U.S. patent application number 10/518870 was filed with the patent office on 2006-02-16 for novel use of ansamycin antibiotics and method of screening novel angiogenesis inhibitor.
Invention is credited to Masayoshi Shichiri, Yujiro Tanaka.
Application Number | 20060035878 10/518870 |
Document ID | / |
Family ID | 30002254 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060035878 |
Kind Code |
A1 |
Shichiri; Masayoshi ; et
al. |
February 16, 2006 |
Novel use of ansamycin antibiotics and method of screening novel
angiogenesis inhibitor
Abstract
The present invention provides novel angiogenesis inhibitors
effective, safe and highly practical for inhibition of angiogenesis
in various diseases, and a method for screening the same. The
active ingredients of the angiogenesis inhibitors of the present
invention comprise ansamycin antibiotics such as rifampicin,
rifamycin SV and 3-formyl rifamycin, which have long been used
extensively as antibacterial agents for treating tuberculosis or
Gram-positive bacterial infections. The active ingredients of the
present invention have excellent angiogenesis-inhibiting activity.
The angiogenesis inhibitor of the present invention is effective
for inhibition of angiogenesis in malignant tumors, diabetic
retinopathy, retinal angiogenesis, inflammatory diseases, and
angiogenesis accompanying cardiovascular remodeling, etc., and can
be used as therapeutic agents against each disease, etc. Further,
the screening method of the present invention makes it possible to
perform an effective screening of angiogenesis-inhibiting
substances by detecting angiogenesis-inhibiting signals based on
gene expression levels.
Inventors: |
Shichiri; Masayoshi; (Tokyo,
JP) ; Tanaka; Yujiro; (Tokyo, JP) |
Correspondence
Address: |
MORGAN & FINNEGAN, L.L.P.
3 WORLD FINANCIAL CENTER
NEW YORK
NY
10281-2101
US
|
Family ID: |
30002254 |
Appl. No.: |
10/518870 |
Filed: |
June 19, 2003 |
PCT Filed: |
June 19, 2003 |
PCT NO: |
PCT/JP03/07813 |
371 Date: |
August 29, 2005 |
Current U.S.
Class: |
514/183 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 35/00 20180101; A61P 43/00 20180101; A61P 9/00 20180101; A61K
31/496 20130101; G01N 33/5011 20130101; A61P 3/10 20180101; A61P
27/02 20180101 |
Class at
Publication: |
514/183 |
International
Class: |
A61K 31/33 20060101
A61K031/33 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 21, 2002 |
JP |
2002-181281 |
Apr 23, 2003 |
JP |
2003-118960 |
Claims
1. An angiogenesis inhibitor containing an ansamycin antibiotic or
a pharmacologically acceptable derivative thereof as an active
ingredient.
2. The angiogenesis inhibitor according to claim 1, wherein the
ansamycin antibiotic is rifampicin, rifamycin SV or 3-formyl
rifamycin.
3. The angiogenesis inhibitor according to claim 1, wherein the
pharmacologically acceptable derivative is a pharmacologically
acceptable salt or a hydrate thereof.
4. The angiogenesis inhibitor according to claim 1, wherein
angiogenesis in a malignant tumor is inhibited.
5. The angiogenesis inhibitor according to claim 1, wherein
angiogenesis in diabetic retinopathy is inhibited.
6. The angiogenesis inhibitor according to claim 1, wherein
angiogenesis in retinal angiogenesis is inhibited.
7. The angiogenesis inhibitor according to claim 1, wherein
angiogenesis in an inflammatory disease is inhibited.
8. The angiogenesis inhibitor according to claim 1, wherein
angiogenesis accompanying cardiovascular remodeling is
inhibited.
9. A method for screening an angiogenesis-inhibiting substance
wherein a test substance is added to cultured vascular endothelial
cells, and an angiogenesis-inhibiting signal based on gene
expression level is detected.
10. The method for screening an angiogenesis-inhibiting substance
according to claim 9, wherein the angiogenesis-inhibiting signal
based on reduced gene express ion level in a cultured cell line is
similar to the change induced by endostatin at a concentration
showing a tumor regression effect.
11. The method for screening an angiogenesis-inhibiting substance
according to claim 9, wherein the angiogenesis-inhibiting signal
based on reduced gene expression level in a cultured cell line
consists of one or more of an immediate early response gene or a
related gene thereof, a growth/cell-cycle-related gene, a cell
adhesion factor, a vasoactive factor, and a vasoactive factor
receptor gene expressed in a vascular endothelial cell.
12. An angiogenesis inhibitor containing an ansamycin antibiotic or
a pharmacologically acceptable derivative thereof as an active
ingredient; wherein the ansamycin antibiotic is rifampicin,
rifamycin SV or 3-formyl rifamycin and the pharmacologically
acceptable derivative is a pharmacologically acceptable salt or a
hydrate thereof.
13. An angiogenesis inhibitor according to claim 2, wherein
angiogenesis in a malignant tumor is inhibited.
14. An angiogenesis inhibitor according to claim 12, wherein
angiogenesis in a malignant tumor is inhibited.
15. The angiogenesis inhibitor according to claim 2, wherein
angiogenesis in diabetic retinopathy is inhibited.
16. The angiogenesis inhibitor according to claim 12, wherein
angiogenesis in diabetic retinopathy is inhibited.
17. The angiogenesis inhibitor according to claim 2, wherein
angiogenesis in retinal angiogenesis is inhibited.
18. The angiogenesis inhibitor according to claim 12, wherein
angiogenesis in retinal angiogenesis is inhibited.
19. The angiogenesis inhibitor according to claim 2, wherein
angiogenesis in an inflammatory disease is inhibited.
20. The angiogenesis inhibitor according to claim 12, wherein
angiogenesis in an inflammatory disease is inhibited.
21. The angiogenesis inhibitor according to claim 2, wherein
angiogenesis accompanying cardiovascular remodeling is
inhibited.
22. The angiogenesis inhibitor according to claim 12, wherein
angiogenesis accompanying cardiovascular remodeling is
inhibited.
23. The method for screening an angiogenesis-inhibiting substance
according to claim 10, wherein the angiogenesis-inhibiting signal
based on reduced gene express ion level in a cultured cell line
consists of one or more of an immediate early response gene or a
related gene thereof, a growth/cell-cycle-related gene, a cell
adhesion factor, a vasoactive factor, and a vasoactive factor
receptor gene expressed in a vascular endothelial cell.
Description
TECHNICAL FIELD
[0001] The present invention relates to an angiogenesis inhibitor,
and more particularly, to a novel angiogenesis inhibitor effective
for inhibition of angiogenesis in malignant tumor, diabetic
retinopathy, retinal angiogenesis, inflammatory diseases,
cardiovascular diseases due to cardiovascular remodeling, etc., and
to a method for screening angiogenesis-inhibiting substances.
BACKGROUND ART
[0002] Angiogenesis is a process in which new blood vessels are
generated from existing blood vessels, and it is known that
angiogenesis is closely involved in an onset and development of
diseases such as malignant (solid) tumor, diabetic retinopathy, or
retinal angiogenesis, inflammatory diseases (rheumatism, etc.). For
example, in order for solid tumors to grow, it is necessary to
secure a supply route of nutrition and oxygen and an elimination
route of wastes by angiogenesis. Angiogenesis plays an important
role for tumor metastasis, an issue special concern for cancer
treatment, since angiogenesis secures the blood supply. As for
diabetic retinopathy, angiogenesis itself is a pathological
condition, and patients lose their eyesight if left untreated.
Therefore, it is considered that inhibition of angiogenesis leads
to prevention/treatment of diseases, and preventing/treating agents
for angiogenesis are currently explored.
[0003] Since angiogenesis promotes various pathological conditions
mentioned above, inhibition of angiogenesis is expected to be
beneficial in prevention/treatment of such conditions. For the
purpose of prevention or treatment of diseases associated with
angiogenesis, studies in search for angiogenesis-inhibiting
substances have been performed. As a result, many
angiogenesis-inhibiting substances have been identified, and for
some of them, clinical usefulness is now under investigation.
[0004] For instance, angiogenesis inhibitors such as endostatin and
angiostatin were once known to be the most potent agents for tumor
dormancy therapy. They were expected to serve as ideal anticancer
drugs with least adverse reactions because their systemic therapy
regressed solid tumors in experimental animals remarkably (Cell,
88, 277-285, 1997) without any acquired resistance as experimental
tumors do not develop resistance to multiple cycles of therapy
unlike conventional anticancer drugs (Nature, 390, 404-407, 4997).
However, in clinical practice, synthesis of an effective dosage of
these high molecular proteins to elicit antitumor effect is
difficult and costly and, consequently, business circles have
already abandoned clinical applications of angiostatin, whose
molecular weight is about 50000.
[0005] Endostatin, with lower molecular weight (about 20000),
attracted attention and its clinical applications have started in
terminal malignant tumor patients in U.S.A. However, its precise
mechanisms of action as well as its receptors had been unknown.
[0006] Endostatin inhibits the proliferation of endothelial cells
and induces apoptosis under reduced serum culture condition (J.
Biol. Chem., 274, 11721-11726, 1999), but since the effect was
limited, and it was difficult to ascribe the potent effect to
regress primary and metastatic cancers. Tumor cells attain
accelerated proliferative characteristics not only by genomic
mutation and deregulated gene expression but also by vigorously
secreting many growth- and angiogenesis-promoting factors in an
autocrine/paracrine fashion; and further, newly generated blood
vessels supply abundant blood flow. In order for endostatin to
inhibit tumor angiogenesis under such circumstances as currently
reported, potent intracellular signals specifically acting on
endothelial cells must be induced. These mechanisms have been
unknown for long time.
[0007] On the other hand, in 1957, P. Sensi et al. of Lepetit
Research Laboratories in Italy separated Streptomyces mediterranei
(later, classified into Nocardio mediterranei) from soil collected
at the coast of the Mediterranean, and obtained rifamycin, an
antibiotic showing antibacterial activity to acid-fast bacteria and
Gram-positive bacteria from the culture liquid thereof. Rifamycin
in the culture liquid is a mixture comprising rifamycins A, B, C,
D, E, etc., and rifamycin O is an oxidized type of rifamycin B.
Rifamycin B and rifamycin O are induced into rifamycin S, and
rifamycin S is reduced to rifamycin SV by ascorbic acid. 3-formyl
rifamycin is made by 3-formylation of rifamycin. Rifampicin is
induced from a substance made by 3-formylation of rifamycin SV.
Rifamycin is collectively called as ansamycin antibiotics because
it has an aromatic ring system to which an aliphatic bridge called
ansa ring is connected.
[0008] In addition, the above-mentioned rifampicin is an ansamycin
antibiotic developed from a collaboration of Ciba-Geigy
(Switzerland) and Lepetit (Italy), and is induced from a substance
made by 3-formylation of rifamycin SV. In other words, rifampicin
is an ansamycin semisynthetic antibiotic having a structure of
3-{[(4-methyl-1-piperazinyl)imino]methyl}rifamycin, and is a
substance which has excellent antituberculosis activity and has
been used widely as an anti-tuberculous drug. Rifampicin has
antibacterial activity not only to Gram-positive bacteria but also
to Gram-negative bacillus, and has been used for brucellosis,
chlamydia infection, and infection of Gram-positive bacteria such
as staphylococcus as well as tuberculosis.
[0009] Rifampicin is synthesized by reacting 3-formyl rifamycin SV
with 1-amino-4-methylpiperazine in tetrahydrofran, and many
synthetic methods including industrial synthetic methods are
disclosed (Japanese Patent Publication Nos. 42-26800, 47-23303,
53-39400, 57-40155, 62-41671, 62-41672, and 62-41673).
[0010] The object of the present invention is to provide a novel
angiogenesis inhibitor which is safe and highly practical, more
particularly, a novel angiogenesis inhibitor which is effective,
safe and highly practical for inhibition of angiogenesis in various
diseases such as malignant tumor, diabetic retinopathy, retinal
angiogenesis and inflammatory diseases. In addition, the present
invention provides a method for screening a novel
angiogenesis-inhibiting substance which serves as an active
ingredient of an angiogenesis inhibitor.
[0011] As a result of intensive search to attain the objective
mentioned above, the present inventors have found that rifampicin,
which has excellent antituberculous and antibacterial activity to
both Gram-positive and negative bacteria and widely used to treat
brucellosis, chlamydia infection, and staphylococcus infection as
well as tuberculosis, has excellent angiogenesis-inhibiting
activity. This led to the completion of the present invention. The
present invention, further confirmed that ansamycin antibiotics
such as rifamycin SV or 3-formyl rifamycin have
angiogenesis-inhibiting activity as well.
[0012] In the present invention, the finding that ansamycin
antibiotics such as rifampicin, etc., have excellent
angiogenesis-inhibiting activity has its origin in the elucidation
of endostatin-induced molecular signals by the present inventors.
Recently, the present inventors have found a molecular mechanism
involved in inhibition of angiogenesis by endostatin (FASEB
Journal. 15, 1044-1053, 2001). Administration of endostatin at
concentrations showing tumor regression in experimental animals
markedly inhibited various immediately early response genes and
apoptosis/cell-cycle/migration-associated genes expressed in
cultured vascular endothelial cells under supplementation with
serum, growth factors and angiogenesis factors.
[0013] As a result of down-regulation of a variety of gene
expression, endostatin causes marginal endothelial cell
proliferation, but marked inhibition of endothelial cell migration.
The molecular responses, which are potent and wide spectrum of gene
down-regulation by endostatin, are designated as
"angiogenesis-inhibiting signals" by a present inventor. By
quantifying mRNA levels using real-time quantitative PCR, it
becomes possible to rapidly identify substances showing potent
signals similar to endostatin among many reagents, and to examine
whether they exert potent inhibition of endothelial cell
migration/proliferation.
[0014] Conventional process of identifying novel
angiogenesis-inhibiting factors required repetition of protein
purification by extracting and fractionating tumor regressive
activity released by tumor themselves from a large amount of body
fluid and/or supernatant of cell cultures. The entire process was
time-consuming and it took long before gene cloning. Further, it
was difficult to synthesize a sufficient dosage of large molecular
weight angiogenesis-inhibiting factors, endostatin and
angiostatin.
[0015] By the method for screening angiogenesis-inhibiting
substances which detects "angiogenesis-inhibiting signals"
constructed by the present inventors, it becomes possible to detect
factors exerting endostatin-type signals among many substances, and
to select a novel angiogenesis-inhibiting factor by examining tumor
regression activity and angiogenesis-inhibiting activity. This
screening method greatly reduces conventional processes, and
further, allows to estimate dosages of peptides/proteins/drugs
required to induce the effect comparable to that of endostatin in
advance.
[0016] The method enables to find novel factors clinically
applicable to a therapy targeting new blood vessel formation in
tumors. Further, when angiogenesis-inhibiting signals by endostatin
are confirmed to correlate with tumor regression activity, in vitro
activity of many synthetic peptides and compounds efficiently
screened can be utilized. A test substance can be added to the
supernatant of cultured vascular endothelial cells to detect
angiogenesis-inhibiting signals, and, it becomes possible to select
efficiently novel angiogenesis inhibitors from many substances.
Peptides/proteins with smaller molecular weight and with structures
applicable easily clinically can be preferentially selected, while
potential side effects may be predicted for ingredients of drugs
and food at effective concentrations to induce antiangiogenesis, in
advance at the stage of screening.
[0017] In the present invention using the above-mentioned screening
method, ansamycin antibiotics such as rifampicin have been found to
induce strong angiogenesis-inhibiting activity among many candidate
substances. Since ansamycins such as rifampicin have been widely
used as antibiotics, their safety is well known and method for
producing and administering them have been established. Therefore,
it is expected that they can be used as highly practical
angiogenesis inhibitors.
DISCLOSURE OF THE INVENTION
[0018] The present invention comprises an angiogenesis inhibitor
containing an ansamycin antibiotic or a pharmacologically
acceptable derivative thereof as an active ingredient ("1"), the
angiogenesis inhibitor according to "1", wherein the ansamycin
antibiotic is rifampicin, rifamycin SV or 3-formyl rifamycin ("2"),
the angiogenesis inhibitor according to "1" or "2", wherein the
pharmacologically acceptable derivative is a pharmacologically
acceptable salt or a hydrate thereof ("3"), the angiogenesis
inhibitor according to any one of "1" to "3", wherein angiogenesis
in malignant tumor is inhibited ("4"), the angiogenesis inhibitor
according to any one of "1" to "3", wherein angiogenesis in
diabetic retinopathy is inhibited ("5"), the angiogenesis inhibitor
according to any one of "1" to "3", wherein angiogenesis in retinal
angiogenesis is inhibited ("6"), the angiogenesis inhibitor
according to any one of "1" to "3", wherein angiogenesis in an
inflammatory disease is inhibited ("7"), and the angiogenesis
inhibitor according to any one of "1" to "3", wherein angiogenesis
accompanying cardiovascular remodeling is inhibited ("8").
[0019] The present invention further comprises a method for
screening an angiogenesis-inhibiting substance wherein a test
substance is added to cultured vascular endothelial cells, and an
angiogenesis-inhibiting signal based on gene expression level is
detected ("9"), the method for screening an angiogenesis-inhibiting
substance according to "9", wherein the angiogenesis-inhibiting
signal based on reduced gene expression level in a cultured cell
line is similar to the change induced by endostatin at a
concentration showing a tumor regression effect ("10"), and the
method for screening an angiogenesis-inhibiting substance according
to "9" or "10", wherein the angiogenesis-inhibiting signal based on
reduced gene expression level in a cultured cell line consists of
one or more of an immediate early response gene or a related gene
thereof, a growth/cell-cycle-related gene, a cell adhesion factor,
a vasoactive factor, and a vasoactive factor receptor gene
expressed in a vascular endothelial cell ("11").
BRIEF DESCRIPTION OF DRAWINGS
[0020] FIG. 1 is a set of views showing angiogenesis-inhibiting
signals induced by addition of various concentrations of rifampicin
to adult human dermal microvascular endothelial cells. A: FAK gene,
B: PECAM-1 gene, C: integrin-.alpha.v gene, D: integrin-.beta.3
gene, E: endothelin-1 gene, F: ET.sub.B gene, G: c-myc gene, H: Flt
gene.
[0021] FIG. 2 is a view showing proliferation-inhibiting activity
of rifampicin to adult human dermal microvascular endothelial cells
with the use of modified 72-hour-proliferation assay (under the
culture in proliferation medium containing 10% fetal bovine
serum).
[0022] FIG. 3 is a view showing the time course of advanced
distance of wounding edge after denudement of confluent culture of
adult human dermal microvascular endothelial cells. Cell migration
is significantly inhibited by the addition of rifampicin to the
culture medium.
[0023] FIG. 4 is a view showing the result of examination whether
oral intake of rifampicin exhibits tumor growth-inhibiting activity
to explanted solid tumors derived from human colon cancer cell line
(CW-2) in nude mice. Tumor volume is significantly inhibited by
oral intake of rifampicin.
[0024] FIG. 5 is a set of views showing relationship between the
changes of plasma .alpha.-fetoprotein levels and long-term
rifampicin administration in two patients with hepatitis C-related
liver cirrhosis combined with pulmonary tuberculosis.
[0025] FIG. 6 is a set of views showing angiogenesis-inhibiting
signals induced by various concentrations of rifampicin, rifamycin
SV and 3-formyl rifamycin in human retinal microvascular
endothelial cells. It is shown that each mRNA amount of c-myc,
integrin-.alpha.v, integrin-.beta.3 is inhibited in a
concentration-dependent manner.
BEST MODE OF CARRYING OUT THE INVENTION
[0026] The present invention comprises angiogenesis inhibitors
containing an ansamycin antibiotic or a pharmacologically
acceptable derivative thereof as an active ingredient. There is no
particular limitation as to the subject of application of the
angiogenesis inhibitor of the present invention, the angiogenesis
inhibitor can be used for inhibiting angiogenesis in malignant
tumors, diabetic retinopathy, retinal angiogenesis and angiogenesis
accompanying inflammatory diseases or cardiovascular remodeling, as
an antitumor agent, a therapeutic agent for diabetic retinopathy,
retinal angiogenesis, or inflammatory diseases, or a therapeutic
agent for arteriosclerosis, angiopathy, etc., respectively.
[0027] The active ingredients of the present invention comprise
ansamycin antibiotics such as rifampicin, rifamycin SV, 3-formyl
rifamycin, etc. The active ingredients of the present invention can
be appropriately converted into, for example, pharmacologically
acceptable derivatives in order to increase water-solubility for
the purpose of easier administration (Japanese Patent Publication
No. 5-44467). As one of the pharmacologically acceptable
derivatives, it is possible to convert the ingredients into the
form of pharmacologically acceptable salts or hydrates thereof,
which are used in formulation of medicines usually. As mentioned
above, any known producing methods can be used for preparing the
active ingredients of the present invention such as rifampicin.
[0028] For administration of the angiogenesis inhibitor of the
present invention, appropriate administration method such as oral
or parenteral administration (intravenous, intramuscular,
subcutaneous administration, or instillation) can be used in
accordance with subjects of administration. In case of oral
administration, the active ingredients of the present invention can
be formulated as a solid or liquid prescribed drug, for instance,
in the form of tablets, granules, capsules, powders, troches,
solutions, suspensions, or emulsions. In case of parenteral
administration, the active ingredients of the present invention can
be prepared as, for example, an injectable prescribed drug by using
an appropriate solvent. Examples of such solvent include water,
aqueous solvents (sodium chloride solution, glucose solution,
etc.), water-miscible solvents (ethyl alcohol, polyethylene glycol,
propylene glycol, etc.), and nonaqueous solvents (corn oil,
cottonseed oil, peanut oil, sesame oil, etc.). When applying them
to diseases such as diabetic retinopathy, the active ingredients of
the present invention can be administered as a formulation of
eyedrops.
[0029] Dosage of the active ingredients of the present invention is
appropriately determined according to subjects and forms of
administration, however, as an example of dosage unit for oral
administration, an amount containing about 50.about.1000 mg of the
active ingredients, preferably, about 150.about.500 mg of the
active ingredients, is exemplified.
[0030] Rifampicin, the active ingredients of the present invention,
is a medicine which have been already used for innumerable patients
including tuberculosis patients, and its dosage regimen and side
effect are well known. Therefore, when using the medicines of the
present invention, dosage form and dosage method based on such
experiences can be used.
[0031] In addition, the present invention contains a method for
screening a novel angiogenesis-inhibiting substance. The method for
screening a novel angiogenesis-inhibiting substance of the present
invention is conducted by adding a test substance with the use of
cultured vascular endothelial cells, and detecting
angiogenesis-inhibiting signals based on gene expression levels. As
the angiogenesis-inhibiting signals, gene expression signals
observed when endostatin is administered at a concentration showing
tumor regression effects can be used. Examples of the
angiogenesis-inhibiting signals based on gene expression include
expression of one or more genes of immediate early response genes,
their related genes, growth/cell-cycle-related genes, cell adhesion
factors, vasoactive factors, and vasoactive factor receptor genes
expressed in vascular endothelial cells.
[0032] Specific examples of the angiogenesis-inhibiting signal
genes include: c-myc, c-fos as immediate early response genes
expressed in vascular endothelial cells; max, mad, mxil as their
related genes; mitogen activated protein kinase-1, mitogen
activated protein kinase-2 as growth/cell-cycle-related genes;
integrin-.alpha.v, integrin-.beta.3 as cell adhesion factor;
endothelin-1 gene as a vasoactive factor; and ET-A, AT1, AT2, as
vasoactive factor receptor genes.
[0033] The method for screening a novel angiogenesis-inhibiting
substance of the present invention to detect
angiogenesis-inhibiting signals can be performed by already
published technique with use of vascular endothelial cells, in
particular, adult human dermal microvascular endothelial cells or
human retinal endothelial cells. The quantification technique of
gene expression is also already known. With the use of real-time
quantitative PCR methods, angiogenesis-inhibiting signals can be
detected by quantifying mRNA levels.
[0034] The present invention will be described more specifically
with examples, but the technical scope of the present invention is
not limited to these.
EXAMPLE 1
Angiogenesis-Inhibiting Signals by Rifampicin
[0035] Exponentially growing adult human dermal microvascular
endothelial cells in the presence of serum/growth
factors/angiogenesis-promoting factors were incubated with various
concentrations of rifampicin for four hours, and extracted RNAs
were subjected to quantification of mRNA of various genes using
LightCycler-based highly sensitive real-time quantitative PCR
(FASEB J., 15, 1044-1053, 2001). The results are shown in FIG. 1.
In the figure, A represents the results of quantification of focal
adhesion kinase gene, B represents those of platelet endothelial
cell adhesion molecule-1 (PECAM-1) genes, C represents those of
integrin-.alpha.v gene which is an adhesive factor, D represents
those of integrin-.beta.3 gene which is another adhesive factor, E
represents those of endothelin-1 gene which is a vasoconstrictive
peptide, F represents those of endothelin receptor subtype B
(ET.sub.B) gene, G represents those of c-myc gene which is an
immediate early response gene, and H represents those of Flt gene
which is a subtype of vascular endothelial growth factor (VEGF)
receptor, respectively.
[0036] As shown in FIG. 1, addition of various concentrations of
rifampicin induced angiogenesis-inhibiting signals showing spectra
similar to those of endostatin.
EXAMPLE 2
Inhibitory Activity of Rifampicin Against Proliferation of Vascular
Endothelial Cells
[0037] The effect of rifampicin on the proliferation of endothelial
cells was examined by modified 72-hour-proliferation assay (Cell,
88, 277-85, 1997).
[0038] Under the culture in growth medium containing 10% fetal
bovine serum, various concentrations of rifampicin were added to
exponentially growing adult human dermal microvascular endothelial
cells for 48.about.72 hours, and the cell number was counted by an
automated blood cell counter.
[0039] The results are shown in FIG. 2. Rifampicin inhibited the
proliferation of adult human dermal microvascular endothelial cells
in a concentration-dependent manner.
EXAMPLE 3
Inhibitory Activity of Rifampicin against Migration of Vascular
Endothelial Cells
[0040] Inhibitory activity of rifampicin against cell migration was
examined by monolayer wounding method (FASEB J., 15, 1044-1053,
2001).
[0041] Confluent culture of adult human dermal microvascular
endothelial cells under the culture in growth medium containing 10%
fetal bovine serum were pretreated with 40 .mu.g/ml of rifampicin
for 24 hours, denuding was conducted by the monolayer wounding
method, photomicrographs were taken chronologically and advanced
distances of wounding edge were measured.
[0042] The results are shown in FIG. 3. The advanced distances of
vascular endothelial cells were markedly decreased by the addition
of 40 .mu.g/ml of rifampicin.
[0043] As shown in FIG. 3, rifampicin inhibited chemotactic
migration of adult human dermal microvascular endothelial
cells.
EXAMPLE 4
Antitumor Effect in Explanted Tumors in Experimental Animals
[0044] Whether oral intake of rifampicin shows regression of
primary solid tumor/metastatic tumors derived from explanted human
colon cancer cell lines (CW-2) in nude mice was examined in
accordance with the original method of O'Reilly et al. (Cell, 79,
315-28, 1994; Cell, 88, 277-85, 1997). The results are shown in
FIG. 4.
[0045] As shown in FIG. 4, oral intake of rifampicin starting when
the size of solid tumors reached 200 mm.sup.3 significantly
inhibited tumor growth in comparison with a group which did not
receive rifampicin.
EXAMPLE 5
Experience of Use in Patients with Hepatoma Associated with
Hepatitis C-Related Liver Cirrhosis
[0046] The present inventor specializing in liver diseases
administered rifampicin to patients with hepatitis C-related liver
cirrhosis associated with lung tuberculosis, and noted a rapid drop
in plasma .alpha.-fetoprotein levels. The present inventors further
noted that hepatoma never recurred during the long term despite the
repeated previous episodes of recurrence. Relationship between the
changes of plasma .alpha.-fetoprotein levels and rifampicin
administration in two patients is shown in FIG. 5.
EXAMPLE 6
Angiogenesis-Inhibiting Signals (The Changes of Gene Expression
Levels) Induced by Addition of Rifampicin, Rifamycin SV and
3-formyl Rifamycin
[0047] Human retinal microvascular endothelial cells were rendered
to grow exponentially in the presence of serum/growth
factors/angiogenesis-promoting factors, and various concentrations
of rifampicin, rifamycin SV and 3-formyl rifamycin were added.
After four hours-incubation, RNA was extracted and subjected to
mRNA quantification of various genes using aforementioned
LightCycler-based highly sensitive real-time quantitative PCR. The
results are shown in FIG. 6. A, B and C represent the
quantification results of c-myc gene; D, E and F represent those of
integrin-.alpha.v gene; G, H and I represent those of
integrin-.beta.3 gene. Further, A, D and G indicate the changes of
mRNA levels after adding rifampicin; B, E and H indicate those
after adding rifamycin SV; C, F and I indicate those after adding
3-formyl rifamycin. As shown in FIG. 6, angiogenesis-inhibiting
signals are markedly elicited by rifampicin in human retinal
microvascular endothelial cells as well, which constitute diabetic
retinopathy. Simultaneously, it is revealed that both rifamycin SV
and 3-formyl rifamycin, derivatives of rifampicin, exhibit similar
activity potently.
INDUSTRIAL APPLICABILITY
[0048] The present invention would provide angiogenesis inhibitors
having potent angiogenesis-inhibiting activity and used for wide
range of diseases including malignant tumor, diabetic retinopathy,
retinal angiogenesis, inflammatory diseases and cardiovascular
diseases, as an antitumor agent, a therapeutic agent for diabetic
retinopathy, retinal angiogenesis, inflammatory diseases, or
cardiovascular diseases, etc., respectively. Because rifampicin,
the active ingredient of the present invention, has been used as an
antibacterial medicine, its safety has been confirmed and methods
for production and administration established, therefore, the
angiogenesis inhibitors of the present invention are promising as
highly practical angiogenesis inhibitors. The present invention
also provides a method for screening a novel
angiogenesis-inhibiting substance which serves as an active
ingredient of angiogenesis inhibitors. The method for screening
angiogenesis-inhibiting substances of the present invention which
detects "angiogenesis-inhibiting signals" based on gene expression,
allows to efficiently detect substances having
angiogenesis-inhibiting activity among many substances whose action
is unknown. This screening method greatly shortens conventional
screening processes, and further, it makes it possible to estimate
dosages of peptides/proteins/drugs required to express the effect
comparable to that of the existing pharmaceutical, endostatin.
* * * * *