U.S. patent application number 12/203661 was filed with the patent office on 2009-01-01 for medicament comprising hgf gene.
This patent application is currently assigned to ANGES MG, INC.. Invention is credited to Ryuichi MORISHITA, Toshikazu Nakamura, Takahiro Ochi, Toshio Ogihara, Tetsuya Tomita.
Application Number | 20090004260 12/203661 |
Document ID | / |
Family ID | 26399523 |
Filed Date | 2009-01-01 |
United States Patent
Application |
20090004260 |
Kind Code |
A1 |
MORISHITA; Ryuichi ; et
al. |
January 1, 2009 |
MEDICAMENT COMPRISING HGF GENE
Abstract
The present invention relates to a method for treating
insufficiency of peripheral circulation or peripheral angiostenosis
in a subject for which HGF is effective, comprising administering
to the area affected by the insufficiency of peripheral circulation
or peripheral angiostenosis a therapeutically effective amount of
an expression vector containing a constitutive promoter operably
linked to a HGF coding sequence, whereby the HGF is expressed,
promoting the growth of vascular endothelial cells but not vascular
smooth muscle cells.
Inventors: |
MORISHITA; Ryuichi;
(Osaka-shi, JP) ; Ogihara; Toshio; (Mino-shi,
JP) ; Nakamura; Toshikazu; (Takatsuki-shi, JP)
; Tomita; Tetsuya; (Toyonaka-shi, JP) ; Ochi;
Takahiro; (Kobe-shi, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W., SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
ANGES MG, INC.
Toyonaka-shi
JP
|
Family ID: |
26399523 |
Appl. No.: |
12/203661 |
Filed: |
September 3, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10615262 |
Jul 9, 2003 |
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12203661 |
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09660522 |
Sep 12, 2000 |
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10615262 |
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09029497 |
Jun 9, 1998 |
6248722 |
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PCT/JP96/02359 |
Aug 22, 1996 |
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09660522 |
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Current U.S.
Class: |
424/450 ;
514/44R |
Current CPC
Class: |
A61K 9/1272 20130101;
A61P 9/10 20180101; A61P 9/00 20180101; A61K 48/00 20130101; A61K
38/1833 20130101; A61K 9/1271 20130101 |
Class at
Publication: |
424/450 ;
514/44 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/7088 20060101 A61K031/7088; A61P 9/00 20060101
A61P009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 29, 1995 |
JP |
07-245475 |
Feb 20, 1996 |
JP |
08-058467 |
Claims
1. A method for treating insufficiency of peripheral circulation or
peripheral angiostenosis in a subject for which HGF is effective,
comprising administering to the area affected by said insufficiency
of peripheral circulation or peripheral angiostenosis a
therapeutically effective amount of an expression vector containing
a constitutive promoter operably linked to a HGF coding sequence,
whereby said HGF is expressed, promoting the growth of vascular
endothelial cells but not vascular smooth muscle cells, thereby
treating the insufficiency of peripheral circulation or peripheral
angiostenosis.
2. The method of claim 1, wherein said expression vector is a
non-viral expression vector containing a HGF coding sequence.
3. The method of claim 2, wherein said non-viral expression vector
is encapsulated in a liposome.
4. The method of claim 3, wherein the membrane of said liposome is
fused with attenuated Sendai virus particles.
5. The method of claim 1, wherein said expression vector is a viral
expression vector containing a HGF coding sequence.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of application Ser. No. 10/615,262
filed Jul. 9, 2003, which is a Divisional Application of U.S.
application Ser. No. 09/660,522 filed Sep. 12, 2000 (abandoned),
which is a Continuation Application of U.S. application Ser. No.
09/029,497 filed Jun. 9, 1998 (U.S. Pat. No. 6,248,722, issued Jun.
19, 2001), which is a National Stage Application filed under
.sctn.371 of PCT Application No. PCT/JP1996/02359 filed Aug. 22,
1996. The entire disclosures of the prior applications, application
Ser. Nos. 10/615,262, 09/660,522, 09/029,497 and PCT/JP1996/02359
are considered part of the disclosure of the accompanying
continuation application and are hereby incorporated by
reference.
TECHNICAL FIELD TO WHICH THE INVENTION PERTAINS
[0002] The present invention relates to a medicament for use in
gene therapy and the like. More particularly, the present invention
relates to a medicament comprising a hepatocyte growth factor (HGF)
gene as well as a liposome containing the HGF gene.
PRIOR ART
[0003] HGF is a physiologically active peptide that exhibits
diverse pharmacological activities. The pharmacological activities
of HGF are described in, e.g., JIKKEN-IGAKU (Experimental
Medicine), Vol. 10, No. 3 (extra issue), 330-339 (1992). In view of
its pharmacological activities, HGF is expected to be useful as: a
drug for the treatment of cirrhosis of the liver or of renal
diseases; epithelial cell growth accelerators; anticancer agents;
agents for the prevention of side effects in cancer therapy; agents
for the treatment of lung disorders, gastrointestinal damages or
cranial nerve disorders; agents for the prevention of side effects
in immunosuppression; collagen degradation accelerators; agents for
the treatment of cartilage disorders, arterial diseases, pulmonary
fibrosis, hepatic diseases, blood coagulopathy, plasma
hypoproteinosis or wounds; agents for the improvement of nervous
disorders; hematopoietic stem cell potentiators; and hair growth
promoters (Japanese Patent KOKAI (Laid-Open) Nos. 4-18028 and
4-49246, EP 492614, Japanese Patent KOKAI (Laid-Open) No. 6-25010,
WO 93/8821, Japanese Patent KOKAI (Laid-Open) Nos. 6-172207,
7-89869 and 6-40934, WO 94/2165, Japanese Patent KOKAI (Laid-Open)
Nos. 6-40935, 6-56692 and 7-41429, WO 93/3061, Japanese Patent
KOKAI (Laid-Open) No. 5-213721, etc.).
[0004] As to gene therapy, extensive studies and investigations
recently have been made all over the world for treatment of
adenosine deaminase deficiency, AIDS, cancer, pustulous fibrosis or
hemophilia, etc.
[0005] However, gene therapy using HGF genes is unknown yet. It is
even unclear if such gene therapy will be effective.
Problems to be Solved by the Invention
[0006] HGF is known to be one of the drugs that have a short half
life in blood. As a result, persistent topical administration for
HGF would be desirable.
[0007] In view of the diverse pharmacological activities of HGF,
HGF is expected to be developed as a drug having extensive
applications to various diseases. On the other hand, when HGF is
systemically administered, side effects might be caused due to the
diverse pharmacological activities of HGF. In addition, when HGF
itself is intravenously administered, HGF has the drawback that a
considerable amount of HGF is retained in the liver, resulting in
reduction of the amount of HGF that reachs the target organ.
Means for Solving the Problems
[0008] The present invention has been made to solve the foregoing
problems. In summary, the present invention relates to:
[0009] (1) a medicament comprising a HGF gene;
[0010] (2) a liposome containing the HGF gene;
[0011] (3) a liposome according to (2), which is a membrane fusion
liposome fused to Sendai virus;
[0012] (4) a medicament comprising the liposome according to (2) or
(3);
[0013] (5) a medicament according to (1) or (4), for use in the
treatment of arterial disorders; and,
[0014] (6) a medicament according to (1) or (4), for use in the
treatment of cartilage injuries.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows expression of HGF in rat coronary endothelial
cells sensitized with the hemagglutinating virus of Japan
(HVJ)-liposome-DNA in Test Example 1.
[0016] In FIG. 2, the (line) graph shows cell growth rate in the
presence or absence of HGF from the HVJ-liposome-cont-sensitized
endothelial cells in Test Example 2, wherein "DSF" designates a
group of endothelial cells sensitized with HVJ-liposome-cont and
"HGF" designates a group of endothelial cells incubated in the
presence of recombinant human HGF at a predetermined concentration.
The bar in FIG. 2 shows the cell growth rate of the
HVJ-liposome-DNA-sensitized endothelial cells in Test Example 2,
wherein "DSF" designates a group of endothelial cells sensitized
with HVJ-liposome-cont and "HGF vector" designates a group of
endothelial cells sensitized with HVJ-liposome-DNA. (*:p<0.05
vs. DSF; **:p<0.01 vs. DSF; #:p<0.05 vs. human recombinant
HGF, 100 ng/ml.)
[0017] FIG. 3 shows the cell growth rate of endothelial cells
sensitized with HVJ-liposome-DNA in the presence or absence of
anti-HGF antibody in Test Example 2, wherein "control" represents a
group of HVJ-liposome-cont-sensitized endothelial cells incubated
in the presence of IgG control; "HGF" represents a group of
HVJ-liposome-DNA-sensitized endothelial cells incubated in the
presence of IgG control; and "HGFab" represents a group of
HVJ-liposome-DNA-sensitized endothelial cells incubated in the
presence of rabbit anti-human HGF antibody. The cell growth rate
(%) is expressed in terms of relative % when the growth rate in the
control group is set as 100.
[0018] FIG. 4 is a graph showing the cell growth effect of culture
supernatant from HVJ-liposome-DNA-sensitized rat vascular smooth
muscle cells (hereinafter often abbreviated as VSMCs) on rat
coronary endothelial cells in Test Example 3, wherein "control"
designates a group to which was added the culture supernatant from
the HVJ-liposome-cont-sensitized rat VSMCs, and "HGF" designates a
group to which was added culture supernatant from the
HVJ-liposome-DNA-sensitized rat VSMCs.
[0019] FIG. 5 is a graph showing the results from Test Example 3 in
which the concentration of HGF in the supernatant from incubated
HVJ-liposome-DNA-sensitized rat VSMCs was determined using
anti-human HGF antibody. In the figure, "no-treatment" represents a
group to which was added culture supernatant from non-sensitized
VSMCs; "control" represents a group to which was added supernatant
from the incubated HVJ-liposome-cont-sensitized rat VSMCs; and
"HGF" represents a group to which was added supernatant from the
incubated HVJ-liposome-DNA-sensitized rat VSMCs.
[0020] FIG. 6 is a graph showing the results from Test Example 3 in
which the concentration of HGF in the supernatant from the
incubated HVJ-liposome-DNA-sensitized rat VSMCs was determined
using anti-rat HGF antibody. In the figure, "no-treatment"
represents a group treated with the culture supernatant of
non-sensitized VSMCs; "control" designates a group treated with the
supernatant from the incubated HVJ-liposome-cont-sensitized rat
VSMCs; and "HGF" designates a group treated with the supernatant
from the incubated HVJ-liposome-DNA-sensitized rat VSMCs.
[0021] FIG. 7 is a graph showing the cell growth effect of the
supernatant from incubated HVJ-liposome-DNA-sensitized rat coronary
endothelial cells on rat coronary endothelial cells in Test Example
4, wherein A, B and C designate, respectively, a group to which was
added supernatant from incubated HVJ-liposome-DNA-sensitized rat
coronary endothelial cells, a group to which was added supernatant
from incubated HVJ-liposome-cont-sensitized rat coronary
endothelial cells, and a group of no-treatment animals.
[0022] FIG. 8 shows the cell growth effect of
HVJ-liposome-DNA-sensitized rat coronary endothelial cells on rat
coronary endothelial cells in the presence of an anti-HGF antibody
in Test Example 4. In the figure, A represents a group to which was
added supernatant from the incubated HVJ-liposome-DNA-sensitized
rat coronary endothelial cells; B represents a group to which was
added supernatant from the incubated HVJ-liposome-cont-sensitized
rat coronary endothelial cells; C represents a group to which was
added supernatant from the incubated HVJ-liposome-DNA-sensitized
rat coronary endothelial cells and wherein anti-HGF antibody was
added to the supernatant; and D represents a group to which was
added supernatant from the incubated HVJ-liposome-DNA-sensitized
rat coronary endothelial cells and wherein control antibody was
added to the supernatant.
[0023] FIG. 9 is a drawing showing the cell growth of endothelial
cells in Test Example 5 when HVJ-liposome-DNA-sensitized human
VSMCs were co-incubated with non-sensitized human endothelial
cells. In the figure, "control" represents a group of human
endothelial cells co-incubated with HVJ-liposome-cont-sensitized
VSMCs, and "HGF" represents a group of human endothelial cells
co-incubated with the supernatant from the incubated
HVJ-liposome-DNA-sensitized VSMCs.
[0024] FIG. 10 indicates the cell growth of endothelial cells in
Test Example 6 when HVJ-liposome-DNA-sensitized rat VSMCs were
co-incubated with non-sensitized rat coronary endothelial cells. In
the figure, "control" represents a group of human endothelial cells
co-incubated with the HVJ-liposome-cont-sensitized VSMCs, and "HGF"
represents a group of human endothelial cells co-incubated with the
culture supernatant from the HVJ-liposome-DNA-sensitized VSMCs.
[0025] FIG. 11 shows an increase in the number of minute blood
vessels in rat heart muscle directly injected with HVJ-liposome-DNA
in Test Example 8, wherein "HGF" denotes the number of minute blood
vessels in rat heart muscle directly injected with
HVJ-liposome-DNA, and "control" denotes the number of minute blood
vessels in rat heart muscle directly injected with
HVJ-liposome-cont.
[0026] FIG. 12 shows drawings at two different magnifications
showing that 3 weeks after administration of HVJ-liposome-DNA into
the joint, development of cartilage-like cells was noted in Test
Example 9, as evidenced by synthesis of Toluidine Blue-stained
proteoglycan.
[0027] FIG. 13 shows drawings at two different magnifications
showing that 4 weeks after administration of HVJ-liposome-DNA into
the joint, development of cartilage-like cells was noted in Test
Example 9, as evidenced by the synthesis of Toluidine Blue-stained
proteoglycan.
[0028] FIG. 14 shows drawings at two different magnifications
showing that even 4 weeks after administration of HVJ-liposome-DNA
(TGF-.beta.) prepared in Comparative Example 2 into the joint, such
development of cartilage-like cells as evidenced by the fact that
synthesis of Toluidine Blue-stained proteoglycan was not observed
in Test Example 9.
[0029] FIG. 15 shows drawings at two different magnifications
showing that even 4 weeks after administration of HVJ-liposome-cont
prepared in Comparative Example 1 into the joint, no such
development of cartilage-like cells as evidenced by synthesis of
Toluidine Blue-stained proteoglycan was observed in Test Example
9.
BEST MODE FOR CARRYING OUT THE INVENTION
[0030] The "HGF gene" employed in the present invention means a
gene capable of expressing HGF. Thus, so long as the polypeptide
expressed has substantially the same effect as that of HGF, the HGF
gene may have a partial deletion, substitution or insertion of the
nucleotide sequence, or may have other nucleotide sequences ligated
therewith at the 5'-terminus and/or 3'-terminus thereof. Typical
examples of such HGF genes include HGF genes as described in
Nature, 342, 440 (1989), Japanese Patent KOKAI (Laid-Open) No.
5-111383, Biohem. Biophys. Res. Commun., 163, 967 (1989), etc.
These genes may be used in the present invention.
[0031] The HGF gene is incorporated into an appropriate vector and
the HGF gene-bearing vector is provided for use. For example, the
HGF gene may be used in the form of a viral vector having the HGF
gene as described hereinafter, or in the form of an appropriate
expression vector having the HGF gene.
[0032] The "pharmaceutical composition" used in the present
invention means a medicament for the treatment or prevention of
human diseases, which is attributed to the pharmacological
activities of HGF. For example, exemplified are medicaments for the
treatment or prevention of the diseases given hereinabove.
[0033] According to the present invention, the HGF gene is
introduced into cells wherein HGF is expressed in those cells to
exhibit the pharmacological actions. Thus, the medicament of the
present invention is effectively applicable to the diseases for
which HGF itself is effective.
[0034] Where the HGF gene is introduced into, e.g., cells, the
growth of vascular endothelial cells is accelerated, while
undesired growth of vascular smooth muscle cells is not
accelerated, as demonstrated in the Examples hereinafter. Moreover,
as demonstrated in the Examples hereinafter, where the HGF gene is
introduced into the heart in in vivo animal tests using rats,
angiogenesis is observed. Therefore, the HGF gene is effective for
the treatment and prevention of arterial disorders, in particular,
various diseases caused by a disturbance which mainly involves
abnormal proliferation of vascular smooth muscle cells (e.g.,
restenosis after percutaneous transluminal coronary angioplasty
(PTCA), arteriosclerosis, insufficiency of peripheral circulation,
etc.), and for the treatment and prevention of diseases such as
myocardial infarction, cardiomyopathy, peripheral angiostenosis,
cardiac insufficiency, etc. HGF itself is also useful for the
treatment and prevention of the diseases as described above, since
HGF promotes the proliferation of vascular endothelial cells but
does not promote the growth of vascular smooth muscle cells. The
pharmacological effects of the HGF gene are attributed to those of
HGF itself.
[0035] As demonstrated in the Examples hereinafter, introduction of
the HGF gene into the joint results in promoting repair of
articular cartilage cells to thereby promote the proliferation of
proteoglycan-synthesizing cells. Therefore, the HGF gene is
effective for the prevention and treatment of various cartilage
injuries such as osteogenetic abnormality, arthritis deformans,
discopathy deformans, fracture repair and restoration
insufficiency, trauma caused by sports, key puncher's disease, etc.
HGF itself is useful for the treatment and prevention of the
diseases described above, since HGF promotes repair and growth of
cartilage cells. The effects of the HGF gene are based on those of
HGF itself.
[0036] A "liposome" is a closed vesicle of lipid bilayer
encapsulating an aqueous compartment. It is known that the lipid
bilayer membrane structure is extremely similar to biological
membranes. To prepare the liposomes of the present invention,
phospholipids are employed. Typical examples of phospholipids are
phosphatidylcholines such as lecithin, lysolecithin, etc.; acidic
phospholipids such as phosphatidylserine, phosphatidylglycerol,
phosphatidylinositol, phosphatidylic acid, etc.; or phospholipids
obtained by replacing an acyl group(s) of these acidic
phospholipids with lauroyl, myristoyl, oleoyl, etc.; and
sphingo-phospholipids such as phosphatidylethanolamine,
sphingomyelin, etc. Neutral lipids such as cholesterol may also be
added to these phospholipids. The liposomes may be prepared, in a
conventional manner, from naturally occurring materials such as
lipids in normal cell membranes. The liposomes containing the HGF
gene of the present invention may be prepared, for example, by
suspending a thin layer of purified phospholipids in a solution
containing the HGF gene and then treating the suspension in a
conventional manner, such as by ultrasonication.
[0037] The liposomes containing the HGF gene of the present
invention may be appropriately fused to viruses, etc. to form
membrane fusion liposomes. In this case, it is preferred to
inactivate viruses, e.g., through ultraviolet irradiation, etc. A
particularly preferred example of the membrane fusion liposome is a
membrane fusion liposome fused with Sendai virus (hemagglutinating
virus of Japan: HVJ). The membrane fusion liposome may be produced
by the methods described in NIKKEI Science, April, 1994, pages
32-38; J. Biol. Chem., 266 (6), 3361-3364 (1991), etc. In more
detail, the HVJ-fused liposome (HVJ-liposome) may be prepared,
e.g., by mixing purified HVJ inactivated by ultraviolet
irradiation, etc. with a liposome suspension containing the HGF
gene vector, gently agitating the mixture and then removing unbound
HVJ by sucrose density gradient centrifugation. The liposomes may
be bound to substances having an affinity to target cells, thereby
enhancing the efficiency of gene introduction into the target
cells. Examples of substances having an affinity for target cells
include ligands such as an antibody, a receptor, etc.
[0038] For introduction of the HGF gene into cells, conventional
methods are employed, which are roughly classified into
introduction via viral vectors and other strategies (NIKKEI
Science, April, 1994, pages 20-45; GEKKAN YAKUJI, 36 (1), 23-48
(1994) and references cited therein). Both types of methods can be
used for preparation of the medicament of the present
invention.
[0039] The former type of method using viral vectors comprises the
step of incorporating the HGF gene into, e.g., a retrovirus, an
adenovirus, an adeno-related virus, a herpes virus, a vaccinia
virus, a poliovirus, a sindbis virus or other RNA virus. Of these
viruses, a retrovirus, an adenovirus and an adeno-related virus are
particularly preferably employed for introduction.
[0040] Examples of the other methods include the liposome method,
lipofectin method, microinjection method, calcium phosphate method,
electroporation method. Of these methods, particularly preferred is
the liposome method.
[0041] For practical use of the HGF gene as a medicament, it is
advantageous to introduce the HGF gene directly into the body (in
vivo method). Alternatively, certain cells are collected from the
human, the HGF gene is then introduced into the cells outside the
body, and cells having the HGF gene introduced therein are returned
to the body (ex vivo method). These methods are described in NIKKEI
Science, April, 1994, pages 20-45; GEKKAN-YAKUJI, 36 (1), 23-48
(1994) and references cited therein. Any of these methods is
suitable, depending upon the disease to be treated, target organs,
etc. and may be applied to the medicament compositions of the
present invention.
[0042] The in vivo method is less costly, less laborious and
therefore more convenient than the ex vivo method, but the latter
method provides a higher efficiency of introduction of the HGF gene
into cells.
[0043] Where the medicament of the present invention is
administered by the in vivo method, the medicament may be
administered through any route appropriate for diseases to be
treated, such as via target organs, etc. The medicament may be
administered intravenously, intraarterially, subcutaneously,
intramuscularly, etc., or directly to the objective organ of the
disease, e.g., kidney, liver, lung, brain, nerve, etc. Direct
administration to the objective site can treat the target organ
selectively. For example, in gene therapy using a gene for
restenosis after PTCA, the composition may be administered
intraarterially (JIKKEN-IGAKU, 12 (extra issue 15), 1298-1933
(1994). Preferably, the medicament of the present invention is
applied at the tip of a balloon used for PTCA and the tip is rubbed
against a blood vessel, whereby the medicament may be introduced
directly into vascular endothelial cells and vascular smooth muscle
cells.
[0044] Where the ex vivo method as described above is used to
introduce the HGF gene, human cells (e.g., lymphocytes or
hematopoietic stem cells) are harvested in a conventional manner,
and the harvested cells are sensitized with the medicament of the
present invention for gene introduction. Thereafter the
HGF-producing cells are inserted back into the human.
[0045] Where the medicament is administered by the in vivo method,
the medicament may take various preparation forms, including the
form of a liquid preparation. In general, the medicament may be
preferably prepared as an injection comprising the HGF gene as an
active ingredient. If necessary and desired, conventional carriers
may be added to the composition. The injection may be prepared in a
conventional manner, e.g., by dissolving the HGF gene in an
appropriate solvent (e.g., sterilized water, a buffered solution, a
physiological saline solution, etc.), filtering the solution
through a filter, etc. for sterilization, filling up a sterile
container with the solution. The medicament may be prepared using
the HGF gene-containing viral vector, instead of the HGF gene
itself. Where the liposomes containing the HGF gene embedded
therein (or HVJ-liposomes) are employed, the medicament may be in
the form of liposome preparations such as a suspension, a frozen
preparation, a centrifugally concentrated frozen preparation,
etc.
[0046] The content of the HGF gene in the medicament may be
appropriately varied depending upon diseases to be treated, target
organs, patients' ages or body weights, etc. However, it is
appropriate to administer in a dose of 0.00001 mg to 100 mg,
preferably 0.001 mg to 10 mg when calculated as the HGF gene. The
dose may be divided into several days or a few months.
EXAMPLES
[0047] Hereinafter the present invention will be described in more
detail with reference to the examples but is not deemed to be
limited thereto. Materials and methods used in the following
examples are outlined below.
Materials and Methods
(1) HGF Expression Vector
[0048] The HGF expression vector was prepared by inserting human
HGF cDNA (2.2 kb, Biochem. Biophys. Res. Commun., 172, 321-327
(1990); Japanese Patent KOKAI (Laid-Open) No. 5-111383) between the
EcORI and NotI sites of a pUC-SR.alpha. expression vector (FEBS,
333, 61-66 (1993)). In this plasmid vector, transcription of HGF
cDNA is regulated by the SRa promoter (Nature, 342, 440-443
(1989)).
(2) Cell Culture
[0049] Rat coronary endothelial cells were isolated from
enzymatically digested heart of 8-week old Sprague-Dawley (SD) rats
by density gradient centrifugation (Transplantation, 57, 1653-1660
(1994)). Rat aortic vascular smooth muscle cells (VSMCs) were
obtained from 12 week old SD rats by enzymatic treatment (J. Clin.
Invest., 93, 355-360 (1994)). These cells were maintained in DMEM
medium supplemented with 10% (vol/vol) fetal calf serum, penicillin
(100 U/ml) and streptomycin (100 .mu./ml). The cells were incubated
at 37.degree. C. in a humidified 95% air-5% CO.sub.2 atmosphere.
The culture medium was routinely changed at 2 day-intervals. Both
immunopathological and morphological observation revealed that
these cells were endothelial cells and smooth muscle cells,
respectively.
[0050] Human aortic endothelial cells (five passages) and human
VSMCs (five passages) were obtained from Kurabo Co. The endothelial
cells were incubated in a manner similar to the above method in
MCDB131 medium supplemented with 5% fetal calf serum, epidermal
growth factor (10 ng/ml), basic fibroblast growth factor (2 ng/ml)
and dexamethasone (1 .mu.M).
[0051] Endothelial cells in the stationary state were prepared
according to the method described in J. Clin. Invest., 86,
1690-1697 (1990), ibid., 94, 824-829 (1994).
(3) Transfection of the HGF Gene Into HVJ-Liposomes In Vitro
[0052] Endothelial cells or VSMCs were inoculated for sensitization
on a 6-well plate in a cell count of 10.sup.6 and proliferated to
reach 80% confluence. The cells were washed 3 times with a balanced
salt solution (137 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH 7.6;
hereinafter abbreviated as "BSS") supplemented with 2 mM calcium
chloride. To the cells was added 1 ml of a solution of the
HVJ-liposome-DNA (containing 2.5 mg of lipids and 10 mg of the
embedded DNA) obtained in Example 1 hereinafter or 1 ml of a
solution of the HVJ-liposome-cont obtained in Comparative Example 1
hereinafter. The resulting mixture was incubated at 4.degree. C.
for 5 minutes and at 37.degree. C. for a further 30 minutes. The
cells were washed and maintained in a fresh medium containing 10%
bovine serum in a CO.sub.2 incubator.
(4) Assay for the HGF Concentrations in Endothelial Cells and
VSMCs
[0053] The concentration of HGF produced from the sensitized
endothelial cells and VSMCs was assayed by ELISA. That is, rat or
human endothelial cells or VSMCs were inoculated on a 6-well plate
(made by Corning) in a cell density of 5.times.10.sup.4
cells/cm.sup.2, followed by incubation for 24 hours. The medium was
replenished 24 hours after the sensitization, and incubation was
continued for a further 48 hours. To investigate if HGF was
released, the sensitized cells (48 hours after sensitization) were
washed and added to 1 ml of a serum-free medium containing
5.times.10.sup.-7 M insulin, 5 .mu.g/ml transferrin and 0.2 mM
ascorbate. After 24 hours, the culture media was collected,
centrifuged at 600 g for 10 minutes and then stored at -20.degree.
C.
[0054] The HGF concentration in the media was determined by an
enzyme immunoassay using an anti-rat HGF antibody or an anti-human
HGF antibody (Exp. Cell Res., 210, 326-335 (1994); Jpn. J. Cancer
Res., 83, 1262-1266 (1992)). A rabbit anti-rat or an anti-human HGF
IgG was coated onto a 96-well plate (made by Corning) at 4.degree.
C. for 15 hours. After blocking with 3% bovine serum
albumin-containing PBS (phosphate buffered saline), the culture
medium was added to each well, and incubation was performed at
25.degree. C. for 2 hours. After washing each well 3 times with PBS
containing 0.025% Tween (PBS-Tween), a biotinated rabbit anti-rat
HGF IgG or an anti-human HGF IgG was added to each well followed by
incubation at 25.degree. C. for 2 hours. After washing with
PBS-Tween, each well was incubated together with horse radish
peroxidase-bound streptoavidin-biotin complex (PBS-Tween solution).
The enzymatic reaction was initiated by adding thereto a substrate
solution (containing 2.5 mM o-phenylenediamine, 100 mM sodium
phosphate, 50 mM citrate and 0.015% hydrogen peroxide). The
reaction was terminated by adding 1 M sulfuric acid to the system.
Absorbance was measured at 490 nm. The anti-human HGF antibody is
reactive only with human HGF, and not with rat HGF. The anti-rat
HGF antibody is reactive solely with rat HGF, and not with human
HGF.
(5) HGF
[0055] The human and rat recombinant HGFs employed were purified
from the culture solution of CHO cells or C-127 cells transfected
with an expression plasmid bearing human or rat HGF cDNA (Cell, 77,
261-271 (1994); J. Clin. Invest., 93, 355-360 (1994)).
(6) Statistical Analysis
[0056] All runs were repeated at least 3 times. Data measured are
shown by mean.+-.standard error. Statistical analysis of the
measured data was made according to Duncan's test.
(7) Hematoxylin-Eosin (HE) Staining and Azan Staining
[0057] Ten days after the gene introduction, the rats having the
HGF gene introduced therein were sacrificed by perfusion with
heparinized physiological saline. Fixation was then made overnight
with a 4% paraformaldehyde PBS solution. After fixation, the tissue
was embedded in paraffin. Slides were prepared and stained with HE
and Azan in a conventional manner. The slides were examined on a
microscope to count the number of microvessels.
Example 1
Preparation of HVJ-Liposomes Containing the HGF Expression
Vector
[0058] Phosphatidylserine, phosphatidylcholine and cholesterol were
mixed with tetrahydrofuran in a weight ratio of 1:4.8:2. By
distilling tetrahydrofuran off through a rotary evaporator, the
lipid mixture (10 mg) was precipitated onto the container wall.
After 96 .mu.g of high mobility group (HMG) 1 nuclear protein
purified from bovine thymus was mixed with a BBS (200 .mu.l)
solution of plasmid DNA (300 .mu.g) at 20.degree. C. for an hour,
the mixture was added to the lipid mixture obtained above. The
resulting liposome-DNA-HMG 1 complex suspension was mixed with a
vortex, ultrasonicated for 3 seconds and then agitated for 30
minutes.
[0059] The purified HVJ (strain Z) was inactivated by UV
irradiation (110 erg/mm.sup.2 sec) for 3 minutes immediately before
use. BSS was added to and mixed with the liposome suspension (0.5
ml, containing 10 mg of the lipids) obtained above and HVJ (20,000
hemagglutinating units) to make the total volume 4 ml. The mixture
was incubated at 4.degree. C. for 10 minutes and gently agitated at
37.degree. C. for a further 30 minutes. The unreacted HVJ was
removed from the HVJ-liposomes by sucrose density gradient
centrifugation. That is, the upper layers in the sucrose density
gradient were collected to give the HVJ-liposomes containing the
HGF expression vector (containing 10 .mu.g/ml of the HGF expression
vector). The HVJ-liposomes containing the HGF expression vector is
hereinafter often referred to as HVJ-liposome-DNA.
Example 2
Administration of the HVJ-Liposome Containing the HGF Expression
Vector To Rats
[0060] The HVJ-liposomes containing the HGF expression vector were
prepared by the method described in the above Example, using 64
.mu.g of HMG 1 nuclear protein and 200 g of plasmid DNA. BSS was
added to and mixed with the liposome suspension (0.5 ml, containing
10 mg of the lipids) and HVJ (35,000 hemagglutinating units) to
make the whole volume 2 ml.
[0061] SD rats (weighing 400-500 g; purchased from Japan Charles
River) were anesthetized with intraperitoneal administration of
sodium pentobarbital (0.1 ml/100 mg), warmed and breathing was
maintained with an automated breather. The rats were subjected to
thoracotomy on the left side. The HVJ-liposome-DNA or
HVJ-liposome-cont (20 .mu.l) was carefully injected directly
through the cardiac apex using a 30 G syringe.
Comparative Example 1
Preparation of HVJ-Liposomes Containing No HGF Expression
Vector
[0062] A vector bearing no HGF gene was treated in the same manner
as described in Example 1 to prepare the HVJ-liposomes containing
no HGF expression vector. The HGF expression vector-free
HVJ-liposomes are hereinafter referred to as HVJ-liposome-cont.
Comparative Example 2
Preparation of HVJ-Liposomes Containing Human TGF-.beta. Expression
Vector
[0063] HVJ-liposomes containing human TGF-.beta. expression vector
were prepared in a manner similar to that described in Example 1
except for using a human TGF-.beta. expression vector.
[0064] The HVJ-liposomes containing human TGF-expression vector are
hereinafter referred to as HVJ-liposome-DNA (TGF-.beta.).
Test Example 1
Expression of HGF in Rat Coronary Endothelial Cells Sensitized With
HVJ-Liposome-DNA
[0065] HVJ-liposome-DNA (concentration of the HGF expression vector
in liposomes: 10 .mu.g/ml) was sensitized to rat coronary
endothelial cells (cell count: 10.sup.6). HGF production was
determined by ELISA. For control, a similar test was conducted
using HVJ-liposome-cont. HGF production was also determined on the
non-sensitized rat coronary endothelial cells (no-treatment group).
The results are shown in FIG. 1 (n=6), wherein "HGF" represents the
group of rat coronary endothelial cells sensitized with
HVJ-liposome-DNA.
[0066] As shown in FIG. 1, the rat coronary endothelial cells
sensitized with HVJ-liposome-DNA produced and secreted HGF at a
high level. On the other hand, HGF production was not substantially
observed either in the intact group or in the group of rat coronary
endothelial cells sensitized with HVJ-liposome-cont.
[0067] Cell counting in the groups tested revealed that the HGF
expression group showed significantly high cell counts.
Test Example 2
Effects of the Sensitized HGF Expression Vector on Proliferation of
Endothelial Cells
[0068] Human endothelial cells were sensitized with
HVJ-liposome-cont. The sensitized cells were incubated in the
presence or absence of exogenously added recombinant human HGF (1,
10 and 100 ng/ml) and the cell growth rate (%) was determined. The
results are shown in FIG. 2 ((line) graph, n=6), wherein "DSF"
represents the group of endothelial cells sensitized with
HVJ-liposome-cont and "HGF" represents the group of endothelial
cells incubated in the presence of recombinant human HGF in a
definite concentration (*:P<0.05, **:P<0.01 for DSF).
[0069] The (line) graph shown in FIG. 2 reveals that the growth of
endothelial cells is promoted by the exogenously added HGF.
[0070] The endothelial cells sensitized with HVJ-liposome-DNA
(concentration: 10 .mu.g/ml) were similarly incubated, the increase
in the number of cells was determined, and the cell growth rate (%)
was calculated. For a control, endothelial cells sensitized with
HVJ-liposome-cont were also incubated, the increase in the number
of cells was determined, and the cell growth rate (%) was
calculated. The results are shown in FIG. 2 (bar, n=6), wherein
"DSF" designates the group of endothelial cells sensitized with
HVJ-liposome-cont and "HGF" designates the group of endothelial
cells sensitized with HVJ-liposome-DNA (* *:P<0.01 for DSF,
#:P<0.05 for HGF, 100 ng/ml).
[0071] As is noted from the bar shown in FIG. 2, the results reveal
that the cell growth rate of the HVJ-liposome-DNA-sensitized
endothelial cells is markedly higher than that of the control group
and significantly high even when compared to that of cells with
exogenously added HGF.
[0072] The aforesaid endothelial cells sensitized with
HVJ-liposome-DNA were incubated in the presence or absence of a
rabbit anti-human HGF antibody. The increase in the number of cells
was determined, and the cell growth rate was calculated. For a
control, the endothelial cells sensitized with HVJ-liposome-cont
were incubated, and the increase in the number of cells was
determined in a similar manner in order to calculate the cell
growth rate. The rabbit anti-human HGF antibody (10 .mu.g/ml) was
purified by the method described in Jpn. J. Cancer Res., 83,
1262-1266 (1992). This antibody is capable of neutralizing the
biological activity of 10 ng/ml at a concentration of 10 .mu.g/ml.
The anti-human HGF antibody reacts only with human HGF and not with
rat HGF, whereas the anti-rat HGF antibody reacts only with rat HGF
and not with human HGF. Normal rabbit serum IgG (10 .mu.g/ml) was
used for the control.
[0073] The results are shown in FIG. 3 (n=6), wherein "control"
designates the group of HVJ-liposome-cont-sensitized endothelial
cells incubated in the presence of IgG control; "HGF" designates
the group of HVJ-liposome-DNA-sensitized endothelial cells
incubated in the presence of IgG control; and "HGFab" designates
the group of HVJ-liposome-DNA-sensitized endothelial cells
incubated in the presence of the rabbit anti-human HGF antibody.
The cell growth rate (%) is expressed in terms of relative % when
the growth rate in the control group is made 100 (*:P<0.01 for
the control group, #:P<0.05 for HGF). As shown in FIG. 3, the
growth of HVJ-liposome-DNA-sensitized endothelial cells was
arrested in the presence of anti-human HGF antibody, and the cell
growth rate was thus substantially the same as that of the control
group. These results clearly demonstrate that HGF is the growth
factor of endothelial cells.
Test Example 3
Effects of the Supernatant of the Incubated
HVJ-Liposome-DNA-Sensitized Rat VSMCs on Rat Coronary Endothelial
Cells
[0074] The supernatant from the incubated
HVJ-liposome-DNA-sensitized rat VSMCs was added to the rat coronary
endothelial cell culture system (cell count: 10.sup.5) during the
stationary phase. After incubation for 3 days, the increase in the
number of endothelial cells was determined. For a control, the
supernatant of the incubated HVJ-liposome-cont-sensitized rat VSMCs
was added to the rat coronary endothelial cells in a similar manner
to that described above, and the increase in the number of
endothelial cells was determined as described above. The results
are shown in FIG. 4 (n=6), wherein "control" indicates the group to
which was added the supernatant from the incubated
HVJ-liposome-cont-sensitized rat VSMCs, and "HGF" represents the
group to which was added the supernatant of the incubated
HVJ-liposome-DNA-sensitized rat VSMCs.
[0075] As shown in FIG. 4, a significant increase in the number of
endothelial cells was observed in the group to which was added the
supernatant of the incubated HVJ-liposome-DNA-sensitized rat
VSMCs.
[0076] The concentration of HGF in the culture supernatant of the
rat VSMCs sensitized with HVJ-liposome-DNA or HVJ-liposome-cont as
described above was assayed by ELISA using an anti-human HGF
antibody and an anti-rat HGF antibody. The HGF concentration in the
culture supernatant of non-sensitized VSMCs was also assayed
(no-treatment group).
[0077] The results obtained using the anti-human HGF antibody and
the anti-rat HGF antibody are shown in FIGS. 5 and 6, respectively
(n=6 in both tests). In the figures, "control" represents the group
to which was added the supernatant from the incubated
HVJ-liposome-cont-sensitized rat VSMCs; and "HGF" represents the
group to which was added the supernatant from the incubated
HVJ-liposome-DNA-sensitized rat VSMCs.
[0078] As shown in FIG. 5, HGF was detected in the supernatant of
the HVJ-liposome-DNA-sensitized rat VSMCs, and the HGF
concentration was significantly higher than that of the control
group.
[0079] FIG. 6 also reveals that rat HGF was further detected in the
supernatant of the HVJ-liposome-DNA-sensitized rat VSMCs, and the
HGF concentration was significantly higher than that of the control
group.
[0080] As observed in FIGS. 5 and 6, no HGF was present in an
amount detectable by ELISA, in both the supernatants of the intact
group and the control group.
Test Example 4
Effects of the Supernatant From the Incubated
HVJ-Liposome-DNA-Sensitized Rat Coronary Endothelial Cells on Rat
Coronary Endothelial Cells
[0081] The supernatant of the incubated HVJ-liposome-DNA-sensitized
rat coronary endothelial cells was added to the rat coronary
endothelial cell culture system (cell count: 10.sup.5) during the
stationary phase. After incubation for 3 days, the increase in the
number of endothelial cells was examined. For the control, the
endothelial cells were incubated in a similar manner, using culture
supernatant from the HVJ-liposome-cont-sensitized rat coronary
endothelial cells, and the increase in the number of endothelial
cells was determined. The results are shown in FIG. 7, wherein A, B
and C represent, respectively, the group to which was added the
culture supernatant of the HVJ-liposome-DNA-sensitized rat coronary
endothelial cells (n=8), the group to which was added the culture
supernatant of the HVJ-liposome-cont-sensitized rat coronary
endothelial cells (n=8), and the no-treatment group (n=15).
[0082] As shown in FIG. 7, a significant increase in the number of
endothelial cells was observed in the group to which was added the
culture supernatant of the HVJ-liposome-DNA-sensitized rat coronary
endothelial cells, whereas in the control group, the cell count was
almost the same as that of the no-treatment group (control group:
0.117.+-.0.002, group A: 0.148.+-.0.03, P<0.01).
[0083] Next, anti-HGF antibody was added to the culture supernatant
of the HVJ-liposome-DNA-sensitized rat coronary endothelial cells.
The increase in the number of endothelial cells was determined as
described above. The results are shown in FIG. 8 (n=8), wherein A
represents the group to which was added the culture supernatant of
the HVJ-liposome-DNA-sensitized rat coronary endothelial cells; B
represents the group to which was added the culture supernatant of
the HVJ-liposome-cont-sensitized rat coronary endothelial cells; C
represents the group to which was added the culture supernatant of
the HVJ-liposome-DNA-sensitized rat coronary endothelial cells and
anti-HGF antibody; and D represents the group to which was added
the supernatant from the incubated HVJ-liposome-DNA-sensitized rat
coronary endothelial cells and a control antibody.
[0084] As shown in FIG. 8, A and C, the cell growth promoting
activity of the culture supernatant of the
HVJ-liposome-DNA-sensitized rat coronary endothelial cells
completely disappeared by adding the anti-HGF antibody thereto. The
results reveal that the cell growth promoting activity of the
culture supernatant of the HVJ-liposome-DNA-sensitized rat coronary
endothelial cells is attributable to HGF.
Test Example 5
Effects of HVJ-Liposome-DNA-Sensitized Human VSMCs on Human
Endothelial Cells
[0085] Human VSMCs were inoculated on a cell culture insert
(manufactured by Coaster, pore diameter of 0.45 .mu.m), and then
grown in DMEM medium supplemented with 10% bovine serum. On the
other hand, human endothelial cells were inoculated on a 6-well
plate and maintained in DMEM medium supplemented with 10% bovine
serum. When the VSMCs reached 80% confluence, the VSMCs were
incubated at 4.degree. C. for 5 minutes and at 37.degree. C. for 30
minutes together with HVJ-liposome-DNA (DNA content in the
liposomes: 10 .mu.g) or with HVJ-liposome-cont. After
sensitization, the insert containing the sensitized VSMCs was added
to each well containing human endothelial cells in the stationary
phase. VSMCs and the endothelial cells were co-incubated for 3 days
in DMEM medium supplemented with 0.5% bovine serum. Thereafter the
cell count was determined with a WST-cell counter kit (manufactured
by Wako Co.). The results are shown in FIG. 9 (n=6). In the figure,
"control" represents the group co-incubated with the
HVJ-liposome-cont-sensitized VSMCs, and "HGF" represents the group
co-incubated with the HVJ-liposome-DNA-sensitized VSMCs.
[0086] The results shown in FIG. 9 reveal that human VSMCs
sensitized with HVJ-liposome-DNA could significantly increase the
growth of non-sensitized human endothelial cells in the stationary
phase.
Test Example 6
Effects of the HVJ-Liposome-DNA-Sensitized Rat VSMCs on Rat
Coronary Endothelial Cells
[0087] The HVJ-liposome-DNA-sensitized rat VSMCs (cell count:
10.sup.6) were co-incubated for 3 days with rat coronary
endothelial cells (cell count: 10.sup.5) in the stationary phase.
Thereafter, the increase in the number of endothelial cells was
determined. For the control, endothelial cells were co-incubated in
a similar manner using the HVJ-liposome-cont-sensitized rat VSMCs,
and the increase in the number of endothelial cells was determined.
The results are shown in FIG. 10 (n=6), wherein "control"
represents the rat VSMCs group sensitized with the
HVJ-liposome-DNA, and "HGF" represents the rat VSMCs group
sensitized with HVJ-liposome-cont.
[0088] As shown in FIG. 10, the growth of the endothelial cells was
stimulated by HGF released from the HVJ-liposome-DNA-sensitized rat
VSMCs, and an increase in the cell count was observed (control
group: 0.126.+-.0.006, HGF group: 0.156.+-.0.01, P<0.05).
Test Example 7
Growth of Rat VSMCs Sensitized With HVJ-Liposome-DNA
[0089] Rat VSMCs sensitized with HVJ-liposome-DNA and rat VSMCs
sensitized with HVJ-liposome-cont were incubated, respectively, to
compare the increase in the cell counts therebetween. Sensitization
with HVJ-liposome-DNA did not affect cell growth at all. The
results reveal that HGF has no cell growth promoting effect on
VSMCs.
Test Example 8
Induction of Angiogenesis in Rat Heart Muscle Directly Injected
With HVJ-Liposome-DNA
[0090] Rat heart muscle directly injected with HVJ-liposome-DNA,
rat heart muscle directly injected with HVJ-liposome-cont and rat
no-treatment heart muscle were stained with HE and Azan,
respectively, and examined on a microscope to count the number of
microvessels. The results are shown in FIG. 11, wherein "HGF"
designates the number of microvessels in rat heart muscle directly
injected with HVJ-liposome-DNA, and "control" designates the number
of microvessels in rat heart muscle directly injected with
HVJ-liposome-cont.
[0091] As is seen from FIG. 11, the number of minute blood vessels
significantly increased in the rat heart muscle injected with
HVJ-liposome-DNA, as compared to that in the rat heart muscle
injected with HVJ-liposome-cont and the rat no-treatment heart
muscle. These results indicate that HGF which is capable of causing
endothelial cells to grow, exhibits angiogenesis activity in
vivo.
Test Example 9
Repair of Articular Cartilage by Directly Introducing
HVJ-Liposome-DNA Into the Joint
[0092] Ten week-old Fischer's rats were injured at the femoral
intercondylaris through the subcartilage using Kirschner's wires of
1.8 mm in diameter. One week after the operation, the
HVJ-liposome-DNA (100 .mu.l/knee) prepared in Example 1 was
introduced directly into the joint. For the control, the
HVJ-liposome-cont prepared in Comparative Example 1 and
HVJ-liposome-DNA (TGF-.beta.) prepared in Comparative Example 2
were administered directly into the joint in the same amount. Rats
were sacrificed 1, 3 and 4 weeks after introduction of these genes,
etc. and the histology of the repaired sites was observed.
[0093] As shown in FIG. 12, the results indicate that the synthesis
of proteoglycan stained with Toluidine Blue was observed 3 weeks
after administration of the HVJ-liposome-DNA into the joint,
displaying the development of cartilage-like cells. Furthermore, as
shown in FIG. 13, 4 weeks after administration of the
HVJ-liposome-DNA into the joint, there was observed a tendency to
further extend the area of developing cartilage-like cells, in
which the synthesis of proteoglycan was observed.
[0094] As shown in FIG. 14, where the HVJ-liposome-DNA (TGF-.beta.)
prepared in Comparative Example 2 was injected into the joint,
development of such cartilage-like cells was not observed even 4
weeks after administration. Furthermore, as shown in FIG. 15, where
the HVJ-liposome-cont prepared in Comparative Example 1 was
injected into the joint, development of such cartilage-like cells
was not observed even 4 weeks after administration.
INDUSTRIAL APPLICABILITY
[0095] The medicament of the present invention provides persistent
therapeutic effects, as compared to HGF itself. Moreover, the
medicament of the present invention may be topically applied to the
target organs so that the effects can be selectively exhibited,
resulting in minimizing the side effects of HGF.
* * * * *