U.S. patent application number 14/843898 was filed with the patent office on 2016-03-03 for pharmaceutical composition and method for regenerating myofibers in the treatment of muscle injuries.
The applicant listed for this patent is LEAD BILLION LIMITED. Invention is credited to Lei CHENG, Ming LI, Hong-Wei LIU.
Application Number | 20160058797 14/843898 |
Document ID | / |
Family ID | 37967417 |
Filed Date | 2016-03-03 |
United States Patent
Application |
20160058797 |
Kind Code |
A1 |
LI; Ming ; et al. |
March 3, 2016 |
PHARMACEUTICAL COMPOSITION AND METHOD FOR REGENERATING MYOFIBERS IN
THE TREATMENT OF MUSCLE INJURIES
Abstract
A pharmaceutical composition and method for regenerating
cardiomyocytes in treating or repairing heart muscle damages or
injuries caused by an ischemic disease. The pharmaceutical
composition contains an active ingredient compound with a backbone
structure of Formula (I). The active ingredient compound is capable
of (a) increasing viability of myogenic precursor cells to enable
said precursor cells to survive through an absolute ischemic
period; (b) reconstituting a damaged blood supply network in said
heart region where said injured muscle is located; and (c)
enhancing cardiomyogenic differentiation efficiency of said
precursor cells down cardiac linage, said steps being performed
simultaneously or in any particular order.
Inventors: |
LI; Ming; (Shatin, HK)
; CHENG; Lei; (Shatin, HK) ; LIU; Hong-Wei;
(Beijing, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LEAD BILLION LIMITED |
Kowloon Bay |
|
HK |
|
|
Family ID: |
37967417 |
Appl. No.: |
14/843898 |
Filed: |
September 2, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11722915 |
Jun 27, 2007 |
9155744 |
|
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PCT/CN2006/002885 |
Oct 27, 2006 |
|
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14843898 |
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60791462 |
Apr 13, 2006 |
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Current U.S.
Class: |
424/93.7 ;
514/33; 514/557 |
Current CPC
Class: |
A61K 31/704 20130101;
A61P 9/04 20180101; C07C 62/32 20130101; A61P 9/10 20180101; A61K
31/192 20130101; A61P 9/00 20180101; A61P 43/00 20180101; A61K
31/56 20130101; A61K 35/34 20130101 |
International
Class: |
A61K 35/34 20060101
A61K035/34; A61K 31/192 20060101 A61K031/192; A61K 31/704 20060101
A61K031/704 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 27, 2005 |
IB |
PCT/IB2005/003191 |
Oct 27, 2005 |
IB |
PCT/IB2005/003202 |
Claims
1. A method of regenerating myocytes or myocardia in the heart of a
mammalian subject suffering an injured heart muscle, comprising a
step of administering an effective amount of a compound with a
backbone structure showing in formula (I) or a functional
derivative of said compound. ##STR00004##
2. The method of claim 1, wherein said compound is said backbone
structure itself without any substitution.
3. The method of claim 1, wherein said compound is selected from
the group consisting of: ##STR00005## ##STR00006##
4. The method of claim 3, wherein said compound is:
##STR00007##
5. The method of claim 4, wherein said injured heart muscle is
caused by an ischemic event.
6. The method of claim 5, wherein said ischemic event is myocardial
infarction.
7. The method of claim 1, wherein said myocytes or myocardia are
regenerated in a process comprising one or more steps of (a)
increasing viability of myogenic precursor cells to enable said
precursor cells to survive through an absolute ischemic period; (b)
reconstituting a damaged blood supply network in said heart region
where said injured muscle is located; and (c) enhancing
cardiomyogenic differentiation efficiency of said precursor cells
down cardiac linage, said steps being performed simultaneously or
in any particular order.
8. The method of claim 7, wherein said myogenic precursor cells are
mesenchymal stem cells coming from bone marrow through blood
circulation.
9. The method of claim 1, further comprising the steps of: (a)
obtaining a plurality of stem cells; (b) contacting said stem cells
with said compound or said functional derivative for a period of
time; and (c) transplanting said cells into an infarcted or damaged
heart tissue of said mammalian subject.
10. The method of claim 1, further comprising the steps of: (a)
formulating said compound or said functional derivative into a
dosage form and (b) systematically administering said compound or
said functional derivative in said dosage form to said mammalian
subject.
11. The method of claim 10, wherein said dosage form is selected
from the group consisting of tablet, capsule, injection solution,
syrup, suspension and powder.
12. The method of claim 1, further comprising the steps of: (a)
culturing a plurality of MSCs or endothelial cells in a culture
medium containing said compound or said functional derivative for a
period of time; (b) collecting said culture medium, containing
secretary proteins from said MSCs or endothelial cells; and (c)
administering or delivering said medium to heart tissues in a
infarct area.
13. The method of claim 1, wherein at least 95% by weight of said
composition is identified compounds.
14. The method of claim 1, further comprising adding a piece of
information on usefulness of said compound, wherein said
information indicates that said compound is beneficial to a human
suffering or having suffered a heart disease.
15. The method of claim 1, further comprising adding a piece of
information on usefulness of said compound, wherein said
information indicates that said compound is beneficial to
regenerate myocytes or myocardia in the heart of said mammalian
subject.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 11/722,915, filed Jun. 27, 2007, which is a
.sctn.371 national stage application of PCT/CN2006/002885, and
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 Oct. 27, 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 regenerating myocytes and myocardium for treating muscle
damages. Particularly, it relates to a pharmaceutical composition
and method for regenerating cardiomyocytes in treating or repairing
heart muscle damages or injuries caused by an ischemic disease.
BACKGROUND OF THE INVENTION
[0003] Myocardial infarction (MI), or heart attack, is a disease
due to interruption of the blood supply to a part of the heart,
causing damage or death of heart muscle cells. It is the leading
cause of death for both men and women over the world. Following
myocardial infarction, there does not seem to be any natural
occurring repairing process capable of generating new
cardiomyocytes to replace the lost muscle cells. Instead, scar
tissues may replace the necrosed myocardium, causing further
deterioration in cardiac function.
[0004] Therapeutic replacement of the necrosed heart tissue with
newly regenerated functional cardiac myocytes is a treatment ideal
that until recently has been unrealistic, because cardiac myocytes
were considered to be terminally differentiated, or in other words,
the heart is a postmitotic nonregenerating organ. This dogma,
however, has recently been challenged by Beltrami et al, and
others, who reported that a population of resident myocytes within
the myocard ia can and do replicate after infarction. In order to
promote and improve the repair for infarcted myocardia,
transplantation of cardiomyocytes or skeletal myoblasts has been
attempted, but has not been very successful in reconstituting
functional myocardia and coronary vessels. Transplantation of adult
bone marrow-derived mesenchymal stem cells (MSCs) for cardiac
repair following myocardial infarction has resulted in some
angiogenesis and myogenesis, but the location of the newly
regenerated cardiac myocytes appeared mostly along the border zone
where the blood supply is relatively less affected .sup.1-3.
[0005] Because acute myocardial infarction (MI) brings rapid
damages or death to myocytes (heart muscle cells), vascular
structures and nonvascular components in the supplied region of the
ventricle, regeneration of new cardiac myocytes to replace the
infarcted myocardia (heart muscular tissues) in the central
infarcted zone (the absolute ischemic region) through a
sub-population of cardiac myocyte growth .sup.4-8 or
transplantation of MSCs .sup.1-3 alone appears to be impossible
without early reestablishment of the blood supply network locally.
This probably explains why regeneration of cardiac myocytes
following MSCs transplantation alone occurred mostly along the
border zone adjacent to the infarct where the blood supply is
largely maintained .sup.1-11. Therefore, the loss of myocardia,
arterioles and capillaries in the central infarct area appeared to
be irreversible, eventually leading to scar formation.
[0006] A more recent study .sup.12 reported that heart
transplantation of MSC pre-modified with exogenous Akt in vitro
produced a better result. Nonetheless, the regenerated cardiac
myocytes could only infiltrate from the border zone into the
scarred area, indicating that overexpression of exogenous Akt,
although enhancing the survival potential of the transplanted MSCs,
itself is insufficient to enable them to survive in central
ischemic regions. Furthermore, even in the less-ischemic border
zones, it was noted that the MSCs-derived regenerating cardiac
myocytes were scattered and seemed to have difficulty to cluster
and form regenerating myocardia. This is probably due to poor
cardiomyogenic differentiation efficiency of the survived
transplanted MSCs. The knowledge that natural cardiomyocyte
reproduction, including differentiation of residential progenitor
myocytes or stem cells recruited from other sources, such as from
endothelial cells or a niche in the bone marrow is insufficient to
balance cardiomyocyte death occurred in acute or chronically
damaged heart, has damped the enthusiasm of the researchers who
thought myocardial regeneration would represent a promising method
of treatment against heart diseases.
[0007] The prior art seems to teach that there are three major
requirements critical for regenerating functional myocytes in the
entire areas of infarcted myocardia: 1) increased viability of the
transplanted cells so that they may survive through the absolute
ischemic period, that is, the period from injection of the donor
cells to formation of new vessels; 2) early reconstitution of the
damaged blood supply network in the infarcted myocardia to sustain
the survival and efficient trafficking of the transplanted cells
and maintain oxygenation and nutrient delivery; and 3) enhanced
cardiomyogenic differentiation efficiency of the transplanted cells
to enable more survived donor cells to differentiate down cardiac
linage.
[0008] Therefore, to realize the therapeutic ideal of replacing
necrosed heart tissues with newly regenerated functional cardiac
myocytes, there is a need for new therapeutic approaches, for
example, an approach using chemical compounds possessing biological
properties that sufficiently satisfy the aforementioned three
requirements in order to serve the therapeutic needs for treating
myocardial infarction.
SUMMARY OF THE INVENTION
[0009] As one object of the present invention, there is provided a
pharmaceutical composition comprising a compound selected from the
group of chemical compounds sharing a common backbone structure of
formula (I). The compounds have potent beneficial therapeutic
effects not only on the survival potential and cardiogenic
differentiation efficiency of MSCs ex vivo, but also on repairing
of MI in vivo. These compounds themselves are known in the art but
they are never known as possessing the above biological activities
and therapeutic effects. They may be isolated from natural
resources, particularly from plants or they may also be obtained
though total or semi-chemical syntheses, with existing or future
developed synthetic techniques. The backbone structure itself
possesses the aforementioned myogenic effects and various variants
can be made from the backbone structure through substitution of one
or more hydrogen atoms at various positions. These variants share
the common backbone skeleton and the myogenic effects. Of course,
they may vary in myogenic potency.
##STR00001##
[0010] The backbone structure of Formula (I) may have one or more
substituents attached. A substituent is an atom or group of atoms
substituted in place of the hydrogen atom. The substitution can be
achieved by means known in the field of organic chemistry. For
example, through a proper design, high through-put combinatorial
synthesis is capable of producing a large library of variants or
derivatives with various substituents attached to various positions
of a backbone structure. The variants or derivatives of formula (I)
may then be selected based on an activity test on mesenchymal stem
cells (MSCs), which can quickly determine whether a particular
variant could enhance proliferation and cardiogenic differentiation
of the cultured MSCs. As used in this application, the term "the
compound of formula (I)" encompasses the backbone compound itself
and its substituted variants with similar biological activities.
Examples of these variants are presented in the following, all of
which possess similar effects in terms of regenerating functional
myocytes as the backbone structure (i.e., the base compound
itself):
##STR00002## ##STR00003##
[0011] Furthermore, as a therapeutic agent, the compound of formula
(I) may be in a form of "functional derivatives" as defined
below.
[0012] It is contemplated, as a person with ordinary skill in the
art would contemplate, that the above compounds 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.
[0013] As used in this 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.
[0014] The pharmaceutical composition of the present invention is
useful for treating myocardial injuries or necrosis caused by a
disease, particularly MI, through regenerating heart tissues. The
pharmaceutical composition may be formulated by conventional means
known to people skilled in the art into a suitable dosage form,
such as tablet, capsule, injection, solution, suspension, powder,
syrup, etc, and be administered to a mammalian subject having
myocardial injuries or necrosis. The formulation techniques are not
part of the present invention and thus are not limitations to the
scope of the present invention.
[0015] The pharmaceutical composition of the present invention may
be formulated in a way suitable for oral administration, systemic
injection, and direct local injection in the heart or implantation
in a body part for long-term slow-releasing.
[0016] In another aspect, present invention provide a method for
treating or ameliorating a pathological condition in a mammal,
where the pathological condition, as judged by people skilled in
medicine, can be alleviated, treated or cured by regenerating
functional cardiomyocytes and where the method comprises
administering to the mammal with the pathological condition a
therapeutically effective amount of a compound of formula (I) and
or its functional derivatives.
[0017] In another aspect, the present invention provides a method
of regenerating functional cardiomyocytes in a mammal who needs to
replace dead or damaged heart tissues caused by a heart disease,
such as, myocardial infarction (MI). This is a cell-transplantation
based therapeutic approach, involving the steps of: (a) obtaining
stem cells, such as MSCs; (b) contacting the stem cells with a
compound of the formula (I) or their functional derivatives to
activate the pathways of cardiogenic differentiation prior to
transplantation and (c) then transplanting the activated cells into
the infarcted heart tissues of the mammal. This therapeutic
approach is capable of achieving the following goals: 1) enhanced
survival potential of the transplanted cells; 2) early
reconstitution of blood supply network, and 3) enhanced
cardiomyogenic differentiation efficiency of the transplanted cells
by ex vivo activation of MSCs forming cardiogenic progenitors prior
to transplantation.
[0018] In another aspect, the present invention provides a method
for treating ischemic heart diseases, particularly MI in mammals,
which comprising the steps: (a) culturing MSCs or endothelial cells
with a compound of formula (I) or their functional derivatives, (b)
gathering the conditioned medium of the treated cells, which
contains secretary proteins that are active in driving heart
infarction repair or cardiogenic differentiation of MSCs, and (c)
administering or delivering the conditioned medium to the heart
tissue in the infarct area.
[0019] In still another aspect, the present invention provides a
research reagent for scientific research on cardiogenic
transdifferentiation of stem cells, such as MSCs. The reagent
comprises one or more compounds of formula (I) or their functional
derivatives. It may be in a solid form or a liquid form. For
example, it may be a solution of DMSO.
[0020] 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
[0021] FIG. 1 outlines the process of isolating Niga-ichigoside Fl
(referred to as "CMF") from the plant of Geum Japonicuin as an
example of making the compound of the present invention.
[0022] FIG. 2 shows the effects of CMF on cardiogenic
differentiation of the MSCs and up-regulation of phospho-Akt1
expression ex vivo.
[0023] FIG. 3 shows the therapeutic effect of a treatment based on
transplantation of CMF-pretreated MSCs.
[0024] FIG. 4 shows distribution ejection fraction (EF) and
fractional shortening (FS) after 2 days and 2 weeks cell
transplantation in three groups of rats (A: normal group; B: MI
group transplanted with CMF-treated MSCs; C: MI group transplanted
with MSCs not treated with CMF).
[0025] FIG. 5 shows the therapeutic effects of CMF on myocardial
infarction (MI) animal model.
[0026] FIG. 6 shows the enhanced proliferation of cultured MSCs and
myocardial regeneration induced by conditioned medium.
[0027] FIG. 7 shows the cellular source for CMF-induced myocardial
regeneration in animal MI model.
DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS
I. Experiment Procedures
[0028] 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.
[0029] For the following discussion, CMF refers to the base
compound (or backbone compound) of the present invention. Its
chemical structure is defined by formula (I) shown in the
above.
[0030] Obtaining Compounds of the Present Invention:
[0031] The compounds can be prepared from plants, although it may
possible to make it through chemical synthesis.
[0032] As an example for illustrating the process of preparing the
compounds from natural resources, the following provides details
involved in CMF's isolation and purification from one plant
species, Geum Japonicum. Other plants that may contain CMF or
variants include, for example, Acaena pinnatifida R. et P.,
Agrimonia pilosa Ledeb, Asparagus filicinus, Ardisia japonica,
Campsis grandiflora, Campylotropis hirtella (Franch. Schindl.),
Caulis Sargentodoxae, Cedrela sinensis, Chaenomeles sinensis
KOEHNE, Debregeasia salicifolia, Eriobotrya japonica calli,
Eriobotrya japonica LINDL. (Rosaceae), Goreishi, Leucoseptrum
stellipillum, Ludwigia octovalvis, Perilla frutescens, Perilla
frutescens (L.) Britt. (Lamiaceae), Physocarpus intermedius
Potentilla multifida L., Poterium ancistroides, Pourouma guianensis
(Moraceae), Rhaponticum uniflorum, Rosa bella Rehd. et Wils., Rosa
laevigata Michx, Rosa rugosa, Rubus alceaefolius Poir, Rubus
allegheniensis, Rubus coreanus, Rubus imperialis, Rubus imperialis
Chum. Schl. (Rosaceae), Rubus sieboldii, Rumex japonicus, Salvia
trijuga Diels, Strasburgeria robusta, Strawberry cv. Houkouwase,
Tiarella polyphylla, Vochysia pacifica Cuatrec, Zanthoxylum
piperitum, etc.
[0033] Isolation of Cardiomyogenic factor (CMF) from Geum
japonicum:
[0034] Referring to FIG. 1, the plant of Geum japonicum 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 dried by
spray drying to yield a powder fraction, which was confirmed for
their specific ability to stimulate cardiogenic differentiation of
MSCs in cell culture in a way described below. It was shown that
n-butanol soluble fraction (GJ-B) could enhance the proliferation
and cardiogenic differentiation of the cultured MSCs in cell
culture systems. The GJ-B fraction was then applied on a column of
Sephadex LH-20 equilibrated with 10% methanol and eluted with
increasing concentration of methanol in water, resolving 7
fractions, GJ-B-1 to GJ-B-7. All the eluted fractions were tested
for their activity with MSC culture systems. Activity test
demonstrated that fraction 6 was most active in enhancing the
cardiogenic differentiation of cultured MSCs. From GJ-B-6, a pure
active compound was further isolated, which is referred to as CMF
through this disclosure. CMF's structure was determined by NMR
analysis and comparison with literature, and shown to be of formula
(I).
[0035] Preparation of MSCs for Transplantation:
[0036] The MSCs were cultured with CMF (10 .mu.g/ml in growth
medium) for 6 days. In parallel, the control MSCs were cultured in
growth medium containing equivalent volume of 5% DMSO. On day 2,
expression of endogenous phospho-Akt1 was assessed by
immunocytochemistry and Western blot. On day 4, myogenic
differentiation was assessed by immunocytochemistry and Western
blot against MEF2, which were further confirmed by
immunocytochemistry and Western blot with an antibody specific to
MHC on day 6. On day 3, both the CMF-pretreated MSCs and the
control MSCs were labeled with CM-DiI in culture and made ready for
transplantation.
[0037] Preparation of Bone Marrow Mesenchymal Stein Cells:
[0038] The tibias/femur bones were removed from Sprague-Dawley (SD)
rats and the bone marrow (BM) was flushed out of the bones with
IMDM culture medium containing 10% heat inactivated FBS (GIBCO) and
1% penicillin/streptomycin. The BM was thoroughly mixed and
centrifuged at 1500 rpm for 5 minutes. The cell pellet was
suspended in 5 ml growth medium. The cell suspension was carefully
put on 5 ml Ficoll solution and centrifuged at 200 rpm for 30 min.
The second layer, which contains BM cells was transferred into a
tube and washed twice with PBS to remove Ficoll (1200 rpm for 5
minutes). The cell pellet was resuspended in IMDM culture medium
containing 10% heat inactivated FBS (GIBCO) and 1%
penicillin/streptomycin antibiotic mixture. After 24 hours culture
in a 37.degree. C. incubator with 5% CO.sub.2, the non-adherent
cells are discarded and the adherent cells are cultured by changing
medium once every 3 days and the cells became nearly confluent
after 14 days of culture. This was the BM cells, referred to as
MSCs in the following, which were used for in vitro and in vivo
studies conducted in the present disclosure.
[0039] Western Blot Analysis:
[0040] Whole cell extracts of the CMF-treated cells or control
cells were prepared by lysing the cells with 3 times packed cell
volume of 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) on
ice for 30 minutes. Protein yield was quantified by Bio-Rad DC
protein assay kit (Bio-Rad). Equal amounts (30 .mu.g) of total
protein were size-fractionated by SDS-PAGE and transferred to PVDM
membranes (Millipore). 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 minutes at room
temperature and probed for 60 minutes with specific primary
antibodies against rat phospho-Akt1 (mouse) or rat MHC (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.
[0041] Transplantation of the CMF Pretreated MSCs to the Heart
Tissue:
[0042] The Sprague-Dawley (SD) rats were used and all animal
procedures were approved by the University Animal Committee on
Animal Welfare. Each rat was anesthetized with intraperitoneal
pentobarbital (50 mg/kg), intubated, and mechanically ventilated
with room air using a Harvard ventilator (model 683). After a left
thoracotomy, myocardial infarction was induced by permanent
ligation of left anterior descending (LAD) coronary artery. The
5.times.10.sup.5 DiI labeled CMF-pretreated MSCs (32 rats)
suspended in saline were injected into three sites of the distal
myocardia (the ischemic region) of the ligated artery immediate
after the ligation respectively (test group). The control rats were
injected with an equivalent amount of DiI labeled non-treated
control MSCs (32 rats) suspended in saline at the same location and
timing. For sham ischemia (32 rats), thoracotomy was performed
without LAD ligation. Sixteen rats subject to no-treatment were set
as normal control.
[0043] Half of the experimental rats from different groups were
sacrificed according to experimental plan on day 7 and day 14
post-infarction after assessment of their heart function by
echocardiography measurements. The hearts of the sacrificed rats
were removed, washed with PBS and photographed respectively. All
the specimens harvested were paraffin embedded and sectioned for
tracing the signals of DiI and examination of revascularization,
infarct size and regeneration of myocardia. If the regenerating
cells were DiI positive, further MHC immonohistochemical staining
was performed to confirm their cardiomyogenic differentiation.
[0044] Colocalization of the DiI label and cardiac-specific marker
expression were examined with a confocal microscope (ZEISS, LSM 510
META). Briefly, the sections were immunohistochemically stained
with rat-specific troponin I antibodies. The confirmation of
cardiomyogenic differentiation of the DiI labeled transplanted MSCs
forming regenerating myocardia was carried out by merging the
DiI-positive cells, indicating their donor cell origin, with the
specific positive staining of cardiac terminal differentiation
marker-troponin I using confocal microscopic examination, implying
their cadiomyogenic differentiation of these transplanted
cells.
[0045] CMF Direct Treatment in MI Model:
[0046] Thirty-two SD rats were randomly divided into four groups:
normal group, sham group, CMF-treated group and non-treated control
group (8 rats each). Rats were anesthetized with intraperitoneal
pentobarbital (50 mg/kg), intubated, and mechanically ventilated
with room air using a Harvard ventilator (model 683). After a left
thoracotomy, myocardial infarction was induced by permanent
ligation of left anterior descending (LAD) coronary artery. CMF in
5% DMSO (0.1 ml, containing 0.1 mg CMF) was injected into the
distal myocardium (the ischemic region) of the ligated artery in 8
rats immediate after the ligation (CMF-treated group). Another 8
rats were injected with an equivalent amount of 5% DMSO at the same
location and timing as non-treated control group. For sham
ischemia, thoracotomy was performed on 8 rats without LAD ligation.
Further 8 rats without any treatment were set as normal group.
[0047] Conditioned Medium Containing Secretary Proteins from MSCs
or Other Cells Induced by CMF:
[0048] The MSCs were treated with 10 ug/ml CMF for 24 hours to
activate/upregulate gene expressions and then washed thoroughly to
remove residue of CMF. Then 5 ml of fresh growth medium was added
to the culture and collected after another 3 days of culturing. The
collected medium was referred to as conditioned medium. The 5 ml of
conditioned medium was condensed to a volume of 1 ml, and was used
as a treatment agent in the heart infarction animal model as
described above. Briefly, after a left thoracotomy and ligation of
LAD, 0.2 ml of the conditioned medium was injected immediately into
the distal part of ligation. Fresh growth medium was used as
control.
[0049] Bone Marrow Replacement with DiI Labeled MSCs:
[0050] Sixteen 5-week old SD rats were used for bone marrow
transplantation. Recipient rats were irritated by 9.5 Gy of gamma
irradiation from a 137Cs source (Elite Grammacell 1000) at a dosage
of 1.140 Gy/min to completely destroy the bone marrow derived stem
cells of the rat. DiI-labeled MSCs (2.times.10.sup.8 cells
suspended in 0.3 ml PBS) were then injected through tail vein
within 2 hours after irritation using a 27-gauge needle. One week
after irritation and transplantation, the rats with DiI-labeled
bone marrow were divided into two groups: one to be treated
directly with CMF and the other as control without treatment. Heart
infarction surgery and treatment scheme were performed as described
above. The experiment was terminated on day 14 post surgery and
treatment for further assessment. Heart specimens of the sacrificed
rats were obtained. All the specimens were traced for DiI positive
cells and their cardiomyogenic differentiation by
immunohistochemical staining with specific antibodies for heart
type troponin I (Santa Cruz) and PCNA (Dako). Specific secondary
antibody conjugated with alkaline phosphatase (Santa Cruz) was used
to visualize the positively stained cells. DiI positive signal was
observed with a fluorescence microscope (Laica)
[0051] Estimation of Infarct Size:
[0052] Left ventricles from experimental rats sacrificed on day 14
were removed and sliced from apex to base in 3 transverse slices.
The slices were fixed in formalin and embedded in paraffin.
Sections (20 .mu.m thickness) of the left ventricle were stained
with Masson's trichrome, which labels collagen blue and myocardium
red. These sections were digitized and all blue staining was
quantified morphometrically. The volume of infarct (mm.sup.3) of a
particular section was calculated based on the thickness of the
slice. The volumes of infarcted tissue for all sections were added
to yield the total volume of the infarct for each particular heart.
All studies were performed by a blinded pathologist.
[0053] Angiogenic Assessment in Infarct Region:
[0054] Vascular density was determined on day 7 postinfarction from
histology sections by counting the number of vessels within the
infarct area using a light microscope under high power field (HPF)
(.times.400). Six random and non-overlapping HPFs within the
infarct filed were used for counting all newly formed vessels in
each section of all experimental hearts. The number of vessels in
each HPF is averaged and expressed as the number of vessels per
HPF.
[0055] Assessment of regenerating cardiac myocytes and myocardia:
The sections from both CMF pretreated MSCs-transplanted and
non-treated MSCs-transplanted groups on day 7 post-ligation were
stained with Ki67 or myosin heavy chain (MHC) antibodies to
identify the regenerating myocardia. Specific secondary antibody
conjugated with alkaline phosphatase was used to visualize the
positive stains. Briefly, paraffin-embedded sections were
microwaved in a 0.1 M EDTA buffer and stained with a polyclonal
rabbit antibody with specificity against rat Ki67 at 1:3,000
dilution (Sant Cruz Biotechnology) and incubated overnight at
4.degree. C. After they were washed, the sections were incubated
with a goat anti-rabbit IgG secondary antibody conjugated with
alkaline phosphatase at 1:200 dilution (Sigma) for 30 min, and the
positive nuclei were visualized as dark blue with a
5-bromo-4-chloro-3-indolylphosphate-p-toluidine-nitro blue
tetrazolium substrate kit (Dako). The immediate neighbor sections
from corresponding paraffin tissue block were incubated overnight
at 4.degree. C. in a 1:50 dilution of rabbit anti-rat MHC (MF20,
Developmental Hybridoma Bank, University of Iowa) antibodies, and
further incubated for 30 minutes at room temperature in a 1:100
dilution of peroxidase-conjugated goat anti-rabbit IgG (Sigma).
After incubation with 1 mg/ml 3,3'-diaminobenzidine (DAB; plus
0.02% H.sub.2O.sub.2), the slides were investigated by microscopic
analysis. The regenerating myocardium area was delineated in the
projected field by a grid containing 42 sampling points.
Approximately, 30-60 calculating points along the border of a
particular regenerating myocardium were selected in each section.
This grid defined an uncompressed tissue area of 62,500
.mu.m.sup.2, which was used to measure the selected 30-60
calculating points in each section. The shapes and volumes of
regenerating myocardia in the central area of infarct were
determined by measuring in each section (50 .mu.m apart) of
approximately 70 sections the shapes and areas occupied by the
regenerating myocardia and section thickness. Integration and
calculation with these variables produced a stereo-structure and
yields the volume of a particular regenerating myocardium in the
central area of the infarct in each section. Values and stereo
structure of all sections of a particular tissue block were added
and computed to obtain the total volume and the full
stereo-structure of the regenerating myocardia.
[0056] Echocardiography Assessment of Myocardial Function:
[0057] Echocardiographic studies were performed using a Sequoia
C256 System (Siemens Medical) with a 15-MHz linear array
transducer. The chest of experimental rats was shaved, the animal
was situated in the supine position on a warming pad, ECG limb
electrodes were placed, and echocardiography was recorded under
controlled anesthesia. Each experimental rat received a baseline
echocardiography before the experimental procedure. Two-dimensional
guided M-mode and two-dimensional (2D) echocardiography images were
recorded from parasternal long- & short-axis views. Left
ventricular (LV) end-systolic and end-diastolic dimensions, as well
as systolic and diastolic wall thickness were measured from the
M-mode tracings by using the leading-edge convention of the
American Society of Echocardiography. LV 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%. Standard formulae were used for
echocardiographic calculations. All data were analyzed offline with
software resident on the ultrasound system at the end of the study.
All measured and calculated indexes were presented as the average
of three to five consecutive measurements.
[0058] Statistics:
[0059] All morphometric data are collected blindly, and the code is
broken at the end of the experiment. Results are presented as
mean.+-.SD computed from the average measurements obtained from
each heart. Statistical significance for comparison between two
measurements is determined using the unpaired two-tailed Student's
t test. Values of P<0.05 are considered to be significant.
II. CMF-Induced Increased Survival Potential and Cardiogenic
Differentiation of MSCs Ex Vivo
[0060] Referring to FIG. 2, following two days treatment with CMF
(10 .mu.g/ml) in the culture, the expression of phospho-Akt1 was
significantly up-regulated compared with the untreated control as
demonstrated by immunocytochemical staining of the cells with
antibodies specific to phospho-Akt1, positive cells being stained
red mainly in the cytoplasm (FIG. 2a: 1). Western blot confirmed
that the increased expression of phospho-Akt1 up to 3-4 folds over
untreated cells (FIG. 2b: 5A). Among the phospho-Akt1 up-regulated
MSCs, more than 90% of them when cultured for 2 additional days
became positively stained with the antibody specific to myocyte
enhancer factor 2 (MEF2), one of the earliest markers for the
cardiogenic lineage, positive cells being stained orange in the
nuclei (FIG. 2a: 2) and confirmed by Western blot (FIG. 2b: 6A),
indicating their commitment to cardiogenic differentiation. It was
noted that the cultured MSCs were not all positively stained by
anti-MEF2 antibody (FIG. 2a: 2), as indicated by the blue nucleus
possibly because not all cultured MSCs were converted down the
cardiogenic differentiation pathway by CMF or because some minor
impurities existed in the preparation of MSCs. Similarly, most of
the cultured MSCs were positively stained with the antibody
specific to heart type myosin heavy chain, positive cells being
stained red in the cytoplasm (FIG. 2a: 3) and confirmed by Western
blot (FIG. 2b: 7A) upon 6 days culture in the presence of CMF,
while control cells were negative stained by all three specific
antibodies (FIGS. 2a: 4 & 2b: Bs). The sequential induction of
MEF2 and MHC expressions confirmed the CMF-induced cardiogenic
differentiation development of MSCs ex vivo. In FIG. 2b, A stands
for CMF-treated sample while B for untreated control.
III. Therapeutic Effect Via Transplantation of MSCs Pretreated with
CMF
[0061] To determine whether the increased survival potential and
cardiogenic differentiation efficiency showed ex vivo in MSCs
treated with CMF prior to transplantation would bring about
significant improvement in repairing of MI in vivo, or in other
words, whether CMF's ex vivo effects have any therapeutic values,
cell transplantation experiments with MI animal model were
performed, where MSCs pre-treated with CMF were implanted in the
areas of infarct. The homing, survival, proliferation,
cardiomyogenic differentiation and maturation of the transplanted
cells were traced by the positive signals of either DiI-florescence
and by immunohistochemical staining for Ki67 and MHC in sections,
which were from the hearts on day 7 and day 14 post infarction and
cell transplantation. As shown in FIG. 3, DiI positive cells on day
7 (FIG. 3: 1) with the characteristic phenotype of a cardiac
myocyte were observed in the whole area of the infarct in the test
myocardium group, indicating their donor cell origin and whole
infarct zone distribution. In control group (untreated with CMF),
only scattered DiI signals around the infarct border could be seen
(FIG. 3: 2). The colocalization of DiI signals (red) and cardiac
specific troponin I expression (green) was observed (FIG. 3: 3-5)
in the whole infarct zone by confocal microscopy. The merged image
of DiI-positive (red) and cardiac specific marker troponin I
expression (green) resulted in yellow-red-green overlapping colors
in the same cells, thus confirming the in vivo cardiomyogenic
differentiation and maturation of the transplanted MSCs pretreated
ex vivo with CMF. It was also noted that a few troponin I positive
cells (green) were not DiI positive (FIGS. 3: 3 & 5), probably
because some regenerating myocytes were not derived from the
transplanted DiI-labeled-MSCs. Similarly, a few DiI positive cells
shown in the light blue circles (FIGS. 3: 4 & 5), were negative
in troponin I immunostaining, indicating a small fraction of the
transplanted cells did not commit cardiogenic differentiation in
vivo, or impurities contained in the preparation of MSCs.
[0062] Formation of new vessels could be detected as early as 12
hours after transplantation and many more newly formed vessels and
capillaries filled with blood cells were observed in the whole
infarct areas in the test group in 24 hours (before any
regenerating cardiac myocytes could be seen) and in 7 days post
infarction (FIG. 3: 1, yellow circles). The density of the newly
formed vessels in the infarct area of the CMF pretreated MSCs
transplanted myocardia was on average 8.+-.2 per high power field
(40.times.) (HPF) on day 7. However, the new vessels were not
DiI-positive, indicating that the cellular source of the vessels
may not be derived from the donor cells. It is contemplated that
the donor MSCs may be activated by CMF-pretreatment to stimulate
and upregulate angiogenesis specific signaling pathways that induce
the expression of certain angiogenic factors, which directly
enhances the process of early revascularization in infarcted
myocardia. By contrast, approximately 3.+-.2 vessels per HPF were
observed in the infarcted myocardia of non-pretreated MSCs
transplantated controls on day 7 (FIG. 3: 2, yellow circles).
[0063] As shown in FIG. 3, a large number of donor cell derived
myocytes were clustered and organized into myocardial-like tissue
in the infarct area, which were positively stained by antibodies
specific to MHC (FIG. 3: 6, blue circles) and Ki67 (FIG. 3: 7, blue
circles), indicating that these transplanted CMF-pretreated MSCs
retained the division ability and committed cardiomyogenic
differentiation after transplantation in vivo. Under a high power
field, these myocardial-like tissues showed the typical morphology
of myocardium, except the size was smaller than undamaged existing
myocytes (FIG. 3: 9, blue circles). These highly organized
regenerating myocardium-like tissues occupied averagely 70.+-.8% of
the total infarct volume on day 7 and replaced the infarcted
myocardia by 80.+-.8.5% on average on day 14 post-infarction in the
test myocardium group (FIG. 3: 8, R, regenerated cardiac myocytes;
N, preexisting normal cardiac myocytes). This replacement of the
infarcted heart tissue was accompanied by significant functional
improvement, as demonstrated in the echocardiography measurements
(FIG. 4 & Table 1). In comparison with the non-pretreated MSCs
transplanted MI group on day 2 and 14 post infarction, ejection
fraction (EF) of the pretreated-MSCs-transplanted MI hearts was
significantly higher (59.79.+-.2.33 vs 52.1.+-.2.54, P=0.03) on day
2, and markedly increased (67.13.+-.2.53 vs 53.3.+-.2.31, P=0.001)
on day 14. Similarly, fraction shortening (FS) of the transplanted
MI heart were significantly higher (29.43.+-.1.35 vs 24.07.+-.1.47,
P=0.01) on day 2 and was significantly increased (31.72.+-.2.57 vs
23.49.+-.1.99, P=0.002) on day 14. The significant improvements in
EF and FS are a solid reflection of the functional recovery of the
cardiac myocytes (Table 1).
TABLE-US-00001 TABLE 1 The distribution of ejection fraction (EF)
and fractional shortening (FS). (mean .+-. _SE): EF (%) FS (%) 2
days.sctn. 14 days 2 days.sctn. 14 days Normal (16) 71.03 .+-. 4.05
68.24 .+-. 4.79 35.65 .+-. 3.99 34.02 .+-. 3.27 Sham (32) 70.45
.+-. 2.67 71.34 .+-. 2.77 36.03 .+-. 2.76 35.86 .+-. 2.13
CMF-pretreated (32) 59.79 .+-. 2.33* 67.13 .+-. 2.53* 29.43 .+-.
1.35* 31.72 .+-. 2.57* MSC control (32) 52.1 .+-. 2.54 53.3 .+-.
2.31 24.07 .+-. 1.47 23.49 .+-. 1.99 .sctn.Sixteen rats for normal
group and 32 rats for sham operated, CMF-pretreated and MSC control
groups respectively. , Eight rats for normal group and 16 rats for
sham operated, CMF-pretreated and MSC control groups respectively.
*EF, P = 0.03 on day 2; P = 0.001 on day 14, and FS, P = 0.01 on
day 2 and P = 0.002 on day 14.
IV. Direct Therapeutic Effects in MI Model without Pre-Treating
MSCs and Transplantation
[0064] Referring to FIG. 5, following direct local injection of CMF
in MI model, it was found two weeks post infarction that in the
control group (without CMF treatment) the myocardium on the distal
part of the ligation site became substantially white on visual
inspection due to ischemic necrosis (FIG. 5: 2). By contrast, the
equivalent part in CMF-treated hearts were relatively red in
appearance probably due to neovascularization (FIG. 5: 1), which
was comparable to the non-ischemic parts of the heart and the sizes
of infarct were significantly smaller than those in the control
hearts (FIG. 5: 2). Moreover, on transect of the infarct area, the
left ventricle walls of the CMF treated hearts (FIG. 5: 3) were
significantly thicker than those in control hearts (FIG. 5: 4).
Histological observations revealed that the infarct sizes in
CMF-treated hearts (n=8) were on average approximately 1/3-1/2
times smaller than those in the control hearts (n=8), as was
calculated by measuring the infarct volume in the left ventricular
free wall on day 14 after ligation. By Masson's Trichrome staining,
it was found that in CMF treated hearts, myocyte-like cell
clusters, which were arranged in almost the same orientation as the
infarcted myocardium or the neighboring viable myocardia, were
distributed in most parts of the whole infarct regions (FIG. 5: 5).
By contrast, the infarcted regions in control hearts were almost
completely occupied by fibrous tissue replacement, leaving almost
no space for any possible cardiomyocytes regeneration, if any (FIG.
5: 6). Under a higher power field, in a sharp contrast to the
overall blue stained fibrous scar in control group (FIG. 5: 8), the
whole infarct areas in the CMF treated group were filled with
regenerating myocyte clusters, well shaped to bear myocardial
morphology with little fibrous tissue in between (FIG. 5: 7),
although the sizes of these regenerating myocytes were smaller than
the neighboring preexisting myocytes, probably because they were
still on the way of maturing.
[0065] Furthermore, echocardiography demonstrated that the
replacement of infarcted heart tissue with structurally integrated
regenerating myocardia and reconstituted vasculatures was
accompanied by significant functional improvement by day 2
post-infarct in CMF-treated hearts, and further improvement by day
14 compared with control hearts, probably due to the growth and
maturation of the regenerated myocardia and vasculatures that
repaired the infarct.
Therapeutic Effect Via Conditioned Medium Induced by CMF-Treated
MSCs
[0066] To determine whether conditioned medium containing certain
induced proteins secreted by CMF-activated-MSCs would bring about
similar effects as CMF direct application or transplantation of the
CMF-pretreated-MSCs to the infarct area, the conditioned medium was
tested with both MSCs culture and heart infarction animal model.
Referring to FIG. 6a, after treatment with conditioned medium for
24 hrs, the proliferation rate of MSCs increased to 120% compared
to control medium (fresh growth medium). Referring to FIG. 6b,
local injection of conditioned medium to the ischemic region of MI
model, cardiomyocyte regeneration was observed. Briefly, many
regenerating cardiomyocytes and many newly formed vessels filled
with blood cells were observed in the whole infarct zone (FIG. 6b:
2) compared with the fibrous replacement in control (FIG. 6b:
1).
V. Cellular Origin of the Regenerated Myocardia after Direct CMF
Treatment
[0067] Referring to FIG. 7, studies on MI model with bone marrow
replacement with DiI labeled MSCs have provided direct evidence
that the cellular origin of the regenerating myocardium were
derived from bone marrow MSCs. One week after bone marrow
replacement, heart infarction surgery was performed as described in
the above. Fourteen days post infarction, it was found that the
whole infarct regions in CMF-treated hearts were well occupied by
DiI labeled cells, which were largely absent in any non-infarcted
regions of having the preexisting viable cardiomyocytes. These DiI
positive cells were clustered together bearing myocardial-like
morphology, but smaller in size compared with pre-existing
cardiomyocytes (FIG. 7: 1). By contrast, only a few scattered DiI
positive cells were observed along the infarct border zone in
control infarcted hearts (FIG. 7: 2). To confirm that these well
organized DiI positive cells were regenerating myocardia,
immunohistochemistry with antibodies specific to troponin I and
PCNA were performed. It was found that numerous DiI positive cells
distributed in the whole infarct zone were positively stained by
specific antibodies for troponin I (FIG. 7: 3) or PCNA (FIG. 7: 4).
These DiI and troponin I or PCNA positively stained cells were
organized into myocardial-like tissue in the whole infarct zone in
CMF treated hearts (FIGS. 7: 3 & 4). Under higher power field,
these regenerating myocardial-like tissues showed the typical
morphology of myocardium with clear intercalated disk connection
between regenerating myocytes, indicating the ultrastructural
maturation of the individual regenerating myocyte into integrated
myocardium (FIG. 7: 5). Without the structural integration between
the regenerating myocytes, and between the regenerating myocardium
and pre-existing viable myocardium, functional integration and
synchronous mechanical activity would not be guaranteed. These
regenerating myocardia occupied averagely 69.3% of the total
infarct volume on day 14 post-infarct. By contrast, in 6 control
hearts, only a few cells were both troponin I and DiI positive and
were scattered around the vessels while the infarction zone was
mainly occupied by fibrous scar (FIG. 7: 6). These results
demonstrated that the CMF induced regenerating myocardium was
functional and derived from bone marrow MSCs.
IV Manufacturing Pharmaceutical Compositions and their Uses in
Treating Ischemic Heart Diseases in Mammals
[0068] 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.
[0069] As used in the instant specification and claims, the term
"plant extract" means a mixture of natural occurring compounds
obtained 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 encompass an identified compound substantially
purified from the plant. 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 amount 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 under a
particular situation.
[0070] 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.
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