U.S. patent application number 11/722911 was filed with the patent office on 2008-05-29 for pharmaceutical composition and method for neoangiogenesis/revascularization useful in treating ischemic heart diseases.
Invention is credited to Lei Cheng, Ming Li, Hong Wei Liu.
Application Number | 20080124388 11/722911 |
Document ID | / |
Family ID | 37967417 |
Filed Date | 2008-05-29 |
United States Patent
Application |
20080124388 |
Kind Code |
A1 |
Li; Ming ; et al. |
May 29, 2008 |
Pharmaceutical Composition And Method For
Neoangiogenesis/Revascularization Useful In Treating Ischemic Heart
Diseases
Abstract
A pharmaceutical composition and a method of treating ischemic
heart diseases by growing new blood vessels that supply oxygen and
nutrients to infarcted heart tissues throughout the entire infarct
zone and for preventing cardiomyocyte apoptosis in ischemic events.
The pharmaceutical composition contains an active ingredient
compound with a backbone structure of formula (I). ##STR00001##
Inventors: |
Li; Ming; (Hong Kong,
CN) ; Cheng; Lei; (Hong Kong, CN) ; Liu; Hong
Wei; (Beijing, CN) |
Correspondence
Address: |
EVAN LAW GROUP LLC
600 WEST JACKSON BLVD., SUITE 625
CHICAGO
IL
60661
US
|
Family ID: |
37967417 |
Appl. No.: |
11/722911 |
Filed: |
October 27, 2006 |
PCT Filed: |
October 27, 2006 |
PCT NO: |
PCT/CN06/02886 |
371 Date: |
June 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60791462 |
Apr 13, 2006 |
|
|
|
Current U.S.
Class: |
424/451 ;
424/464; 514/25 |
Current CPC
Class: |
A61K 31/192 20130101;
A61P 9/10 20180101; A61K 31/704 20130101; A61P 43/00 20180101; A61P
9/04 20180101; A61P 9/00 20180101; A61K 35/34 20130101; C07C 62/32
20130101; A61K 31/56 20130101 |
Class at
Publication: |
424/451 ; 514/25;
424/464 |
International
Class: |
A61K 31/7024 20060101
A61K031/7024; A61K 9/48 20060101 A61K009/48; A61K 9/20 20060101
A61K009/20 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 27, 2005 |
IB |
2005-003191 |
Oct 27, 2005 |
IB |
2005-003202 |
Claims
1. A pharmaceutical composition, which comprises a pharmaceutically
acceptable excipient and an effective amount of a compound with a
backbone structure showing in formula (I) and does not contain any
extract of a plant, said composition being formulated for treating
an ischemic heart disease: ##STR00003##
2. The pharmaceutical composition of claim 1, wherein at least 95%
by weight of said composition is identified compounds and said
compound is of said backbone structure itself without
substitution.
3. The pharmaceutical composition of claim 1, which is accompanied
by a piece of information stating that said composition is useful
for treating an ischemic heart disease.
4. The pharmaceutical composition of claim 3, which is formulated
in a pharmaceutical dosage form and packaged into a container and
said information is shown on said container or in an insert or
pamphlet included in said container.
5. The pharmaceutical composition of claim 4, wherein said dosage
form is selected from the group consisting of tablet, capsule,
injection, suspension, solution, powder, and syrup.
6. A method of treating an ischemic disease in a mammalian subject,
comprising a step of administering to said mammalian subject an
effective amount of a compound of formula (I) or a functional
derivative of said compound.
7. The method of claim 6, wherein said compound or functional
derivative exerts a therapeutic effect by revascularization in an
infarcted heart tissue of said mammalian subject.
8. The method of claim 7, where said revascularization occurs
within 24 to 72 hours following a treatment with said compound or
functional derivative.
9. The method of claim 6, wherein said ischemic diseases is
ischemic heart diseases.
10. The method of claim 6, wherein said ischemic disease is caused
by atherosclerosis of coronary arteries.
11. A method for revascularization in infarcted myocardia of a
mammalian subject, comprising a step of treating said infarcted
myocardia with a compound of formula (I) or a functional derivative
of said compound.
12. The method of claim 11, wherein said compound or functional
derivative of said compound up-regulates expressions of VEGF and
bFGF.
13. The method of claim 11, wherein said compound or functional
derivative of said compound is injected directly into tissues in
said infarcted myocardia.
14. The method of claim 11, wherein said compound or functional
derivative of said compound is delivered to tissues in said
infarcted myocardia via oral administration.
15. The method of claim 11, wherein said compound or functional
derivative of said compound is delivered to tissues in said
infarcted myocardia via subcutaneous injection, intramuscular
injection, or intravenous infusion.
16. The pharmaceutical composition of claim 5, wherein said
mammalian subject is a human patient.
17. The pharmaceutical composition of claim 16, wherein said dosage
form is injection.
18. A pharmaceutical product, comprising the pharmaceutical
composition of claim 1, a container and a piece of information on
usefulness of said pharmaceutical composition, said information
indicating that said pharmaceutical composition is beneficial to a
human suffering or having suffered an ischemic heart disease.
19. The pharmaceutical product of claim 18, wherein said
information is shown an outside surface of said container.
20. The pharmaceutical product, wherein said information is shown
in a pamphlet or an insert contained in said container.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/791,462, filed Apr. 13, 2006, the contents of
which are hereby incorporated by reference. The application further
claims priority to PCT Application Nos. PCT/IB2005/003202 and
PCT/IB2005/003191, both filed Nov. 8, 2005, the contents of which
are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to a pharmaceutical composition and a
method of treating ischemic heart diseases. Particularly, it
relates to a pharmaceutical composition and method for growing new
blood vessels that supply oxygen and nutrients to infarcted heart
tissues throughout the entire infarct zone and for preventing
cardiomyocyte apoptosis in ischemic events.
BACKGROUND OF THE INVENTION
[0003] Ischemic heart diseases including coronary heart disease and
heart infarction are diseases due to insufficient coronary blood
supply or interruption of the blood supply to a part of the heart,
causing damages or death of heart muscle cells. It is the leading
cause of death for both men and women over the world. For example,
about 1.5 million Americans suffer a heart attack each year (that's
about one heart attack every 20 seconds) and millions suffer from
ischeinic heart diseases.
[0004] During remodeling progress post infarction,
neoangiogenesis/revascularization to the infarcted heart tissues is
insufficient to keep pace with the tissue growth required for
contractile compensation and is unable to support the greater
demands of the hypertrophied but viable myocardium, especially the
myocardium along the border zone of the infarct-the cardiomyocytes
at risk. The relative lack of oxygen and nutrients to the
hypertrophied myocytes might be an important etiological factor in
the death of otherwise viable myocardium, resulting in progressive
infarct extension and fibrous replacement. Therefore, the most
direct way to rescue the cardiac myocytes at risk apparently is to
establish a new blood supply at an early stage that would allow
circulating stem cells, nutrients and growth factors, in addition
to oxygenation, to be delivered to the infarct zone. Restoration of
coronary blood flow by rapid angiogenesis should offer a direct and
effective therapeutic modality to intractable ischemic heart
diseases.
[0005] Although therapeutic angiogenesis has been studied
intensively as an alternative treatment for ischemic vascular
diseases using growth factors such as VEGF, aFGF, bFGF or PDGF,
these factors take weeks to act.sup.1-6, while myocardial necrosis
due to coronary occlusion occurs very rapidly within a matter of
hours.sup.5, 7, 8. The consequence is that fibrous tissue grows
rapidly despite the ischemic condition, which replaces the
infarcted heart tissues and leaves little room for any newly
regenerated myocyte replacement. Up to now, there is no drug and
therapeutic method available that can promote early reconstitution
of the damaged coronary vasculature with newly formed vessels.
[0006] Therefore, to realize the therapeutic value of angiogenesis
in combating ischemic heart diseases, there is a need for chemical
compounds possessing biological properties that can sufficiently
promote early growth of new blood vessels in the infarct zone to
quickly restore the coronary blood circulation once an ischemic
event occurs.
SUMMARY OF THE INVENTION
[0007] As one object of the present invention, there is provided a
pharmaceutical composition for treating ischemic heart diseases
which comprises one or more chemical compounds sharing a common
backbone structure of formula (I), i.e., the compounds derived by
substituting one or m o r e hydrogen atoms at various positions of
the backbone structure of formula (I). The base compound, i.e., the
backbone structure of formula (I) itself without any substitution,
has shown potent beneficial therapeutic effects in treating
ischemic heart diseases by promoting angiogenesis and protecting
against endothelial apoptosis, resulting in revascularization in
infarcted myocardia and prevention of further ischemic death of the
cardiomyocytes. The base compound is referred to as "Ga"
hereinafter. The compounds are known in the art but they are never
known as possessing the above biological activities and therapeutic
effects. In fact, the tannins, to which Ga belongs, are
conventionally reviewed as non-active ingredients and in the
process of identifying the active ingredients in herbal medicines
researchers routinely discard the tannins as debris. Ga may be
isolated from natural resources, particularly from plants or they
may, with existing or future developed synthetic techniques, be
obtained through total or semi-chemical syntheses.
##STR00002##
[0008] The backbone compound of formula I (also referred to as Ga
in this application) can have substituents at various positions and
retain similar biological activities as the backbone compound Ga. A
substituent is an atom or group of atoms substituted in place of
the hydrogen atom. The substitution can be achieved by methods
known in the field of organic chemistry. As used in this
application, the term "a compound of formula I" encompasses the
backbone compound itself and its substituted variants with similar
biological activities.
[0009] It is contemplated, as a person with ordinary skill in the
art would contemplate, that the above backbone compound or its
substituted variant may be made in various possible racemic,
enantiomeric or diastereoisomeric isomer forms, may form salts with
mineral and organic acids, and may also form derivatives such as
N-oxides, prodrugs, bioisosteres. "Prodrug" means an inactive form
of the compound due to the attachment of one or more specialized
protective groups used in a transient manner to alter or to
eliminate undesirable properties in the parent molecule, which is
metabolized or converted into the active compound inside the body
(in vivo) once administered. "Bioisostere" means a compound
resulting from the exchange of an atom or of a group of atoms with
another, broadly similar, atom or group of atoms. The objective of
a bioisosteric replacement is to create a new compound with similar
biological properties to the parent compound. The bioisosteric
replacement may be physicochemically or topologically based. Making
suitable prodrugs, bioisosteres, N-oxides, pharmaceutically
acceptable salts or various isomers from a known compound (such as
those disclosed in this specification) are within the ordinary
skill of the art. Therefore, the present invention contemplates all
suitable isomer forms, salts and derivatives of the above disclosed
compounds.
[0010] As used in the present application, the term "functional
derivative" means a prodrug, bioisostere, N-oxide, pharmaceutically
acceptable salt or various isomer from the above-disclosed specific
compound, which may be advantageous in one or more aspects compared
with the parent compound. Making functional derivatives may be
laborious, but some of the technologies involved are well known in
the art. Various high-throughput chemical synthetic methods are
available. For example, combinatorial chemistry has resulted in the
rapid expansion of compound libraries, which when coupled with
various highly efficient bio-screening technologies can lead to
efficient discovering and isolating useful functional
derivatives.
[0011] The pharmaceutical composition may be formulated by
conventional means known to people skilled in the pharmaceutical
industry into a suitable dosage form, such as tablet, capsules,
injection, solution, suspension, powder, syrup, etc, and be
administered to a mammalian subject suffering coronary heart
disease or myocardial infarction (MI) in a suitable manner. The
formulation techniques are not part of the present invention and
thus are not limitations to the scope of the present invention.
[0012] In another aspect, the present invention provides a method
of promoting revascularization in dead or damaged heart tissues
caused by an ischemic heart disease, such as, for example,
atherosclerosis of coronary arteries in a mammalian subject. The
method comprises a step of administering an effective amount of a
compound of formula (I) or its functional derivative to the
mammalian subject.
[0013] In still another aspect, present invention provides a method
for treating, ameliorating or curing a pathological condition in a
mammal, where the pathological condition, as judged by people
skilled in medicine, can be treated or alleviated by up-regulating
the expressions of angiogenic factors (VEGF and FGF) that promotes
early revascularization in infarcted myocardium, and/or by inducing
anti-apoptotic protein expression that inhibits apoptotic death of
cardiomyocytes in the infarcted hearts and prevents the progressive
extending of further ischemic injury and limiting infarct size. The
method comprises a step of administering an effective amount of a
compound of formula (I) or its functional derivative to the
mammal.
[0014] The various features of novelty which characterize the
invention are pointed out with particularity in the claims annexed
to and forming a part of this disclosure. For a better
understanding of the invention, its operating advantages, and
specific objects attained by its use, reference should be made to
the drawings and the following description in which there are
illustrated and described preferred embodiments of the
invention.
BRIEF DESCRIPTION OF DRAWINGS
[0015] FIG. 1 outlines the process of isolating Ga from the plant
of Geum Japonicum as an example of making the compound of the
present invention.
[0016] FIG. 2 shows the effect of early neovascularization of the
infarcted myocardium following Ga treatment. 1: two days after left
anterior descending coronary artery (LAD) ligation and Ga
injection; 2: two day control heart; 3: seven days after LAD
ligation and Ga injection; 4: seven days control heart; 5: RT-PCR
analysis and 6: Western blot analysis, showing significantly
up-regulated gene expressions of VEGFb and VEGFc in the Ga treated
heart tissues (A standing for VEGFb, B for VEGFc, G for GAPDH, C
for control group, T for Ga treated group, M for molecular
marker).
[0017] FIG. 3 shows the Ga-induced effect on survival potential and
infarct size. 1: seven days after LAD ligation (control); 2: seven
days after LAD ligation (Ga treated); 3: Western blot analysis
showing increased expressions of phospho-Akt1 with Ga treatment; 4:
Western blot analysis showing increased expressions of Bcl2 with Ga
treatment (C and T standing for control group and Ga treated group,
respectively); 5: trichrome staining of the rat myocardium at
2-week post infarct (control); and 6: trichrome staining of the rat
myocardium at 2-week post infarct (Ga treated), showing
significantly reduced infarct size and increased mass of viable
myocardium within the anterior wall.
DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS
I. Experimental Procedures
[0018] All protocols used in the present invention conformed to the
Guide for the Care and Use of Laboratory Animals published by the
U.S. National Institutes of Health, and were approved by the Animal
Experimental Ethical Committee of The Chinese University of Hong
Kong.
[0019] Isolation of Ga from Geum Japonicum: For the experiments
disclosed in the following, Ga was obtained from the plant of Geum
Japonicum. Referring to FIG. 1, the plant was collected from
Guizhou Province of China in August was dried (10 kg) and
percolated with 70% ethanol (100 L) at room temperature for 3 days
twice. The extract was combined and spray-dried to yield a solid
residue (1 kg). The solid residue was suspended in 10 liter
H.sub.2O and successively partitioned with chloroform (10 L) twice,
then n-butanol (10 L) twice to produce the corresponding fractions.
The n-butanol (GJ-B) soluble fraction was filtered and spray dried
to yield a powder fraction. It was shown that n-BuOH soluble
fraction could significantly enhance the proliferation of
HCAECs-hunan coronary artery endothelial cells (Clonetics, Inc.)
and stimulate rapid neovascularization in infarct zone of MI animal
model. The n-BuOH soluble fraction was applied on a column of
Sephadex LH-20 equilibrated with 10% methanol and eluted with
increasing concentration of methanol in water, resolving 7
fractions. Fraction 3, eluted with approximately 50% methanol,
showed the potent activity in stimulating significant angiogenesis
in infarcted myocardium. This fraction 3 containing tannins was
used to test its healing effects on a MI animal model. The
structures of the active compounds contained in this active
fraction were determined by NMR analysis. Of course, as Ga is a
known natural occurring compound, it may be obtained from other
plants and produces satisfactory results.
[0020] Animals, surgical procedures: Male Sprague-Dawley (SD) rats,
weighing 250-300 g were used. Following proper anesthesia, a left
thoracotoiny was performed on the animals, the pericardium was
opened and the left anterior descending (LAD) coronary artery was
ligated. Ga dissolved in PBS (0.1 ml, containing 0.3 mg Ga) was
injected into the distal myocardium (the presumed ischemic region)
of the ligated artery immediately after the ligation in the test
group (having 60 rats, i.e., n=60). An equivalent volume of PBS was
injected to the corresponding location of the rats in the control
group (n=60). Fifteen rats of each group were euthanatized on day
2, 7, 14 and 30 post-infarct for morphological and functional
assessment. For the sham group (n=6), left thoracotomy was
performed and the pericardium was opened but with no LAD ligation.
For the normal control group (n=6), the rats were not subject o any
surgical procedures and treatments.
[0021] Measurement of neovascularization in the infarct zone: Left
ventricles from the rats sacrificed on day 2 and 7 post-infarction
were removed and sliced from apex to base in 3 transverse slices.
The slices were fixed in formalin and embedded in paraffin.
Vascular density was determined on the histology section samples by
counting the number of vessels within the infarct zone using a
light microscope under a high power field (HPF) (.times.400). Eight
random and non-overlapping HPFs within the infarct filed were used
for counting all the vessels in each section. The number of vessels
in each HPF was averaged and expressed as the number of vessels per
HPF. Vascular counts were performed by two investigators in a blind
fashion.
[0022] Measurement of myocyte apoptosis by TUNEL assay of paraffiin
tissue sections: The TUNEL assay method was used for in situ
detection of apoptosis at the single-cell level.sup.9. Rat
myocardial infarction tissue sections were obtained from both the
test group and the control group on day 7 post-infarction. After
general deparaffinization and rehydration, tissues were digested
with Proteinase K (Dako) for 15 minutes and incubated with TdT
(Roche) and Biotin-16-dUTP (Roche) for 60 minutes at 37.degree. C.
After incubation with SP-HRP (Roche) for 20 minutes, the TUNEL
staining was visualized with DAB (Dako), which stained the nuclei
(with DNA fragmentation stained brown). Tissue sections were
examined microscopically at a high power field (.times.400) and at
least 100 cells were counted in a minimum of 10 HPF. The number of
the apoptotic myocytes per HPF was referred to as the apoptotic
index.
[0023] Estimation of the myocardial infarction: The hearts of the
rats, sacrificed on day 14 post infarction, were removed and
sectioned from apex to base in three to four transverse slices and
embedded in paraffin. Thin sections (5 .mu.m thick) were cut from
each slide and stained with H&E staining and Masson's trichrome
(Sigma, USA), which labels collagen blue and myocardium red. These
sections from all slices were projected onto a screen for
computer-assisted planimetry (ImageJ 1.34S, Wayne Rasband, National
Institutes of Health, USA). The endocardial and epicardial
circumferences as well as the length of the scar were measured for
each slice. The infarcted portion of the left ventricle was
calculated from these measurements and the ratio of scar length to
ventricular circumference of the endocardium and epicardium of the
slices was expressed as a percentage to define the infarct
size.sup.9,10,11.
[0024] Echocardiography Assessment of Myocardial Function: In all,
118 SD rats received baseline echocardiography before any
experimental procedures. Echocardiography was recorded under
controlled anesthesia using a S10-MHz phased-array transducer and
GE VingMed Vivid 7 system. M-mode tracing and 2-dimensional (2D)
echocardiography images were recorded from the parasternal long-
and short-axis views. Short axis view was at the papillary muscles
level. Left ventricular end-diastolic (LVDA) and end-systolic
(LVSA) areas were planimetered from the parasternal long axis and
LV end-diastolic and end-systolic volumes (LVEDV and LVESV) were
calculated by the M-mode method. LV ejection fraction (LVEF) and
fractional shortening (FS) were derived from LV cross-sectional
area in 2D short axis view: EF=[(LVEDV-LVESV)/LVEDV].times.100% and
FS=[(LVDA-LVSA)/LVDA].times.100% .sup.12. Standard formulae were
used for echocardiographic calculations.
[0025] RT-PCR analysis of survival associated gene expressions: A
small slice from the above prepared infarcted myocardial tissue
were put into liquid nitrogen immediately after incision and stored
at -80.degree. C. According to manufacturer's instructions, total
RNA was isolated using Qiagen RNeasy Mini Kit (Catalog Number
74104, Qiagen, Germany), dissolved in 20-30 .mu.l RNase free water
and stored at -80.degree. C. The integrity of the ribosomal RNA and
DNA contamination was checked routinely using formaldehyde
denaturing RNA gel electrophoresis (1.2%) before proceeding with
the further analysis. Protein contamination and concentration of
the total RNA was assessed by determining the ratio OD260:0D280
spectrophotometrically (Eppendorf BioPhotometer, Hamburg,
Germany).
[0026] Western BlotAnalysis. About 50 mg of the above prepared
infarcted myocardial tissue were grinded to powder in liquid
nitrogen. 1 mL lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1% Nonidet P-40, 10% glycerol, 200 mM NaF, 20 mM
sodium pyrophosphate, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 200
mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate)
was added to the powder and put on ice for 30 min. Protein yield
was quantified by Bio-Rad DC protein assay kit (Bio-Rad). Equal
amounts (10 g) of total protein were size-fractionated by SDS-PAGE
and transferred to PVDF membranes (Amersham, USA). The blots were
blocked with phosphate-buffered saline plus 0.1% (vol/vol) Tween 20
(PBST) containing 5% (wt/vol) milk powder (PBSTM) for 30 min at
room temperature and probed for 60 min with specific primary
antibodies against rat phospho-Akt1 (mouse, Santa Cruz) or rat
Bcl-2 (mouse, Sigma-Aldrich), diluted 1:1000 in PBSTM. After
washing extensively in PBST, the blots were probed by horseradish
peroxidase-coupled anti-mouse IgG (Amersham Biosciences) (1/1000
dilution in PBSTM, 60 min), extensively washed with PBST, and
developed by chemiluminescence.
[0027] Biostatistics: All morphometric data were collected blindly.
Results are presented as mean.+-.SD computed from the average
measurements obtained from each heart. Statistical significance for
comparison between two measurements was determined using the
unpaired two-tailed Student's t test. Values of P<0.05 were
considered to be significant.
II. Ga-Induced Revascularization in Infarcted Myocardium
[0028] Referring to FIG. 2, histology studies revealed that many
vessels were newly formed throughout the entire infarct zone,
including the central areas and the border zones on day 2 post
infarction (FIG. 2:1), where the newly formed vessels are pointed
to by red arrowheads). Some of the newly formed vessels were filled
with blood cells and others were still at the early stage of the
vessel regeneration development and displayed as a lumen like
structure without filling of blood cells. The capillary density in
the infarct zone of the Ga treated myocardium was on average 18
(18.+-.3.9) filling with blood cells and 8 (8.+-.2.8) lumen-like
structures per HPF, calculated from 8 randomly selected view fields
on each of the 15 slides from 15 Ga treated hearts on day 2 (FIG.
2: 1). By contrast, fewer blood vessels (5.+-.2.1 per HPF) with an
inflammatory cell infiltration were observed in the infarct zone in
the control myocardium on day 2 post MI (FIG. 2: 2). In Ga treated
hearts, on day 7 post MI, the newly formed blood vessels filled
with blood cells remained (11.+-.3.6) throughout the infarct zone
but the lumen-like structures were not observed (FIG. 2: 3). By
contrast, the control samples showed mainly fibrous tissue
replacement of the infarcted myocardium with only a few of blood
vessels (3.+-.1.2) at 7-day post infarction (FIG. 2: 4). RT-PCR and
Western blots analysis demonstrated that the Ga-induced
revascularization within 24 hours in infarcted myocardium was
concomitantly accompanied with the up-regulated gene expressions of
VEGF and bFGF in the corresponding heart tissues. The expressions
of VEGF and FGF in the Ga-treated myocardium were increased to 1.8
and 2.2 folds respectively (FIG. 2: 5 & 6, T) compared with
their expressions in non-treated myocardium of control group (FIG.
2: 5 & 6, C).
III. Ga-Enhanced Survival Potential and Reduction of Infarct
Size
[0029] Referring to FIG. 3, seven days after LAD ligation, the
myocytes at risk along peri-infarct rim of the controls (FIG. 3: 1)
showed distorted and irregular shapes compared with the myocytes at
distal part of the heart. By contrast, the myocytes at the
peri-infarct rim of the Ga-treated hearts showed a regular shape
(FIG. 3: 2) and the myofibers remain healthy and not as narrow and
thin as in the non-treated heart. With the staining of TUNEL, it
was found that number of apoptotic myocytes detected in the
Ga-treated left ventricle myocardium (FIG. 3: 2) was approximately
3-fold lower compared with the non-treated controls (per high power
field: 1.70.+-.0.18 versus 5.04.+-.0.75, P<0.001; FIG. 3: 1).
These differences were particularly evident within the peri-infarct
rim, where the irregularly shaped myocytes in the control hearts
had the highest number of apoptotic nuclei, which were stained
brown. Most of the apoptotic nuclei were observed at the
peri-infarct rim rather than the myocytes distal to the infarct
zone. Furthermore, significantly higher density of capillaries
surrounded by the myocytes with much less apoptotic nuclei was
found in the infarct zone of the Ga-treated hearts. By contrast,
significantly lower density of capillaries and more apoptotic
nuclei were observed in the non-treated hearts of the control
group. Together, these results indicate that the angiogenesis
induced by Ga-treatment prevented an extending pro-apoptotic
process in both myocytes and endothelial cells, enhanced survival
of the viable myocytes and endothelial cells within the
peri-infarct zone and consequently improved myocardial function. In
order to determine whether the Ga-induced anti-apoptotic effect on
the viable myocytes at risk was through expressions of
anti-apoptotic proteins, western blots analysis were performed. It
was demonstrated that the Ga-induced prevention of extending
pro-apoptotic process of heart tissue at risk were concomitantly
accompanied by increased gene expressions of key survival factors.
The expressions of Akt1 (FIG. 3: 3, T) and Bcl2 (FIG. 3: 4, T) were
increased by 3.3 and 2.8 folds respectively compared with the heart
tissues in the control group (FIG. 3 & 4, C).
[0030] In order to investigate whether the increased survival
potential of the viable myocytes and endothelial cells within the
peri-infarct zone induced by Ga would result in reduction of
infarct size, the infarct sizes of different animal groups were
measured. As shown in FIG. 3, the mean proportion of collagenous
deposition or scar tissue/left ventricular myocardium (as defined
by Masson's Trichrome stain) was 27.44% in rats treated by Ga (FIG.
3: 5), compared with 39.53% for those in the control group (FIG. 3:
6)14-day post infarction, indicating that Ga-enhanced survival
potential of both myocytes and endothelial cells significantly
increased the mass of viable myocardium within the anterior free
wall of left ventricles. The Ga-treatment-induced reconstitution of
damaged coronary vasculature and reduction of the infarct size were
accompanied by significant functional improvement, as demonstrated
in the echocardiography measurements where, in comparison with
non-treated control MI hearts on day 7 and 14 post infarction,
ejection fraction (EF) of the Ga-treated MI hearts was
significantly higher (55.68.+-.2.63 vs 49.67.+-.2.78, P=0.03) on
day 7, and significantly increased (60.11.+-.2.66 vs 48.26.+-.2.55,
P=0.001) on day 14. Similarly, fraction shortening (FS) of the
Ga-trated MI heart were significantly higher (27.33.+-.1.63 vs
22.17.+-.1.67, P=0.01) on day 7 and was significantly increased
(29.87.+-.2.66 vs 21.35.+-.2.08, P=0.002) on day 14.
[0031] In summary, the above examples demonstrate that Ga is
capable of up-regulating the expressions of VEGF and bFGF for early
reconstitution of blood supply network, inducing expression of
anti-apoptotic proteins-Akt1 and Bcl2 for preventing apoptotic
death of cardiomyocytes at risk, and bringing about significant
functional improvement of the heart suffering an ischemic event.
Thus, Ga provides a new dimension, as a therapeutic angiogenesis
medicine, in the treatment of ischemic heart diseases.
IV Manufacturing Pharmaceutical Compositions and their Uses in
Treating Ischemic Heart Diseases in Mammals
[0032] Once the effective chemical compound is identified and
partially or substantially pure preparations of the compound are
obtained either by isolating the compound from natural resources
such as plants or by chemical synthesis, various pharmaceutical
compositions or formulations can be fabricated from partially or
substantially pure compound using existing processes or future
developed processes in the industry. Specific processes of making
pharmaceutical formulations and dosage forms (including, but not
limited to, tablet, capsule, injection, syrup) from chemical
compounds are not part of the invention and people of ordinary
skill in the art of the pharmaceutical industry are capable of
applying one or more processes established in the industry to the
practice of the present invention. Alternatively, people of
ordinary skill in the art may modify the existing conventional
processes to better suit the compounds of the present invention.
For example, the patent or patent application databases provided at
USPTO official website contain rich resources concerning making
pharmaceutical formulations and products from effective chemical
compounds. Another useful source of information is Handbook of
Pharmaceutical Manufacturing Formulations, edited by Sarfaraz K.
Niazi and sold by Culinary & Hospitality Industry Publications
Services.
[0033] As used in the instant specification and claims, the term
"plant extract" means a mixture of natural occurring compounds
obtained via an extracting process from parts of a plant, where at
least 10% of the total dried mass is unidentified compounds. In
other words, a plant extract does not mean an identified compound
substantially purified from the plant. The extracting process
typically involves a step of immersing raw plant part(s) in a
solvent (commonly, water and/or an organic solvent) for a
predetermined length of time, optionally separating the solution
from the plant debris and then removing the solvent from the
solution, to afford an extract, which may further optionally
undergo concentration and/or partial purification. The term
"pharmaceutical excipient" means an ingredient contained in a drug
formulation that is not a medicinally active constituent. The term
"an effective amount" refers to the amount that is sufficient to
elicit a therapeutic effect on the treated subject. Effective doses
will vary, as recognized by those skilled in the art, depending on
the types of diseases treated, route of administration, excipient
usage, and the possibility of co-usage with other therapeutic
treatment. A person skilled in the art may determine an effective
amount in a particular situation using conventional method known in
the art.
V. References
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[0046] While there have been described and pointed out fundamental
novel features of the invention as applied to a preferred
embodiment thereof, it will be understood that various omissions
and substitutions and changes, in the form and details of the
embodiments illustrated, may be made by those skilled in the art
without departing from the spirit of the invention. The invention
is not limited by the embodiments described above which are
presented as examples only but can be modified in various ways
within the scope of protection defined by the appended patent
claims.
* * * * *