U.S. patent application number 13/393062 was filed with the patent office on 2012-06-21 for drug composition for angiogenesis therapy.
Invention is credited to Keiichi Hukuda, Takashi Kawakami, Tadashi Tanabe.
Application Number | 20120156170 13/393062 |
Document ID | / |
Family ID | 43627595 |
Filed Date | 2012-06-21 |
United States Patent
Application |
20120156170 |
Kind Code |
A1 |
Tanabe; Tadashi ; et
al. |
June 21, 2012 |
DRUG COMPOSITION FOR ANGIOGENESIS THERAPY
Abstract
The present invention relates to: drug compositions of
angiogenesis therapy that contains gene coding for human
prostacyclin synthase (hPGIS) synthesizing prostaglandin I.sub.2
with activities of vasodialation and/or anti-platelet aggregation;
drug compositions contain adeno-associated virus (AAV) inserted
with gene for angiogenesis factors. The administration of the drug
compositions into the aimed treatment region results in transfer of
AAV type 1-hPGIS to skeletal muscles and induces a notable
expression of human PGIS gene in skeletal muscles. The PGI.sub.2 is
produced by mediation of the gene expression in the muscle cells,
secreted, induces vessel-protective, neovascularization and
anti-platelet aggregation actions, which lead to an improvement in
vascular ischemia.
Inventors: |
Tanabe; Tadashi; (Osaka,
JP) ; Hukuda; Keiichi; (Tokyo, JP) ; Kawakami;
Takashi; (Tokyo, JP) |
Family ID: |
43627595 |
Appl. No.: |
13/393062 |
Filed: |
August 30, 2010 |
PCT Filed: |
August 30, 2010 |
PCT NO: |
PCT/JP2010/005321 |
371 Date: |
February 28, 2012 |
Current U.S.
Class: |
424/93.2 |
Current CPC
Class: |
A61K 38/1866 20130101;
A61K 48/005 20130101; A61P 9/00 20180101; C12N 2750/14132 20130101;
C12N 2799/025 20130101; C12N 2750/14143 20130101; C12N 7/00
20130101; A61K 45/06 20130101; A61K 38/52 20130101; A61K 31/5585
20130101; A61P 9/08 20180101; A61K 38/1833 20130101; A61P 9/10
20180101; A61K 35/76 20130101; A61P 7/02 20180101; A61P 43/00
20180101; A61K 38/1825 20130101; A61K 35/13 20130101; C12Y
503/99004 20130101; A61P 35/00 20180101; A61K 38/1866 20130101;
A61K 2300/00 20130101; A61K 38/1825 20130101; A61K 2300/00
20130101; A61K 38/1833 20130101; A61K 2300/00 20130101; A61K 38/52
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/93.2 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61P 7/02 20060101 A61P007/02; A61P 9/08 20060101
A61P009/08; A61P 35/00 20060101 A61P035/00; A61P 9/00 20060101
A61P009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 31, 2009 |
JP |
2009-200900 |
Claims
1. A drug composition of angiogenesis therapy used for treatment
and prevention of peripheral arterial disease comprising; an active
component, an adeno-associated virus (AAA) inserted with a PGIS
gene producing a prostaglandin I2 (PGI2) which induces one or more
of the activities selected from the group consisting of
vasodialation, anti-platelet aggregation and angiogenesis.
2. The drug composition of angiogenesis therapy according to claim
1, wherein the adeno-associated virus (AAV) is further inserted
with a gene coding for an angiogenesis factor.
3. A drug composition of angiogenesis therapy used for treatment
and prevention of peripheral arterial disease comprising; an active
component, a first adeno-associated virus (AAV) inserted with a
PGIS gene producing a prostaglandin I2 (PGI2) which induces one or
more of the activities selected from the group consisting of
vasodialation, anti-platelet aggregation and angiogenesis, and a
second adeno-associated virus (AAV) inserted with a gene coding for
an angiogenesis factor.
4. The drug composition of angiogenesis therapy according to any of
claims 1 to 3, wherein the adeno-associated virus (AAV) is of the
type selected from the group consisting of type 1, 2, 5 and 8.
5. The drug composition of angiogenesis therapy according to any of
claims 1 to 3, wherein the adeno-associated virus (AAV) is of the
type selected from the group consisting of type 1 and 2.
6. The drug composition of angiogenesis therapy according to any of
claims 2 to 3, wherein the gene coding for an angiogenesis factor
is a gene coding for a material selected from the group consisting
of a protein, a peptide and a part thereof capable of inducing
angiogenesis.
7. The drug composition of angiogenesis therapy according to any of
claims 1, wherein the gene coding for an angiogenesis factor is a
vascular endotherial growth factor (VEGF).
8. The drug composition of angiogenesis therapy according to any of
claims 1, further comprising a pharmaceutically permissible carrier
containing the adeno-associated virus (AVV).
Description
BACKGROUND
[0001] The present invention relates to reinforcing materials for
angiogenesis activities, which are useful pharmaceutical
compositions for angiogenesis therapy
[0002] Western life-style and aging society of Japan yearly cause
an marked increase in number of patients suffering from peripheral
arterial disease (PAD), which is based on arteriosclerosis brought
by risk factors such as aging, smoking, diabetes, hypertension and
metabolic disorders of lipids. According to the report from
American Heart Association, furthermore, popularization of
noninvasive examination tests for PAD detected a contract rate of
about 2-3% below age of 50 years and that of about 20% over 75
years old. And approximately about 40% PAD patients are known to
show no indications.
[0003] Presently, there are, as therapeutic methods for PAD,
treatments with drugs such as anti-platelets, vasodilator, and
prostaglandins, or with vascular stent, and surgical bypass
operation. However, effects by therapy with drugs are limited, and
treatment with vascular stent or surgical bypass operation, the
first choice of treatment for serious patients, are not always
adopted. As PAD resistant to known therapeutic methods critical
limb ischemia (CLI) is known. CLI is defined as the state
accompanying chronic ischemic pain at rest and ischemic skin
disorder (ulcer and gangrene). Prognosis of CLI is gloomy. At one
year after the onset of CLI it is known that 25% of patients lose a
leg(s) and 25% of patients are dead.
[0004] In recent clinical research various cell therapy and gene
therapy as neovascular treatments of PAD. Bone marrow mononuclear
cells and peripheral mononuclear cells are used for the cell
therapy. Recombinant angiogenesis factors are used for the gene
therapy. These factors used for the gene therapy, for example, are
vascular endotherial growth factor (VEGF), fibroblast growth factor
(FGF), and hepatic growth factor (HGF). These genes for
angiogenesis factors enhances angiopoiesis in ischemic tissues by
expressing these factors. And by the use of these factors it was
possibly ascertained that development of collateral blood vessels
in model animals of heart or limb ischemia is promoted and/or
enhanced (Circulation 90, 11-228-11-234 (1994).
[0005] Therapy for vasculopathy with above-mentioned angiogenesis
factors is designated as "angiogenesis therapy". More recently,
using gene for above-mentioned angiogenesis factors therapeutic
angiogenesis studies in ischemic and arterial diseases have been
actively carried out.
[0006] Furthermore, a drug composition of angiogenesis therapy is
one described in WO2002/000258 (patent reference 1). The drug
composition of angiogenesis therapy mentioned in patent reference 1
is the one containing gene for HGF or VEGF and prostacyclin
synthase gene as effective components.
CITATION LIST
Patent Literature
[0007] PLT 1: WO2002/000258
SUMMARY
Technical Problem
[0008] By the way, the above-mentioned therapy using angiogenesis
factors are not effective in all cases of PAD and their and
long-term effectiveness and noxious events are also not known.
Especially, the incident and/or growth of tumors caused by the gene
therapy with growth factors can't be negligible. Therefore,
developments of new therapy have been desired for solving the
above-mentioned problems.
[0009] In pathologic state vascular endothelium is injured and
decreases in vasodialators such as nitric oxide, prostaglandin,
endothelium-derived hyperpolarization factor (EDHF) and VEGF, and
increases in vasoconstrictors, angoutensin-2, endothelin-1 etc.
these vascular factors are can be useful for the treatment of PAD.
Actually prostanoid is used for the clinical treatment of PAD and
pulmonary arterial hypertention.
[0010] Therefore, the present inventors gave attention to
prostacyclin synthase (PGIS), the synthase for prostacyclin
(prostaglandin I.sub.2) with activities of anti-platelet
aggregation, vasodialation, and angiogenesis.
[0011] PGIS gene is a gene encoding an enzyme catalyzing the
bio-synthesis of a remedy showing high effectiveness in treatment
of experimental models of pulmonary hypertension and
endoterium-injured arterioscrelosis. The metabite shows activities
inducing anti-platelet aggregation, vasodialation, and
angiogenesis. The gene therapy raises a long-term expression of the
gene in local lesion, so that noxious side effects by the products
always become issues. Clinical research with growth factors is
possibly accompanied by occurrence and development of tumors. In
the clinical cases with PGI.sub.2 drugs, however, there has been no
report on tumorigenesis, and the safety of PGI.sub.2 drugs is high
because of their vasodialation and vaso-protective activities.
Similarly, adeno-associated virus vector is judged to be the most
suitable vector for the gene therapy due to the safe and active
gene expression maintained for a relatively long-time.
[0012] In above-mentioned clinical research on critical limb
ischemia plasmid or aveno virus are used as an expression vector.
It is, however, with plasmid vectors that the strong and long gene
expression is difficult to be obtained and that the frequent
administration is necessary to maintain the expression level.
Although a strong gene expression is obtained with the adenovirus
vector, it has a short effect and the strong immunogenicity, and
shows some concern about its safety. Conventionally, virus vectors
have high transgenic activities, but their pathogenicities are
often discussed in the human use. On the other hand AAV vectors
deserve the attention because of a high efficiency and long-term
expression in nondividing cells such as muscle and nerve cells, and
their nonpathogenicity to human.
[0013] Thus, present inventors, using AAV as a gene transfer
vector, carried out the gene transfer in skeletal muscle of human
prostacyclin synthase synthesizing prostaglandin I.sub.2 (PG
I.sub.2) resulting in improvement of the condition of pulmonary
hypertension model mouse. In this experiment intense expression of
the transferred PGIS gene was recognized in the local skeletal
muscle receiving the gene transfer. It is obtained from this
experiment the information that the PGIS gene transfer is hopeful
in treatment of limb ischemia.
[0014] In addition, for the gene therapy not only selection of the
cure gene but also a sort of the vector for gene transfer
influences the efficiency of gene transfer, namely the effect of
treatment.
[0015] The vector for gene therapy is expected to express long time
in a possibly high level of the cure gene in the lesion. However,
many plasmids or non viral vectors were in the past gene therapy
studies and with these vectors strong and long-term expression on
the cure gene was difficult.
[0016] Incidentally, in case of adeno-associated virus (AVV) as a
virus vector it has an strong tropism for skeletal muscle cells and
can achieve a strong and long-term cure gene expression. This AVV
is used in the gene therapy for human hemophilia, cryptic fibrosis,
Parkinson disease, and its high safety and effective is verified.
By the use of AVV achieving a strong and long-term efficient gene
expression, a long-term expression of the cure gene is attained and
a possibility of its clinical application is expected.
[0017] Present inventors, based on the above-mentioned evidence,
reached to the completion on the present invention.
Solution of Problems
[0018] The present invention is concerned with the drug composition
of angiogenesis used for the treatment or prevention of peripheral
arterial disease. This drug composition contains, as the active
component, the adeno-associated virus (AVV) inserted with human
gene for PGIS producing prostaglandin I.sub.2 which at least
induces the activities of anti-platelet aggregation, vasodialation,
and/or angiogenesis.
[0019] Particularly, this drug composition of angiogenesis therapy
is characteristic of the recombinant adeno-associated virus (AVV)
inserted with the gene for production of a factor inducing
angiogenesis.
[0020] Noteworthy, the present investigation is concerned with the
drug composition of angiogenesis used for the treatment or
prevention of peripheral arterial disease, which contains the
adeno-associated virus (AVV) inserted with human gene for PGIS
producing prostaglandin I.sub.2 which at least induces the
activities of anti-platelet aggregation, vasodialation, and/or
angiogenesis or the recombinant adeno-associated virus (AVV)
inserted with the gene for the angiogenesis factor.
[0021] Here, the use of types 1 or 2 is efficient as the
adeno-associated virus (AVV).
[0022] The drug composition for angiogenesis therapy in the present
invention includes a pharmaceutically permissible carrier such as
the recombinant adeno-associated virus (AVV) inserted with the gene
for human PGIS or human PGIS and the angiogenesis factor.
[0023] The gene coding for the angiogenesis factor used in the drug
composition for angiogenesis therapy of the present invention is
possibly the entire or a part of the gene coding for a protein or a
peptide, which can newly induces vasculogenesis.
[0024] Especially, as the gene for angiogenesis factor, vascular
endotherial growth factor (VEGF) is acceptable.
[0025] The present invention, as described previously, presents a
drug composition for angiogenesis therapy useful in treatment or
prevention of peripheral arterial disease, which includes human
gene for PGIS (hPGIS) producing prostaglandin I.sub.2 which induces
the activities of vasodialation, and/or anti-platelet aggregation,
and the recombinant adeno-associated virus (AVV) inserted with the
gene for the angiogenesis factor
[0026] In use of the present angiogenesis therapy, the "gene for
angiogenesis" inserted in adeno-associated virus (AAV) is,
similarly to the human PGIS gene, the gene coding for the entire or
a part of a protein or a peptide, which newly induces
vasculogenesis and shows either activity of inhibition of platelet
aggregation, vasodialation, angiogenesis. Namely listed are gene
for endotherial growth factor (VEGF), VEGF-2, acidic FGF (aFGF),
basic FGF (bFGF), TGF-.alpha., TGF-.beta., platelet-derived
endotherial growth factor (PD-ECGF), platelet-derived growth
factor, tumor necrosis factor-.alpha. (TNF-.alpha.), insulin-like
growth factor, angiopoieti-1 etc. Additionally candidates are the
gene for HIF-1 and ets family translation factors including ets-1
which regulate the gene expression of VEGF. The genetic sequences
of their gene mentioned above are registered in public data bases
and are easily cloned by a man of skill in related fields.
[0027] Blow is an example of gene of VGEF. "VEGF gene" in the
present invention indicates the gene coding for VEGF protein and,
in some case, simply designates the expression vector (? plasmid)
encoding the VEGF gene. As concrete illustration a cDNA for VEGF is
inserted into the virus vector mentioned after. Regarding human
VEGF four subtypes (VEGF121, VEGF165, VEGF189, VEGF206) formed by
alternative splicing in transcription of the human gene have been
reported (Science, 219, 983, (1983); J. Clin. Invest., 84, 1470
(1989); Biochem. Biophys. Res. Commun, 161, 851 (1989)). Although
it is possible in the present invention, that an either subtype of
VEGF gene is used for constructing the expression vector, the gene
for VEGF165 with the biologically strongest activity is preferable.
In addition even the VEGF gene subjected to modifications are
classified as the VGEF gene as far as they express the active
protein with an angiogenesis activity.
[0028] The gene for VGF165 is easily cloned or modified by a man of
skill in related fields according to information on the sequence
registered in the literature (i.e. Science, 246, 1306 (1989)) or
data bases.
[0029] The activity expressed by VEGF or its modified gene can be
examined in vitro by their growth effect on vascular endotherial
cells as described in WO97/07824.
[0030] Previously described gene for angiogenesis factors can be
used singly or in combination with multiple gene.
[0031] Here, PGI.sub.2 produced by hPGIS shows the activities of
vasodialation, accentuation of vasopermiability and anti-platelet
aggregation and a combination of gene for PGIS and an angiogenesis
factor brings a more remarkable combination effect.
[0032] On the other hand, in the present invention, as "a material
with activity of vasodialation" is used PGI.sub.2 or preferably its
analogs. Additionally, as a "material with activity of
anti-platelet aggregation" is also used PGI.sub.2 or preferably its
analogs.
[0033] In the present invention, "the material having activities of
vasodialation and/or anti-platelet aggregation" is defined as the
compound which produces PGI.sub.2 showing activities of
vasodialation and/or anti-platelet aggregation, or enhances the
production of PGI.sub.2. Properly it indicates the compound which
enhances synthesis, production of induction of prostaglandin
(I.sub.2?). Although the suitable compound can be any material
including gene, proteins, low molecular weight chemicals etc., the
gene for PGIS is utilized as the compound induces the enzyme
synthesizing PGI.sub.2. The nucleotide sequence of the gene for
PGIS are registered in public data bases and its gene is easily
cloned by a man of skill in related fields.
[0034] Blow is an example of gene of human PGIS gene (hPGIS). In
the present investigation "PGIS gene" indicates the gene coding for
PGIS. Incidentally in some case PGIS gene constructed into an
expression plasmid is also described as "PGIS gene". Actually shown
as an example is the clone obtained by inserting the PGIS cDNA
recorded in B.B.R.C., Vol. 200, No. 3, p 1728-1734 (1994) and
WO95/30013 into a proper expression vector. Furthermore, as well as
in case of previously described VEGF, the expression constructs
with modified PGIS gene are included in the PGIS gene as far as
they are the gene coding for protein with the function of PGIS in
the present investigation.
[0035] The present PGIS gene, as well as the VEGF gene, is also
easily cloned on the basis of the sequence information described in
the previously described reference and registered in public data
bases by a man of skill in related fields and its modification of
the clone is easily carried out. The expected activity of the
protein coded by the present gene can be examined by assaying
products, for example, using an enzyme immunoassay using 6-keto
Prostaglandin F1.alpha. enzyme immunoassay kit (Cayman, Co.,
catalog #515211), or thin-layer chromatography detecting the
products of prostacyclin synthase.
[0036] PGI.sub.2 as the compound with activities of vasodialation
and/or anti-platelet aggregation or PGI.sub.2-inducing materials
can be singly used or in combination of multiple compounds.
[0037] In relation to the present invention, the administration
method, conditions and doses of drug composition of angiogenesis
therapy are described below.
[0038] 1) When used are PGI.sub.2 with activities of vasodialation
and/or anti-platelet aggregation, and PGIS gene induces the
production of PGI.sub.2, the gene coding for angiogenesis in use of
gene for an angiogenesis factor described above, and the gene
coding for angiogenesis in use of combination of gene for PGIS and
an angiogenesis factor(s), namely in combination of two or more
gene, i.e. the gene for PGIS and VEGF, they need to be in the form
of drug for gene therapy.
[0039] When the present drug for angiogenesis is administered to a
patient, an administration form with the virus vector is given as
an example. Experimental manual books particularly describe the
methods for preparation and administration of the drug (Separate
Volume Jikken-igaku, Fundamental Technique for Gene Therapy (in
Japanese), Yodosha Co, 1997; Separate Volume Jikken-igaku,
Experimental Mehods for Gene Transfer and Expression Analyses (in
Japanese), Yodosha Co, 1997; Handbook for R&D of Gene Therapy
(in Japanese), ed. The Japan Society of Gene Therapy, NTS Co.,
1999). Below the methods for preparation and administration of the
drug are explained in detail.
[0040] By the way, in the present invention, the objective gene is
inserted into the adeno-associated virus (AVV), and can be
transfered into the target cells by infection of the recombinant
virus (AVV) constructed by insertion of the gene into the
adeno-associated virus (AVV) as a vector. Here, types 1, 2, 5, 8 of
adeno-associated virus (AVV) are suitable, but types 1 or 2 are
preferred.
[0041] In the present invention the vector is used for the purpose
of transfer of the gene into the target region of the lesion. This
selection of adeno-associated virus (AVV) as a vector is due to the
efficient transfer of gene into non-dividing cells such as muscle,
nerve and liver cells etc. and to the possible long-term expression
maintained in these target cells.
[0042] Furthermore, the gene therapy drug in the present invention
consisting of drug composition of angiogenesis is preferred to be
type 1 or 2 of adeno-associated virus (AVV) with an efficient
transfer activity of the gene into non-dividing cells such as
muscle, nerve and liver cells etc.
[0043] Then as the transfer method of the gene-therapeutic drug to
the patient, there are two methods: the in vivo method where the
gene-therapeutic drug is directly delivered into the body; the ex
vivo method where it is ex vivo transfected into a specified cells
isolated from the human body and returned to the body (Nikkei
Science (in Japanese), April issue, 1994, pp. 20-45; Gekkanyakuji
(in Japanese), 36(1), 23-48, 1994; Separate Volume Jikken-igaku,
12(15), 1994; Handbook for R&D of Gene Therapy (in Japanese),
ed. The Japan Society of Gene Therapy, NTS Co., 1999). The in vivo
method is preferred in the present invention. When the drug for
gene therapy is given to the patient, it is delivered in the
pathway dependent on the disease as the therapeutic target and the
target organ etc. For example the drug for gene therapy is injected
into veins, arteries, hypodermic or intracutaneous tissues, and
muscles, or directly given to the tissues with the lesion.
[0044] As the dose form, various types of dose forms, such as a
liquid dose, suitable to their above mentioned administration form
can be adoptable. For example, the injection as the dose form
containing the active ingredient of a gene can be prepared by
common methods. The injection is possibly prepared as follows: the
ingredient, for example, is dissolved in a suitable solvent (a
buffer such as PBS, saline, sterilized water etc) if necessary,
subjected to mechanical sterilization by filtration, and filled in
sterilized vessels. A commonly used carrier can be added to the
injection. And in use of a liposome such as the HVJ-liposome, the
injection can be a suspension, a frozen form or a frozen drug after
centrifugal concentration.
[0045] And in order to an easy distribution of the gene around the
site of the lesion the drug can be embedded around the lesion or
slowly and continuously delivered with an osmotic pump.
[0046] Incidentally the content of the active pharmaceutical
ingredient (API) of the gene can be adjusted according to the
disease as the treatment aim, age of patient, body weight etc., is
generally in a range of 0.001-100 mg, preferably 0.01-10 mg, which
is suitably given to a patient once per days or months.
[0047] 2) When used are PGI.sub.2 with activities of vasodialation
and/or anti-platelet aggregation, and PGIS gene induces the
production of PGI.sub.2, the gene coding for angiogenesis in use of
gene for an angiogenesis factor described above, and the gene
coding for angiogenesis in use of combination of gene for
angiogenesis, and a low-molecular eight chemical, protein, peptide
etc., they need to be in the form of drug for gene therapy. On the
hand, low-molecular weight chemicals etc. in form of the general
drug compositions are administered orally or parenterally. As a
typical combination is considerably a combined use of VGEF gene and
a PGI.sub.2 analog.
[0048] The above described drug compositions of low-molecular eight
chemicals, proteins etc. as active ingredients are explained
below.
[0049] When drugs with activities of vasodialation and/or
anti-platelet aggregation (anti-platelet drug) are commercially
available, their administration methods and dose can be determined
according to their protocols. Generally, the dose form and
administration method are as follows.
[0050] Namely, when the drug is orally administered, it is done in
the dose form usually used in the specified field. When the drug is
parenterally dispensed, it is administered as the dose form of a
focal administration drug, an intrarectal administration drug, an
injection, a nasogastric drug etc.
[0051] As the oral administration drug or focal administration
drug, for example, the dose form of capsules, tablets, pills,
powder, drop, suppository, liquid formulation etc. is conceived.
For example, a sterilized solution, suspension, and emulsion etc,
such as water, a water-propylene glycol mixed solution, a buffered
solution, 0.4% saline etc. are conceived as the injection form. The
dose form of cream, ointment, lotion, transdermal absorption agent
etc. can be chosen as a focal administration drug.
[0052] The above dose forms are prepared to the formulations with
pharmaceutically tolerably excipients and additives, by common
methods employed in the pharmaceutical field. Pharmaceutically
tolerably excipients and additives are carriers, binders, incenses,
buffering agents, thickening agents, tinctions, stabilizers,
emulsifiers, dispersing agents, suspensions, antiseptics,
pH-adjusting agents, tonicity-adjusting agents infiltrate agents
etc. In addition, pharmaceutically tolerably carriers for example,
are magnesium carbonate, lactose, pectin, starch, methylcellulose
etc.
[0053] When the previously described drug composition is given to
the patient, it can be delivered in the pathway dependent on the
disease as the therapeutic target and the target organ etc. For
example it may be injected into veins, arteries, hypodermic or
intracutaneous tissues, and muscles, or directly given to the
tissues with the lesion. Additionally the drug composition is also
possible to be administered orally or in its suppository form.
Incidentally dose and administration frequency of the drug
composition is depends on the symptom, age, body weight of a
patient, and the administration form. Generally the dose per an
adult is in a range of 0.001-100 mg a day, preferably 0.01-10 mg,
which is suitably given to a patient once or multiple times
divided.
[0054] The above mentioned drug compositions with a low-molecular
weight chemical or protein as an active ingredient can be
administered together with the gene-therapy drug containing the
gene for angiogenesis, or separately at regular intervals.
Advantageous Effects of Invention
[0055] The drug composition of angiogenesis therapy in the present
investigation is described above. The present drug composition is
useful if treatment or prevention of the peripheral arterial
disease, which needs the angiogenesis therapy.
BRIEF DESCRIPTION OF THE FIGURES
[0056] FIG. 1 shows subcutaneous blood perfusion by the laser
Doppler method of ischemic limbs treated with adeno-associated
virus-mediated PGIS gene (AAV-PGIS group), saline (control group),
adeno-associated virus-mediated EGFP gene (AAV-EGFP (enhanced green
fluorescence protein) group) and the corresponding macroscopic
images.
[0057] FIG. 2 shows qualitative analyses of perfusion rates of
ischemic left limbs treated with adeno-associated virus-mediated
PGIS gene (AAV-PGIS), saline (control), adeno-associated
virus-mediated EGFP gene (AAV-EGFP) in comparison with the
respective control non-ischemic right limbs (n=12)
[0058] FIG. 3 indicates the improvement in limb necrosis rate of
AAV-PGIS group mice in comparison with those of control and
AAV-EGFP group mice.
[0059] FIG. 4 shows quantitative RT-PCR analysis of the expression
of human PGIS, mouse PGIS, mouse VEGF, and a mouse receptor for
VEGF, FLK-1 relative to mouse glyceraldehydes-3-phosphate
dehydrogenase (GAPDH) mouse in mouse left thigh muscles received an
injection of AAV-PGIS, control (saline) or AAV-EGFR Expression of
hPGIS (A), mGAPDH (B), mVEGF (C), and mFLK-1(D) in left limb
skeletal muscles were examined by quantitative RT-PCR (n=5).
[0060] FIG. 5 shows the western blot analysis of the expression of
human PGIS (hPGIS) in ischemic muscles received an injection of
control (saline), AAV-EGFP or AAV-PGIS.
[0061] FIG. 6 shows immunostaining images of human PGIS (A) and
mouse von Willebrand factor (vWF) (B), and relative capillary
vessel area in hind limb muscles received an injection of control
(saline), AAV-EGFP or AAV-PGIS. (A) Human PGIS expressed in hind
limb muscles received an injection of control (saline), AAV-EGFP or
AAV-PGIS was immunostained by anti-human PGIS antibody. (B) Mouse
vWF as a neovascularization marker expressed in hind limb muscles
in the three groups was immunostained by Anti-vWF antibody. (C) vWF
positive area of the images in FIG. 6B was quantitatively
analyzed.
DETAILED DESCRIPTION
[0062] Below the present invention is concretely explained with a
practice example, but the present invention is not restricted by
this practice example.
[0063] Construction of Plasmids and AAV Vectors
[0064] The expression vector for human PGIS was constructed as
described previously. In brief, the blunted HindIII/BamHI fragment
of the full-length human PGIS cDNA was ligated into the blunted
XhoI site of the pUC-CAGGS expression plasmid. To verify that the
pUC/PGIS construct encoded a biologically active PGIS protein,
pUC/PGIS was transfected into NIH3T3 cells, and conversion of
[.sup.14C]-PGH.sub.2 to 6-keto-[.sup.14C]PGF1.alpha. was measured.
pUC-CAGGS vector lacking the insert served as the control vector.
Human PGIS genes were then inserted into AAV-CAG plasmids, and
AAV-hPGIS vectors were constructed. AAV-EGFP (enhanced green
fluorescent protein) vector for control experiments was also
prepared as described previously.
[0065] Murine Model of Hind Limb Ischemia
[0066] Eight-week-old male BALB/c nude mice (Japan CLEA) were
anesthetized with diethyl ether, and the skin was incised over the
femoral artery in the mid-portion of the left hind limb. The
femoral artery was then gently isolated, and the proximal portion
of the artery was ligated with 7-0 silk ligatures. All experimental
procedures and protocols were reviewed and approved by the Animal
Care and Use Committee of the Keio University School of Medicine,
and they conformed to the NIH Guide for the Care and Use of
Laboratory Animals.
[0067] Experimental Protocols
[0068] Mice were divided into 3 groups (control, AAV-EGFP and
AAV-hPGIS). The AAV-hPGIS group was administered AAV type1-hPGIS
(1.0.times.10.sup.11) into left thigh muscle. The control group was
administered the vehicle (0.9% saline) into the left thigh muscle.
As negative control, the AAV-EGFP group was administered AAV
type1-EGFP (1.0.times.10.sup.11) into same site. After 1 week of
gene transfer, the left femoral artery of the mice was ligated to
produce hind limb ischemia model. After 2 week of ligation, the
incidence of limb necrosis was evaluated and blood perfusion rate
of the mice was measured using Laser Doppler perfusion imaging
(LDPI) system. The left thigh muscles were excised and utilitied
for further analysis.
[0069] To examine the continuity of the expression of human PGIS
mRNA, each 4 to 5 mice in the groups of hPGIS, control, and
AAV-EGFP were administered AAV type1-hPGIS (1.0.times.10.sup.11,
v.g./body), into left thigh muscle of C57/BALB6 mice (8
week-old)
Laser Doppler Blood Perfusion Analysis
[0070] The three groups (AAV-hPGIS, CONTROL and AAV-EGFP) of mice
are subjected to ischemia in left hind limbs, and the blood
perfusion rate in the ischemic left (L) and normal right (R) hind
limb was measured with a Laser Doppler perfusion imaging (LDPI)
system (Moor LDI, Moor Instruments, USA). The measurement was
carried out by taking LDPI of perfusion state of ischemic hind
limbs in each group. The results are shown in FIG. 1.
[0071] In FIG. 1 optical (Macro) and the corresponding Laser
Doppler (LDP) perfusion images obtained by measurement with LDPI
system.
[0072] In FIG. 1 the images obtained by LDPI method are represented
in color. The index for color images representing perfusion state
shown below. The index from left to right changes from dark blue to
red. Low or nil blood perfusion was displayed as dark blue, and the
highest perfusion intervals were displayed as red up to white
(maximum).
[0073] FIG. 2 shows quantitative analysis of the blood perfusion
rate of the ischemic limb in groups of AAV-PGIS, Control and
AAV-EGFP, compared with the non-ischemic control limb (n=12).
Representative Laser Doppler perfusion images in each group.
(B)
[0074] In FIG. 2 data are mean.+-.SD. *P<0.05; ns, not
significant.
Quantitative RT-PCR
[0075] RNA extraction and quantitative RT-PCR were performed as
described previously. Quantitative RT-PCR was performed with TaqMan
probes (Applied Biosystems): human PGIS (Hs00168766_m1), mouse PGIS
(Mm00447271_m1), mouse VEGF (Mm01281449_m1), FLK-1 (Mm01222419_m1),
FLT-1 (Mm00438971m1), and Rodent GAPDH. The mRNA levels were
normalized by comparison to GAPDH mRNA. In FIG. 4 data are
mean.+-.SD. *P<0.05; ns, not significant.
Western Blot Analysis
[0076] Membrane fractions of skeletal muscle were prepared by
homogenization of thigh muscle in ice-cold buffer as described
Immunodetection was performed on membrane extracts with an antibody
to human PGIS. The results are shown in FIG. 5.
Histological Analysis
[0077] Samples were fixed with 10% formalin, embedded with
paraffin, and cut into 4-.mu.m sections. The sections were stained
with hematoxylin and eosin. In addition, thigh muscle sections were
stained with anti-human PGIS antibody. The results are shown in
FIG. 6, which clearly indicates that the remarkable expression of
hPGIS mRNA in group of AAV-hPGIS mice with administration of
AAV-hPGIS contributed to angiogenesis.
[0078] In FIG. 6(A) illustrates microscopic images showing
expression of hPGIS in left hind limb muscles in groups of
AAV-hPGIS, Control and AAV-EGFR The each muscle in (A) was stained
by immnohistochemical methods. (B) Typical examples of microscopic
images of von Willebrand factor (vWF)-stained left hind limb
muscles mouse. (C) The results of quantitative analysis of
microvessels in left hind limb muscles subjected to
immunofluorecence staining of vWF. In FIG. 6C data are mean.+-.SD.
*P<0.05; ns, not significant.
Density of Microvessels
[0079] Animals were sacrificed under anesthesia, and perfusion
fixation was performed with 4% paraformaldehyde. The thigh muscles
were excised and embedded with OTC compound. Frozen sections (7
.mu.m) were cut from the tissue specimens Immunofluorescent
staining for endothelial cells was carried out with anti-von
Willebrand factor (vWF) antibody (DAKO). Nuclei were stained with
TOTO-3 (Molecular Probes). The density of microvessels was
calculated from the number of vWF-positive vessels. All confocal
microscopy was carried out on a LSM 510 META (Carl Zeiss, Jena,
Germany). The capillary density was the ratio between the total
area of capillary vessels and the total skeletal muscle area, each
measured by Image J software. The data for each mouse were
calculated from 30 serial sections.
Statistical Analysis
[0080] All data are presented as the mean.+-.SD. Statistical
significance was evaluated using the unpaired Student t test.
Comparisons among more than three groups were performed using
analysis of variance. P<0.05 was considered significant.
[0081] (Experimental Results)
[0082] Duration o f hPGIS expression after administration of AAV
type1-hPGIS vector
[0083] By the present inventors etc. were examined the duration of
expression efficiency by AAV type1-hPGIS vector using 8-weeks
control mice (C57/BL/6). Mice were administered AAV type1-hPGIS
vector into the left thigh muscle. For analysis the left thigh
muscle from mice treated with before and after 2, 4, 8, 12 weeks
vector administration. The results, as analyzed by quantitative
RT-PCR, indicates a strong human PGIS mRNA expression from 2 weeks
after gene transfer. The strong expression was maintained even
after 12 weeks. The results are shown in Table 1.
[0084] Table 1 Change in expression of human PGIS after
administration of AAV1-human PGIS vector (1.0.times.10.sup.11
(v.g./body)) into left thigh muscle
TABLE-US-00001 TABLE 1 Amount of Number of expressed human Term of
administration treated mouse PGIS mRNA*.sup.1 Before administration
8 .sup. 0.003 .+-. 0.005*.sup.2 2 weeks after administration 4 1220
.+-. 4880 of AAV-human PGIS 4 weeks after administration 5 6310
.+-. 9470 of AAV-human PGIS 8 weeks after administration 5 4648
.+-. 2640 of AAV-human PGIS 12 weeks after administration 5 3370
.+-. 1980 of AAV-human PGIS *.sup.1Relative amount of hPGIS mRNA to
mouse GAPDH mRNA. *.sup.2Mean value .+-. SD. *.sup.3mice were
administered AAV1-human PGIS vector (1.0 .times. 10.sup.11
(v.g./body)).
Improvement of Hind Limb Ischemia
[0085] To treat limb ischemia model mice, the present inventor et
al administered AAV type1-hPGIS into left thigh muscle and examined
the degree of improvement in the ischemia two weeks after the
limb-ischemia operation. In examination the seriousness of limb
ischemia was divided into three groups. As shown in FIG. 3, foot or
finger necrosis was observed in 63% and 69% mice in groups of
Control and AAV-EGFP treated with AAV type1-EGFP, respectively. But
the rate of necrosis in AAV-hPGIS group mice received an
administration of AAV type1-hPGIS, was significantly lowered to 19%
and improved. Furthermore, frequency of tertiary foot necrosis in
AAV-hPGIS was lowest in the three groups described above. These
results indicate the administration of AAV type1-hPGIS in skeletal
muscles is effective in limb ischemia.
Laser Doppler Blood Perfusion Analysis
[0086] Using laser Doppler blood perfusion (LDP) the blood flow in
limb muscle tissues of AAV-hPGIS, Control, and AAV-EGFP group mice
was analyzed percutaneously. In the LDP images shown in FIG. 1, the
blood perfusion of left thigh muscle tissues in the lesion side of
mice was lowered in Control and AAV-EGFP groups compared with
AAV-hPGIS group. Additionally quantitative analysis (blood
perfusion rate) by LDP method indicates 73, 54 or 118% of perfusion
rates in the left lesion side of mice as referred to those in
respective right healthy sides in the three groups of Control,
AAV-EGFP and AAV-hPGIS (see FIG. 2). It is likely from the remark
of LDP images that the administration of AAV-hPGIS improves the
blood perfusion.
Analysis of mRNA in Ischemic Muscles
[0087] Expression of mRNA in ischemic limb tissues was analyzed
using quantitative RT-PCR. Compared with Control and AAV-EGFP
groups, a remarkably significant expression of human PGIS gene was
observed in AAV-hPGIS group (see FIG. 4A) and the expression of
intrinsic mouse PGIS gene was not significantly different among the
three group mice. Furthermore, expression of murine mRNA for VEGF,
FLK-1 and FLT-1 was analyzed to study neovascularization in the
ischemic limb tissues. Consequently, it was significant that
expression of VEGF mRNA in AAV-PGIS group was twice compared with
that in the other two groups (See FIG. 4C). Although expression of
mRNA for FLK-1 and FLT-1 in AAV-PGIS group has a increasing
tendency compared that in the other two groups, the difference was
not statistically significant among them (see FIGS. 4D and 4E).
Hereinbefore, strong expression of hPGIS was confirmed in skeletal
muscles. Additionally, expression of VEGF mRNA increased in
AAV-PGIS group.
Western Blot Analysis of Ischemic Muscles
[0088] Furthermore, gene expression at protein level was examined
using western blot analysis. Western blots confirmed hPGIS protein
in AAV-hPGIS administration group (see FIG. 5).
Expression of hPGIS in Skeletal Muscles
[0089] Moreover, histological examination of the skeletal muscle of
the region administered AAV-PGIS was carried out. Imunostaining
with anti-human PGIS antibody confirmed expression of hPGIS
gene/protein in muscle cells in AAV-hPGIS administration group (see
FIG. 6), and none of immunological changes such as infiltration of
inflammatory cells was observed among the three groups administered
AAV-PGIS, Control and AAV-EPDGF.
Angiogenesis in Skeletal Muscles
[0090] Additionally to examine improvement at the level of ischemic
muscle cells, skeletal muscles were subjected to immunostaining
with anti-vWF antibody. In confocal microscopy the increase in
vEF-positive vessels was observed in AAV-hPGIS group compared with
Control and AAV-EGFP groups, suggesting an increase in capillary
density (see FIGS. 6A and 6B). Thus, the density of vVW-positive
microvessels was quantitatively analyzed using Image J software.
The capillary density was expressed as the ratio between the total
area of capillary vessels and the total skeletal muscle area. It
was statistically significant that the density of vVW-positive
microvessels in AAV-hPGIS group was 6.5 folds higher than that in
Control and AAV-EGFP groups (see FIG. 6C). Elastica van Gieson
(EGV) stain of skeletal muscles was also carried out, but any
significant change was not observed among the above three
groups.
[0091] As described above, neovascularization at the capillary
level occurred in the ischemic skeletal muscles administered
AAV-PGIS, suggesting reduction of ischemic injury of skeletal
muscles.
[0092] It is shown from the facts described above that improvement
of hind limb ischemia is obtained by transfer of human PGIS gene in
ischemic skeletal muscles using AAV type 1 vector. Although many
serotypes are known as AVV, AAV type 1 is known to have an ability
of the strongest and long-term expression in skeletal muscles. It
is reported by the present inventor et al that AAV type 1-human
PGIS (AAV-hPGIS) shows a long-term strong gene transfer efficiency
in the in vitro and in vivo control experiments. Additionally gene
expression of AAV type 1 is limited in the local site of
administration, and it is not found in remote organs. This
characteristic of the expression can reduce the side effect of the
gene therapy in remote organs, and is useful in the treatment of
patients suffering from serious limb ischemia, which are
complicated with arteriosclerosis-induced diseases in other
organs.
[0093] As it is clear from previously described experimental
results, the rate of necrosis in AAV-hPGIS group mice received an
administration of AAV type1-hPGIS, was significantly lowered and
frequency of tertiary foot necrosis was lowest in AAV-hPGIS.
Furthermore, in quantitative analysis by LDP the perfusion rate in
AAV-hPGIS group caused a statistically significant increase
compared with those in the two groups without administration of
AAV-hPGIS. LDP image suggests that the development of collateral
vessels contribute to the improvement of blood perfusion in
ischemic limbs, because disruption of blood perfusion in femoral
arteries by ligation, namely the blockage of femoral arteries was
confirmed in all groups.
[0094] Subsequently, gene expression was examined with skeletal
muscles, the administration site of AAV-hPGIS. The expression of
PGIS gene was analyzed by quantitative RT-PCR. A remarkable gene
expression of external human PGIS was observed with skeletal
muscles in AAV-hPGIS group, and was ten thousand-folds compared
with those of the control groups. An enhanced two-fold gene
expression of intrinsic VEGF was also observed with skeletal
muscles in AAV-hPGIS group, suggesting induction of VEGF by
expression of external human PGIS. Using immunostaining of hPGIS a
remarkable expression of human PGIS protein was observed in
skeletal cells mediated AAV-hPGIS. In addition, any inflammatory
changes and tumor formation of muscles was not found by the AAV
viral administration. The findings prove the safety of AAV-PGIS,
and coincide with the fact that AAV has the lowest immunogenicity
among virus vectors.
[0095] Furthermore, by the present inventors skeletal muscles was
subjected to immnostaining with anti-vWF antibody in order to
examine whether neovascularization occurs or not. In AAV-PGIS group
neovascularization was confirmed at the capillary level.
Incidentally EVG-staining for elastic fibers in skeletal muscles
was carried out and significant changes were not observed in all
treated groups, suggesting that the neovascularization at capillary
level occurs in skeletal muscles administered AAV-hPGIS.
[0096] It is demonstrated from results mentioned above that
transfer of AAV-hPGIS into skeletal muscles improves limb ischemia
by a remarkable expression of human PGIS in skeletal muscle cells,
leading to production in the muscle cells and secretion of
PGI.sub.2 meditated by the expression, which induces activities of
vessel protection, induction of neovascularization etc. And it is
noteworthy that the effects mentioned above were achieved by single
administration. Previous clinical and basic research reports reveal
that the strong continuous gene expression could not be maintained
due to plasmids, non-viral vectors etc. without a property of
long-term expression. Thus, with use of these vectors the
multiple-time administration is necessary to keep the gene
expression. The highly safe virus vectors such as the AAV vector
achieving a long-term and strong expression can be ideal for
clinical applications
* * * * *