
[0027] In the above formula, R.sup.1 to R.sup.3 may be identical
with or different from one another and may be any one of hydrogen,
C1 to C16 alkyl, acyloxymethyl, or trimethyltridecyl, and n may be
an integer of 1 to 2.
[0028] In the bicyclic pyridinol derivative, R.sup.1 may be any one
of hydrogen, methyl, ethyl, or n-C.sub.16H.sub.33, R.sup.2 may be
any one of hydrogen or methyl, R.sup.3 is one of hydrogen, methyl,
n-butyl, t-butyl, acyloxymethyl, or trimethyltridecyl
##STR00006##
and n may be an integer of 1 to 2.
[0029] Further, the bicyclic pyridinol derivative may be preferably
a compound expressed by the following Chemical Formula 2 and more
preferably a compound expressed by the following Chemical Formula
3:
##STR00007##
[0030] In the above formula, n may be an integer of 1 to 2.
##STR00008##
[0031] The diseases caused by angiogenesis may be selected from the
group consisting of rheumatic arthritis, osteoarthritis, septic
arthritis, psoriasis, corneal ulcer, senile macular degeneration,
diabetic retinopathy, proliferative vitreous body retinopathy,
premature retinopathy, ocular inflammation, conical cornea,
Sjogren's syndrome, myopia eye tumor, cornea graft rejection,
abnormal wound healing, bone disease, proteinuria, abdominal aortic
aneurysm disease, regressive cartilage loss due to traumatic joint
injury, demyelinating disease of nervous system, hepatic cirrhosis,
glomerular disease, premature rupture of embryonic membrane,
inflammatory bowel disease, periodontal membrane disease,
arteriosclerosis, restenosis, inflammatory disease of central
nervous system, Alzheimer's disease, skin aging, and infiltration
metastasis of cancer.
[0032] A bicyclic pyridinol derivative of the present invention can
be effectively used as a drug for preventing and treating diseases
caused by angiogenesis since it inhibits angiogenesis induced by an
angiogenesis inducer such as a vascular endothelial growth factor
in a chicken chorioallantoic membrane model.
[0033] Doses and ways of application of a pharmaceutical
composition containing a bicyclic pyridinol derivative of the
present invention may vary depending on the formulations and the
use purposes thereof.
[0034] The pharmaceutical composition containing a bicyclic
pyridinol derivative of the present invention may contain a
bicyclic pyridinol derivative in the amount of 0.1 to 50 weight %
with respect to the total weight of the composition.
[0035] Further, the pharmaceutical composition containing a
bicyclic pyridinol derivative of the present invention may further
contain suitable carriers, excipients, or diluents commonly used in
preparing pharmaceutical compositions.
[0036] Examples of the carriers, excipients, or diluents may
include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,
erythritol, maltitol, starch, acacia rubber, alginate, gelatin,
calcium phosphate, calcium silicate, cellulose, methyl cellulose,
microcrystalline cellulose, polyvinylpyrrolidone, water,
methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium
stearate, and mineral oil.
[0037] The pharmaceutical composition containing a bicyclic
pyridinol derivative of the present invention may be prepared in
oral formulations including powders, granules, tablets, capsules,
suspensions, emulsions, syrups, aerosols, and the like, agents for
external application, suppository and sterilizing injection
solutions by respective methods commonly used.
[0038] In the case of being formulated, the pharmaceutical
composition is prepared using diluents or excipients such as
fillers, extenders, bonding agents, humectants, disintegrants,
surfactants, etc. Solid dosage forms for oral administration
include tablets, pills, powders, granules, capsules, and the like.
Such solid dosages are prepared by mixing the compound with at
least one excipient, such as starch, calcium carbonate, sucrose or
lactose, and gelatin.
[0039] In addition to simple excipients, lubricants such as
magnesium stearate, and talc may be used. Liquid dosage forms for
oral administration, such as suspensions, internal solutions,
emulsions, and syrups, may contain commonly used simple diluents,
for example, water and liquid paraffin, as well as various
excipients, for example, humectants, sweeteners, aromatics,
preservatives, and the like. Dosage forms for parenteral
administration include sterilized aqueous solutions, non-aqueous
solvents, suspensions, emulsions, lyophilized agents,
suppositories, and the like. Non-aqueous solvents and suspensions
may be prepared using propylene glycol, polyethylene glycol,
vegetable oils such as olive oil, or injectable esters such as
ethyl oleate. As bases for suppositories, witepsol, macrogol, Tween
61, cacao oil, laurinum, and glycerogelatine can be used.
[0040] The dosages of the bicyclic pyridinol derivative of the
present invention may vary depending on age, sex, and weight of a
patient. 0.001 to 100 mg/kg or preferably, 0.01 to 10 mg/kg of the
bicyclic pyridinol derivative can be administrated once or several
times a day. Moreover, the dosages of the compound may be increased
and decreased depending on an administration path, severity of
disease, sex, weight, age, and the like. Accordingly, the dosages
do not limit the scope of the present invention in any aspect.
[0041] The pharmaceutical composition can be administrated to
mammals such as rats, mice, livestock, and humans through various
paths. For example, it can be administered by any kinds of
predictable administration methods such as oral, rectal or
intravenous, intramuscular, cutaneous, intrauterine or
intracerebroventricular injection administrations.
[0042] The bicyclic pyridinol derivative according to the present
invention has 50% lethality (LC.sub.50) of 2 g/kg or more with
secured stability and thus can be used in the pharmaceutical
composition of the present invention.
[Mode]
[0043] Hereinafter, the present invention will be explained in more
detail with reference to the following Examples. However, the
present invention is not limited to these Examples.
Example 1
Synthesis of Bicyclic Pyridinol Derivative
[0044] The following Table 1 lists and FIG. 1 illustrates bicyclic
pyridinol derivatives according to an example, and each of the
bicyclic pyridinol derivatives were prepared by synthesizing the
compounds by the methods described in references, respectively.
##STR00009##
TABLE-US-00001 TABLE 1 Compound N R.sup.1 R.sup.2 R.sup.3 Reference
1 2 H H H Chem. Eur. J., 2010, 16, 14106-14114 2 2 Me H H Chem.
Eur. J., 2010, 16, 14106-14114 3 2 n-C.sub.16H.sub.33 H H Chem.
Eur. J., 2010, 16, 14106-14114 4 2 H Me Me Synthesis, 2005,
1397-1404 5 2 Me Me Me Synthesis, 2005, 1397-1404, J. Am. Chem.
Soc., 2007, 129, 10211-10219 6 2 Me Me n-Bu J. Am. Chem. Soc.,
2007, 129, 10211-10219 7 2 Me H Me J. Am. Chem. Soc., 2007, 129,
10211-10219 8 2 Me H n-Bu J. Am. Chem. Soc., 2007, 129, 10211-10219
9 2 Me H t-Bu J. Am. Chem. Soc., 2007, 129, 10211-10219 10 1 Et H H
Org. Biomol. Chem., 2011, in press. DOI: 10.1039/c1ob05144j 11 1 Et
Me CH.sub.2OCOMe Org. Biomol. Chem., 2011, in press. DOI:
10.1039/c1ob05144j 12 1 Me Me C16* Org. Biomol. Chem., 2011, 9,
1749-1755
[0045] Me represents methyl, Et represents ethyl, Bu represents
butyl, and C16*
represents
##STR00010##
Experimental Example 1
Angiogenesis Inhibitory Effects Via CAM Assay
[0046] To examine angiogenesis inhibitory effects in vivo, a
chorioallantoic membrane (CAM) assay was carried out (Nguyen M et
al., Microvascular Res., 47, pp 31-40, 1994).
[0047] Fertilized chicken eggs were cultured keeping the
temperature at 37.degree. C. and the relative humidity at 55%. On
the tenth day, the first small hole was made in the region of air
sac and the second hole was dug in the flat region of egg, where a
window was to be made, using a hypodermic needle (Greencross
Medical Science, Korea).
[0048] Each egg was deflated through the first hole in the region
of the air sac so that the chorioallantoic membrane (CAM) was split
from the egg shell. Subsequently, a window was made by cutting this
region using a grinding wheel (Multipro 395JA, Dremel, Mexico).
[0049] Then, a whatman filter disc #1 (Whatman Inc. USA) was
treated with 3 mg/ml of cortisone acetate and dried. The filter
disc was drenched with a vascular endothelial growth factor (VEGF)
in a concentration of 20 ng/CAM.
[0050] The filter disc was put on the vessels through the
previously made window, and a compound 12 of Example was dissolved
in dimethylsulfoxide (DMSO) and diluted with phosphate buffered
saline (PBS) to treat by concentrations.
[0051] After 3 days from the drug treatment, the CAMs, on which the
filter disc was placed, were separated and washed with PBS to take
images using a stereomicroscope (Stemi SV6 stereomicroscope, Carl
Zeiss, Germany) and Image-Pro Plus software (Media Cybernetics;
Silver Spring, Md., USA). Then, the number of branches was counted
and the result data were analyzed.
[0052] As a result thereof, as listed in the following Table 2, it
could be confirmed that an increase in neovascularization induced
by the VEGF was decreased in a concentration-dependent manner due
to the treatment with the compound 12 according to the present
invention. In particular, according to comparison in angiogenesis
inhibitory effect between the compound 12 and SU4312, batimastat,
and .alpha.-tocopherol, the compound 12 had an excellent
angiogenesis inhibitory effect capable of inhibiting angiogenesis
in a lower concentration than the other compounds.
TABLE-US-00002 TABLE 2 Number of branches/ Inhibition rate
Experimental group Main vessel (%) PBS 14.4 .+-. 2.7 -- VEGF (20
ng/CAM) 52.2 .+-. 2.7 -- VEGF (20 ng/ SU4312 24 .+-. 4.6.sup.# 74.6
.+-. 12.1 CAM)+ (1 .mu.g/CAM, 378 .mu.M) Batimastat 19.9 .+-.
2.87.sup.# 78.4 .+-. 7.4 (1 .mu.g/CAM, 209 .mu.M)
.alpha.-tocopherol 32.3 .+-. 3.5 45.1 .+-. 9.3 (3 ng/CAM, 0.7
.mu.M) .alpha.-tocopherol 24.3 .+-. 2.7.sup.# 64.4 .+-. 14.2 (100
ng/CAM, 23 .mu.M) Compound 12 28 .+-. 4.7.sup.# 64.0 .+-. 12.5
(0.004 ng/CAM, 1 nM) Compound 12 25.2 .+-. 2.4.sup.# 71.4 .+-. 6.4
(0.04 ng/CAM, 10 nM)
[0053] Further, IC.sub.50 was obtained by treating the compound 12
with a CAM by concentrations, and the IC.sub.50 was 0.7 nM (FIG.
2).
Experimental Example 2
VEGF-Induced Growth Inhibitory Effect in HUVEC Cells
[0054] HUVEC cells were cultured in a flask coated with 0.2%
gelatin. Then, the HUVEC cells were cultured in an endothelial cell
basal medium-2 (EBM-2, Clonetics, San Diego, Calif.). This EBM-2
contained fetal bovine serum (FBS), hydrocortisone, a human basic
fibroblast growth factor (hFGF-B), a vascular endothelial growth
factor (VEGF), a human recombinant insulin-like growth factor
(R3-IGF-1), ascorbic acid, a human epidermal growth factor (hEGF),
GA-1000, and heparin. The HUVEC cells were cultured for 4 to 5 days
and injected into a 24-well plate in a density of 5.times.10.sup.4
cells/well, and the media of the respective wells were set at 1 ml.
In order to test the effect of the compound 12 in endothelial cells
induced by the VEGF, the HUVEC cells were treated with a mixture of
the VEGF and the compound 12 and cultured for 24 hours, and, then,
100 .mu.l of MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide; 5 g MTT/l in H.sub.2O) was added thereto and
further cultured for 4 hours. Then, 200 .mu.l of dimethylsulfoxide
(DMSO) was added to the respective wells containing the
corresponding cells and mixed with a pipette to dissolve the
reduced MTT crystals. Relative cell survival rates were measured by
scanning with a microplate reader (Molecular Devices, Menlo Park,
Calif.) having 540 nm filter. As illustrated in FIG. 3, the
compound 12 of the present invention reduced VEGF-induced cell
growth in a concentration-dependent manner.
Experimental Example 3
Chemostatic Migration Inhibitory Effect in HUVEC Cells
[0055] In order to examine a chemostatic migration inhibitory
effect of the compound of the present invention, the following in
vitro experiment was carried out using HUVEC cells (Mi-Sung Kim et
al., Cancer Research, 63, 5454-5461, 2003; Sang-Oh Yoon et al., The
Journal of Biological Chemistry, 276, 20085-20092, 2001; Sonia
Zorzet et al., The Journal of Pharmacology and Experimental
Therapeutics, 295, 927-933, 2000). A culture plate applied hereto
was a 24-well plate (Corning Costar, Cambridge, Mass.) including a
polycarbonate filter having a plurality of pores of 8 mm in size.
The bottom side of the polycarbonate filter was coated with 20 l of
type I collagen in a concentration of 0.5 mg/ml, and the top side
thereof was coated with 20 l of matrigel (BD Bioscience, Bedford,
Mass.) in a concentration of 1.5 mg/ml. Here, the lower region of
the polycarbonate filter was filled with a medium including 2% FBS,
and the HUVEC cells were inoculated into the upper region of the
polycarbonate filter. In this case, the compound 12 was dissolved
in dimethyl sulfoxide (DMSO) and diluted with phosphate buffered
saline (PBS) by concentrations (1, 3, 10 .mu.M). The HUVEC cells
inoculated as such were cultured at 37.degree. C. for 24 hours.
Subsequently, the cells infiltrating the bottom side of the
polycarbonate filter were fixed with methanol and stained with
hematoxylin and eosin. FIG. 4 provides photomicrographs taken at a
magnification of 200 times of the bottom side of the polycarbonate
filter in order to check an infiltration inhibitory effect of the
compound 12 on HUVEC cells. Herein, in the case of a treatment with
the compound 12 of the present invention, it could be seen that
cancer cells infiltrating the bottom side of the polycarbonate
filter was remarkably decreased as compared with the control group,
and, thus, it could be seen that the compound 12 of the present
invention reduced infiltration of the HUVEC cells in a
concentration-dependent manner.
Experimental Example 4
Measurement of Reactive Oxygen Species (ROS) Scavenging Effect in
HUVEC Cells
[0056] In order to measure an ROS scavenging effect in HUVEC cells
induced by a VEGF, 2',7'-dichlorofluorescein diacetate (DCF-DA) was
used. When the ROS was present in cells, the DCF-DA was oxidized to
fluorescent DCF exhibiting green fluorescence. The HUVEC cells were
injected into 8-well plates coated with 0.2% gelatin in a density
of 1.times.10.sup.5 cells/well and then cultured for 24 hours. A
pre-treatment was carried out to the compound 12 for 3 hours and
induced by the VEGF for 15 minutes and washed with PBS (pH 7.4)
three times. Then, 10 .mu.M DCF-DA was put into an EBM-2 and used
to treat in darkroom for 30 minutes, and then washed again with the
PBS three times. A fluorescence degree of the cells was measured
using a fluorescence microscope. FIG. 5 provides photomicrographs
taken at a magnification of 400 times of cells in order to check an
ROS scavenging effect of the compound 12 of the present invention.
Herein, in the case of a treatment with the compound 12 of the
present invention, it could be seen that a fluorescence degree of
the cells was remarkably decreased as compared with the VEGF. FIG.
6 and FIG. 7 illustrate an observation result of the ROS scavenging
effect of the compound 12 by treating the adult retinal pigment
epithelium (ARPE)-19 cell line with 4-hydroxynonenal (4-HNE) or
angiotensin II, which is a risk factor of macular degeneration.
Likewise, it could be seen that a fluorescence degree of the cells
was remarkably decreased as compared with the control group. The
4-hydroxynonenal (4-HNE) or angiotensin II, which is a risk factor
of macular degeneration caused cell damage by generation of ROS in
ARPE cell and caused damage of a Bruch's membrane and angiogenesis,
resulting in macular degeneration and loss of eyesight. Based on
the above experimental result, the compound 12 strongly inhibits
generation of ROS of 4-hydroxynonenal (4-HNE) or angiotensin II and
thus can be expected to be effective in treating macular
degeneration.
Experimental Example 5
Angiogenesis Caused by Tumorigenesis and Tumor Growth Inhibitory
Effect
[0057] FIG. 8 illustrates an observation result of angiogenesis
caused by tumorigenesis by inoculating A549 lung cancer cells to a
CAM and a tumor growth inhibitory effect. Fertilized chicken eggs
were cultured, and on the ninth day, a window was made in the same
manner, and instead of a disc, cancer cells were mixed with
matrigel at a ratio of 1:1 and treated with the compound 12 and
inoculated in a density of 1.5.times.10.sup.6 cells/CAM. After 5
days from the inoculation, a CAM portion, in which tumor was
formed, was separated and washed with PBS to take images using a
stereomicroscope (Stemi SV6 stereomicroscope, Carl Zeiss, Germany)
and Image-Pro Plus software (Media Cybernetics; Silver Spring, Md.,
USA). Then, the number of branches was counted and the result data
were analyzed. As a result thereof, the compound 12 of the present
invention inhibited angiogenesis caused by tumorigenesis and also
inhibited tumor growth in a significantly low concentration as
compared with .alpha.-tocopherol as the control group.
Experimental Example 6
Toxicity Experiment
[0058] The compound 12 was suspended in a 0.5% methyl cellulose
solution and orally administered to male Balb/c mice with a single
dose of 0.5 g/kg, 1 g/kg, and 2 g/kg. Then, the survival rate and
weights of the mice were checked for 7 days.
[0059] After the administration, whether the animal was dead or
not, clinical symptoms, changes in weight were observed, and a
hematological test and a blood chemical test were carried out.
Further, the mice were autopsied, and then, whether or not
abdominal and chest organs are abnormal was observed with the naked
eye.
[0060] As a result thereof, any especially remarkable clinical
symptoms were not observed from the animals, and no animal was
dead. No change in toxicity was observed from the weight change,
the hematological test, the blood chemical test, and the postmortem
report.
[0061] According to the above result, the compounds of the present
invention with a dose of up to 2 g/kg did not exhibit a change in
toxicity in the mice, and, thus, they were determined as safe
substances having a median lethal dose (LD.sub.50) of more than 2
g/kg for oral administration.
[0062] Hereinafter, the formulation examples of the composition
containing the compound 12 according to the present invention will
be explained, however, which should not be limited thereto. Rather,
these examples are provided no more than to explain the present
invention in more detail.
Formulation Example 1
Preparation of Powders
[0063] 20 mg of the compound 12, 100 mg of lactose, and 10 mg of
talc were mixed with one another and packed in an airtight bag,
thus preparing a powder.
Formulation Example 2
Preparation of Tablets
[0064] 20 mg of the compound 12, 100 mg of corn starch, 100 mg of
lactose, and 2 mg of magnesium stearate were mixed with one another
and tableted according to a conventional method of preparing
tablets, thus preparing a tablet.
Formulation Example 3
Preparation of Capsules
[0065] 10 mg of the compound 12, 100 mg of corn starch, 100 mg of
lactose, and 2 mg of magnesium stearate were mixed with one another
and packed in a gelatin capsule according to a conventional method
of preparing capsules, thus preparing a capsule.
Formulation Example 4
Preparation of Injections
[0066] 10 mg of the compound 12, sterile distilled water for
injection in a proper amount, and a pH regulator in a proper amount
were mixed with one another, and then, an injection was prepared to
2 ml of an ampoule containing the above ingredients according to a
conventional method of preparing injections.
Formulation Example 5
Preparation of Ointments
[0067] 10 mg of the compound 12, 250 mg of PEG-4000, 650 mg of
PEG-400, 10 mg of white vaseline, 1.44 mg of Methyl
para-oxybenzoate, 0.18 mg of Propyl para-oxybenzoate and a balance
of distilled water were mixed with one another, and then, an
ointment was prepared according to a conventional method of
preparing ointments.
[0068] Specific parts of the present invention have been described
in detail, and, thus, it is obvious to those skilled in the art
that such detailed descriptions are provided as preferable aspects
but do not limit the scope of the present invention. Therefore, the
scope of the present invention should be considered in light of the
appended claims and equivalent arrangements.
* * * * *